Single-cell spatial transcriptomics uncovers niches that govern response to PD-1/PD-L1 blockade in cutaneous squamous cell carcinoma

Neoadjuvant PD-1/PD-L1 blockade demonstrates robust efficacy in cutaneous squamous cell carcinoma, yet a significant proportion of patients do not achieve a major pathological response for unclear reasons. Here, we characterize spatially resolved gene expression profiles before, during, and after treatment in cutaneous squamous cell carcinoma (cSCC) patients enrolled on two pivotal phase II clinical trials of neoadjuvant PD-1/PD-L1 blockade. Using single-cell RNA-based spatial molecular imaging, we profiled more than 56 million transcripts from over 250,000 cells across 27 patients. Traditional cell type clustering uncovered five broad cell types (keratinocytes, fibroblasts, B-lineage cells, myeloid cells, and a mixed dendritic/T/NK compartment). Genes associated with interferon signaling and metabolic pathways were significantly upregulated in responders, but bulk gene expression alone did not fully distinguish responders from non-responders. We then applied a spatial clustering approach, which revealed six distinct spatial niches defined by their differential gene expression signatures. Notably, these spatial niches were differentially enriched in responders versus non-responders, suggesting niche-specific mechanisms of immunotherapy sensitivity and resistance. In-depth analyses of individual patients demonstrated that these spatial niches correspond to diverse mechanisms of immune evasion, including impaired interferon-stimulated antigen presentation, immunosuppressive myeloid infiltration, or epithelial–mesenchymal transition. Taken together, our study provides a comprehensive single-cell spatial transcriptomic atlas of cSCC evolution during PD-1/PD-L1 blockade, highlighting the heterogeneity of resistance mechanisms. These findings illuminate potential targets for rational combination therapies and underscore the importance of spatial context in understanding and overcoming immunotherapy resistance in cSCC.