Functional interrogation of pancreatic cancer resident microbes reveals their role in host modulation

Tumor resident microbes are a well-recognized component of the tumor microenvironment. Microbial subcellular location across tumors along with their functionality remains to be determined. Bulk microbial profiling techniques lack subcellular and spatial resolution and ultimately cannot distinguish between microbial signals or live microbial presence. To address these limitations, we performed orthogonal methods for functional microbial-host characterization. We first developed advanced quantitative fluorescent imaging methodologies that allows visualization of microbial cellular compartmentalization across three different tumor types (total n=30). Using this methodology, we performed spatial microbial transcriptomics at the regional and single cell levels to determine microbial distribution and to interrogate microbial regulation of tumor cell signaling in human pancreatic tumors (n=55). To confirm presence of viable microbes, we performed multiplexed culturomics of patient tumors and normal adjacent tissue specimens (n=80), followed by Whole Genomic Sequencing (WGS) analysis. We tested the effect of the isolated clinical strains on tumor cell signaling pathways with in vitro co-culture assays, and upon genetic fluorescent labelling we defined their role on in vivo tumor growth in murine models. These experiments confirmed their role in promoting tumors, driving resistance to therapeutics and modulation of host signaling mechanisms. Overall, our results identified several pathways under microbial regulation within cancer cells that can drive immune evasion through impaired antigen presentation. In summary, using multiple complimentary novel methodologies we characterize the microbial niche of tumors (MiNT) that uncover microbial regulation of host cell signaling and patient outcomes. Microbial modulatory approaches may be needed to reverse resistance to therapies in pancreatic cancer.