Colorectal cancer (CRC) is a leading cause of cancer-related deaths worldwide and the morbidity and mortality of CRC are still rising (1). The mechanism of the initiation and progression of CRC has not been fully elucidated yet, but it is generally believed to be the result of the extensive and complex interaction between genetic and environmental factors. Epidemiological data have demonstrated that adverse environmental exposures, including overweight and obesity, high-fat diet (HFD), smoking and high consumption of alcohol, perform predominant roles in carcinogenesis (2, 3). Significantly, these controllable factors could have a strong impact on the structure and function of gut microbiota.

The microbiota that inhabits the human gut is a key factor in maintaining the stability of the intestinal microecosystem by participating in the immune regulation, substance metabolism, nutrient absorption in the human body, directly or indirectly (4). Usually, the gut microbiome engages in mutualistic relationships with the host, whereas it may mediate the occurrence and development of some diseases, which depends on both environment and the susceptibility of the host. Due to next generation sequencing technologies, such as 16S rRNA, 18S rRNA, internal transcribed spacer (ITS) sequencing, shotgun metagenomic sequencing, metatranscriptomic sequencing and virome sequencing, identifying the characterization of host-microbiota interactions has tremendous progress and the intestinal microbiota is indicated to be closely related to CRC (5, 6). The proposed pathogens which are candidates for CRC mainly include Fusobacterium nucleatum, Escherichia coli, Bacteroides fragilis, Streptococcus bovis, Enterococcus faecalis and Peptostreptococcus anaerobius (7). Also, the influence of the vaster microbial community, particularly its metabolome, has been connected to CRC ( Table 1 ). The gut microbiota, the “new organ” of the human organism, has an enormous metabolic potential (8), and its metabolic capacity greatly exceeds that of human cells (9). About 50% of the metabolites found in feces and urine are derived from, or modified by the gut microbiota (10). Metabolomics, which is the qualitative and quantitative assessment of the metabolites (small molecules<1.5 kDa) in cells, tissues, organs, or biological fluids, has discovered thousands of microbiota-derived metabolites and further expanded our knowledge on the effects of specific metabolites in carcinogenic or cancer-promoting activity (11, 12). Common gut microbiota-derived metabolites mainly include amino acids and their by-products, lipids and lipid-like metabolites, bile acids (BAs) derivatives, and other metabolites produced by the degradation of carnitine and choline (11, 13, 14). These metabolites are altered in CRC patients and some of them have been confirmed to have both local and systemic effects on promoting the risk, initiation and progression of CRC ( Table 2 ).

The current review highlighted the recent advances on the mechanisms by which detrimental metabolites from gut microbiota modulated the development and progression of CRC. Meanwhile, multiple potential therapeutic approaches for CRC were summarized. Targeting small molecule metabolites may contribute to providing promising therapeutic strategies for CRC.

The potential genetic changes of CRC have been well confirmed up to now. At least three major molecular pathways can lead to CRC (chromosomal instability pathway, microsatellite instability and CpG island methylation pathway). However, in addition to the existence of several essential mutations, the occurrence and development of CRC also depend on the close interaction of mutagenized cells with the tumor microenvironment (TME) (45). During the development of CRC, tumor-promoting inflammation induced by gut microbiota usually involves the first two, characterized by the exaggerated production of cytokines by resident innate immune cells and the establishment of an immunosuppressive TME. Aberrant inflammation is considered as one of the hallmarks of cancer. During the development of CRC, tumor-promoting inflammation induced by gut microbiota usually involves the first two, characterized by the exaggerated production of cytokines by resident innate immune cells and the establishment of an immunosuppressive TME (45–47). These proinflammatory mediators interact with epithelia to compromise barrier function and further amplify the response by recruiting and activating additional immune cells. The breakdown of the intestinal barrier triggers the invasion of pathogenic microorganisms and their metabolites from the intestinal lumen, and leads to contact between intestinal epithelial cells (IECs) and components of the microbiota that might have pro-tumorigenic characteristics. Ensuring sustained immune response may trigger a cascade of inflammatory changes and promote carcinogenesis (47–49). The causal and complicit roles of microbes in cancer through their effect on the host’s immune system is defined as the immuno-oncology-microbiome (IOM) axis (50).

Pattern recognition receptors (PRRs) are essential components of the host immune system, which can recognize the conserved molecular patterns specific to microorganisms called pathogen-associated molecular patterns (PAMPs) such as lipopolysaccharide (LPS) to trigger diverse innate immune responses (51). The sub-families of PRRs mainly include several types of recognition receptors: the toll-like receptors (TLRs), the NOD-like receptors (NLR), C-type lectin receptors (CLRs), and RIG-I-like receptors (RLRs) (51). Intestinal mucosal epithelial cells and immune cells recognize intestinal microorganisms and their products mainly through TLRs, which are regulated by the intestinal microbiota and by circadian rhythmicity (52–55). In spite of different receptors, the vast majority of TLRs’ signaling is initiated by the myeloid differentiation primary-response protein-88 (MyD88) and leads to activation of downstream signaling pathways and recruitment of transcription factors such as nuclear factor-Kappa B (NF-κB), mitogen-associated protein kinase (MAPK), and interferon (IFN) regulatory factors and subsequent generation of cytokines and chemokines. The expression of TLR2 in colon cancer is significantly upregulated and the TLR2 agonists significantly enhance the proliferation, migration, and invasion of CRC cells (56). TLR4 can produce trophic factors and vascular growth factors through the TLR4/MyD88/NF-κB signaling pathway (57) and promote tumor proliferation through TLR4/Cyclooxygenase 2 (COX2)/prostaglandin E2 (PGE2). TLR9 could induce downstream signals to recruit inflammatory factors, such as IL(interleukin)-8, TGF-β, PGE2, and other immunosuppressive molecules, leading to the continuous state of inflammation, the escape of tumor immunity, and the unlimited proliferation of tumor cells (58).

Immune elimination and immune escape are hallmarks of cancer. The TME is usually at an immunosuppressive state. A range of microbial derivatives and metabolites can mediate host immune disorders via affecting the differentiation, proliferation, maturation and effector function of innate and adaptive immune cells, thereby influencing tumor surveillance (47). Polyamine synthesis is essential to induce cytotoxic activity and T-cell proliferation. Supplementation with spermidine or L-arginine promotes homeostatic differentiation of Treg cells with a beneficial role in the context of gut inflammation (59). However, polyamines possibly have opposing roles depending on their concentrations, since elevated polyamine levels in cancer have been shown to diminish the antitumor immune responses by resulting in various defects in immune cell function, including inhibition of lymphocyte proliferation (60–62). Moreover, TME rich in fatty acids can inhibit the function of effector T cells and M1-polarization of macrophages, and facilitate the differentiation of T regulatory cells (Tregs) and M2-like macrophages (63). Notably, only a relatively narrow segment of the studies has elucidated the reciprocal interaction between metabolites and tumor immunity, and their influences on immunosuppression remain poorly characterized.

One of the primary modes of interaction between the gut microbiota and the CRC is through metabolites. Specific classes of detrimental microbiota-derived metabolites such as Trimethylamine-N-oxide (TMAO), BAs, hydrogen sulfide (H₂S), N-nitroso compounds (NOCs) form a complex metabolic network related to the etiology and severity of CRC ( Figure 1 ). Once these metabolites pass the mucosal barrier, they can act directly on IECs or influence immune responses in the intestinal stroma, trigger the release of pro-inflammatory signals, such as tumor necrosis factor (TNF) and IL-17, or lead to immunosuppression in the TME, which can further promote tumorigenesis. Furthermore, microbial metabolites can induce tumorigenesis by inducing DNA damage and activating the intracellular tumorigenic signaling pathways.

Metabolomics has been increasingly used to identify biomarkers in disease with great potential for clinical translation. With relevant technological advances and new analytical and bioinformatics tools development, metabolomics can already provide sensitive and highly reproducible platforms allowing the quantification of the known compound or comprehensive analysis of all the measurable analytes in a sample (107). A large amount of metabolomics data has been accumulated to be used to investigate the interaction between the host and microorganisms from the perspective of metabolism. It was found that several gut microbiome-associated serum metabolites (GMSM) have changed significantly through integrated analysis of the serum metabolites and fecal metagenomics of patients with CRC and adenoma, which can efficiently discriminate patients with CRC and adenoma from normal individuals (108). Dysregulation of these metabolites indicates the possibility of common diagnostic biomarkers for CRC. However, there is still a need for better non-invasive biomarkers for CRC, especially for the early stages of the disease, including the adenoma step and the initial CRC stages. The content of several classes of bioactive lipids, including polyunsaturated fatty acids, secondary BAs and sphingolipids (109, 110) increased in adenoma patients. Most of these metabolites show directionally consistent changes in patients with CRC, indicating that these changes may represent early events of carcinogenesis (111). Rao J et al. pointed out that three metabolites (hydroquinone, leucenol and sphingomyelin) are positively and significantly correlated with CEA and/or CA 19-9, which may be potential biomarkers for advanced CRC (112). Furthermore, dynamic shifts of microbial metabolism have been confirmed in patients with different stages of colorectal neoplasia. Yachida et al. showed that branched-chain amino acids and phenylalanine were significantly increased in patients with intramucosal carcinomas, and BAs were significantly elevated both in patients with multiple polypoid adenomas or intramucosal carcinomas (113). These data provided evidence for the functional importance of the gut metabolome in CRC and implied a potential role of the gut microbiota-derived metabolites in the early diagnosis and prognosis of CRC. The term “pathogenic function (pathofunction)” has been proposed to evaluate specific features of host bacterial communities based on the detrimental metabolites produced by the gut microbiota. The pathofunction of the gut microbiota may contribute to the development of precision treatment directing gut microbiota to increase host health. However, universal biomarkers for CRC detection have not been identified due to the high variability of the microbiota and their metabolites between individuals. A standardized sample preparation and metabolomics analysis methods warrant further exploration.

In view of the importance of the mechanistic link between detrimental gut microbiota-derived metabolites and CRC, targeted metabolomics has been a remarkable research topic in recent years. There have been several beneficial metabolites [i.e., short-chain fatty acids (SCFAs)] profiling studies for preventing and treating CRC, however, fewer studies have been involved in CRC therapeutic explorations by targeting detrimental microbial-derived metabolites. In our review, we put effort to mention those studies which have linked regulating excess detrimental microbial-derived metabolites in the diagnosis, prevention and treatment of CRC. Pay more attention to how to use these studies for clinical application has great significance for the diagnosis, prognosis, therapeutics, and prevention of CRC.

Diet is an important environmental determinant of metabolism. Diet-mediated changes in whole-body metabolism and systemic nutrient availability can affect the overall performance of TME (2, 114). Metabolites derived from gut microbes are the key executors of diet affecting the host physiology, either inhibiting or accelerating tumor growth. Red and processed meat might increase CRC risk by increasing the formation of detrimental microbial metabolites such as TMAO, NOCs and HCAs. On the contrary, dietary fiber (DF) enriched in fruits, vegetables and whole grains is associated with a lower risk of CRC (115). A comprehensive meta-analysis of prospective studies and clinical trials provided convincing evidence that fiber intake was related to the risk of CRC. Dose-response relationships suggested greater effects on CRC risk reduction for higher intakes of DF (116). Importantly, DF can protect against CRC by altering not only microbial composition but also the concentration and availability of metabolites. The link between DF and BAs may be identified as a potential mechanism to explain the protective role of DF in CRC. In a comparative study in African American individuals, switching from low-fiber, high-fat to high-fiber, low-fat diet resulted in significant improvement in the microbiota and metabolome associated with CRC risk. Further exploration revealed that it was mainly due to an increase of saccharolytic fermentation and butyrogenesis and inhibition of secondary BAs synthesis (117). Consistent with this, a recent study investigated concentrations of fecal and serum BAs in vegans and omnivores, showing a vegan diet low in fat and high in fiber was positively correlated with fecal BA concentrations (118). Additionally, Li et al. have suggested that supplying the DF diet to mice can reduce TMA and TMAO outputs via inhibiting intestinal microbial TMA lyase activity (119). Thus, DF intervention for individuals with various clinical statuses is expected to become an emerging treatment against CRC.

Probiotics are well-known biologically active candidates for the treatment of several diseases and unhealthy conditions by positively regulating the gut microbiota (120). Lactobacilli, Clostridium butyricum and Lactobacillus rhamnosus GG (LGG) are some of the most studied and well-characterized among probiotics. On the one hand, probiotics are commensal live bacteria that reduce intestinal inflammation and improve intestinal barrier function to achieve the prevention and treatment of CRC. On the other hand, probiotics can remove carcinogenic metabolites and toxins from the gastrointestinal tract, compete with spoilage bacteria and pathogenic bacteria and maintain the balance of gut microbiota (121, 122). Detrimental metabolites are mainly derived from the gut microbiota, such as Bacteroides, Clostridium (clusters XIVa and XI), Eubacterium, Enterobacter, Campylobacter jejuni and SRB. Supplementing probiotics can reduce the colonization of these bacteria, which may reduce the level of detrimental metabolites in the intestine or body, and thus inhibit intestinal tumor development. Probiotics can regulate intestinal bacteria related to proteolysis, reduce harmful protein fermentation, thus reducing the toxicity of metabolites (123). It was found that supplementing with Lactobacillus or Bifidobacterium or both can significantly reduce serum cholesterol. The main mechanism is the use of bile salt hydrolyzing enzymes to decouple the bile, and then precipitation of cholesterol along with the decoupled bile (124, 125). Moreover, Our previous studies have found that Clostridium butyricum antagonized against HFD-induced intestinal carcinogenesis and decreased the production of secondary BAs via down-regulating the abundance of Clostridium (126).

Fructooligosaccharides and galactooligosaccharides and inulin are frequently reported prebiotics. Prebiotics are non-digestible selectively fermented DF that can be used as substrates by gut microbiota to promote the metabolism of lipids, proteins and minerals, and produce metabolites that are potentially protective of gut functionality (127). In addition, prebiotics promote favorable bacteria of the indigenous intestinal flora of humans, or also improve the survival of probiotics that have been ingested at the same time, thereby indirectly reducing potentially pathogenic bacteria and/or detrimental metabolites in the intestine. Interestingly, the introduction of prebiotics improves the viability of probiotics. Compared with used alone, the combination of prebiotics and probiotics has been reported to reduce the expression of genes involved in carcinogenic pathways and drug resistance, and decrease the levels of the metabolite lactate, thus disturbing the growth of cancer cells and benefiting for CRC treatment (128). However, clinical trials evaluating the efficacy of probiotics for CRC are still limited and there are no specific criteria for selecting probiotics or prebiotics, thus systematic evaluation of different strains is required.

Fecal microbial transplantation (FMT) is an emerging treatment scheme for transplanting gut microbiota from healthy donors to patients through various channels, so as to restore the intestinal microbial diversity of patients and achieve the therapeutic purpose (129). The significant differences in gut microbiota composition between cancer patients and healthy individuals show the therapeutic potentials of microbiota modulation. It is reasonable to support the hypothesis that FMT is prospective in cancer management as well as cancer-treatment associated complications, which is being actively explored (130, 131). The natural gut microbiome could promote host fitness, eliminate excessive inflammation and protect against mutagen/inflammation-induced colorectal tumorigenesis (132). FMT affects metabolism by resolving dysbiosis, or directly transferring primary and secondary BAs, which may underlie the observed effects of FMT in CRC. At this stage, the uncertainty and safety of the efficacy of FMT still should not be ignored. In-depth experiments and clinical trials are essential for its development and improvement.

Therapeutic approaches that use live tumor-targeting bacteria are ushering in a new era of cancer therapy. Bacteria can be engineered to act as therapeutic delivery payloads following simple genetic rules or complex synthetic bioengineering principles to realize the functions of delivering drug molecule or immunoregulatory factors to tumors, destroying tumor matrix, and silencing tumor genes (133). Compared with non-precision approaches such as the use of probiotics, engineered bacteria can be more accurately mitigate the effects of detrimental microbial-derived metabolites that are significant in the pathogenesis of CRC. A recent study developed engineered Escherichia colistrain that could convert systemic ammonia into L-arginine in a mouse model, thus exhibiting the potential for microbiota-based therapeutics to more precisely regulate metabolism (134). Engineered bacteria can be applied either as a monotherapy or a complement to other anticancer therapies to obtain improved antitumor activities. Some of them have passed clinical tests and shown greatly encouraging results. In fact, the success of therapy depends on the functional stability, clinical potency and safety of the engineered bacteria (135). Meanwhile, we must identify appropriate gut-adapted strains and consider performance metrics when deploying such bacteria in vivo (136).

Phage therapy may be a promising therapeutic tool for modulating the gut microbiota and gut metabolome as well as stimulating systemic anticancer immune response (137). Bacteriophages are viruses ubiquitous in the human intestinal tract that infect and replicate within bacteria and usually have species-level specificity. By directly knockdowning their bacterial targets or further inducing cascading effects on non-susceptible species through inter-bacterial interactions, the therapeutic effect of bacteriophages can be utilized to modulate the gut microbiota, which in turn has a potential impact on gut metabolome such as significant changes in bile salts (138, 139). The link between phage and microbial metabolites provides an interesting therapeutic avenue. Manipulation of microbiota by lytic phage can be used to selectively reduce detrimental microbial metabolites (140). For instance, phage predation of E. faecalis reduced tyramine, which can induce ileal contractions (141).

The value of chemoprevention strategies for preventing early stages or recurrence of CRC or new polyp formation has been widely noted in recent decades. Several studies to date have supported the chemopreventive potency of some promising agents, particularly those targeting metabolic pathways. Inhibition of the formation of detrimental metabolites appears, therefore, to be a rational target in chemoprevention.

Ursodeoxycholic acid (UDCA) is one of the BAs that has different effects on CRC compared with DCA. A population-based study indicated that the use of UDCA was associated with a lower risk of CRC (142). UDCA treatment affected the microbial community composition in men, as manifested by increased abundance of Faecalibacterium prausnitzii and decreased Ruminococcus gnavus, which partially explained the UDCA action to inhibit adenoma development (143). Additionally, the hydrophobicity of BAs may be an important determinant of their carcinogenic properties (144). One possible biologic mechanism by which UDCA acts therapeutically may be to increase the hydrophilicity of the biliary pool, dilute the concentration of toxic secondary BAs in the biliary pool such as DCA and lithocholic acid (LCA) and then prevent colonic neoplastic transformation (145). However, only low-dose UDCA is recommended for the prevention of colorectal neoplasia. The hypothesis that UDCA may increase the risk of developing neoplasia was first proposed by Eaton et al., who showed that high-dose UDCA can increase the incidence of colorectal neoplasia in patients with ulcerative colitis and primary sclerosing cholangitis (146). Obviously, more studies are needed for UDCA applications.

CRC and diabetes mellitus share various clinical risk factors leading to an interest in anti-diabetic agents as potential chemopreventive drugs for CRC. Metformin, as the first-line oral medicine for type 2 diabetes, has been shown to significantly reduce colorectal adenoma and cancer incidence and improving survival outcomes (147, 148). In the context of CRC, the protective effects of metformin are likely multi-factorial. One of the antitumor effects of metformin is an indirect effect resulting from systemic metabolic changes, including decreases in plasma glucose. Metformin had strong effects on the gut microbiome, and this was reversed when the drug was removed (149). Exposure to metformin modulated gut microbiota in patients with type 2 diabetes and increased the concentration of BA glycoursodeoxycholic acid and tauroursodeoxycholic acid, which were known antagonists of the FXR. These changes inhibited intestinal FXR signaling and improved metabolic dysfunction, hinting the alterations in microbiome composition and the subsequent impact on secondary BAs production may be an important factor in the prevention of CRC by metformin (150). The other is a direct effect on tumor cells. The liver kinase B1 (LKB1)-dependent activation of AMPK and reduction of mammalian target of rapamycin (mTOR) activity may be significant contributors to the inhibitory effects of metformin on cancer cell growth and proliferation (151).

The polyamine metabolic pathway is a potential target for cancer chemoprevention. A polyamine-blocking therapy (PBT) by targeting their synthesis and transport can exert antitumor effects. It not only altered the levels of polyamines in many tumors, but also relieves the immunosuppression in the TME, characterized by an increase in granzyme B+, IFN-γ+ CD8+ T-cells, and decreased immunosuppressive cell levels (60, 152). Ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase (AdoMetDC), as the rate-limiting enzyme of polyamine biosynthesis, are the key factors in the regulation of polyamine levels. Difluoromethylornithine (DFMO) is an irreversible inhibitor of ODC and recognized as a chemopreventive and chemotherapeutic agent to decreases cancer hallmarks including enhanced cell proliferation and apoptosis resistance (153). To date, several phase II and Phase II studies on DFMO in the treatment of CRC have been reported. DFMO may prevent CRC by reversing Ca²⁺ channel remodeling, activating a synergistic re-expression of aberrantly silenced tumor-suppressor genes and modulating DNA hypomethylation (154, 155). To inhibit polyamine synthesis, many AdoMetDC inhibitors have been discovered from the first-generation inhibitor methylglyoxal bis (MGBG) to the third-generation inhibitor AbeAdo and tested in clinical trials treating cancers (156). However, none of them has been finally approved for clinical use due to low efficiency or strong side effects. In addition, given that the polyamine transport system is upregulated in cancers, a range of polyamine analogs and polyamine-like structures have been synthesized. They can interrupt polyamine biosynthesis and compete for uptake, and thus reduce the normal polyamine content required for cell growth (157).

A convergence of basic research, epidemiological and clinical studies is illuminating the contribution of detrimental gut microbiota-derived metabolites to the initiation and progression of CRC. The microbial metabolites described above, specifically TMAO, BAs, H₂S and NOCs, work as critical signaling molecules that mediate crosstalk between the microbes and host, and play pivotal roles in colorectal carcinogenesis. They represent potential biomarkers for the early diagnosis and prognosis of CRC.

Although it is known that the detrimental gut microbiota metabolites contribute to intestinal malignant lesions in numerous ways, most of them have not yet been functionally characterized. In particular, the in-depth molecular mechanisms involved in the interaction between metabolites and CRC, as well as direct synergy between metabolites remain to be elucidated. Of note, there are still controversial findings that parts of the detrimental metabolites exhibit both the properties of anti-carcinogenic and pro-tumorigenic, which may depend on many factors, such as their luminal concentrations, the duration of the colonic stasis, interactions with other metabolites and tumor developmental stage. However, based on large-scale epidemiological studies and clinical outcome trials, we still believe that targeted regulation of detrimental metabolites to eliminate or reduce their concentration is expected to be effective strategies for the prevention and treatment of CRC. Furthermore, more studies are necessary to ensure the functional stability, clinical potency and safety of management measures.

WZ, YA, and XQ were the major contributors to the writing and revision of the manuscript. XMW, XYW, and HH performed the literature search and data analysis. XS and TL critically revised the manuscript. BW supervised the entire project. XH and HC were involved in the study design and the critical review and revision of the manuscript. All authors contributed to the article and approved the submitted version.

This work was supported by the grants from the National Natural Science Foundation of China (82070545 and 81970477) and the Key Project of Science and Technology Pillar Program of Tianjin (20YFZCSY00020).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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