Abstract
Over the past two decades, the gut microbiome has emerged as a crucial factor in the development and progression of colorectal cancer. While associations between the microbiome and modulation of the tumour microenvironment are well-established, our understanding of the specific mechanistic links between tumour-resident bacteria and carcinogenesis remains limited. The research described in this thesis aimed to investigate the microbial landscape of colorectal polyps. The findings provide further evidence to support the hypothesis that tumour-resident bacteria are a driver of colorectal carcinogenesis. This thesis builds upon previous research from our group, that identified lipopolysaccharide (LPS) derived from F. periodonticum and B. fragilis as potential links between the microbiome and immune microenvironment. This thesis aimed to further the complex interplay between the microbiome, tumour microenvironment, and colorectal cancer. Particular emphasis was placed on characterisation of bacterial LPS and their functional ability to modulate immune responses and epigenetic landscapes, as well as examining the bacterial and molecular profiles of colorectal polyps.
F. periodonticum and B. fragilis LPS contained unique lipid A structures compared to known structures from the same species. F. periodonticum lipid A had higher acylation than B. fragilis’ Lipid A, suggesting a more potent biological effect. F. periodonticum LPS was a potent agonist of TLR4 and TLR2, while B. fragilis LPS only affected TLR2. Pre-incubation of these LPS species reduced their stimulatory ability on TLR2. F. periodonticum LPS strongly induced PD-L1 expression in immune and cancer cells, whilst the effect of B. fragilis LPS was relatively small. This may contribute to immune evasion and therefore has potential therapeutic applications.
Altered DNA methylation is a cancer hallmark. Some colorectal tumour subtypes show higher degrees of methylation (CpG island methylator phenotype (CIMP) positive) and have distinct bacterial profiles. F. periodonticum LPS induced extensive promoter hyper- and hypomethylation in HT29 colorectal cancer cells, including of tumour suppressor genes. B. fragilis LPS induced more hypomethylation than hypermethylation, aligning with its association with non-CIMP colorectal tumours. This work demonstrates that LPS modulates the methylation profiles of colorectal cancer cells, demonstrating a link between the microbiome and epigenetic alterations.
While bacterial profiles within colorectal tumours are well defined, few studies have examined tissue-resident bacteria in colorectal polyps. Sessile serrated lesions (SSLs) were associated with an abundance of commensal bacteria and an enrichment of immune-related gene-sets, compared to adenomas, which had a low abundance of commensal bacteria and were associated with increased cell-cycle and DNA repair gene-sets.
The breakdown of the gut barrier and bacterial translocation is characteristic of colorectal cancer, but the timing is unclear. Some commensal gut bacteria improve intestinal barrier function, but this has not been investigated in colorectal polyps. Using LPS binding protein (LBP) as a marker for barrier dysfunction and microbial translocation, the relationship between LBP and tissue-resident bacteria in colorectal polyps was investigated. Patients with SSLs or adenomas showed increased serum LBP concentration, suggesting gut barrier dysfunction may be an early event in colorectal carcinogenesis. Uncultured Faecalibacterium and Bacteroides clarus were associated with a low serum LBP concentration, while a decreased abundance of these bacteria was associated with a higher serum LBP concentration, identifying a potential link between the microbiome and gut barrier integrity in polyps.
This work has demonstrated that F. periodonticum and B. fragilis derived LPS provide mechanistic links between the microbiome and colorectal cancer cells. Although not identical with respect to their cellular effects, they both modulate the immune landscape and epigenetic profiles of colorectal cancer cells. Furthermore, this research has highlighted distinct bacterial profiles and molecular characteristics of colorectal polyps, and identified a potential link between bacteria and gut barrier function.