Abstract 5062: CAFs in breast cancer circulation: The influence of cancer cell intrinsic mechanisms

Background: Metastatic disease is the primary cause of breast cancer (BC) mortality. Tumor-stromal cell interactions play a pivotal in tumor initiation, progression and metastasis. Cancer-associated fibroblasts (CAFs) are the majority of stroma in BC and are critical to BC tumorigenicity and malignancy. Metastasis occurs due to the transport of circulating tumor cells (CTC) and clusters of CTCs through the vasculature. We recently identified circulating CAFs (cCAFs) as a novel circulating biomarker associated with metastatic BC. cCAFs not only circulate individually, in BC patient blood and in blood from both spontaneous and xenograft murine BC models, but cCAFs are also found in clusters with CTCs. In this study, we examine the egress of CAFs and follow them through to metastatic sites, and evaluate the ability of BC cell subtype/metastatic propensity to influence cCAF and cCAF/CTC cluster egress. Methods: We used NSG mice with orthotopic xenograft implantation of BC cells, primary CAF cell lines, or co-implantation of BC and CAF cell lines. We used two different BC cell lines, the nonmetastatic BC cell line, MCF-7, and the highly metastatic primary BC cell line, DT28. We also employed the MMTV-PyMT spontaneous model of BC metastasis to evaluate cCAFs and CTCs in a preclinical model. Mice were sacrificed at specific time points, and cardiac blood was collected to ascertain the temporal dynamics of cCAF and CTC presence. Blood was filtered using the faCTChecker microfluidic filtration instrument (Circulogix). Filters were enumerated by IF for cCAFs, CTCs, and cCAF/CTC co-clusters. To explore BC-intrinsic factors that influence cCAF and cCAF/CTC cluster egress, we modeled egress from the primary tumor in vitro using transendothelial cell migration assays, where the chemoattractant for CAF cells was conditioned media from the different BC cells lines. Results: In both spontaneous and orthotopic xenograft models of BC, cCAFs, CTCs, and cCAF/CTCs appear early in tumor development. cCAF/CTC clusters increase in correlation with tumor burden and metastasis. CAFs injected alone in orthotopic xenografts are able to egress independently of BC cells and cCAFs and cCAF clusters were seen; however, co-inoculation with BC cells resulted in substantially higher numbers of both individual cCAFs and cCAF clusters, and now co-clusters were seen. cCAFs appear about 4 days post-injection, and precede appearance of CTCs and CAF/CTC clusters. CAFs co-injected with BC cells into the MFP appear at metastatic sites. In in vitro transendothelial assays, BC secreted factors that potentiate CAF egress are identified as well as the if cCAF/CTC clusters egress as clusters or cluster in the circulation. Conclusion: Although CAFs are highly motile and cCAFs precede CTCs into the circulation, BC subtype influences the ability of CAFs to egress. Targeting cCAF egress and/or cCAF/BC cell clusters provides novel avenues to prevent or treat BC metastasis. Citation Format: Utsav Sharma, Philip Miller, Kelsie Medina Saenz, Angela Spartz, Marc Lippman, Dorraya El-Ashry. CAFs in breast cancer circulation: The influence of cancer cell intrinsic mechanisms [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5062.