Abstract 2912: Changes in mouse bladder cancer microbiome associated with exposure to bladder carcinogen N-butyl-N-(4-hydroxybutyl) nitrosamine
Authors: Ginwala Rashida , Franklin Jodie , White James R. , Satyal Uttam , Srivastava Abhishek , Abbosh Philip H. 
Published: July 2021

Background: BBN (N-butyl-N-(4-hydroxybuytl) nitrosamine) is the most commonly employed carcinogen in the classical animal model of bladder cancer based on chemical carcinogenesis of the bladder urothelium. BBN closely recapitulates the histological and genetic characteristics of human bladder cancer and conceptually simulates tobacco exposure as the leading cause of bladder cancer. Little is known about microbial changes that occur during bladder carcinogenesis, but the microbiome is increasingly recognized as an important dimension in both carcinogenesis and cancer treatment. We hypothesized that the bladder tissue microbiome would become altered during the course of BBN carcinogenesis and sought to annotate these changes using 16S metagenomics.

Methods: Starting at 8-12 weeks of age, male (N=33) and female (N=22) C57BL/6 mice were administered 0.05% BBN in drinking water for 12 weeks then switched to regular water. Control mice drank regular water. Bladder was collected pre-exposure, after 6 and 12 weeks of exposure, and at the time of tumor identification (tumors typically detectable between 14-22 weeks from start of treatment using this model). DNA extracted from the bladders was subjected to PCR amplification using primers for the V3-V4 regions of the bacterial 16S rRNA.

Results: Bladder tissue 16S rRNA deep sequencing revealed significant differences in alpha-diversity metrics between the BBN and control groups at 12 weeks including PD_whole_tree, observed species, Fisher's alpha and Margalef indices (p&lt;0.05 for all). BBN treatment appeared to lower bacterial diversity in the bladder. The alpha-diversity scores remained significantly reduced at the time of bladder tumor harvest in the BBN group (p&lt;0.05). Verrucomicrobia was identified as the most abundant phylum across BBN-administered groups. Akkermansia, Clostridium XI, and Veillonella were the most enriched genera identified in the BBN mice. The mucin-degrading bacterial species Akkermansia muciniphila began to be enriched in the BBN group starting at 6 weeks of treatment (control v BBN, p=0.05). Functional assessment of the microbiome using PICRUSt revealed significant upregulation of genes belonging to the pathways related to flagellar assembly and bacterial motility, which could facilitate tumor colonization through intracellular accumulation, and intratumoral motility. Interestingly, coincident with the cessation of BBN treatment at 12 weeks, the differences between the BBN and control groups began to be less pronounced at later time points than those seen at 6 weeks, suggesting a restoration of microbial community upon BBN removal despite the carcinogenesis cascade having started.

Conclusions: Pronounced microbial changes occur within the bladder and bladder tumors induced by BBN in the murine model. This annotation lays the groundwork for potential therapeutic manipulations.

Citation Format: Rashida Ginwala, Jodie Franklin, James R. White, Uttam Satyal, Abhishek Srivastava, Philip H. Abbosh. Changes in mouse bladder cancer microbiome associated with exposure to bladder carcinogen N-butyl-N-(4-hydroxybutyl) nitrosamine [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2912.