Ccr5 Upregulation In Two Subtypes Of Tumor Associated Circulating Cells Predict Worse Prognosis In Metastatic Breast Cancer.

Abstract Background: CCR5 is a motility chemokine receptor implicated in tumor progression, whose activation and endocytosis may identify highly aggressive breast cancer (BC) subtypes likely to spread via the circulatory system. We first studied the activation and endocytosis of CCR5 in response to its ligand CCL5 (RANTES) in the model BC cell line MDA-MB231. We then screened two types of circulating tumor-associated cells (TACs) with known negative clinical outcomes, 1) circulating tumor cells (CTCs) and 2) cancer-associated macrophage-like (CAMLs) cells, to evaluate CCR5 upregulation in relation to disease progression in metastatic breast cancer (mBC). Methods: MB231 cells were used to visualize CCR5 activation within the cell's surface and intracellular space using an anti-CCR5 antibody after stimulation with RANTES. MB231 cells were stained with LAMP-1A to verify co-localization of CCR5 and RANTES to endosomes. Anonymized peripheral blood samples from (n=54) mBC patients were obtained in accordance with local IRB regulations and with informed consent. TACs were isolated using a low-flow CellSieve Microfiltration system and stained for Cytokeratin, CD45, and CCR5. CCR5 expression signal and localization were evaluated on all TACs via fluorescence microscopy. Results: CCR5 in MB231 cells appeared as ~1μm clusters known as “CCR5 pools”, which can be found on the cell's surface, within the cytoplasm, and in the nuclear space. CCR5 pools were upregulated in the presence of RANTES, which appears to relate to endocytosis and nuclear translocation. Within the TACs of mBCs, we found similar patterns of CCR5 pooling with 70% of patients (n=38) having CCR5+ CAMLs and 41% (n=22) having CCR5+ CTCs. It was found that higher numbers of CCR5 pools (≥10pools/cell) were correlated to a 2-fold increase in both CTCs and CAMLs in circulation. In the analysis of outcome data, it was determined that patients having ≥10 CCR5 pools on their CAMLs was a highly significant predictor of worse progression free survival (PFS) and overall survival (OS) (PFS HR=4.8, CI 95% 1.9-11.6, p=0.0015) and (OS HR=3.6, CI 95% 1.3-10.0, p=0.0331). In CTCs ≥10 pools/cell was not a significant predictor for worse PFS and OS. However, ≥5 CCR5 pools/cell significantly predicted for worse PFS but not OS (PFS HR=2.9, CI 95% 1.2-7.4, p=0.0418 and OS HR=3.3, CI 95% 1.1-9.3, p= 0.0539). Conclusion: We observed the activation and endocytosis of CCR5 pools using RANTES in MD-MB231 cell lines, and then demonstrated that this upregulated motility pathway appears to be conserved in migratory cells in mBC patients. Further, this upregulation of CCR5 appears to correlate with worse clinical outcomes, which may provide an explanation for how tumor-associated cells move into circulation and acquire metastatic potential. Citation Format: Ashvathi Raghavakaimal, Massimo Cristofanilli, Cha-Mei Tang, R. K. Alpaugh, Kirby P. Gardener, Saranya Chumsri, Daniel L. Adams. CCR5 upregulation in two subtypes of tumor associated circulating cells predict worse prognosis in metastatic breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 589.