CAF-Driven EMT and ECM Remodeling Programs Promote Mesothelioma Progression

Background: The aggressive progression of mesothelioma is driven not only by intrinsic tumor cell plasticity but also by dynamic interactions between tumor cells and the surrounding stromal microenvironment; however, the mechanisms by which stromal populations regulate epithelial–mesenchymal transition (EMT), tumor evolution, and therapeutic resistance remain unclear. Methods: We integrated longitudinal transcriptomic profiling with single-cell RNA sequencing in a murine intraperitoneal mesothelioma model spanning disease progression from week 0 to week 8 to identify stromal-EMT programs associated with tumor progression. In vitro fibroblast–tumor co-culture systems were used to assess how different fibroblast-to-tumor cell ratios influence transcriptional reprogramming and mesenchymal transition. Results: Single-cell analysis revealed marked stromal heterogeneity, identifying eight distinct cancer-associated fibroblast (CAF) subtypes: myCAF, mCAF, iCAF, TGF-βCAF, vCAF, plCAF, apCAF, and meCAF. These subsets showed specialized transcriptional programs associated with developmental signaling and metabolic adaptation. Functional analyses demonstrated coordinated intercellular communication across six interconnected modules, including mesenchymal transition, fibrotic remodeling, TGF-β/metabolic adaptation, adhesion signaling, WNT activation, and inflammatory crosstalk. Notably, mCAF, TGF-βCAF, and plCAF populations showed transcriptional convergence, suggesting cooperative formation of a desmoplastic, immunoregulatory niche. Co-culture experiments confirmed that fibroblast-derived signaling induces ratio-dependent transcriptional changes, promoting a shift from epithelioid to mesenchymal phenotypes. Conclusions: These findings highlight stromal regulation of EMT as a key driver of mesothelioma progression, sarcomatoid transition, and therapy resistance, and identify tumor–stroma signaling networks as potential therapeutic targets.