Spatial Immune Profiling and AI-Based Classifiers Identify Predictors of BCG Therapy Outcomes in High-Risk Non-Muscle-Invasive Bladder Cancer

<b>Background/Objectives</b>: High recurrence rates and intensive lifelong surveillance make bladder cancer among the costliest malignancies to treat. Although Bacillus Calmette-Guérin (BCG) immunotherapy is the standard treatment for high-risk non-muscle-invasive bladder cancer (NMIBC), up to 50% of patients fail to respond, and predictive biomarkers are lacking. Molecular profiling has established three BCG response subtypes (BRS1-3), with BRS3 characterized by an immunosuppressive, BCG-resistant phenotype; however, these features have not been validated at single-cell spatial resolution. <b>Methods</b>: We applied imaging mass cytometry (IMC) to 82 BCG-treated high-risk NMIBC samples and performed (i) single-cell IMC with unsupervised clustering to identify phenotypic cell clusters and quantify cluster abundances and (ii) a convolutional neural network-based gated attention multiple instance learning model trained on IMC images (IMC-GA-MIL) to predict BCG response. Cluster abundances were summarized using II (immune composition within the immune compartment), TT (tumor phenotypic composition), and IT (immune/stromal abundance relative to tumor cells) indices. <b>Results</b>: Single-cell IMC identified 18 distinct phenotypic cell clusters. In BCG responders, immune cells localized within the tumor compartment were enriched and independently protective (HR 0.67, 95% CI 0.49-0.92). BCG nonresponse was associated with a higher abundance of fibroblast-dominant clusters relative to tumor cells (IT index). Plasma cell-dominant clusters were the strongest predictors of progression (II index HR 2.28, 95% CI 1.37-3.79; IT index HR 1.25, 95% CI 1.06-1.48). The IMC-GA-MIL model predicted BCG response with 90% accuracy (9/10) and identified myeloid- and T-cell-associated marker patterns involving CD14, CD11b, CD68, CD8, and FOXP3 as the most informative contributors. <b>Conclusions</b>: Spatial single-cell profiling and IMC-GA-MIL identify spatial immune and stromal features associated with BCG failure. However, findings from both analyses should be considered exploratory and will require validation in larger, independent cohorts.