Elucidate the Role of Tumor Isolated Enterococcus faecalis and Its Metabolic Products on CRC onset and GVB Impairment

Colorectal cancer (CRC) is the third leading cause of cancer-related mortality worldwide and 50% of patients with localized disease ultimately develop distant metastases. Both genetic and environmental factors play a role in the development of the disease, with western diet and lifestyle representing one of the major risks. Recently, deep-sequencing technology revealed a strict correlation between colonic dysbiosis and CRC development, suggesting that the gut microbiota represents the link in the diet-cancer relationship. Members of the resident microbiota can have pro-tumorigenic features and contribute to CRC onset by impacting gut barrier integrity or via the activation of immune responses. Moreover, bacteria may take part in CRC progression, further enhancing carcinogenesis or even promoting metastasis formation. To better identify those bacteria that may be involved in CRC, we took advantage of the APCMin/+C3aRKO mice, a murine model of spontaneous colon tumorigenesis. These mice develop fewer tumors in the small intestine compared to the APCMin/+ model and are characterized by increased tumor burden in the colon and an impaired gut vascular barrier (GVB), resembling human CRC. By whole metagenomic analysis we observed a statistically significant enrichment of Enterococcus faecalis (EFSC1) in the tumors of APCMin/+C3aRKO mice compared to the healthy mucosa. Therefore, we wondered if EFSC1 isolated from tumors might have a direct role in primary tumor onset as well as GVB impairment, which occurs early during carcinogenesis and represents one of the first steps in metastatic spreading. We found that, in C57BL6/J WT mice, EFSC1 and its metabolic products impacted gut homeostasis, reducing the colonic mucus layer, and disrupting both epithelial and endothelial barriers, suggesting its possible role in carcinogenesis. To further verify this hypothesis, we first treated APCMin/+C3arKO mice with EFSC1 or its metabolites and confirmed that the increased presence of either the bacterium or the metabolites increased tumor multiplicity in the colon. Then, we tested the role of EFSC1 metabolites on colon tumorigenesis in the APCMin/+ model. In accordance with the results obtained in the previous model, we observed a higher number of neoplastic cells, increased tumor size, and selective recruitment of macrophages in the EFSC1 6 metabolites treated group compared to the control. We confirmed that the detrimental effect on gut barrier integrity was specific for EFSC1, as a different Enterococcus faecalis species (EFSC2) failed to have the same effect in vivo. In addition, metagenomic and metabolomic analysis of both strains showed differences in terms of gene content and metabolic profile. Increased fatty acid biosynthesis and metabolism pathways in EFSC1 support the idea that fatty acids or specific products of their metabolism may be responsible for the role of EFSC1 on colon tumorigenesis. Collectively, these results suggest that the metabolites produced by EFSC1 induce changes in gut homeostasis and, together with mutations in APC genes, boost the formation of primary tumors in the colon. Moreover, EFSC1 metabolites damage the GVB enhancing vascular permeability, enabling the hematogenous dissemination of cancer cells. Hence, we speculate that the identification of EFSC1 specific metabolites may be clinically relevant; as works, targeting these metabolites could be a new therapeutic intervention in CRC treatment.