Abstract P3-01-28: Circulating cancer associated fibroblasts (cCAFs) enhance CTC extravasation and establishment of metastases
Authors: Spartz Angela , Troness Benjamin , Sharma Utsav , El-Ashry Dorraya 
Published: March 2020

Introduction: Breast cancer metastasis is a multi-step process that involves cellular migration, invasion, travel through the circulation, extravasation and secondary site proliferation. Circulating tumor cell (CTC) travel is thought to be via rolling on vascular endothelium, and CTC clusters have been shown to have a shorter circulation half-life than single CTCs due to increased entrapment within capillaries. We have previously shown that CTCs cluster with circulating cancer associated fibroblasts (cCAFs) in breast cancer patients and this observation is recapitulated in the mouse PYMT breast cancer model. Circulating CAF/CTC clusters are seen in patients of all stages and in all PyMT mice with tumors, before overt metastases. 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 metastatic seeding.

Methods: Xenografts: In vitro co-clusters were produced by culturing 75,000 human CAF23 cells and 75,000 MDA231(Ffluc) cells in ultra-low attachment plates for 18 hrs. Clusters were injected via tail vein into NSG mice and metastatic growth was monitored by bioluminescent imaging. Endothelial binding assays: In vitro clusters were made as for xenografts except that CAFs and BC cells were labeled with Cell Tracker dyes. Clusters were 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: Co-clusters were made as above with Cell Tracker labeled MDA231 and CAF23 cells and injected via tail vein into FVB mice. Lung endothelia was labeled by tail vein injection of FITC-lectin an hour before lungs were extracted and imaged by confocal microscopy.

Results: Xenograft experiments demonstrate that within the first week, CAF23/MDA231(Ffluc) co-clusters produce durable, proliferative lung metastases faster than MDA231(Ffluc) monoclusters. This early difference in metastatic growth suggests that CAFs help cancer cells establish metastases faster, possibly by enhancing the binding and rolling of CTCs along the vascular endothelium and/or by aiding extravasation into the lung parenchyma. In vitro binding experiments revealed that CAF23/MDA231 co-clusters bind to HUVEC endothelial cells better than MDA231 monoclusters under static conditions. Similar results were observed with the aggressive basal cell line, DT28. To determine the effect of shear stress, we performed endothelial binding assays in an orbital shaker at 100 rpm (used to mimic oscillatory and continuous flow shear stress), and under these conditions, CAFs continue to bind to HUVECs at significantly higher numbers than MDA231 cells. CAF binding increased two-fold when endothelial e-selectin expression was induced with IL1-β. Immunofluorescence and flow cytometry showed that CAFs express the CA19-9 antigen, sialyl-Lewis A (sLeA), which acts as an e-selectin ligand; binding to e-selectin being the first step in cancer cell rolling and extravasation. Using our ex-vivo extravasation imaging protocol, we observed that significantly more CAF23/MDA231 co-clusters than MDA231 monoclusters were in the process of extravasation and/or had extravasated into the lung parenchyma at 8 and 24 hrs post-injection.

Conclusions: Together, these experiments point to CAFs in co-clusters as critical enhancers of endothelial binding and extravasation, which may ultimately result in quicker establishment of metastases and enhanced survival of CTCs in clusters. Future studies will focus on identifying the molecular mechanisms in these processes facilitated by cCAFs that could be amenable to therapeutic targeting.

Citation Format: Angela Spartz, Benjamin Troness, Utsav Sharma, Dorraya El-Ashry. Circulating cancer associated fibroblasts (cCAFs) enhance CTC extravasation and establishment of metastases [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P3-01-28.