Summary This multi-Pl R01 is submitted in response to NCI FOA PAR-20-062 Co-infection and Cancer and includes 2 seasoned, collaborative Pis with complementary expertise in microbial carcinogenesis (Richard Peek and James Fox). H. pylori confers the highest known risk for gastric adenocarcinoma. However, studies by the CoPis and others have demonstrated that gastric microbiota populations affect cancer risk in synergy with H. pylori. We have full access to a unique longitudinal, prospective human cohort in Colombia where gastric adenocarcinoma and H. pylori infections are endemic. Full clinical, endoscopic, and histologic data are available at baseline and at each interval follow-up out to 26 years, including frozen gastric tissue for microbiota analysis and culture from the 20- and 26-year timepoints from persons who either progressed histologically or remained stable, providing a unique opportunity to define disease mechanisms. We also have access to prospectively obtained gastric tissue from patients at Stanford with or without premalignant lesions, allowing us to extend these studies into a US population. Our laboratories have developed models that closely recapitulate the gastric niche to define mechanisms of carcinogenesis within the context of H. pylori and the microbiota. Dr. Fox has utilized germ-free (GF) INS-GAS mice to demonstrate 1) H. pylori accelerates carcinogenesis in mice harboring a gastric microbiota compared to GF mice, and 2) colonization with bacteria differentially represented in high vs. low cancer risk populations modifies the ability of H. pylori to induce gastric injury. Dr. Peek has developed complex primary gastroid:macrophage:T cell systems to demonstrate that H. pylori drives oncogenic signaling in a cell- and strain-specific manner and that non-H. pylori gastric species successfully colonize these models. Collectively, our scientific scope, available resources including cutting-edge metabolomics, and innovative model systems will allow us to fully address the hypothesis that progression to gastric cancer is influenced not only by H. pylori virulence constituents, but also by prolonged interactions with members of the gastric microbiota. which can modulate immune responses. We will address this hypothesis via these Aims. Aim 1: Identify and define components of the gastric microbiota in persons who did or did not progress towards gastric cancer using Whole Metagenome Shotgun (WMS} sequencing Aim 2: Utilize innovative ex vivo systems to define the carcinogenic potential of gastric microbiota species and prioritize candidates for more definitive in vivo studies of gastric cancer Aim 3: Utilize novel germ-free rodent models and metabolomics to define causality of high priority gastric microbiota species and corresponding immune responses linked to disease progression within the gastric niche