SPP1⁺ Macrophage-POSTN⁺ Fibroblast-Endothelial Triad Dictates Immunotherapy Response in Bladder Cancer

Although immunotherapy has shown promise in improving outcomes for bladder cancer (BCa) patients, treatment responses remain highly variable. A comparative examination of the tumor microenvironment (TME) between responders and non-responders may reveal key resistance mechanisms and identify potential therapeutic targets. We integrated spatial transcriptomics, single-cell RNA sequencing, and multiplexed immunofluorescence to characterize spatial structures within the TME that influence response to anti-PD-1 therapy in BCa patients. In non-responders, we observed an accumulation of stem-like malignant epithelial cells with high MYBL2 expression near the tumor boundary. Furthermore, we identified a spatial triad structure-composed of SPP1⁺ tumor-associated macrophages (TAMs), POSTN⁺ cancer-associated fibroblasts (CAFs), and endothelial cells-located at the tumor periphery. This structure was associated with T-cell exclusion and reduced efficacy of immune checkpoint blockade. In a preclinical model, inhibiting SPP1 enhanced the response to anti-PD-1 therapy, resulting in reduced CAF infiltration and increased recruitment of cytotoxic T cells. Our study reveals a triad cellular structure mediated by SPP1⁺ TAMs, POSTN⁺ CAFs, and endothelial cells that contribute to immunotherapy resistance in BCa. Targeting this structure, particularly through SPP1 blockade, represents a promising strategy to augment the efficacy of immune checkpoint inhibitors.