Biofilms and core pathogens shape the tumour microenvironment and immune phenotype in colorectal cancer

Objective Growing evidence links bacterial dysbiosis with colorectal cancer (CRC) carcinogenesis, characterized by an increased presence of core pathogens such as Bacteroides fragilis and Fusobacterium nucleatum . Here, we characterized the in situ biogeography and transcriptional interactions between bacteria and the host in mucosal colon biopsies. Design The influence of CRC core pathogens and biofilms on the tumour microenvironment (TME) was investigated in biopsies from patients with and without CRC (paired normal tissue and healthy tissue biopsies) using fluorescence in situ hybridization and dual-RNA sequencing. Results Tissue-invasive, mixed-species biofilms enriched for B. fragilis and F. nucleatum were observed in CRC tissue, especially in right-sided tumours. Fusobacterium spp. was associated with increased bacterial biomass and inflammatory response in CRC samples. CRC samples with high bacterial activity demonstrated increased expression of pro-inflammatory cytokines, defensins, matrix-metalloproteases, and immunomodulatory factors. In contrast, the gene expression profiles of CRC samples with low bacterial activity resembled healthy tissue samples. Moreover, immune cell profiling showed that B. fragilis and F. nucleatum modulated the TME and correlated with increased infiltration of neutrophils and CD4+ T-cells. Overall, bacterial activity was critical for the immune phenotype and correlated with the infiltration of several immune cell subtypes, including M2 macrophages and regulatory T-cells. Conclusion Biofilms and core pathogens shape the TME and immune phenotype in CRC. Our results support that Fusobacterium spp . may provide a future therapeutic target to reduce biofilms and the inflammatory response in the TME while highlighting the importance of widening the scope of bacterial pathogenesis in CRC beyond core pathogens. ### Competing Interest Statement The authors have declared no competing interest.