Multiple studies concur that CTCs - the “seeds” of fatal metastasis - intravasate into the bloodstream throughout the early stages of cancer promoting generation of micro-metastatic reservoirs, some of which ultimately evolve to metastatic tumors. However, while it may take decades for CTCs to progress from cells in circulation to clinically detectable metastases, once detected, the tumor usually grows at an exponential rate. This difference in growth dynamics at two ends of the metastatic spectrum should also be reflected in the behavior of CTCs shed from these organ sites. Having a focus towards breast cancer brain-colonizing CTCs and their properties, we hypothesized that a gradual evolution of dormant CTCs into mitotically active CTCs with brain-metastatic potency is central to the progression of primary breast cancer to brain metastasis.

We isolated CD44+/CD24- (stem-like) and Pan-Cytokeratin+ (epithelial) breast cancer CTCs from peripheral blood of two groups of breast cancer patients – five with brain metastases (BCBM) (MRI-detectable) and five without brain metastases (No BCBM). CTCs were isolated by multi-parametric flow cytometry and the expression of CTC-specific antigen was confirmed by immuno-cytochemistry and DEPArrayTM platform. Intra-cellular flow cytometry showed that No BCBM patients had 3-fold higher mitotically dormant (Ki67-) CTCs compared to BCBM patients. Whole genome expression arrays demonstrated low generalized transcriptional activity in CTCs suggestive of mitotic/metabolic dormancy. Comparison of CTC transcriptomes with ER+/PR+, HER2+ and triple negative breast cancers identified a unique CTC gene signature valid not only across the three molecular subtypes but also independent of the molecular subtype patients were initially diagnosed as. Pathway analyses predicted increased activation of pluripotency-related pathways along with decreased protein translational machinery and proliferative pathways in CTCs. Further, to dissect mechanisms of metastatic reactivation in the brain microenvironment, we compared the CTC transcriptome in BCBM vs No BCBM patients and identified a 126 gene signature potentiated in BCBM CTCs. Subsequent pathway analyses revealed upregulation of known CTC pathways like Notch along with novel hematopoietic and immune evasion networks. Cellular and functional annotations of cell migration and chemotaxis were significantly activated in BCBM along with pro-inflammatory (TNF, IL1β, NF-kB), immunomodulatory chemokines (CXCL8, CXCR4, CD86) and mitogenic growth factors (PDGF-BB). Lastly, lack of cell surface expression of Urokinase Plasminogen Activator Receptor (uPAR) and integrin beta-1 (int-β1) were used to identify CTCs in a state of metastatic dormancy. Whole genome sequencing showed significantly higher incidence of genomic mutations in proliferating CTCs compared to their dormant counterparts.

We provide first time evidence of signaling pathways keenly implicated in breast cancer CTC biology based on comprehensive analyses of CTC transcriptomes. We discovered the existence of CTCs in a state of metabolic/mitotic dormancy and identified genomic instability as a switch for CTCs to revert to their proliferative phenotype.

Citation Format: Boral D, Vishnoi M, Liu HN, Yin W, Hong DS, Scamardo AT, Marchetti D. The evolution of breast cancer circulating tumor cells mediating brain metastasis [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P1-01-08.