Cancer-associated fibroblasts (CAFs) shape the tumor microenvironment (TME), influencing immune cell (IC) infiltration and anti-tumor activity. Three major CAF subtypes are identified in cutaneous carcinomas. Immunomodulatory CAFs (iCAFs), enriched in late-stage tumors, express cytokines and chemokines that promote IC recruitment and activation. Matrix CAFs (mCAFs) produce extracellular matrix and ensheath tumor nests, restricting IC entry. Myofibroblast-like CAFs (myoCAFs) resemble pericytes and contractile fibroblasts and likely support tissue remodeling and tumor vasculature.  

Keratinocyte cancers, including basal cell carcinomas (BCCs) and squamous cell carcinomas (SCCs), were profiled by Imaging Mass Cytometry using a panel targeting major myeloid, lymphoid and CAF subsets, including activation and exhaustion markers. Image analysis, clustering, and spatial mapping were performed with HALO, Seurat, and Squidpy, enabling high-resolution definition of CAF niches.  

SCCs harbored more immune cells than BCCs, particularly lymphocytes. Tumor immune phenotypes—immune-desert, -excluded, and -inflamed—correlated with CAF composition: inflamed SCCs were enriched in iCAFs, whereas immune-excluded BCCs and SCCs contained more mCAFs and myoCAFs; infiltrative BCCs were particularly enriched in myoCAFs. Spatial analysis mapped iCAFs adjacent to lymphocytes, while myoCAFs formed fibroblast-rich stromal regions largely devoid of immune cells. These findings indicate that specific CAF subtypes differentially regulate immune infiltration and activation, suggesting that targeting specific CAF subsets could enhance immunotherapy efficacy in cutaneous keratinocyte carcinomas and could be possibly translated to other keratinocyte cancers.