Cross-talk between Cancer-Associated Fibroblasts and Triple Negative Breast Cancer (TNBC) cells in heterotypic clusters

Circulating Cancer-Associated Fibroblasts (cCAFs) have been discovered in circulating tumor cell clusters from all stages of disease progression in breast cancer patients. As the most abundant non-cancerous cell type in the tumor microenvironment (TME), CAFs impart many of the tumor promoting functions defined by the hallmarks of cancer such as: proliferation, invasion, migration, metastasis, stemness, immunosuppression, altered cancer cell metabolism, and drug resistance. Human primary CAF cell lines can form co-clusters in vitro with human breast cancer cells when grown in ultra-low attachment conditions. We have previously used these co-clusters to show that they promote metastasis in the mouse tail vein model. In this study, we seek to understand the cross-talk that occurs between CAFs and TNBC cells when they are present in heterotypic co-clusters compared to TNBC monoclusters or CAF monoclusters. We created samples of mono-clusters and co-clusters grown in 3D conditions for 48 hrs, then disrupted the clusters into a single cell suspension, and subsequently processed them for Single-cell RNA sequencing. Comparisons of breast cancer cells grown as mono-clusters versus co-clusters revealed differentially expressed genes (DEGs) that are up and down regulated in the presence of CAFs. Integration Pathway Analysis and Gene Ontology analysis revealed that CAFs increase expression of several components of the fibrinolysis pathway in breast cancer cells, such as uPA/PLAU and SERPINE1. Upstream regulators identified included TGFbeta. We also looked at DEGs in CAFs when in co-clusters versus mono-clusters and found that when in co-clusters, CAFs become even more CAF-like with the upregulation of genes such as collagens, integrins, and FN1. This study provided us with candidates to study in the process of CAF-mediated breast cancer cell extravasation, such as uPA/PLAU. Pathways identified support our conclusions that circulating CAFs are important players in the metastatic process.