An immune-competent, durable MB49-Luc orthotopic bladder cancer model demonstrates bladder-specific tumor immunity for translational immunotherapy research

Background and Objective: Bladder cancer remains a significant health concern in the United States, with approximately 85,000 new cases and 17,000 deaths annually. Nearly 70% of cases present as non-muscle-invasive bladder cancer (NMIBC), commonly treated with transurethral resection followed by intravesical immunotherapy. However, limited and inconsistent responses to current therapies underscore ongoing clinical challenges. To enable effective therapeutic development, a reliable, immune-competent, and longitudinally trackable preclinical model capable of representing the unique immune and physiological features of bladder cancer is essential. Despite decades of effort to refine syngeneic orthotopic bladder cancer models in mice, low tumor take rates and limited in-life study duration remain major challenges. Here, we present a syngeneic orthotopic bladder cancer model with extended in-life monitoring capacity that recapitulates bladder tumor immunity and enables robust immunotherapy evaluation. Method and Result: A luciferase-expressing MB49 cell line was generated via lentiviral transduction. Female C57BL/6 mice were implanted intravesically to establish an orthotopic tumor model. In-life bioluminescence imaging (BLI) using IVIS revealed stable and quantifiable bladder-localized tumor signals for more than 21 days, with a 100% tumor take rate. Tumor presence in the bladder lumen was confirmed by H&E histopathology. Tumor-bearing bladders collected on Days 10 and 21 underwent next-generation sequencing (NGS) and subsequent enrichment analysis. Differential gene expression revealed biomarker signatures reflective of early- and late-stage tumor burden, along with dynamic immune-profiling changes. Pathway enrichment analysis linked these gene-level differences to functional networks governing immune regulation, tumor progression, and bladder cancer physiology. Conclusion: We established an optimized MB49-Luc orthotopic bladder cancer model that preserves immune relevance, provides a prolonged imaging window, and demonstrates distinct molecular transitions during tumor progression. This long-duration, immune-competent platform enables rigorous evaluation of intravesical and immuno-oncology therapeutics and offers meaningful translational insights to support next-generation NMIBC immunotherapy development.