Abstract 6524: In vivo selection of tumoral microbiota identified pro-metastatic anaerobes: A novel experimental platform allowing mechanistic studies leading to the discovery of bacteria-derived molecular therapeutic targets

Abstract Introduction: Tumoral microbiota is a strong predictor of gastrointestinal cancer outcome. However, our efforts in identifying bacterial molecular drivers of cancer progression are hindered by 1. the lack of efficient anaerobic bacteria culturing system, 2. >90% of human gut microbiota are anaerobes. This predicament limits our capability to conduct mechanistic studies in tumor microbiota research. To fill this gap, we sought to establish a novel experimental system allowing the propagation of metastasis-associated tumoral microbiota by taking advantage of the curated culturable human gut microbiota library. Methods: We established a gnotobiotic system following to a sequential oral gavage of the curated human gut microbiota library in germ-free C57BL/6 mice. Subsequently, the mice underwent intrapancreatic injections of syngeneic KPC pancreatic cancer cells. Liver metastatic foci, primary pancreatic cancer, and the duodenum with their associated bacteria were subject to 16s sequencing and direct culture-based propagation. Results: To validate the feasibility of our experimental platform in identifying bacterial species despite the expected low biomass, we conducted proof-of-concept experiments by intraspenically injecting KPC cancer cell lines in wild-type C57BL/6 mice. Liver metastatic foci, primary pancreas tumors, and the duodenum were subject to 16s sequencing and direct culture. Bray-Curtis dissimilarity matrix-based PCoA plot revealed an apparent dissimilarity between samples from tumor-bearing mice and the corresponding samples from non-tumor-bearing mock-surgery mice in a sample-site agnostic manner, suggesting a systemic impact from the tumor-bearing state. In the tumor-bearing duodenum and liver samples, there was systemic depletion of the Bacteroidetes phylum. Conversely, there is the enrichment of the Firmicutes phylum. Within the Firmicutes phylum, Clostridia species were particularly enriched in the tumor-bearing duodenum and liver metastatic samples, suggesting a biological pressure in the hypoxic tumors and hypoxic liver microenvironment favoring obligate anaerobes such as Clostridia species. Conclusion: Our pilot studies suggest the presence of tumor microenvironment-driven biological selection of bacteria. The observed symbiosis in metastasizing tumors between obligate anaerobes such as Clostridia and cancer cells in the hypoxic tumor microenvironment warrants further functional validation, which is uniquely feasible in our proposed system. In summary, we established a novel experimental platform allowing tumor microbiota mechanistic studies to determine bacteria-derived molecular drivers of cancer progression and metastasis. Citation Format: Adriana Zingone, Senthil Muthuswamy, Norihiro Yamaguchi. In vivo selection of tumoral microbiota identified pro-metastatic anaerobes: A novel experimental platform allowing mechanistic studies leading to the discovery of bacteria-derived molecular therapeutic targets [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 6524.