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

Abstract

Introduction: Bladder cancer is a heterogenous disease and the emerging knowledge on molecular classification of bladder tumors may impact 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-hydroxybutyl)-nitrosamine (BBN) in the drinking water. Tumors were excised and serially passed by subcutaneous implantation into sex-matched syngeneic C57BL/6 hosts. Eight lines were named BBN-induced Urothelium Roswell Park (BURP) tumor lines. 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.

Discussion: The BURP 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.

Keywords: BBN carcinogen-induced; CIBERSORT; N-butly-N-(4-hydroxybutyl)-nitrosamine; bladder cancer molecular subtype; cisplatin response; immune tumor microenvironment; immune-intact mouse model; male and female sex disparity.

Figure 1

Graphical representation of methods. Created in BioRender.com.

Figure 2

Generation of histologically diverse BURP tumor lines. H&E of original high-grade muscle-invasive tumors (BBN Donor Tumor) in male and female C57BL/6 mice exposed to 0.1% BBN. Representative H&E from each established BURP tumor lines from each original BBN Donor Tumor, Scale bar is 100µm.

Figure 3

Molecular characterization of BURP tumor lines. (A) Multidimensional scaling (MDS) analysis of gene expression in the eight BURP tumor lines. Each data point represents an individual tumor with different colors representing different BURP tumor lines. Male lines are indicated by a triangle and female lines by a circle. The dotted lines mark three different clusters. The x and y axis depict eigenvalues in 2 dimensions (Dim1 and Dim2). (B) Heatmap of Molecular Consensus classifiers from Kamoun et al., 2020 for the BURP tumor lines. The color represents scaled correlation coefficient values between the BURP tumor line transcriptome data and the Consensus classifiers. Red represents higher correlation, and blue represents lower correlation.

Figure 4

IHC of differentiation markers in the BURP lines with Ba/Sq molecular subtype. Serial sections of BURP-12, -17, -18, -21, -24, -25 were stained with H&E, TP63 IHC, FOXA1 IHC, and GATA3 IHC. Blue bar indicates male models and pink bar indicates female models. Scale bar is 50µm.

Figure 5

IHC of differentiation markers in the BURP-16SR and BURP-19NE lines. Serial sections were stained with H&E, TP63 IHC, FOXA1 IHC, GATA3 IHC, Synaptophysin IHC, and Vimentin IHC. Normal bladder control from BURP-16SR allograft is a control for the IHC staining. Scale bar is 50µm.

Figure 6

Immune contexture of BURP models. Heatmap of scaled estimated fraction of 25 immune cell signatures across the eight BURP tumor lines. The immune signatures are estimated by applying CIBERSORT deconvolution method using the ImuCC input signature. Red represents a higher scaled estimated fraction and blue represents a lower scaled estimated fraction.

Figure 7

Comparison of the estimated fraction of different immune populations in different BURP tumor lines lines obtained by CIBERSORT. (A) polarized forms of macrophages. (B) naïve and memory B-cells. (C) DC immature and activated. (D) naïve, activated, memory CD8Tcells (E) naive and memory CD4 T-cells. (F) CD4+ T cell helper subpopulations.

Figure 8

Response of BURP-16SR and BURP-24BaSq tumor lines to cisplatin treatment and regulon activity. (A, B) Cisplatin treatment (10mg/kg, IV) was initiated when tumors reached 200 mm³. Mice were treated on days 0, 7, 14, and 21 (indicated by blue arrows) and tumor growth is determined by caliper measurements twice a week. BURP-16SR n=10 each cohort, * growth curve p<0.01 unpaired t-test. (B) BURP-24BaSq n=9 cisplatin and n=8 vehicle cohorts, NS= not significant difference in growth curve p=0.10 unpaired t-test. (C) Unsupervised clustering of regulon activity of BURP-16SR and BURP-24BaSq models. The regulons evaluated are identified to be relevant to bladder cancer as represented in Kamoun et al. The color represents differential regulon activity, with red representing active status and blue representing inactive status.