Abstract PO038: Circulating cancer associated fibroblasts (cCAFs) enhance CTC extravasation and establishment of metastases

Abstract

Abstract Background: Cancer associated fibroblasts (CAFs) are the most abundant non-cancerous cell component of the breast cancer (BC) TME and have been shown to participate in most all steps of metastasis. We have previously shown that CTCs cluster with circulating CAFs (cCAFs) in BC patients at all disease stages, and this observation is recapitulated in the mouse PYMT BC model. Our central hypothesis is that cCAF/CTC co-clusters comprise the functional metastatic unit, and here we sought to determine the role of cCAFs in co-clusters on CTC extravasation and metastatic seeding. Methods: Xenografts: In vitro co-clusters were produced by culturing human CAF23 cells and MDA231(Ffluc) cells in ULA plates for 18 hrs. Clusters were injected via tail vein into NSG mice and metastatic growth was monitored by BLI. Endothelial binding assays: In vitro clusters, single CAFs or single BC cells were labeled with Cell Tracker dyes, pipetted onto confluent HUVEC monolayers, and allowed to bind for 30 minutes before gentle washing. Bound cells were counted on a fluorescent microscope. Ex vivo extravasation assays: Cell Tracker labeled co-clusters were injected via tail vein into FVB mice. One hour before sacrifice, FITC-lectin was injected via tail vein to label lung endothelia. Lungs were then extracted and imaged by confocal microscopy. Results/Discussion: Xenograft experiments demonstrate that within the first week, CAF23/MDA231 co-clusters produce durable, proliferative lung metastases faster than MDA231 monoclusters. This early difference in metastatic growth suggests that CAFs help BC cells establish metastases faster, possibly by aiding extravasation into the lung parenchyma. Using our ex vivo model of lung extravasation, we saw that significantly more CAF23/MDA231 co-clusters than MDA231 monoclusters were in the process of and/or had extravasated into the lung parenchyma at 8 and 24 hrs post-injection. In vitro binding experiments revealed that HUVEC endothelial cells bind more CAF23/BC co-clusters than BC monoclusters. Similarly, HUVEC cells bound significantly more single CAF23 or CAF19 cells than BC cells. CAF binding increased when endothelial e-selectin expression was induced with IL1-b; binding to e-selectin being the first step in cancer cell rolling and extravasation. Immunofluorescence and flow cytometry showed that CAFs express the CA19-9 antigen, sialyl-Lewis A (sLeA), which acts as an e-selectin ligand. Tannic acid, an inhibitor of the chemokine SDF1/CXCL12, decreased binding, as did 5mM EDTA. CD44 siRNA had no effect on binding whereas depletion of hyaluronin, a ligand of CD44, had a significant effect on CAFs but not on BC cells. Together, these experiments point to CAFs in co-clusters as critical enhancers of endothelial binding and extravasation, which may ultimately result in faster establishment of metastases and enhanced survival of CTCs. Future investigations will focus on therapeutic targeting of the mechanisms we have identified to block cCAF/CTC extravasation and metastasis. Citation Format: Angela Spartz, Benjamin Troness, Utsav Sharma, Geneva Taylor, Dorraya El Ashry. Circulating cancer associated fibroblasts (cCAFs) enhance CTC extravasation and establishment of metastases [abstract]. In: Proceedings of the AACR Virtual Special Conference on the Evolving Tumor Microenvironment in Cancer Progression: Mechanisms and Emerging Therapeutic Opportunities; in association with the Tumor Microenvironment (TME) Working Group; 2021 Jan 11-12. Philadelphia (PA): AACR; Cancer Res 2021;81(5 Suppl):Abstract nr PO038.