Multi-omic spatial atlas shows Deleted inMalignantBrain Tumours 1 (DMBT1) glycoprotein is lost in colonic dysplasia II

Colorectal cancer(CRC) is responsible for 50,000 deaths annually in the United States. Emerging evidence supports a causal role for pro-carcinogenic bacteria in the colonic microbiome. We previously showed toxigenic C. difficile from human CRC-associated bacterial biofilms accelerates tumourigenesis in ApcMin/+ mice, both in specific pathogen-free mice and in gnotobiotic mice colonised with a defined consortium of bacteria. To further understand host-microbe interactions during colonic tumourigenesis, we combined single-cell RNA-sequencing (scRNA-seq), spatial transcriptomics, and immunofluorescence to define the molecular spatial organization of colonic dysplasia in our consortium model with or without C. difficile. Our data show a striking bipartite regulation of Deleted in Malignant Brain Tumours 1 (DMBT1) in the inflamed versus dysplastic colon. From scRNA-seq, differential gene expression analysis of normal absorptive colonocytes at 2 weeks post-inoculation showed DMBT1 upregulated by C. difficile compared to colonocytes from mice without C. difficile exposure. In contrast, our spatial transcriptomic analysis showed DMBT1 dramatically downregulated in dysplastic foci compared with normal-adjacent tissue. We further integrated our datasets to generate custom colonic dysplasia scores and ligand-receptor mapping. Validation with immunofluorescence showed DMBT1 protein downregulated in dysplastic foci from three mouse models of colonic tumourigenesis. Finally, we demonstrated downregulation of DMBT1 mRNA and protein in adenomatous dysplasia from human samples. Together, our data reveal cell type-specific regulation of DMBT1, a potential mechanistic link between bacteria and colonic tumourigenesis. Overall design: C57Bl/6 mice with the ApcMin/+ (?716 Apc) genotype were bred germ-free. At age 8-10 weeks, adult males in the C. difficile experimental group were inoculated with a single gavage of the 29-member bacterial consortium described by Drewes et al, (2022 Cancer Discovery) along with 104 spores of C. difficile strain Cd_3728T. The control group was given a single gavage of the same consortium but without C. difficile (n = 3 mice/group). Two weeks after inoculation, all mice were euthanised humanely for tissue harvesting and scRNA-seq.