Microbial dynamics with CRC progression: a study of the mucosal microbiota at multiple sites in cancers, adenomatous polyps, and healthy controls

Colorectal cancer (CRC) is one of the most common cancers in Norway and worldwide. The incidence of CRC has increased significantly in recent years, and Norway now has one of the world’s highest incidences of colorectal cancer, with 4550 new cases reported in 2021 [1].

Over the last decade, emerging evidence suggests involvement of the gut microbiota in initiation and progression of CRC. Already in 2012, two independent groups showed significantly higher levels of the anaerobic oral commensal Fusobacterium nucleatum in tumor tissues compared to healthy tissues [2, 3]. Following this, F. nucleatum has repeatedly been reported in tumor samples and has also been associated with CRC cell proliferation, tumor invasion, and lymph node metastases [4–6].

Several studies have further shown that larger communities of pathogenic microorganisms are involved in progression of CRC, specifically through establishment of densely populated biofilms [7, 8]. Biofilms have been linked to CRC development through enhanced epithelial permeability, tissue inflammation, and bacterial invasion of the intestinal epithelium [9]. Bacteria that frequently have been associated with CRC biofilms are Fusobacterium, Peptostreptococcus, Campylobacter, Parvimonas, Streptococcus, and Granulicatella, among others [10–14]. The documented microbiota in CRC is not consistent between studies, apart from the repeated detection of F. nucleatum. Although F. nucleatum is clearly associated with CRC, very few studies have investigated different F. nucleatum subspecies associated with CRC [15–17]. F. nucleatum is a heterogeneous species, currently divided into four subspecies, F. nucleatum ssp. nucleatum, ssp. animalis, ssp. vincentii, and ssp. polymorphum [18]. It has been proposed that these are sufficiently divergent to be characterized as different species [19]. To gain a deeper understanding of F. nucleatum in CRC progression, it could be important to identify which subspecies are involved in CRC. Additionally, if F. nucleatum is to be utilized as a biomarker for CRC, identifying the different F. nucleatum subspecies is crucial.

The role of F. nucleatum as a driver of disease initiation or a passenger able to colonize or infect cancer tissues and contribute to disease progression has been debated [20]. Several studies argue that F. nucleatum appears at the tumor site after cancer development, attaching to Gal-GalNAc receptor that is overexpressed in tumor cells [21, 22]. Conversely, Tomkovich et al. showed that human bacterial biofilms present in non-neoplastic tissues were able to initiate CRC in a mouse model and suggested that bacterial biofilms could be important progenitors for CRC [23]. Most studies that have investigated the tumor microbiome composition have examined biopsies from the tumor sites and adjacent healthy tissues [8, 12]. Flemer et al. showed that a CRC-associated microbiota was found also in adjacent healthy tissues 2–30 cm away from the tumor and argued that a CRC distinctive microbiota was established prior to CRC development [24]. Unraveling the microbiota in larger parts of the colon could show if the changes indeed are local or widespread throughout the colon. Furthermore, patients with adenomatous polyps are valuable when studying CRC progression. Although only 5% of adenomatous polyps progress to cancerous tumors, most of the colorectal cancers evolve through adenomatous polyps, and the risk increases with polyp size [25, 26]. It is therefore important to examine the microbial composition in polyps to assess if there are any microbial signatures already at the polyp stage.

In the present study, we have characterized the mucosal microbiome in colonic biopsies from different sampling sites (ascending colon, tumor or polyp, adjacent healthy tissue, and colon sigmoideum) from subjects of cancer, adenomatous polyps, and controls through 16S rRNA amplicon sequencing. Furthermore, Fusobacterium-positive tumor biopsies were subjected to MinION nanopore sequencing to characterize the Fusobacterium subspecies. The aim of the study was to investigate microbial composition in different sampling sites, in order to profile the microbial dynamics with CRC progression.

Colorectal cancer is a heterogeneous disease associated with the environmental factors such as high-fat diet, obesity, lifestyle, and composition of the gut microbiome [34–37]. The present study was initiated to explore the microbial composition at different sampling sites of the colon from subjects of cancer, adenomatous polyps, and controls in a small but well-defined Norwegian cohort.

Our study showed higher alpha diversity in the cancer group and adenomatous polyp group compared to healthy controls, suggesting that these two groups consist of more diverse and evenly present microbiota than the healthy controls. Zhao et al. reported that patients with more oral-related microbiota had lower alpha diversity [38]. However, we could not see any association between oral-related microbiota and alpha diversity in our study. Studies are conflicting when reporting the relationship between gut microbiota in biopsies and alpha diversity. Thomas et al. demonstrated higher species diversity in rectal biopsy samples from cancer patients compared to non-cancer controls [39]. Microbial diversity in fecal samples has both been either reported to be decreased in CRC patients compared to healthy controls [40, 41] or to show no differences between controls and cancer patients [17]. It is worth noting that, although most of the studies use 16S rRNA gene sequencing for gut microbiome analysis, there are differences in the choice of the target variable region, sequencing platform used, and databases used for taxonomic assignment. In addition, low sequencing depth can lead to loss of rare species [42]. These factors may have led to sequencing bias and the differences in alpha diversity.

Beta diversity analyses revealed significantly different bacterial compositions between the patient groups in our study. Comparison of microbial composition in the cancer, adenomatous polyp, and control groups illustrates that some taxa appear to be specific to the cancer group, while others are increased in both cancer and polyp patients. Higher abundance of Phascolarctobacterium, B. vulgatus, B. plebeius, B. eggerthii, Tyzzerella, Desulfovibrio, Eubacterium coprostanoligenes, and Frisingicoccus in cancer patients and patients with adenomatous polyps compared to healthy controls may suggest that these taxa could provide a favorable environment for polyp formation and initial stages of cancer. Other studies that examined tumor-associated bacterial taxa also reported elevated abundances of Phascolarctobacterium, Desulfovibrio, and Bacteroides in cancer patients compared to healthy controls. Desulfovibrio, a Gram-negative sulfate-reducing bacterium, may contribute to mucosal inflammation through hydrogen sulfide production [43]. Toxin-producing strains of Bacteroides fragilis have often been associated with CRC and even suggested as a keystone pathogen for CRC development [39, 44]. In the present study, three other Bacteroides species were increased in polyps and cancers compared to healthy controls. B. vulgatus has been shown to decrease in abundance after CRC surgery and could potentially have a role in CRC [45]. Yachida et al. investigated the tumor microbiome in different stages of CRC and showed that Phascolarctobacterium was increased in early stages of colorectal disease [46]. Its role in disease development has yet to be described. Phascolarctobacterium is a Gram-negative anaerobic bacterium known to produce the short-chain fatty acid propionate and may therefore have beneficial effects for the intestinal mucosa [44]. Eubacterium coprostanoligenes was also significantly increased in polyps and cancers compared to controls in this study. This Gram-positive anaerobic bacterium has cholesterol-reducing properties [47]. To our knowledge, this species has not previously been associated with polyps or CRC; however, its close relative Eubacterium rectale has been described as a “driver” bacterium for CRC initiation by promoting colitis [48]. Further studies on species level are required to determine the role of these bacteria in CRC progression.

Fusobacterium, Parvimonas, Gemella, Granulicatella, Leptotrichia, Peptostreptococcus, Campylobacter, Porphyromonas, Selenomonas, and Prevotella were enriched in cancer patients compared to patients with adenomatous polyps and controls, suggesting they either have a role in cancer development or are favored in the cancerous state. Although higher in abundance in cancers, Prevotella were present in relatively high quantities at all sample sites in all patient groups. These Gram-negative anaerobic commensals are known to colonize several mucosal sites in the human body. Prevotella target peptides and amino acids for their digestion, resulting in ammonia production which would raise the local pH and neutralize hostile and acidic environment pH. This could facilitate the establishment of a more acid-intolerant bacterial flora [49], and an increase of Prevotella could possibly be important to favor a microbiota with oncogenic properties. Furthermore, P. intermedia has been shown to enhance the migration and invasion of CRC cells in conjunction with F. nucleatum [50] and could therefore be important in advanced stages of CRC.

The nine additional taxa that were significantly increased in cancer patients were all bacteria that are normally part of the oral microbiota. Biofilm-associated bacteria of oral origin are involved in many extra-oral diseases and have recently been associated with colorectal cancer [7, 14, 51]. Warren et al. identified co-occurrence of Fusobacterium, Leptotrichia, and Campylobacter in tumor samples, whereas Flemer et al. found several operational taxonomic units (OTUs) related to oral cavity bacteria in a subset of cancer samples, namely, Fusobacterium, Porphyromonas, Anaerococcus, Prevotella, Granulicatella, and Parvimonas [24, 51]. CRC biofilms do not necessarily consist of the same species in every patient, but it may appear that multispecies communities are required for invasion of tumor cells [7]. CRC biofilms have been described as essential for disruption of the colonic epithelial barrier [9, 52].

Comparison of the taxa present at different sampling sites in cancer patients showed that of the biofilm-associated genera, only Fusobacterium and Gemella were significantly different at the tumor site. This could suggest that the growth of Fusobacterium and Gemella is particularly favored in colorectal tumors. Fusobacterium is known for its cancer-promoting effect mediated by its virulence factor FadA. FadA is a adhesion protein that interacts with E-cadherin expressed on the epithelial cells resulting in activation of the β-catenin signaling pathway [5]. Gemella are Gram-positive facultative anaerobic cocci described as early colonizers of biofilms and have been associated with extra-oral opportunistic infections such as endocarditis. Case reports have described an association of Gemella endocarditis with underlying colonic malignancy [53, 54]. G. morbillorum was shown to be a promising biomarker for CRC [55]. Although significantly higher in tumor tissues, Fusobacterium and Gemella were also identified at non-neoplastic sites. The remaining biofilm-related bacterial taxa were present in similar quantities at all sites. This observation was also done by Flemer et al. and Dejea et al. who showed that the biofilm-associated bacteria were not restricted to cancerous tissues [9, 24]. Our analyses were extended to larger parts of the colon and illustrate that the taxa are present at all sampling sites. This supports the idea that the establishment of a cancer-associated microbiota appears prior to tumor development.

The anaerobic Gram-positive and opportunistic pathogen Parvimonas micra has recently been shown to promote CRC tumorigenesis in multiple mouse models. The pro-tumorigenic effect of P. micra was associated with altered immune responses and enhanced secretion of inflammatory cytokines in the gut [56]. P. micra has already been suggested as a non-invasive biomarker for colorectal cancer in a number of studies, including a Swedish cohort, closely related to the Norwegian population [57]. We found that the biofilm-associated taxa were not significantly different in adenomatous polyp patients compared to healthy controls. However, inspection of taxonomy data showed the presence of one or more of the biofilm-associated taxa in some polyp samples at several sampling sites. This may suggest a beginning of CRC-associated biofilm formation and that these polyps could potentially develop into tumors if the above-mentioned taxa have cancer promoting properties.

On the other hand, we cannot rule out that contamination between samples during colonoscopy may contribute to this finding. During colonoscopy, samples were first taken from ascending colon, then from tumor, adjacent tissue, and finally from colon sigmoideum. However, the findings of oral biofilm-associated taxa in the ascending colon strengthen the validity of our results.

We identified lower abundance of Parabacteroides at the tumor site compared to off-tumor sites in the cancer group. A lower abundance of Parabacteroides at tumor sites suggests that these bacteria succumb in the inflamed microenvironment. Similar to our findings, other studies report a depletion of Parabacteroides in inflammatory bowel disease patients compared to control patients or in inflamed tissue compared to uninflamed tissue [58, 59].

In the present study, Fusobacterium was detected in 21 of 25 tumor biopsies from cancer patients, confirming that Fusobacterium is associated with CRC. We identified F. nucleatum spp. animalis as the most common subspecies present in tumor tissues. This is in line with Ye et al. [15] and Bi et al. [60], who both found F. nucleatum ssp. animalis to be the dominating subspecies on colorectal tumors. In a review of oral bacteria in extra-oral infections, Han and Wang showed that selected subtypes of a given species, such as F. nucleatum ssp. animalis, are more prone to extra-oral translocations [61]. It is not known if this subtype possesses specific virulence factors or oncogenic properties that are not present in the other subspecies. The gene encoding the well-known adhesion protein FadA is present in all F. nucleatum subspecies, as well as other oral Fusobacterium spp. [62]. It is unknown if expression of the protein in different strains and environments could potentially differ. A possible explanation for the dominance of subspecies animalis on colorectal tumors may simply be overabundance in the oral cavity of cancer patients, but studies have shown that all four subspecies are present in the oral cavity of healthy individuals [63]. Although subspecies animalis was the most prevalent subspecies in the present study, an important finding was that five cancer patient samples contained other F. nucleatum subspecies and even other Fusobacterium species: F. pseudoperiodonticum, F. necrophorum, and F. gonidiaformans. This illustrates that a CRC biomarker assay targeting Fusobacterium should be able to detect additional Fusobacterium species. Large variations in detection rates of Fusobacterium spp. have been reported in different studies, ranging from 9 to 100% [64]. Both methodological and biological variations could explain these differences. In our previous study using qPCR with NusG as a qPCR target, we detected Fusobacterium nucleatum in 13 of 21 tumors, illustrating that some Fusobacterium-positive samples were not detected by the qPCR assay [27] and showing clearly a need for design of PCR assays capable of detecting of several Fusobacterium species.

We are aware of the limitations of this study such as small number of patients in each group and none accounting for confounders. Considering run-to-run variation, runs 1 and 2 contained the same samples, while run 3 had new samples; this may contribute to the difference shown in the diversity analysis between the three runs.

In conclusion, we have demonstrated enrichment of several bacteria with CRC progression. In particular, several biofilm-associated bacteria were increased in cancer patients. These are promising targets for detection of colorectal cancer. Another group of bacteria was enriched in both cancer and polyps, suggesting that they may have a role in polyp formation and initial stages of CRC. Although F. nucleatum ssp. animalis was the most prevalent in this study, other F. nucleatum subspecies and Fusobacterium species were detected. This illustrates that a pan-Fusobacterium PCR assay should be considered for detection of colorectal cancer. Further investigation and confirmation of these findings in a larger population will make it possible to develop microbial-based diagnosis for CRC.