Introduction:

Approximately 80% of patients with advanced bladder cancer do not respond to immune checkpoint inhibitor (ICI) immunotherapy. Therefore, there is an urgent unmet need to develop clinically relevant preclinical models for studying therapeutic resistance in immunocompetent mice. We sought to develop a syngeneic triple knockout (TKO, Trp53, Pten, Rb1) bladder cancer model that results in urothelial tumors in immunocompetent C57BL/6J mice.

Methods:

Fresh mouse bladder urothelial cells from triple-flox (Trp53 ^f/f, Pten ^f/f, Rb1^f/f) mice were dissociated for organoid cultures. Urothelial organoid cells were transfected ex vivo with Ad5CMVCre (Adenovirus Cre recombinase driven by CMV promoter). PCR and western blotting were used for validating the gene deletions in the TKO organoids. The TKO organoids were subcutaneously injected into immunocompetent C57BL/6J and treated with anti-PD1 antibodies. The widely accepted MB49 cells were also injected as a parallel comparison. Immunohistochemistry (IHC) of urothelial markers and immune cell markers (CD8 and PD1) were performed.

Results:

Cre recombination was detected in nearly 100% of TKO organoids as indicated by an mT/mG (membrane tdTomato/membrane EGFP) reporter gene. All three gene deletions (Trp53, Pten, Rb1) were confirmed by PCR and Western blotting analysis in the TKO organoids. Both subcutaneous and orthotopic xenografts demonstrated the expression of urothelial markers (CK7, CK5 and p63), whereas the MB49 xenografts demonstrated no expression of such markers (Figure 1). CD8 (5.4%) cytotoxic T lymphocytes and PD-1 expression (3.8%) were detected in TKO xenografts. Anti–PD-1 immunotherapy demonstrated a mixed pattern of treatment responses (Responders vs Non-Responders) in individual tumors (n=9).

Conclusion:

We generated a syngeneic mouse organoid line with specific gene deletions that constantly establishes urothelial tumors in immunocompetent C57BL/6J mice. Further molecular characterization of tumor cells and microenvironment from ICI resistant tumors may reveal molecular mechanisms of immunotherapy resistance and novel therapeutic targets.

Funding: This research work was supported in part by NIH Grants, K08CA252161(Li, Q), P30CA016056 (NCI Cancer Center Core Support Grant), the Roswell Park Alliance Foundation and the Friends of Urology Foundation.