Nonmuscle-invasive bladder cancer (NMIBC) accounts for ∼75% of bladder cancer diagnoses [1], but 10% to 30% of NMIBC cases will recur or progress to the muscle-invasive form of bladder cancer (MIBC) [2]. Recurrence and progression can be prevented through transurethral resection of all visible tumors, which, for intermediate- and high-risk NMIBC patients, is usually followed by intravesical adjuvant therapy with the Bacillus Calmette-Guérin (BCG) vaccine [1,2]. Intravesical adjuvant therapy most commonly comprises 6 weekly BCG instillations (BCG induction phase), followed by cycles of 3 weekly BCG instillations on months 3, 6, and 12 for intermediate-risk patients or on months 3, 6, 12, 18, 24, 30, and 36 for high-risk patients (BCG maintenance phase) [3]. This regimen (induction + maintenance) reduces the risk of NMIBC recurrence and progression to MIBC by 40% and 27%, respectively [4,5].

Although the use of BCG as an adjuvant immunotherapy for NMIBC was approved by FDA in 1990, its mechanisms of action are not yet completely understood. The current understanding of BCG's mechanism of action involves BCG internalization by bladder cancer cells, enhanced antigen presentation, cytokine production, and immune-cell recruitment, leading to immune-mediated toxicity against cancer cells [6]. However, adjuvant BCG is not always effective [7], suggesting its mechanism of action has additional overseen factors. Identifying these additional factors is critical to maximize BCG indication in patients more likely to benefit from therapy and to develop combined therapeutic approaches for NMIBC.

Since the discovery that the healthy bladder is not sterile, evidence is mounting regarding the link between bladder microbes and urological conditions [8], including bladder cancer [9]. However, associations between bladder microbiota and bladder cancer have been contradictory, and we are still far from underpinning the role of bladder-dwelling microbes in bladder cancer pathogenesis and response to treatment. This is especially pressing for NMIBC patients treated with BCG, as the introduction of live bacteria in the bladder may affect the bladder microbial ecosystem, with an outcome that is likely linked to BCG efficacy, as proposed previously [10].

Most studies investigating the association between bladder microbiota and bladder cancer are cross-sectional case-control studies [9], with a single longitudinal study evaluating the impact of intravesical therapy with BCG or chemotherapy on the bladder microbiota of NMIBC patients [11]. In addition, the majority of the aforementioned cross-sectional case-control studies evaluated the urinary microbiota as a whole using voided urine samples rather than specifically analyzing the bladder microbiota [9], which can only be assessed by urine collection via transurethral catheterization or suprapubic aspiration [12].

In this study, we collected catheterized urine samples from males with intermediate/high-risk NMIBC and nononcological male patients to compare cross-sectionally their bladder microbiota and identify bladder microbiota differences specific to bladder cancer patients. We also collected urine samples from NMIBC patients during and after BCG induction to evaluate longitudinally the impact of BCG intravesical instillations on the bladder microbiota and the influence of the bladder microbiota on BCG efficacy.