Phenotypic and functional characterization of Circulating Tumor Cells (CTCs) from breast cancer patients with emphasis on triple negative (ER-/PR-/ HER2-) breast cancer (TNBC)

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Βάρδας, Βασίλειος

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Triple-negative breast cancer (TNBC) stands out as the most aggressive breast cancer subtype, characterized by a lack of expression of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2). Despite its aggressive nature, TNBC faces a significant dearth of targeted therapies, highlighting the pressing need for the identification of novel biomarkers. Notably, metastasis is responsible for approximately 90% of breast cancer-related deaths, with a pivotal role attributed to Circulating Tumor Cells (CTCs). Particularly, metastatic dissemination is associated with the presence of CTCs and Disseminated Tumor Cells (DTCs) in peripheral blood and bone marrow, respectively. However, metastasis is still not well understood and is largely incurable. Hence, there is an urgent need to explore the mechanisms that govern metastatic spread of cancer cells. Microtentacles (McTNs), are tubulin-based protrusions of the plasma membrane playing a pivotal role in the metastatic cascade. They are also associated with the Epithelial-to-Mesenchymal Transition (EMT) pathway. In this context, the detection and characterization of CTCs exhibiting an EMT phenotype is very important. These cells are of particular interest due to their potential association with high-risk prognosis and increased susceptibility to relapse and mortality. Traditionally, methods reliant on epithelial markers may inadvertently underestimate CTC numbers, necessitating a shift toward more comprehensive approaches. Moreover, BRCA1 mutations have been implicated in the predisposition to TNBC. The intricate interplay between BRCA1, PARP, and the DNA damage repair pathways further amplifies the significance of these factors in TNBC pathogenesis. Against this backdrop, our research aimed to contribute to the understanding of TNBC by examining the phenotype of CTCs in a cohort of 64 TNBC patients. An additional group of 30 hormonal-positive (luminal) patients were included in our analysis to provide a comparative context. Our investigation was focused on elucidating the expression profiles of key biomarkers, such as immune checkpoints(PD-L1, CTLA-4) and EMT-related molecules (GLU, VIM), in CTCs. These markers proved to be valuable indicators of disease severity and clinical outcomes. Furthermore, our study employed advanced technologies, including the FDA-approved CellSearch system to explore vimentin expression and the EMT phenotype in CTCs. This analysis, both in early and metastatic patients, revealed a novel (CK-VIM+CD45-) phenotype which enabled the detection of elusive cancer cells, offering insights into breast cancer prognosis and therapeutic strategies. Additionally, our research extended beyond mere characterization to functional analysis. Leveraging the innovative TetherChips technology from the University of Maryland, our study explored the metastatic potential through McTN formation, and biomarker expression in viable CTCs isolated from TNBC patients post-treated with Vinorelbine (Vin), an FDA-approved drug. The rapid effects observed within just one hour on McTN formation, expression of key biomarkers, and apoptosis, suggest Vinorelbine as a potential strategy to inhibit metastatic spread in TNBC patients. Our findings also highlighted the importance of the functional analysis of CTCs, using TetherChips technology as a valuable tool for real-time investigation of drug efficacy on isolated, cultured CTCs. Furthermore, our investigation examined the effects of a new Combrestatin A4 (CA-4) analog on TNBC cell lines (MDA-MB-231 and MDA-MB-436), assessing viability, cytoskeletal organization (McTNs), migration capacity, and expression of important biomarkers (PD-L1, ΑΑ-TUBULIN, and VIM). The investigation of mutations in BRCA1 exon 20 and their correlation with PARP-1 expression in CTCs of TNBC patients added another layer of depth to our research. High nuclear expression of PARP-1 and the presence of BRCA1 mutations were identified as distinctive features of TNBC CTCs, offering biomarkers linked to patients' clinical outcomes and potentially the PARP-1 targeted therapies. In conclusion, our comprehensive research not only advances the understanding of TNBC but also opens promising avenues for assessing important biomarkers and new insights into personalized treatment approaches. The comprehensive knowledge gained from this study contributes to the evolving landscape of TNBC research, paving the way to target metastatic dissemination.



Liquid biopsy, Breast cancer, Triple negative breast cancer, CTLA-4, PD-L1, Circulating tumor cells, Detyrosinated α-tubulin (GLU), Vimentin (VIM), Luminal, EMT, Immune checkpoints, Metastasis, Vinorelbine, BRCA1, PARP-1, Combrestatin A4 (CA-4) analog