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

Background Neoadjuvant PD-1/PD-L1 blockade yields robust efficacy in advanced cutaneous squamous cell carcinoma (cSCC), yet many patients fail to achieve a complete or major pathologic response. The reasons why some patients experience response but others do not are unclear. Methods We profiled cSCC specimens before, after 1 dose, and after 3–4 doses of PD-1/PD-L1 blockade to uncover resistance mechanisms and predict therapeutic response. In total, 27 patients across three cohorts, including two phase II trials, were studied. We created 1.7 mm tissue-core microarrays and performed single-cell spatial transcriptomics, including spatial clustering, gene-set enrichment, and spatial correlation analyses. Results After profiling all samples, six distinct spatial niches emerged, each differentially enriched in responders versus non-responders. A high antigen presentation niche, B/plasma cell enriched niche, and inflammatory keratinocyte niche were more frequent in responders, whereas proliferative keratinocyte, low antigen presentation myeloid, and fibroblast-rich epithelial–mesenchymal transition niches prevailed in non-responders. Notably, spatial niche profiling on pretreatment samples outperformed PD-L1 status in predicting pathologic response. Each niche displayed unique gene coexpression modules, suggesting niche-specific resistance mechanisms. Individual tumor analyses revealed varied immune evasion strategies, including defective interferon-induced antigen presentation, immunosuppressive myeloid environments, and epithelial–mesenchymal transition. Conclusions Our single-cell spatial transcriptomic approach identifies six spatial niches that predict immunotherapy response better than PD-L1 status using only 1.7 mm tissue cores and may inform the development of biomarkers. Our results further underscore the heterogeneity of resistance mechanisms among cSCC patients, highlighting the need for tailored therapeutic strategies.