Transcriptomic profiling of circulating tumor cells from metastatic breast cancer patients reveals new hints in their biological features and phenotypic heterogeneity

The study of circulating tumor cells (CTCs) provides critical insights into the biological mechanisms underlying metastasis. This study aims to demonstrate the applicability of an integrated DEPArray-based phenotypic analysis combined with transcriptomic characterization to investigate the biology of CTCs isolated from 10 patients with metastatic breast cancer (MBC). The transcriptional profiles of CTCs were consistent with both the cancer type and epithelial characteristics. Gene set enrichment analysis identified pathways associated with synapse organization and calcium channel activity. Furthermore, distinct gene expression profiles were linked to specific metastatic sites, particularly bone metastases. We also report a rare and understudied population of CTCs, characterized by the co-expression of epithelial and leukocyte markers, observed exclusively in patient-derived samples and not in blood samples from healthy volunteers spiked with SKBR-3 and MCF-7 cell lines. This suggests that these double-positive CTCs (dpCTCs) may have a specific role in the metastatic process. The transcriptomic characterization of these rare CTCs enhances our understanding of their biology and potential involvement in metastasis. As a pilot study, our findings underscore the potential of CTC-based transcriptomics as a valuable tool for advancing clinical and biological understanding of MBC, particularly regarding metastatic mechanisms and organotropism. Moreover, it provides preliminary insights into dpCTCs, a poorly characterized population that may play a pivotal role in metastasis but remains largely unexplored. These findings could pave the way for developing targeted therapies aimed at the pathways driving metastasis and for improving patient monitoring through CTC profiling.