By Luis Enrique Cortés-Hernández & Catherine Alix-Panabières
Cancer is a progressive disease, this means that cancer cells have the capacity to replicate significantly faster than normal cells, thus all of their internal cellular mechanisms become oriented to the only selfish aim of replication and survival1. In a similar fashion to animal populations, once resources for proliferation start lacking, individuals are forced to migrate; cancer cells might migrate in search of nutrients as well2. Despite that this analogy might represent one of the reasons by which cancer cells migrate, the research in the field of circulating tumor cell (CTC’s) have shown that metastatic cellular mechanisms are variable and depends on cancer type, stage, exposition to therapeutic agents, time of clonal evolution, circadian cycle; and different mechanisms can be present at the same given time within a tumor3,4. For example, CTC’s might originate from carcinoma cells that have acquired features related to migration, like those expressed during embryogenesis, where epithelial genes are down-regulated to switch into a mesenchymal phenotype5. In contrast, other CTC’s might reach the circulation by simply being directly exposed to the bloodstream; in fact, the physical manipulation of prostate cancer by invasive methods have shown to increase the detection of CTC’s in blood, potentially this might be the case in other cancer types and other situations, for example during accidents6.
However, it is not because a cancer cell becomes a CTC that means this tumor cell holds the capability to colonize other organs. Several experiments performed by Dr. Fidler in 19707, demonstrated that most cancer cells lack the capability to survive in the bloodstream8. In our team, we have shown that the key feature for the metastatic cascade is the survival of CTC’s in the blood circulation9. In this aspect, functional studies of CTC’s are fundamental in order to understand which CTCs are the metastatic initiators (or viable ones) for the metastatic cascade and which ones are not. Moreover, in vitro expansion of CTC’s can help to elucidate the cellular mechanism behind a viable metastasis-initiator CTC. As an example, the establishment of the only series of colon cancer CTC lines in our group has allowed the identification of how the treatment pressure had an impact on the clonal evolution with the selection of different subpopulations of aggressive CTC’s with cellular adaptations to thrive even under and after chemotherapy10,11.
What makes a CTC viable to achieve the metastatic cascade?
So far there is not a simple answer for this question. Nevertheless, a viable CTC must have, at least, three key features: 1) structural flexibility and the right signals to migrate among the tumor microenvironment and then through the small blood capillaries, 2) survival capabilities with anoikis resistance and immune system escape in the bloodstream, 3) stemness properties to self-renew in distant organs to recreate a new tumor named metastasis. Moreover, the force or the weakness of cancer patients can facilitate each one of these key features, like chronic diseases that affect the efficacy of the immune system to reduce the survival of CTC’s in blood.
In order to obtain a higher plasticity, CTCs from carcinomas can lose epithelial proteins on their surface that keep them attached to each other, thus the epithelial-mesenchymal transition (EMT) has been suggested as the main mechanism involved in the generation of viable CTC’s5. However, not all CTC’s undergo the full EMT, as this process should keep a balance between E and M12. Indeed, CTC’s that fully become mesenchymal might not be able to revert to the full epithelial phenotype, a must for the CTC’s to colonize a distant organ. Moreover, CTC clusters might be not related to EMT and depend on other mechanisms13,14. Interestingly, these CTC clusters have been shown to be more complex than just a homotypic microemboli of tumor cells. Indeed, neutrophils can be involved in heterotypic microemboli as a kind of circulating tumor microenviroment15. This complexity might reduce the structural flexibility of CTC’s but in exchange clearly increase the survival capabilities. Single and clustered CTC’s can use different mechanisms to protect themselves and survive during their journey in the bloodstream. Some examples could be the downregulation of specific antigenic surface markers or the full coverage with circulating platelets16. Independently to how a CTC is actively released by a tumor or how it survives, a viable CTC must be able to self-renew and to initiate a new tumor; therefore, stem-like cell features are key hallmarks in this process17. All the aforementioned features and mechanisms probably act simultaneously and synergistically to increase significantly the probabilities of successful metastasis.
How can CTC’s be detected?
Currently, many technologies exist to detect CTC’s: they use physical or biological properties to discriminate CTC’s from non-cancer cells in the bloodstream or other biological fluids18. The enrichment step is a key to detect CTC’s in the best conditions and should be selected with the minimal biases. For instance, methods that use size and/or deformability as a parameter to detect CTC’s will neglect small CTC’s or CTC’s with high phenotypic flexibility; methods that use the depletion of non-wanted cells should capture all CTC’s, even if the sample is less than pure. Methods that use specific surface markers like EpCAM in the CellSearch® system (Menarini-Silicon Biosystem©, Italy) can theoretically detect all EpCAMpos-CTC’s with high sensitivity. Interestingly, viable CTC’s expressing EpCAM is associated with stemness19. Of course, the use of specific markers as a positive selection might miss CTC’s that have downregulated these markers. Other methods can be more suitable for the detection of single viable CTC’s, for instance, the innovative quantitative and qualitative EPIDROP technology (EPIspot in a DROP) which combines the identification of a total number of viable CTC’s within a few hours and a subset of functional CTC’s that can secrete (e.g. Prostatic Specific Antigen – PSA in prostate cancer), release or shed (e.g. membrane receptor) any specific proteins3,20. Using a functional assay adds new evident parameters that will help to identify the clinically relevant CTC’s in cancer patients.
Are CTC’s introduced in clinic practice for cancer patients?
Despite the existing technologies for the CTC detection/characterization, there are relatively few clinical trials demonstrating the clinical utility: whether a positive or a negative result could change the clinical managements of cancer patient. As of today, the only technology cleared by the Food & Drugs Administration (FDA) for cancer prognosis is the CellSearch® system. Moreover, this the only technology to show clinical utility of the enumeration of CTC’s in metastatic breast cancer hormone receptor positive and HER2-negative, by defining whether or not a patient gets benefits in hormone therapy or chemotherapy21. Today, we need to initiate more interventional clinical trials to introduce CTC’s in the clinical routine. There is an urgent need for the identification of clinical scenarios where the CTC analysis offers clear benefits to cancer patients. The CTC analysis can go further than the enumeration and even detect viable CTC’s with specific tumor targetable markers. One example is the detection of PD-L122,23 on CTC’s, we can highlight here a current French clinical trial ALCINA2 (NCT02866149) focusing on the clinical relevance of liquid biopsy in non-small cell lung cancer in the context of immunotherapy. Another example is the detection of androgen receptor variant 7 (AR-v7) which is a marker of endocrine therapy resistance in prostate cancers24. It is crucial to identify specific markers on CTC’s with the right clinical question to propose the corresponding targeted therapy as precision medicine to cancer patients.
Where CTC’s stand out in the liquid biopsy field?
There is not a universal definition for what is a liquid biopsy test, however, this term could apply, at least in cancer, to identify circulating cancer-derived biomarkers as well as immune cells or tumor microenvironment-derived biomarkers in the body fluids25. During the last decade, an emphasis in early diagnosis of cancer has been made, mainly by the detection of circulating cell-free DNA (cfDNA) or circulating tumor DNA (ctDNA). Moreover, multi-cancer detection methods have shown promising results for cancer screening26,27. However, these methods still face the difficult challenge to demonstrate their clinical utility. Indeed, the natural occurring mutations in healthy tissues28 mostly in aging persons could yield false positive results; in addition, these methods could even diagnose pre-malignant lesions that might never fully develop in cancer. To measure the real risk of these theoretical problems, clinical trials using methods to detect cancer should prove their clinical relevance such as the increase of the overall survival (OS) with a minimal risk for false positive cases. This is rather a difficult task as early interventions might only show proof of increased OS after years or decades due to the naturally long evolution of some cancer types, and patient and diagnosis delays. For CTC’s, their use in early cancer diagnosis have been hampered by biological and technological limitations, as CTC’s are rare events in blood. Nevertheless, the theoretical detection of CTC’s is an undoubtedly a sign of a potentially metastatic tumor, so their detection could theoretically reduce the number of unnecessary invasive diagnostic or therapeutic interventions.
Despite the, yet, unmet applications of liquid biopsy for early diagnosis, both ctDNA and CTC’s have shown their clinical validity as real-time follow-up in progression and identification of targetable markers such as the aforementioned use of CTC’s in breast cancer or the evaluation of EGFR mutation status in non-small cell lung cancer using ctDNA. Moreover, other circulating biomarkers such as extracellular vesicles (exosomes), could expand the number of applications in this field. This field will lead us to include combinatory approaches to use the right liquid biopsy analytes for the right purpose for the right cancer type at the right stage29.
Since the coining of the term “liquid biopsy” in 201030, fundamental and clinical research in this field has exponentially increased. Considered as a key discovery milestone in cancer by the journal Nature31, liquid biopsy is going to change the way that clinicians diagnose cancer and manage patients in the following years. More work needs to be done to answer to all potential issues related to the clinical utility of liquid biopsy. Patients, clinicians, institutions, governments and regularity agencies can be assured that minimal invasive methods like the liquid biopsy will offer precision healthcare and economic benefits for the society in a soon future. There are still questions remaining about the metastatic cascade, and despite its complexity, functional studies of CTC’s and ongoing clinical trials will show us the way in which these mechanisms can soon decipher and defeat cancer in the near future.
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Dr. Alix-Panabières is an associate professor and the director of the laboratory of rare human circulating cells (LCCRH) at the University Medical Center of Montpellier and the CREEC – CANECEV CNRS, University Montpellier, France. She is working in the liquid biopsy field for 23 years and is the expert for the EPISPOT assay, a technology that has been recently improved to detect functional CTCs at the single cell level (EPIDROP). Importantly, Drs. Alix-Panabières & Pantel coined for the first time the term ‘Liquid Biopsy’ in 2010 (Trends Mol Med). She has authored >125 scientific publications during the last years and 15 book chapters, she is the inventor of 3 patents in the field of liquid biopsy. She is involved in basic sciences (establishment of CTC lines), technological developments and many national and international clinical trials in liquid biopsy. With her colleagues in France, she demonstrated for the first time the clinical utility of CTCs in metastatic breast cancer (Bidard et al. JAMA Oncol 2021). She established many fruitful collaborations all over the world, in Europe, USA, South America, Asia and Australia, and got many national and European grants during the last decade and is actively involved in the European Society of Liquid Biopsy (ELBS). It was a great honor for her to receive the Gallet et Breton Cancer Prize, the highest honor conferred by the French Academy of Medicine in November 2012 for her huge work in the field of liquid biopsy, the 2017 AACR Award for the most cited scientific article in 2015 (Cayrefourcq et al. Cancer Res). In October 2021, she received the International Liquid Biopsy Society (ISLB) Award for her Lifetime Achievement as well as the prestigious Alexandr Savchuk Award in January 2022, during the ‘15ème biennale Monégasque de Cancérologie’, both in consideration of her huge contribution in the field during her career and her commitment in the development of the liquid biopsy research.
Luis Enrique Cortés Hernández is a medical doctor from the Autonomous University of Nuevo Leon in Mexico. He obtained a Masters in comparative vertebrate morphology at the University of Antwerp, Belgium. Currently, he is a PhD student at University of Montpellier, France, and works in the Laboratory of Rare Human Circulating Cells under the supervision of Dr. Catherine Alix-Panabières.