Hypothesis / aims of study
Intravesical Bacillus Calmette–Guérin (BCG) immunotherapy is the gold standard adjuvant treatment for non-muscle invasive bladder cancer (NMIBC) following tumour resection [1]. However, up to 70% of patients develop bothersome lower urinary tract symptoms (LUTS), including urgency and frequency, significantly impairing quality of life [2]. The mechanisms underlying these symptoms remain poorly understood, limiting targeted therapeutic strategies [3]. We hypothesised that BCG treatment induces LUTS via inflammation-driven alterations in bladder contractile function. The aim of this study was to characterise bladder functional and molecular changes in a preclinical BCG model and to evaluate the safety and efficacy of the β3-adrenoceptor agonist, mirabegron, as a potential therapy for BCG-induced LUTS.
Study design, materials and methods
An in vivo BCG mouse model was established using female C57BL/6 mice receiving weekly intravesical instillations of BCG (1.2 x 107 CFU/mouse/week) or phosphate-buffered saline (sham) for six weeks. Bladders were analysed one day after the final instillation using flow cytometry (n = 8/group) and bulk RNA sequencing (n = 3/group) to assess immune cell infiltration and transcriptional changes. Additionally, ex vivo bladder compliance and the frequency of spontaneous contractions during bladder distension (0-50 mmHg) were assessed (n = 3/group).
In vitro studies were conducted in human and murine bladder cancer cell lines (UM-UC3, T24, MB49) to assess the effects of mirabegron (1–30 µM) on BCG-induced cytotoxicity, senescence, and internalisation.
Bladder function was assessed ex vivo using organ bath contractility experiments in sham, BCG, and BCG + mirabegron groups (n = 7/group). Mirabegron was administered via drinking water (10 µg/mL) throughout the BCG treatment period.
Results
BCG treatment in mice induced a robust immune response, with significant increases in CD45⁺ leukocytes, CD4⁺ T cells, neutrophils, and natural killer cells, alongside enrichment of inflammatory HALLMARK pathways relevant to BCG efficacy including interferon (IFN)-α response, IFN-γ response, and inflammatory response. RNA sequencing also revealed significant downregulation of β3-adrenoceptor (Adrb3) expression in BCG-treated mice compared to sham (p = 0.002, log₂FC = –1.26).
Functionally, BCG treated mice exhibited increased spontaneous bladder contractile activity ex vivo (p < 0.01). However, there were no significant differences between BCG and BCG + mirabegron groups in carbachol- or potassium chloride-induced contractile responses (p > 0.05).
In vitro, mirabegron did not alter BCG-induced cytotoxicity, senescence, and internalisation across UM-UC3, T24, and MB49 cells (p > 0.05)
Interpretation of results
The observed increase in immune cell infiltration, and enrichment of inflammatory pathways indicates that this model recapitulates key features of the inflammatory response associated with BCG therapy. BCG treatment decreased β3-adrenoceptor expression and increased spontaneous bladder activity during bladder distension ex vivo, suggesting inflammation-associated alterations in bladder function that may contribute to LUTS. However, chronic mirabegron treatment during BCG exposure did not modify agonist-induced contractility, suggesting that BCG-induced effects on detrusor contractile function during distension may be driven by neurogenic pathways rather than direct changes in myogenic contractility of the detrusor. Importantly, mirabegron did not compromise BCG-induced anti-tumour activity in vitro, supporting its safety as a potential adjunct therapy in patients with BCG-induced LUTS.