A High Density of Impaired Tertiary Lymphoid Structures Is Associated with Higher Tumor Stage and Recurrence in Non–Muscle-Invasive Bladder Cancer

Tertiary lymphoid structures (TLS) have emerged as critical immune niches within the tumor microenvironment across various cancers. However, their structural organization and functional roles in non-muscle-invasive bladder cancer (NMIBC) remain poorly characterized. In this study, we comprehensively characterized TLS in NMIBC using en bloc resected primary diagnostic specimens, which enabled high-resolution spatial mapping and quantitative histological assessment across a large cohort of primary tumors (97 patients). Compared with TLS in inflammation-driven cystitis, tumor-associated TLS showed heterogeneous spatial organization, altered immune composition, and disrupted follicular dendritic cell (FDC) networks with reduced high endothelial venules density. TLS density increased with NMIBC grade and stage and correlated with higher recurrence risk and shorter progression-free survival in multivariable analysis. Paired analysis of primary and recurrent tumors showed a reduction in FDC density and a shift from M1 to M2 differentiation in tumor recurrences, suggesting the development of an immunosuppressive microenvironment. Single-cell RNA sequencing of TLS from NMIBC and cystitis samples revealed impaired germinal center activity in tumor-associated TLS, with asynchronous B-cell maturation and reduced B-cell interactions with both T follicular helper cells and FDC. Tumor-associated TLS also showed increased myeloid infiltration and altered dendritic-cell function, including reduced MHC class II antigen presentation and downregulation of co-stimulatory (CD86-CD28) and migration-related (CD99) pathways compared to TLS from cystitis samples. Overall, we found that NMIBC-associated TLS are structurally and functionally impaired, which may limit effective local immune responses and suggests they may be relevant as biomarkers and targets for immunomodulatory therapy in bladder cancer.