Kvich, L.; Fritz, B. G.; Zschach, H.; Terkelsen, T.; Raskov, H.; Hoest-Rasmussen, K.; Jakobsen, M. R.; Gheorghe, A. G.; Gogenur, I.; Bjarnsholt, T.

Objective: Increasing evidence links bacterial dysbiosis with colorectal cancer (CRC) carcinogenesis, with enrichment of core pathogens such as Bacteroides fragilis and Fusobacterium nucleatum. Here, we characterized the in situ biogeography and the molecular interplay between bacteria and the host at the transcriptional level 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: Distinct, tissue-invasive, mixed-species biofilms characterized CRC tissue with B. fragilis and F. nucleatum enrichment, especially in right-sided tumours. Notably, Fusobacterium spp. was associated with increased bacterial biomass and inflammatory response in CRC samples. CRC samples with high bacterial activity had increased expression of proinflammatory cytokines, defensins, matrix-metalloproteases, and immunomodulatory factors, contrasting with samples with low bacterial activity with an expression profile resembling those of healthy tissue samples. Moreover, immune cell profiling showed that B. fragilis and F. nucleatum were essential modulators of the TME and correlated with the infiltration of neutrophils and CD4+ T-cells. Overall, bacterial activity was critical for the immune phenotype and correlated with the infiltration of various immune cell subtypes, including M2 macrophages and regulatory T-cells, highlighting the importance of widening the scope beyond CRC core pathogens. Conclusion: Biofilms and core pathogens shape the TME and immune phenotype in CRC. Our results support that Fusobacterium spp. may provide a therapeutic target to reduce biofilms and the inflammatory response in the TME.