Spatial atlas highlights contribution of C. difficile in early-stage colorectal cancer

Background and AimsIntratumoral heterogeneity with respect to both host and microbial components has emerged as an important contributor to colorectal cancer (CRC) biology. Although the overall taxonomy and abundance of the CRC tumor microbiome have become well characterized, much less is known about bacterial niches within the tumor microenvironment (TME). Escherichia coli and Fusobacterium nucleatum are highly prevalent and abundant species linked to CRC. Less abundant organisms, like Clostridioides difficile and Enterocloster aldenensis, are emerging as potentially important CRC-associated bacteria. Additionally, many studies are confounded by preoperative oral antibiotics or the inclusion of late-stage cancers, both of which may alter the gut microbiome. To better understand the spatial relationships between bacteria and CRC, we generated a molecular atlas based on surgically resected tissue specimens collected from a unique cohort of early-stage CRC patients in whom oral antibiotics were not administered preoperatively. MethodsFrom 20 CRC specimens, we performed matched histopathological analysis, fluorescence in situ hybridization (FISH), whole exome sequencing (WES), 16S rRNA amplicon bacterial DNA sequencing, codetection by indexing (CODEX) multiplex immunofluorescence, and spatial transcriptomics of 756 regions among the specimens. We used 16S rRNA amplicon sequencing data to design and experimentally validate custom bacterial probes that were applied to the spatial transcriptomics. Human colonic organoids were used to validate the relationship between Clostridioides difficile toxin B (TcdB) and an E-twenty-six family transcription factor, ELF3. ResultsWe identified eight intratumoral bacterial niches consisting of a variety of bacterial species, and each niche was associated with unique tumor features. We further defined tumor gene expression patterns correlating with individual bacterial species and show that C. difficile has a disproportionate impact on the tumor transcriptome given its relatively low abundance. Specifically, TcdB induces nuclear localization of ELF3, increased cytosolic {beta}-catenin protein, and upregulated WNT signaling. ConclusionOur study integrates multi-omics to identify bacterial species with biological and spatial relevance in CRC regardless of abundance. These findings will enable further studies to define diagnostic and therapeutic targets for bacteria-associated CRC.