Cancer cell-derived extracellular matrix promotes differentiation of fibroblasts into cancer-associated fibroblasts

Breast cancer is the most common cancer and the leading cause of cancer-related mortality in women. In addition to cancer cells, the bulk of a breast tumor comprises a range of stromal cell types, including fibroblasts. Cancer-associated fibroblasts (CAF) are crucial players in the tumor microenvironment; however, the process by which fibroblasts differentiate into CAFs is not fully understood. Extracellular matrix (ECM) is known to modulate cell phenotypes. Decellularized ECM (dECM) is a useful tool for studying in-vitro cell-ECM interactions. Yet, whether cancer cell-derived ECM (ccECM) has a role in CAF formation is not known. Here, we optimized the culture duration (5 days) and the extraction method (freeze-thaw) for obtaining ccECM. We confirmed the presence of ccECM using coomassie blue staining and scanning electron microscopy. We showed that ccECM contained fibronectin and laminin using immunofluorescence staining. In addition, we showed that the presence of ccECM but not glass surface or TGFβ promoted the initial adhesion of fibroblasts, as expected. Finally, using quantitative immunofluorescence microscopy, we demonstrated that in contrast to fibroblasts cultured on glass surfaces in the presence and absence of TGFβ, fibroblasts cultured on ccECM showed increased expression of CAF markers vimentin (2.8 fold), FAP (3.4 fold) and PDGFR β (1.8 fold), but not FSP1/s100A4. Overall, our results indicate that ccECM promotes the differentiation of fibroblasts into CAFs.