Investigating the impact of regulatory B cells and regulatory B cell-related genes on bladder cancer progression and immunotherapeutic sensitivity.

Background: Regulatory B cells (Bregs), a specialized subset of B cells that modulate immune responses and maintain immune tolerance in malignant tumors, have not been extensively investigated in the context of bladder cancer (BLCA). This study aims to elucidate the roles of Bregs and Breg-related genes in BLCA.

Methods: We assessed Breg infiltration levels in 34 pairs of BLCA and corresponding paracancerous tissues using immunohistochemical staining. We conducted transwell and wound healing assays to evaluate the impact of Bregs on the malignant phenotype of SW780 and T24 cells. Breg-related genes were identified through gene sets and transcriptional analysis. The TCGA-BLCA cohort served as the training set, while the IMvigor210 and 5 GEO cohorts were used as external validation sets. We employed LASSO regression and random forest for feature selection and developed a risk signature using Cox regression. Primary validation of the risk signature was performed through immunohistochemical staining and RT-qPCR experiments using the 34 local BLCA samples. Additionally, we employed transfection assays and flow cytometry to investigate Breg expansion ability and immunosuppressive functions.

Results: Breg levels in BLCA tissues were significantly elevated compared to paracancerous tissues (P < 0.05) and positively correlated with tumor malignancy (P < 0.05). Co-incubation of SW780 and T24 cells with Bregs resulted in enhanced invasion and migration abilities (all P < 0.05). We identified 27 Breg-related genes, including CD96, OAS1, and CSH1, which were integrated into the risk signature. This signature demonstrated robust prognostic classification across the 6 cohorts (pooled HR = 2.25, 95% CI = 1.52-3.33). Moreover, the signature exhibited positive associations with advanced tumor stage (P < 0.001) and Breg infiltration ratios (P < 0.05) in the local samples. Furthermore, the signature successfully predicted immunotherapeutic sensitivity in three cohorts (all P < 0.05). Knockdown of CSH1 in B cells increased Breg phenotype and enhanced suppressive ability against CD8 + T cells (all P < 0.05).

Conclusions: Bregs play a pro-tumor role in the development of BLCA. The Breg-related gene signature established in this study holds great potential as a valuable tool for evaluating prognosis and predicting immunotherapeutic response in BLCA patients.