Syngeneic model of carcinogen-induced tumor mimics basal/squamous, stromal-rich, and neuroendocrine molecular and immunological features of muscle-invasive bladder cancer

Background Bladder cancer is a heterogenous disease and the emerging knowledge on molecular classification of bladder tumors could have impact to drive treatment decisions based on molecular subtype. Pre-clinical models representing each subtype are needed to test novel therapies. Carcinogen-induced bladder cancer models represent heterogeneous, immune-competent, pre-clinical testing options with many features found in the human disease. Methods Invasive bladder tumors were induced in C57BL/6 mice when continuously exposed to N-butyl-N-(4-hydroxbutyl nitrosamine) (BBN) in the drinking water. Tumors were excised and serially passed by subcutaneous implantation into sex-matched syngeneic C57BL/6 hosts. Eight tumor lines were developed and named BBN-induced Urothelium Roswell Park (BURP) tumor lines. The BURP lines were characterized by applying consensus molecular classification to RNA expression, histopathology, and immune profiles by CIBERSORT. Two lines were further characterized for cisplatin response. Results Eight BURP tumor lines were established with 3 male and 3 female BURP tumor lines, having the basal/squamous (BaSq) molecular phenotype and morphology. BURP-16SR was established from a male mouse and has a stromal-rich (SR) molecular phenotype and a sarcomatoid carcinoma morphology. BURP-19NE was established from a male mouse and has a neuroendocrine (NE)-like molecular phenotype and poorly differentiated morphology. The established BURP tumor lines have unique immune profiles with fewer immune infiltrates compared to their originating BBN-induced tumors. The immune profiles of the BURP tumor lines capture some of the features observed in the molecular classifications of human bladder cancer. BURP-16SR growth was inhibited by cisplatin treatment, while BURP-24BaSq did not respond to cisplatin. Conclusions The BURP tumor lines represent several molecular classifications, including basal/squamous, stroma-rich, and NE-like. The stroma-rich (BURP-16SR) and NE-like (BURP-19NE) represent unique immunocompetent models that can be used to test novel treatments in these less common bladder cancer subtypes. Six basal/squamous tumor lines were established from both male and female mice. Overall, the BURP tumor lines have less heterogeneity than the carcinogen-induced tumors and can be used to evaluate treatment response without the confounding mixed response often observed in heterogeneous tumors. Additionally, basal/squamous tumor lines were established and maintained in both male and female mice, thereby allowing these tumor lines to be used to compare differential treatment responses between sexes. ### Competing Interest Statement The authors have declared no competing interest. * BaSq : basal/squamous BBN : N-butyl-N-(4-hydroxbutyl nitrosamine) BSA : Bovine serum albumin BURP : BBN-induced Urothelium Roswell Park EMT : Epithelial mesenchyme transition ETM : Experimental tumor model DCs : Dendritic cells GSR : Genomic shared resource IACUC : Institutional Animal Care and Use Committee IHC : Immunohistochemistry LASR : Laboratory Animal Shared Resource LumNS : Luminal Non-Specified LumP : Luminal Papillary LumU : Luminal Unstable MIBC : Muscle-invasive bladder cancer MDS : Multidimensional scaling NE : neuroendocrine NMIBC : Non-muscle invasive bladder cancer PDX : Patient-derived xenograft RIN : RNA integrity number SR : Stromal-rich TILs : tumor-infiltrating lymphocytes Tregs : T regulatory cells