basic research examples

25 Basic Research Examples

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basic research examples and definition, explained below

Basic research is research that focuses on expanding human knowledge, without obvious practical applications.

For a scholarly definition, we can turn to Grimsgaard (2023):

“Basic research, also called pure, theoretical or fundamental research, tends to focus more on ‘big picture’ topics, such as increasing the scientific knowledge base around a particular topic.”

It is contrasted with applied research , which “seeks to solve real world problems” (Lehmann, 2023).

Generally, basis research has no clear economic or market value, meaning it tends to take place in universities rather than private organizations. Nevertheless, this blue-skies basic research can lead to enormous technological breakthroughs that forms the foundation for future applied research .

Basic Research Examples

Physics: Understanding the properties of neutrinos.
Medicine: Investigating the role of gut microbiota in mental health.
Anthropology: Studying the social structures of ancient civilizations.
Biology: Exploring the mechanism of CRISPR-Cas9 gene editing.
Psychology: Understanding the cognitive development in infants.
Chemistry: Researching new catalytic processes for organic synthesis.
Astronomy: Investigating the life cycle of stars.
Sociology: Exploring the impacts of social media on society.
Ecology: Studying the biodiversity in rainforests.
Computer Science: Developing new algorithms for machine learning.
Mathematics: Exploring new approaches to number theory.
Economics: Investigating the causes and effects of inflation.
Linguistics: Researching the evolution of languages over time.
Political Science: Studying the effects of political campaigns on voter behavior.
Geology: Investigating the formation of mountain ranges.
Architecture: Studying ancient building techniques and materials.
Education: Researching the impact of remote learning on academic performance.
History: Investigating trade routes in the medieval period.
Literature: Analyzing symbolism in 19th-century novels.
Philosophy: Exploring concepts of justice in different cultures.
Environmental Science: Studying the impact of plastics on marine life.
Genetics: Investigating the role of specific genes in aging.
Engineering: Researching materials for improving battery technology.
Art History: Investigating the influence of politics on Renaissance art.
Agricultural Science: Studying the impact of pest management practices on crop yield.

Case Studies

1. understanding the structure of the atom.

The study of atomic structure began in the early 1800s, with John Dalton’s atomic theory suggesting that atoms were indivisible and indestructible. However, it was not until the 20th century that Ernest Rutherford’s gold foil experiment led to the discovery of the nucleus and the proposal of the planetary model of the atom, which was further refined by Niels Bohr and eventually led to the quantum mechanical model, showing that electrons move in orbital shells around the nucleus.

Research Context:

Topic: Investigating the structure and behavior of atoms.
Purpose: Understand the fundamental particles (protons, neutrons, and electrons) and forces that govern atomic behavior.
Methodology: Utilize particle accelerators, theoretical models, and experimental physics.
Significance: Fundamental understanding of atomic structures has paved the way for numerous technological and scientific breakthroughs, such as the development of nuclear energy and advancements in chemistry and materials science.

Outcomes and Further Developments:

Discovery and exploration of subatomic particles like quarks.
Development of quantum mechanics and quantum field theory.
Subsequent advancements in various scientific fields, such as nuclear physics, chemistry, and nanotechnology.

2. Researching the Human Genome

The Human Genome Project, an international research effort that began in 1990, aimed to sequence and map all of the genes – collectively known as the genome – of humans. Completed in 2003, it represented a monumental achievement in science, providing researchers with powerful tools to understand the genetic factors in human disease, paving the way for new strategies for diagnosis, treatment, and prevention.

Topic: Investigating the structure, function, and mapping of the human genome.
Purpose: Understand the genetic makeup of humans, identify genes, and learn how they work.
Methodology: Techniques like DNA sequencing, genetic mapping, and computational biology.
Significance: Foundational for various advancements in genetics, medicine, and biology, providing insights into diseases, development, and evolution.
Completion of the Human Genome Project, which mapped the entire human genome.
Advancements in personalized medicine, genetic testing, and gene therapy.
Development of CRISPR technology, enabling precise genetic editing.

Basic Research vs Applied Research

Basic research focuses on expanding knowledge and understanding fundamental concepts without immediate practical application, while applied research focuses on solving specific, practical problems using the knowledge gained from basic research (Akcigit, Hanley & Serrano-Velarde, 2021).

A simple comparison of definitions is below:

Basic research seeks to gain greater knowledge or understanding of the fundamental aspects of phenomena.
Applied research seeks to solve practical problems the researcher or their stakeholders are facing.

A researcher might choose basic research over applied if their primary motivation is to expand the boundaries of human knowledge and contribute to academic theories, whilst they might favor applied research if they are more interested in achieving immediate solutions, innovations, or enhancements impacting real-world scenarios (Akcigit, Hanley & Serrano-Velarde, 2021; Baetu, 2016).

To learn more about applied research, check out my article on applied research.

Basic Research: Disappearing in 21st Century Universities?

In the 1980s, universities increasingly came under pressure to prove their specific financial value to society. This has only intensified over the decades. So, whereas once universities were preoccupied with basic research, there’s been a big push toward academic-industry collaborations where research demonstrates its economic value, rather than its cultural or intellectual value, to society. This may, on the one hand, help make universities relevant to today’s world. But on the other hand, it may interfere with the blue skies research that could identify and solve the bigger, less financially pressing, questions and problems of our ages (Bentley, Gulbrandsen & Kyvik, 2015).

Pros and Cons of Basic Research

The primary advantage of basic research is that it generates knowledge and understanding of fundamental principles that can later serve as a foundation for technological advancement or social betterment.

It can lead to groundbreaking discoveries, stimulate creativity, and drive scientific innovation by satisfying human curiosity (Akcigit, Hanley & Serrano-Velarde, 2021; Baetu, 2016).

It is also often the catalyst for training the next generation of scientists and researchers.

However, basic research can be time-consuming, expensive, and its outcomes may not always be directly observable or immediately beneficial.

This is why it’s often left to government-funded research institutes and universities to conduct this sort of research. As Binswanger (2014) argues, “basic research constitutes, for the most part, a common good which cannot be sold profitably on markets.

Furthermore, its value is often underestimated because the applications are not immediately apparent or tangible.

Below is a summary of some advantages and disadvantages of basic research:


Expands fundamental knowledge and understanding	May not have immediate practical applications (Hanley & Serrano-Velarde, 2021; Lehmann, 2023)
Drives technological and scientific innovation	Can be expensive and resource-intensive
Enables future applied research (Wild & Diggines, 2009)	Outcomes can be uncertain
Can lead to unexpected discoveries	May be deemed less prioritized during economic downturns
Enhances educational processes	Can be time-consuming (Abeysekera, 2019)
Promotes intellectual growth and stimulation	Research may become obsolete or be disproven in the future
Addresses curiosity and theoretical questions	May require specialized knowledge or equipment
Can inform policy and guide future research (Baetu, 2016; Lehmann, 2023)	Results might not be directly applicable or translatable to real-world problems (Akcigit, Hanley & Serrano-Velarde, 2021)
Encourages development of new methodologies	Ethical concerns may arise during the research
Boosts global knowledge and international collaboration	Competition for funding can hinder collaboration and data sharing

Abeysekera, A. (2019). Basic research and applied research. Journal of the National Science Foundation of Sri Lanka , 47 (3).

Akcigit, U., Hanley, D., & Serrano-Velarde, N. (2021). Back to basics: Basic research spillovers, innovation policy, and growth. The Review of Economic Studies , 88 (1), 1-43.

Baetu, T. M. (2016). The ‘big picture’: the problem of extrapolation in basic research. The British Journal for the Philosophy of Science.

Bentley, P. J., Gulbrandsen, M., & Kyvik, S. (2015). The relationship between basic and applied research in universities. Higher Education , 70 , 689-709. ( Source )

Binswanger, M. (2014). How nonsense became excellence: forcing professors to publish. In Welpe, I. M., Wollersheim, J., Osterloh, M., & Ringelhan, S. (Eds.), Incentives and Performance: Governance of Research Organizations . Springer International Publishing.

Grimsgaard, W. (2023). Design and strategy: a step by step guide . New York: Taylor & Francis.

Lehmann, W. (2023). Social Media Theory and Communications Practice . London: Taylor & Francis.

Wiid, J., & Diggines, C. (2009). Marketing Research . Juta.

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Basic Research: What it is with examples

In building knowledge, there are many stages and methodologies to generate insights that contribute to its understanding and advancement; basic research and applied research are usually the most effective on this path.

Understanding research allows us to understand all the properties of a specific science or phenomenon at a fundamental level. Some examples are branches such as sociology, humanities, and other scientific fields; below, we will tell you everything you need to know about this type of research and its possible applications.

What is Basic Research?

Basic Research is a type of research used in the scientific field to understand and extend our knowledge about a specific phenomenon or field. It is also accepted as pure investigation or fundamental research .

This type of research contributes to the intellectual body of knowledge. Basic research is concerned with the generalization of a theory in a branch of knowledge; its purpose is usually to generate data that confirm or refute the initial thesis of the study.

It can also be called foundational research; many things get built on this foundation, and more practical applications are made.

Basic Research vs. Applied Research

Basic Research finds its counterpart and complement in applied research. They are two handy research methods when generating and giving a utility to the generated data. There are very marked differences, and understanding them will allow you to understand the path followed to create new knowledge.

The most important difference between basic research and applied research lies in the objective of each. It seeks to expand the information and understanding of the object of study, while applied research aims to provide a solution to the problem studied.

The relationship between these two types of research is usually very close since the methodologies used are often quite similar; the significant change is found in the initial and final point of the investigation.

Basic Research Examples

There can be many examples of basic research; here are some of them:

A study of how stress affects labor productivity.
Studying the best factors of pricing strategies.
Understand the client’s level of satisfaction before certain interactions with the company providing solutions.
The understanding of the leadership style of a particular company.

Advantages & Disadvantages

Basic research is critical for expanding the pool of knowledge in any discipline. The introductory course usually does not have a strict period, and the researcher’s concern commonly guides them. The conclusion of the fundamental course is generally applicable in a wide range of cases and plots.

At the same time, the basic study has disadvantages as well. The findings of this type of study have limited or no constructive conclusions. In another sense, fundamental studies do not resolve complex and definite business problems, but it does help you understand them better.

Taking actions and decisions based on the results of this type of research will increase the impact these insights may have on the problem studied if that is the purpose.

LEARN ABOUT: Theoretical Research

How to do basic research?

This process follows the same steps as a standard research methodology. The most crucial point is to define a thesis or theory that involves a perfectly defined case study; this can be a phenomenon or a research problem observed in a particular place.

There are many types of research, such as longitudinal studies , observational research , and exploratory studies. So the first thing you should do is determine if you can obtain the desired result with research or if it is better to opt for another type of research.

Once you have determined your research methodology, the data collection process begins, also depending on your type of study; sometimes, you can collect the data passively through observation or experimentation. On other occasions, intervene directly and collect quantitative information with tools such as surveys.

Platforms like QuestionPro will help you have a wide range of functions and tools to carry out your research; its survey software has helped students and professionals obtain all the information necessary to generate high-value insights.

In addition, it has a data analysis suite with which you can analyze all this information using all kinds of reports for a more straightforward interpretation of the final results.

QuestionPro is much more than survey software ; we have a solution for each specific problem and industry. We also offer data management platforms such as our research data repository called Insights Hub.

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Basic Research in Psychology

Basic research—also known as fundamental or pure research—refers to study and research meant to increase our scientific knowledge base. This type of research is often purely theoretical, with the intent of increasing our understanding of certain phenomena or behavior. In contrast with applied research, basic research doesn't seek to solve or treat these problems.

Basic Research Examples

Basic research in psychology might explore:

Whether stress levels influence how often students engage in academic cheating
How caffeine consumption affects the brain
Whether men or women are more likely to be diagnosed with depression
How attachment styles among children of divorced parents compare to those raised by married parents

In all of these examples, the goal is merely to increase knowledge on a topic, not to come up with a practical solution to a problem.

The Link Between Basic and Applied Research

As Stanovich (2007) noted, many practical solutions to real-world problems have emerged directly from basic research. For this reason, the distinction between basic research and applied research is often simply a matter of time. As social psychologist Kurt Lewin once observed, "There is nothing so practical as a good theory."

For example, researchers might conduct basic research on how stress levels impact students academically, emotionally, and socially. The results of these theoretical explorations might lead to further studies designed to solve specific problems. Researchers might initially observe that students with high stress levels are more prone to dropping out of college before graduating. These first studies are examples of basic research designed to learn more about the topic.

As a result, scientists might then design research to determine what interventions might best lower these stress levels. Such studies would be examples of applied research. The purpose of applied research is specifically focused on solving a real problem that exists in the world. Thanks to the foundations established by basic research, psychologists can then design interventions that will help students effectively manage their stress levels , with the hopes of improving college retention rates.

Why Basic Research Is Important

The possible applications of basic research might not be obvious right away. During the earliest phases of basic research, scientists might not even be able to see how the information gleaned from theoretical research might ever apply to real-world problems. However, this foundational knowledge is essential. By learning as much as possible about a topic, researchers are able to gather what they need to know about an issue to fully understand the impact it may have.

"For example, early neuroscientists conducted basic research studies to understand how neurons function. The applications of this knowledge were not clear until much later when neuroscientists better understood how this neural functioning affected behavior," explained author Dawn M. McBride in her text The Process of Research in Psychology . "The understanding of the basic knowledge of neural functioning became useful in helping individuals with disorders long after this research had been completed."

Basic Research Methods

Basic research relies on many types of investigatory tools. These include observation, case studies, experiments, focus groups, surveys, interviews—anything that increases the scope of knowledge on the topic at hand.

Frequently Asked Questions

Psychologists interested in social behavior often undertake basic research. Social/community psychologists engaging in basic research are not trying to solve particular problems; rather, they want to learn more about why humans act the way they do.

Basic research is an effort to expand the scope of knowledge on a topic. Applied research uses such knowledge to solve specific problems.

An effective basic research problem statement outlines the importance of the topic; the study's significance and methods; what the research is investigating; how the results will be reported; and what the research will probably require.

Basic research might investigate, for example, the relationship between academic stress levels and cheating; how caffeine affects the brain; depression incidence in men vs. women; or attachment styles among children of divorced and married parents.

By learning as much as possible about a topic, researchers can come to fully understand the impact it may have. This knowledge can then become the basis of applied research to solve a particular problem within the topic area.

Stanovich KE. How to Think Straight About Psychology . 8th edition. Boston, MA: Pearson Allyn and Bacon; 2007.

McCain KW. “Nothing as practical as a good theory” Does Lewin's Maxim still have salience in the applied social sciences? Proceedings of the Association for Information Science and Technology . 2015;52(1):1-4. doi:10.1002/pra2.2015.145052010077

McBride DM. The Process of Research in Psychology . 3rd edition . Thousand Oaks, CA: Sage Publications; 2015.

Committee on Department of Defense Basic Research. APPENDIX D: Definitions of basic, applied, and fundamental research . In: Assessment of Department of Defense Basic Research. Washington, D.C.: The National Academic Press; 2005.

By Kendra Cherry, MSEd Kendra Cherry, MS, is a psychosocial rehabilitation specialist, psychology educator, and author of the "Everything Psychology Book."

Basic Research

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Basic research can be defined as systematic inquiry that involves a quest for some fundamental scientific aspects of phenomena without any specific practical applications in mind. The pay-off of basic research is often uncertain and, once published, difficult to appropriate. Accordingly, the social returns to basic research exceed the private returns, rendering it a ‘public good’. Basic research results in contributions to the world stock of scientific knowledge. It ultimately supports long-term economic growth, increased productivity and subsequent practical applications on a global basis.

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Research: Meaning and Purpose

Scientific Relevance

Arrow, K. 1962. Economic welfare and the allocation of resources for invention. In The rate and direction of inventive activity . Princeton: Princeton University Press.

Google Scholar

Bergman, E.M. 1990. The economic impact of industry-funded university R&D. Research Policy 19: 340–355.

Bush, V. 1945. Science: The endless frontier. A report to the president . Washington: US Government Printing Office. Available at http://www.nsf.gov/od/lpa/nsf50/vbush1945.htm

Cockburn, I., and R. Henderson. 1998. Absorptive capacity, coauthoring behavior, and the organization of research in drug discovery. Journal of Industrial Economics 46: 157–182.

Article Google Scholar

David, P., D. Mowery, and W.E. Steinmueller. 1992. Analysing the economic payoffs from basic research. Economics, Innovation and New Technology 2: 73–90.

Gibbons, M., and R. Johnston. 1974. The roles of science in technological innovation. Research Policy 3: 220–242.

Griliches, Z. 1986. Productivity, R&D, and basic research at the firm level in the 1970s. American Economic Review 76: 141–154.

Kline, S.J., and N. Rosenberg. 1986. An overview of innovation. In The positive sum strategy: Harnessing technology for economic growth , ed. R. Landau and N. Rosenberg. Washington, DC: National Academy Press.

Kuhn, R. 1962. The structure of scientific revolutions . Chicago: University of Chicago Press.

Mansfield, E. 1980a. Comments on returns to research and development expenditures. In New developments in productivity measurement , NBER Studies in Income and Wealth, vol. 44, ed. J.W. Kendrick and B.N. Vaccara. New York: National Bureau of Economic Research.

Mansfield, E. 1980b. Basic research and productivity increase in manufacturing. American Economic Review 70: 863–873.

March, J.G. 1991. Exploration and exploitation in organizational learning. Organization Science 2: 71–87.

Mowery, D., and N. Rosenberg. 1989. Technology and the pursuit of economic growth . Cambridge: Cambridge University Press.

Book Google Scholar

Nelson, R. 1959. The simple economics of basic scientific research. Journal of Political Economy 67: 297–306.

Nelson, R. 1962. The link between science and invention: The case of the transistor. In The rate and direction of inventive activity , ed. R. Nelson. Princeton: Princeton University Press.

Nelson, R. 1987. Understanding technical change as an evolutionary process . Amsterdam: North-Holland.

Nelson, R. 1990. Capitalism as an engine of progress. Research Policy 19: 193–214.

NSF (National Science Foundation). 1953. What is basic research? Available at http://www.nsf.gov/pubs/1953/annualreports/ar_1953_sec6.pdf . Accessed 17 Feb 2014.

NSF (National Science Foundation). 2010. Definitions: Research and development (R&D) definitions. In Globalization of science and engineering research , 9. Arlington: National Science Board Publication. Available at http://www.nsf.gov/statistics/nsb1003/pdf/nsb1003.pdf

OECD (Organisation for Economic Co-operation and Development). 1994. The measurement of scientific and technological activities: Standard practice for surveys of research and experimental development , Frascati Manual 1993. Paris: OECD.

Pavitt, K. 1991. What makes basic research economically useful? Research Policy 20: 109–119.

Rosenberg, N. 1982. Inside the Black Box: Technology and economics . Cambridge: Cambridge University Press.

Rosenberg, N. 1990. Why do firms do basic research (with their own money)? Research Policy 19: 165–174.

Rosenberg, N. 1992. Scientific instrumentation and university research. Research Policy 21: 381–390.

Teece, D.J. 1989. Inter-organizational requirements of the innovation process. Managerial and Decision Economics 10: 35–42.

Teece, D.J. 2003. Industrial research. In Dictionary of American history , 3rd ed, ed. S.I. Kutler. New York: Gale/Charles Scribner.

Trajtenberg, M., Henderson, R. and Jaffe, A. 1992. Ivory tower versus corporate lab: An empirical study of basic research and appropriability . National Bureau of Economic Research Working Paper, No. 4146 (August), Cambridge, MA.

Von Hippel, E. 1988. The sources of innovation . New York: Oxford University Press.

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Leih, S., Teece, D.J. (2018). Basic Research. In: Augier, M., Teece, D.J. (eds) The Palgrave Encyclopedia of Strategic Management. Palgrave Macmillan, London. https://doi.org/10.1057/978-1-137-00772-8_332

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Basic vs. applied research

Coding qualitative data for valuable insights

What is the difference between applied research and basic research?

Examples of basic research vs. applied research, basic vs. applied research: a comparative analysis, the interplay between basic and applied research, introduction.

Basic and applied research look at existing knowledge and create new knowledge in different ways. They share the same basic principles of contributing to knowledge through research findings, but their aims and objectives are distinctly different.

In the vast realm of scientific inquiry, research stands as the cornerstone for advancement, driving our understanding of the world and fostering innovation. At its core, research can be bifurcated into two primary types: applied and basic research . While both serve pivotal roles in contributing to our collective knowledge, they operate with distinct objectives and outcomes.

Any approach that is called basic research delves into the foundational principles and theories of science. It is driven by a researcher's curiosity and the aspiration to expand the frontiers of understanding. The primary goal isn't to solve an immediate problem but to garner knowledge for the sake of understanding.

On the other hand, applied research focuses on analysis intended to solve practical problems. Conducting applied research means seeking solutions to specific, tangible challenges that society or industries face. Using the principles derived from basic research, applied research aims to bring about real-world impact and deliver pragmatic solutions.

Basic research

Basic research, often called "pure" or "fundamental" research , is characterized by its intrinsic quest to unravel the mysteries of nature and society. It is an investigation into the very core of phenomena, aiming to discover new principles, theories, or facts without an immediate application in mind. This kind of research is often propelled by the researcher's curiosity, a thirst to understand the "why" and "how" of things, rather than the "what can we do with it."

Basic research has a relatively broad scope and aims to enhance the existing body of knowledge in a particular field. It's not about creating a new product, improving a process, or solving a current societal problem. Instead, it's about laying the groundwork for future investigations, paving the way for applied research to build upon. Basic research poses questions like, "What are the fundamental principles of this phenomenon?" or "How does this process work at different levels?"

Such goals provide the essential framework upon which much of our modern understanding and technological advancement rests. Without the exploratory and explanatory nature of basic research, the foundational knowledge needed to drive innovation would be missing.

Applied research

While basic research focuses on curiosity and the pursuit of knowledge for its own sake, applied research takes a different approach by examining how real-world phenomena or outcomes can be altered. At its core, applied research is oriented towards identifying practical solutions to specific problems. Its primary objective is not just to add to the existing knowledge base but to leverage that knowledge to develop solutions, innovations, or interventions that can be directly applied in the real world.

Applied research is deeply rooted in real-world issues. Whether it's finding a cure for a specific disease, developing a new technological solution for environmental challenges, or creating strategies to improve education in underprivileged communities, the primary goal is to generate practical outcomes that can be directly implemented. Its relevance is often immediately apparent, as it's tailored to answer particular challenges faced by society, industries, or organizations.

The line between basic and applied research can sometimes blur, especially when foundational discoveries from basic research lead directly to tangible applications. However, the main distinction lies in the intent: while basic research seeks to understand the fundamental nature of phenomena, applied research aims to harness that understanding for tangible benefits.

Applied research is invaluable as it accelerates the transition of theoretical knowledge into practical, impactful solutions. Through applied research, the abstract findings of basic research are transformed into actionable insights, tools, and technologies that shape our daily lives and address pressing challenges.

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Research in the social sciences encompasses a broad spectrum of topics, ranging from understanding human behavior and societal structures to exploring the dynamics of interpersonal relationships. Basic and applied research methods in the social sciences offer unique insights into these areas. Let's delve into some examples to understand their distinct approaches.

Basic research examples

The social construction of reality

A classic area of investigation in sociology is understanding how societies construct reality. This kind of research delves deep into the ways cultures, languages, and institutions shape our understanding of the world. It doesn't immediately aim to solve societal problems but provides essential insights into how perceptions and beliefs are formed. Research methods often used for this type of study include in-depth interviews , participant observations , and ethnographic studies .

Attachment theory in psychology

Attachment theory seeks to understand the deep emotional and physical attachment between a child and at least one primary caregiver. It delves into the nature of attachment and its implications for personal development. The research often involves longitudinal studies that observe behaviors over extended periods.

Applied research examples

Interventions for at-risk youth

Applied researchers might design programs or interventions to help at-risk youth, building on the foundational knowledge of psychology, sociology, and education. The research might involve evaluating the effectiveness of a particular program, using methods like surveys , focus groups , and pre-and-post assessments.

Communication strategies for public health

Understanding human behavior is crucial for successful public health campaigns. Researchers might study the best ways to communicate vital health information to various populations, especially in times of crisis like pandemics. Methods often include A/B testing of messages, surveys to assess message efficacy, and observational studies to gauge real-world behavior following communication campaigns.

The distinction between basic and applied research is not just a matter of intent or outcome; it also encompasses differences in methodologies , scopes, and approaches. Let's undertake a comparative analysis to illuminate these distinctions further, particularly in the context of the social sciences.

Purpose and motivation

Basic research is motivated by the quest for knowledge. It seeks to answer fundamental questions about human behavior, societal structures, and the interplay between various social factors. The driving force here is curiosity. In contrast, applied research is driven by the need to address specific societal or practical problems. Its purpose is to take the theoretical knowledge derived from basic research and convert it into actionable solutions.

Methodological approaches

It's important to acknowledge that there is no one universal research method that can address all potential research inquiries. Moreover, the same research methods, such as conducting interviews or engaging in inductive and deductive reasoning , can be utilized in basic and applied research, but they will differ in their scope and objectives. While applied research is more experimental or confirmatory, a basic research approach is often exploratory or explanatory in nature. Basic research methods include ethnography , in-depth interviews , or longitudinal studies to gain a deep understanding of a topic. The focus is on generating theories and understanding patterns.

Applied research, on the other hand, often employs more structured and targeted methodologies. Surveys , experiments, and evaluations are commonly used to verify propositions, assess the efficacy of interventions, or gauge public opinion. The approach is more pragmatic, seeking results that can inform decisions and guide actions.

Outcomes and results

Basic research outcomes are usually theoretical contributions: new concepts, theories, or insights into existing phenomena. The results expand the academic literature and provide a foundation for future studies.

Applied research results in tangible solutions or recommendations. The outcomes might include a new social program, policy recommendations, interventions, or communication strategies. The results are geared towards immediate implementation and often have direct implications for organizations, governments, or communities.

The discourse on basic and applied research often sets them apart, emphasizing their distinct objectives and methodologies. However, it's crucial to recognize that these research types aren't isolated from each other. They coexist in a symbiotic relationship, where the findings from basic research often provide the foundational knowledge for applied research, and the results of applied research can inspire further basic investigations.

The transition of knowledge

One of the most notable instances of the interplay is how basic research's findings become the bedrock for applied research projects. For example, a basic research study on cognitive development in children might reveal specific patterns or stages. An applied researcher, recognizing the implications of these findings, could then design educational interventions tailored to these developmental stages.

How one complements the other

Basic research pushes the boundaries of our understanding, expanding the horizon of what we know. Applied research, on the other hand, can reframe this expansive knowledge and make it relevant and actionable for society's immediate needs.

But the relationship is reciprocal. Applied research can also highlight gaps in our understanding, pointing out areas where basic research is needed. For instance, if an intervention designed based on current knowledge fails to achieve its intended results, it signals to basic researchers that there might be underlying factors or dynamics not yet understood.

The dynamic continuum

Instead of viewing basic and applied research as two separate entities, it's more accurate to see them as points on a continuum. The knowledge generated by basic research flows towards applied projects, which in turn can inspire further basic investigations. This dynamic loop ensures that research in the social sciences remains both grounded in fundamental understanding and relevant to real-world challenges.

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National Research Council (US) Committee to Update Science, Medicine, and Animals. Science, Medicine, and Animals. Washington (DC): National Academies Press (US); 2004.

Science, Medicine, and Animals.

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The Concept of Basic Research

A nimal research is also important in another type of research, called basic research. Basic research experiments are performed to further scientific knowledge without an obvious or immediate benefit. The goal of basic research is to understand the function of newly discovered molecules and cells, strange phenomena, or little-understood processes. In spite of the fact that there may be no obvious value when the experiments are performed, many times this new knowledge leads to breakthrough methods and treatments years or decades later. For example, chemists developed a tool called a nuclear magnetic resonance (NMR) machine to determine the structure of chemicals. When it was developed, it had no obvious applications in medicine; however, scientists eventually realized that the NMR machine could be hooked up to a computer to make a magnetic resonance imagery (MRI) machine. The MRI machine takes pictures of the bone and internal tissues of the body without the use of radioactivity. Other examples of basic research that have led to important advances in medicine are the discovery of DNA (leading to cancer treatments) and neurotransmitters (leading to antidepressants and antiseizure medications). However, there are many other instances where basic research, some of which has been done on animals, has not yet resulted in any practical benefit to humans or animals.

NMR (nuclear magnetic resonance)—a machine that measures the vibration of atoms exposed to magnetic fields. Scientists use this machine to study the physical, chemical, and biological properties of matter.

MRI (magnetic resonance imaging)—a machine that produces pictures of the bone and internal tissues of the body.

Cite this Page National Research Council (US) Committee to Update Science, Medicine, and Animals. Science, Medicine, and Animals. Washington (DC): National Academies Press (US); 2004. The Concept of Basic Research.
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Science, Medicine, and Animals (2004)

Chapter: the concept of basic research, the concept of basic research.

MRI (magnetic resonance imaging)—a machine that produces pictures of the bone and internal tissues of the body.

Science, Medicine, and Animals explains the role that animals play in biomedical research and the ways in which scientists, governments, and citizens have tried to balance the experimental use of animals with a concern for all living creatures. An accompanying Teacher's Guide is available to help teachers of middle and high school students use Science, Medicine, and Animals in the classroom. As students examine the issues in Science, Medicine, and Animals, they will gain a greater understanding of the goals of biomedical research and the real-world practice of the scientific method in general.

Science, Medicine, and Animals and the Teacher's Guide were written by the Institute for Laboratory Animal Research and published by the National Research Council of the National Academies. The report was reviewed by a committee made up of experts and scholars with diverse perspectives, including members of the U.S. Department of Agriculture, National Institutes of Health, the Humane Society of the United States, and the American Society for the Prevention of Cruelty to Animals. The Teacher's Guide was reviewed by members of the National Academies' Teacher Associates Network.

Science, Medicine, and Animals is recommended by the National Science Teacher's Association .

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Understanding basic research

Last updated

8 February 2023

Reviewed by

Cathy Heath

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Research is an essential activity for all of us. It's how we foster curiosity, gather information, learn about the world, and find solutions. There are many different types of research, but they all fall into one of two categories: basic and applied.

This article will look at basic research, what it is, how we use it, and how it compares to applied research.

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What is basic research?

Basic research is all about information-gathering and answering What, Why, and How research questions. It involves learning the facts about a subject, finding out why things happen, perhaps by investigation or observation, and then using this expanded knowledge to better understand the topic.

Basic research is also called fundamental, foundational, or pure research.

Where is basic research used?
What are examples of basic research?

Studying how a client moves through the sales funnel process

Examining the components of a cell

Analyzing performance before and after coffee consumption

Looking at how stress affects productivity

Determining which areas of a country are driest and wettest

Understanding how a doctor makes a mental health diagnosis

What are basic research methods?

You can conduct basic research using several different methods. The best method will depend on what you are studying and what questions you are trying to answer.

Some methods available for basic research include:

Experimentation

Conducting a survey or interview

Observation

What is the value of basic research?

Basic research teaches us about the world around us. It helps us gather more primary data about a subject, which we can use later in applied research. Without that basic information on a subject, we wouldn't have the data we need to make informed decisions.

That's why basic research is often called foundational research . The information we acquire through basic research gives us a foundation of knowledge we can build on in the future.

Is basic research qualitative or quantitative?

Basic research can be qualitative, quantitative, or both.

Quantitative data deals with quantities or numbers. You could chart this data on a graph, using measurements such as:

Other numerical data

Qualitative data deals with qualities . It focuses more on the language and sentiment found in:

Interview responses

Observations about human behavior

Verbal answers to surveys

You can get qualitative and quantitative data from basic research, depending on what you are researching and the methods you use.

Basic research vs. applied research

Where basic research gathers information and data on a subject, applied research uses that data to look for answers to questions. Applied research takes the data obtained in basic research and applies it to answer a question and provide a possible solution.

There are three types of applied research:

Evaluation research: determining how well something is working or what it’s worth

Action research: understanding how to improve a process, for example in education or business

Research and development: looking at new products and services that businesses can offer consumers to solve a problem

Applied research often hinges on data collected during basic research. For example, you might gather data on how customers move through the sales funnel. The information you learn about the customer journey comes from your basic research, which you might gather through website analytics and customer interviews.

Now you want to know the best method for bringing more people into the sales funnel. You could use applied research to determine if it's better to increase your digital ad spending, send more emails, or use telemarketing to capture more sales leads.

Where is applied research used?

As applied research is solution-based, agencies concerned with medical research, psychology, and education all use it to improve lives. For businesses, applied research is the foundation of research and development (R&D) departments that are looking to create new consumer products and services.

You can also use applied research in your everyday life. You might use basic research to gather data on how caffeine affects your sleeping patterns, then use applied research techniques to determine how to get a better night's sleep.

Examples of applied research

Examples of applied research include:

Identifying new products to help aging seniors stay safe at home

Looking at ways to treat a medical condition safely

Determining how to make products last longer

Examining the best ways to prevent Type 2 diabetes

Understanding how to engage teens in math classes

How to market a product to Gen Z

Understanding how businesses could help reduce their impact on climate change

What is the value of applied research?

Applied research's value lies in its ability to help us solve problems with data-backed solutions. However, without comprehensive basic research at the outset, we wouldn't have the information we need to find those solutions. This illustrates why basic research and applied research often work hand in hand, supporting research efforts and validating better outcomes.

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Methodology

Research Methods | Definitions, Types, Examples

Research methods are specific procedures for collecting and analyzing data. Developing your research methods is an integral part of your research design . When planning your methods, there are two key decisions you will make.

First, decide how you will collect data . Your methods depend on what type of data you need to answer your research question :

Qualitative vs. quantitative : Will your data take the form of words or numbers?
Primary vs. secondary : Will you collect original data yourself, or will you use data that has already been collected by someone else?
Descriptive vs. experimental : Will you take measurements of something as it is, or will you perform an experiment?

Second, decide how you will analyze the data .

For quantitative data, you can use statistical analysis methods to test relationships between variables.
For qualitative data, you can use methods such as thematic analysis to interpret patterns and meanings in the data.

Methods for collecting data, examples of data collection methods, methods for analyzing data, examples of data analysis methods, other interesting articles, frequently asked questions about research methods.

Data is the information that you collect for the purposes of answering your research question . The type of data you need depends on the aims of your research.

Qualitative vs. quantitative data

Your choice of qualitative or quantitative data collection depends on the type of knowledge you want to develop.

For questions about ideas, experiences and meanings, or to study something that can’t be described numerically, collect qualitative data .

If you want to develop a more mechanistic understanding of a topic, or your research involves hypothesis testing , collect quantitative data .


Qualitative		to broader populations. .
Quantitative		.

You can also take a mixed methods approach , where you use both qualitative and quantitative research methods.

Primary vs. secondary research

Primary research is any original data that you collect yourself for the purposes of answering your research question (e.g. through surveys , observations and experiments ). Secondary research is data that has already been collected by other researchers (e.g. in a government census or previous scientific studies).

If you are exploring a novel research question, you’ll probably need to collect primary data . But if you want to synthesize existing knowledge, analyze historical trends, or identify patterns on a large scale, secondary data might be a better choice.


Primary	.	methods.
Secondary

Descriptive vs. experimental data

In descriptive research , you collect data about your study subject without intervening. The validity of your research will depend on your sampling method .

In experimental research , you systematically intervene in a process and measure the outcome. The validity of your research will depend on your experimental design .

To conduct an experiment, you need to be able to vary your independent variable , precisely measure your dependent variable, and control for confounding variables . If it’s practically and ethically possible, this method is the best choice for answering questions about cause and effect.


Descriptive		. .
Experimental

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Research methods for collecting data
Research method	Primary or secondary?	Qualitative or quantitative?	When to use
	Primary	Quantitative	To test cause-and-effect relationships.
	Primary	Quantitative	To understand general characteristics of a population.
Interview/focus group	Primary	Qualitative	To gain more in-depth understanding of a topic.
Observation	Primary	Either	To understand how something occurs in its natural setting.
	Secondary	Either	To situate your research in an existing body of work, or to evaluate trends within a research topic.
	Either	Either	To gain an in-depth understanding of a specific group or context, or when you don’t have the resources for a large study.

Your data analysis methods will depend on the type of data you collect and how you prepare it for analysis.

Data can often be analyzed both quantitatively and qualitatively. For example, survey responses could be analyzed qualitatively by studying the meanings of responses or quantitatively by studying the frequencies of responses.

Qualitative analysis methods

Qualitative analysis is used to understand words, ideas, and experiences. You can use it to interpret data that was collected:

From open-ended surveys and interviews , literature reviews , case studies , ethnographies , and other sources that use text rather than numbers.
Using non-probability sampling methods .

Qualitative analysis tends to be quite flexible and relies on the researcher’s judgement, so you have to reflect carefully on your choices and assumptions and be careful to avoid research bias .

Quantitative analysis methods

Quantitative analysis uses numbers and statistics to understand frequencies, averages and correlations (in descriptive studies) or cause-and-effect relationships (in experiments).

You can use quantitative analysis to interpret data that was collected either:

During an experiment .
Using probability sampling methods .

Because the data is collected and analyzed in a statistically valid way, the results of quantitative analysis can be easily standardized and shared among researchers.

Research methods for analyzing data
Research method	Qualitative or quantitative?	When to use
	Quantitative	To analyze data collected in a statistically valid manner (e.g. from experiments, surveys, and observations).
Meta-analysis	Quantitative	To statistically analyze the results of a large collection of studies. Can only be applied to studies that collected data in a statistically valid manner.
	Qualitative	To analyze data collected from interviews, , or textual sources. To understand general themes in the data and how they are communicated.
	Either	To analyze large volumes of textual or visual data collected from surveys, literature reviews, or other sources. Can be quantitative (i.e. frequencies of words) or qualitative (i.e. meanings of words).

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If you want to know more about statistics , methodology , or research bias , make sure to check out some of our other articles with explanations and examples.

Chi square test of independence
Statistical power
Descriptive statistics
Degrees of freedom
Pearson correlation
Null hypothesis
Double-blind study
Case-control study
Research ethics
Data collection
Hypothesis testing
Structured interviews

Research bias

Hawthorne effect
Unconscious bias
Recall bias
Halo effect
Self-serving bias
Information bias

Quantitative research deals with numbers and statistics, while qualitative research deals with words and meanings.

Quantitative methods allow you to systematically measure variables and test hypotheses . Qualitative methods allow you to explore concepts and experiences in more detail.

In mixed methods research , you use both qualitative and quantitative data collection and analysis methods to answer your research question .

A sample is a subset of individuals from a larger population . Sampling means selecting the group that you will actually collect data from in your research. For example, if you are researching the opinions of students in your university, you could survey a sample of 100 students.

In statistics, sampling allows you to test a hypothesis about the characteristics of a population.

The research methods you use depend on the type of data you need to answer your research question .

If you want to measure something or test a hypothesis , use quantitative methods . If you want to explore ideas, thoughts and meanings, use qualitative methods .
If you want to analyze a large amount of readily-available data, use secondary data. If you want data specific to your purposes with control over how it is generated, collect primary data.
If you want to establish cause-and-effect relationships between variables , use experimental methods. If you want to understand the characteristics of a research subject, use descriptive methods.

Methodology refers to the overarching strategy and rationale of your research project . It involves studying the methods used in your field and the theories or principles behind them, in order to develop an approach that matches your objectives.

Methods are the specific tools and procedures you use to collect and analyze data (for example, experiments, surveys , and statistical tests ).

In shorter scientific papers, where the aim is to report the findings of a specific study, you might simply describe what you did in a methods section .

In a longer or more complex research project, such as a thesis or dissertation , you will probably include a methodology section , where you explain your approach to answering the research questions and cite relevant sources to support your choice of methods.

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Psychological Theories

What are psychological theories.

Psychological theories are systematic explanations of human mental processes and behaviors, developed through both empirical research and field observation. These theories provide frameworks for understanding how and why people think, feel, and act the way they do, and guide both academic research and practical applications in areas like therapy, education, marketing, and public policy.

The Basic Idea

Theory, meet practice.

TDL is an applied research consultancy. In our work, we leverage the insights of diverse fields—from psychology and economics to machine learning and behavioral data science—to sculpt targeted solutions to nuanced problems.

If you’ve ever snoozed your alarm and then been late to work or school, you may look back and ask yourself: why did I do that? Maybe your bus driver saw you running for the bus and chose to keep on driving, and you ask yourself: what was going through his mind? Or maybe the day turned out okay, because you came home to your loving partner, and you thought to yourself: why am I so compatible with this person but not others? To begin unpacking these questions and more, we can turn to psychological theories to help us understand ourselves and the people around us.

Psychological theories are systematic frameworks for understanding, predicting, and explaining human behavior and mental processes. These theories include everything from cognitive theories , which focus on mental processes such as perception and memory, to behavioral theories , which examine the relationship between stimuli and responses. Although there are many ways to explain how and why we are the way we are, it is the constant testing and refining of different psychological theories that guides research and helps us to consistently improve our understanding of humans—both within academia and beyond.

Most Influential Psychological Theories:

Psychoanalytic Theory (Sigmund Freud): Focuses on the influence of the unconscious mind on behavior and uses concepts like the id, ego, superego, and psychosexual stages of development.
Behaviorism (John B. Watson, B.F. Skinner): Emphasizes the study of observable behaviors and the role of environmental stimuli in shaping behavior, including classical conditioning and operant conditioning.
Cognitive Development Theory (Jean Piaget): Explains how children's thinking evolves as they grow, identifying four stages of cognitive development (sensorimotor, preoperational, concrete operational, and formal operational).
Humanistic Psychology (Carl Rogers, Abraham Maslow): Emphasizes individual potential, self-actualization, and the importance of personal growth and free will. This also includes Maslow's hierarchy of needs and Rogers' client-centered therapy.
Social Learning Theory (Albert Bandura): Proposes that people learn behaviors through observation, imitation, and modeling, rather than solely through direct reinforcement and uses reciprocal determinism, where behavior, personal factors, and the environment interact and influence each other.

The world makes much less sense than you think. The coherence comes mostly from the way your mind works. – Daniel Kahneman

Cognition: Mental processes involved in gaining knowledge and comprehension, including thinking, knowing, remembering, judging, and problem-solving.

Psychoanalysis: A therapy developed aimed at exploring the unconscious mind to understand and treat psychological disorders.

Positive Reinforcement : In behaviorism, the process of encouraging or establishing a pattern of behavior by offering a reward when the desired behavior is exhibited.

Montessori Method: An educational approach developed by Dr. Maria Montessori that emphasizes self-directed learning, hands-on activities, creativity, and a love for learning and collaborative play in a child-centered environment.

Herzberg's Motivation Theory: Also known as the two-factor theory, this theory proposes that job satisfaction is influenced by two distinct sets of factors: hygiene factors and motivator factors. Hygiene factors pertain to external conditions such as the workplace environment and salary, while motivator factors are typically intangible elements like receiving recognition or opportunities for personal growth.

As long as there have been humans, there have been questions about why we think and act the way we do. There have likely been countless theories developed throughout time that we have no written record of, such as those developed by native peoples. For many, the known history of psychological theories goes back to ancient philosophical inquiries about the human mind and behavior. Early thinkers like Plato and Aristotle laid the groundwork for understanding the mind's complexities, focusing on issues of perception, memory, and motivation. Hundreds of years later, the 17th century marked a significant shift with the rise of empiricism, championed by philosophers like John Locke who proposed that knowledge is derived from sensory experience. This idea laid the foundation for later psychological theories that emphasize the role of the environment in shaping behavior. 1

The formal birth of psychology as a scientific discipline is often credited to Wilhelm Wundt, who established the first psychology laboratory in Leipzig, Germany, in 1879. Wundt's work marked the beginning of experimental psychology, where he used introspection to explore the structure of the conscious mind. His approach, known as structuralism, aimed to break down mental processes into their most basic components. Around the same time, William James in the United States was developing his own approach called functionalism, which focused on the purpose of consciousness and behavior in helping individuals adapt to their environment. These early schools of thought laid the groundwork for more complex psychological theories. 1

The early 20th century saw the emergence of several influential psychological theories that have shaped the field as we know it today. Sigmund Freud's psychoanalytic theory introduced the idea of the unconscious mind and the role of early childhood experiences in shaping personality (he’s the guy who you usually picture talking to a patient while they lay on a couch, discovering a repressed memory). While Freud's ideas were revolutionary, they were (and still are) controversial, spurring the development of alternative theories. Behaviorism, led by John Watson and later B.F. Skinner, rejected the introspective methods of the past and focused instead on observable behavior, emphasizing the role of environmental stimuli in shaping actions. Behaviorism dominated psychology from the 1920s to the 1950s, particularly in the United States, influencing everything from education to advertising. 1

Toward the mid-20th century emerged a cognitive revolution, as cognitive psychologists began challenging the behaviorist movement by reintroducing the importance of mental processes. Pioneers like Jean Piaget and Noam Chomsky argued that studying internal cognitive processes (like thinking, memory, and language) was crucial for a complete understanding of behavior. This shift led to the development of cognitive psychology, which remains one of the most prominent areas of the field today. Additionally, humanistic psychology, which was considered more of a ‘counter-movement’ to behaviorism, emerged during this time, with figures like Carl Rogers and Abraham Maslow emphasizing personal growth, self-actualization, and the inherent goodness of people. All of these diverse perspectives have contributed to a more comprehensive and multifaceted understanding of human behavior, continuing to influence psychological research and practice today. 2

Plato (c. 427-347 BCE) : Greek philosopher from Athens known for his works on philosophy and the mind, including the theory of forms and the allegory of the cave.

Aristotle (384-322 BCE) : Greek philosopher from Stagira who studied under Plato and is renowned for his contributions to logic, metaphysics, and psychology, particularly his theory of the soul.

John Locke (1632-1704) : English philosopher, often called the "father of liberalism," known for his theory of empiricism, which posits that knowledge is derived from sensory experience.

Wilhelm Wundt (1832-1920) : German psychologist, often regarded as the "father of modern psychology," who established the first psychology laboratory and developed the theory of structuralism.

William James (1842-1910) : American philosopher and psychologist, known as the "father of American psychology," who founded the school of functionalism and authored the first psychology textbook, The Principles of Psychology.

Sigmund Freud (1856-1939) : Austrian neurologist and the founder of psychoanalysis, known for his theories on the unconscious mind, psychosexual development, and defense mechanisms.

John B. Watson (1878-1958) : American psychologist, best known for founding behaviorism, which focuses on the study of observable behavior rather than internal mental processes.

B.F. Skinner (1904-1990) : American psychologist and behaviorist, known for developing the theory of operant conditioning and his work on reinforcement and punishment.

Jean Piaget (1896-1980) : Swiss psychologist, famous for his theory of cognitive development, which outlines how children's thinking evolves through distinct stages.

Noam Chomsky (b. 1928) : American linguist, philosopher, and cognitive scientist, known for his theory of universal grammar and his critique of behaviorism in language acquisition.

Carl Rogers (1902-1987) : American psychologist, one of the founders of humanistic psychology, known for his client-centered therapy and emphasis on self-actualization.

Abraham Maslow (1908-1970) : American psychologist, best known for creating Maslow's hierarchy of needs, a theory that outlines the stages of human motivation from basic needs to self-actualization.

Consequences

The impact of psychological theories extends far beyond academic research; scientifically understanding our own thoughts and behavior has influenced almost all aspects of modern life, shaping practices in education, marketing, public policy, and business.

In the realm of education, psychological theories have revolutionized teaching and learning methodologies. For example, certain developmental theories have provided educators with insights into how children think and learn at different stages. Jean Piaget’s theory of cognitive development suggests that children progress through specific stages of cognitive growth, with each stage characterized by distinct thinking patterns. 3 Understanding these stages allows teachers to adjust their teaching strategies to better match the cognitive capacity of their students.

Meanwhile, Lev Vygotsky’s concept of the zone of proximal development explains the gap between what a student can learn on their own versus with help. This theory posits that the role of education is to provide children with experiences that are in their proximal development stage, encouraging and advancing individual learning through social interaction. Students can solve problems independently, applying knowledge from conversations with peers and teachers to gradually develop the skills to perform tasks without direct help. 4 Understanding this theory has helped shape the role of teachers in the classroom; sometimes, it’s more about putting students in a situation where they can teach themselves to succeed rather than being explicitly taught.

For better or worse, psychological theories have also left a huge mark on the field of marketing and consumer behavior. Behaviorism, with its focus on conditioning and reinforcement, has been particularly influential in understanding and shaping consumer habits. Marketers have applied principles of operant conditioning to design reward systems—like loyalty programs or memberships—that encourage repeat purchases by reinforcing desired behaviors. The understanding of cognitive biases, such as the availability heuristic and the framing effect , has also allowed many marketers to craft persuasive messages that influence buyer decision-making. All of these psychological insights have helped businesses increase sales and foster brand loyalty—potentially misleading or even taking advantage of customers.

Public policy

Public policy is another major area where psychological theories have had significant consequences. Understanding human behavior has helped policymakers create more effective campaigns to improve public health, environmental protection, education systems, and programs that better address social issues such as poverty and crime. The application of small interventions that capitalize on our biases in a nonrestrictive way (often referred to as “ nudging ”), has gained prominence in recent years. This approach, popularized by Richard Thaler and Cass Sunstein, is based on the idea that small changes in the way choices are presented can have a substantial impact on behavior, like automatically enrolling employees in retirement savings plans, with the option to opt-out. A change as simple as this has been shown to dramatically increase participation rates in retirement plans.

Organizations

If you’re in a traditional workplace, you may have experienced the influence of psychological theories in your office without realizing it. Organizational behavior and human resource management have been hugely shaped by Maslow’s hierarchy of needs and Herzberg’s two-factor theory. Both of these theories have changed how companies understand and manage employee motivation and satisfaction. Maslow’s theory suggests that employees (and all humans) are motivated by a hierarchy of needs, starting with basic physiological needs like food and water and progressing to self-actualization. Thus, companies that recognize and address these needs by providing a safe working environment, opportunities for social interaction, and chances for personal growth, are more likely to foster a motivated and productive workforce (perhaps this is why having free coffee and snacks in the office is so motivating?). Herzberg’s two-factor theory, which distinguishes between hygiene factors (e.g., salary, work conditions) and motivators (e.g., recognition, achievement), has also guided organizations in designing jobs and work environments to maximize employee satisfaction.

Controversies

Psychological theories, while important to our understanding of human behavior, are theories, not facts. We can still only speculate and make educated guesses as to how and why our brains function the way they do. Many of the theories we’ve previously developed haven’t withstood the test of time, or at least not without serious criticism.

Psychoanalytic Theory

One of the most contentious figures in psychology is Freud, whose psychoanalytic theory has faced huge criticism. Although Freud's focus on the unconscious mind and his theories of psychosexual development were groundbreaking at the time, they were also largely unscientific and overly deterministic. Critics argue that many of Freud’s ideas, such as the Oedipus complex (briefly, that boys are repressing attraction to their moms and jealousy of their fathers’ genitals) and the emphasis on sexual drives, lack empirical support and are difficult if not impossible to test systematically. His theories have also been accused of being culturally biased, reflecting the patriarchal and sexually repressive values of his time, which are largely not applicable (or at least hopefully less so) today.

Behaviorism

Behaviorism, another major psychological theory, has also faced significant criticism, particularly for its reductionist approach. Watson and Skinner’s theory focuses exclusively on observable external behaviors and their environmental conditioning, largely ignoring the importance of internal mental processes like emotion. To no surprise, humans have a lot going on under the surface, and not taking this into account can be problematic. That’s because we are much more than machines reacting directly to the environment around us, and internal or unobservable processes (like our preferences, hunger, hormones, and social upbringing) can have dramatic influences on the way we react to stimuli. This critique helped propel the cognitive revolution to take into account the other missing piece of the puzzle: the mind.

Cognitive Theory

Enter cognitive psychology, which has also been criticized for its heavy reliance on computational models of the mind, which can oversimplify the complexities of human cognition—after all, the brain is not a computer. Since these models usually compare the mind to a computer processing information, they’ve also been accused of neglecting the emotional, social, and cultural factors that influence thought and behavior. Thus, cognitive psychology, just like behaviorism before it, tends to focus on “universal” principles—when, of course, there are huge individual differences among people, places, and cultures.

Humanistic Psychology

Humanistic psychology emerged as a response to both behaviorism and psychoanalysis to introduce a more optimistic view of human nature—but hasn’t escaped its share of criticisms. While prominent figures like Rogers and Maslow emphasized the potential for personal growth and self-actualization, proposing that people are inherently good and capable of achieving their full potential, many critics argue that this perspective is overly idealistic. Much like Freud’s psychoanalysis, it can be incredibly hard to test humanistic psychology empirically which can make it even harder to find acceptance from the scientific community. Also, due to the focus on the individual’s growth and self-fulfillment, it can be easy to overlook the social and structural factors that limit personal development; essentially, the individual is responsible for their own shortcomings or inadequacies, ignoring systemic issues like poverty, discrimination, and lack of access to education.

Cultural Context

Much of the existing psychology research is on a WEIRD (Western, Educated, Industrialized, Rich, and Democratic) population, which has raised concerns about the generalizability of its findings to non-WEIRD populations. All theories involve some sort of bias, and no population will ever be truly representative. The role of psychological theories in perpetuating or challenging societal norms is a controversial subject because, while the field has contributed to understanding and reducing prejudice, critics argue that some research in social psychology specifically can inadvertently reinforce stereotypes or fail to account for cultural and contextual differences.

For example, the application of Maslow’s hierarchy of needs discussed earlier posits that individuals prioritize their needs in a specific order, starting with basic physiological needs and moving up through safety, love and belonging, esteem, and self-actualization. This model aligns well with WEIRD cultures, which tend to emphasize individualism and personal achievement since cultural values prioritize personal growth and self-fulfillment. However, in more collectivist cultures, where community, family, and social harmony are often prioritized over individual needs, Maslow’s hierarchy doesn’t fit quite as well. In addition, studies that categorize people into rigid social groups based on race, gender, or socioeconomic status may unintentionally perpetuate the very biases they aim to study. These critiques have led to a growing recognition of the need for more culturally sensitive research and a broader, more inclusive approach to studying behavior.

Behavioral Psychology in Education

Behavioral psychology—particularly the principles of operant conditioning—has been instrumental in shaping modern educational practices. Operant conditioning, a theory developed by Skinner, posits that behavior is influenced by the consequences that follow it. Specifically, reinforcement increases the likelihood of a behavior being repeated, while punishment decreases it. This approach can be applied to classroom management when teachers try to promote positive behavior that leads to academic achievement.

For instance, many schools implement reward systems like tokens or points that use positive reinforcement to encourage good behavior like completing homework on time, participating in class discussions, or staying silent during reading times. Usually, these tokens are traded in for rewards like extra recess time, which can help keep kids motivated. We can also see how punishment systems like detention or extra homework can be used to discourage unwanted or disruptive behavior. All of these systems are designed based on our understanding of different psychological theories, from how we best learn to which tools are appropriate for which ages, and have informed interventions for students with special needs, behavioral disorders, or learning disabilities.

The Montessori Method is an educational approach that is based on principles from Piaget’s developmental psychology and Vygotsky’s zone of proximal learning principles. Montessori classrooms emphasize self-directed learning, hands-on activities, and collaborative play, giving kids the chance to explore their environment at their own pace and in their own way. Educational materials are designed to support the children’s developmental stages and foster independence, curiosity, and a love of learning: key components of the developmental theory.

Psychological Theories in Therapy

One of the most direct applications of psychological theories is in the field of clinical psychology, where theories of human behavior and mental processes have informed the development of many therapeutic techniques. For example, many people are familiar with cognitive-behavioral therapy (CBT) , as it’s one of the most widely used therapeutic approaches today. This type of therapy is grounded in cognitive theory by helping individuals identify and change distorted thinking patterns that lead to negative emotions and behaviors. This theory-based approach has proven effective in treating a variety of psychological disorders, including depression, anxiety, and PTSD. 5

Understanding psychological theories is crucial for developing effective therapeutic approaches to provide the foundational knowledge needed to comprehend complex mechanisms underlying mental health issues. These theories can offer structured frameworks that guide therapists in assessing, diagnosing, and treating various psychological conditions and help them address the specific needs of individuals. Since so much of the work is built on tested theory, treatment can be both scientifically grounded and practically effective.

About the Author

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Annika Steele

Annika completed her Masters at the London School of Economics in an interdisciplinary program combining behavioral science, behavioral economics, social psychology, and sustainability. Professionally, she’s applied data-driven insights in project management, consulting, data analytics, and policy proposal. Passionate about the power of psychology to influence an array of social systems, her research has looked at reproductive health, animal welfare, and perfectionism in female distance runners.

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Market Research Plan

In 1970, food and drink sales of the US restaurant industry reached only 42.8 billion US dollars, which is way behind the 745.61 billion US dollar sales of 2015. According to the statistic posted in statista, this number should grow in the next few years. In fact, the website reported that from the 2015’s over 14 million employees of the restaurant industry, it should increase up to 16 million in 2026. However, as a result of this growth, there will be possibilities that the market will be saturated and more competitive. Thus, as a business owner, you will need to gear up and gain an edge to stand out in the market. By conducting market research for a restaurant, you can prepare your business to become more competitive and strategic, which will ensure its success.

What Do You Need to Know About Market Research?

Market research is an essential component of a business plan which aims to get information concerning the target market of a business. Through this study, you will determine the chances of a proposed service or new product to survive in the market. As part of market research, you need to develop a research plan.

What is Market Research Plan?

In general, market research plan is the foundation of a detailed research proposal . This document contains the initial thoughts about the research project that you are planning to take place logically and concisely, which is a crucial content of market research. Simply put, by obtaining a market research plan, you can thoroughly examine how your product or service will proceed in a specific domain.

2+ Market Research Plan Examples

Conducting market research will give significant benefits to your business. However, to materialize it, you may need to ensure that you build your market research plan correctly. Below is a list of the market research plan samples and templates that you can use as a guide.

1. Market Research Plan Template

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2. Sample Market Research Plan Example

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3. Basic Market Research Plan Example

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4. Market Research Business Plan Example

Size: 600 KB

How to Develop a Strong Market Research Project Plan?

Now that you know how a marketing research plan should look, make a secure market research plan by following the steps below:

1. Set Goals and Objectives

What do you want to attain with your research? Your goals and objectives should answer that question. You can start by forming a general marketing goal . You will, then, make it more specific. This goal will help you focus and direct the entire research process to make the best data-driven marketing decisions. To determine the most critical issue, you may conduct qualitative research . This research methodology ensures that you address the issue that really requires an urgent solution.

2. Determine Your Target Respondents and Appropriate Distribution Method

In this step, you will identify the right people to get the information that you need to create the right decision for your marketing goals. After that, list down the best possible ways for the data gathering. For example, your target market is veterans. You may want to use more appropriate channels such as direct mails, phone, or personal interview. Once you have chosen the most appropriate data collection method, create an outline that will allow your team to get the most relevant information from your target market or audience.

4. Brainstorm for the Right Questions

In deciding the right questions for your marketing research, it is crucial to keep your study goals in mind. Only include items that are relevant to the study to come up with the best business decisions. Asking the wrong questions may lead to inadequate conclusions. Data-driven solutions mostly obtained through quantitative research questions. You can still use qualitative research questions but make it minimal to avoid making the respondents bored and held up, which can lead to survey abandonment. As much as possible, make your survey short and answerable in less than 5 minutes. Otherwise, you may want to find an alternative option in getting the desired data. Also, it would help if you will consider other factors in building the right questions. Refrain from asking sensitive, personal, and offensive questions. To do it, research your target audience.

5. Analyze the Data

Start this step by cleaning your survey data. To do it, filter out any low-quality responses. These items can affect your decision-making negatively. Basing on the set standards, remove the outlier responses. To do that, determine if the respondents answered in the desired format. If not, especially if it has become a trend, disqualify the question or conduct another data-gathering or investigation for this question. In this process, you will also find out if the answers of the participants are contributing to your research goals. At the end of this stage, you will, then, share your findings. To effectively show your results, you can use data visualization methods such as charts, graphs, and infographics.

6. Create a Data-Driven Marketing Decisions

Now that you have the necessary market research data, you can come up with a data-driven decision. Whether you are running a pharmaceutical firm or a corporal business such as Coca Cola, you can develop a new marketing campaign and other relevant business actions without unnecessary worries since you have directly reached out to your target market.

In a market that is becoming more competitive, creating a market research plan for a new product of your business can give you an advantage and an edge over your opponents. This type of method will also save your time, effort, and money because it allows you to determine the proper actions that you can take towards the corporate goals in terms of marketing and other relevant sectors.

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Review Article
Open access
Published: 02 September 2024

Circulating tumor cells: from new biological insights to clinical practice

Xuyu Gu 1 na1 ,
Shiyou Wei ORCID: orcid.org/0000-0002-1783-0372 2 na1 &

Signal Transduction and Targeted Therapy volume 9 , Article number: 226 ( 2024 ) Cite this article

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Cancer metabolism

The primary reason for high mortality rates among cancer patients is metastasis, where tumor cells migrate through the bloodstream from the original site to other parts of the body. Recent advancements in technology have significantly enhanced our comprehension of the mechanisms behind the bloodborne spread of circulating tumor cells (CTCs). One critical process, DNA methylation, regulates gene expression and chromosome stability, thus maintaining dynamic equilibrium in the body. Global hypomethylation and locus-specific hypermethylation are examples of changes in DNA methylation patterns that are pivotal to carcinogenesis. This comprehensive review first provides an overview of the various processes that contribute to the formation of CTCs, including epithelial-mesenchymal transition (EMT), immune surveillance, and colonization. We then conduct an in-depth analysis of how modifications in DNA methylation within CTCs impact each of these critical stages during CTC dissemination. Furthermore, we explored potential clinical implications of changes in DNA methylation in CTCs for patients with cancer. By understanding these epigenetic modifications, we can gain insights into the metastatic process and identify new biomarkers for early detection, prognosis, and targeted therapies. This review aims to bridge the gap between basic research and clinical application, highlighting the significance of DNA methylation in the context of cancer metastasis and offering new avenues for improving patient outcomes.

Epigenomic analysis reveals a unique DNA methylation program of metastasis-competent circulating tumor cells in colorectal cancer

Circulating tumor cells: biology and clinical significance

Liquid biopsy: one cell at a time

Introduction.

The progression of cancer from a localized tumor to a widespread metastatic disease is a complex and multifaceted process, making it cancer patients’ leading cause of death. 1 The driving force behind this process is CTCs, which break from the primary tumor site and spread through the bloodstream to colonize other organs. 2 The study of CTCs has garnered considerable attention as it opens new avenues for understanding the intricacies of metastasis, offering potential markers for early detection and targets for therapeutic intervention. 3

Recent technological advancements in genomics and molecular biology have significantly enhanced our capability to scrutinize the biological mechanisms underpinning CTC dissemination. 4 Among the myriad of molecular processes implicated in this journey, DNA methylation emerges as a pivotal regulatory mechanism influencing gene expression and chromosome stability. 5 Cellular differentiation, growth, and adaptation to environmental changes all depend on DNA methylation, a reversible epigenetic alteration that adds methyl groups to the DNA molecule. 6 For their roles in the development and propagation of cancer, changes in DNA methylation patterns are becoming more and more recognized. 7

Hypomethylation across the entire genome, alongside hypermethylation of specific gene loci, has been implicated in the disruption of genomic integrity and the silencing of tumor suppressor genes, respectively. 8 , 9 These epigenetic changes are fundamental to the transformation of normal cells into malignant ones, affecting their ability to proliferate, evade immune detection, and metastasize. 10 This comprehensive review delves into the mechanisms of CTC formation, highlighting processes such as EMT, immune system evasion, and the colonization of distant tissues, all of which are critical to the metastatic cascade. Furthermore, this review aims to dissect the role of DNA methylation modifications within CTCs, examining how these alterations influence the aforementioned stages of CTC dissemination. After comprehending the epigenetic landscape of CTCs, researchers can find new targets for therapeutic intervention as well as biomarkers for cancer diagnosis and prognosis. The modulation of DNA methylation in CTCs, in particular, presents a promising path for the development of targeted therapies intended to prevent the spread of cancer.

Biology of CTCs

CTCs enter the peripheral circulation from primary or metastatic lesions either spontaneously or as a result of therapeutic manipulation. 11 The notion of CTCs was initially introduced by Ashworth in 1969, and these cells can be categorized into three groups: epithelial, mesenchymal, and hybrid. 12 , 13 , 14 , 15 While most CTCs can be eliminated by the host immune system, a subset of highly active and metastatic CTCs may evade immune clearance, ultimately resulting in the establishment of microscopic cancer foci, tumor recurrence, and metastasis. 16 , 17 , 18 , 19 , 20 , 21 Thus, CTCs are regarded as a plausible origin of fatal metastatic disease in individuals (Fig. 1 ).

Route and location of CTCs Metastasis. Circulating tumor cells (CTCs) are the fundamental constituents of liquid biopsy, functioning as the cornerstones of this approach. a These neoplastic cells are shed naturally from primary or metastatic tumors and circulate within the bloodstream, b serving as the “seeds” of tumors that can potentially result in fatal metastasis of various sites. The figure was created with BioRender.com

However, there are several obstacles that CTCs must overcome in order to successfully detach from the primary tumor tissue, including: 1) Adhesion: Extracellular matrix (ECM) and surrounding cells in the tissue of the primary tumor are both firmly adhered to by cancer cells, which are typically highly sticky. CTCs must overcome these adhesion forces in order to detach and enter the lymphatic or bloodstream system. 2) Invasion: Cancer cells must also be able to invade the neighboring tissue to reach the lymphatic or blood vessels. This requires the cells to degrade the ECM and surrounding tissues. 3) Shear stress: Once in the bloodstream or lymphatic system, CTCs are subjected to significant shear stress due to the movement of fluid. This can cause mechanical damage to the cells and can also trigger apoptosis (cell death). 4) Immune system surveillance: The immune system is constantly surveilling the bloodstream and lymphatic system for foreign or abnormal cells. CTCs must avoid being detected by the immune system in order to survive and potentially form secondary tumors. Overall, the process of CTC detachment and dissemination is complex and involves many different factors. Understanding these obstacles and how CTCs overcome them is a crucial area of research on the biology of cancer and may lead to new therapies for preventing or treating cancer metastasis.

Molecular markers of CTCs

CTCs have been found in a wide range of cancer types using a comprehensive panel of molecular markers. Table 1 provides a summary of CTC-associated markers utilized in different cancers. 22 , 23 The leading marker for CTCs is epithelial cell adhesion molecule (EpCAM), a “universal” epithelial marker for tumors, since the majority of tumors originate in epithelial tissues. 24 Different cancer types 25 express EpCAM differently, and EpCAM-based CTC detection systems are widely employed for tumors with significant EpCAM expression, like prostate and breast cancers. Numerous studies have demonstrated that CTCs in prostate and breast cancers are EpCAM-positive, indicating their prognostic value in the disease’s early and metastatic stages. 26 , 27 Other epithelial-derived cancers, including pancreatic, 28 colorectal, 29 and hepatocellular cancers, 30 also exhibit significant detection rates of EpCAM-positive CTCs. Similar to early distant metastases, the presence of these EpCAM-positive CTCs is associated with poorer patient survival. 29 , 31 , 32 However, there are limitations to using EpCAM as a CTC marker. Tumors that are low-expression or EpCAM-negative, including neurogenic cancers, are unable to be treated with this method. CTCs can undergo EMT, a process during which epithelial markers, including EpCAM, are downregulated. The rate of EpCAM-positive CTC detection is affected by this downregulation. Numerous studies have shown the clinical usefulness of EpCAM-positive CTCs, despite uncertainties about the efficacy of EpCAM-based technology for detecting all CTCs. 33 Positive EpCAM CTCs make up a significant subset of all CTCs, indicating that they could serve as a trustworthy biomarker for cancer prognosis and therapy efficacy when necessary. EpCAM-positive CTCs are likely to miss critical biological information associated with EpCAM-negative CTCs, leading to an underestimation of the actual number of CTCs within the population. This phenomenon has been observed in certain types of cancer such as NSCLC (NSCLC), the number of EpCAM-negative CTCs is significantly higher than that of EpCAM-positive CTCs. 34 However, the usage of both mesenchymal and epithelial cells, along with marker-independent detection techniques, can enhance the suboptimal isolation of CTCs by EpCAM-based technologies. For example, the use of fluorescent-magnetic nanoparticles with a dual-antibody interface that targets both N-cadherin and EpCAM in breast cancer has accelerated the identification of CTCs and increased the efficiency of CTC isolation. 35 , 36 The identification of both epithelial and non-conventional CTCs, which lacked leukocyte and epithelial markers, enhanced the CTC positivity rate in a single-cell assay for CTC detection in biliary tract cancer. 37

Molecular changes accompany the EMT in cancer cells. Among these are the downregulation of epithelial indicators like ZO-1, claudins, and occludins and the upregulation of mesenchymal markers like Vimentin, N-cadherin, fibroblast-specific protein 1, and fibronectin. 38 EMT is regulated by EMT-related transcription factors, mainly from the TWIST, ZEB, and SNAIL families. 39 All these EMT-related molecules have the potential to be utilized in targeting methods for EMT-associated CTCs. Many EMT-related molecules, however, are nuclear or cytoplasmic proteins, limiting their application in current membrane-based CTC detection techniques. The usage of proteins like E-cadherin, Vimentin, and TWIST has been common in the past because of their detectability in traditional CTC detection methods, including as flow cytometry sorting, immunostaining, and fluorescence in situ hybridization (FISH) staining. 40 Nevertheless, the advent of single-cell CTC sequencing technologies 41 holds the promise of more comprehensively revealing the EMT status of CTCs, encompassing all EMT-related molecular alterations at the RNA level.

Numerous studies have proposed that either differentiated progenitor cells or somatic stem cells may be the source of cancer stem cells (CSCs) with tumor initiating capacities. Evidence suggests that this specific subpopulation of cancer cells may be responsible for relapse and metastasis. 42 If the majority of cancer cells are unable to form new tumors and only the rarest CSCs may travel to cause metastatic disease, therefore the main goal of CTC research is to identify and eliminate this circulating-CSC population. After identifying and isolating CSCs within CTCs, it would be possible to combat residual cancer more effectively. Two hypotheses can be taken into consideration to clarify the origin of circulating CSCs. First, cancerous somatic stem cells EMT and exit the main tumor into the circulation. These cells are known as mesenchymal CSCs. Second, these cells can be the result of fully differentiated cancer cells that have undergone EMT pathways to acquire migratory properties. 43 Regardless of the pathway, the end result is the dispersal of CSCs, which move in the direction of niches via intermediary cells that display a combination of mesenchymal, epithelial, and stemness characteristics. This could explain the diversity of surface markers and/or transcription factors observed in tumor-initiating cells within CTCs. The heterogeneity of CTCs, originating from either EMT in CSCs or differentiated cancer cells, underscores the challenge of selecting significant identification markers. This complexity is further compounded by the specific organ of tumor origin. During EMT, cancer cells acquire stemness characteristics, transforming into mesenchymal stem cancer cells. The MET, which is a reverse process, transforms them into epithelial stem cancer cells. ALDH1 expression, initially observed in 1–2% of normal breast epithelial cells, was first documented in breast cancer tissue by Ginestier et al. 44 Barriere et al. found in a clinical study for early breast cancer diagnosis that blood samples from patients with non-metastatic breast cancer had a stemness CTC population. Out of 130 patients, 17 had ALDH1 markers and 49 were CTC-positive. 45 CD44, a cell surface glycoprotein implicated in metastasis and cell migration, 46 characterizes a subpopulation of breast CSCs capable of intravasating into the bloodstream, identified by the CD44 + /CD24 low/– /Lin – markers. 47 In colorectal cancer, CSCs expressing CD44v6 are associated with metastatic potential. 48 Additionally, CSCs have decreased levels of certain glycosphingolipids that are important for cell growth and motility, such as Fuc-(n) Lc4Cer and Gb3Cer, while elevated levels of GD2, GD3, GM2, and GD1a are seen. These gangliosides have the potential to function as markers of CTC stemness. Specifically, GD2 is associated with the CD44high CD24low phenotype linked to CSCs, and GD3 similarly indicates stemness features, although GD1a remains a putative marker. 49 , 50 Recent research has also linked the CSCs phenotype to high ABCG2 transporter expression levels. This transporter is responsible for removing a variety of xenobiotics, including chemotherapy drugs. This chemoresistance implies that ABCG2 expression and stemness are closely associated. It has been found that CSCs from lung, pancreatic, and retinoblastoma tumors exhibit high levels of ABCG2. 51 , 52 Additional biomarkers have been found to be markers for CTCs in a variety of cancers, each with unique therapeutic implications. These include human epidermal growth factor receptor-2 (HER-2) 53 , 54 ; estrogen receptor 55 , 56 , 57 ; prostate-specific membrane antigen 58 , 59 ; folate receptor 60 , 61 ; and survivin. 62 Table 1 provides specifics on these CTC markers. The majority of these markers match the unique molecular markers of the primary tumor. However, the expression of specific markers in CTCs often differs from that of the primary tumor. For example, there is about a 15% 63 discrepancy in HER-2 gene amplification between primary breast tumors and CTCs, indicating that genetic instability is most likely the cause of clonal selection or acquisition. CTC detection technologies are dependent on a number of melanoma cell adhesion molecules, which are highly specific molecular markers for melanoma and include HMW-MAA, 62 , 64 MART-1, 65 , 66 CD146, 67 , 68 and MAGE A. 66 , 67 , 69 , 70 , 71 Melanoma is a kind of skin cancer that starts in melanocytes. The diverse range of CTC markers underscores the heterogeneity of CTCs across various cancer types. Even within a single patient, CTCs display spatio-temporal heterogeneity, possibly attributable to spatially distinct microenvironments within the bloodstream and temporal variations in response to therapy. As such, characterizing the whole population of CTCs using the few molecular markers that are currently accessible is difficult. Furthermore, CTC markers vary over the course of cancer treatment and at different stages of the disease.

The biological progress of CTCs development

Epithelial cells typically undergo anoikis, a form of programmed cell death, upon detachment from their surrounding environment. This characteristic makes metastatic seeding generally inefficient. 72 , 73 , 74 This raises an important question: what characteristics allow CTCs to metastasize successfully? Enhanced survival and tumor-seeding ability appear to be restricted to a small subset of cells initiating tumors or metastases with stem-like properties. 75 EMT has been proposed as an essential factor for metastasis since it increases both invasive potential and contact-independent survival (i.e., resistance to anoikis). 75 , 76 , 77 Preclinical studies have shown that EMT transcription factors, like TWIST and SNAIL, enhance motility and invasiveness in vitro while suppressing cell-cell adhesion. 78 , 79 Silencing SNAIL and TWIST, however, can reduce metastasis in vivo but does not totally prevent it, raising doubts about the absolute requirement of EMT-related transcriptional regulators in the metastatic process. 80 , 81 Interestingly, it has been found that EMT can inhibit the effective seeding of metastatic tumors by deleting E-cadherin or modifying TWIST expression in several EMT lineage tracing models. 80 , 82 The observed plasticity of the epithelium-to-mesenchymal transition raises the possibility that this change is fluid and transitional rather than binary and irreversible, acting more as a catalyst for metastasis than as a direct cause. 83 , 84 Consequently, CTCs have been seen to exhibit an intermediate degree of EMT, which is linked to their adaptability, stem cell-like qualities, poor response to therapy, and progression of the disease. 76 , 84 , 85 There have been multiple hybrid states found at the invasive boundary of patient tumor tissues from common carcinoma types and xenografts. 84 , 86 , 87 Their functional role in invasion, invasion, and metastatic colonization requires further studies.

There may be concurrent existence of metastasis pathways that are both plasticity-dependent and plasticity-independent. 88 The discovery of collectively moving, highly metastatic clusters of cell contact-dependent CTCs, which were first reported several decades ago, offers a significant refutation of the idea that EMT is necessary for metastasis. 89 , 90 Metastatic colonies are polyclonal, 91 , 92 , 93 , 94 and subclones interact synergistically, 95 , 96 suggesting that heterogeneous CTC clusters 97 , 98 as well as solitary CTCs are involved in cancer propagation. Despite making up a small portion of all CTC events in peripheral circulation, multiple studies have demonstrated that CTC clusters have up to a 100-fold higher potential for metastasis than individual CTCs. 82 , 97 , 98 , 99 Intratumor hypoxia is shown to upregulate genes producing cell adhesion proteins in patient samples and mice models, which promotes collective CTC cluster shedding. 98 Homotypic clustering has been shown to improve a number of cellular characteristics, such as the overexpression of stem-like traits 100 , 101 via the hypomethylation of binding sites for transcription factors OCT4, SOX2, and NANOG 102 and the methylation of metastasis suppressor genes. 5 Clustering can also be augmented by circulating galectin 3 or cancer-associated MUC1, 100 homotypic ICAM interactions, 103 or CD44 interacting with PAK2. 98 This clustering can also enhance survival and self-renewal capacity through CDK6 or increased size or number of desmosomes and hemidesmosomes. 97 , 104 CD44 was among the first markers discovered to detect breast cancer cells with elevated tumor-initiating capacity in solid tumors. 47 Later, it was demonstrated that CD44, along with MET, epithelial EpCAM, and CD47, could identify subpopulations of breast cancer that were highly metastatic. 76 Importantly, CD44 expression should not be employed as a CTC marker alone because it is widely expressed in the hematopoietic cell compartment. 105 Previously thought to be specific to stem-like cells, CK14 expression is more common in CTC clusters than in individual CTCs and is required for distant metastasis. 106 EpCAM – , HER2 + , EGFR + , heparanase (HPSE) + , and NOTCH1 + are stem-like phenotypic signatures in CTCs that have been shown to provide competence for brain and lung metastasis. 104

Heterotypic clusters, which include platelets, myeloid cells, and cancer-associated fibroblasts (CAFs), as well as homotypic clusters formed by CTCs, can potentially form between CTCs or other cell types. 107 , 108 , 109 , 110 , 111 The rapid interaction of CTCs with platelets in circulation 112 enhances plasticity and metastasis-initiating capacity 107 through mechanisms like RhoA–MYPT1–PP1-mediated YAP1 signaling 113 and increased vascular permeability via platelet-derived ATP–P2Y2 interaction. 114 The glycoprotein A additionally, protecting against T cell-mediated clearance and NK cell-mediated clearance, respectively, are repetitions predominant (GARP)–TGFβ axis 115 and platelet-derived major histocompatibility complex class I. 116 Barbara Maria Szczerba et al. demonstrated that neutrophils also participate in forming heterotypic CTC clusters. 117 Neutrophils are drawn to CTCs by chemotaxis that is dependent on CXCL5 and CXCL7 111 and connects with them via adhesion mediated by vascular cell adhesion molecule 1 (VCAM1), which enhances their potential for proliferation and metastasis. 111 Through the formation of neutrophil extracellular traps or the secretion of matrix metalloproteinases and IL-1β, neutrophils facilitate adhesion and extravasation. 108 In addition, neutrophils protect CTCs from immune surveillance 118 , 119 ; macrophages and myeloid-derived suppressor cells in CTC clusters also provide this protection. 118 , 120 It has been demonstrated that heterotypic adherens junctions, mediated by N-cadherin in stromal CAFs and E-cadherin in invasive cancer cells, promote collective invasion. Here, CAFs serve as invasive and migrating leader cells, 110 , 119 facilitating metastasis by allowing tumors to “bring their own soil”. 121 Mathematical models indicate that the form of clusters influences CTC behavior during circulation in addition to the biological consequences of clustering. Compact clusters usually flow closer to the endothelium wall compared to linear clusters. 122 However, clusters move in a “single chain” structure as they pass through narrow capillaries. 109 , 123 , 124 , 125 The metastatic potential of CTCs is significantly impacted by the phenotypic plasticity and clustering differences, which highlights potential strategies for inhibiting the metastatic process.

Furthermore, the dynamics of CTC propagation are becoming recognized as an equally important component for the dissemination of tumor cells. 111 , 126 Chronotherapy has been used to study and evaluate the clinical application of the circadian rhythm’s function in tumor onset 127 , 128 and growth dynamics. 129 , 130 This therapeutic approach seeks to optimize therapy timing in order to increase the effectiveness of antitumor drugs. 131 , 132 However, it has only recently been established how circadian rhythm impacts CTC release and the dissemination of metastatic disease. 111 , 126 The way that CTCs are currently detected frequently assumes that peripheral blood counts remain stable throughout the day, which may lead to inconsistent results and make it more difficult to use CTCs as a liquid biopsy analyte in clinical settings. Fluorescence in vivo flow cytometry studies in orthotopic mice models of human prostate cancer have suggested circadian rhythmicity. 133 The temporal dynamics of CTC intravasation, demonstrating significant circadian rhythm-based variations both in breast cancer patients and in mouse models. CTC counts are highest during sleep because to these variations, which are caused by rhythmic changes in hormone levels, such as melatonin. 128

The role and mechanism of CTC in drug resistance

The manifestation of an EMT phenotype in CTCs has been associated with therapeutic resistance and tumor relapse. For example, in patients with metastatic colorectal cancer, overexpression of CSV and plastin 3 in EMT-positive CTCs has been associated with drug resistance. 134 CTC survival is improved by the acquisition of stem cell characteristics during EMT, which promotes enhanced migratory and invasive abilities as well as resistance to therapy. Tumor relapse following targeted therapy can be driven by CTCs with stem cell features, 135 such as in colorectal cancer, 136 where drug-resistant CTCs may act as metastasis-initiating cells, driving tumors to become more aggressive as a result of anticancer drugs’ selection pressure. Targeted therapy-induced tumor relapse in melanoma patients has been linked to phenotypic switching of CTCs to a less differentiated state. 137 Therapies resistance in breast, prostate, pancreatic, and NSCLC have also been linked to phenotypic switching. 138 Therapeutic resistance mechanisms seen in primary tumor cells include target mutation or inactivation, improved genomic DNA repair, faster drug efflux, upregulation of markers associated with quiescence, downregulation of markers associated with proliferative activity, and inhibition of the formation of oxygen radicals, 139 are also evident in CTCs. Tumor cells can separate from the original tumor and circulate in the blood as clusters of tiny cells, challenging the idea that CTCs with EMT and/or stem cell phenotypes are the sole type that initiates metastasis (i.e., groups of >2 CTCs and up to large micro-emboli). 97 Metastatic potential and drug resistance may be influenced by CTC clusters. The absence of proliferation in CTC clusters, as indicated by Ki67 staining, has been linked to their resistance to cytotoxic therapies. 140 Significantly reduced overall survival following systemic therapy is observed in PDAC patients who harbor a high number of CTC clusters (more than 30 clusters per 2 mL of blood). 141 Expanding CTCs from patients into cell lines or xenografts may reveal insights into their therapeutic resistance and present an opportunity for targeted therapy. 142 However, high CTC numbers and several months of establishment make CTC-derived cell lines or xenografts impractical for clinical use. Furthermore, prolonged passage of CTCs in culture is likely to induce irreversible adaptation and clonal expansion. 143

Stem cell and EMT markers exhibit frequent overexpression in CTCs of metastatic breast cancer. 144 Recently, within breast tumors, a subpopulation displaying a stem cell-like phenotype characterized by CD44 positivity and CD24 negativity has been delineated. 47 This subpopulation potentially disseminates into the bloodstream, evading therapeutic measures, 145 and demonstrates an expression profile associated with metastatic recurrence. 146 The modulation of HER2 signaling has been reported to augment cancer stem cell reservoir, potentially necessary for its maintenance; a significant association between HER2-like tumors and stem cells has been observed. 147 A breast cancer stem cell-like phenotype, exhibiting higher resistance to treatment and reduced proliferation in circulation, is suggested by preliminary evidence. 148 ALDH1, a marker indicative of both normal and neoplastic breast stem cells, 144 has been found to be overexpressed in 70% of CTCs, correlating with therapy resistance. A substantial proportion of CTCs display detectable levels of at least one EMT marker such as TWIST, AKT2, and PI3Kalpha, in addition to ALDH1, thereby delineating a highly tumorigenic subset of EMT-associated breast CSCs. 44 This subset of CTCs has clinical value since it predicts drug resistance and a poor result for patients with metastatic cancer. 44 Notably, ALDH1 and EMT markers were discernible even prior to CTC detection in circulation, as evidenced by their positivity in RT-PCR for transcripts such as HER2, MUC1, and EpCAM. 44 Consistently, stem cell-like phenotypes, particularly CD44 + CD24 −/low and ALDH1 high CD24 −/low , have been found in 35.2% and 17.7% of CTCs, respectively. 63 Among ALDH1-highly positive CTCs, the subset expressing CD44 + CD2 4−/low exhibits heightened tumorigenic potential. 149 Though ALDH1-positive cells constitute just 5% of cells in tumors expressing ALDH1, ALDH1 positivity is associated with a high histological grade and poor clinical outcomes. 149

According to the cancer stem cell model, putative cancer stem cells 44 are necessary for tumor growth and drug resistance, and their existence should be associated with a worsened prognosis, 149 albeit data on this matter are still inconsistent. 44 , 150 , 151 Because of their capacity to self-renew and resistance to chemotherapeutic drugs, eradicating these cells during therapy poses challenges. Notably, patients’ chances of responding to chemotherapy are poorer when their CTCs are ALDH1-positive. 44 It has recently been shown that CTCs express one or more multidrug resistance-related proteins (MRPs) in 86% of metastatic breast cancer patients, with patients exhibiting positive MRP-positive CTCs having considerably shorter progression-free intervals. 57 , 144 , 152 , 153 Further investigations are warranted to elucidate the association between the presence of CD44 + CD24 − or ALDH1 high CD24 −/low CTCs and clinical trajectory as well as disease progression.

CTC isolation and identification

For CTCs to be used as a liquid biopsy analyte in clinical settings, it is essential to adopt impartial, affordable, quick, and effective capture technologies that can reliably isolate adequate numbers of CTCs. In order to facilitate the development of data necessary for precise patient stratification and therapeutic decision-making, these capture approaches must also be compatible with functional assays and cutting-edge sequencing technologies. For probing the biology and vulnerabilities of metastatic cancer at the molecular and functional levels, CTCs provide a wide variety of scope when they are isolated in a viable state. CTCs have a unique role as a liquid biopsy analyte because they can represent aggressive subclones with greater metastatic potential. There is conjecture that their molecular and phenotypic analysis may provide more pertinent insight than traditional tissue biopsies (which involve the random subclone isolation) 154 , 155 or other circulating analytes like circulating tumor DNA (which mainly identify dying subclones). However, further exploration is warranted. The ease of access provided by minimally invasive blood draws may allow for regular, long-term assessments of the effectiveness of treatment interventions, which may also make early cancer detection or recurrence possible. 156 As a result, CTCs become an optimal biomarker repository for personalized therapy and real-time clinical applications. However, it is still challenging to obtain CTCs because of their paucity, and effective CTC enrichment is required for reliable downstream analysis and applications.

In the previous ten years, numerous technical progress has been made to improve CTC analysis and detection 157 , 158 by utilizing distinct characteristics and phenotypes of CTCs. These developments can be broadly categorized into antigen-dependent and antigen-independent techniques. The most widely used strategies currently in use help to promote positive selection by using antigens that are expressed on CTCs and minimally expressed on other circulating cells. This strategy is frequently combined with CD45-based negative selection to reduce the number of hematopoietic cells and enhance discrimination. It is notable that the CellSearch system (Menarini Silicon Biosystems, Italy) and AdnaTest CTC Select (QIAGEN, Germany), which have been approved by the US Food and Drug Administration (FDA), utilize immunomagnetic selection based on EpCAM expression. 159 , 160 To increase sensitivity and specificity, respectively, more markers are used, such as pan-CK and CD45. For CTC capture, antibody-coated magnetic beads are used in the Magnetic-Activated Cell Separation (MACS) method (Miltenyi Biotec, Germany). 85 Additionally, the Geometrically Enhanced Differential Immunocapture method combines microfluidics with different antibodies according to the kind of tumor (tumor includes, for instance, HER2 in breast cancer and PSMA in prostate cancer) and positive for cytokeratin for counting. 161

Physical characteristics including size, charge, density, or elasticity are used by antigen-agnostic detection systems to enrich CTCs. Detection of CTCs based on physical properties is facilitated by several methods, including density gradient centrifugation, filter-based devices, capture surfaces, and microfluidic systems. Notable examples of microfluidic systems are ISET (Rarecells Diagnostics, France), Smart Biosurface Slides, CTC-iChip (TellBio, USA), and the FDA-approved Parsortix (ANGLE, UK). 162 , 163 To improve sensitivity and specificity even more, multimodality techniques are being developed. For example, Isoflux (Fluxion Biosciences, USA) combines immunomagnetic beads, 164 with flow control, and the Cyttel system (CYTTEL Biosciences, China) is an image-based detection tool that identifies CTCs by combining immunohistochemistry, fluorescence in situ hybridization, and centrifugation in that order. With the use of the microfluidic platforms Parsortix and CTC-iChip, marker-based positive and negative selection (such as EGFR, HER2, CD45, and EpCAM) can be combined with imaging and micromanipulation to identify pure CTC subsets. 5 , 111 , 162 , 165

Novel in vivo CTC detection tools have emerged to address the challenge of low CTC levels in peripheral blood samples. CTCs can be directly extracted from the bloodstream using methods such as intravascular CTC-catching guidewires coated with EpCAM-directed antibodies, as exemplified by the CellCollector device from GILUPI in Germany. 166 When combined with antigen-dependent selection, cytopheresis facilitates the enrichment of cell fractions from vast blood volumes and offers promise for the isolation of CTCs. 167 The adoption of this approach into routine clinical practice may pose challenges due to its lengthy and invasive nature, alongside potential vascular health issues in heavily treated cancer patients. Studies comparing different methods of accessing the vasculature shows that patients with early-stage NSCLC have higher CTC counts in tumor-draining vessels than in peripheral locations. 156 , 168 For early-stage cancer patients undergoing surgery, this concept provides an attractive opportunity for liquid biopsy. Despite its significance, it remains impractical to use these findings to routine CTC evaluation or to patients with advanced illness who do not undergo surgery.

Advances in capture methods have enabled thorough molecular and functional studies of CTCs at both bulk and single-cell levels, epigenomic, exploring genomic, proteomic, transcriptomic, and functional properties. This has extended research on these cells beyond mere counting to comprehensive analysis. Since these advances have been extensively reviewed, we will focus primarily on aspects related to cell multi-omics and the functional evaluation of CTCs. 2 , 157 , 169 , 170 For instance, assessing drug responses alongside single-cell examination of individual CTCs and CTC clusters can uncover biological dependencies and potential targets for therapy. 5 CTC proteins and secreted factors are being characterized using microfluidic platforms that employ single-cell resolution mass spectrometry and bead-based immunoassays. 171 , 172 , 173 A key barrier to ex vivo CTC cultures’ clinical applicability is their low success rates, despite the fact that experimental proof-of-principle studies 174 , 175 have demonstrated their efficacy. Although the feasibility and therapeutic relevance of CTC capture and downstream analysis have been shown, most of the above approaches are not currently used as routine. Existing CTC technologies face limitations that necessitate resolution for robust integration into clinical settings. These issues encompass the necessity for a more profound comprehension of epitope expression and plasticity, along with challenges associated with cell loss caused by variations in size and deformability, low purity of CTCs, device blockage, the need for substantial blood volumes, time limitations, and difficulties with automation. Additional challenges include the need to improve functional assays, such as enhancing culture methods and developing CTC-derived xenografts, and ensuring robust validation of molecular analyses. This involves addressing issues like stochastic variations, limited sequencing coverage, biases in amplification, high error rates, and variability in bioinformatics approaches. 2 If these challenges are overcome, CTCs may become prominent sources of comprehensive biomarkers that are minimally invasive and personalized.

Metastasis of CTCs

Stephen Paget first proposed the “seed theory” or the soil and seed hypothesis of metastasis in 1889. 176 According to the hypothesis, cancer cells (seeds) have a selective affinity for certain organs (soil) and that the capacity of cancer cells to establish colonies in distant locations is determined not only by the characteristics of the cells themselves but also by the specific microenvironment of the secondary site. According to the seed theory, cancer cells must go through a sequence of steps in order to successfully metastasize to a distant site. encompass the cancer cells’ ability to invade the nearby tissue, penetrate the bloodstream or lymphatic system, survive while circulating, extravasate into the secondary site, and establish a new colony of cancer cells.

Initiation of metastasis

According to the “seed theory”, metastasis typically entails two distinct steps. The first step entails a tumor cell’s detachment from its original site and subsequent circulation through the bloodstream or lymphatic system to a distant location. The second step involves the successful colonization of the tumor cell in the distant site, which requires the cancer cell to be able to proliferate and establish a secondary tumor. 177 CTCs are crucial for the process of tumor metastasis, and it is believed that a specific subgroup of CTCs found in patient blood is what initiates the metastatic cascade. 76 During the early stages of cancer, cellular properties like adhesion and stroma formation act as physical obstacles that impede distant metastasis. To overcome these barriers, carcinoma cells must boost their motility within the stroma and gain access to the bloodstream through either active or passive entry mediated by either the EMT or non-EMT pathways. CTCs that are viable in the bloodstream have the potential to arrest at various locations, including the secondary metastatic sites, or new distant locations. 178 , 179 , 180 , 181 Upon arrest, these CTCs have the potential to undergo MET, which can facilitate extravasation and allow the cells to either enter a dormant state or colonize and engraft at the site (Fig. 2 ). CTCs with stem-like properties, demonstrating increased resistance to anoikis and invasive potential, are identified as crucial for successful metastatic dissemination due to their phenotypic heterogeneity. 182 Notably, CTC clusters, characterized by polyclonality and enhanced metastatic potential, exhibit superior survival in circulation compared to individual CTCs. 183 These clusters are formed through homotypic interactions among tumor cells or heterotypic interactions involving other cell types such as platelets or myeloid cells. 184 The formation of these clusters not only boosts the proliferation and survival of CTCs in the bloodstream but also enhances their metastatic efficiency by aiding in immune evasion and seeding competency at distant sites (Fig. 3 ). Key factors like intratumor hypoxia and platelet interactions are implicated in promoting CTC cluster formation and the initiation of metastasis. 185

Schematic representation of EMT-associated mechanisms supporting CTC survival and early metastasis. a CTCs are liberated into the bloodstream by means of epithelial-mesenchymal transition (EMT)-related mechanisms, including individual cell or collective migration, intravasation, as well as passive processes such as the detachment of isolated tumor cells or clumps through damaged blood vessels. Certain individual or clustered CTCs have the ability to survive in the bloodstream and form metastases in secondary organs. This is due to their possession of characteristics that are elevated in EMT-induced cells, and that facilitate their survival in the bloodstream and establishment of metastases. Specifically, b the Fas/FasL signaling pathway plays a crucial role in tumorigenesis, where impairment in cancer cells can lead to resistance to apoptosis and contribute to tumor progression and CTCs generation, c EMT progression is regulated by signaling pathways such as integrin and TGF-β that can cooperate to induce downstream responses to promote CTCs survival and anti-apoptosis properties. The figure was created with BioRender.com

Mechanisms of CTCs cluster generation and the potential CTC subpopulations survival in the blood circulation. CTCs that undergo EMT express Tissue Factor (TF), which significantly contributes to platelet activation. These coagulation-dependent mechanisms initiate the formation of a fibrin/platelet-rich cocoon around tumor cells, believed to offer protection from shear stress, anoikis, and immune attack. The formation of the aforementioned cocoon is crucial for CTC seeding and early establishment. Additionally, neutrophils have been observed to physically interact with tumor cells and platelets, thereby promoting tumor cell survival and proliferation. Furthermore, neutrophils aid in the adhesion of CTCs to the vascular wall. Neutrophils, through their capacity to capture tumor cells in Neutrophil Extracellular Traps (NETs)—structures that also facilitate coagulation events - aid in the formation of a protective and anchoring scaffold that supports CTC survival. This process facilitates the arrest of CTCs in capillaries and early phases of metastatic establishment. The coagulation- and neutrophil-dependent shielding mechanisms described above safeguard CTCs from immune destruction. Furthermore, CTCs, especially those that undergo EMT, possess an increased ability to evade immune surveillance. One mechanism that contributes to this is the expression of immune checkpoint proteins such as PD-L1, which likely enhances their resistance to cytotoxic immune cells. After a possible period of dormancy, CTCs can eventually resume growth and initiate secondary tumors. The figure was created with BioRender.com

EMT of CTCs

Through a process called EMT, tumor cells can acquire on mesenchymal characteristics like enhanced motility and invasiveness. 186 , 187 , 188 The dynamic and intricate process of EMT in cancer cells involves changes in gene expression and the activation of various signaling pathways. During the early stages of EMT, cancer cells undergo a transformation in which they lose their epithelial traits, such as intercellular adhesion and polarity, and gain mesenchymal characteristics to acquire an invasive phenotype. 189 , 190 Alongside this, a number of genes show changed expression, including those related to ECM remodeling, cytoskeletal organization, and cell adhesion. 191 , 192 , 193 As the EMT process progresses, cancer cells may undergo additional changes, such as the activation of stemness, which can lead to tumor heterogeneity and treatment resistance. EMT can also facilitate the formation of metastases, as mesenchymal-like cancer cells are better able to invade neighboring tissues and enter the bloodstream or lymphatic system. 194 , 195 The progression of EMT in cancer cells is influenced by various factors such as signaling pathways (TGF-β, WNT, and NOTCH, etc.) including the TME, such as hypoxia and inflammation. 38 , 196 Furthermore, EMT is regulated by a number of transcription factors, such as Twist, Slug, and Snail, which have the ability to either activate or inhibit the expression of genes connected to EMT. 197 , 198 , 199 In general, EMT in cancer cells is a dynamic and complex process that is orchestrated by multiple signaling pathways and transcription factors. Understanding the mechanisms that drive EMT in cancer cells may provide new ideas for the management of cancer.

Initiation of EMT

Several signaling pathways, such as TGF-β, WNT, and NOTCH signaling, initiate the process of EMT. These pathways trigger transcription factors, such as Twist, Snail, Slug, and zinc finger E-box binding homeobox 1 (ZEB1), that promote mesenchymal genes and suppress epithelial genes. 200 , 201 , 202 By reducing cell-cell interaction, EMT-related transcription factors including Snail and TWIST promote motility and invasiveness in vitro. Nevertheless, knockdown of these factors could only partially limit metastasis in vivo, which challenges the necessity of them for the metastatic process. 80 , 81 Interestingly, loss of E-cadherin or TWIST modulation was found to prevent the successful metastatic seeding of in several EMT lineage tracing models. 82 Many studies have investigated several genes associated with EMT in CTCs. Among the commonly studied EMT target genes, Vimentin is often included. 203 , 204 , 205 Vimentin, primarily expressed in mesenchymal cells as a type III intermediate filament, is a well-known indicator of EMT and is frequently studied in relation to CTCs. Moreover, it has been associated with tumor metastasis, including promoting CTC survival and tumor cell migration. 206 , 207 EMT has been observed to affect the expression of a variety of epithelial adhesion molecules, thereby modifying intercellular interactions. A typical example is the modulation of adhesion molecules, which involves suppression of E-cadherin and up-regulation of N-cadherin. This change in expression has been linked to the EMT process, 208 , 209 and both E-cadherin and N-cadherin, molecules that modulate cell-cell interactions and whose expression is altered in EMTs, are often evaluated in CTCs. The adhesion molecule EpCAM, previously utilized in CTC studies for enrichment purposes, is another EMT target gene that is frequently investigated in CTC research. 210 , 211

Disassembly of cell–cell junctions

EMT begins with the disruption of tight junctions, adherens junctions, and desmosomes, which hold epithelial cells together. 111 , 212 , 213 In order for tumor cells to separate from the main tumor and infiltrate nearby tissues, this step entails the breakdown of the intercellular adhesion molecules that hold epithelial cells together. One key player in the disassembly of cell-cell junctions during EMT is Snail. 214 Snail suppresses the E-cadherin expression, which is a key component of adherens junctions that hold epithelial cells together. By recruiting co-repressors such histone deacetylases (HDACs) and chromatin remodeling factors, snail is able to repress this gene by attaching to E-box elements in the E-cadherin promoter region. 63 , 215 This results in the E-cadherin silencing and the subsequent loss of cell-cell adhesion. Other transcription factors such as ZEB1 and Slug also exerts crucial functions in the disassembly of cell-cell junctions during EMT. Slug represses E-cadherin expression by binding with its promoter and recruiting HDACs and other co-repressors. ZEB1 upregulates mesenchymal genes including Vimentin and fibronectin while suppressing the expression of E-cadherin, further supporting the mesenchymal phenotype of cancer cells. 216 , 217 A role for signaling pathways in the disassembly of cell-cell junctions during EMT has also been studied in previous research. For instance, it is known that the TGF-β pathway induces EMT in different forms of cancer by increasing the expression of associated transcription factors that inhibit E-cadherin. 218 , 219 The Notch and Wnt pathways have also correlated with EMT and cell-cell junction disassembly in different cancer types. 114 , 220 , 221 In conclusion, the disassembly of cell-cell junctions is a complex process that entails the dysregulation of multiple signaling pathways and transcriptional networks. The development of novel therapies for the control and prevention of metastatic cancer may result from improving our understanding of the molecular mechanisms underlying this process.

Remodeling of the ECM

During EMT, cancer cells undergo changes that allow them to break down the ECM and invade surrounding tissues. 191 Several studies have investigated the molecular mechanisms underlying this process in various cancer types. One of the key players in ECM remodeling during EMT is the matrix metalloproteinases (MMPs). MMPs are a class of zinc-dependent endopeptidases that can degrade collagens, laminins, and proteoglycans, among various ECM components. 222 , 223 , 224 MMPs are upregulated during EMT in numerous cancer types, and their expression is manipulated by a number of signaling pathways and transcription factors, including Twist and Snail. 225 , 226 Other proteases have also been implicated in ECM remodeling during EMT, including urokinase-type plasminogen activator (uPA) and its receptor. 227 , 228 uPA is an enzyme belonging to the class of serine proteases that can activate plasminogen, leading to the degradation of ECM components. 229 uPA expression is upregulated during EMT in various cancer types, and its expression is governed by various transcription factors such as Snail and ZEB1. 230 , 231 The remodeling of the ECM during EMT also involves changes in the expression and activity of integrins, a group of transmembrane receptors that regulate cell-ECM interactions. A variety of signaling pathways and transcription factors, such as TGF-β and Snail, orchestrate the upregulation of integrin expression during EMT in various cancer types. 232 Integrins can promote the invasiveness of cancer cells by activating downstream pathways such as phosphatidylinositol 3-kinase (PI3K) and focal adhesion kinase. 233 , 234 Studies have also shown the involvement of non-coding RNAs (nc), including long nc RNAs (lncRNAs) and microRNAs (miRNAs), in ECM remodeling during EMT. For example, miR-29b has been shown to inhibit the expression of MMP2 and MMP9, leading to the suppressed ECM degradation and inhibited EMT in lung cancer cells. Similarly, the lncRNA MALAT1 has been demonstrated to trigger ECM remodeling and EMT in bladder cancer cells by manipulating the expression of MMP2 and uPA. 235 , 236 , 237

Non-EMT with CTCs

Recent studies utilizing transgenic mouse models has indicated that invasion and metastasis may happen without the involvement of EMT. These findings support the concept that the initiation of metastasis may not necessarily require EMT. 80 , 81 The deletion of EMT-related transcription in transgenic mouse models suggests that EMT inhibition does not affect the development of systemic metastasis. 81 Moreover, it has been reported by Godinho et al. that cancer cell invasion can also be triggered by centrosome amplification. This occurs through the disruption of cell-cell adhesion and an increase in actin polymerization dependent on Arp2/3. 238 Moreover, passive infiltration, which is caused by external forces such as mechanical stress and tumor growth, can lead to the genesis of “accidental” CTCs in the bloodstream. 239 , 240 The disseminated cancer cells that arise from invasion not mediated by EMT can reside either as individual cells or clusters and maintain their epithelial phenotype. 80 , 239

CTC clusters pose a higher risk for cancer metastasis compared to individual CTCs, and they also have unique properties that make them resistant to chemotherapy and immune surveillance. Though the mechanism of CTC cluster formation remains elusive, several studies have shed light on the process. A study by Aceto et al. investigated the role of E-cadherin, a cell adhesion molecule, in CTC cluster formation. They found that E-cadherin was downregulated in single CTCs, but it was upregulated in CTC clusters. Furthermore, they showed that blocking E-cadherin expression prevented CTC cluster formation, suggesting that E-cadherin plays a crucial role in CTC cluster formation. 241 , 242 Gasic et al. was one of the first to suggest that platelets might be involved in CTC cluster formation. 84 They found that platelets accumulated around CTCs in the bloodstream, and they speculated that platelets might facilitate CTC cluster formation. More recent studies have confirmed this hypothesis. For instance, a study by Haemmerl found that platelets promoted CTC cluster formation by activating the TGF-β signaling pathway, which upregulates EMT genes and downregulates cell adhesion molecules. 243 , 244 The role of integrin in CTC cluster formation. They found that CTCs overexpressed several integrins, including integrin α5β1, which promoted CTC cluster formation by facilitating cell-cell adhesion. 245 , 246 The role of chemokines, a group of signaling proteins that recruit immune cells to the location of inflammation or infection, in CTC cluster formation. CTCs secreted several chemokines, including CXCL1 and CXCL2, which promoted CTC cluster formation by attracting neutrophils, a type of immune cell, to the site of CTCs. 247 , 248 The role of hypoxia, a condition of low oxygen levels, in CTC cluster formation. Hypoxia upregulated the expression of a transcription factor called HIF-1α, which promoted CTC cluster formation by upregulating EMT genes and downregulating cell adhesion molecules. The role of physical forces, such as shear stress and hydrodynamic forces, in CTC cluster formation. CTCs were more likely to form clusters in areas of low shear stress and high hydrodynamic forces, such as in the corners of blood vessels. 249 , 250 , 251 In summary, CTC cluster formation is a multifactorial and complex process that entails multiple biological and physical factors. Despite our incomplete comprehension of the mechanism of CTC cluster formation, technological advancements have facilitated a more precise and comprehensive examination of CTCs and CTC clusters, offering new potential for understanding and treating metastatic cancer.

When epithelial cells detach from their surrounding tissue, they usually undergo programmed cell death known as anoikis, which limits the efficacy of metastatic seeding. 72 , 73 , 74 However, a small proportion of tumor- or metastasis-initiating cells with stem-like characteristics have been found to have an enhanced ability to survive and seed tumors. 47 , 75 , 85 EMT is considered as a prerequisite for metastatic dissemination, as it enhances contact-independent survival and invasive potential. 78 , 79 This indicates that EMT may function as a modulator rather than a promoter of metastasis. Meanwhile, a moderate level of EMT has been observed in CTCs, which links to stem-like properties, plasticity, reduced response to treatment and disease development. 76 , 85 On the invasive edge of xenografts and patient tumor tissues of common carcinoma types, intermediate EMT stages have been seen. 84 , 86 , 87 However, further investigation is needed to determine their functions in invasion, and metastatic dissemination.

The investigation of EMT within the context of CTCs is a field that is rapidly evolving, with numerous essential factors being explored. Commonly examined within CTCs are EMT-associated genes such as Vimentin, adhesion molecules, and core transcription factors. The analysis of signaling pathways triggered by membrane receptors and Receptor Tyrosine Kinases (RTKs) is a common practice in CTC studies, as these pathways, such as EGFR, TGF-β, Notch, c-Met, and Wnt, are crucial in the regulation of EMT. The investigation of Axl, an RTK associated with EMT, is gaining momentum in CTC studies, given that clinical trials are currently evaluating commercial inhibitors for Axl. It is noteworthy that EMT is associated with cancer-stem cells, and the majority of EMT-related markers examined in CTCs affect CTC survival and the ability to metastasize. Numerous studies have provided evidence of the heterogeneity of EMT-associated molecules present in the CTC population and have identified hybrid epithelial/mesenchymal (E/M) phenotypes in lung cancer. Table 2 highlights these findings.

Intravasation of CTCs

Intravasation, which involves the entry of cancer cells into the bloodstream, can occur through either active or passive mechanisms. The exact mechanism utilized by the tumor cells depends on various factors, including the tumor types, the micro-environment, and the integrity of the blood vessels. 252 Active intravasation occurs when tumor cells or clusters actively migrate into the bloodstream through EMT. Passive intravasation, on the other hand, happens when single cells or groups of cells separate from the main promise and enter the bloodstream as a result of broken tumor blood vessels brought on by tumor growth or treatment. 181 After entering the bloodstream, CTCs are subjected to various obstacles that can threaten their survival, including shear stress caused by blood flow, anoikis, all of which can potentially lead to their elimination. 253 Tumor cells can gain access to the bloodstream through either a blood vessel or a lymphatic vessel, which can be influenced by several factors such as accessibility, physical constraints, and the existence of active mechanisms that lure cells to specific types of vessels. 254 , 255 Additionally, tumor cells can utilize lymphatic vessels as a pathway to enter the bloodstream since these vessels ultimately empty into the major thoracic duct. 254 , 256 Although lymphatic vessels can aid in the entry of certain cancer cells into the blood circulatory system, current evidence is limited, and cancer cells may encounter dead ends in lymphatic deposits. This observation highlights the degree of concurrent dissemination from the primary tumor. 75 , 257 , 258 Alternatively, cancer cells are likely to spread primarily through the bloodstream for distant metastasis. In this process, lymphatic fluid passes through a sequence of lymph nodes, which frequently serve as the primary sites of metastasis. 252 , 259

Colonization of CTCs

The colonization of tumor cells is the final step of the metastasis process and involves the establishment of tumor cells in a distant organ. 232 , 260 , 261 The colonization process is complex and involves several steps that are regulated by multiple signaling pathways and molecular networks. This process requires the re-activation of epithelial factors and the suppression of mesenchymal ones, a process known as MET. Several studies have investigated the molecular mechanisms underlying this process in various cancer types. One of the key players in the colonization of tumor cells is p63, which is a member of the p53 group of tumor suppressors. P63 is upregulated during MET in a variety of cancer types and is essential for the maintenance of epithelial stem cells. 262 P63 promotes the reactivation of epithelial genes like E-cadherin and cytokeratins, while suppressing mesenchymal genes like Vimentin and fibronectin. 263 Twist and Snail have also been implicated in the regulation of MET and the colonization of tumor cells. Twist is upregulated during EMT and downregulated during MET. 264 Twist improves the mesenchymal phenotype of tumor cells by suppressing E-cadherin expression and increasing N-cadherin and fibronectin expression. 265 Snail, on the other hand, promotes EMT and invasion by inhibiting E-cadherin expression, but its role in MET and colonization is less clear. Several signaling pathways also participated in the regulation of MET and tumor cell the colonization. 266 , 267 The TGF-β pathway, for example, can induce EMT in various cancer types but can also promote MET and colonization by activating the expression of p63 and other epithelial genes. 268 Other signaling pathways, such as Notch, Hedgehog, and Wnt, have also been implicated in the regulation of MET and colonization in various cancer types. 269 , 270 During metastasis, CTCs and the target organ microenvironment engage in a critical dialog that determines the fate of metastatic colonization. This interaction encompasses various components such as ECM proteins, immune cells, and soluble factors, which collectively influence the survival, dormancy, and growth of CTCs. Key processes include the EMT, which CTCs undergo to establish new tumors, and the evasion of immune surveillance. The microenvironment’s role extends to either supporting CTC colonization through the provision of growth factors or hindering it by presenting physical barriers and immune challenges. 271 , 272

The CTCs under immune system surveillance

The mechanism by which CTCs overcome immune system surveillance is not completely understood, but there are several possible ways in which they may evade detection by the immune system. One explanation is that CTCs suppress or mask the expression of surface antigens that immune cells often recognize as abnormal or foreign. This can make the CTCs less visible to the immune system and allow them to escape detection. Another possibility is that CTCs produce factors that suppress or inhibit the immune response, such as cytokines or chemokines that recruit immune cells to the tumor microenvironment but then suppress their function. For example, studies have shown that CTCs can produce the cytokine TGF-β, which can suppress the activity of T cells and other immune cells (Fig. 4 ). Finally, CTCs may be able to evade immune surveillance by mimicking normal cells or using other mechanisms to avoid detection. For example, some CTCs may express proteins or molecules that are normally present in healthy cells, leading to a challenge for the immune system in differentiating them from healthy cells. 273 , 274

Immune-escape mechanisms of CTCs in the peripheral blood. The diagram depicts the various mechanisms employed by CTCs to circumvent the immune system and the interactions that take place between CTCs and immune cells in the peripheral blood. The interaction between circulating tumor cells (CTCs) and natural killer (NK) cells is a prominent area of investigation, given that CTCs secrete LDH5 and shed the MICA/MICB ligand via ADAM10, which inhibits recognition and elimination of CTCs through NK cell-mediated lysis. The diagram portrays three mechanisms employed by CTCs to evade recognition by NK cells and T cells through MHC I molecules. These strategies include masking MHC I recognition by TCR via cytokeratins (CK8, CD18, and CK19) bound to the cell surface, gaining a “pseudo-normal” phenotype through membrane transfer from platelets to CTCs, and reducing or eliminating MHC I expression altogether. The interaction between CTCs and natural killer (NK) cells is a key area of focus, as the release of LDH5 and shedding of MICA/MICB ligands by ADAM10 from CTCs prevents their recognition and elimination via NK cell-mediated lysis. Additionally, LDH5 enhances NKG2D ligand expression on circulating monocytes, which in turn reduces NKG2D expression on NK cells. Furthermore, the diagram illustrates other strategies employed by CTCs to evade the immune system, such as the upregulation of the inhibitory immune checkpoint molecule PD-L1, expression of the “don’t eat me” signaling receptor CD47 and altered expression of apoptotic proteins FAS and/or FASL. The figure was created with BioRender.com

Immune evasion through antigen loss

CTCs can downregulate or lose expression of antigens normally recognized by the immune system, making them less visible to immune cells. MHC class I molecules, for example, are required for T cell recognition, and their expression is downregulated by certain CTCs. For example, in 2012, researchers found that the expression of EpCAM, a common antigen used for CTC detection, was downregulated in CTCs from breast cancer patients who had received chemotherapy. 275 , 276 Similarly, a study reported that CTCs from breast patients who received anti-HER-2 therapy reduced their expression of HER-2, another common antigen. 277 , 278 , 279

CTCs secreted immunosuppressive factors

CTCs have the ability to secrete immunosuppressive factors including TGF-β and IL-10, which can inhibit immune cell activation and function and create an immunosuppressive milieu that shields CTCs from immune attack. A previous work demonstrated that CTCs from breast cancer patients secrete high levels of TGF-β, which can and promote tumor growth and metastasis because of immune cell inactivation. 280 , 281 , 282 Similarly, another study showed that CTCs from melanoma patients release IL-1, which has the ability inhibit T cell activation and proliferation, a crucial part of the immune system’s defense against cancer. 103 , 283 , 284 Besides, Zhao et al. reported that CTCs from lung cancer patients secrete CCL2, which can attract immune-suppressive cells to the tumor microenvironment, thereby suppressing the immune response. Then, a clinical trial demonstrated that CTCs from prostate cancer patients secrete GAL-3, which can induce the apoptosis of tumor-killing immune cells, thereby blocking the immune response against cancer. 285 , 286 , 287 In order to promote angiogenesis and suppress the immune response, CTCs from breast cancer patient have also been reported to secrete VEGF. 288 , 289 , 290 Also, CTCs from colorectal cancer patients secreted PGE2 and led to immune suppression and tumor progression as well. 291 , 292 Moreover, CTCs from melanoma patients secrete IL-10, which can inhibit the proliferation and promote tumor growth and metastasis by activating T cells. 293 , 294 A study showed that CTCs from lung cancer patients expressed high levels of programmed cell death ligand 1(PD-L1), which inhibited the activity of immune cells and promoted immune evasion. 295 , 296 , 297 , 298

Immunological checkpoint (IC) proteins

CTCs have the ability to express IC proteins, such as PD-L1, which bind to the receptors on immune cells to prevent the immune cells from activating and functioning. This can prevent immune cells from attacking CTCs and allow them to escape immune surveillance. CTCs from patients with advanced colorectal, breast, or prostate cancer exhibit IC proteins such PD-L1 and CTLA-4, which can promote immune evasion and support in tumor growth, according to Krebs MG. 297 , 298 , 299 Maheswaran et al. demonstrated that CTCs from lung cancer patients express PD-L1 and other IC proteins, and that PD-L1 expression is linked to poor prognosis. 300 , 301 Gkountela et al. reported that CTCs from breast cancer patients express PD-L1, which is linked to resistance to chemotherapy. 302 Zhang W et al. showed that CTCs from gastric cancer patients express PD-L1 and TIM-3, which is associated with advanced stage and poor prognosis. 303 Sanmamed et al. demonstrated that CTCs from melanoma patients express PD-L1 and other IC proteins, and that PD-L1 expression leads to treatment resistance and poor outcome. 304 Similar results have been observed in patients with breast cancer. 305 Another study revealed that CTCs from lung cancer patients who received IC inhibitor therapy have downregulated PD-L1 expression. 306 , 307 , 308 The dual inhibition strategy of blocking PD-L1 and CD47 on CTCs has been found to significantly enhance the effectiveness of IC therapy. By enabling the immune system to more efficiently target and eliminate CTCs, this approach holds promise for reducing the risk of tumor recurrence and metastasis. Preclinical models have demonstrated that this combination therapy is more effective, indicating its potential for successful clinical application in cancer treatment. 309

Production of extracellular vesicles

By promoting the detachment of CTCs from the original site, extracellular vesicles (EVs) play a vital role in the metastatic process. EVs enhance the shedding of CTCs and protect them in circulation, thereby influencing their metastatic direction. The detachment of CTCs from the main site is facilitated by EVs, which also contribute to EMT and ECM remodeling, promote angiogenesis, and improve vascular permeability. In addition, EVs protect CTCs by activating platelets, inducing immunosuppression, and determining the organotropism of metastasis, thus influencing the colonization of CTCs in distant organs. 310 EVs released by CTCs can interact with immune cells and modulate their function, creating an environment that favors CTC survival and growth. Armstrong AJ showed that CTCs from prostate cancer patients produce extracellular vesicles that can suppress the immune response by suppressing the T cell and NK cell activity. 311 , 312 , 313 Keklikoglou demonstrated that CTCs from breast cancer patients produce extracellular vesicles that can promote immune evasion by inducing the differentiation of monocytes into immunosuppressive macrophages. 314 , 315 , 316 Whiteside TL et al. discussed the role of extracellular vesicles produced by CTCs and other tumor cells in promoting immune suppression and tumor progression. 317 Peinado H et al. showed that CTCs from breast cancer patients produce extracellular vesicles that can promote metastasis by modifying the microenvironment at distant sites and suppressing the immune response. 318 Liu C et al. demonstrated that extracellular vesicles produced by CTCs from lung cancer patients can induce immunosuppression by blocking the activity of NK and T cells. 319 Melo SA et al. showed that CTCs from pancreatic cancer patients produce extracellular vesicles that can promote immune evasion by inducing the differentiation of monocytes into immunosuppressive macrophages. 320 Lu J et al. demonstrated that extracellular vesicles produced by CTCs from lung cancer patients can induce immunosuppression. 321 , 322 Hoshino et al. showed that CTCs from melanoma patients produce extracellular vesicles that can promote immune evasion by inhibiting the activity of tumor-killing immune cells, and that targeting these vesicles can enhance the effectiveness of immunotherapy. 323

Formation of immune-resistant micro-metastases

CTCs can establish small metastatic lesions that are immune-resistant. These lesions can create a reservoir of CTCs that can escape immune surveillance and facilitate further metastatic spread. ICAM1 is essential for the development of CTC clusters and their trans-endothelial migration in lung metastases of BRCA. It has been found to facilitate metastasis more effectively in clusters as opposed to single cells, thereby contributing to a decrease in overall survival rates. In lung metastases of patient-derived xenografts of TNBC, the expression of ICAM1 is noticeably elevated. The inhibition of ICAM1 has been shown to impede the lung colonization of TNBC cells by disrupting the formation of tumor cell clusters, suggesting that ICAM1 could serve as a promising therapeutic target for inhibiting metastasis initiation in TNBC. 100 Janssen LME showed that CTCs can form immune-resistant micro-metastases by secreting extracellular vesicles that modulate the immune response and create a favorable microenvironment for metastatic growth. 324 Krebs et al. demonstrated that CTCs can form micro-metastases that are resistant to chemotherapy and immune surveillance in breast cancer patients. 325 Focusing on the formation of osimertinib-resistant micro-metastases following treatment, a study described an orthotopic model of lung cancer. This model provides a platform for analyzing resistance mechanisms and evaluating new therapeutic strategies against metastases of NSCLC. 326 Kallergi et al. showed that CTCs can form micro-metastases that are resistant to chemotherapy and immune surveillance in breast cancer patients, and that targeting the immune system can improve treatment outcomes. 326 Aceto N et al. showed that CTCs can form micro-metastases that are resistant to chemotherapy and immune surveillance in breast cancer patients, and that targeting the immune system can improve treatment outcomes. 327 Bidard FC et al. demonstrated that CTCs can form micro-metastases that are resistant to chemotherapy and immune surveillance in breast cancer patients, and that targeting the immune system can improve treatment outcomes. 328

Platelets coordinate with CTCs

It has been indicated that the lifespan of certain CTCs is brief, as a majority of them are undetectable within 24 h after primary tumor excision. 329 Research reported that the interaction of CTCs with other blood components, particularly platelets, significantly affects their survival and potential for metastasis. 329 CTCs shortly after entering the bloodstream, are known to create a thrombus rich in platelets around them, serving as a protective shield against shear stress and immune response. Furthermore, this thrombus promotes the attachment of CTCs to the endothelial lining of blood vessels, enabling extravasation. 121 , 309 , 330 Upon entering the bloodstream, CTCs can form a shield of platelets through the action of platelet tissue factor. The formation of a thrombus rich in platelets can offer protection to CTCs against shear stress and immune system attacks, while also aiding in the attachment of tumor cells to the blood vessel wall and extravasation. Moreover, studies have shown that activated platelets can transfer the MHC to CTCs, enabling them to evade immune surveillance by mimicking host cells. 117 , 331 In addition, platelets have been shown to reduce the recognition and elimination of tumor cells by NK cells. Platelets can release soluble factors that impair the cytotoxicity of NK cells and promote the expansion of regulatory T cells, which further interfere with the immune responses. Platelets can also inhibit the recognition and destruction of tumor cells by immune cells, as they express molecules that interact with receptors on immune cells and prevent their activation. One such molecule is CD47, which can bind to SIRPα on macrophages and inhibit their engulfment of tumor cells. These mechanisms collectively enhance the survival and dissemination of CTCs. 117 , 243

Moreover, the transfer of MHC molecules by platelets is a mechanism by which CTCs can evade immune surveillance. 117 , 332 Platelets have the ability to impede the identification and destruction of neoplastic cells by NK cells. 18 , 322 , 333 The invasiveness and metastasis of CTCs can be enhanced by platelets, which release TGFβ and promote the EMT in these cells. This phenomenon has been demonstrated in previous research. 107 , 334 CTCs have been found to interact with different types of leukocytes such as, monocytes, and macrophages, which can enhance the survival of CTCs and facilitate their interaction with endothelial cells, leading to extravasation. 21 , 107

Role of epigenetic modifications in deciphering the properties of CTCs

Recent research has highlighted that significant epigenetic alterations in normal cells can lead to the acquisition of a malignant phenotype. 335 Epigenetics involves the investigation of inheritable alterations in gene expression that occur without modifications to the DNA sequence itself. 336 A number of epigenetic modifications, including histone acetylation and methylation patterns, microRNA-mediated gene regulation, hypomethylation of oncogenes, hypermethylation of tumor suppressor genes, and others, greatly contribute to the progression of cancer. 337 Understanding the intratumor heterogeneity and gaining insight into tumor-specific epigenetic markers linked to metastasis may be facilitated by mapping the epigenetic landscape of CTCs. This approach may also identify subpopulations of CTCs capable of metastatic dissemination. The main areas of epigenetic research that apply to this scenario are gene regulation and DNA methylation.

DNA methylation

DNA methylation is the addition of methyl groups to DNA’s cytosine residues, and it is essential for regulating gene expression. DNA methylation patterns that deviate from the normal or expected patterns have been linked to various health conditions, including cancer. Several studies have shown that CTCs have distinct DNA methylation profiles compared to primary tumors, which could be useful in detecting early-stage cancer, predicting cancer prognosis, and developing targeted therapies. 338 , 339

CTCs exhibit unique DNA methylation patterns that differ from primary tumor. 339 For instance, in breast cancer patients, CTCs had increased DNA methylation levels compared to primary tumors. 121 They identified several differentially methylated genes that were specific to CTCs, including the FBLN1, FBN2, and IGFBP6 genes. A prognostic factor for cancer may be the DNA methylation status of CTCs, as these genes have been linked to the progression and metastasis of cancer. Similarly, in prostate cancer, CTCs had unique DNA methylation patterns compared to primary tumors. 340 They identified several differentially methylated genes, including the FOXA2, HOXA9, and PTEN genes. These genes are involved in cancer progression, invasiveness, and metastasis. Additionally, they discovered a correlation between the DNA methylation status of CTCs and patient survival, suggesting that this biomarker may have applications in the prediction of cancer prognosis. Moreover, DNA methylation patterns of CTCs can also differ based on the origin of the tumor. For instance, a study by Xu et al. compared DNA methylation profiles of CTCs separated from lung cancer and pancreatic cancer patients. 341 They found that CTCs from lung cancer patients exhibited higher DNA methylation levels compared to CTCs from PAAD patients. Furthermore, they identified differentially methylated genes specific to each cancer type, such as the RASSF1A gene in lung cancer CTCs and the TGFBI gene in pancreatic cancer CTCs. This evidence support that the DNA methylation pattern of CTCs could be used to distinguish between different cancer types and aid in personalized cancer treatment. 342

In addition, DNA methylation patterns of CTCs can also be influenced by treatment. DNA methylation patterns of CTCs changed after chemotherapy in breast cancer patients. 343 They identified several genes, including the CDH1 and ZEB1 genes, which had differentially methylated CpG sites before and after chemotherapy. These results imply that monitoring alterations in the DNA methylation patterns of CTCs could be utilized to evaluate the effectiveness of cancer treatments.

Further research is required to unravel the gene methylation landscape of CTCs and how these methylation changes contribute to CTC-mediated metastasis. Such studies can reveal the tumor-specific epigenetic characteristics related to metastasis and the heterogeneity of tumor populations. 344 , 345 Gkountela et al. analyzed the DNA methylation patterns of CTCs from BRCA patients and tumor xenograft models in NSG mice and discovered numerous differentially methylated regions (DMRs) in CTCs. 5 , 346 CTCs showed lower DNA methylation levels of genes such as JUN, MIXL1, SHOX2, and MEF2C, which are often enriched in various types of cancer. CTC clustering was associated with low methylation levels of TFBSs that regulate and proliferation- and stemness-related genes, such as OCT4, MANOG, SOX2, and SIN3A. 17 , 97 , 347 This was accompanied by hypermethylation and H3K27me3 repression of target gene promoters and bodies of PRC2 targets, including SUZ12 and EED, collectively increasing the hematogenous metastatic potential of CTC clusters. 348 , 349 In a study comparing the DNA methylation of CTCs from cancer patients and normal blood controls, abnormal methylation changes were found in multiple genes, and were associated with resistance to sunitinib. This ample evidence suggests that abnormal gene methylation in CTCs may play a crucial role in their clustering and promote distant metastatic ability (Table 3 ).

Aberrant DNA methylation leading to mutation and inactivation of oncogenes or tumor suppressor genes

TSGs play a critical role in orchestrating cellular processes and maintaining genomic stability. Their mutations can cause excessive cell proliferation, which can contribute to the development of tumors. They are also in responsible for causing apoptosis, repairing DNA damage, and regulating cell division. One example of a TSG is hMLH1, which is involved in DNA mismatch repair. 350 Studies have shown that hMLH1 gene mutations and Microsatellite Instability are positively correlated with hMLH1 promoter hypermethylation. The Knudson hypothesis proposes that mutations in both alleles are necessary to inactivate TSGs, and epigenetic modifications, such as the silencing of one allele through promoter hypermethylation, can satisfy this requirement. 351

Some patients showed evidence of epigenetic silencing of the MGMT gene by promoter hypermethylation, which was connected to a higher frequency of activating mutations in KRAS, a proto-oncogene frequently mutated in colorectal cancer. It has been indicated that hypermethylation of the MGMT promoter may contribute to the development of colorectal cancer by promoting KRAS mutations through the inactivation of a DNA repair gene. A study of 244 patients with colorectal cancer analyzed the effects of epigenetic alterations on proto-oncogenes by examining the MGMT gene. The study found that the promoter hypermethylation-mediated MGMT inactivation was linked with a rise in guanine-to-adenine mutations in K-Ras oncogenes. The MGMT gene expression helps to prevent such transitions in the Ras genes. 352 The aforementioned discoveries underscore the essential function of epigenetic modifications in stimulating proto-oncogenes and consequently, advancing the growth and advancement of cancer. A study revealed that lung cancer CTCs demonstrate a distinctive DNA methylation pattern in comparison to primary tumors and normal tissues. This signature is characterized by a substantial reduction in CTC DNA methylation overall, indicating a process of gradual demethylation that progresses from primary tumors to normal tissues and ultimately to CTCs. This underscores the dynamic epigenetic alterations linked to cancer metastasis and spread. Notably, this demethylation phenomenon is observed throughout various genomic regions, encompassing promoters, gene bodies, introns, and intergenic regions. There is a noticeable decrease in methylation at CpG-poor promoters in CTCs when contrasted with primary tumors. 338 We have summarized the different mechanisms of DNA methylation modifications in CTCs to promote metastasis in Fig. 5 .

Different DNA methylation modifications operating in CTCs to promote metastasis. The process by which methylation promotes detachment and invasiveness of CTCs is intricate and involves both hypermethylation and hypomethylation of various genes. a Specifically, hypermethylation of tumor suppressor genes (TSGs) and metastasis-associated genes (MSGs) triggers the detachment of CTCs and contributes to their enhanced proliferative capacity. CTC clusters exhibit distinct DNA methylation profiles compared to single CTCs, featuring hypomethylation of binding sites for transcription factors like OCT4, NANOG, SOX2, and SIN3A, b which are stemness-related transcription factors play crucial roles in the pluripotency network of induced pluripotent stem cells (iPSCs). The figure was created with BioRender.com

Hypomethylation regulation of CTC clusters and increases metastatic potential

The sustained proliferation and self-renewal capacities of embryonic stem cells require the maintenance of stemness and proliferation properties. 353 It has been indicated that stemness-related genes may be entailed in the dissemination of cancer cells. However, the precise mechanisms underlying the ability of CTCs to disseminate and form metastases are not yet fully elucidated, and their survival in the bloodstream is critical for this process. A research study on breast cancer patients aimed to examine the metastasis of CTCs by investigating their ability to form clusters. In the study, single CTCs and CTC clusters from patients with progressing breast cancer were isolated using Parsortix microfluidic technology. Subsequently, whole-genome bisulfite sequencing was conducted on single-cell resolution matched CTCs, which were selected from eight out of a total of 43 analyzed samples (19%). The sequencing outcomes revealed that clusters had hypomethylation of stemness-related genes, specifically SOX2, NANOG, and SIN3A, compared to sing CTCs. This observation facilitated the identification of potential therapeutic targets that might diminish the metastatic potential of the CTC clusters. Specifically, the researchers identified the alteration of methylation patterns in the promoter of the related genes by a Na + /K + ATPase inhibitor led to the separation of the clusters and the reduced tumor metastasis in mice. 5

A study identified DMRs between CTCs and primary lung cancer samples, revealing a significant number of hypomethylated DMRs in CTCs. This trend towards decreased DNA methylation at aberrantly methylated loci in CTCs suggests a potential mechanism underlying their unique characteristics. A potential function in regulating crucial physiological processes such cell proliferation, differentiation, and apoptosis was discovered in the hypomethylated regions of CTCs, which were found to be highly enriched for transcription factor binding sites (TFBSs). Consequently, the evidence supports the idea that hypomethylation in CTCs may play a crucial role in the formation of CTC clusters and enhance their metastatic potential by influencing gene expression related to EMT and other pathways critical for cancer progression. 338 The significance of this study lies in its use of genome-wide sequencing-based methylation profiling for CTCs, making it the first of its kind. This emphasizes the importance of generating comprehensive methylation patterns for CTCs in the context of broader biological research. The incorporation of advanced technology in this research signifies a significant advancement in the current understanding of metastasis and the recognition of novel drug targets.

Hypermethylation regulation the invasiveness of CTCs

The process of detachment of CTCs from their primary tumors and their subsequent migration to distant organs is a pivotal event in the progression of metastasis. To facilitate this process, the cells require the ability to detach and enhance their plasticity, allowing them to penetrate through capillaries and settle in different organs. The mechanism that plays a crucial role in this process is known as EMT, which endows the cells with increased invasiveness and motility. 354 Utilizing peripheral blood samples from 52 patients with metastatic colorectal cancer, the researchers extracted CTCs and examined the methylation patterns of two crucial EMT genes, Vimentin and SFRP2, to investigate the function of EMT in CTC invasiveness. The findings of the study indicated that the selected genes were heavily methylated in the isolated CTCs as compared with the healthy tissue samples. The study demonstrated that the Vimentin gene was suppressed by DNA methylation, causing a disruption in the formation of the cytoskeleton, and consequently promoting increased plasticity and invasiveness of the cells. In contrast, the study revealed that the suppression of the SFRP2 gene by methylation activated the Wnt signaling pathway, which led to increased invasiveness of the CTCs. 355 In another study, researchers evaluated the methylation status of E-cadherin, an EMT suppressor gene that is responsible for preserving the epithelial phenotype. The methylation pattern was assessed in CTCs obtained from six individuals diagnosed with metastatic prostate cancer. The findings indicated that E-cadherin was highly methylated in the CTCs from the cancer patients. Furthermore, the study presented an association between E-cadherin hypermethylation and an increased invasiveness of CTCs. 356 The role of EMT in CTCs is crucial for their adaptability and survival in the bloodstream during the early stages of metastatic colonization, as highlighted in a recent review. Therefore, a comprehensive analysis of EMT in CTCs is essential for informing personalized medicine strategies that target specific aspects of this biological process. 178 The research conducted by Zavridou et al. offers compelling evidence for the effectiveness of the size-based method in addressing the heterogeneity of CTCs. 357

DNA methylation and CTCs immune escape

The dysregulation of DNMTs and TET enzymes can profoundly influence gene expression and contribute to transcriptional silencing or activation in various pathologies, such as cancer. 358 Studies have provided evidence indicating that the dysregulation of TET enzymes and DNMTs can have significant impacts on gene expression, leading to transcriptional activation or silencing in several pathological states, including cancer. Studies have shown that breast cancer and colorectal cancer patients exhibit increased expression of TET enzymes and decreased expression of DNMTs in both their tumor tissues and circulation, which correlates with DNA hypomethylation and upregulation of IC molecules or their ligands 359 , 360 (Fig. 6 ).

DNA methylation regulation in CTCs related Immunity. DNA methylation play key roles in adaptive immune response, including dendritic cell development and T cell priming and activation. a Recent studies revealed the contributions of chromatin remodeling responding to cytotoxic attack in tumor cells and exhaustion phenotype in tumor infiltrating CD8 + T cells. b CTLA-4 DNA hypermethylation significantly correlated with a poor response to treatment, highlighting the potential of CTLA-4 methylation as a predictive biomarker for therapy outcomes. c In tumor cells, DNA modifications affects production of tumor antigens, silencing of anti-tumor cytokines, and induction of the PD-L1 checkpoint. d In NK cells, the methylation status of killer Ig-like receptors (KIR) CpG islands is crucial for maintaining clonal KIR expression and modulating NK cell recognition and lysis of abnormal cells. The figure was created with BioRender.com

Furthermore, it has been demonstrated that restoring normal DNA methylation patterns can enhance the therapeutic efficacy of various cancer treatments. Specifically, the use of epigenetic modifiers that target DNA methylation in combination with chemotherapy, radiotherapy, and immunotherapy has shown increased treatment effect on preclinical models and early clinical trials of several cancers, including breast, prostate, and colon cancer. 361 , 362 , 363 Based on the outcomes of diverse preclinical models and preliminary clinical trials, it appears that epigenetic modulators aimed at DNA methylation may have the ability to function as adjunctive therapy to conventional cancer treatments. Nevertheless, further large-scale clinical trials are required to verify these findings and establish the optimal combination of treatments for different cancer types. To sum up, there is a correlation between abnormal DNA methylation patterns in cancer cells and immune evasion, tumorigenesis, and resistance to cancer treatments. Therefore, DNA methylation patterns may be a crucial target in cancer therapy, and reinstating regular DNA methylation patterns could be a promising strategy to enhance the therapeutic efficacy and overcome resistance mechanisms.

The evidence provided by these studies suggests that the control of ICs and IC ligand expression in cancer patients, particularly in breast and colorectal cancers, is critically influenced by DNA methylation. According to the study, significant upregulation of ICs, including PD-L1 and TIGIT, as well as other ICs/IC ligands in both circulation and tumor tissues, can be attributed to DNA hypomethylation. The significance of DNA methylation in predicting the effectiveness of immunotherapeutic treatments is highlighted by the correlation between overall hypomethylation and inadequate clinical responses. Based on these findings, therapeutic manipulation of DNA methylation has the potential to serve as an effective strategy for enhancing the outcomes of immunotherapies. However, further investigation is demanded to fully understand the mechanism by which DNA methylation impacts the expression of ICs and to develop efficient therapeutic approaches that target these epigenetic alterations. 364

DNA methylation alterations have been linked to the expression of several immunomodulatory genes in breast and colorectal cancer tissues. A study demonstrated that the hypomethylation of CpG islands led to the upregulation of PD-1, CTLA-4, and TIM-3 in breast cancer. 359 Moreover, the study observed complete hypomethylation of LAG-3 gene promoter regions in both breast tumor tissues and their corresponding normal tissue. This suggests that DNA methylation is not entailed in the elevation of these genes in breast cancer. 359 According to another study, DNA hypomethylation is responsible for the overexpression of CTLA-4 and TIGIT in human colorectal cancer tissues. 360 By contrast, in colorectal tumor tissues, the PD-1, PD-L1, galectin-9, and TIM-3 overexpression was not found to be associated with DNA methylation, according to the study. 360 Marwitz et al. reported that the PD-1 and CTLA-4 upregulation in tumor tissues of patients with lung cancer is attributed to DNA hypomethylation. 365 However, the study did not find any relationship between the elevated PD-L1 expression and DNA methylation in the tumor tissues. 365 Conversely, DNA hypomethylation was discovered to be the underlying cause of the upregulation of PD-L1 expression in HNSCC tumor tissues. 366 In a study by Goltz et al., the promoter methylation of PD-L1 was predictive of favorable prognosis in several cancer types, including colorectal cancer, HNSCC, and acute myeloid leukemia. 367 , 368 DNA hypomethylation was identified as the cause of the increased expression of CTLA-4, PD-1, PD-L1, and PD-L2 in patients with lower-grade gliomas, according to a study by Rover et al. Overall, these findings support that DNA hypomethylation is entailed in the upregulation of IC molecules and ligands in different types of cancers, although the specific genes that are regulated by DNA methylation may vary across different cancer types.

DNA methylation of CTCs cluster formation

Recent research has suggested that there may be a relationship between CTC cluster formation and DNA methylation. One study found that DNA methylation changes in CTCs may contribute to the CTC cluster formation, and that these changes may be associated with increased metastatic potential. Specifically, the researchers found that methylation changes in genes associated with cell adhesion and motility were more frequent in CTC clusters than in single CTCs. Jang et al. investigates the DNA methylation status of CTCs in gastric cancer patients and its correlation with CTC cluster formation. 369 The authors find that DNA methylation alterations in CTCs are associated with increased cluster formation and suggest that targeting these alterations may be a potential therapeutic approach. 370 , 371 , 372 Gkountela et al. demonstrates that CTC clustering induces DNA methylation changes that promote metastatic seeding. The authors claim that targeting these epigenetic changes may be a potential therapeutic strategy for preventing metastasis. 373 , 374 Zhang et al. demonstrate that DNA methylation changes in CTCs are involved in their ability to form brain metastases in breast cancer patients. The authors suggest that targeting these epigenetic alterations may represent a potential strategy for preventing brain metastases. 376 Huang et al. investigates the correlation between DNA methylation in CTCs and their ability to resist EGFR inhibitors in lung adenocarcinoma patients. 253 The findings of the authors indicate that targeting DNA methylation abnormalities in CTCs may enhance treatment response, as these variations are linked to higher resistance to EGFR inhibitors. 375 , 376 Overall, while the relationship between CTC cluster formation and DNA methylation is still not fully understood, the current evidence suggests that changes in DNA methylation may be involved in the progression of metastatic cancer.

Histone modification

Histones are essential components of the dynamic architecture of chromatin. The octameric core of these proteins is formed by the assembly of two copies of each histone variation, including H3, H4, H2A, and H2B. This structure serves as a pool around which a DNA sequence consisting of 146 base pairs coils elaborately. 377 The globular structures of histones are enriched in basic amino acids like arginine and lysine in their tails, which serve as excellent locations for a wide range of covalent posttranslational modifications (PTMs). The chromatin landscape is altered by these chemical modifications, which also generate docking sites for proteins that control chromatin functionality. 378 These modifications also modify the interaction between histones and DNA. 379 Extensive studies has shown phosphorylation, methylation, and acetylation as the central histone modifications. All of these PTMs, however, add to the complexity of the regulation of gene expression by expanding the histone code. These additional PTMs include crotonylation, lactylation, citrullination, ubiquitination, and adenosine diphosphate (ADP)-ribosylation. 380 Enzymes known as “writers”, “readers”, and “erasers”, whose dysregulation is usually linked to cancer, meticulously control this dynamic epigenetic gene landscape. 381 Decoding the histone modification patterns presents a challenge since, even in identical cellular contexts, same configurations can produce divergent biological responses. 378 Abnormalities in this molecular communication have the potential to cause oncogenic transformation, 382 alter gene regulatory networks, and upset cellular equilibrium. Understanding this molecular dialog is critical for both understanding and fighting the molecular bases of CTC metastasis. It is also fundamental to elucidating cellular physiology.

Dysregulation in the landscape of histone modifications on TSG and oncogenes

By regulating transcriptional activity, histone alterations play a critical role in the initiation and proliferation of tumors. This often results in the upregulation of oncogenes and the downregulation of tumor suppressor genes. The erratic patterns of H3K27me3, which have a substantial influence on genomic stability, are a notable example of this dysregulation385. 383 The enhancer of EZH2 gene, which encodes the methyltransferase responsible for the specific histone modification H3K27me3, can be influenced by recurring mutations that may alter the levels of this modification. 384 The transcriptional landscape undergoes a significant reconfiguration during tumorigenesis. After being found to be tumor suppressors in a variety of cancers, 385 CBP/p300 has lately come to light as essential regulators of transcriptional activation mediated by enhancers and super-enhancers, especially with regard to important oncogenes. 386 , 387 In ALL, binding sites for the MYB transcription factor are created upstream of the TAL1 oncogene by heterozygous somatic mutations. This MYB interaction attracts CBP, resulting in the formation of a super-enhancer that promotes cell transformation and leukemogenic expression by driving the overexpression of TAL1. 388 Similar to this, p300 has been associated with a substantial reprogramming of super-enhancers in HCC, which results in the upregulation of critical oncogenes such MYC, MYCN, and CCND1, which promotes cancer cell proliferation both in vitro and in vivo. 389

Due to their important role in tumorigenesis, chromatin remodeling complexes (CRCs), especially the SWI/SNF family, are crucial in the DNA damage response (DDR). More than 20% of cancers have mutations in the SWI/SNF complex genes, highlighting the significance of these genes in the genesis of cancer. 390 Gene mutations can be caused by environmental factors such as UV light and gamma radiation that damage DNA. Prompt damage detection, repair signaling, repair factor mobilization, and cellular fate direction towards apoptosis or senescence are done by the DDR machinery. The historical background of DDR deficits in carcinogenesis is well established, having begun with the finding of chromosomal abnormalities in genes and continuing through the revelation that inadequate telomere maintenance catalyzes genomic instability and the detection of important tumor-suppressive functions of DDR components. 391 , 392 , 393 The SWI/SNF complexes affect DNA repair pathways by increasing nucleosome mobility through ATPase activity, which facilitates DDR. There are several roles that SWI/SNF subunits play in DDR. Some directly recruit DDR proteins, whereas others alter the chromatin architecture at DNA damage sites. 394 , 395 , 396 The PBAF and cBAF complexes are associated with DNA repair processes such as homologous recombination (HR) and non-homologous end joining. 337 , 397 To be more precise, DNA lesions are attracted by SMARCA4 and the cBAF-exclusive ARID1A, which help in double-strand break resolution and repair. 398 Poly-ADP ribose polymerase 1 (PARP1) and SMARCA4 have been shown to collaborate at damage sites, promoting chromatin remodeling to lower nucleosome density and assisting in the repair process. 396 SMARCA4 and ARID1A deficiencies are linked to mitotic abnormalities and erratic chromosomal segregation, suggesting that these proteins also play roles in DNA decatenation and telomere cohesion. 399 DDR also involves PBRM1, a component of PBAF. It is involved in centromeric cohesion maintenance, which is essential for genomic integrity, and transcriptional suppression at double-strand breaks to speed DNA lesion repair. 400 CHD1L plays a key role in the regulation of checkpoint control following DNA damage. It facilitates the movement of nucleosomes, which is promoted by PARP1, and also regulates checkpoint activities. 401 A lack of CHD1L impairs the accessibility of chromatin and the recruitment of repair factors, resulting in increased sensitivity to PARP. 402 The multifaceted involvement of cancer highlights their pivotal role in maintaining genomic integrity and preventing cancer. Understanding these complex interactions and mechanisms not only elucidates cellular physiology but also provides insights into potential therapeutic targets for cancer treatment.

Topologically Associating Domains (TADs) borders breaking down in the oncogenic landscape represent a fundamental abnormality. These disruptions are often caused by structural variations or damaged CCCTC-binding factor (CTCF) interaction due to changes in DNA methylation. 403 The proto-oncogene TAL1 is activated by microdeletions that obliterate TAD boundaries in T-ALL. 403 Broken genomic insulation in gliomas and gastrointestinal stromal tumors interferes with CTCF anchoring at loop structures. Like with PDGFRA and FGF4, this results in oncogene activation and ectopic enhancer-promoter crosstalk. 404 , 405 An explanation for the observed correlation between increased CCNE1 expression in gastric cancer primary tumors and CCNE1 rearrangement in response to changed TAD borders and interactions has been found. 406 Because the promoter-enhancer looping dynamics are dysregulated, this rearrangement promotes oncogenicity. An example of such a dynamic is the interaction between the MYC gene and the lncRNA PVT1 promoter. The PVT1 promoter blocks the promoter-enhancer looping of MYC, which in healthy cells decreases MYC expression competitively. On the other hand, malignant transformation often silences the PVT1 promoter by structural or epigenetic changes, re-establishing MYC’s enhancer-gene interaction and quickening tumorigenesis. 407 The loss of a single CTCF allele, which has been linked to oncogenic drivers in cancers including breast and endometrial, has supported CTCF’s role as a tumor suppressor through numerous genetic aberrations. 408 , 409 , 410 Prostate, ovarian, and breast cancers 411 frequently have hemizygous deletions of CTCF, while kidney and endometrial cancers with an allelic loss of CTCF are linked to lower patient survival. 412 , 413 Since hypermethylation of CpG islands is associated with a decrease in CTCF binding, this tumor-suppressing mechanism may entail the regulation of DNA methylation patterns. On top of that, PD-L1 upregulation is associated with CTCF deficiency, which allows cancer cells to evade immune monitoring. 414

Histone H3 and H4 acetylation patterns have become characteristic indicators of cancer cells, and dysregulation in the histone modification landscape is becoming more and more associated with metastasis. Histone acetylation states in cancer are impacted by metabolic reprogramming, which modifies the absolute amounts of acetyl-CoA and the ratio of acetyl-CoA to coenzyme A. Because of its role in acetyl-CoA gene rating through the ligation of acetate and CoA, ACSS2 can cause HIF-2 to become acetylated, which inhibits EMT under hypoxic conditions in HCC. 415 Overexpression of ACSS2, which promotes acetylation of H3K27 in the ATG5 promoter region, is used to achieve reduced breast cancer cell proliferation, migration, and invasion. This, in turn, ensures the maintenance of autophagic flow. 416 ACOT12, also referred as cytoplasmic acetyl-CoA hydrolase, is a predominant liver enzyme that selectively hydrolyzes the thioester bond of acetyl-CoA, gene rating acetate and CoA. 417 Reduced levels of ACOT12 in HCC lead to increased levels of acetyl-CoA, which in turn promotes the acetylation of H3K9 and H3K56. This acetylation process facilitates the EMT mediated by TWIST2. 418 , 419

Acetyl groups are transferred from acetyl-CoA to lysine residues by histone acetyltransferases (HATs). This process helps neutralize the positive charge on histones, thereby loosening the interaction between histones and DNA. As a result, genes become more accessible to transcription factors, facilitating gene expression. 420 GCN5, the initial identified HAT, controls a diverse array of biological processes including cellular proliferation, gene expression, and metabolism. It has also been demonstrated to play a role in the growth and metastasis of cancer cells. 421 GCN5 is recruited to the Runx2 promoter to sustain H3K27ac levels, which leads to the upregulation of Runx2 and promotes lung metastasis in osteosarcoma. 422 The biological function of HDACs in cancer is well-established. HDACs have the ability to both promote and inhibit tumor metastasis. Specific types, mainly through facilitating the downregulation of E-cadherin, have been linked to the proliferation and potential for metastasis of a variety of cancers. These include HDAC1, HDAC2, HDAC4, HDAC5, and HDAC6. 423 , 424 Simultaneously, HDAC8 has been identified as a new TGF-β pathway regulator. It works by transcriptionally suppressing SIRT7 via specific chromatin remodeling. Lung cancer metastasis is accelerated by this HDAC8 activity, which functions as a cofactor to the SMAD3/4 complex and triggers the activation of TGF-β signaling. 425 Histone H3K27 can be maintained in a deacetylated state by recruiting HDAC1 to the promoter of the DUSP2 gene. This results in DUSP2 being silenced and elevated MMP2 levels, promoting metastasis of nasopharyngeal metastesis. 426 In colorectal cancer, HDAC11 suppresses metastasis by downregulating MMP3 expression. This occurs through the reduction of histone H3K9 acetylation at the MMP promoter. 427 On the progression and metastasis of breast cancer, HDAC11 may have divergent effects. The survival and proliferation of tumors within the lymph nodes can be enhanced by increased HDAC11 expression, whereas a migratory phenotype is promoted by reduced HDAC11 expression, which significantly increases migration from the lymph nodes to distant organs. 428

Dysregulation in the landscape of histone modifications in CTCs immune escape

CTCs employ various strategies to evade immune surveillance by cytotoxic T cells. These mechanisms include suppressing T-cell activation, altering the expression of MHC-I at both transcriptional and posttranscriptional levels, reducing the levels of TAAs, and overexpressing IC molecules such as PD-L1 and GAL-9. 429 Downregulation of MHC-I molecules hinders CD8 + T-cell activation against TAAs presented by CTCs. The WNT/β-catenin signaling pathway regulates STT3, which glycosylates and stabilizes PD-L1, leading to increased PD-L1 levels on CTCs and facilitating evasion from cytotoxic T-cell immune surveillance. 430

In hypoxic conditions within the TME, PD-L1 and VEGF expression is elevated in CTCs. VEGF promotes the expression of TIM-3, the T-cell inhibitory receptor. Interaction between overexpressed PD-L1 and Gal-9 on CTCs with their corresponding receptors (e.g., PD-1 and TIM-3) on T cells suppresses T-cell proliferation, reduces cytokine production, induces T-cell exhaustion, and ultimately enables CTC evasion from cytotoxic T-cell activity. 429 , 431 Mature dendritic cells (DCs) are pivotal in initiating T-cell-mediated immune responses by presenting TAAs and expressing costimulatory molecules. 432 However, CTCs employ diverse mechanisms to inhibit DC-mediated antitumor responses, including the release of TGF-β. Specifically, CTCs hinder the recruitment of CD103+ DCs to tumors, 433 impair their maturation, promote differentiation into immunosuppressive regulatory DCs, 434 and induce the development of PD-1+ DCs that deactivate CD8 + T cells. 435 Through its negative regulation of DCs, macrophages, and T cells expressing their receptors, CD200, an increased immune checkpoint in CLL, BRCA, NSCLC, and COAD, develops immunological tolerance. 436 The interaction between TAMs and CTCs promotes CTC survival and the establishment of an immunosuppressive TME. 437 Numerous cells, including fibroblasts, endothelial cells, and immune cells, make up the CTC niche within the TME. These cells are enriched with factors like periostin (OSF-2, an osteoblast-specific factor), TGF-β, and colony-stimulating factor 1. 438 These factors drive the polarization of macrophages towards an immunosuppressive M2 or TAM phenotype. 439 TAM-secreted molecules like WNT, TGF-β, and VEGF promote cancer stemness, create an immunosuppressive TME, facilitate EMT, and support cancer metastasis. 440 Additionally, TAMs enhance PD-L1 expression on CTCs and PD-1 on T cells, thus dampening T-cell-mediated cytotoxicity. 441

MDSCs play a pivotal role in the TME by secreting cytokines and chemokines that foster an immunosuppressive niche, thereby impairing the efficacy of immunotherapy. 442 Granulocyte-macrophage colony-stimulating factors are released by CTCs via the mTOR signaling pathway, promoting MDSC infiltration into tumors. 443 Within the TME, MDSCs release IL-6 and nitric oxide, which epigenetically upregulate CTCs markers such as EpCAM, while also facilitating the activation of Tregs through TGF-β release. 444 , 445 By interacting with the immunosuppressive TME, Tregs, an immunosuppressive T-cell fraction, promote CTC immune evasion, suppressing the effects of cancer immunotherapy. CTC-derived TGF-β facilitates Treg infiltration into tumors, correlating with poorer survival rates. 446 Additionally, CTCs upregulate CCL1 expression through epigenetic mechanisms that decrease H3K27me3 levels at the CCL1 promoter, enhancing Treg recruitment to the TME. 447 Moreover, CTCs evade T-cell-induced apoptosis by inducing the differentiation of uncommitted CD4 + T cells into Tregs. 448 Specifically, Tregs in the hypoxic TME release VEGF, promoting CTCs stemness, angiogenesis, and EMT. 449 , 450 Natural killer (NK) cells express receptors like NKG2D, FASL, and TRAIL, which selectively target and eliminate MHC-I-negative CTCs. 451 NK cell-mediated cytotoxicity has been observed in MHC-I-negative colon and ovarian CTCs expressing NKG2DL and ligands for activating receptors NKp30 and NKp44. 452 However, CTCs from some ovarian and renal carcinoma patients upregulate MHC-I molecules, reducing susceptibility to NK cell-mediated lysis. 444 , 445 Interestingly, latent competent cancer cells expressing SOX2/SOX9 induce dormant CTCs (or latency-competent cancer cells) that downregulate NKG2DL through a unique mechanism involving WNT inhibitor DKK1, thereby evading NK cell-mediated immunity. 446 Epigenetic modifications, such as H3K27 acetylation, are enforced by CBP and p300 in the regulatory regions of genes crucial to Treg cell and MDSC survival and function. Through upregulating these genes, CBP/p300 promotes the growth of tumors by inhibiting lymphocyte activation, proliferation, and immunity mediated by cytotoxic T-cells. 453 Additionally, it has become clear that the sirtuin family of NAD+-dependent deacetylases, which includes mitochondrial SIRT3, SIRT4, and SIRT5, is essential for regulating epigenetic changes like as acetylation, demalonylation, and desuccinylation. 454 It has been demonstrated that SIRT4 loss enhances breast cancer stem cells’ capacity for self-renewal, which is essential for nutritional catabolism. 455 Increased cancer cell proliferation has been associated with desuccinylation and consequently decreased activity of succinate dehydrogenase (SDH), which has been linked to high levels of SIRT5 activity. In contrast, SDH hyper-succinylation and reactivation result from silencing SIRT5, which inhibits the growth of cancer cells. 456 Interferon serves as a potent cytokine with antitumor properties, inhibiting the expansion of CTCs and suppressing their tumor-initiating capabilities. 448 IFN-stimulated genes also play a role in overcoming chemoresistance in CTCs. 448 However, CTCs can develop resistance mechanisms against IFN’s antitumor effects, thereby promoting their survival and inducing the expression of CTC markers while evading immune surveillance through IFN signaling pathways. 447 , 449 The dual role of IFNs in cancer therapy may vary depending on the duration and concentration of IFN exposure. 450 , 451 Suboptimal type-I IFN signaling triggered by immunogenic cell death (ICD) does not consistently result in effective anticancer immunity. Instead, it can paradoxically promote tumor progression by increasing the population CTCs equipped with enhanced immune evasion capabilities. 452 Under specific conditions, elevated levels of IFN-γ have been demonstrated to trigger apoptosis in NSCLC through activation of the JAK1/STAT1/caspase pathway. Conversely, low concentrations of IFN-γ can enhance the properties of CTCs through the ICAM1/PI3K/AKT/NOTCH1 pathway, potentially contributing to tumor progression. 457 Overall, the interplay between various immune cells and CTCs fosters an immunosuppressive TME that facilitates immune evasion, thereby leading to adaptive resistance against cancer immunotherapy.

Clinical research progress targeting CTCs and therapeutic advances

Current methodologies for eradicating metastasis mirror those utilized for primary tumors: focusing on proliferation and tumorigenesis rather than directly addressing the metastatic cascade. 458 , 459 Surgical intervention or systemic treatments for primary tumors may not always eradicate the source of metastasis if dissemination have already happened. 460 , 461 , 462 , 463 The majority of anticancer agents undergo initial evaluation in metastatic contexts before being repurposed for adjuvant therapy to deter metastatic spread, albeit with only moderate success. 464 The scarcity of agents specifically targeting metastasis is being confronted by numerous preclinical investigations and considerations for future clinical trial frameworks. 1 , 459 Theoretically, therapies aimed at CTCs at different points in the metastatic process could halt the progression of metastatic cancers as CTCs are the cause of metastatic cancers and may come from different subpopulations within tumors.

CTCs in clinical trials

As a preventive approach against metastasis in preclinical models, addressing hypoxia-induced cluster release with vascular normalization-inducing drugs (e.g., ephrin B2 Fc chimaera protein, modulating VEGFR signaling) has been proposed. 465 The PLK1 inhibitor BI 2536 also impedes CTC intravasation, 165 suggesting its clinical utility in mitigating metastatic dissemination. 466 Targeting integrins, cadherins, cell-surface glycoproteins, 467 invadopodia (e.g., through N-WASP inhibition), 468 , 469 or employing antibodies against CD36, P-selectin, αIIbβ3, and α6β1 integrins could prevent intravasation and extravasation. HPSE, which facilitates ICAM1-mediated cell adherence in CTC clusters, is the target of one newly developed drugs class. 470 Additionally, urokinase and Na + /K + -ATPase inhibitors like digoxin exhibit promise in dissociating CTC clusters, leading to metastasis suppression in animal models. 5 Digoxin is now under studied in a phase I trial to determine its potential to disrupt CTC clusters in patients with advanced or metastatic breast cancer ( NCT03928210 ). Through cell-cell dissociation, heterotypic clustering may also be disrupted. Blocking key platelet receptors on CTCs, like glycoprotein Ib–IX–V and glycoprotein VI, reduces the potential for metastasis by interfering with platelet–cancer cell interactions. 108 Similarly, targeting VCAM1 on CTC–neutrophil clusters retards proliferation and metastatic efficiency. 111 , 471 , 472 , 473 Alternatively, utilizing CTCs’ VCAM1-mediated affinity for neutrophils for immune-based targeting could imitate the lethal activity of NK cells by energizing neutrophils with nanoscale liposomes that carry TRAIL and E-selectin. 474 Metabolic vulnerabilities could be exploited by either increasing oxidative stress or inhibiting pyruvate metabolism. 475 , 476 , 477 Immune checkpoint inhibitors have the ability to identify CTCs for T cell destruction, 478 and when combined with immune checkpoint inhibitors, dual targeting of HER2 or EpCAM results in improved cancer cell killing as compared to monotherapy. 479 , 480 Additionally, by employing mechanically disrupted CTCs as nanolysates, CTCs can be used as a source to produce cancer vaccines. Harnessing the homing capacity of CTCs to TME could be therapeutically advantageous by identifying homing signals and delivering therapeutic payloads. 126 , 481 Systemically administered CTCs engineered to express the prodrug-converting enzyme cytosine deaminase-uracil phosphoribosyl transferase can convert non-toxic 5′-fluorocytosine into the cytotoxic compound 5′-fluoruridine monophosphate. This conversion causes the CTCs to attach to the neoplastic tissues, where they kill the surrounding cancer cells. Lastly, given the rhythmic nature of CTC release into the bloodstream, 126 , 481 making the most of the current therapeutic opportunities by timing chronotherapy-based designs to coincide with CTC production peaks could optimize efficacy. Although these methods show promise in future research on CTCs, there is a lack of specific tools for dealing with metastatic cells, and the metastatic process is complicated, so testing their effectiveness in clinical settings will necessitate innovative and bold trial designs.

The results of clinical trials have demonstrated that CTCs are found in peripheral blood in all major carcinoma types, and their significance for prognosis in colorectal, breast, prostate, and small- and non-small-cell lung malignancies has been validated. 482 , 483 When a patient is first diagnosed with metastatic breast cancer, higher CTC counts before starting treatment are predictive indicators of shorter disease-free and total survival times. 484 , 485 , 486 For patients with colorectal 487 and prostate 488 , 489 cancer, a negative relationship between pretreatment CTC counts and clinical prognosis has also been reported. Significantly, numerous studies have illustrated that fluctuations in CTC counts following treatment administration offer more robust prognostic insights compared to baseline CTC levels. The persistence of CTCs post-therapy correlates with a poorer prognosis. 490 , 491 , 492 In patients receiving therapy, evaluation of CTC cluster abundance significantly improves the prognostic value in addition to single-CTC counts. 493 However, since most of the studies used antigen-dependent CTC methods, enumeration carries the risk of producing false-negative results in this situation.

CTC counts can be detected 7–9 weeks before the disease’s clinical manifestation. This suggests that CTC analysis in patients may help predict the likelihood of minimal residual disease and relapse in later stages, 163 of the illness 163 , 494 and serve as a tool for early cancer diagnosis. In NSCLC, CTCs taken following surgery showed a strong mutational concordance with metastatic tumors found 10 months later (91%). 156 Although CTCs are useful for risk assessment, there has been limited success in using CTCs for therapeutic patient stratification. This includes monitoring treatment response over time and identifying the onset of therapy resistance in many clinical trials. 491 , 495 , 496 The METABREAST STIC disease trial highlighted scenarios where CTC count could provide useful advice for therapeutic decisions, even though the interventional SWOG-S0500 trial did not show an advantage of CTC count-guided intervention compared to physician’s choice upon disease development. 495 The benefit of therapy selection based on CTC molecular features has been explored in a number of interventional studies. 497 , 498 In two proof-of-principle studies, trastuzumab–emtansine or lapatinib (HER2-targeted treatments) were used to target HER2-positive CTCs in HER2-negative metastatic breast cancer. 498 , 499 Although one trial (DETECT III) still awaits completion, the studies have only found a little benefit thus far. 499 Results from patients receiving endocrine therapy for metastatic prostate cancer are predicted by the expression of AR-V7 in CTCs (PROPHECY trial). 500 , 501 A phase II trial was then started to explore how the microtubule inhibitor cabazitaxel affected patients with AR-V7-positive CTCs and metastatic castration-resistant prostate cancer. However, the European Society for Medical Oncology guidelines do not support AR-V7 testing in this context due to the recent negative outcome of that trial, as it offers no advantage over current decision algorithms. 502 , 503 , 504

In conclusion, CTCs are now included in both the seventh edition of the AJCC Cancer Staging Manual and the fifth version of the WHO Classification of Tumours: Breast Tumours. The designation “cM0 (i+)“ indicates the absence of overt metastasis but the presence of tumor cells in the blood, bone marrow, or lymph nodes. Important cancer societies have yet to incorporate CTCs into their clinical practice recommendations, including the European Society for Medical Oncology and the American Society for Clinical Oncology. Arguably, CTCs’ actual potential lies in their ability to represent highly metastatic tumor subclones and in their abundance as modern biomarkers for molecular and functional studies. CTCs, as living cells, can be cultured outside of the body and analyzed for drugs response, which could offer significant insight to inform treatment choices in a timely manner. 175 , 505 , 506

Despite these advancements, significant enhancements to such workflows are necessary for their transition to clinical application. Innovative, prospective, randomized interventional trials are required to determine whether the use of CTCs as diagnostic aids offers clear benefits over standard-of-care (SOC) methods for specific cancer types. Future validation efforts should give priority to the predicted strengths of CTCs, including their ability to detect little residual illness, express clinically actionable targets for therapy selection, and provide longitudinal follow-up.

Diagnostic and prognostic value of methylation patterns of CTCs

The investigation of DNA methylation patterns in CTCs spans multiple cancer types, offering insights into their potential as biomarkers for diagnosis, prognosis, and therapeutic targeting. 97 , 126 Madhavan et al. highlighted the importance of methylation patterns in circulating cell-free DNA as markers of tumor progression and response to treatment in their investigation of CTC DNA’s potential as a prognostic diagnostic in metastatic breast cancer. 507 Cabel et al. investigated the role of CTCs and circulating tumor DNA in prostate cancer, focusing on DNA methylation markers as potential tools for assessing disease progression and therapeutic response. 508 Widschwendter et al. demonstrated that the methylation status of circulating tumor DNA can be used as a non-invasive diagnostic marker for ovarian cancer, enabling early detection and therapeutic response monitoring. 509 Powrózek et al. examined SHOX2 gene methylation in CTCs of NSCLC patients and discovered that SHOX2 methylation could function as a non-invasive biomarker for NSCLC prognosis and diagnosis. This suggests the possible utility of methylation analysis in CTCs for clinical purposes. 510 The potential of circulating tumor DNA to identify EGFR mutations in lung cancer patients was examined by Hulbert et al. 511 In identifying individuals who may benefit from treatment with tyrosine kinase inhibitors, the study demonstrated the utility of circulating tumor DNA as a feasible alternative to invasive tissue biopsies. Furthermore, the research shed light on the expansion of genetic and epigenetic profiling using circulating tumor material, such as exploring DNA methylation patterns. This highlights the growing importance of utilizing non-invasive biomarkers for precision medicine applications in oncology. 511 Mazor et al. demonstrated how the methylation patterns of circulating tumor DNA in lung cancer patients can serve as indicators of tumor burden and heterogeneity. 512 Their study underlines the potential of DNA methylation profiling of circulating tumor DNA, encompassing CTC-derived DNA, in providing crucial prognostic and diagnostic insights. 512 Aberrant DNA methylation profiles have been detected in CTCs from cancer patients, and these patterns have shown promise as diagnostic or prognostic biomarker. For instance, Wong et al.’s study, which examined the DNA methylation patterns of CTCs from lung cancer patients, found a correlation between patient survival and the methylation levels of specific genes. 513 , 514 Specifically, high methylation of HOXA9 and LMX1A genes was associated with poor overall survival, while high methylation levels of the IGFBP3 gene were associated with better overall survival. 515 , 516 Another study by Wu et al. investigated the DNA methylation patterns of CTCs from patients with lung adenocarcinoma and identified differentially methylated genes that were associated with metastasis. 5 , 111 , 126 They identified that the methylation levels of GABRB2, CLDN3, and SFRP1 were conspicuously different between CTCs from patients with and without metastasis. 517 In addition to their potential as diagnostic or prognostic biomarkers, DNA methylation patterns in CTCs may also have therapeutic implications. For instance, a study by Chen et al. suggested that treatment with a DNA-demethylating agent called decitabine reduced the metastasis of CTCs from lung cancer patients by reversing the aberrant DNA methylation patterns in these cells. 332 , 518 The DNA methylation profile of CTCs has demonstrated a significant potential for lung cancer diagnosis, prognosis, and treatment (Table 4 ). 495 , 519 , 520 , 521 , 522 , 523 , 524 , 525

DNA methylation detection in CTCs

The principal methodology utilized in liquid biopsy is the identification of DNA methylation within the circulatory system. Circulating tumor DNA has better sensitivity and specificity for cancer screening than traditional tumor markers, especially in the early stages of the disease. The sensitivity of Vimentin gene methylation in serum for the diagnosis of CRC was found by Atsushi Shirahata et al. to be significantly higher (57.1% vs. 14.3%) for diagnosing CRC in situ (Stage 0) than for the tumor marker CEA (32.6% vs. 33.1%). 432 Similar to this, the FDA has approved SEPT9, a circulating tumor DNA test, as an effective early non-invasive screening method for colorectal cancer. 526 , 527 SEPT9, also referred to as MSF, was originally discovered by Osaka et al. 530 They observed that MSF acts as a proto-oncogene, promoting leukemia upon fusion with the MLL gene. 528 Further research has shown that SEPT9 generates 18 transcriptional products, each contributing uniquely to the development and progression of cancer. 529 Specifically, the SEPT9 gene, particularly the SEPT9_v2 variant, functions as a tumor suppressor in colorectal cancer. 530 Hypermethylation of the SEPT9_v2 promoter region, leading to decreased SEPT9 gene expression, is a defining characteristic of colorectal cancer (CRC) and is closely associated with the progression from adenoma to atypical hyperplasia to CRC. In their 2021 study, Guoxiang Cai et al. developed a classifier named “ColonAiQ”, which incorporates six circulating tumor DNA markers (SEPT9, SEPT9 region 2, BCAT1, IKZF1, BCAN, VAV3). 531 Their findings showed that “ColonAiQ” was able to achieve a greater detection rate in both early and advanced CRC than fecal immunochemical tests, CEA, and SEPT9. 531 Additionally, the “ColonAiQ” classifier predicted early postoperative recurrence and poor prognosis of CRC, with patients exhibiting higher ColonAiQ risk scores more likely to experience early postoperative recurrence. 531 Using whole-genome bisulfite sequencing technology, Liu et al. conducted a prospective clinical trial to examine tissue and circulating tumor DNA methylation in breast cancer. 423 They found methylation patterns that differed between cancer patients and those with benign tumors, and they developed a diagnostic model. The detection of breast cancer was significantly improved by this combined diagnostic models. One benefit of the study is that it uses whole-genome bisulfite sequencing to identify differential methylation sites in detail. This method measures the average degree of DNA methylation at each CpG site in the target genome. However, this method does not precisely pinpoint methylation sites. A variety of methods have been used in investigations to identify site-specific DNA methylation, including digital PCR, pyrosequencing, bisulfite sequencing, and methylation-specific PCR. 532 , 533 , 534 The study’s examination of methylation differences in tissues and circulating tumor DNA from cancer and benign lesion patients enables the exclusion of non-cancer-related methylation sites in circulating tumor DNA. Differential diagnosis on patients with benign masses improves the detection of tumor-specific sites in contrast to many other studies that employ healthy controls. Additionally, combining imaging examinations (e.g., ultrasound, mammography) with circulating tumor DNA testing improves breast cancer detection sensitivity and specificity, reducing unnecessary invasive procedures. This prospective clinical trial focused on early-stage cancer lesions, offering insights distinct from studies utilizing advanced cancer tissues, which might not be as effective for early cancer screening. Nevertheless, the study had limitations, such as unmatched patient age, smoking, and other influencing factors between case and control groups, potentially introducing heterogeneity and affecting site detection efficacy. 535 , 536 Moreover, the study’s small sample size (10 tissue pairs) diminished locus selection efficiency, likely influenced by the high cost of whole-genome sequencing. Therefore, developing cost-effective sequencing technologies is crucial for early cancer screening. Colonoscopy, ultrasonography, mammography, and low-dose chest CT have become widely used and have greatly improved early screening for lung cancer, breast cancer, and colorectal cancer, as well as early diagnosis rates and patient prognoses. 537 , 538 , 539 However, effective screening tools for pancreatic cancer (PC) remain lacking. Nine studies on blood-based DNA methylation biomarkers for early PC diagnosis, all published in the last decade, have been retrieved. 533 , 540 , 541 , 542 , 543 , 544 Notably, three studies identified ADAMTS1 methylation as a liquid biopsy marker for PC, achieving high diagnostic efficacy (sensitivity >80%, specificity >85%). 540 , 545 , 546 Keiko Shinjo et al. also demonstrated the diagnostic capability of a five-DNA molecule panel, including ADAMTS2, for PC, with sensitivity and specificity of 68 and 86%, respectively, when combined with KRAS mutation. 542 The ADAMTS family, comprising 19 members, plays crucial roles in arthritis, cardiovascular diseases, and cancer, 547 particularly in regulating ECM structure and function. Given the abundant stroma in PC, certain ADAMTS family subtypes may serve as effective biomarkers for PC diagnosis, warranting further investigation. 548 The circulating tumor DNA methylation model is also pivotal in prognosis. Mastoraki et al. discovered that non-small cell lung cancer patients with methylation in the KMT2C promoter region of circulating tumor DNA experienced poorer overall survival (OS) and disease-free survival ( P = 0.017 and P < 0.001, respectively). 549 Promising results have been found in numerous studies examining the role of circulating tumor DNA in predicting the prognosis of different cancers, including colorectal, prostate, and ovarian cancer. 549 , 550 , 551 , 552 , 553 , 554 , 555 Establishing a prognostic model based on blood-based DNA methylation facilitates early identification of high-risk groups, enabling timely and effective intervention, while non-high-risk individuals may undergo milder treatment or follow-up, thus minimizing unnecessary invasive treatments and conserving medical resources. Despite the utility of circulating tumor DNA methylation in reflecting patient prognosis across various cancers, challenges remain in developing prognostic methylation markers. The lack of consensus on detection methods and the difficulty in identifying universally accepted prognostic methylation sites contribute to these challenges. In order to determine prognosis and choose adjuvant treatment, DNA gene mutation data derived from tumor tissue or blood is widely used. The clinical application of assessing the methylation status of the MGMT gene promoter in gliomas, which indicates tumor response to temozolomide chemotherapy and patient prognosis, has been successful. 556 , 557 Unfortunately, this application is based on tumor tissue-derived MGMT gene promoter methylation status, with no mature clinical studies on circulating tumor DNA.

Therapeutic targeting of methylation in CTCs

To evaluate the effectiveness of treatment and track any tumor recurrence, tumor markers and cross-sectional exams are widely used in the postoperative follow-up of cancer patients. After surgery, a decrease in serum tumor markers indicates effective treatment, whereas an increase indicates a possibility of metastasis or recurrence. 558 , 559 Tumor marker detection has been shown to predict tumor recurrence up to six months ahead of cross-sectional imaging 560 ; however, higher tumor markers are correlated with tumor burden and may not be visible in the early stages of relapse. 561 Other factors can also elevate tumor marker levels; for example, CA19-9, a reliable marker for monitoring postoperative PAAD recurrence, can be elevated due to pancreatic inflammation, obstructive jaundice, and persistent diabetes. 558 Approximately 8–10% of the population are Lewis-negative, and over 70% of Lewis-negative PAAD patients exhibit low CA19-9 expression, rendering this marker ineffective for prognosis in these patients who typically have worse outcomes. 562 Because it is noninvasive, highly reproducible, and sensitive, the detection of circulating tumor DNA methylation has become a crucial method for dynamically monitoring tumor response after treatment. 563 In order to highlight the importance of circulating tumor DNA in cancer surveillance, Michail Ignatiadis et al. introduced the term “circulating tumor DNA relapse”. 564 Nakayama et al. found that P16INK4a methylation is a sensitive marker of colorectal cancer recurrence, highlighting the crucial role circulating tumor DNA plays in the postoperative follow-up of CRC patients. 565 In their study of 21 CRC patients, 13 exhibited elevated P16INK4a methylation pre-surgery, with all patients showing decreased methylation levels within 2 weeks post-surgery, except for two with residual metastases or subsequent relapse. In these two patients, there was noticeably higher P16INK4a methylation at relapse; this was not the case in the individuals without tumor recurrence. 565 Jin et al. found that cfDNA follow-up could detect colon cancer recurrence early, with circulating tumor DNA reappearing in 70% of patients (14 cases) before recurrence, approximately eight months earlier than imaging suggested. 566 Therefore, early tumor relapse detection is made possible by dynamic monitoring of circulating tumor DNA methylation, which aids in clinical decision-making. The tumor information provided by circulating tumor DNA aids in guiding subsequent targeted therapy selection. Tumor heterogeneity partially explains the poor response to antitumor therapy, with new clonal subtypes forming during tumor progression, a significant factor in therapeutic inefficacy. 567 Circulating tumor DNA detection provides great reproducibility when compared to standard tissue biopsy, which decreases the effects of tumor tissue heterogeneity and allows for timely treatment plan adjustments and dynamic monitoring of therapy response. However, compared to traditional tissue exams, circulating tumor DNA extraction and sequencing involves greater technical demands and expenses.

Current research primarily focuses on mutation information within circulating tumor DNA. Detailed gene mutation analysis can elucidate the cancer molecular landscape, potentially leading to more suitable treatment options. However, studies on using circulating tumor DNA methylation for therapeutic target selection are scarce. This disparity might result from the theory that tumor responses rather than tumor causes are the reason for variations in the circulating tumor DNA methylation state in cancer patients. More research is needed in this area. DNA methylation in tissues is critical for tumor suppressor gene inactivation and tumorigenesis. Cancers including pancreatic, breast, and bladder cancer have demonstrated benefit from targeted therapy of DNA methylation, notably when DNA methyltransferase inhibitors are used. 568 , 569 , 570

Therapeutic targeting of histone modification enzyme in CTCs

Beyond broad-spectrum modifiers, drugs designed to target particular mutations within enzymes that modify the epigenome are becoming part of the landscape of epigenetic therapies. One such drug is tazemetostat, a selective inhibitor that specifically targets the EZH2 mutation. EZH2, the PRC2 complex’s catalytic component, regulates transcriptional repression via H3K27 trimethylation. Overexpression of EZH2 is associated with poor prognosis and increased malignancy in various cancers, 571 prompting its exploration as a therapeutic target. Based on phase 2 trial results that demonstrated a 69% objective response rate (ORR) in patients with EZH2 mutations, compared to 35% in those with wild-type EZH2, Tazemetostat was approved by the FDA. 572 Dual inhibitors that target both EZH1 and EZH2 have been found to be more effective than selective EZH2 inhibition in reducing cellular H3K27me3 levels and increasing anticancer effects in mouse models of hematologic malignancies. 573 In a phase 2 trial, the dual inhibitor valemetostat demonstrated promise in treating adult T-cell leukemia/lymphoma, with a 48% ORR. 574 Currently, a phase 1 trial for metastatic urothelial cancer is exploring the possible synergistic effects of valemetostat with ipilimumab. In a similar vein, DOT1L, the sole H3K79 methyltransferase, has been studied as a possible therapeutic target in tumors containing MLL gene rearrangements, particularly in cases of acute leukemia. 575 Even though the outcomes of early clinical trials of DOT1L inhibitors were inconsistent, pinometostat may make juvenile AML cells more sensitive to the multikinase inhibitor sorafenib, opening the door to novel therapeutic options. 576 These developments are poised to reshape the therapeutic landscape for pediatric AML and highlight the evolving precision in targeting specific epigenetic mutations for cancer therapy.

The advancement of cancer has been linked to abnormal LSD1 amplification and activity. 577 LSD1’s role in transcriptional repression involves removing methylation from H3K4me1/2, a marker of gene activation. 578 Preclinical evidence of differentiation and growth attenuation has prompted the evaluation of LSD1 inhibitors, including pulrodemstat (CC-90011), iadademstat, seclidemstat, and GSK2879552, in a number of clinical trials. Results from early phase studies, notably those employing pulrodemstat for solid tumors and non-Hodgkin lymphoma, show significant anti-neoplastic effects, particularly in neuroendocrine tumors. 579 Several non-histone proteins, including as DNMT1, are impacted by LSD1 in addition to histones. 580 LSD1-mediated demethylation of DNMT1 is critical for its stabilization and the maintenance of global DNA methylation patterns. 581 LSD1 inhibitors have the potential to be used in combination therapy for hematological malignancies. A clinical trial, with the ClinicalTrials.gov code NCT04734990, is presently exploring the use of seclidemstat in combination with azacytidine for the treatment of chronic myelomonocytic leukemia. Preclinical studies have also demonstrated that by raising tumor immunogenicity and T-cell infiltration, LSD1 inhibition can improve the effectiveness of immune checkpoint blockade. Clinical trials exploring combination therapies have been started to optimize the effects of immunotherapy, particularly in tumor forms with previously limited responses, including lung cancer. 582 , 583 These developments underscore the expanding potential of LSD1 inhibitors in both standalone and combination treatments across a spectrum of cancers.

HDACs have zinc-enriched active sites, which HDAC inhibitors (HDACi) bind to in order to impede their function and maintain a hyperacetylated state of chromatin that promotes a transcriptionally active configuration. 584 In order to treat cutaneous T-cell lymphoma, the FDA approved vorinostat, one of the first-generation HDACi, in 2006. This approval was based on clinical trials demonstrating ORRs of ~30%. 585 Similar to DNMT inhibitors, HDACi have shown synergistic effects in preclinical studies when combined with other anticancer agents, leading to the strategic design of combination clinical trials. HDAC inhibitors not only enhance the expression of PD-L1, potentially priming tumors for immunotherapy, but also reduce populations of Tregs, which can bolster immune responses against tumors. 586 Vorinostat’s capacity to make hormone-resistant ER-positive breast tumors more susceptible to apoptosis has been validated in preclinical studies, indicating that it may be used in combination with antiestrogen drugs to improve therapeutic results in hormone therapy. 587 In 2014, Belinostat, a second-generation HDAC inhibitor, obtained expedited FDA approval for the treatment of peripheral T-cell lymphoma based on the results of a single-arm trial with 120 patients. 588 To address toxicity concerns related to earlier generations of HDACi, selectivity against specific members of the HDAC family has been improved through advancements in HDAC inhibition. A benzamide derivative called entinostat has shown promise as a strong and specific inhibitor of class I and IV HDACs. Low-dose azacitidine plus entinostat has been explored in clinical studies for patients with advanced breast cancer and recurrent metastatic NSCLC, particularly in those who have had extensive prior treatment. 589 , 590 These studies reflect ongoing efforts to optimize the efficacy and safety profiles of HDAC inhibitors in cancer therapy.

Proteins called bromodomain and extraterminal (BET) domains are important chromatin dynamics regulators and have become promising targets in cancer research. The BRD2, BRD3, BRD4, and BRDT proteins belong to the BET protein family and use their bromodomains to identify acetylated lysine residues on histones. This recognition initiates chromatin remodeling and gene expression by recruiting additional transcriptional effectors. 591 A key factor in identifying the oncogenic functions of BET proteins has been the emergence of small-molecule BET inhibitors like JQ1. These inhibitors disrupt the binding of BET proteins to acetylated histones, thereby modulating the expression of key oncogenes implicated in cancer progression. 592 , 593 Despite their initial promise in preclinical models, the clinical translation of BET inhibitors has been limited by pharmacokinetic challenges, including short half-lives and poor oral bioavailability. Tyrosine kinase inhibitors like lapatinib can be resistant to BET inhibitors like JQ1 and I-BET151 in TNBC. In order to prolong the therapeutic response, they achieve this by suppressing the production of certain kinases which drive resistance mechanisms. 594 , 595 Furthermore, homologous recombination, a crucial DNA damage repair pathway, is disrupted by BET inhibitors by the transcription of proteins involved. This disruption has significant implications for cancer therapy, particularly in combination approaches. When PARP inhibitors and BET inhibitors are used together, tumors that are proficient in homologous recombination may become more sensitive to the PARP inhibitors and may eventually acquire resistance to them. 596 , 597 Clinical investigations have been prompted by the synergistic benefits between PARP and BET inhibition observed in preclinical studies, particularly in breast and ovarian cancers. In order to provide novel avenues for targeted cancer therapy, these studies aim to validate these results in patient populations. 598 , 599 This strategic combination approach highlights the potential of BET inhibitors to enhance the efficacy of existing therapies and to address resistance mechanisms in cancer treatment.

Conclusion and perspective

Cancers remain the highest number of cancer-related deaths globally. CTCs are tumor cells that have separated from the primary tumor and entered the lymphatic or circulatory system. This allows the tumor cells to spread throughout the body and result in the formation of new tumors. According to studies, patients with lung cancer can have CTCs in their blood, and the number of CTCs in a patient’s blood is correlated with the disease’s progression and chances of metastasis. CTCs have been found in the blood of lung cancer patients even before a tumor has been identified by conventional methods, providing evidence that they may be a valuable tool for the early identification and monitoring of lung cancer.

DNA methylation is the addition of methyl groups to the cytosine residues of DNA, and it is necessary for regulation the expression of certain genes. Numerous cancer types, including lung cancer, have been related to abnormal DNA methylation patterns at the onset and progression of the disease. It has been discovered that DNA methylation has a role in both immune surveillance and metastasis in the setting of CTCs. 600 The immune system’s method of identifying and getting rid of cancer cells is called immune surveillance. However, by DNA methylation, CTCs can suppress the expression of immune-related genes, enabling them to elude the immune system and persist in the bloodstream. For example, some CTCs have been found to have DNA hypermethylation of genes that are involved in antigen processing and presentation, which may help them escape recognition and elimination by the immune system. Furthermore, DNA methylation has also been involved in the metastasis of CTCs. The EMT process, for example, is essential for cancer cell invasion and metastasis, and it has been linked to DNA hypermethylation of E-cadherin, a gene involved in cell adhesion and migration. Additionally, it has been found that the metastatic potential of CTCs 273 , 601 is influenced by hypomethylation of genes related to DNA repair and cell cycle regulation. 273 , 601

There are several limitations that must be addressed before CTC analysis can be widely employed in the clinic, despite the fact that it has showed promise in the diagnosis and treatment of lung cancer. One of the major limitations is the low sensitivity of CTC detection methods. The number of CTCs in the bloodstream is usually very low, and current methods for CTC detection and isolation may miss a significant proportion of these cells. This may limit the clinical value of CTC analysis and lead to false negative results. The diversity of CTCs presents another difficulty. In contrast to the primary tumor or other CTCs, CTCs are a diverse population of cells that may exhibit various phenotypic and genetic characteristics. This heterogeneity can make it difficult to identify and isolate CTCs that are representative of the entire tumor and to develop targeted therapies. 602 , 603 Furthermore, CTCs are often found in a dormant state, meaning they are not actively dividing or producing detectable levels of tumor markers. This can make it challenging to monitor the response of the tumor to therapy using CTC analysis alone. Finally, there are technical challenges correlated with CTC analysis, such as the need for specialized equipment and expertise, which may limit the availability and accessibility of this technology to all patients. Addressing these limitations may require the development of new technologies and methods for the detection and analysis of CTCs as well as an improved comprehension of CTC biology and role in lung cancer. 604 , 605

This review has encapsulated the obstacles surrounding the genesis of CTCs in the context of cancers, as well as the effect of epigenetics modifications on CTCs pertaining to EMT, immune surveillance, cluster formation, and colonization. The epigenetic modifications resulting from DNA methylation in CTCs may serve as a key to unlock the underlying mechanisms of metastasis in lung cancer, and holds significant promise in the areas of lung cancer diagnosis, prognosis, and treatment.

Ganesh, K. & Massagué, J. Targeting metastatic cancer. Nat. Med. 27 , 34–44 (2021).

Article CAS PubMed PubMed Central Google Scholar

Castro-Giner, F. & Aceto, N. Tracking cancer progression: from circulating tumor cells to metastasis. Genome Med. 12 , 31 (2020).

Article PubMed PubMed Central Google Scholar

Lawrence, R., Watters, M., Davies, C. R., Pantel, K. & Lu, Y. J. Circulating tumour cells for early detection of clinically relevant cancer. Nat. Rev. Clin. Oncol. 20 , 487–500 (2023).

Article PubMed Google Scholar

Lin, D. et al. Circulating tumor cells: biology and clinical significance. Signal Transduct. Target Ther. 6 , 404 (2021).

Gkountela, S. et al. Circulating tumor cell clustering shapes DNA methylation to enable metastasis seeding. Cell 176 , 98–112.e114 (2019).

Mattei, A. L., Bailly, N. & Meissner, A. DNA methylation: a historical perspective. Trends Genet. 38 , 676–707 (2022).

Article CAS PubMed Google Scholar

Papanicolau-Sengos, A. & Aldape, K. DNA methylation profiling: an emerging paradigm for cancer diagnosis. Annu. Rev. Pathol. 17 , 295–321 (2022).

Guo, H. et al. DNA hypomethylation silences anti-tumor immune genes in early prostate cancer and CTCs. Cell 186 , 2765–2782.e2728 (2023).

Li, Y., Liang, J. & Hou, P. Hypermethylation in gastric cancer. Clin. Chim. Acta 448 , 124–132 (2015).

Ehrlich, M. et al. Hypomethylation and hypermethylation of DNA in Wilms tumors. Oncogene 21 , 6694–6702 (2002).

Praharaj, P. P., Bhutia, S. K., Nagrath, S., Bitting, R. L. & Deep, G. Circulating tumor cell-derived organoids: current challenges and promises in medical research and precision medicine. Biochim. Biophys. Acta Rev. Cancer 1869 , 117–127 (2018).

Gwark, S. et al. Analysis of the serial circulating tumor cell count during neoadjuvant chemotherapy in breast cancer patients. Sci. Rep. 10 , 17466 (2020).

Tellez-Gabriel, M., Knutsen, E. & Perander, M. Current status of circulating tumor cells, circulating tumor DNA, and exosomes in breast cancer liquid biopsies. Int. J. Mol. Sci. 21 , 9457 (2020).

Vismara, M., Reduzzi, C., Daidone, M. G. & Cappelletti, V. Circulating tumor cells (CTCs) heterogeneity in metastatic breast cancer: different approaches for different needs. Adv. Exp. Med. Biol. 1220 , 81–91 (2020).

Piñeiro, R., Martínez-Pena, I. & López-López, R. Relevance of CTC clusters in breast cancer metastasis. Adv. Exp. Med. Biol. 1220 , 93–115 (2020).

Cui, T. et al. DDB2 represses ovarian cancer cell dedifferentiation by suppressing ALDH1A1. Cell Death Dis. 9 , 561 (2018).

Singh, M., Yelle, N., Venugopal, C. & Singh, S. K. EMT: Mechanisms and therapeutic implications. Pharm. Ther. 182 , 80–94 (2018).

Article CAS Google Scholar

Zhang, Q. et al. Circulating tumor cells in hepatocellular carcinoma: single-cell based analysis, preclinical models, and clinical applications. Theranostics 10 , 12060–12071 (2020).

Deng, Z., Wu, S., Wang, Y. & Shi, D. Circulating tumor cell isolation for cancer diagnosis and prognosis. EBioMedicine 83 , 104237 (2022).

Tayoun, T. et al. CTC-derived models: a window into the seeding capacity of circulating tumor cells (CTCs). Cells 8 , 1145(2019).

Ward, M. P. et al. Platelets, immune cells and the coagulation cascade; friend or foe of the circulating tumour cell? Mol. Cancer 20 , 59 (2021).

Lianidou, E. S. & Markou, A. Circulating tumor cells in breast cancer: detection systems, molecular characterization, and future challenges. Clin. Chem. 57 , 1242–1255 (2011).

Thanh Huong, P. et al. Emerging role of circulating tumor cells in gastric cancer. Cancers 12 , 695 (2020).

Gires, O., Pan, M., Schinke, H., Canis, M. & Baeuerle, P. A. Expression and function of epithelial cell adhesion molecule EpCAM: where are we after 40 years? Cancer Metastasis Rev. 39 , 969–987 (2020).

Popovic, D., Vucic, D. & Dikic, I. Ubiquitination in disease pathogenesis and treatment. Nat. Med. 20 , 1242–1253 (2014).

Criscitiello, C., Sotiriou, C. & Ignatiadis, M. Circulating tumor cells and emerging blood biomarkers in breast cancer. Curr. Opin. Oncol. 22 , 552–558 (2010).

Gorin, M. A. et al. Circulating tumour cells as biomarkers of prostate, bladder, and kidney cancer. Nat. Rev. Urol. 14 , 90–97 (2017).

Varillas, J. I. et al. Microfluidic isolation of circulating tumor cells and cancer stem-like cells from patients with pancreatic ductal adenocarcinoma. Theranostics 9 , 1417–1425 (2019).

Marcuello, M. et al. Circulating biomarkers for early detection and clinical management of colorectal cancer. Mol. Asp. Med. 69 , 107–122 (2019).

Xia, W. et al. In vivo coinstantaneous identification of hepatocellular carcinoma circulating tumor cells by dual-targeting magnetic-fluorescent nanobeads. Nano Lett. 21 , 634–641 (2021).

Gall, T. M. H., Belete, S., Khanderia, E., Frampton, A. E. & Jiao, L. R. Circulating tumor cells and cell-free DNA in pancreatic ductal adenocarcinoma. Am. J. Pathol. 189 , 71–81 (2019).

Ye, Q., Ling, S., Zheng, S. & Xu, X. Liquid biopsy in hepatocellular carcinoma: circulating tumor cells and circulating tumor DNA. Mol. Cancer 18 , 114 (2019).

Eslami, S. Z., Cortés-Hernández, L. E. & Alix-Panabières, C. Epithelial cell adhesion molecule: an anchor to isolate clinically relevant circulating tumor cells. Cells 9 ,1836 (2020).

Wang, J. et al. Label-free isolation and mRNA detection of circulating tumor cells from patients with metastatic lung cancer for disease diagnosis and monitoring therapeutic efficacy. Anal. Chem. 87 , 11893–11900 (2015).

Wang, Z. et al. High-efficiency isolation and rapid identification of heterogeneous circulating tumor cells (CTCs) using dual-antibody-modified fluorescent-magnetic nanoparticles. ACS Appl. Mater. Interfaces 11 , 39586–39593 (2019).

Wang, Z. et al. Antifouling hydrogel-coated magnetic nanoparticles for selective isolation and recovery of circulating tumor cells. J. Mater. Chem. B 9 , 677–682 (2021).

Reduzzi, C. et al. A novel circulating tumor cell subpopulation for treatment monitoring and molecular characterization in biliary tract cancer. Int. J. Cancer 146 , 3495–3503 (2020).

Lee, S. Y. et al. Induction of metastasis, cancer stem cell phenotype, and oncogenic metabolism in cancer cells by ionizing radiation. Mol. Cancer 16 , 10 (2017).

Mittal, V. Epithelial mesenchymal transition in tumor metastasis. Annu. Rev. Pathol. 13 , 395–412 (2018).

Okabe, T. et al. Mesenchymal characteristics and predictive biomarkers on circulating tumor cells for therapeutic strategy. Cancers 12 , 3588 (2020).

Xu, J., Liao, K., Yang, X., Wu, C. & Wu, W. Using single-cell sequencing technology to detect circulating tumor cells in solid tumors. Mol. Cancer 20 , 104 (2021).

Sun, S. & Qiu, X. S. Cancer stem cells and tumor metastasis. J. Cancer Res. Ther. 9 , S150–S152 (2013).

Liu, S. et al. Breast cancer stem cells transition between epithelial and mesenchymal states reflective of their normal counterparts. Stem Cell Rep. 2 , 78–91 (2014).

Ginestier, C. et al. ALDH1 is a marker of normal and malignant human mammary stem cells and a predictor of poor clinical outcome. Cell Stem Cell 1 , 555–567 (2007).

Barriere, G., Riouallon, A., Renaudie, J., Tartary, M. & Rigaud, M. Mesenchymal characterization: alternative to simple CTC detection in two clinical trials. Anticancer Res. 32 , 3363–3369 (2012).

PubMed Google Scholar

Balic, M. et al. Most early disseminated cancer cells detected in bone marrow of breast cancer patients have a putative breast cancer stem cell phenotype. Clin. Cancer Res. 12 , 5615–5621 (2006).

Al-Hajj, M., Wicha, M. S., Benito-Hernandez, A., Morrison, S. J. & Clarke, M. F. Prospective identification of tumorigenic breast cancer cells. Proc. Natl Acad. Sci. USA 100 , 3983–3988 (2003).

Todaro, M. et al. CD44v6 is a marker of constitutive and reprogrammed cancer stem cells driving colon cancer metastasis. Cell Stem Cell 14 , 342–356 (2014).

Battula, V. L. et al. Ganglioside GD2 identifies breast cancer stem cells and promotes tumorigenesis. J. Clin. Investig. 122 , 2066–2078 (2012).

Liang, Y. J. et al. Differential expression profiles of glycosphingolipids in human breast cancer stem cells vs. cancer non-stem cells. Proc. Natl Acad. Sci. USA 110 , 4968–4973 (2013).

Ho, M. M., Ng, A. V., Lam, S. & Hung, J. Y. Side population in human lung cancer cell lines and tumors is enriched with stem-like cancer cells. Cancer Res. 67 , 4827–4833 (2007).

Seigel, G. M., Campbell, L. M., Narayan, M. & Gonzalez-Fernandez, F. Cancer stem cell characteristics in retinoblastoma. Mol. Vis. 11 , 729–737 (2005).

CAS PubMed Google Scholar

Mishima, Y. et al. Detection of HER2 amplification in circulating tumor cells of HER2-negative gastric cancer patients. Target Oncol. 12 , 341–351 (2017).

Wülfing, P. et al. HER2-positive circulating tumor cells indicate poor clinical outcome in stage I to III breast cancer patients. Clin. Cancer Res. 12 , 1715–1720 (2006).

Beije, N. et al. Prognostic impact of HER2 and ER status of circulating tumor cells in metastatic breast cancer patients with a HER2-negative primary tumor. Neoplasia 18 , 647–653 (2016).

Forsare, C. et al. Evolution of estrogen receptor status from primary tumors to metastasis and serially collected circulating tumor cells. Int. J. Mol. Sci. 21 , 2885 (2020).

Gradilone, A. et al. Circulating tumor cells (CTCs) in metastatic breast cancer (MBC): prognosis, drug resistance and phenotypic characterization. Ann. Oncol. 22 , 86–92 (2011).

Todenhöfer, T. et al. Preliminary experience on the use of the Adnatest® system for detection of circulating tumor cells in prostate cancer patients. Anticancer Res. 32 , 3507–3513 (2012).

Yin, C. et al. Molecular profiling of pooled circulating tumor cells from prostate cancer patients using a dual-antibody-functionalized microfluidic device. Anal. Chem. 90 , 3744–3751 (2018).

Chen, L. et al. Combined use of EpCAM and FRα enables the high-efficiency capture of circulating tumor cells in non-small cell lung cancer. Sci. Rep. 8 , 1188 (2018).

Wei, S. et al. Effect of vein-first vs artery-first surgical technique on circulating tumor cells and survival in patients with non-small cell lung cancer: a randomized clinical trial and registry-based propensity score matching analysis. JAMA Surg. 154 , e190972 (2019).

Cao, W. et al. Using detection of survivin-expressing circulating tumor cells in peripheral blood to predict tumor recurrence following curative resection of gastric cancer. J. Surg. Oncol. 103 , 110–115 (2011).

Steinemann, G. et al. Antitumor and antiangiogenic activity of the novel chimeric inhibitor animacroxam in testicular germ cell cancer. Mol. Oncol. 13 , 2679–2696 (2019).

Krishnamurthy, S. et al. Discordance in HER2 gene amplification in circulating and disseminated tumor cells in patients with operable breast cancer. Cancer Med. 2 , 226–233 (2013).

Bidard, F. C. et al. Detection rate and prognostic value of circulating tumor cells and circulating tumor DNA in metastatic uveal melanoma. Int. J. Cancer 134 , 1207–1213 (2014).

Lucci, A. et al. Circulating tumor cells and early relapse in node-positive melanoma. Clin. Cancer Res. 26 , 1886–1895 (2020).

Hoshimoto, S. et al. Assessment of prognostic circulating tumor cells in a phase III trial of adjuvant immunotherapy after complete resection of stage IV melanoma. Ann. Surg. 255 , 357–362 (2012).

Kiniwa, Y. et al. Usefulness of monitoring circulating tumor cells as a therapeutic biomarker in melanoma with BRAF mutation. BMC Cancer 21 , 287 (2021).

Hall, C. S. et al. Circulating tumor cells in stage IV melanoma patients. J. Am. Coll. Surg. 227 , 116–124 (2018).

Hoshimoto, S. et al. Association between circulating tumor cells and prognosis in patients with stage III melanoma with sentinel lymph node metastasis in a phase III international multicenter trial. J. Clin. Oncol. 30 , 3819–3826 (2012).

Lin, S. Y. et al. Prospective molecular profiling of circulating tumor cells from patients with melanoma receiving combinatorial immunotherapy. Clin. Chem. 66 , 169–177 (2020).

Chambers, A. F., Groom, A. C. & MacDonald, I. C. Dissemination and growth of cancer cells in metastatic sites. Nat. Rev. Cancer 2 , 563–572 (2002).

Mehlen, P. & Puisieux, A. Metastasis: a question of life or death. Nat. Rev. Cancer 6 , 449–458 (2006).

Merino, D. et al. Barcoding reveals complex clonal behavior in patient-derived xenografts of metastatic triple negative breast cancer. Nat. Commun. 10 , 766 (2019).

Lambert, A. W., Pattabiraman, D. R. & Weinberg, R. A. Emerging biological principles of metastasis. Cell 168 , 670–691 (2017).

Baccelli, I. et al. Identification of a population of blood circulating tumor cells from breast cancer patients that initiates metastasis in a xenograft assay. Nat. Biotechnol. 31 , 539–544 (2013).

Mani, S. A. et al. The epithelial-mesenchymal transition generates cells with properties of stem cells. Cell 133 , 704–715 (2008).

Batlle, E. et al. The transcription factor snail is a repressor of E-cadherin gene expression in epithelial tumour cells. Nat. Cell Biol. 2 , 84–89 (2000).

Yang, J. et al. Twist, a master regulator of morphogenesis, plays an essential role in tumor metastasis. Cell 117 , 927–939 (2004).

Fischer, K. R. et al. Epithelial-to-mesenchymal transition is not required for lung metastasis but contributes to chemoresistance. Nature 527 , 472–476 (2015).

Zheng, X. et al. Epithelial-to-mesenchymal transition is dispensable for metastasis but induces chemoresistance in pancreatic cancer. Nature 527 , 525–530 (2015).

Padmanaban, V. et al. E-cadherin is required for metastasis in multiple models of breast cancer. Nature 573 , 439–444 (2019).

Tsai, J. H., Donaher, J. L., Murphy, D. A., Chau, S. & Yang, J. Spatiotemporal regulation of epithelial-mesenchymal transition is essential for squamous cell carcinoma metastasis. Cancer Cell 22 , 725–736 (2012).

Yu, M. et al. Circulating breast tumor cells exhibit dynamic changes in epithelial and mesenchymal composition. Science 339 , 580–584 (2013).

Giordano, A. et al. Epithelial-mesenchymal transition and stem cell markers in patients with HER2-positive metastatic breast cancer. Mol. Cancer Ther. 11 , 2526–2534 (2012).

Rhim, A. D. et al. EMT and dissemination precede pancreatic tumor formation. Cell 148 , 349–361 (2012).

Strauss, R. et al. Analysis of epithelial and mesenchymal markers in ovarian cancer reveals phenotypic heterogeneity and plasticity. PLoS One 6 , e16186 (2011).

Brabletz, T. To differentiate or not–routes towards metastasis. Nat. Rev. Cancer 12 , 425–436 (2012).

Friedl, P., Locker, J., Sahai, E. & Segall, J. E. Classifying collective cancer cell invasion. Nat. Cell Biol. 14 , 777–783 (2012).

Liotta, L. A., Saidel, M. G. & Kleinerman, J. The significance of hematogenous tumor cell clumps in the metastatic process. Cancer Res. 36 , 889–894 (1976).

Cleary, A. S., Leonard, T. L., Gestl, S. A. & Gunther, E. J. Tumour cell heterogeneity maintained by cooperating subclones in Wnt-driven mammary cancers. Nature 508 , 113–117 (2014).

Gundem, G. et al. The evolutionary history of lethal metastatic prostate cancer. Nature 520 , 353–357 (2015).

Maddipati, R. & Stanger, B. Z. Pancreatic cancer metastases harbor evidence of polyclonality. Cancer Discov. 5 , 1086–1097 (2015).

McFadden, D. G. et al. Genetic and clonal dissection of murine small cell lung carcinoma progression by genome sequencing. Cell 156 , 1298–1311 (2014).

Eirew, P. et al. Dynamics of genomic clones in breast cancer patient xenografts at single-cell resolution. Nature 518 , 422–426 (2015).

Miller, B. E., Miller, F. R. & Heppner, G. H. Interactions between tumor subpopulations affecting their sensitivity to the antineoplastic agents cyclophosphamide and methotrexate. Cancer Res. 41 , 4378–4381 (1981).

Aceto, N. et al. Circulating tumor cell clusters are oligoclonal precursors of breast cancer metastasis. Cell 158 , 1110–1122 (2014).

Liu, X. et al. Homophilic CD44 interactions mediate tumor cell aggregation and polyclonal metastasis in patient-derived breast cancer models. Cancer Discov. 9 , 96–113 (2019).

Aceto, N., Toner, M., Maheswaran, S. & Haber, D. A. En route to metastasis: circulating tumor cell clusters and epithelial-to-mesenchymal transition. Trends Cancer 1 , 44–52 (2015).

Taftaf, R. et al. ICAM1 initiates CTC cluster formation and trans-endothelial migration in lung metastasis of breast cancer. Nat. Commun. 12 , 4867 (2021).

Chimonidou, M. et al. DNA methylation of tumor suppressor and metastasis suppressor genes in circulating tumor cells. Clin. Chem. 57 , 1169–1177 (2011).

Zhao, Q. et al. Interaction between circulating galectin-3 and cancer-associated MUC1 enhances tumour cell homotypic aggregation and prevents anoikis. Mol. Cancer 9 , 154 (2010).

Li, C. et al. Neural EGFL like 1 as a potential pro-chondrogenic, anti-inflammatory dual-functional disease-modifying osteoarthritis drug. Biomaterials 226 , 119541 (2020).

Cheung, K. J. et al. Polyclonal breast cancer metastases arise from collective dissemination of keratin 14-expressing tumor cell clusters. Proc. Natl Acad. Sci. USA 113 , E854–E863 (2016).

Zhang, L. et al. The identification and characterization of breast cancer CTCs competent for brain metastasis. Sci. Transl. Med. 5 , 180ra148 (2013).

Article Google Scholar

Haynes, B. F., Telen, M. J., Hale, L. P. & Denning, S. M. CD44–a molecule involved in leukocyte adherence and T-cell activation. Immunol. Today 10 , 423–428 (1989).

Labelle, M., Begum, S. & Hynes, R. O. Direct signaling between platelets and cancer cells induces an epithelial-mesenchymal-like transition and promotes metastasis. Cancer Cell 20 , 576–590 (2011).

Labelle, M., Begum, S. & Hynes, R. O. Platelets guide the formation of early metastatic niches. Proc. Natl Acad. Sci. USA 111 , E3053–E3061 (2014).

Labernadie, A. et al. A mechanically active heterotypic E-cadherin/N-cadherin adhesion enables fibroblasts to drive cancer cell invasion. Nat. Cell Biol. 19 , 224–237 (2017).

Sprouse, M. L. et al. PMN-MDSCs enhance CTC metastatic properties through reciprocal interactions via ROS/Notch/nodal signaling. Int. J. Mol. Sci. 20 , 1916 (2019).

Szczerba, B. M. et al. Neutrophils escort circulating tumour cells to enable cell cycle progression. Nature 566 , 553–557 (2019).

Labelle, M. & Hynes, R. O. The initial hours of metastasis: the importance of cooperative host-tumor cell interactions during hematogenous dissemination. Cancer Discov. 2 , 1091–1099 (2012).

Haemmerle, M. et al. Platelets reduce anoikis and promote metastasis by activating YAP1 signaling. Nat. Commun. 8 , 310 (2017).

Russell, J. O. & Monga, S. P. Wnt/β-catenin signaling in liver development, homeostasis, and pathobiology. Annu Rev. Pathol. 13 , 351–378 (2018).

Schumacher, D., Strilic, B., Sivaraj, K. K., Wettschureck, N. & Offermanns, S. Platelet-derived nucleotides promote tumor-cell transendothelial migration and metastasis via P2Y2 receptor. Cancer Cell 24 , 130–137 (2013).

Rachidi, S. et al. Platelets subvert T cell immunity against cancer via GARP-TGFβ axis. Sci. Immunol. 2 , eaai7911 (2017).

Placke, T. et al. Platelet-derived MHC class I confers a pseudonormal phenotype to cancer cells that subverts the antitumor reactivity of natural killer immune cells. Cancer Res. 72 , 440–448 (2012).

Cools-Lartigue, J. et al. Neutrophil extracellular traps sequester circulating tumor cells and promote metastasis. J. Clin. Investig. 123 , 3446–3458 (2013).

Spiegel, A. et al. Neutrophils suppress intraluminal NK cell-mediated tumor cell clearance and enhance extravasation of disseminated carcinoma cells. Cancer Discov. 6 , 630–649 (2016).

Coffelt, S. B. et al. IL-17-producing γδ T cells and neutrophils conspire to promote breast cancer metastasis. Nature 522 , 345–348 (2015).

Schuster, E. et al. Better together: circulating tumor cell clustering in metastatic cancer. Trends Cancer 7 , 1020–1032 (2021).

Gaggioli, C. et al. Fibroblast-led collective invasion of carcinoma cells with differing roles for RhoGTPases in leading and following cells. Nat. Cell Biol. 9 , 1392–1400 (2007).

Au, S. H. et al. Clusters of circulating tumor cells traverse capillary-sized vessels. Proc. Natl Acad. Sci. USA 113 , 4947–4952 (2016).

Duda, D. G. et al. Malignant cells facilitate lung metastasis by bringing their own soil. Proc. Natl Acad. Sci. USA 107 , 21677–21682 (2010).

Rejniak, K. A. Circulating tumor cells: when a solid tumor meets a fluid microenvironment. Adv. Exp. Med. Biol. 936 , 93–106 (2016).

Diamantopoulou, Z. et al. The metastatic spread of breast cancer accelerates during sleep. Nature 607 , 156–162 (2022).

Schernhammer, E. S. et al. Rotating night shifts and risk of breast cancer in women participating in the nurses’ health study. J. Natl Cancer Inst. 93 , 1563–1568 (2001).

Zhu, X. et al. In vivo flow cytometry reveals a circadian rhythm of circulating tumor cells. Light Sci. Appl. 10 , 110 (2021).

Lauriola, M. et al. Diurnal suppression of EGFR signalling by glucocorticoids and implications for tumour progression and treatment. Nat. Commun. 5 , 5073 (2014).

Travis, R. C. et al. Night shift work and breast cancer incidence: three prospective studies and meta-analysis of published studies. J. Natl Cancer Inst. 108 , djw169 (2016).

Giacchetti, S. et al. Phase III trial comparing 4-day chronomodulated therapy versus 2-day conventional delivery of fluorouracil, leucovorin, and oxaliplatin as first-line chemotherapy of metastatic colorectal cancer: the European Organisation for Research and Treatment of Cancer Chronotherapy Group. J. Clin. Oncol. 24 , 3562–3569 (2006).

Sephton, S. E. et al. Diurnal cortisol rhythm as a predictor of lung cancer survival. Brain Behav. Immun. 30 , S163–S170 (2013).

Lévi, F., Zidani, R. & Misset, J. L. Randomised multicentre trial of chronotherapy with oxaliplatin, fluorouracil, and folinic acid in metastatic colorectal cancer. International Organization for Cancer Chronotherapy. Lancet 350 , 681–686 (1997).

Yokobori, T. et al. Plastin3 is a novel marker for circulating tumor cells undergoing the epithelial-mesenchymal transition and is associated with colorectal cancer prognosis. Cancer Res. 73 , 2059–2069 (2013).

Adorno-Cruz, V. et al. Cancer stem cells: targeting the roots of cancer, seeds of metastasis, and sources of therapy resistance. Cancer Res. 75 , 924–929 (2015).

de Sousa e Melo, F. et al. A distinct role for Lgr5(+) stem cells in primary and metastatic colon cancer. Nature 543 , 676–680 (2017).

Tsao, S. C. et al. Characterising the phenotypic evolution of circulating tumour cells during treatment. Nat. Commun. 9 , 1482 (2018).

Hata, A. N. et al. Tumor cells can follow distinct evolutionary paths to become resistant to epidermal growth factor receptor inhibition. Nat. Med. 22 , 262–269 (2016).

Paoletti, C. et al. Development of circulating tumor cell-endocrine therapy index in patients with hormone receptor-positive breast cancer. Clin. Cancer Res. 21 , 2487–2498 (2015).

Hou, J. M. et al. Clinical significance and molecular characteristics of circulating tumor cells and circulating tumor microemboli in patients with small-cell lung cancer. J. Clin. Oncol. 30 , 525–532 (2012).

Chang, M. C. et al. Clinical significance of circulating tumor microemboli as a prognostic marker in patients with pancreatic ductal adenocarcinoma. Clin. Chem. 62 , 505–513 (2016).

Hodgkinson, C. L. et al. Tumorigenicity and genetic profiling of circulating tumor cells in small-cell lung cancer. Nat. Med. 20 , 897–903 (2014).

Kilgour, E., Rothwell, D. G., Brady, G. & Dive, C. Liquid biopsy-based biomarkers of treatment response and resistance. Cancer Cell 37 , 485–495 (2020).

Aktas, B. et al. Stem cell and epithelial-mesenchymal transition markers are frequently overexpressed in circulating tumor cells of metastatic breast cancer patients. Breast Cancer Res. 11 , R46 (2009).

Pantel, K., Brakenhoff, R. H. & Brandt, B. Detection, clinical relevance and specific biological properties of disseminating tumour cells. Nat. Rev. Cancer 8 , 329–340 (2008).

Liu, R. et al. The prognostic role of a gene signature from tumorigenic breast-cancer cells. N. Engl. J. Med. 356 , 217–226 (2007).

Korkaya, H., Paulson, A., Iovino, F. & Wicha, M. S. HER2 regulates the mammary stem/progenitor cell population driving tumorigenesis and invasion. Oncogene 27 , 6120–6130 (2008).

Papadaki, M. A. et al. Co-expression of putative stemness and epithelial-to-mesenchymal transition markers on single circulating tumour cells from patients with early and metastatic breast cancer. BMC Cancer 14 , 651 (2014).

Theodoropoulos, P. A. et al. Circulating tumor cells with a putative stem cell phenotype in peripheral blood of patients with breast cancer. Cancer Lett. 288 , 99–106 (2010).

Abraham, B. K. et al. Prevalence of CD44+/CD24-/low cells in breast cancer may not be associated with clinical outcome but may favor distant metastasis. Clin. Cancer Res. 11 , 1154–1159 (2005).

Trumpp, A. & Wiestler, O. D. Mechanisms of disease: cancer stem cells–targeting the evil twin. Nat. Clin. Pract. Oncol. 5 , 337–347 (2008).

Honeth, G. et al. The CD44+/CD24- phenotype is enriched in basal-like breast tumors. Breast Cancer Res. 10 , R53 (2008).

Mylona, E. et al. The clinicopathologic and prognostic significance of CD44+/CD24(-/low) and CD44-/CD24+ tumor cells in invasive breast carcinomas. Hum. Pathol. 39 , 1096–1102 (2008).

Jamal-Hanjani, M. et al. Tracking the evolution of non-small-cell lung cancer. N. Engl. J. Med. 376 , 2109–2121 (2017).

Turajlic, S. et al. Deterministic evolutionary trajectories influence primary tumor growth: TRACERx renal. Cell 173 , 595–610.e511 (2018).

Chemi, F. et al. Pulmonary venous circulating tumor cell dissemination before tumor resection and disease relapse. Nat. Med. 25 , 1534–1539 (2019).

Visal, T. H., den Hollander, P., Cristofanilli, M. & Mani, S. A. Circulating tumour cells in the -omics era: how far are we from achieving the ‘singularity’? Br. J. Cancer 127 , 173–184 (2022).

Pantel, K. & Alix-Panabières, C. Liquid biopsy and minimal residual disease—latest advances and implications for cure. Nat. Rev. Clin. Oncol. 16 , 409–424 (2019).

Müller, V. et al. Prognostic impact of circulating tumor cells assessed with the CellSearch System™ and AdnaTest Breast™ in metastatic breast cancer patients: the DETECT study. Breast Cancer Res. 14 , R118 (2012).

Wang, L. et al. Promise and limits of the CellSearch platform for evaluating pharmacodynamics in circulating tumor cells. Semin Oncol. 43 , 464–475 (2016).

Gleghorn, J. P. et al. Capture of circulating tumor cells from whole blood of prostate cancer patients using geometrically enhanced differential immunocapture (GEDI) and a prostate-specific antibody. Lab Chip 10 , 27–29 (2010).

Karabacak, N. M. et al. Microfluidic, marker-free isolation of circulating tumor cells from blood samples. Nat. Protoc. 9 , 694–710 (2014).

Krol, I. et al. Detection of clustered circulating tumour cells in early breast cancer. Br. J. Cancer 125 , 23–27 (2021).

Harb, W. et al. Mutational analysis of circulating tumor cells using a novel microfluidic collection device and qPCR assay. Transl. Oncol. 6 , 528–538 (2013).

Donato, C. et al. Hypoxia triggers the intravasation of clustered circulating tumor cells. Cell Rep. 32 , 108105 (2020).

Saucedo-Zeni, N. et al. A novel method for the in vivo isolation of circulating tumor cells from peripheral blood of cancer patients using a functionalized and structured medical wire. Int. J. Oncol. 41 , 1241–1250 (2012).

PubMed PubMed Central Google Scholar

Eifler, R. L. et al. Enrichment of circulating tumor cells from a large blood volume using leukapheresis and elutriation: proof of concept. Cytom. B Clin. Cytom. 80 , 100–111 (2011).

Crosbie, P. A. et al. Circulating tumor cells detected in the tumor-draining pulmonary vein are associated with disease recurrence after surgical resection of NSCLC. J. Thorac. Oncol. 11 , 1793–1797 (2016).

Wang, Y. & Navin, N. E. Advances and applications of single-cell sequencing technologies. Mol. Cell 58 , 598–609 (2015).

Gawad, C., Koh, W. & Quake, S. R. Single-cell genome sequencing: current state of the science. Nat. Rev. Genet. 17 , 175–188 (2016).

Abouleila, Y. et al. Live single cell mass spectrometry reveals cancer-specific metabolic profiles of circulating tumor cells. Cancer Sci. 110 , 697–706 (2019).

Donato, C., Buczak, K., Schmidt, A. & Aceto, N. Mass spectrometry analysis of circulating breast cancer cells from a Xenograft mouse model. STAR Protoc. 2 , 100480 (2021).

Armbrecht, L. et al. Quantification of protein secretion from circulating tumor cells in microfluidic chambers. Adv. Sci. 7 , 1903237 (2020).

Yu, M. et al. Cancer therapy. Ex vivo culture of circulating breast tumor cells for individualized testing of drug susceptibility. Science 345 , 216–220 (2014).

Khoo, B. L. et al. Expansion of patient-derived circulating tumor cells from liquid biopsies using a CTC microfluidic culture device. Nat. Protoc. 13 , 34–58 (2018).

Fokas, E., Engenhart-Cabillic, R., Daniilidis, K., Rose, F. & An, H. X. Metastasis: the seed and soil theory gains identity. Cancer Metastasis Rev. 26 , 705–715 (2007).

Chaffer, C. L. & Weinberg, R. A. A perspective on cancer cell metastasis. Science 331 , 1559–1564 (2011).

Genna, A. et al. EMT-Associated heterogeneity in circulating tumor cells: sticky friends on the road to metastasis. Cancers 12 , 1632 (2020).

Silva Paiva, R., Gomes, I., Casimiro, S., Fernandes, I. & Costa, L. c-Met expression in renal cell carcinoma with bone metastases. J. Bone Oncol. 25 , 100315 (2020).

D’Oronzo, S., Brown, J. & Coleman, R. The role of biomarkers in the management of bone-homing malignancies. J. Bone Oncol. 9 , 1–9 (2017).

Majidpoor, J. & Mortezaee, K. Steps in metastasis: an updated review. Med Oncol. 38 , 3 (2021).

Zhu, L. et al. Characterization of stem-like circulating tumor cells in pancreatic cancer. Diagnostics 10 , 305 (2020).

Fabisiewicz, A. & Grzybowska, E. CTC clusters in cancer progression and metastasis. Med. Oncol. 34 , 12 (2017).

Obenauf, A. C. & Massague, J. Surviving at a distance: organ-specific metastasis. Trends Cancer 1 , 76–91 (2015).

Ring, A., Nguyen-Strauli, B. D., Wicki, A. & Aceto, N. Biology, vulnerabilities and clinical applications of circulating tumour cells. Nat. Rev. Cancer 23 , 95–111 (2023).

Novikov, N. M., Zolotaryova, S. Y., Gautreau, A. M. & Denisov, E. V. Mutational drivers of cancer cell migration and invasion. Br. J. Cancer 124 , 102–114 (2021).

Trepat, X., Chen, Z. & Jacobson, K. Cell migration. Compr. Physiol. 2 , 2369–2392 (2012).

Fanfone, D. et al. Confined migration promotes cancer metastasis through resistance to anoikis and increased invasiveness. Elife 11 , e73150 (2022).

Muscella, A., Vetrugno, C., Cossa, L. G. & Marsigliante, S. TGF-β1 activates RSC96 Schwann cells migration and invasion through MMP-2 and MMP-9 activities. J. Neurochem. 153 , 525–538 (2020).

Wan, G. et al. SLFN5 suppresses cancer cell migration and invasion by inhibiting MT1-MMP expression via AKT/GSK-3β/β-catenin pathway. Cell Signal 59 , 1–12 (2019).

Bonnans, C., Chou, J. & Werb, Z. Remodelling the extracellular matrix in development and disease. Nat. Rev. Mol. Cell Biol. 15 , 786–801 (2014).

Theocharis, A. D., Skandalis, S. S., Gialeli, C. & Karamanos, N. K. Extracellular matrix structure. Adv. Drug Deliv. Rev. 97 , 4–27 (2016).

Insua-Rodríguez, J. & Oskarsson, T. The extracellular matrix in breast cancer. Adv. Drug Deliv. Rev. 97 , 41–55 (2016).

Lambert, A. W. & Weinberg, R. A. Linking EMT programmes to normal and neoplastic epithelial stem cells. Nat. Rev. Cancer 21 , 325–338 (2021).

Zhou, P. et al. The epithelial to mesenchymal transition (EMT) and cancer stem cells: implication for treatment resistance in pancreatic cancer. Mol. Cancer 16 , 52 (2017).

Deshmukh, A. P. et al. Identification of EMT signaling cross-talk and gene regulatory networks by single-cell RNA sequencing. Proc. Natl Acad. Sci. USA 118 , e2102050118 (2021).

Meng, J. et al. USP5 promotes epithelial-mesenchymal transition by stabilizing SLUG in hepatocellular carcinoma. Theranostics 9 , 573–587 (2019).

Dai, X. et al. CBP-mediated Slug acetylation stabilizes Slug and promotes EMT and migration of breast cancer cells. Sci. China Life Sci. 64 , 563–574 (2021).

Yoon, H. et al. Cancer-associated fibroblast secretion of PDGFC promotes gastrointestinal stromal tumor growth and metastasis. Oncogene 40 , 1957–1973 (2021).

Ingruber, J. et al. KLF4, Slug and EMT in head and neck squamous cell carcinoma. Cells 10 , 539(2021).

Schinke, H. et al. SLUG-related partial epithelial-to-mesenchymal transition is a transcriptomic prognosticator of head and neck cancer survival. Mol. Oncol. 16 , 347–367 (2022).

Cho, E. S., Kang, H. E., Kim, N. H. & Yook, J. I. Therapeutic implications of cancer epithelial-mesenchymal transition (EMT). Arch. Pharm. Res. 42 , 14–24 (2019).

Kuburich, N. A., den Hollander, P., Pietz, J. T. & Mani, S. A. Vimentin and cytokeratin: good alone, bad together. Semin. Cancer Biol. 86 , 816–826 (2022).

Satelli, A. & Li, S. Vimentin in cancer and its potential as a molecular target for cancer therapy. Cell Mol. Life Sci. 68 , 3033–3046 (2011).

Serrano-Gomez, S. J., Maziveyi, M. & Alahari, S. K. Regulation of epithelial-mesenchymal transition through epigenetic and post-translational modifications. Mol. Cancer 15 , 18 (2016).

Francart, M. E. et al. Epithelial-mesenchymal plasticity and circulating tumor cells: travel companions to metastases. Dev. Dyn. 247 , 432–450 (2018).

Nowak, E. & Bednarek, I. Aspects of the epigenetic regulation of emt related to cancer metastasis. Cells 10 , 3435 (2021).

Loh, C. Y. et al. The E-cadherin and N-cadherin switch in epithelial-to-mesenchymal transition: signaling, therapeutic implications, and challenges. Cells 8 , 1118 (2019).

Imran, M. et al. Luteolin, a flavonoid, as an anticancer agent: a review. Biomed. Pharmacother. 112 , 108612 (2019).

Keller, L., Werner, S. & Pantel, K. Biology and clinical relevance of EpCAM. Cell Stress 3 , 165–180 (2019).

Usman, S. et al. Vimentin is at the heart of epithelial-mesenchymal transition (EMT) mediated metastasis. Cancers 13 (2021).

Friedl, P. & Alexander, S. Cancer invasion and the microenvironment: plasticity and reciprocity. Cell 147 , 992–1009 (2011).

Friedl, P. & Mayor, R. Tuning collective cell migration by cell-cell junction regulation. Cold Spring Harb. Perspect. Biol. 9 , a029199 (2017).

Shankar, J. & Nabi, I. R. Actin cytoskeleton regulation of epithelial mesenchymal transition in metastatic cancer cells. PLoS One 10 , e0119954 (2015).

Bordin, M., D’Atri, F., Guillemot, L. & Citi, S. Histone deacetylase inhibitors up-regulate the expression of tight junction proteins. Mol. Cancer Res. 2 , 692–701 (2004).

Samulitis, B. K. et al. Gemcitabine resistant pancreatic cancer cell lines acquire an invasive phenotype with collateral hypersensitivity to histone deacetylase inhibitors. Cancer Biol. Ther. 16 , 43–51 (2015).

Rolando, M. et al. Contractile actin cables induced by Bacillus anthracis lethal toxin depend on the histone acetylation machinery. Cytoskeleton 72 , 542–556 (2015).

Jiang, J. et al. Redox regulation in tumor cell epithelial-mesenchymal transition: molecular basis and therapeutic strategy. Signal Transduct. Target Ther. 2 , 17036 (2017).

Ottaviani, S. et al. TGF-β induces miR-100 and miR-125b but blocks let-7a through LIN28B controlling PDAC progression. Nat. Commun. 9 , 1845 (2018).

Przybyla, L., Lakins, J. N. & Weaver, V. M. Tissue mechanics orchestrate Wnt-dependent human embryonic stem cell differentiation. Cell Stem Cell 19 , 462–475 (2016).

Li, Z. et al. Hotspot ESR1 mutations are multimodal and contextual modulators of breast cancer metastasis. Cancer Res. 82 , 1321–1339 (2022).

Nagase, H., Visse, R. & Murphy, G. Structure and function of matrix metalloproteinases and TIMPs. Cardiovasc Res. 69 , 562–573 (2006).

Niland, S., Riscanevo, A. X. & Eble, J. A. Matrix metalloproteinases shape the tumor microenvironment in cancer progression. Int. J. Mol. Sci. 23 , 146 (2021).

Zhang, Y. et al. Melatonin modulates IL-1β-induced extracellular matrix remodeling in human nucleus pulposus cells and attenuates rat intervertebral disc degeneration and inflammation. Aging 11 , 10499–10512 (2019).

Magnan, L. et al. In vivo remodeling of human cell-assembled extracellular matrix yarns. Biomaterials 273 , 120815 (2021).

Jafari, G. et al. Genetics of extracellular matrix remodeling during organ growth using the Caenorhabditis elegans pharynx model. Genetics 186 , 969–982 (2010).

Gucciardo, F., Pirson, S., Baudin, L., Lebeau, A. & Noël, A. uPARAP/Endo180: a multifaceted protein of mesenchymal cells. Cell Mol. Life Sci. 79 , 255 (2022).

Kunze, P. et al. Multiphoton microscopy reveals DAPK1-dependent extracellular matrix remodeling in a chorioallantoic membrane (CAM) model. Cancers 14 , 2364 (2022).

Shmakova, A. A. et al. Urokinase receptor uPAR downregulation in neuroblastoma leads to dormancy, chemoresistance and metastasis. Cancers 14 , 994 (2022).

Ismail, A. A., Shaker, B. T. & Bajou, K. The plasminogen-activator plasmin system in physiological and pathophysiological angiogenesis. Int. J. Mol. Sci. 23 , 337 (2021).

Ingram, K. G., Curtis, C. D., Silasi-Mansat, R., Lupu, F. & Griffin, C. T. The NuRD chromatin-remodeling enzyme CHD4 promotes embryonic vascular integrity by transcriptionally regulating extracellular matrix proteolysis. PLoS Genet. 9 , e1004031 (2013).

Hamidi, H. & Ivaska, J. Every step of the way: integrins in cancer progression and metastasis. Nat. Rev. Cancer 18 , 533–548 (2018).

Vining, K. H. et al. Mechanical checkpoint regulates monocyte differentiation in fibrotic niches. Nat. Mater. 21 , 939–950 (2022).

Wang, X. et al. Succinylation inhibits the enzymatic hydrolysis of the extracellular matrix protein fibrillin 1 and promotes gastric cancer progression. Adv. Sci. 9 , e2200546 (2022).

Leiva, O. et al. The role of extracellular matrix stiffness in megakaryocyte and platelet development and function. Am. J. Hematol. 93 , 430–441 (2018).

Schüler, S. C. et al. Extensive remodeling of the extracellular matrix during aging contributes to age-dependent impairments of muscle stem cell functionality. Cell Rep. 35 , 109223 (2021).

Hynes, R. O. & Naba, A. Overview of the matrisome–an inventory of extracellular matrix constituents and functions. Cold Spring Harb. Perspect. Biol. 4 , a004903 (2012).

Godinho, S. A. et al. Oncogene-like induction of cellular invasion from centrosome amplification. Nature 510 , 167–171 (2014).

Sawabata, N. et al. Perioperative circulating tumor cells in surgical patients with non-small cell lung cancer: does surgical manipulation dislodge cancer cells thus allowing them to pass into the peripheral blood? Surg. Today 46 , 1402–1409 (2016).

Camara, O. et al. Seeding of epithelial cells into circulation during surgery for breast cancer: the fate of malignant and benign mobilized cells. World J. Surg. Oncol. 4 , 67 (2006).

Hapach, L. A. et al. Phenotypic heterogeneity and metastasis of breast cancer cells. Cancer Res 81 , 3649–3663 (2021).

Chang, P. H. et al. Interplay between desmoglein2 and hypoxia controls metastasis in breast cancer. Proc. Natl Acad. Sci. USA 118 , e2014408118 (2021).

Jin, K., Luo, Z., Zhang, B. & Pang, Z. Biomimetic nanoparticles for inflammation targeting. Acta Pharm. Sin. B 8 , 23–33 (2018).

Kapeleris, J. et al. Cancer stemness contributes to cluster formation of colon cancer cells and high metastatic potentials. Clin. Exp. Pharm. Physiol. 47 , 838–847 (2020).

Martínez-León, E. et al. Fibronectin stimulates human sperm capacitation through the cyclic AMP/protein kinase A pathway. Hum. Reprod. 30 , 2138–2151 (2015).

Castellano, L. et al. Evaluation of α5β1 integrin as a candidate marker for fertility in bull sperm samples. Theriogenology 168 , 66–74 (2021).

Kim, M. Y. et al. Tumor self-seeding by circulating cancer cells. Cell 139 , 1315–1326 (2009).

Divella, R. et al. Circulating levels of transforming growth factor-βeta (TGF-β) and chemokine (C-X-C motif) ligand-1 (CXCL1) as predictors of distant seeding of circulating tumor cells in patients with metastatic breast cancer. Anticancer Res. 33 , 1491–1497 (2013).

Varol, R. et al. Acousto-holographic reconstruction of whole-cell stiffness maps. Nat. Commun. 13 , 7351 (2022).

Hynes, W. F. et al. Examining metastatic behavior within 3D bioprinted vasculature for the validation of a 3D computational flow model. Sci. Adv. 6 , eabb3308 (2020).

Dong, C., Wang, H., Zhang, Z., Zhang, T. & Liu, B. Carboxybetaine methacrylate oligomer modified nylon for circulating tumor cells capture. J. Colloid Interface Sci. 432 , 135–143 (2014).

Yang, M. H., Imrali, A. & Heeschen, C. Circulating cancer stem cells: the importance to select. Chin. J. Cancer Res 27 , 437–449 (2015).

CAS PubMed PubMed Central Google Scholar

Huang, Q. et al. Fluid shear stress and tumor metastasis. Am. J. Cancer Res 8 , 763–777 (2018).

Wong, S. Y. & Hynes, R. O. Lymphatic or hematogenous dissemination: how does a metastatic tumor cell decide? Cell Cycle 5 , 812–817 (2006).

Dianat-Moghadam, H. et al. The role of circulating tumor cells in the metastatic cascade: biology, technical challenges, and clinical relevance. Cancers 12 , 867 (2020).

Badia-Ramentol, J., Linares, J., Gómez-Llonin, A. & Calon, A. Minimal residual disease, metastasis and immunity. Biomolecules 11 , 130(2021).

Li, H. et al. Tissue factor: a neglected role in cancer biology. J. Thromb. Thrombolysis 54 , 97–108 (2022).

Lian, S., Xie, X., Lu, Y. & Jia, L. Checkpoint CD47 function on tumor metastasis and immune therapy. Onco Targets Ther. 12 , 9105–9114 (2019).

Lozar, T., Gersak, K., Cemazar, M., Kuhar, C. G. & Jesenko, T. The biology and clinical potential of circulating tumor cells. Radio. Oncol. 53 , 131–147 (2019).

Nguyen, D. X., Bos, P. D. & Massagué, J. Metastasis: from dissemination to organ-specific colonization. Nat. Rev. Cancer 9 , 274–284 (2009).

Liu, S. J., Dang, H. X., Lim, D. A., Feng, F. Y. & Maher, C. A. Long noncoding RNAs in cancer metastasis. Nat. Rev. Cancer 21 , 446–460 (2021).

Tucci, P. et al. Loss of p63 and its microRNA-205 target results in enhanced cell migration and metastasis in prostate cancer. Proc. Natl Acad. Sci. USA 109 , 15312–15317 (2012).

Vasilaki, E. et al. Ras and TGF-β signaling enhance cancer progression by promoting the ΔNp63 transcriptional program. Sci. Signal 9 , ra84 (2016).

Bakir, B., Chiarella, A. M., Pitarresi, J. R. & Rustgi, A. K. EMT, MET, plasticity, and tumor metastasis. Trends Cell Biol. 30 , 764–776 (2020).

Lee, Y. C. et al. The dynamic roles of the bladder tumour microenvironment. Nat. Rev. Urol. 19 , 515–533 (2022).

Zhang, K. L. et al. Organ-specific cholesterol metabolic aberration fuels liver metastasis of colorectal cancer. Theranostics 11 , 6560–6572 (2021).

Tsai, J. H. & Yang, J. Epithelial-mesenchymal plasticity in carcinoma metastasis. Genes Dev. 27 , 2192–2206 (2013).

Nimmakayala, R. K. et al. Metabolic programming of distinct cancer stem cells promotes metastasis of pancreatic ductal adenocarcinoma. Oncogene 40 , 215–231 (2021).

Fedele, M., Sgarra, R., Battista, S., Cerchia, L. & Manfioletti, G. The epithelial-mesenchymal transition at the crossroads between metabolism and tumor progression. Int. J. Mol. Sci. 23 , 800 (2022).

Basturk, O. et al. A revised classification system and recommendations from the baltimore consensus meeting for neoplastic precursor lesions in the pancreas. Am. J. Surg. Pathol. 39 , 1730–1741 (2015).

Peinado, H. et al. Pre-metastatic niches: organ-specific homes for metastases. Nat. Rev. Cancer 17 , 302–317 (2017).

Massague, J. & Obenauf, A. C. Metastatic colonization by circulating tumour cells. Nature 529 , 298–306 (2016).

Sun, Y. F. et al. Dissecting spatial heterogeneity and the immune-evasion mechanism of CTCs by single-cell RNA-seq in hepatocellular carcinoma. Nat. Commun. 12 , 4091 (2021).

Ebright, R. Y. et al. Deregulation of ribosomal protein expression and translation promotes breast cancer metastasis. Science 367 , 1468–1473 (2020).

Austin, R. G., Huang, T. J., Wu, M., Armstrong, A. J. & Zhang, T. Clinical utility of non-EpCAM based circulating tumor cell assays. Adv. Drug Deliv. Rev. 125 , 132–142 (2018).

Agnoletto, C. et al. Heterogeneous expression of EPCAM in human circulating tumour cells from patient-derived xenografts. Biomark. Res. 6 , 31 (2018).

Ligthart, S. T. et al. Unbiased quantitative assessment of Her-2 expression of circulating tumor cells in patients with metastatic and non-metastatic breast cancer. Ann. Oncol. 24 , 1231–1238 (2013).

Kallergi, G. et al. Expression of truncated human epidermal growth factor receptor 2 on circulating tumor cells of breast cancer patients. Breast Cancer Res. 17 , 113 (2015).

Müller, V. et al. Prognostic relevance of the HER2 status of circulating tumor cells in metastatic breast cancer patients screened for participation in the DETECT study program. ESMO Open 6 , 100299 (2021).

Zhang, Z. et al. YOD1 serves as a potential prognostic biomarker for pancreatic cancer. Cancer Cell Int. 22 , 203 (2022).

Braun, A. C. et al. Circulating tumor cells in desmoid tumors: new perspectives. Front. Oncol. 11 , 622626 (2021).

Mego, M. et al. Vitamin D and circulating tumor cells in primary breast cancer. Front. Oncol. 12 , 950451 (2022).

Lu, Y. et al. S-Nitrosocaptopril prevents cancer metastasis in vivo by creating the hostile bloodstream microenvironment against circulating tumor cells. Pharm. Res. 139 , 535–549 (2019).

Silva-Carvalho, A. et al. GVHD-derived plasma as a priming strategy of mesenchymal stem cells. Stem Cell Res. Ther. 11 , 156 (2020).

Wei, C. et al. Crosstalk between cancer cells and tumor associated macrophages is required for mesenchymal circulating tumor cell-mediated colorectal cancer metastasis. Mol. Cancer 18 , 64 (2019).

Klusa, D. et al. Dynamics of CXCR4 positive circulating tumor cells in prostate cancer patients during radiotherapy. Int. J. Cancer 152 , 2639–2654 (2023).

Mehdipour, P., Javan, F., Jouibari, M. F., Khaleghi, M. & Mehrazin, M. Evolutionary model of brain tumor circulating cells: cellular galaxy. World J. Clin. Oncol. 12 , 13–30 (2021).

Jung, Y. Y., Ha, I. J., Um, J. Y., Sethi, G. & Ahn, K. S. Fangchinoline diminishes STAT3 activation by stimulating oxidative stress and targeting SHP-1 protein in multiple myeloma model. J. Adv. Res. 35 , 245–257 (2022).

Huang, Q. et al. Shear stress activates ATOH8 via autocrine VEGF promoting glycolysis dependent-survival of colorectal cancer cells in the circulation. J. Exp. Clin. Cancer Res. 39 , 25 (2020).

Strati, A. et al. A comprehensive molecular analysis of in vivo isolated EpCAM-positive circulating tumor cells in breast cancer. Clin. Chem. 67 , 1395–1405 (2021).

Balamurugan, K. et al. Stabilization of E-cadherin adhesions by COX-2/GSK3β signaling is a targetable pathway in metastatic breast cancer. JCI Insight 8 , e156057 (2023).

Mathenge, E. G. et al. Core needle biopsy of breast cancer tumors increases distant metastases in a mouse model. Neoplasia 16 , 950–960 (2014).

Christensen, E. et al. Longitudinal cytokine expression during IMRT for prostate cancer and acute treatment toxicity. Clin. Cancer Res. 15 , 5576–5583 (2009).

Long, Y. et al. Self-delivery micellar nanoparticles prevent premetastatic niche formation by interfering with the early recruitment and vascular destruction of granulocytic myeloid-derived suppressor cells. Nano Lett. 20 , 2219–2229 (2020).

Liu, M. et al. Prognostic significance of PD-L1 expression on cell-surface vimentin-positive circulating tumor cells in gastric cancer patients. Mol. Oncol. 14 , 865–881 (2020).

Wang, X. et al. CTC immune escape mediated by PD-L1. Med. Hypotheses 93 , 138–139 (2016).

Yılmaz, M., Kaplan, F., Mender, I., Gryaznov, S. M. & Dikmen, Z. G. Cancer stem cells and anti-tumor immunity. Curr Stem Cell Res. Ther. 18 , 445–459 (2022).

Darga, E. P. et al. PD-L1 expression on circulating tumor cells and platelets in patients with metastatic breast cancer. PLoS One 16 , e0260124 (2021).

Zhang, T. et al. Expression of immune checkpoints on circulating tumor cells in men with metastatic prostate cancer. Biomark. Res. 9 , 14 (2021).

Ao, H., Xin, Z. & Jian, Z. Liquid biopsy to identify biomarkers for immunotherapy in hepatocellular carcinoma. Biomark. Res. 9 , 91 (2021).

Indini, A., Rijavec, E. & Grossi, F. Circulating biomarkers of response and toxicity of immunotherapy in advanced non-small cell lung cancer (NSCLC): a comprehensive review. Cancers 13 , 1794 (2021).

Mohammad Alizadeh, A. H., Hajilooi, M., Ranjbar, M., Fallahian, F. & Mousavi, S. M. Cytotoxic T-lymphocyte antigen 4 gene polymorphisms and susceptibility to chronic hepatitis B. World J. Gastroenterol. 12 , 630–635 (2006).

Wang, X. et al. Cell by cell immuno- and cancer marker profiling of non-small cell lung cancer tissue: checkpoint marker expression on CD103+, CD4+ T-cells predicts circulating tumor cells. Transl. Oncol. 14 , 100953 (2021).

Arnoletti, J. P. et al. Pancreatic Ductal Adenocarcinoma (PDAC) circulating tumor cells influence myeloid cell differentiation to support their survival and immunoresistance in portal vein circulation. PLoS One 17 , e0265725 (2022).

Po, J. W. et al. Immunomagnetic isolation of circulating melanoma cells and detection of PD-L1 status. PLoS One 14 , e0211866 (2019).

Bootsma, M. et al. Longitudinal molecular profiling of circulating tumor cells in metastatic renal cell carcinoma. J. Clin. Oncol. 40 , 3633–3641 (2022).

Kong, D. et al. Correlation between PD-L1 expression ON CTCs and prognosis of patients with cancer: a systematic review and meta-analysis. Oncoimmunology 10 , 1938476 (2021).

Bergmann, S. et al. Evaluation of PD-L1 expression on circulating tumor cells (CTCs) in patients with advanced urothelial carcinoma (UC). Oncoimmunology 9 , 1738798 (2020).

Zhong, X. et al. Circulating tumor cells in cancer patients: developments and clinical applications for immunotherapy. Mol. Cancer 19 , 15 (2020).

Guo, S. et al. The role of extracellular vesicles in circulating tumor cell-mediated distant metastasis. Mol. Cancer 22 , 193 (2023).

Kang, Y. T. et al. On-chip biogenesis of circulating NK cell-derived exosomes in non-small cell lung cancer exhibits antitumoral activity. Adv. Sci. 8 , 2003747 (2021).

Termini, R. et al. Circulating tumor and immune cells for minimally invasive risk stratification of smoldering multiple myeloma. Clin. Cancer Res. 28 , 4771–4781 (2022).

Lou, C., Wu, K., Shi, J., Dai, Z. & Xu, Q. N-cadherin protects oral cancer cells from NK cell killing in the circulation by inducing NK cell functional exhaustion via the KLRG1 receptor. J. Immunother. Cancer 10 , e005061 (2022).

Yu, W. et al. Exosome-based liquid biopsies in cancer: opportunities and challenges. Ann. Oncol. 32 , 466–477 (2021).

Wagner, K. T., Nash, T. R., Liu, B., Vunjak-Novakovic, G. & Radisic, M. Extracellular vesicles in cardiac regeneration: potential applications for tissues-on-a-chip. Trends Biotechnol. 39 , 755–773 (2021).

Jerabkova-Roda, K., Dupas, A., Osmani, N., Hyenne, V. & Goetz, J. G. Circulating extracellular vesicles and tumor cells: sticky partners in metastasis. Trends Cancer 8 , 799–805 (2022).

Chen, V. L., Xu, D., Wicha, M. S., Lok, A. S. & Parikh, N. D. Utility of liquid biopsy analysis in detection of hepatocellular carcinoma, determination of prognosis, and disease monitoring: a systematic review. Clin. Gastroenterol. Hepatol. 18 , 2879–2902.e2879 (2020).

Raza, A. et al. Dynamic liquid biopsy components as predictive and prognostic biomarkers in colorectal cancer. J. Exp. Clin. Cancer Res. 41 , 99 (2022).

Vaidyanathan, R., Soon, R. H., Zhang, P., Jiang, K. & Lim, C. T. Cancer diagnosis: from tumor to liquid biopsy and beyond. Lab. Chip 19 , 11–34 (2018).

Brozos-Vázquez, E. M. et al. Immunotherapy in nonsmall-cell lung cancer: current status and future prospects for liquid biopsy. Cancer Immunol. Immunother. 70 , 1177–1188 (2021).

Lo, H. C. et al. Resistance to natural killer cell immunosurveillance confers a selective advantage to polyclonal metastasis. Nat. Cancer 1 , 709–722 (2020).

Pereira-Veiga, T., Schneegans, S., Pantel, K. & Wikman, H. Circulating tumor cell-blood cell crosstalk: biology and clinical relevance. Cell Rep. 40 , 111298 (2022).

Poulet, G., Massias, J. & Taly, V. Liquid biopsy: general concepts. Acta Cytol. 63 , 449–455 (2019).

Razmara, A. M. et al. Tumor shedding and metastatic progression after tumor excision in patient-derived orthotopic xenograft models of triple-negative breast cancer. Clin. Exp. Metastasis 37 , 413–424 (2020).

Toss, A., Mu, Z., Fernandez, S. & Cristofanilli, M. CTC enumeration and characterization: moving toward personalized medicine. Ann. Transl. Med. 2 , 108 (2014).

von Felden, J. et al. Circulating tumor cells as liquid biomarker for high HCC recurrence risk after curative liver resection. Oncotarget 8 , 89978–89987 (2017).

Beinse, G. et al. Circulating tumor cell count and thrombosis in metastatic breast cancer. J. Thromb. Haemost. 15 , 1981–1988 (2017).

Bidard, F. C. et al. Clinical application of circulating tumor cells in breast cancer: overview of the current interventional trials. Cancer Metastasis Rev. 32 , 179–188 (2013).

Stott, S. L. et al. Isolation and characterization of circulating tumor cells from patients with localized and metastatic prostate cancer. Sci. Transl. Med. 2 , 25ra23 (2010).

Nieswandt, B., Hafner, M., Echtenacher, B. & Männel, D. N. Lysis of tumor cells by natural killer cells in mice is impeded by platelets. Cancer Res. 59 , 1295–1300 (1999).

Lizama, B. N. & Chu, C. T. Neuronal autophagy and mitophagy in Parkinson’s disease. Mol. Asp. Med. 82 , 100972 (2021).

Nikanjam, M., Kato, S. & Kurzrock, R. Liquid biopsy: current technology and clinical applications. J. Hematol. Oncol. 15 , 131 (2022).

Fridrichova, I., Kalinkova, L. & Ciernikova, S. Clinical relevancy of circulating tumor cells in breast cancer: epithelial or mesenchymal characteristics, single cells or clusters? Int. J. Mol. Sci. 23 , 12141 (2022).

Ma, S. et al. Current progress in CAR-T cell therapy for solid tumors. Int. J. Biol. Sci. 15 , 2548–2560 (2019).

Patel, S. A. & Vanharanta, S. Epigenetic determinants of metastasis. Mol. Oncol. 11 , 79–96 (2017).

Pal, S. & Tyler, J. K. Epigenetics and aging. Sci. Adv. 2 , e1600584 (2016).

Chabanon, R. M., Morel, D. & Postel-Vinay, S. Exploiting epigenetic vulnerabilities in solid tumors: novel therapeutic opportunities in the treatment of SWI/SNF-defective cancers. Semin. Cancer Biol. 61 , 180–198 (2020).

Zhao, L., Wu, X., Zheng, J. & Dong, D. DNA methylome profiling of circulating tumor cells in lung cancer at single base-pair resolution. Oncogene 40 , 1884–1895 (2021).

Chen, H. et al. Single-cell DNA methylome analysis of circulating tumor cells. Chin. J. Cancer Res. 33 , 391–404 (2021).

Li, J., Gregory, S. G., Garcia-Blanco, M. A. & Armstrong, A. J. Using circulating tumor cells to inform on prostate cancer biology and clinical utility. Crit. Rev. Clin. Lab. Sci. 52 , 191–210 (2015).

Xiong, G. et al. Hsp47 promotes cancer metastasis by enhancing collagen-dependent cancer cell-platelet interaction. Proc. Natl Acad. Sci. USA 117 , 3748–3758 (2020).

Ntzifa, A. et al. DNA methylation analysis in plasma cell-free DNA and paired CTCs of NSCLC patients before and after osimertinib treatment. Cancers 13 , 5974 (2021).

Sun, K. et al. Ultrasensitive nanopore sensing of mucin 1 and circulating tumor cells in whole blood of breast cancer patients by analyte-triggered triplex-DNA release. ACS Appl. Mater. Interfaces 13 , 21030–21039 (2021).

Gerlinger, M. et al. Intratumor heterogeneity and branched evolution revealed by multiregion sequencing. N. Engl. J. Med. 366 , 883–892 (2012).

McGranahan, N. & Swanton, C. Clonal heterogeneity and tumor evolution: past, present, and the future. Cell 168 , 613–628 (2017).

Yu, M. Metastasis stemming from circulating tumor cell clusters. Trends Cell Biol. 29 , 275–276 (2019).

Bertamini, L. et al. High levels of circulating tumor plasma cells as a key hallmark of aggressive disease in transplant-eligible patients with newly diagnosed multiple myeloma. J. Clin. Oncol. 40 , 3120–3131 (2022).

Yuan, H. et al. SETD2 restricts prostate cancer metastasis by integrating EZH2 and AMPK signaling pathways. Cancer Cell 38 , 350–365.e357 (2020).

Duan, R., Du, W. & Guo, W. EZH2: a novel target for cancer treatment. J. Hematol. Oncol. 13 , 104 (2020).

Hemminki, A. et al. Loss of the wild type MLH1 gene is a feature of hereditary nonpolyposis colorectal cancer. Nat. Genet. 8 , 405–410 (1994).

Galm, O., Herman, J. G. & Baylin, S. B. The fundamental role of epigenetics in hematopoietic malignancies. Blood Rev. 20 , 1–13 (2006).

Esteller, M. et al. Inactivation of the DNA repair gene O6-methylguanine-DNA methyltransferase by promoter hypermethylation is associated with G to A mutations in K-ras in colorectal tumorigenesis. Cancer Res. 60 , 2368–2371 (2000).

Hadjimichael, C. et al. Common stemness regulators of embryonic and cancer stem cells. World J. Stem Cells 7 , 1150–1184 (2015).

Yang, J. et al. Guidelines and definitions for research on epithelial-mesenchymal transition. Nat. Rev. Mol. Cell Biol. 21 , 341–352 (2020).

Lyberopoulou, A. et al. Identification of methylation profiles of cancer-related genes in circulating tumor cells population. Anticancer Res. 37 , 1105–1112 (2017).

Pixberg, C. F. et al. Analysis of DNA methylation in single circulating tumor cells. Oncogene 36 , 3223–3231 (2017).

Zavridou, M. et al. Direct comparison of size-dependent versus EpCAM-dependent CTC enrichment at the gene expression and DNA methylation level in head and neck squamous cell carcinoma. Sci. Rep. 10 , 6551 (2020).

Robertson, K. D. DNA methylation, methyltransferases, and cancer. Oncogene 20 , 3139–3155 (2001).

Sasidharan Nair, V. et al. DNA methylation and repressive H3K9 and H3K27 trimethylation in the promoter regions of PD-1, CTLA-4, TIM-3, LAG-3, TIGIT, and PD-L1 genes in human primary breast cancer. Clin. Epigenet. 10 , 78 (2018).

Sasidharan Nair, V., Toor, S. M., Taha, R. Z., Shaath, H. & Elkord, E. DNA methylation and repressive histones in the promoters of PD-1, CTLA-4, TIM-3, LAG-3, TIGIT, PD-L1, and galectin-9 genes in human colorectal cancer. Clin. Epigenet. 10 , 104 (2018).

McCracken, L. C. et al. Development of a physical shoulder simulator for the training of basic arthroscopic skills. Int. J. Med. Robot. 14 (2018).

Andersen, F. H. [Not Available]. Tidsskr Nor Laegeforen 137 (2017).

Ota, M. & Fukuda, M. Highly efficient on-chip excitation of orthogonal-polarized gap plasmons for a dense polarization multiplexing circuit. Opt. Express 26 , 21778–21783 (2018).

Elashi, A. A., Sasidharan Nair, V., Taha, R. Z., Shaath, H. & Elkord, E. DNA methylation of immune checkpoints in the peripheral blood of breast and colorectal cancer patients. Oncoimmunology 8 , e1542918 (2019).

Marwitz, S. et al. Epigenetic modifications of the immune-checkpoint genes CTLA4 and PDCD1 in non-small cell lung cancer results in increased expression. Clin. Epigenet. 9 , 51 (2017).

Franzen, A. et al. PD-L1 (CD274) and PD-L2 (PDCD1LG2) promoter methylation is associated with HPV infection and transcriptional repression in head and neck squamous cell carcinomas. Oncotarget 9 , 641–650 (2018).

Goltz, D., Gevensleben, H., Dietrich, J. & Dietrich, D. PD-L1 (CD274) promoter methylation predicts survival in colorectal cancer patients. Oncoimmunology 6 , e1257454 (2017).

Goltz, D. et al. PD-L1 (CD274) promoter methylation predicts survival in patients with acute myeloid leukemia. Leukemia 31 , 738–743 (2017).

Jiang, J. H. et al. Circulating tumor cell methylation profiles reveal the classification and evolution of non-small cell lung cancer. Transl. Lung Cancer Res. 11 , 224–237 (2022).

Cheng, S. B. et al. Three-dimensional scaffold chip with thermosensitive coating for capture and reversible release of individual and cluster of circulating tumor cells. Anal. Chem. 89 , 7924–7932 (2017).

Cao, Y., Gao, H. L., Chen, J. N., Chen, Z. Y. & Yang, L. Identification and characterization of conjugated linolenic acid isomers by Ag+-HPLC and NMR. J. Agric. Food Chem. 54 , 9004–9009 (2006).

Puga, A. V. et al. From mono- to poly-substituted frameworks: a way of tuning the acidic character of C(c)-H in o-carborane derivatives. Chemistry 15 , 9755–9763 (2009).

Xu, L. H. et al. First report of pear blossom blast caused by Pseudomonas syringae pv. syringae in China. Plant Dis. 92 , 832 (2008).

Feng, H. et al. Homogeneous pancreatic cancer spheroids mimic growth pattern of circulating tumor cell clusters and macrometastases: displaying heterogeneity and crater-like structure on inner layer. J. Cancer Res. Clin. Oncol. 143 , 1771–1786 (2017).

Liu, X. et al. EGFR inhibition blocks cancer stem cell clustering and lung metastasis of triple negative breast cancer. Theranostics 11 , 6632–6643 (2021).

Woo, H. J. et al. Continuous centrifugal microfluidics (CCM) isolates heterogeneous circulating tumor cells via full automation. Theranostics 12 , 3676–3689 (2022).

Ma, F., Jiang, S. & Zhang, C. Y. Recent advances in histone modification and histone modifying enzyme assays. Expert Rev. Mol. Diagn. 19 , 27–36 (2019).

Dawson, M. A. & Kouzarides, T. Cancer epigenetics: from mechanism to therapy. Cell 150 , 12–27 (2012).

Bhaumik, S. R., Smith, E. & Shilatifard, A. Covalent modifications of histones during development and disease pathogenesis. Nat. Struct. Mol. Biol. 14 , 1008–1016 (2007).

Kebede, A. F., Schneider, R. & Daujat, S. Novel types and sites of histone modifications emerge as players in the transcriptional regulation contest. FEBS J. 282 , 1658–1674 (2015).

Feinberg, A. P., Koldobskiy, M. A. & Göndör, A. Epigenetic modulators, modifiers and mediators in cancer aetiology and progression. Nat. Rev. Genet. 17 , 284–299 (2016).

Bannister, A. J. & Kouzarides, T. Regulation of chromatin by histone modifications. Cell Res. 21 , 381–395 (2011).

Verma, A. et al. EZH2-H3K27me3 mediated KRT14 upregulation promotes TNBC peritoneal metastasis. Nat. Commun. 13 , 7344 (2022).

Piunti, A. & Shilatifard, A. Epigenetic balance of gene expression by Polycomb and COMPASS families. Science 352 , aad9780 (2016).

Kang-Decker, N. et al. Loss of CBP causes T cell lymphomagenesis in synergy with p27Kip1 insufficiency. Cancer Cell 5 , 177–189 (2004).

Narita, T. et al. Enhancers are activated by p300/CBP activity-dependent PIC assembly, RNAPII recruitment, and pause release. Mol. Cell 81 , 2166–2182.e2166 (2021).

Hogg, S. J. et al. Targeting histone acetylation dynamics and oncogenic transcription by catalytic P300/CBP inhibition. Mol. Cell 81 , 2183–2200.e2113 (2021).

Mansour, M. R. et al. Oncogene regulation. An oncogenic super-enhancer formed through somatic mutation of a noncoding intergenic element. Science 346 , 1373–1377 (2014).

Tsang, F. H. et al. Aberrant super-enhancer landscape in human hepatocellular carcinoma. Hepatology 69 , 2502–2517 (2019).

Kadoch, C. & Crabtree, G. R. Mammalian SWI/SNF chromatin remodeling complexes and cancer: mechanistic insights gained from human genomics. Sci. Adv. 1 , e1500447 (2015).

Stern, C. Boveri and the early days of genetics. Nature 166 , 446 (1950).

Lazzerini-Denchi, E. & Sfeir, A. Stop pulling my strings—what telomeres taught us about the DNA damage response. Nat. Rev. Mol. Cell Biol. 17 , 364–378 (2016).

Li, S. K. H. & Martin, A. Mismatch repair and colon cancer: mechanisms and therapies explored. Trends Mol. Med. 22 , 274–289 (2016).

Ogiwara, H. et al. Histone acetylation by CBP and p300 at double-strand break sites facilitates SWI/SNF chromatin remodeling and the recruitment of non-homologous end joining factors. Oncogene 30 , 2135–2146 (2011).

Qi, W. et al. BRG1 promotes the repair of DNA double-strand breaks by facilitating the replacement of RPA with RAD51. J. Cell Sci. 128 , 317–330 (2015).

Chen, Y. et al. A PARP1-BRG1-SIRT1 axis promotes HR repair by reducing nucleosome density at DNA damage sites. Nucleic Acids Res. 47 , 8563–8580 (2019).

Brownlee, P. M., Meisenberg, C. & Downs, J. A. The SWI/SNF chromatin remodelling complex: its role in maintaining genome stability and preventing tumourigenesis. DNA Repair 32 , 127–133 (2015).

Watanabe, R. et al. SWI/SNF factors required for cellular resistance to DNA damage include ARID1A and ARID1B and show interdependent protein stability. Cancer Res. 74 , 2465–2475 (2014).

Zhao, B. et al. ARID1A promotes genomic stability through protecting telomere cohesion. Nat. Commun. 10 , 4067 (2019).

Kakarougkas, A. et al. Requirement for PBAF in transcriptional repression and repair at DNA breaks in actively transcribed regions of chromatin. Mol. Cell 55 , 723–732 (2014).

Ahel, D. et al. Poly(ADP-ribose)-dependent regulation of DNA repair by the chromatin remodeling enzyme ALC1. Science 325 , 1240–1243 (2009).

Verma, P. et al. ALC1 links chromatin accessibility to PARP inhibitor response in homologous recombination-deficient cells. Nat. Cell Biol. 23 , 160–171 (2021).

Hnisz, D. et al. Activation of proto-oncogenes by disruption of chromosome neighborhoods. Science 351 , 1454–1458 (2016).

Flavahan, W. A. et al. Insulator dysfunction and oncogene activation in IDH mutant gliomas. Nature 529 , 110–114 (2016).

Ooi, W. F. et al. Integrated paired-end enhancer profiling and whole-genome sequencing reveals recurrent CCNE1 and IGF2 enhancer hijacking in primary gastric adenocarcinoma. Gut 69 , 1039–1052 (2020).

Cho, S. W. et al. Promoter of lncRNA gene PVT1 is a tumorsuppressor DNA boundary element. Cell 173 , 1398–1412.e1322 (2018).

Rubio-Perez, C. et al. In silico prescription of anticancer drugs to cohorts of 28 tumor types reveals targeting opportunities. Cancer Cell 27 , 382–396 (2015).

Marshall, A. D. et al. CTCF genetic alterations in endometrial carcinoma are pro-tumorigenic. Oncogene 36 , 4100–4110 (2017).

Gonzalez-Perez, A. et al. IntOGen-mutations identifies cancer drivers across tumor types. Nat. Methods 10 , 1081–1082 (2013).

Damaschke, N. A. et al. CTCF loss mediates unique DNA hypermethylation landscapes in human cancers. Clin. Epigenet. 12 , 80 (2020).

Kemp, C. J. et al. CTCF haploinsufficiency destabilizes DNA methylation and predisposes to cancer. Cell Rep. 7 , 1020–1029 (2014).

Uhlen, M. et al. A pathology atlas of the human cancer transcriptome. Science 357 , eaan2507 (2017).

Martin, T. D. et al. The adaptive immune system is a major driver of selection for tumor suppressor gene inactivation. Science 373 , 1327–1335 (2021).

Oreskovic, E. et al. Genetic analysis of cancer drivers reveals cohesin and CTCF as suppressors of PD-L1. Proc. Natl Acad. Sci. USA 119 , e2120540119 (2022).

Sun, L. et al. Decreased expression of acetyl-CoA synthase 2 promotes metastasis and predicts poor prognosis in hepatocellular carcinoma. Cancer Sci. 108 , 1338–1346 (2017).

Liang, Y. et al. Cadmium promotes breast cancer cell proliferation, migration and invasion by inhibiting ACSS2/ATG5-mediated autophagy. Environ. Pollut. 273 , 116504 (2021).

Horibata, Y., Ando, H., Itoh, M. & Sugimoto, H. Enzymatic and transcriptional regulation of the cytoplasmic acetyl-CoA hydrolase ACOT12. J. Lipid Res. 54 , 2049–2059 (2013).

Lu, M. et al. ACOT12-dependent alteration of Acetyl-CoA drives hepatocellular carcinoma metastasis by epigenetic induction of epithelial-mesenchymal transition. Cell Metab. 29 , 886–900.e885 (2019).

Zhou, X. et al. ACOT12-mediated acetyl-CoA hydrolysis suppresses intrahepatic cholangiocarcinoma metastasis by inhibiting epithelial-mesenchymal transition. J. Cancer 13 , 1734–1744 (2022).

Shen, Y., Wei, W. & Zhou, D. X. Histone acetylation enzymes coordinate metabolism and gene expression. Trends Plant Sci. 20 , 614–621 (2015).

Haque, M. E. et al. The GCN5: its biological functions and therapeutic potentials. Clin. Sci. 135 , 231–257 (2021).

Wang, X. et al. Targeting the CK1α/CBX4 axis for metastasis in osteosarcoma. Nat. Commun. 11 , 1141 (2020).

Liu, J. et al. Both HDAC5 and HDAC6 are required for the proliferation and metastasis of melanoma cells. J. Transl. Med 14 , 7 (2016).

Cheng, C. et al. HDAC4 promotes nasopharyngeal carcinoma progression and serves as a therapeutic target. Cell Death Dis. 12 , 137 (2021).

Tang, X. et al. HDAC8 cooperates with SMAD3/4 complex to suppress SIRT7 and promote cell survival and migration. Nucleic Acids Res. 48 , 2912–2923 (2020).

Ding, S. et al. DNTTIP1 promotes nasopharyngeal carcinoma metastasis via recruiting HDAC1 to DUSP2 promoter and activating ERK signaling pathway. EBioMedicine 81 , 104100 (2022).

Wen, Y. et al. Histone deacetylase (HDAC) 11 inhibits matrix metalloproteinase (MMP) 3 expression to suppress colorectal cancer metastasis. J. Cancer 13 , 1923–1932 (2022).

Leslie, P. L. et al. Histone deacetylase 11 inhibition promotes breast cancer metastasis from lymph nodes. Nat. Commun. 10 , 4192 (2019).

Bayik, D. & Lathia, J. D. Cancer stem cell-immune cell crosstalk in tumour progression. Nat. Rev. Cancer 21 , 526–536 (2021).

Hsu, J. M. et al. STT3-dependent PD-L1 accumulation on cancer stem cells promotes immune evasion. Nat. Commun. 9 , 1908 (2018).

Meder, L. et al. Combined VEGF and PD-L1 blockade displays synergistic treatment effects in an autochthonous mouse model of small cell lung cancer. Cancer Res. 78 , 4270–4281 (2018).

Shirahata, A. & Hibi, K. Serum vimentin methylation as a potential marker for colorectal cancer. Anticancer Res. 34 , 4121–4125 (2014).

Welte, T. et al. Oncogenic mTOR signalling recruits myeloid-derived suppressor cells to promote tumour initiation. Nat. Cell Biol. 18 , 632–644 (2016).

Ai, L. et al. Myeloid-derived suppressor cells endow stem-like qualities to multiple myeloma cells by inducing piRNA-823 expression and DNMT3B activation. Mol. Cancer 18 , 88 (2019).

Lee, C. R. et al. Myeloid-derived suppressor cells are controlled by regulatory T cells via TGF-β during murine colitis. Cell Rep. 17 , 3219–3232 (2016).

Liu, S. et al. Regulatory T cells promote glioma cell stemness through TGF-β-NF-κB-IL6-STAT3 signaling. Cancer Immunol. Immunother. 70 , 2601–2616 (2021).

Xu, Y. et al. Sox2 communicates with Tregs through CCL1 to promote the stemness property of breast cancer cells. Stem Cells 35 , 2351–2365 (2017).

Rezalotfi, A., Ahmadian, E., Aazami, H., Solgi, G. & Ebrahimi, M. Gastric cancer stem cells effect on Th17/Treg balance; a bench to beside perspective. Front Oncol. 9 , 226 (2019).

Oh, E., Hong, J. & Yun, C. O. Regulatory T cells induce metastasis by increasing Tgf-β and enhancing the epithelial–mesenchymal transition. Cells 8 , 1387(2019).

Beck, B. et al. A vascular niche and a VEGF-Nrp1 loop regulate the initiation and stemness of skin tumours. Nature 478 , 399–403 (2011).

Castriconi, R. et al. NK cells recognize and kill human glioblastoma cells with stem cell-like properties. J. Immunol. 182 , 3530–3539 (2009).

Tallerico, R. et al. Human NK cells selective targeting of colon cancer-initiating cells: a role for natural cytotoxicity receptors and MHC class I molecules. J. Immunol. 190 , 2381–2390 (2013).

Jewett, A. et al. Natural killer cells preferentially target cancer stem cells; role of monocytes in protection against NK cell-mediated lysis of cancer stem cells. Curr. Drug Deliv. 9 , 5–16 (2012).

Akhter, M. Z. et al. Aggressive serous epithelial ovarian cancer is potentially propagated by EpCAM(+)CD45(+) phenotype. Oncogene 37 , 2089–2103 (2018).

Zhong, Y. et al. Spheres derived from the human SK-RC-42 renal cell carcinoma cell line are enriched in cancer stem cells. Cancer Lett. 299 , 150–160 (2010).

Malladi, S. et al. Metastatic latency and immune evasion through autocrine inhibition of WNT. Cell 165 , 45–60 (2016).

Sainz, B. Jr, Martín, B., Tatari, M., Heeschen, C. & Guerra, S. ISG15 is a critical microenvironmental factor for pancreatic cancer stem cells. Cancer Res. 74 , 7309–7320 (2014).

Martin-Hijano, L. & Sainz, B. Jr The interactions between cancer stem cells and the innate interferon signaling pathway. Front. Immunol. 11 , 526 (2020).

Zhu, Y. et al. Influence of interferon-α on the expression of the cancer stem cell markers in pancreatic carcinoma cells. Exp. Cell Res. 324 , 146–156 (2014).

Wilson, E. B. et al. Blockade of chronic type I interferon signaling to control persistent LCMV infection. Science 340 , 202–207 (2013).

Teijaro, J. R. et al. Persistent LCMV infection is controlled by blockade of type I interferon signaling. Science 340 , 207–211 (2013).

Musella, M. et al. Type I IFNs promote cancer cell stemness by triggering the epigenetic regulator KDM1B. Nat. Immunol. 23 , 1379–1392 (2022).

de Almeida Nagata, D. E. et al. Regulation of tumor-associated myeloid cell activity by CBP/EP300 bromodomain modulation of H3K27 acetylation. Cell Rep. 27 , 269–281.e264 (2019).

Szade, K. et al. Where hematopoietic stem cells live: the bone marrow niche. Antioxid. Redox Signal 29 , 191–204 (2018).

Du, L. et al. Loss of sirt4 promotes the self-renewal of breast cancer stem cells. Theranostics 10 , 9458–9476 (2020).

Ma, Y. et al. SIRT5-mediated SDHA desuccinylation promotes clear cell renal cell carcinoma tumorigenesis. Free Radic. Biol. Med. 134 , 458–467 (2019).

Song, M. et al. Low-Dose IFNγ Induces Tumor Cell Stemness in Tumor Microenvironment of Non-Small Cell Lung Cancer. Cancer Res. 79 , 3737–3748 (2019).

Eisenhauer, E. A. et al. New response evaluation criteria in solid tumours: revised RECIST guideline (version 1.1). Eur. J. Cancer 45 , 228–247 (2009).

Anderson, R. L. et al. A framework for the development of effective anti-metastatic agents. Nat. Rev. Clin. Oncol. 16 , 185–204 (2019).

Zhao, Z. M. et al. Early and multiple origins of metastatic lineages within primary tumors. Proc. Natl Acad. Sci. USA 113 , 2140–2145 (2016).

Hosseini, H. et al. Early dissemination seeds metastasis in breast cancer. Nature 540 , 552–558 (2016).

Harper, K. L. et al. Mechanism of early dissemination and metastasis in Her2(+) mammary cancer. Nature 540 , 588–592 (2016).

Hüsemann, Y. et al. Systemic spread is an early step in breast cancer. Cancer Cell 13 , 58–68 (2008).

Parsons, S., Maldonado, E. B. & Prasad, V. Comparison of drugs used for adjuvant and metastatic therapy of colon, breast, and non-small cell lung cancers. JAMA Netw. Open 3 , e202488 (2020).

Groppa, E. et al. EphrinB2/EphB4 signaling regulates non-sprouting angiogenesis by VEGF. EMBO Rep. 19 , e45054 (2018).

Scheidmann, M. C. et al. An in vivo CRISPR screen identifies stepwise genetic dependencies of metastatic progression. Cancer Res 82 , 681–694 (2022).

Gligorijevic, B. et al. N-WASP-mediated invadopodium formation is involved in intravasation and lung metastasis of mammary tumors. J. Cell Sci. 125 , 724–734 (2012).

Xu, X. R., Yousef, G. M. & Ni, H. Cancer and platelet crosstalk: opportunities and challenges for aspirin and other antiplatelet agents. Blood 131 , 1777–1789 (2018).

Leong, H. S. et al. Invadopodia are required for cancer cell extravasation and are a therapeutic target for metastasis. Cell Rep. 8 , 1558–1570 (2014).

Choi, J. W. et al. Urokinase exerts antimetastatic effects by dissociating clusters of circulating tumor cells. Cancer Res. 75 , 4474–4482 (2015).

Mitchell, M. J., Wayne, E., Rana, K., Schaffer, C. B. & King, M. R. TRAIL-coated leukocytes that kill cancer cells in the circulation. Proc. Natl Acad. Sci. USA 111 , 930–935 (2014).

Cooke, N. M., Spillane, C. D., Sheils, O., O’Leary, J. & Kenny, D. Aspirin and P2Y12 inhibition attenuate platelet-induced ovarian cancer cell invasion. BMC Cancer 15 , 627 (2015).

Bambace, N. M. & Holmes, C. E. The platelet contribution to cancer progression. J. Thromb. Haemost. 9 , 237–249 (2011).

Elia, I., Doglioni, G. & Fendt, S. M. Metabolic hallmarks of metastasis formation. Trends Cell Biol. 28 , 673–684 (2018).

Mazel, M. et al. Frequent expression of PD-L1 on circulating breast cancer cells. Mol. Oncol. 9 , 1773–1782 (2015).

Elia, I. et al. Breast cancer cells rely on environmental pyruvate to shape the metastatic niche. Nature 568 , 117–121 (2019).

Elia, I. et al. Proline metabolism supports metastasis formation and could be inhibited to selectively target metastasizing cancer cells. Nat. Commun. 8 , 15267 (2017).

Chen, H. N. et al. EpCAM signaling promotes tumor progression and protein stability of PD-L1 through the EGFR pathway. Cancer Res. 80 , 5035–5050 (2020).

Müller, P. et al. Trastuzumab emtansine (T-DM1) renders HER2+ breast cancer highly susceptible to CTLA-4/PD-1 blockade. Sci. Transl. Med. 7 , 315ra188 (2015).

Dombroski, J. A., Jyotsana, N., Crews, D. W., Zhang, Z. & King, M. R. Fabrication and characterization of tumor nano-lysate as a preventative vaccine for breast cancer. Langmuir 36 , 6531–6539 (2020).

Parkins, K. M. et al. Engineering circulating tumor cells as novel cancer theranostics. Theranostics 10 , 7925–7937 (2020).

Scher, H. I. et al. Circulating tumour cells as prognostic markers in progressive, castration-resistant prostate cancer: a reanalysis of IMMC38 trial data. Lancet Oncol. 10 , 233–239 (2009).

Aggarwal, C. et al. Relationship among circulating tumor cells, CEA and overall survival in patients with metastatic colorectal cancer. Ann. Oncol. 24 , 420–428 (2013).

Sparano, J. et al. Association of circulating tumor cells with late recurrence of estrogen receptor-positive breast cancer: a secondary analysis of a randomized clinical trial. JAMA Oncol. 4 , 1700–1706 (2018).

Rack, B. et al. Circulating tumor cells predict survival in early average-to-high risk breast cancer patients. J. Natl. Cancer Inst. 106 , dju066(2014).

Cristofanilli, M. et al. Circulating tumor cells, disease progression, and survival in metastatic breast cancer. N. Engl. J. Med. 351 , 781–791 (2004).

Cohen, S. J. et al. Isolation and characterization of circulating tumor cells in patients with metastatic colorectal cancer. Clin. Colorectal Cancer 6 , 125–132 (2006).

de Bono, J. S. et al. Circulating tumor cells predict survival benefit from treatment in metastatic castration-resistant prostate cancer. Clin. Cancer Res. 14 , 6302–6309 (2008).

Danila, D. C. et al. Circulating tumor cell number and prognosis in progressive castration-resistant prostate cancer. Clin. Cancer Res. 13 , 7053–7058 (2007).

Wallwiener, M. et al. Serial enumeration of circulating tumor cells predicts treatment response and prognosis in metastatic breast cancer: a prospective study in 393 patients. BMC Cancer 14 , 512 (2014).

Smerage, J. B. et al. Circulating tumor cells and response to chemotherapy in metastatic breast cancer: SWOG S0500. J. Clin. Oncol. 32 , 3483–3489 (2014).

Liu, M. C. et al. First-line doublet chemotherapy for metastatic triple-negative breast cancer: circulating tumor cell analysis of the tnAcity trial. Cancer Manag. Res. 11 , 10427–10433 (2019).

Liu, M. C. et al. Circulating tumor cells: a useful predictor of treatment efficacy in metastatic breast cancer. J. Clin. Oncol. 27 , 5153–5159 (2009).

Thery, L. et al. Circulating tumor cells in early breast cancer. JNCI Cancer Spectr. 3 , pkz026 (2019).

Bidard, F. C. et al. Efficacy of circulating tumor cell count-driven vs clinician-driven first-line therapy choice in hormone receptor-positive, ERBB2-negative metastatic breast cancer: the STIC CTC randomized clinical trial. JAMA Oncol. 7 , 34–41 (2021).

Cabel, L. et al. Clinical utility of circulating tumour cell-based monitoring of late-line chemotherapy for metastatic breast cancer: the randomised CirCe01 trial. Br. J. Cancer 124 , 1207–1213 (2021).

Georgoulias, V. et al. Trastuzumab decreases the incidence of clinical relapses in patients with early breast cancer presenting chemotherapy-resistant CK-19mRNA-positive circulating tumor cells: results of a randomized phase II study. Ann. Oncol. 23 , 1744–1750 (2012).

Jacot, W. et al. Actionability of HER2-amplified circulating tumor cells in HER2-negative metastatic breast cancer: the CirCe T-DM1 trial. Breast Cancer Res. 21 , 121 (2019).

Pestrin, M. et al. Final results of a multicenter phase II clinical trial evaluating the activity of single-agent lapatinib in patients with HER2-negative metastatic breast cancer and HER2-positive circulating tumor cells. A proof-of-concept study. Breast Cancer Res. Treat. 134 , 283–289 (2012).

Armstrong, A. J. et al. Prospective multicenter validation of androgen receptor splice variant 7 and hormone therapy resistance in high-risk castration-resistant prostate cancer: the PROPHECY study. J. Clin. Oncol. 37 , 1120–1129 (2019).

Antonarakis, E. S. et al. AR-V7 and resistance to enzalutamide and abiraterone in prostate cancer. N. Engl. J. Med. 371 , 1028–1038 (2014).

Parker, C. et al. Prostate cancer: ESMO Clinical Practice Guidelines for diagnosis, treatment and follow-up. Ann. Oncol. 31 , 1119–1134 (2020).

Belderbos, B. P. S. et al. Associations between AR-V7 status in circulating tumour cells, circulating tumour cell count and survival in men with metastatic castration-resistant prostate cancer. Eur. J. Cancer 121 , 48–54 (2019).

Isebia, K. T. et al. CABA-V7: a prospective biomarker selected trial of cabazitaxel treatment in AR-V7 positive prostate cancer patients. Eur. J. Cancer 177 , 33–44 (2022).

Khoo, B. L. et al. Liquid biopsy and therapeutic response: circulating tumor cell cultures for evaluation of anticancer treatment. Sci. Adv. 2 , e1600274 (2016).

Khoo, B. L. et al. Short-term expansion of breast circulating cancer cells predicts response to anti-cancer therapy. Oncotarget 6 , 15578–15593 (2015).

Madhavan, D. et al. Circulating miRNAs as surrogate markers for circulating tumor cells and prognostic markers in metastatic breast cancer. Clin. Cancer Res. 18 , 5972–5982 (2012).

Cabel, L. et al. Circulating tumor cells: clinical validity and utility. Int. J. Clin. Oncol. 22 , 421–430 (2017).

Widschwendter, M. et al. The potential of circulating tumor DNA methylation analysis for the early detection and management of ovarian cancer. Genome Med. 9 , 116 (2017).

Kneip, C. et al. SHOX2 DNA methylation is a biomarker for the diagnosis of lung cancer in plasma. J. Thorac. Oncol. 6 , 1632–1638 (2011).

Hulbert, A. et al. Early detection of lung cancer using DNA promoter hypermethylation in plasma and sputum. Clin. Cancer Res. 23 , 1998–2005 (2017).

Mazor, T., Pankov, A., Song, J. S. & Costello, J. F. Intratumoral Heterogeneity of the Epigenome. Cancer Cell 29 , 440–451 (2016).

Suvilesh, K. N. et al. Tumorigenic circulating tumor cells from xenograft mouse models of non-metastatic NSCLC patients reveal distinct single cell heterogeneity and drug responses. Mol. Cancer 21 , 73 (2022).

Han, H. J. et al. Fibronectin regulates anoikis resistance via cell aggregate formation. Cancer Lett. 508 , 59–72 (2021).

Wang, C. et al. HOTAIR lncRNA SNPs rs920778 and rs1899663 are associated with smoking, male gender, and squamous cell carcinoma in a Chinese lung cancer population. Acta Pharm. Sin. 39 , 1797–1803 (2018).

Onstenk, W. et al. Molecular characteristics of circulating tumor cells resemble the liver metastasis more closely than the primary tumor in metastatic colorectal cancer. Oncotarget 7 , 59058–59069 (2016).

Barros-Filho, M. C., Marchi, F. A., Pinto, C. A., Rogatto, S. R. & Kowalski, L. P. High diagnostic accuracy based on CLDN10, HMGA2, and LAMB3 transcripts in papillary thyroid carcinoma. J. Clin. Endocrinol. Metab. 100 , E890–E899 (2015).

Vasantharajan, S. S. et al. The epigenetic landscape of circulating tumour cells. Biochim. Biophys. Acta Rev. Cancer 1875 , 188514 (2021).

Dong, S. et al. Hypermethylated PCDHGB7 as a universal cancer only marker and its application in early cervical cancer screening. Clin. Transl. Med. 11 , e457 (2021).

Dubois, F., Bergot, E., Zalcman, G. & Levallet, G. RASSF1A, puppeteer of cellular homeostasis, fights tumorigenesis, and metastasis-an updated review. Cell Death Dis. 10 , 928 (2019).

Hiddinga, B. I., Pauwels, P., Janssens, A. & van Meerbeeck, J. P. O. 6)-Methylguanine-DNA methyltransferase (MGMT): a drugable target in lung cancer? Lung Cancer 107 , 91–99 (2017).

Punnoose, E. A. et al. PTEN loss in circulating tumour cells correlates with PTEN loss in fresh tumour tissue from castration-resistant prostate cancer patients. Br. J. Cancer 113 , 1225–1233 (2015).

Keller, L. & Pantel, K. Unravelling tumour heterogeneity by single-cell profiling of circulating tumour cells. Nat. Rev. Cancer 19 , 553–567 (2019).

Frenel, J. S. et al. Efficacy of subsequent chemotherapy for patients with BRCA1/2-mutated recurrent epithelial ovarian cancer progressing on olaparib versus placebo maintenance: post-hoc analyses of the SOLO2/ENGOT Ov-21 trial. Ann. Oncol. 33 , 1021–1028 (2022).

Koyanagi, K. et al. Association of circulating tumor cells with serum tumor-related methylated DNA in peripheral blood of melanoma patients. Cancer Res. 66 , 6111–6117 (2006).

Warton, K. & Samimi, G. Methylation of cell-free circulating DNA in the diagnosis of cancer. Front. Mol. Biosci. 2 , 13 (2015).

Church, T. R. et al. Prospective evaluation of methylated SEPT9 in plasma for detection of asymptomatic colorectal cancer. Gut 63 , 317–325 (2014).

Osaka, M., Rowley, J. D. & Zeleznik-Le, N. J. MSF (MLL septin-like fusion), a fusion partner gene of MLL, in a therapy-related acute myeloid leukemia with a t(11;17)(q23;q25). Proc. Natl Acad. Sci. USA 96 , 6428–6433 (1999).

Sun, J., Zheng, M. Y., Li, Y. W. & Zhang, S. W. Structure and function of Septin 9 and its role in human malignant tumors. World J. Gastrointest. Oncol. 12 , 619–631 (2020).

Song, L. & Li, Y. SEPT9: a specific circulating biomarker for colorectal cancer. Adv. Clin. Chem. 72 , 171–204 (2015).

Cai, G. et al. A multilocus blood-based assay targeting circulating tumor DNA methylation enables early detection and early relapse prediction of colorectal cancer. Gastroenterology 161 , 2053–2056.e2052 (2021).

Roperch, J. P. et al. Aberrant methylation of NPY, PENK, and WIF1 as a promising marker for blood-based diagnosis of colorectal cancer. BMC Cancer 13 , 566 (2013).

Park, J. K. et al. The role of quantitative NPTX2 hypermethylation as a novel serum diagnostic marker in pancreatic cancer. Pancreas 41 , 95–101 (2012).

Matsui, S. et al. Methylation of the SEPT9_v2 promoter as a novel marker for the detection of circulating tumor DNA in breast cancer patients. Oncol. Rep. 36 , 2225–2235 (2016).

Moore, S. R. et al. Distinctions between sex and time in patterns of DNA methylation across puberty. BMC Genom. 21 , 389 (2020).

Bell, C. G. et al. DNA methylation aging clocks: challenges and recommendations. Genome Biol. 20 , 249 (2019).

Mazzone, P. J. et al. Screening for lung cancer: CHEST guideline and expert panel report. Chest 153 , 954–985 (2018).

Cho, N. et al. Breast cancer screening with mammography plus ultrasonography or magnetic resonance imaging in women 50 years or younger at diagnosis and treated with breast conservation therapy. JAMA Oncol. 3 , 1495–1502 (2017).

Gupta, S. Screening for colorectal cancer. Hematol. Oncol. Clin. North Am. 36 , 393–414 (2022).

Yi, J. M. et al. Novel methylation biomarker panel for the early detection of pancreatic cancer. Clin. Cancer Res. 19 , 6544–6555 (2013).

Singh, N. et al. Clinical significance of promoter methylation status of tumor suppressor genes in circulating DNA of pancreatic cancer patients. J. Cancer Res. Clin. Oncol. 146 , 897–907 (2020).

Shinjo, K. et al. A novel sensitive detection method for DNA methylation in circulating free DNA of pancreatic cancer. PLoS One 15 , e0233782 (2020).

Schott, S. et al. HYAL2 methylation in peripheral blood as a potential marker for the detection of pancreatic cancer: a case control study. Oncotarget 8 , 67614–67625 (2017).

Fujimoto, Y. et al. Combination of CA19-9 and blood free-circulating methylated RUNX3 may be useful to diagnose stage I pancreatic cancer. Oncology 99 , 234–239 (2021).

Ying, L. et al. Methylation-based cell-free DNA signature for early detection of pancreatic cancer. Pancreas 50 , 1267–1273 (2021).

Eissa, M. A. L. et al. Promoter methylation of ADAMTS1 and BNC1 as potential biomarkers for early detection of pancreatic cancer in blood. Clin. Epigenet. 11 , 59 (2019).

Kelwick, R., Desanlis, I., Wheeler, G. N. & Edwards, D. R. The ADAMTS (A Disintegrin and Metalloproteinase with Thrombospondin motifs) family. Genome Biol. 16 , 113 (2015).

Kota, J., Hancock, J., Kwon, J. & Korc, M. Pancreatic cancer: stroma and its current and emerging targeted therapies. Cancer Lett. 391 , 38–49 (2017).

Mastoraki, S. et al. KMT2C promoter methylation in plasma-circulating tumor DNA is a prognostic biomarker in non-small cell lung cancer. Mol. Oncol. 15 , 2412–2422 (2021).

Liu, Y. et al. Methylation of serum SST gene is an independent prognostic marker in colorectal cancer. Am. J. Cancer Res. 6 , 2098–2108 (2016).

Fujita, N. et al. Methylated DNA and total DNA in serum detected by one-step methylation-specific PCR is predictive of poor prognosis for breast cancer patients. Oncology 83 , 273–282 (2012).

Tserpeli, V. et al. Prognostic significance of SLFN11 methylation in plasma cell-free DNA in advanced high-grade serous ovarian cancer. Cancers 14 (2021).

Matthaios, D. et al. Methylation status of the APC and RASSF1A promoter in cell-free circulating DNA and its prognostic role in patients with colorectal cancer. Oncol. Lett. 12 , 748–756 (2016).

Karamitrousis, E. I. et al. Prognostic role of RASSF1A, SOX17 and Wif-1 promoter methylation status in cell-free DNA of advanced gastric cancer patients. Technol. Cancer Res. Treat. 20 , 1533033820973279 (2021).

Deng, Q. K., Lei, Y. G., Lin, Y. L., Ma, J. G. & Li, W. P. Prognostic value of protocadherin10 (PCDH10) methylation in serum of prostate cancer patients. Med. Sci. Monit. 22 , 516–521 (2016).

Mansouri, A. et al. MGMT promoter methylation status testing to guide therapy for glioblastoma: refining the approach based on emerging evidence and current challenges. Neuro Oncol. 21 , 167–178 (2019).

Fuster-Garcia, E. et al. MGMT methylation may benefit overall survival in patients with moderately vascularized glioblastomas. Eur. Radio. 31 , 1738–1747 (2021).

Luo, G. et al. Roles of CA19-9 in pancreatic cancer: biomarker, predictor and promoter. Biochim. Biophys. Acta Rev. Cancer 1875 , 188409 (2021).

Xu, X., Xiao, Y., Hong, B., Hao, B. & Qian, Y. Combined detection of CA19-9 and B7-H4 in the diagnosis and prognosis of pancreatic cancer. Cancer Biomark. 25 , 251–257 (2019).

Rieser, C. J. et al. CA19-9 on postoperative surveillance in pancreatic ductal adenocarcinoma: predicting recurrence and changing prognosis over time. Ann. Surg. Oncol. 25 , 3483–3491 (2018).

Mujica, V. R., Barkin, J. S. & Go, V. L. Acute pancreatitis secondary to pancreatic carcinoma. Study Group Participants. Pancreas 21 , 329–332 (2000).

Luo, G. et al. New observations on the utility of CA19-9 as a biomarker in Lewis negative patients with pancreatic cancer. Pancreatology 18 , 971–976 (2018).

Stastny, I. et al. Aberrantly methylated cfDNA in body fluids as a promising diagnostic tool for early detection of breast cancer. Clin. Breast Cancer 20 , e711–e722 (2020).

Ignatiadis, M., Sledge, G. W. & Jeffrey, S. S. Liquid biopsy enters the clinic—implementation issues and future challenges. Nat. Rev. Clin. Oncol. 18 , 297–312 (2021).

Nakayama, G. et al. p16INK4a methylation in serum as a follow-up marker for recurrence of colorectal cancer. Anticancer Res. 31 , 1643–1646 (2011).

Jin, S. et al. Efficient detection and post-surgical monitoring of colon cancer with a multi-marker DNA methylation liquid biopsy. Proc. Natl Acad. Sci. USA 118 , e2017421118 (2021).

Hutóczki, G., Virga, J., Birkó, Z. & Klekner, A. Novel concepts of glioblastoma therapy concerning its heterogeneity. Int. J. Mol. Sci. 22 , 10005 (2021).

Nunes, S. P., Henrique, R., Jerónimo, C. & Paramio, J. M. DNA methylation as a therapeutic target for bladder cancer. Cells 9 , 1850 (2020).

Wong, K. K. DNMT1 as a therapeutic target in pancreatic cancer: mechanisms and clinical implications. Cell Oncol. 43 , 779–792 (2020).

Wong, K. K. DNMT1: a key drug target in triple-negative breast cancer. Semin. Cancer Biol. 72 , 198–213 (2021).

Morin, R. D. et al. Somatic mutations altering EZH2 (Tyr641) in follicular and diffuse large B-cell lymphomas of germinal-center origin. Nat. Genet. 42 , 181–185 (2010).

Morschhauser, F. et al. Tazemetostat for patients with relapsed or refractory follicular lymphoma: an open-label, single-arm, multicentre, phase 2 trial. Lancet Oncol. 21 , 1433–1442 (2020).

Honma, D. et al. Novel orally bioavailable EZH1/2 dual inhibitors with greater antitumor efficacy than an EZH2 selective inhibitor. Cancer Sci. 108 , 2069–2078 (2017).

Izutsu, K. et al. An open-label, single-arm phase 2 trial of valemetostat for relapsed or refractory adult T-cell leukemia/lymphoma. Blood 141 , 1159–1168 (2023).

Okada, Y. et al. hDOT1L links histone methylation to leukemogenesis. Cell 121 , 167–178 (2005).

Lonetti, A. et al. Inhibition of methyltransferase DOT1L sensitizes to sorafenib treatment AML cells irrespective of MLL-rearrangements: a novel therapeutic strategy for pediatric AML. Cancers 12 , 1972 (2020).

Shen, D. D. et al. LSD1 deletion decreases exosomal PD-L1 and restores T-cell response in gastric cancer. Mol. Cancer 21 , 75 (2022).

Fang, Y., Liao, G. & Yu, B. LSD1/KDM1A inhibitors in clinical trials: advances and prospects. J. Hematol. Oncol. 12 , 129 (2019).

Hollebecque, A. et al. Phase I study of lysine-specific demethylase 1 inhibitor, CC-90011, in patients with advanced solid tumors and relapsed/refractory non-hodgkin lymphoma. Clin. Cancer Res. 27 , 438–446 (2021).

Perillo, B., Tramontano, A., Pezone, A. & Migliaccio, A. LSD1: more than demethylation of histone lysine residues. Exp. Mol. Med. 52 , 1936–1947 (2020).

Wang, J. et al. The lysine demethylase LSD1 (KDM1) is required for maintenance of global DNA methylation. Nat. Genet. 41 , 125–129 (2009).

Xu, S., Wang, X., Yang, Y., Li, Y. & Wu, S. LSD1 silencing contributes to enhanced efficacy of anti-CD47/PD-L1 immunotherapy in cervical cancer. Cell Death Dis. 12 , 282 (2021).

Qin, Y. et al. Inhibition of histone lysine-specific demethylase 1 elicits breast tumor immunity and enhances antitumor efficacy of immune checkpoint blockade. Oncogene 38 , 390–405 (2019).

Park, J. W. & Han, J. W. Targeting epigenetics for cancer therapy. Arch. Pharm. Res. 42 , 159–170 (2019).

Mann, B. S., Johnson, J. R., Cohen, M. H., Justice, R. & Pazdur, R. FDA approval summary: vorinostat for treatment of advanced primary cutaneous T-cell lymphoma. Oncologist 12 , 1247–1252 (2007).

Terranova-Barberio, M. et al. HDAC inhibition potentiates immunotherapy in triple-negative breast cancer. Oncotarget 8 , 114156–114172 (2017).

Thomas, S., Thurn, K. T., Biçaku, E., Marchion, D. C. & Münster, P. N. Addition of a histone deacetylase inhibitor redirects tamoxifen-treated breast cancer cells into apoptosis, which is opposed by the induction of autophagy. Breast Cancer Res. Treat. 130 , 437–447 (2011).

O’Connor, O. A. et al. Belinostat in patients with relapsed or refractory peripheral T-cell lymphoma: results of the pivotal phase II BELIEF (CLN-19) study. J. Clin. Oncol. 33 , 2492–2499 (2015).

Juergens, R. A. et al. Combination epigenetic therapy has efficacy in patients with refractory advanced non-small cell lung cancer. Cancer Discov. 1 , 598–607 (2011).

Connolly, R. M. et al. Combination epigenetic therapy in advanced breast cancer with 5-azacitidine and entinostat: a phase II national cancer institute/stand Up to cancer study. Clin. Cancer Res. 23 , 2691–2701 (2017).

Faivre, E. J. et al. Selective inhibition of the BD2 bromodomain of BET proteins in prostate cancer. Nature 578 , 306–310 (2020).

Feng, M., Xu, H., Zhou, W. & Pan, Y. The BRD4 inhibitor JQ1 augments the antitumor efficacy of abemaciclib in preclinical models of gastric carcinoma. J. Exp. Clin. Cancer Res. 42 , 44 (2023).

Pang, Y. et al. The BRD4 inhibitor JQ1 suppresses tumor growth by reducing c-Myc expression in endometrial cancer. J. Transl. Med. 20 , 336 (2022).

Stuhlmiller, T. J. et al. Inhibition of lapatinib-induced kinome reprogramming in ERBB2-positive breast cancer by targeting BET family bromodomains. Cell Rep. 11 , 390–404 (2015).

Leonard, B. et al. BET inhibition overcomes receptor tyrosine kinase-mediated cetuximab resistance in HNSCC. Cancer Res. 78 , 4331–4343 (2018).

Sun, C. et al. BRD4 inhibition is synthetic lethal with PARP inhibitors through the induction of homologous recombination deficiency. Cancer Cell 33 , 401–416.e408 (2018).

Fiorentino, F. P. et al. BET-inhibitor I-BET762 and PARP-inhibitor talazoparib synergy in small cell lung cancer cells. Int. J. Mol. Sci. 21 , 9595 (2020).

Yang, L. et al. Repression of BET activity sensitizes homologous recombination-proficient cancers to PARP inhibition. Sci. Transl. Med. 9 , eaal1645 (2017).

Karakashev, S. et al. BET bromodomain inhibition synergizes with PARP inhibitor in epithelial ovarian cancer. Cell Rep. 21 , 3398–3405 (2017).

Powell, A. A. et al. Single cell profiling of circulating tumor cells: transcriptional heterogeneity and diversity from breast cancer cell lines. PLoS One 7 , e33788 (2012).

Steinert, G. et al. Immune escape and survival mechanisms in circulating tumor cells of colorectal cancer. Cancer Res. 74 , 1694–1704 (2014).

Adams, D. L. et al. Sequential tracking of PD-L1 expression and RAD50 induction in circulating tumor and stromal cells of lung cancer patients undergoing radiotherapy. Clin. Cancer Res. 23 , 5948–5958 (2017).

Papadaki, M. A. et al. Clinical relevance of immune checkpoints on circulating tumor cells in breast cancer. Cancers 12 , 376 (2020).

Morales, M. et al. RARRES3 suppresses breast cancer lung metastasis by regulating adhesion and differentiation. EMBO Mol. Med. 6 , 865–881 (2014).

Hsu, T. H. et al. Involvement of RARRES3 in the regulation of Wnt proteins acylation and signaling activities in human breast cancer cells. Cell Death Differ. 22 , 801–814 (2015).

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Acknowledgements

This work was supported by the National Natural Science Foundation of China (No. 82372155), Project of Key Supported Disciplines by Shanghai Municipal Health Commission (grant number: 2023ZDFC0204), Program of Shanghai Academic Research Leader (No. 21XD1402800), and Development Fund for the Department of Anesthesiology, Shanghai Pulmonary Hospital.

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Department of Oncology, Shanghai Pulmonary Hospital, School of Medicine, Tongji University, Shanghai, China

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Shiyou Wei & Xin Lv

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Xin Lv, Xuyu Gu, and Shiyou Wei proposed the study conception. Xuyu Gu and Shiyou Wei searched the references. Xuyu Gu assisted with the chart making. Xuyu Gu and Shiyou Wei drafted the manuscript. All authors read and approved the final manuscript.

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Gu, X., Wei, S. & Lv, X. Circulating tumor cells: from new biological insights to clinical practice. Sig Transduct Target Ther 9 , 226 (2024). https://doi.org/10.1038/s41392-024-01938-6

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DOI : https://doi.org/10.1038/s41392-024-01938-6

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Learn what basic research is, how it differs from applied research, and what types, methods, and examples of basic research exist. Explore the data collection and analysis methods, the research methodology, and the applications of basic research in various fields.
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Basic research is a type of research that aims to expand knowledge and understanding of a subject or phenomenon. Learn how to conduct basic research in different fields, such as education, science, psychology and health, and see examples of data collection methods.
Basic Research: What it is with examples
Basic research is a type of scientific investigation that aims to expand and generalize knowledge about a phenomenon or field. Learn the difference between basic and applied research, see some examples, and discover how to do basic research with QuestionPro.
Basic research
Basic research is a type of scientific research that aims to improve scientific theories and understanding of natural or other phenomena. It often fuels technological innovations and conservation efforts, and is funded mainly by the federal government and done mainly at universities and institutes.
Basic Research: Definition, Examples
Learn what basic research is, how it differs from applied research, and why it is important for science and society. See examples of basic research questions and methods in various fields.
Basic Research, Its Application and Benefits
Basic research is essential for solving global challenges and advancing science and society. The statement provides examples of how basic research has led to technological progress, improved health, and enhanced understanding of the natural world and human behavior.
Basic Research in Psychology: Definition and Examples
Basic research is study and research meant to increase our scientific knowledge base. Learn how basic research differs from applied research, why it is important, and see examples of basic research topics in psychology.
What is Basic Research?
Basic research or fundamental research is an essential component to building scientific knowledge. Learn the characteristics, importance, and examples of basic research in various fields such as education, psychology, and health.
Basic Research
Basic research is systematic inquiry that seeks to understand the fundamental aspects of phenomena without specific applications in mind. It produces public goods that support long-term economic growth and innovation, but it is difficult to capture and appropriate.
Types of Research Designs Compared
Learn how to choose between different types of research based on your research aims, data, sampling, timescale, and location. Find out the key differences and examples of basic research and other types of research.
Basic vs Applied Research
Basic research examples. The social construction of reality. A classic area of investigation in sociology is understanding how societies construct reality. This kind of research delves deep into the ways cultures, languages, and institutions shape our understanding of the world. It doesn't immediately aim to solve societal problems but provides ...
The Concept of Basic Research
Other examples of basic research that have led to important advances in medicine are the discovery of DNA (leading to cancer treatments) and neurotransmitters (leading to antidepressants and antiseizure medications). However, there are many other instances where basic research, some of which has been done on animals, has not yet resulted in any ...
The Concept of Basic Research
A nimal research is also important in another type of research, called basic research. Basic research experiments are performed to further scientific knowledge without an obvious or immediate benefit. The goal of basic research is to understand the function of newly discovered molecules and cells, strange phenomena, or little-understood processes.
Basic vs. Applied Research: What's the Difference?
Basic research, or fundamental research, focuses on expanding the existing knowledge of a certain subject or occurence. This type of research examines data to find the unknown and fulfill a sense of curiosity. Usually, basic research seeks to answer "how," "what" and "why" when explaining occurrences. Examples of basic research Here are some ...
Basic vs. Applied Research: Key Differences
Basic research (sometimes called fundamental or pure) advances scientific knowledge to completely understand a subject, topic, or phenomenon. It's conducted to satisfy curiosity or develop a full body of knowledge on a specific subject. Basic research is used to bring about a fundamental understanding of the world, different behaviors, and is the foundation of knowledge in the scientific ...
Understanding Basic Research vs. Applied Research
Basic research vs. applied research. Where basic research gathers information and data on a subject, applied research uses that data to look for answers to questions. Applied research takes the data obtained in basic research and applies it to answer a question and provide a possible solution. There are three types of applied research:
Basic and Applied Research
Basic research is a little less direct than applied research, so we will look at two different examples. The first basic research example is a common type: evaluation .
10 Research Question Examples to Guide your Research Project
The first question asks for a ready-made solution, and is not focused or researchable. The second question is a clearer comparative question, but note that it may not be practically feasible. For a smaller research project or thesis, it could be narrowed down further to focus on the effectiveness of drunk driving laws in just one or two countries.
What is Basic Research and Why is it Useful?
As such, a steady stream of new basic research discoveries is required to fuel new, innovative applied research. The COVID-19 mRNA vaccines (made by Moderna and Pfizer-BioNTech) are good examples of how basic research lays the foundations for innovative technologies to improve human health.
Understanding Applied and Basic Research
Other examples of basic research with practical implications include: Discovery of x-rays which led to studying bone fractures; Discovery of chlorpromazine, a drug used in the treatment of ...
Research Methods
Learn how to choose and apply research methods for collecting and analyzing data. Compare qualitative and quantitative, primary and secondary, descriptive and experimental methods with examples and pros and cons.
Basic vs Applied Research: 15 Key Differences
Theory Formulation. Basic research aims at formulating theories that explain research findings and in the process, improve a body of knowledge while applied research aims at arriving at research findings that can solve practical problems. Basic research focuses on principles and theories while applied research focuses on solutions.
Ultimate Guide to Primary Market Research: Methods, Examples, and Tips
For example, Zappos enhances customer personalization by using feedback from customer surveys and in-person interviews to understand their needs and preferences better. ... What are the two basic kinds of information gathered in primary research? The two basic kinds of information gathered in primary research are exploratory information and ...
Psychological Theories
Cognition: Mental processes involved in gaining knowledge and comprehension, including thinking, knowing, remembering, judging, and problem-solving. Psychoanalysis: A therapy developed aimed at exploring the unconscious mind to understand and treat psychological disorders. Positive Reinforcement: In behaviorism, the process of encouraging or establishing a pattern of behavior by offering a ...
Market Research Plan
2+ Market Research Plan Examples. Conducting market research will give significant benefits to your business. However, to materialize it, you may need to ensure that you build your market research plan correctly. Below is a list of the market research plan samples and templates that you can use as a guide. 1. Market Research Plan Template
Circulating tumor cells: from new biological insights to clinical
This review aims to bridge the gap between basic research and clinical application, highlighting the significance of DNA methylation in the context of cancer metastasis and offering new avenues ...

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