Basal cell carcinoma (BCC) and cutaneous squamous cell carcinoma (SCC) are the most common keratinocyte-derived malignancies, yet they differ markedly in invasiveness, metastatic potential, and immune contexture. Although cancer-associated fibroblasts (CAFs) are increasingly recognized as key regulators of tumor architecture and tumor immunity, the spatial organization of distinct CAF subtypes in cutaneous carcinomas and their functional relationship with immune cells remains incompletely understood. Using a 33-plex imaging mass cytometry (IMC) panel, we profiled 28 regions of interest (ROIs) from 17 human BCC and SCC specimens, encompassing more than 739,000 single cells, and integrated these data with RNA fluorescence in situ hybridization (RNA-FISH), immunohistochemistry (IHC), multiplex immunofluorescence, and in vitro functional assays. We identified four fibroblast populations, including immunomodulatory CAFs (iCAFs), matrix CAFs (mCAFs), myofibroblast-like CAFs (myoCAFs), and reticular fibroblasts (retFIBs), and found that aggressive tumor subtypes were characterized by increased stromal area, extracellular matrix deposition, and altered CAF composition. CAF composition differed most prominently across BCC subtypes, with nodular BCC enriched for mCAFs and infiltrative BCC showing increased myoCAF density, consistent with a shift toward a contractile stromal program. Spatial analyses revealed distinct CAF-immune niches: iCAFs localized to immune-cell-rich, inflamed niches enriched for activated and/or exhaustion-associated immune-cell marker programs, whereas myoCAFs occupied fibroblast-dense, immune-poor niches with globally reduced immune activation. mCAFs were preferentially associated with immune cell accumulation in the stroma and spatial immune compartmentalization, with limited immune cell presence within tumor nests. At the invasive front, CAF-immune coupling was highly subset-dependent, with iCAFs linked to antigen-experienced T-cell states and myoCAFs linked to immune exclusion. In vitro, patient-derived CAF cultures from myoCAF-rich biopsies showed enhanced collagen-gel contraction, with cultures enriched for MCAM+ CAFs displaying increased contractile capacity. Aggressive tumor variants displayed increased stromal nuclear YAP/TAZ, while complementary single-cell pathway analysis supported a mechanically remodeled stromal microenvironment in which mCAFs contribute ECM/matrix-remodeling programs and RGS5/myoCAF-like populations show enhanced mechanotransduction-associated signaling, rather than a uniform CAF-wide increase in canonical YAP/TAZ transcriptional output. Together, these findings define spatially organized CAF programs in cutaneous carcinomas and identify myoCAF-rich stromal niches as a recurrent feature of aggressive, immune-repressed tumor architecture. These results nominate CAF composition as a biomarker of immune architecture and a potential determinant of therapeutic response.
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