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

ABSTRACT

Extensive research has explored the role of gut microbiota in colorectal cancer (CRC). Nonetheless, metatranscriptomic studies investigating the in situ functional implications of host-microbe interactions in CRC are scarce. Therefore, we characterized the influence of CRC core pathogens and biofilms on the tumor microenvironment (TME) in 40 CRC, paired normal, and healthy tissue biopsies using fluorescence in situ hybridization (FISH) and dual-RNA sequencing. FISH revealed that Fusobacterium spp. was associated with increased bacterial biomass and inflammatory response in CRC samples. Dual-RNA sequencing demonstrated increased expression of pro-inflammatory cytokines, defensins, matrix-metalloproteases, and immunomodulatory factors in CRC samples with high bacterial activity. In addition, bacterial activity correlated with the infiltration of several immune cell subtypes, including M2 macrophages and regulatory T-cells in CRC samples. Specifically, Bacteroides fragilis and Fusobacterium nucleatum correlated with the infiltration of neutrophils and CD4⁺ T-cells, respectively. The collective bacterial activity/biomass appeared to exert a more significant influence on the TME than core pathogens, underscoring the intricate interplay between gut microbiota and CRC. These results emphasize how biofilms and core pathogens shape the immune phenotype and TME in CRC while highlighting the need to extend the bacterial scope beyond CRC pathogens to advance our understanding and identify treatment targets.

KEYWORDS:

• Colorectal cancer (CRC)
• biofilms
• Fusobacterium nucleatum
• Bacteroides fragilis
• in situ hybridization, fluorescence
• sequence analysis, RNA

Acknowledgments

We want to thank Veronica Drejer for her help with RNA purification and preparation of samples for RNA sequencing. We also thank Britt Capellen from the Center of Surgical Science for the initial sample preparation before RNA purification. The illustration of the sampling area in Figure 1 was created with Biorender.com.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Author contributions

Conceptualization, L.K., I.G., and T.B.; methodology, L.K., B.G.F, I.G., and T.B.; investigation, L.K., B.G.F., M.R.J., and A.G.G.; data curation, L.K., B.G.F., H.Z., T.B.T., and K.H-R.; data analysis, L.K. B.G.F., T.B.T., and H.Z.; generation of figures, L.K. B.G.F., T.B.T., and H.Z.; writing – original draft, L.K.; writing – review & editing, All authors.; supervision, H.R., I.G., and T.B.; project administration, L.K.; funding acquisition, I.G. and T.B.

Data availability statement

The raw sequencing data and gene count tables produced in this study are not accessible to the public, as such an action would infringe upon patient consent and ethical regulations stipulated by the authorities in Denmark. Processed data devoid of sensitive information and original code supporting the results of this study is available in the Supplementary Information as well as in a Zenodo repository (DOI: https://zenodo.org/doi/10.5281/zenodo.10997467). Additional information is available upon reasonable request, and in the case of raw sequencing data, appropriate permissions are required for data accession.

Supplementary material

Supplemental data for this article can be accessed online at https://doi.org/10.1080/19490976.2024.2350156

Additional information

Funding

This work was supported by the Novo Nordisk Foundation [Tandem program #NNF19OC0054390 to T.B.]. Also, we would like to acknowledge Greater Copenhagen Health Science Partners (GCHSP) for their financial support.