Multiplex Immunofluorescence-defined CXCL13-associated TLS-like Niches Predict Outcome in Bladder Cancer

PURPOSE: Spatially organized tertiary lymphoid structure (TLS)-like niches may provide prognostic and therapeutic information beyond individual immune mediators such as CXCL13 in bladder cancer (BC). We evaluated the clinical and translational significance of multiplex immunofluorescence-defined TLS-like structures (mTLS) in BC. EXPERIMENTAL DESIGN: We analyzed 469 clinically annotated BC tumors using 41-plex multiplex immunofluorescence with the PhenoCycler platform. Bulk RNA sequencing, CIBERSORTx deconvolution, Cox modeling with CXCL13 as a continuous variable, and spatial neighborhood analysis were used to define mTLS-associated immune programs. Orthotopic BC mouse models treated with cisplatin and/or recombinant Cxcl13 followed by anti-PD-L1 blockade were analyzed by flow cytometry. RESULTS: mTLS were identified in 22.2% of tumors and were not apparent on routine histopathology. mTLS positivity was associated with immune activation, improved survival, and favorable response patterns in a small PD-L1 blockade-treated subset. In Cox models, mTLS remained independently associated with outcome, whereas continuous CXCL13 expression was not significant. Spatial profiling showed CXCL13/CXCR5-enriched B- and T-cell neighborhoods, including CXCL13-expressing and CXCR5-positive CD8+ T-cell subsets. In mouse models, cisplatin plus exogenous Cxcl13 was associated with lymphocyte infiltration, enrichment of CD8+ PD-1+ and CD8+ Cxcr5+ Cxcl13+ T-cell states, and sustained tumor control after PD-L1 blockade. CONCLUSIONS: mIF-defined mTLS identify a clinically relevant spatial immune architecture linked to favorable outcome and therapeutic responsiveness in BC. These findings support prospective evaluation of mTLS as a spatial biomarker for risk stratification and treatment sequencing, with CXCL13/CXCR5 features interpreted as contextual companion markers rather than standalone determinants.