Identification of Small-Molecule Inhibitors Targeting Different Signaling Pathways in Cancer-Associated Fibroblast Reprogramming under Tumor-Stroma Interaction.

Cancer-associated fibroblasts (CAFs) interact reciprocally with tumor cells through various signaling pathways in many cancer types, including cutaneous squamous cell carcinoma. Among normal fibroblast subtypes, papillary fibroblasts (PFs) and reticular fibroblasts (RFs) respond distinctly to tumor cell signaling, eventuating the differentiation of RFs rather than PFs into CAFs. The regulation of subtype differentiation in fibroblasts remains poorly explored. In this study, we assessed the differences between PFs, RFs, and CAFs and examined the effects of small-molecule inhibitors targeting the TGFβ, phosphoinositide 3-kinase/protein kinase B/mTOR, and NOTCH pathways on the tumor-promoting property of CAFs and CAF reprogramming in 2-dimensional and 3-dimensional cultures. Blocking TGFβ and phosphoinositide 3-kinase strongly deactivated and concurrently induced a PF phenotype in RFs and CAFs. Three-dimensional coculturing of a cutaneous squamous cell carcinoma cell line MET2 with RFs or CAFs led to enhanced tumor invasion, RF-CAF transition, and cytokine production, which were further repressed by blocking TGFβ and phosphoinositide 3-kinase/mTOR pathways but not NOTCH pathway. In conclusion, the study identified biomarkers for PFs, RFs, and CAFs and displayed different effects of blocking key signaling pathways in CAFs and tumor cell-CAF interplay. These findings prompted a CAF-to-PF therapeutic strategy and provided perspectives of using included inhibitors in CAF-based cancer therapy.