Modeling primary immunotherapy resistance in metastatic bladder cancer: a syngeneic, bioluminescent mouse model

Approximately 90% of bladder cancer deaths are due to distant metastases rather than local tumor effects. The current first-line systemic treatment for metastatic bladder cancer (mBC) is chemoimmunotherapy or immunotherapy with pembrolizumab plus enfortumab vedotin (EV). However, most mBC patients treated with chemoimmunotherapy or pembrolizumab/EV do not respond or eventually relapse, highlighting the critical need for robust immunocompetent animal models to elucidate the mechanisms of primary and acquired resistance. We previously generated a syngeneic murine cell line CMV-TRP (triple knockout of Trp53, Rb1, Pten) via ex vivo transduction with adenovirus (Ad5CMVCre). To establish an mBC model, the TKO cells were effectively labeled with a lentiviral luciferase and GFP double-expressing reporter and injected into tail veins of C57 BL/6J mice. Tail vein injection of TKO cells reliably established distant metastases with lung and bone lesions. In immunotherapy experiments, mice injected with TKO-labeled cell lines were randomly treated with an anti-PD-1 or control IgG2a antibody. All mice developed lung and/or bone (hind limb or sacrum) metastases. There was no difference in tumor bioluminescence between the control group and anti-PD-1 group (median proton/second 6.94 × 10⁸ vs. 4.32 × 10⁸, p = 0.85). Kaplan-Meier analysis showed no difference in median survival between the control group and anti-PD-1 group (19 days vs. 20 days, p = 0.47). Histology and immunohistochemical profile of lung and bone metastases revealed high-grade basal-like urothelial carcinoma, closely resembling the profile observed in subcutaneous tumor. No significant changes in immune cell infiltrations (CD4⁺, CD8⁺, or F4/80⁺) between groups may explain anti-PD-1 immunotherapy resistance. Therefore, the novel TKO metastatic model represents a useful and reproducible tool for studying tumor-cell dissemination, bone/lung metastasis, and the underlying mechanisms of anti-PD-1 immunotherapy resistance.