A landscape of patient-derived cancer-associated fibroblast signals in endometrial cancers.

ABSTRACT

In conversation with endometrial tumor cells, the endometrial cancer-associated fibroblasts (CAFs) are the "partners in crime" of uterine neoplasm's highly heterogeneous tumor microenvironment (TME). We designed a laboratory-friendly method to culture endometrial CAFs on a patient-to-patient basis for studying the CAF-TME and CAF-tumor cell interaction(s). Here, we present a comprehensive characterization of endometrial CAFs derived from patients' tumor tissues (T) and tumor-adjacent normal tissues (N). We used more than 80 T and N from 53 consecutive consented patients with endometrial cancers at the Avera Cancer Institute. We derived TCAF and NCAF in a non-enzymatic feeder-layer culture and characterized their expression of markers by qRT-PCR, flow cytometry, immunocytochemistry, immunofluorescence, and Western blot. Although similar in the expression pattern of EpCAM-/CK18-/vimentin+ as in ovarian CAFs, endometrial NCAFs, and TCAFs characteristically presented dual morphology in culture. Endometrial CAFs were EpCAM-/CK18-/CD45-/CD31-/SMA+/TE-7+/PDGFRA+/CXCL12+/Meflin+/CD155+/CD90+ with patient-specific positivity for S100A4/FAP/PD-L1/CD44. Endometrial CAFs expressed mRNAs for signaling proteins of several pathways and receptor-ligands, including (1) cell cycle pathway, (2) TGF pathway, (3) FGF pathway, (4) Wnt-beta-catenin pathway, (5) HER pathway, (6) tyrosine kinase receptor ligands, and (7) steroid receptors. We tested the hypoxic response of CAFs to show that endometrial CAFs upregulate MMP1 in a HIF-1a-independent manner. In trying to delineate the relationship between expressions of CAF markers and T-cells in the tumor tissue, we observed that FAP-positive CAFs that are derived from CD4/CD8 positive tumor tissue expressed CXCL12 mRNA. The data indicate the role of the CXCL12-CXCR4 pathway of the CAF-rich stroma in the lymphocytic infiltration of the tumor. We demonstrate that endometrial CAFs can be cultured in an enzymatic-digestion-independent manner, and their signaling landscape can be mapped toward understanding CAF-TME dialogue. Our data will help unearth the functional relevance of endometrial CAFs in the context of clinical outcomes and designing CAF-inclusive therapy in the future.