The dynamics of the inflammatory response during BBN-induced bladder carcinogenesis in mice

Abstract

BackgroundBladder cancer (BC) is the most common malignant disease of the urinary tract. Recurrent high grade non muscle invasive BC carries a serious risk for progression and subsequent metastases. The most common preclinical mouse model for bladder cancer relies on administration of N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN) to mice. BBN-induced tumors in mice recapitulate the histology of human BC and were characterized with an overexpression of markers typical for basal-like cancer subtype in addition to a high mutational burden with frequent mutations in Trp53, similar to human muscle invasive BC.MethodsBladder cancer was induced in C57BL/6J male mice by administering the BBN in the drinking water. A thorough histopathological analysis of bladder specimen during and post BBN treatment was performed at 2, 4, 16, 20 and 25 weeks. RNA sequencing and qPCR was performed to assess the levels of expression of immunologically relevant genes at 2 weeks and 20 weeks during and post BBN treatment.ResultsWe characterized the dynamics of the inflammatory response in the BBN-induced BC in mice. The treatment with BBN had gradually induced a robust inflammation in the first 2 weeks of administration, however, the inflammatory response was progressively silenced in the following weeks of the treatment, until the progression of the primary carcinoma. Tumors at 20 weeks were characterized with a marked upregulation of IL18 when compared to premalignant inflammatory response at 2 weeks. In accordance with this, we observed an increase in expression of IFNγ-responsive genes coupled to a pronounced lymphocytic infiltrate during the early stages of malignant transformation in bladder. Similar to human basal-like BC, BBN-induced murine tumors displayed an upregulated expression of immunoinhibitory molecules such as CTLA-4, PD-L1, and IDO1 which can lead to cytotoxic resistance and tumor escape.ConclusionsDespite the recent advances in bladder cancer therapy which include the use of checkpoint inhibitors, the treatment options for patients with locally advanced and metastatic BC remain limited. BBN-induced BC in mice displays an immunological profile which shares similarities with human MIBC thus representing an optimal model for preclinical studies on immunomodulation in management of BC.

Single-cell RNAseq and longitudinal proteomic analysis of a novel semi-spontaneous urothelial cancer model reveals tumor cell heterogeneity and pretumoral urine protein alterations

“…Among pre-clinical models used to study urothelial cancer, are wild type animals exposed to OH-BBN [N-butyl-N-(4-hydroxybutyl) nitrosamine] [17], genetically engineered murine (GEM) models [18] or allografted tumorigenic cell lines such as MB49 (DMBA-in vitro transformed urothelial cells) [19] and MBT-2 (FANFT-induced bladder tumor in C3H/He mouse) [20]. The subtype and grade of OH-BBN induced tumors depend on genetic background, strain and sex [16,21]. In wild type C57BL/6 mice, prolonged exposure to OH-BBN (20 weeks) is required to induce MIBC that represents the molecular subtypes and mutational landscape found in humans [22][23][24].…”

“…In wild type C57BL/6 mice, prolonged exposure to OH-BBN (20 weeks) is required to induce MIBC that represents the molecular subtypes and mutational landscape found in humans [22][23][24]. However, commonly only male mice of the C57BL/6 strain are used in such studies, disregarding the sex-biased disease outcome and response to therapy [16,21,22]. On the other hand, most GEM models acquire urothelial hyperplasia and papillary non-progressive NMIBC instead of MIBC [25], and do not reflect the heterogeneous mutational landscape and gene expression profile of human tumors [16,22,23].…”

The influence of age and sex on the pre-treatment immune microenvironment of a carcinogen induced murine model of bladder cancer

“…In a recent report, Degoricija et al, showed the immunologic alterations and higher expression of immune checkpoint molecules in tumors from young male mice at 20 weeks following exposure to BBN carcinogen. 31 Although this is one of the few reports that have characterized some local inflammatory changes accompanying carcinogens, this study was conducted in young male mice. Based on our observation on increased PD-L1 protein expression in the urothelial and endothelial cells, it can be speculated that this may result from BBN induced DNA damage and activation of cellular IFN pathways.…”

Ppargdrives luminal differentiation and luminal tumor formation in the urothelium

“…BBN is a nitrosamine that is metabolized in the liver to N-butyl-N-(3-carboxypropyl)nitrosamine (BCPN), which is found in tobacco products (Bonfanti et al, 1988;Cohen, 1998;Mirvish, 1995). Five months of exposure to BBN induces basal subtype tumors, however short exposure induces a potent inflammatory response that resolves by about one month and recedes as tumors form (Degoricija et al, 2019).…”