Abstract 30: Establishment of a chemical carcinogen-induced bladder cancer model using healthy canine bladder organoids

Abstract Background Canine bladder cancer (BC) accounts for approximately 2% of all spontaneous canine cancers, and the majority (over 90%) of these are moderate to highly invasive bladder cancers. In terms of biological behavior, canine BC is said to be similar to the cytological and molecular characteristics of human muscle-invasive BC, especially high-grade invasive transitional cell carcinoma. In recent years, cancer research in dogs has been used to define genetic and environmental risks and evaluate prevention and treatment approaches that will benefit not only dogs but also humans. An organoid is a miniaturized and simplified version of an organ produced ex vivo in three dimensions, mimicking the key functional, structural, and biological complexity of the actual organ. Organoids are attracting attention as complementary models to traditional animal models, and are often used in human and animal cancer research. In the current study, we developed a carcinogen-induced BC organoid model using organoids generated from healthy canine bladder tissue. Materials and methods Canine normal bladder organoids (CNBOs) were established using bladder epithelial cells collected using a urinary catheter from a healthy beagle dog in a non-invasive manner. The CNBOs were treated with 1 µM 2-Acetylaminofluorene (2-AAF), a known bladder carcinogen, and then transplanted subcutaneously into immunodeficient mice. As a result, a mass was formed, confirmed to be a benign bladder tumor (canine bladder benign tumor: CBB) by histopathological analysis. When organoids were developed again from CBB (CBBO) and transplanted under the skin of immunodeficient mice, the tumor was reformed. The re-formed tumor was confirmed to be BC (Canine bladder carcinoma: CBC), and organoids were created from CBC (CBCO), and comparative analysis was conducted on CNBO, CBBO, and CBCO. Results The expression of CK5 and UPK3A, was confirmed in CNBO, CBBO, and CBCO. In addition, observations using electron microscopy revealed findings suggestive of a normal bladder in CNBO and findings suggestive of a malignant tumor in CBCO. RNA sequencing analysis showed significant differences in gene expression in CNBO, CBBO, and CBCO. Gene set enrichment analysis revealed that the epithelial-mesenchymal transition pathway was enhanced in CBCO compared to CNBO. CNBO showed high cell proliferation in cell proliferation assays. The anti-cancer drug sensitivity test showed different sensitivities to treatment with vinblastine, mitoxantrone, carboplatin, and lapatinib. Discussions In this study, chemical carcinogen-induced canine BC organoids were generated by treating CNBO with bladder carcinogen (2-AAF) and transplanting it into mice twice. In the future, we plan to perform cancer-related gene mutation analysis and proteomic analysis of CNBO, CBBO, and CBCO to clarify further the detailed molecular mechanisms of carcinogenesis. Citation Format: Yuko Nagashima, Haru Yamamoto, Mohamed Elbadawy, Amira Abugomaa, Tatsuya Usui, Kazuaki Sasaki. Establishment of a chemical carcinogen-induced bladder cancer model using healthy canine bladder organoids [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 30.