Development and characterization of a novel autochthonous semi-spontaneous bladder cancer model by pathological evaluation, single cell sequencing and proteomic profiling

Abstract

There is scarce knowledge of the molecular events of recurrence and progression in non-muscle invasive bladder cancer (NMIBC). The heterogeneity of patients’ clinical disease impacts the use of molecular tools and hampers therapeutic strategies. To improve the understanding of tumor development and progression in the native microenvironment, we induced and profiled an autochthonous bladder cancer model. 0.05% OH-BBN was administrated in drinking water to Hgf-Cdk4(R24C) mice for 10 weeks. Histopathology evaluation of tissue, single cell RNA sequencing, and urine and serum proteomics by the Proximity Extension Assay were performed. Therapeutic studies (anti-PD1, CpG ODN) were performed at the NMIBC stage and on a transplanted novel cell line. Exposure of transgenic animals to OH-BBN for 10 weeks led to urothelial NMIBC that differed from exposed wt C57BL/6 mice, while females developed T1 tumors faster than males. Tumors were of Tis/T1 stage with squamous differentiation and within 10 weeks progressed to muscle invasive bladder cancer (MIBC). Single cell sequencing displayed three malignant or premalignant cell clusters with distinct molecular profiles trending towards the luminal or basal subtype and genomic instability. Carcinogen induced bladders presented a clear change in neutrophil, macrophage and fibroblast populations. Anti-PD1 monotherapy at NMIBC did not protect animals from progression, but a possible delay in tumor growth was seen in females. Sex differences in proteomic profiles of urine and serum were obvious, however indicators of myeloid cell recruitment to the tumor microenvironment were shared. Subcutaneous tumors of an outgrown Hgf-Cdk4(R24C) tumor derived cell line were significantly controlled using CpG ODN treatment only in female mice. Hgf-Cdk4(R24C) mice develop NMIBC upon OH-BBN exposure and progress to MIBC. Female animals present more rapid tumor establishment than males. Single cell RNA sequencing revealed the coexistence of heterogeneous tumor cell populations. Sex specific differences appeared in tumor growth, proteomic profiling and therapeutic outcome and should be further explored.