Maintaining Myofibroblastic-Like Cancer-Associated Fibroblasts by Cancer Stemness Signal Transduction Feedback Loop

Background: Myofibroblast-like cancer-associated fibroblasts (myCAF) in the tumor microenvironment (TME) promote cancer stemness, growth, and metastasis. Cancer cell-derived osteopontin (OPN) has been reported as a biomarker related to malignant cancer growth. In this study, we confirm that cancer cell stemness is required for the maintenance of an OPN-induced myCAF phenotype. Methods: MDA-MB-231 or HepG2 cells and Sox2 knockout variants were co-cultured with human mesenchymal stem cells (MSC). In selected instances, the OPN bioactivity inhibitor OPN-R3 aptamer (APT), OPN-R3 mutant aptamer (MuAPT), or cancer cell stemness inhibitor BBI-608 were added separately. MDA-MB-231 cancer stemness and myCAF markers were quantified by real-time PCR. Stemness-lacking cancer cell mice models were created to confirm that stemness is required for the maintenance of the OPN-induced myCAF phenotype in vivo. Results: In an MDA-MB-231 co-culture system, myCAF and stemness markers increased. Osteopontin and stemness blockade in this co-culture system decreased both myCAF and stemness marker expression, but OPN blockade after 72 hours had no effect. In contrast, when BBI608 was added at 72 hours, myCAF markers were abated after 36-hour treatment. Replacing wildtype with MDA-MB-231(-/-sox2) in co-cultures at 72 hours decreased myCAF marker expression to baseline despite the Western blot confirming the presence of OPN. Conversely, replacing MDA-MB-231(-/-sox2) cells with wildtype increased myCAF marker expression to a level equivalent to the MDA-MB-231+MSC co-culture system. In vivo osteopontin blockade diminished stemness and myCAF marker expression and stemness lacking cancer cell models, indicated by decreasing myCAF presence. Experiments were repeated in a HepG2 cell line with identical results. Conclusions: Cancer and myCAF crosstalk increases myCAF maintenance and cancer cell stemness. In this study using human breast and liver cancer cell lines, maintenance of the OPN-induced myCAF phenotype also requires cancer stemness. This indicates that the myCAF phenotype requires two distinct signaling pathways: initiation and maintenance.