Urinary microbiome and metabolomic profiling reveal propionic acid as an enhancer of BCG immunotherapy in bladder cancer.

Bladder cancer (BC) is the tenth most common malignancy worldwide, with non-muscle invasive bladder cancer (NMIBC) accounting for approximately 75% of cases, and Bacillus Calmette-Guérin (BCG) remaining the standard intravesical therapy. In light of global BCG shortages, this study aimed to identify potential strategies to enhance BCG efficacy by integrating urinary microbiome profiling with targeted short-chain fatty acid (SCFA) metabolomics. Urine samples collected from BC patients before and after BCG instillation were analyzed to determine BCG-associated microbial and metabolic changes, and the functional role of the key metabolite propionic acid (PA) was evaluated through in vitro experiments using MB49 and MBT2 murine BC cell lines and in vivo mouse BC models treated with BCG plus PA. Flow cytometry was used to quantify tumor-infiltrating CD4+ and CD8+ T cells to elucidate immune-related mechanisms. BCG treatment significantly reshaped urinary microbial composition, dominant flora, and fatty acid metabolic patterns, and notably increased urinary PA levels. Functional assays demonstrated that PA markedly augmented the antitumor activity of BCG, potentially by promoting CD4+/CD8+ T-cell infiltration into tumor tissues. Collectively, these findings indicate that BCG modulates the urinary microbiome and SCFA metabolic landscape in BC patients, and that PA enhances BCG efficacy by boosting T-cell-mediated immune responses, highlighting PA-BCG combination therapy as a promising therapeutic strategy for BC.