CD206+CD14– Skin-resident Macrophages and DC–T Cell clusters are spatial features characterizing non-Relapsing Cutaneous Squamous Cell Carcinoma

Abstract Current histopathological classifications do not reliably distinguish patients with primary cutaneous squamous cell carcinoma (cSCC) at risk of relapse from those with non-relapsing tumors. This underscores the need for molecular signatures capable of stratifying patients during primary tumor resection. Here, we used high-dimensional imaging mass cytometry (IMC) and a 39-antibody panel to define the immune landscape of twenty primary cSCC with distinct clinical outcomes, four relapsing cSCC and their perilesional skins. Computational analysis of spatially-resolved single-cell data from forty-seven IMC images identified twelve immune-cell subsets that discriminated primary cSCC from perilesional skin. Regulatory T cells, cytotoxic CD8+ T lymphocytes, tumor-associated macrophages and neutrophils characterized tumors while 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 non-relapsing 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 the skin-resident macrophages had phagocytic properties, suggesting a role in tumor antigen processing. Additionally, neighborhood profiling revealed DC-LAMP+ dendritic cells (DC) 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 DC–T cell clusters as features differentiating non-relapsing cSCC from primary cSCC at risk of relapse. These data have the potential to guide the identification of prognostic biomarkers for cSCC.