CD206+CD14− Skin-Resident Macrophages and DC–T Cell Clusters Are Spatial Features Characterizing Nonrelapsing Cutaneous Squamous Cell Carcinoma

Abstract Current histopathologic classifications do not reliably distinguish patients with primary cutaneous squamous cell carcinomas (cSCC) at risk of relapse from those with nonrelapsing tumors. This underscores the need for molecular signatures capable of stratifying patients during primary tumor resection. In this study, we used high-dimensional imaging mass cytometry and a 39-antibody panel to define the immune landscape of 20 primary cSCC with distinct clinical outcomes, four relapsing cSCC, and their perilesional skins. Computational analysis of spatially resolved single-cell data from 47 imaging mass cytometry images identified 12 immune-cell subsets that discriminated primary cSCC from perilesional skin. Regulatory T cells, cytotoxic CD8+ T lymphocytes, and tumor-associated macrophages and neutrophils characterized tumors, whereas Langerhans cells and skin-resident macrophages defined perilesional skin. Skin-resident macrophages were characterized by the expression of CD206, CD11c, and HLA-DR and the absence of CD14. These cells infiltrated tumors from nonrelapsing patients more efficiently. We found a higher density of proliferating, mature, and cytotoxic cells within this macrophage subset, consistent with the absence of relapse. Spectral flow cytometry analysis on fresh tumor biopsies revealed that the skin-resident macrophages had phagocytic properties, suggesting a role in tumor antigen processing. Additionally, neighborhood profiling revealed that DC-LAMP+ dendritic cells were in close proximity with helper and cytotoxic T lymphocytes in primary cSCC from patients without relapse, indicative of active adaptive immunity. Our findings identify phagocytic skin-resident macrophages and dendritic cell–T cell clusters as features differentiating nonrelapsing cSCC from primary cSCC at risk of relapse. These data have the potential to guide the identification of prognostic biomarkers for cSCC.