Colorectal cancer (CRC) is the second most common cancer in men and the third in women globally [1]. It is also the second most commonly diagnosed cancer after gastric cancer in South Korea, with 30,000 new diagnoses and 8,700 deaths each year [2].

There are many long-term and late effects during the treatment of CRC, including chronic peripheral neuropathy, secondary cancers, bowel dysfunction such as nausea and diarrhea, and psychological issues such as depression and anxiety [3]. Most current guidelines focus on diagnosis and treatment of the tumor itself, but there is also a need for a “better adjunctive care” during the colon cancer treatment, and one of them is the “modulation of gut microbiota.”

The human intestine is an organ inhabited by billions of microorganisms, of which 10 [14] are in the colon [4,5]. These microorganisms are collectively referred to as “microbiota.” Their genes are called “microbiome” or “the 2nd genome” of humans. The importance of the gut microbiome in colorectal carcinogenesis is relatively well known. Active research is underway on the effects of microbial modulation through pre- and probiotics during cancer treatment.

This review is aimed to attract attention regarding the beneficial effects of intestinal microbial modulation during the treatment course of CRC. A deeper understanding of this topic will help the treatment of CRC patients. In the long term, it will help us develop tailored therapy according to characteristics of patient-specific intestinal microbiota and their immunologic states.

“All disease begins in the gut.” As mentioned in the 3rd century BC by Hippocrates, the gut microbiome plays surprisingly diverse roles. They contribute to the body’s energy metabolism, synthesis of vitamins and other essential nutrients, signaling the endocrine system, preventing colonization of harmful bacteria, regulating the immune system, and contributing to the metabolism of xenobiotic compounds [6,7].

Maintaining the intestinal mucosa’s immunological homeostasis begins with a challenging task of discriminating rare pathogenic species from billions of harmless microbes. During this process, both innate and adaptive immune responses prevent colonization of pathogens and induce local and systemic inflammatory responses to foreign microbial and dietary antigens. Gut-associated lymphoid tissue is the backbone of immune surveillance and defense mechanism. It consists of Peyer’s patch, appendix, isolated lymphoid follicle, and mesenteric lymph nodes. It is responsible for both activation and suppression of the mucosal immune system. The presence of intestinal bacteria is also essential for the development and maturation of the gut-associated lymphoid tissue [8].

When pathogens or symbiotic bacteria cross the epithelial barrier and enter the host, they first encounter macrophages. Antigens that are not phagocytosed by macrophages are captured by intestinal dendritic cells and migrated from the intestine to mesenteric lymph nodes, triggering a differentiation process that leads to the production of regulatory T cells (Treg), T-helper 17 (Th17), and IgA secreting B-cells [9]. According to a study using germ-free mice, the intestinal microbial community plays an important role in forming adequate mucosal immunity. Compared to specific pathogen-free mice, germ-free mice had fewer intraepithelial lymphocytes, less sIgA secretion in the lamina propria, and fewer Treg production [10].

Foxp3+ Treg cells play a key role in intestinal immune tolerance mechanisms [11]. CD4+Foxp3+ Treg cells derived from naive CD4+ T cells in both the thymus (nTreg) and intestine (iTreg) can help maintain immune unresponsiveness to autoantigens and suppress excessive immune reactions that might be harmful to the host. In an environment where Treg cells are abnormally present, proper immune tolerance is not induced, resulting in hypersensitive reactions [12,13]. Bacteroides fragilis, a type of symbiotic bacteria, can produce polysaccharide A, inhibit interleukin17 (IL-17) production from Th17 cells, and enhance the activity of iTregs, leading to anti-inflammatory effects. polysaccharide A can also induce the transformation of CD4+ T cells into Foxp3+ Tregs. It can also down-regulate the production of pro-inflammatory Th17 cells [14,15]. B. fragilis is known to be able to improve colitis in a mouse model. However, Bacteroides spp. are thought to have ambivalence as B. fragilis toxin (BFT) can cause inflammatory bowel disease by altering the function of epithelial tight junctions [16,17].

Th17 cells in the mucous membrane of the small intestine are vital in protecting the mucosal surface from microbial pathogens. However, they are also notorious for inducing autoimmune inflammation when they are activated by IL-23 [18,19]. Th17 cells seem to have an opposite function of Tregs [20-23]. Recently, it has been reported that Th17 cells are a unique CD4+ T-helper subset characterized by the production of IL-17, which can promote inflammation against a variety of pathogens. Specific intestinal microflora that can induce small intestinal Th17 cells are known as “segmented filamentous bacteria.” [24,25]

Another role of intestinal microbes is that they can ferment polysaccharides (such as resistant starch, oligosaccharides, inulin, etc.) that humans cannot normally digest or absorb, resulting in the production of short-chain fatty acids (SCFA) [26]. These SCFAs also contribute to the activation of several types of immune cells and play an important role in the differentiation of Treg cells [27,28].

As described above, it has been suggested that modulating the gut microbiome may prevent or worsen various types of inflammatory and allergic diseases because it can alter the differentiation of immunologic cells and modulate the production of SCFA by promoting colonization of beneficial bacteria [29,30].

The gut microbiota has a large diversity of microbial populations composed of bacteria, archaea, eukaryotes, and viruses. Both tissue and fecal samples provide information on the structure of bacterial populations. Analyzing tissue samples will show a more direct relationship between colon cancer’s pathophysiology and the gut microbiota. However, since sampling the intestinal mucosa is invasive, research using biopsy samples from normal mucosa is particularly limited. Therefore, most studies rely on fecal samples to analyze the distribution and diversity of intestinal microorganisms.

Most bacteria that reside in the gut are strictly anaerobic, and therefore they cannot be grown or cultured. The ability to identify bacteria using culture-independent methods was a huge advance in the microbiome field. Sequencing V1-V3 or V3-V5 variable regions of bacterial 16S ribosomal RNA has become a standard method for identifying bacterial populations or operational taxonomic units known to generally represent bacterial species whose sequences share more than 97% identities to each other [31]. Using the 16s rRNA target gene sequencing method and the recently introduced shotgun metagenomic sequencing, we can obtain more detailed information on the intestinal microbiome.

The gut microbiota not only promotes intestinal homeostasis and anti-tumor responses, but also contributes to genotoxic effects that can lead to carcinogenesis by causing chronic dysregulated inflammation [32]. Whether the gut microbiota will form a healthy symbiosis relationship with the host or promote colon cancer ultimately depends on the composition of gut microbiota and the balance between harmful bacterial populations within the microbiome. However, whether this “dysbiosis” might precede or cause CRC remains unclear. Studies on mice strongly suggest that gut microbiota may modulate susceptibility to CRC and may serve as both early diagnostic biomarkers and therapeutic targets. Such clinical research should be done considering the influence of each individual’s race, lifestyle, diet, sample type, location of the tissue sampled, and gut microbial ecosystem. Several notable shifts in the phylum level have been reported in the intestinal bacterial community of CRC patients. Among them, Bacteroides, Fusobacteria, and Proteobacteria are increased while Firmicutes are decreased in both intestinal mucosa and feces of the colon cancer patients [33,34]. More specifically, enterotoxic strains of B. fragilis and polyketide synthase (PKS) positive strains of Escherichia coli, Fusobacterium nucleatum are 3 most well-known strains in colorectal tumorigenesis (Fig. 1) [35,36].

Colonization of enterotoxin positive BFT (ETBF) has long been studied to be associated with diarrhea and gastrointestinal inflammation. ETBF also induced early carcinogenesis in mice models. ETBF coats tumors and recruits other bacteria to form a biofilm. Recently, increased colonization ETBF biofilms coating early human CRCs was confirmed [37]. BFT induces Th-17 mediated colitis and IL17-dependent carcinogenic inflammation through an accumulation of Treg cells.

E. coli species can be divided into 4 phylotypes (A, B1, B2, and D). Bonnet et al. [38] and Raisch et al. [39] confirmed that mucous membranes of cancer patients were much more abundant in E. coli subgroup B2. When B2 phylotype E. coli are incubated in vitro with various epithelial cell lines, they can arrest the epithelial cell cycle and force them to enter senescence [40-42]. Such effect is due to a group of compounds collectively named cyclomodulins that can introduce double-strand DNA breaks in target cells. These cyclomodulins include cytolethal distending toxin, cytotoxic necrotizing factor, and the best known “colibactin” produced by the PKS locus [40,43,44]. Colibactin is most likely a combination of hybrid molecules containing both a peptide and a polyketide produced in the gut by PKS positive E. coli [45]. Transient infection of cultured epithelial cells with PKS positive E. coli can induce chromosomal aberrations and increase mutation frequency rates. Correspondingly, in animal models of carcinogenesis, exposure to PKS can induce DNA strand breaks and lead to tumor generation [36,46].

While many independent studies have identified specific operational taxonomic units that can differentiate between healthy and CRC patients, F. nucleatum related to periodontal disease have gained attention due to their association with CRC [47,48]. Its prevalence has been reported to be gradually increasing as the disease progresses from colon polyp to CRC [48]. Studies in mice have shown that F. nucleatum can directly promote tumor growth [49]. F. nucleatum can reduce natural killer cell-mediated tumor killing by interacting with receptor TIGIT (T-cell immunoglobulin and ITIM domain) and inhibiting anti-tumorous natural killer cell activity [50]. In addition, E-cadherin-mediated interactions with CRC epithelial cells can induce cell proliferation via Wnt signaling and avoid immune surveillance [51]. Interestingly, there are reports that F. nucleatum is specifically related to microsatellite instability high tumors and that colonization of F. nucleatum is associated with relatively shorter cancer survival [51], making it suitable for use as a biomarker. Indeed, CRC with high loads of F. nucleatum, cells are more resistant to oxaliplatin by activating autophagy through Toll-like receptor 4 expressed on CRC cells [52]. Fap2 and FadA of F. nucleatum are proteins facilitate the binding to TIGIT and E-cadherin and enriches tumor proliferation. They could be used as potential targets for treating and detecting early CRC. However, since F. nucleatum is not universally present in all CRCs and its DNA is only detected in about 13% of all CRC cases, the use of F. nucleatum-derived proteins as a treatment target should be individualized.

Questions remain on how and when these tumor-related microbial act in the carcinogenesis of CRC species (sequentially, tandemly, or simultaneously). In addition, potential species such as Streptococcus gallolyticus and Enterococcus faecalis should be investigated. We must emphasize that further research to understand how the microbial species’ arrivals and departures in intestinal mucosa affect the tumor and its progression as time passes through is desperately needed beyond simply listing which species coexist with CRC.

The role of microbiome in colorectal carcinogenesis is evident and its potential as a treatment target is also promising. In summary, probiotic strains such as B. infantis, LGG, L. acidophilus and L. casei are expected to play an adjunctive role in the future treatment of CRC through cancer cell immunomodulation and chemoprotective effects. However, before it can be acknowledged as an established anticancer therapy, the following points should be elucidated before modulating intestinal ecosystem in CRC patients: types of probiotic/prebiotic strains we can choose, their optimal concentrations, duration of therapy, and supplementation method (by dietary habit change, oral pills of pro-, pre-, synbiotics) to bring out the best clinical outcomes. In addition, prospective clinical studies revealing how the gut flora and colon cancer interact over time are needed to explore the exact mechanism for their effectiveness.