Obligate anaerobes in the gut drive pancreatic cancer metastasis

Introduction: Tumoral microbiota is a strong predictor of gastrointestinal cancer outcome. However, our efforts to identify bacterial molecular drivers of cancer progression are hindered by 1) the lack of an efficient anaerobic bacterial culturing system and 2) the fact that >90% of the 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 to propagate metastasis-associated tumoral microbiota by leveraging a curated, culturable human gut microbiota library. Methods: We established a gnotobiotic system by sequentially administering the curated human gut microbiota library via oral gavage 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 intrapancreatically 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. A Bray-Curtis dissimilarity matrix-based PCoA plot revealed apparent dissimilarity between tumor-bearing mouse samples and corresponding mock-surgery non-tumor-bearing samples, in a sample-site-agnostic manner, suggesting a systemic impact of the tumor-bearing state. Clostridium species were particularly enriched in primary pancreatic tumors and liver metastatic samples, suggesting biological pressure in hypoxic tumors and the hypoxic liver microenvironment that favors obligate anaerobes. Co-injections of KPC cancer cells and Clostridium species promoted liver metastasis more than 10-fold. Moreover, Clostridium-conditioned media also enhanced liver metastasis, suggesting a critical role for bacteria-derived soluble factors in regulating metastasis. Conclusion: The observed symbiosis between metastasizing pancreatic cancer cells and obligate anaerobes warrants further searches of similar co-opted bacteria-cancer interactions in other cancers. Our discovery of pro-metastatic bacteria-derived soluble factors paves the way for developing bacteria-derived, yet live microorganism-free, scalable therapeutics by blocking their interactions with host cells. In summary, we identified novel bacteria-derived soluble factors that strongly drive cancer metastasis.