Characterization of microbial and host requirements for colonic biofilm assembly and biofilm-mediated colon tumorigenesis

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Sporadic colorectal cancer (CRC) remains a global health burden as one of the leading causes of cancer and cancer-related deaths, and, strikingly, is also emerging as an acute health crisis in younger patients in the US under the age of 50. These data suggest that novel efforts to identify contributors to the changing landscape of CRC are urgently needed. In this R00 proposal, we seek to dissect the assembly and tumorigenic mechanisms behind invasive, polymicrobial colonic biofilms, which were previously established in the Sears laboratory to be a nearly universal feature of CRC tumors proximal to the hepatic flexure. Biofilms were also identified on a subset (10-15%) of healthy patients without CRC, in whom biofilms were associated with early, procarcinogenic changes, positing a role for bacterial biofilms in CRC development. These biofilms were also associated with elevations in polyamine metabolites in mass spectrometry-based analyses and induced a strong Interleukin-17 (IL-17) response in the mice by one week, a cytokine signature associated with a worse prognosis in human CRC. Tissue slurries made from human biofilms re-assembled into biofilms in the distal mouse colon; some - but not all – of these slurries were directly tumorigenic in germ-free ApcMin/+ mice by 10-15 weeks. Extensive culturing of one tumorigenic slurry following inoculation into mice revealed 35 bacterial isolates that, when re-inoculated into additional mice, recapitulated the tumorigenesis of the original patient tumor slurry. Our specific aims seek to build on these preliminary studies. Specific Aim (SA) 1 will identify the critical bacterial species within the 35 isolate mixture that are responsible for biofilm formation and tumorigenesis. SA2 will test the hypothesis that IL-17 and other immune parameters are critical mediators of biofilm formation and biofilm-mediated tumorigenesis utilizing a combination of single-cell RNA sequencing, flow cytometry, and qRT-PCR. Therapeutic modulation with an IL-17 antibody or knockout mice will be used to confirm the requirement of IL-17 in biofilm formation and biofilm-mediated tumorigenesis. SA3 will utilize advanced mass spectrometry methods to determine metabolites associated with biofilms in both patient and mouse tissues and the localization of these metabolites in select samples. The experiments in this proposal will provide valuable information regarding the host and microbial requirements for biofilm formation and biofilm-mediated tumorigenesis that may lead to potential therapeutic targets or biomarkers for biofilms.