Integrated single-cell transcriptomic, proteomic, and tissue-level analysis defines cancer-associated fibroblast programs in pseudomyxoma peritonei

Pseudomyxoma peritonei (PMP) is an ultra-rare mucinous neoplasm with extensive stromal remodeling, yet the role of cancer-associated fibroblasts (CAFs) remains poorly defined. A PMP-derived CAF culture was established and characterized through multiple omics and phenotypic assays. Single-cell transcriptomic profiling revealed two distinct CAF populations: fibroblast-derived CAFs enriched for characteristic CAF functionalities and mesothelial-derived CAFs expressing mesothelial markers and pathways linked to metabolism and motility, consistent with mesothelial-to-mesenchymal transition. PMP CAFs exhibit baseline invasive and contractile properties and acquire an inflammatory CAF-like phenotype upon TNF-α/IL-1β stimulation, accompanied by altered proteomic and secretome profiles. Conditioned media from PMP CAFs further promoted macrophage polarization, highlighting their role in shaping the immune microenvironment. In conclusion, our findings reveal a previously uncharacterized role for CAF in PMP, highlighting their contribution to immune modulation in this extremely scarce peritoneal malignancy.