Abstract SY21-01: Molecular and functional characterization of circulating tumor cells in carcinoma patients

Abstract

Abstract “Real-time liquid biopsy” was introduced in 2010 for the analysis of circulating tumor cells (CTCs) in the blood of cancer patients (1), and this new diagnostic approach has received enormous attention because of its obvious clinical implications for personalized medicine. CTCs are derived from primary tumors and metastatic lesions and, therefore, harbor important information on the molecular characteristics relevant to tumor progression and cancer therapy. Analyses of CTCs have paved new diagnostic avenues with obvious clinical implications for personalized medicine. Key areas of clinical applications of CTCs include detection of cancer, prediction of prognosis in patients with curable disease, monitoring of systemic therapies, and stratification of patients based on the detection of therapeutic targets or resistance mechanisms (2). Much progresses has been made in the development of various technologies to enrich and detect CTCs; however, the discovery and validation of new CTC biomarkers are still in its infancy (3). Different types of CTC characterization studies have been carried out: (i) descriptive studies on isolated individual CTCs requiring a specific isolation process and a process of whole-genome or transcriptome amplification, which are informative on intrapatient heterogeneity; (ii) descriptive studies providing information at the individual cell level, which require only an enrichment but no single-cell isolation; (iii) functional studies on pools of total CTCs used for cell culture and/or xenografts (4). Since current assays cannot distinguish between apoptotic and viable CTCs, it is possible to apply the EPISPOT assay that detects proteins secreted/released/shed from viable epithelial cancer cells (5). Cells are cultured for a short time on a membrane coated with antibodies that capture the secreted/released/shed proteins that are subsequently detected by secondary antibodies labeled with fluorochromes. This functional assay detects viable CTCs at the single-cell level and has been used on hundreds of patients with different tumor types including epithelial tumors (breast, prostate, ovarian, and colon cancer) and melanomas, showing its clinical relevance (6). The fluoro-EPISPOT assay has been optimized for single CTCs, is now named EPIDROP, and should be a more reliable and sensitive technology than those already existing in this field of expertise, allowing the analysis of proteome and secretome simultaneously of unique functional CTCs or of clusters of CTCs. In addition, testing different drugs in an individual patient might lead to a personalized "oncogram" that might help to improve the clinical management of cancer patients. Moreover, CTC research opens a new avenue for understanding the biology of metastasis in cancer patients. Understanding the molecular mechanisms that regulate the biology of metastasis-competent CTCs is of utmost importance in unraveling the formation of metastases and tumor relapse in patients with cancer (3). However, an in-depth investigation of CTCs is hampered by the very low number of these cells, especially in the blood of colorectal cancer patients. Thus, the establishment of cell cultures and permanent cell lines from CTCs has become the most challenging task over the past year. In 2015, Cayrefourcq et al. described for the first time the establishment of a permanent cell line from CTCs of one metastatic colon cancer patient (7). The cell line, designated "CTC-MCC-41," is stable for more than three years and cells have been characterized at the genome, transcriptome, proteome, and secretome levels. This thorough analysis showed that CTC-MCC-41 cells resemble characteristics of the original tumor cells in the colon cancer patient and display a stable phenotype characterized by an intermediate epithelial/mesenchymal phenotype, stem-cell like properties and an osteomimetic signature indicating a bone marrow origin. Functional studies showed that CTC-MCC-41 cells induced rapidly in vitro endothelial cell tube formation and in vivo tumors after xenografting in immunodeficient mice. Subsequently, Alix-Panabières et al. determined the molecular bases underlying differences between this colon CTC line and well-described cancer cell lines derived from primary tumors and from metastatic sites (8). The results showed clearly that the CTC-MCC-41 displayed a very specific transcription program. Interestingly, among the 1,624 transcripts exclusively upregulated in CTC-MCC-41, key genes related to energy metabolism, DNA repair, and stemness genes were observed. Furthermore, blood samples collected during the course of the systemic therapy and tumor progression from the same metastatic colon cancer patient who gave rise to the CTC-MCC1-41 line have allowed the acquisition of 8 additional colon CTC lines. To our best knowledge, there is no other report on the in vitro expansion of CTCs from cancer patients during the course of their disease and treatment. This unique biologic material represents a chance to understand the clonal selection and resistance mechanisms during tumor progression and cancer treatment. In conclusion, functional analyses of CTCs provide insights for the discovery of new biomarkers to identify the most aggressive CTC subpopulations with stem cell properties. CTC lines could contribute to the development of new drugs to eradicate metastasis-initiator CTCs causing relapses and cancer-related death in individuals with cancer.