Immune senescence and antigen presentation defects in BCG-unresponsive bladder cancer.

823 Background: Intravesical Bacillus Calmette-Guérin (BCG) remains the cornerstone therapy for high-risk non–muscle invasive bladder cancer (NMIBC). However, up to 40% of patients experience recurrence or progression following adequate BCG therapy. Identifying molecular determinants of BCG unresponsiveness is crucial for patient stratification and development of alternative intravesical immunotherapies. We aimed to define tumor immune microenvironmental features of BCG unresponsiveness using single-cell and bulk transcriptomic approaches and to validate these findings in independent multi-institutional datasets. Methods: Pre-treatment tumors from 10 high-risk NMIBC patients (5 BCG-responsive, 5 BCG-unresponsive) underwent single-cell RNA sequencing (scRNA-seq). Unsupervised clustering and cell type annotation were performed to identify distinct tumor microenvironmental populations. Differential pathway and gene set enrichment analyses were used to explore key functional differences between groups. Transcriptomic modules derived from scRNA-seq were validated using our institutional microarray cohort (YUHS, n = 54) and multiple external cohorts (CBNUH, LEED, UROMOL, BRS; total > 350 patients) through gene set enrichment, deconvolution, and survival analyses. Results: Thirteen major cell populations were identified. BCG-unresponsive tumors exhibited an increased proportion of GZMK ⁺ CD8⁺ T cells characterized by senescence-like features (elevated GZMK , CXCR4 , reduced GZMB , CXCL13 ) and decreased proliferative activity. Plasmacytoid dendritic cells were markedly depleted, with concomitant suppression of interferon signaling and epithelial antigen presentation pathways. The interferon and antigen presentation modules derived from scRNA-seq were consistently reproduced in both our YUHS microarray data and the external validation cohorts, where high module activity significantly stratified recurrence-free survival (HR = 0.45, p < 0.01). Conclusions: BCG-unresponsive NMIBC exhibits an “immune senescence” tumor microenvironment, defined by dysfunctional GZMK ⁺ CD8⁺ T cells, dendritic cell depletion, and defective epithelial antigen presentation. The reproducibility of these features across multi-cohort transcriptomic datasets underscores their translational value as potential biomarkers for predicting BCG response and guiding development of novel intravesical immunotherapy strategies.