Functional PD-1/PD-L1 engagement defines a spatial biomarker of immunotherapy response

Effective predictive biomarkers for immune checkpoint inhibitor (ICI) therapy remain an unmet need across solid tumors. Here, we present an integrated spatial proteomics workflow that combines in situ proximity ligation assay with multiplexed immunofluorescence to directly resolve PD-1/PD-L1 signaling events at the level of defined cellular phenotypes and their spatial organization within intact tumor tissue. Applied as a proof-of-concept to tumor samples from patients with metastatic urothelial carcinoma treated with pembrolizumab, this approach reveals that PD-1/PD-L1 interactions specifically involving cytotoxic CD8 T cells are significantly enriched in complete responders, while such interactions are rare in patients with progressive disease. This interaction-defined T cell subset achieves superior discrimination of clinical response compared to single-marker PD-L1 expression or immune cell abundance alone. By integrating direct detection of protein-protein interactions with high-dimensional single-cell phenotyping, our workflow provides a mechanistically informed, spatially resolved biomarker of functional immune engagement. Beyond urothelial carcinoma, this platform establishes a generalizable framework for translating spatial signaling biology into predictive tools for immunotherapy response across tumor types. One Sentence SummaryIn situ detection of PD1/PD-L1 interactions between cancer cells and cytotoxic T cells predicts response to immunotherapy in urothelial carcinoma