Microbiota-driven immunometabolic regulation in colorectal cancer: Mechanisms and therapeutic opportunities

Abstract

Colorectal cancer (CRC) is a highly prevalent and lethal malignancy worldwide, marked by a multifaceted pathogenesis that involves various genetic, environmental, and lifestyle factors. Recent studies have increasingly underscored the significant role of gut microbiota in CRC, particularly focusing on how these microorganisms can modulate the host’s immune and metabolic processes. Metabolites produced by gut microbiota function as crucial signaling molecules that can have profound impacts on immune cell activity, contributing to the remodeling of the tumor microenvironment (TME). This, in turn, can influence critical aspects of cancer biology, including tumor initiation, progression, and the efficacy of therapeutic interventions. This review delves into the complex interactions that exist between gut microbiota and immunometabolism within the context of CRC, paying particular attention to how microbiota-mediated regulation of immunometabolism can affect the TME. Additionally, we explore the potential applications of these insights in enhancing the effectiveness of radiotherapy for CRC treatment. By synthesizing the latest findings from both clinical and preclinical studies, we aim to highlight the prospects of targeting gut microbiota as a novel therapeutic strategy, thereby offering a theoretical framework and fresh perspectives for precision treatment approaches in CRC management.

Key Words: Colorectal cancer; Gut microbiota; Immunometabolism; Tumor microenvironment; Metabolic reprogramming

Core Tip: The treatment of colorectal cancer (CRC) faces challenges of drug resistance mediated by the tumor microenvironment (TME). This review proposes that the gut microbiota is a core regulator in reshaping CRC immune metabolism and TME. We have established an integrated framework of the “microbiota-immune metabolism-TME” axis, systematically elaborating on the mechanisms of CRC occurrence, development, and treatment resistance from the micro level to the macro level. This perspective provides new ideas for deconstructing complexities such as clinical drug resistance. Based on this, we further explore translational strategies targeting the microbiota to reverse immunosuppression, enhance the efficacy of radiotherapy, chemotherapy, and immunotherapy, offering innovative theoretical basis and intervention prospects for the precise treatment of CRC.

INTRODUCTION

Colorectal cancer (CRC) has the second-highest global incidence and mortality rates of all cancers[1]. A key challenge in treating CRC is that complex immunometabolic changes within tumors can induce immunosuppression, limiting the effectiveness of immunotherapy. Although the 5-year survival rate for CRC has improved over time, rising from 50% in the 1970s to 65% in 2012-2018, a concerning trend has emerged: Incidence rates among individuals aged 15-50 years have increased by 80%-100% in recent years[2]. This rise in early-onset CRC presents significant new challenges for treatment. In the era of precision medicine, immunotherapy has gained widespread attention as a complement to conventional surgery, radiotherapy, and chemotherapy. Its principal advantage is the precise targeting of tumor cells, which minimizes damage to healthy tissues[3]. However, immunotherapy is effective only in a limited subset of patients. A key predictive biomarker is high microsatellite instability, but this status is found in only about 15% of all CRC cases and a mere 4% of metastatic cases[3-7]. The tumor microenvironment (TME) is a critical factor in this limited response. Understanding the dynamic interactions between the cellular and non-cellular components of the CRC TME is essential for improving the clinical efficacy of immunotherapy.

Metabolic reprogramming is a hallmark of CRC[8] (Figure 1) and is closely linked to the low immunotherapy response rates observed in this cancer. For instance, intestinal stem cells, which can initiate CRC, possess unique metabolic traits that confer resistance to conventional therapies[9]. A primary metabolic alteration in CRC cells is a heightened reliance on glycolysis, known as the Warburg effect, even in the presence of oxygen[10]. Concurrently, alterations in lipid metabolism channel glycolytic intermediates into de novo lipid synthesis[11]. This accumulated lipid provides building blocks for new membranes and facilitates signaling activities, thereby supporting tumor initiation and progression[12]. Collectively, these metabolic shifts—including imbalanced glycolysis/oxidative phosphorylation (OXPHOS) and enhanced lipid synthesis—create a TME characterized by acidity, hypoxia, and nutrient depletion. This harsh environment directly impairs the metabolic pathways and anti-tumor functions of infiltrating immune cells[13].

Figure 1 Metabolic reprogramming in cancer cells. The metabolic reprogramming mechanism in cancer cells involves a series of changes in core molecules and metabolic pathways aimed at meeting their energy, nutrient, and biosynthetic material demands for rapid growth and uncontrolled proliferation. Warburg effect is a hallmark of metabolic reprogramming in cancer cells. While normal cells primarily produce energy through oxidative phosphorylation, cancer cells tend to ferment glucose into lactic acid, even in the presence of oxygen. This process allows cancer cells to generate fewer ATP but provides more biosynthetic materials needed for growth and division. Glucose metabolism: Cancer cells significantly increase their uptake and metabolism of glucose to meet their high energy demands. This includes glycolytic pathways where glucose is broken down into pyruvate and lactic acid, producing a small amount of ATP. Core molecules involved in this process include the regulation of PFK-1 and the regulation of the pentose phosphate pathway. When cancer cells are exposed to low oxygen conditions, HIF-1α protein is activated, prompting cells to use lactic acid fermentation for metabolism to enhance their survival and growth. HIF-1α activation also leads to the upregulation of glucose metabolism genes such as GLUT1 and lactate dehydrogenase. AMPK activated when energy supply is insufficient. AMPK inhibits cell growth and promotes energy production by inhibiting mTORC1, slowing down protein synthesis, and promoting glucose uptake and oxidation to restore energy balance. PI3K/Akt/mTOR pathway plays a crucial role in the metabolic reprogramming of cancer cells. Activation of Akt promotes highly enhanced glycolysis, while mTOR regulates cell growth and metabolism. p53 is a suppressive protein that regulates the cell cycle and metabolism. In some cases, the loss of p53 in cancer cells leads to metabolic reprogramming, including increased glycolysis and glucose uptake. Lipid metabolism-related core molecules include lipid synthesis enzymes such as fatty acid synthase and enzymes related to lipid breakdown. Cancer cells tend to increase lipid synthesis to meet their membrane component requirements. Cancer cells often exhibit alterations in amino acid uptake and metabolism. Core molecules include mTORC1 and GCN2, which play important roles in amino acid synthesis and metabolism. Serine metabolism-related molecules including serine synthesis enzymes and pyruvate kinase, are involved in serine metabolism, providing essential metabolic products for growth and biosynthesis. This Figure was drawn by Figdraw (Supplementary material). MCT: Monocarboxylate transporter; FA: Fatty acid; FAS: Fatty acid synthase; FATP: Fatty acid transport protein; TCA: Tricarboxylic acid; ROS: Reactive oxygen species; OXPHOS: Oxidative phosphorylation; SOD: Superoxide dismutase; SDH: Succinate dehydrogenase; FH: Fumarate hydratase; FAO: Fatty acid oxidation; OAA: Oxaloacetate.

Driven by these metabolic alterations, along with genetic and epigenetic changes, CRC evolves robust immune evasion mechanisms. These are typified by the exhaustion of T cells and the polarization of macrophages towards a pro-tumor (M2) phenotype[14-17]. The gut microbiota is now recognized as a key regulator of immunometabolism and a potent modifier of the TME in CRC. For example, in advanced CRC, microbial-derived ammonia can accumulate and trigger metabolic reprogramming in T cells, promoting their exhaustion and contributing to immunotherapy resistance[18]. Given its direct anatomical connection to the colon and its demonstrated role in carcinogenesis, the gut microbiota holds unique significance in CRC, distinguishing it from many other cancer types.

The role of the gut microbiota in the initiation and progression of CRC is now widely acknowledged. As a major symbiotic community, the composition and function of the gut microbiota are critical for host health. An imbalance, known as dysbiosis, is strongly linked to CRC development[19]. Specific pro-oncogenic species include Fusobacterium nucleatum (F. nucleatum), certain strains of Escherichia coli (E. coli), and enterotoxigenic Bacteroides fragilis (B. fragilis)[20]. Furthermore, modulating the gut microbiota shows promise for improving immunotherapy efficacy and reducing radiotherapy-related toxicity[21]. For instance, the compound ginsenoside Rh4 exerts anti-CRC effects in a microbiota-dependent manner by altering bile acid metabolism and increasing levels of ursodeoxycholic acid (UDCA)[22]. Therefore, a deeper understanding of the gut microbiota's interaction with host immunometabolism is crucial for developing novel therapeutic strategies for CRC.

Therefore, investigating the changes in the relationship between CRC, the immune system, and the gut microbiota during the development and progression of CRC is of great clinical significance. The primary objective of this review is to integrate and synthesize recent research on the interplay between the gut microbiota and immunometabolism phenomena in CRC. Compared with existing literature on CRC microbiota or immune metabolism, we not only outlined the roles of both in the development of CRC from a “microscopic” perspective respectively, but also explored their mutual influence and combined effect from a “macroscopic perspective”. By placing the single characteristics of CRC cells in the broader context of the TME, we more systematically and coherently analyzed and demonstrated the mechanisms underlying the occurrence and development of CRC, which coincides with deconstructing the complexity of various important mechanisms affecting prognosis represented by “drug resistance” in the actual clinical treatment process.

To ensure a comprehensive and reproducible overview of the current knowledge, a systematic literature search was conducted. Primary searches were performed in the electronic databases PubMed, Web of Science, and Scopus for articles published between January 1984 and October 2025. The search strategy employed a combination of keywords and MeSH terms related to three core themes: (1) Colorectal cancer (“colorectal cancer”, “CRC”, “colon cancer”, “rectal cancer”); (2) Gut microbiota (“gut microbiome”, “intestinal flora”, “microbiota”); and (3) Immunometabolism and tumor microenvironment (“immunometabolism”, “tumor microenvironment”, “TME”, “immunotherapy”, “metabolic reprogramming”). Boolean operators (AND, OR) were used to link these concepts. The initial search results were screened based on their titles and abstracts to identify relevant studies. The full texts of the shortlisted articles were then thoroughly assessed against predefined inclusion criteria (original research or review articles in English focusing on the interplay between gut microbiota, immunometabolism, and CRC) and exclusion criteria (studies not relevant to the core focus, non-English publications, or conference abstracts). Any disagreements during the selection process were resolved through consensus among the authors. Finally, a total of 2010 documents were included in the analysis. In addition, we will outline the current progress in the clinical translation of research related to the gut microbiota and immunometabolism, further elucidating promising innovative therapeutic approaches from a clinical perspective. We hope that our study can contribute to advancements in the field of immunotherapy for CRC.

IMMUNOMETABOLISM IN CRC

The role of immune cells in CRC

A conspicuous infiltration of T cells stands as a hallmark feature of CRC[23], yet their functional fate is dictated by a complex interplay with other immune constituents within the TME. Staging based on CD3⁺/CD8⁺ T cell density can provide better prognostic stratification compared with traditional staging, and it has the potential to become a promising independent prognostic biomarker[24]. The mechanisms that cause immunosuppression can be divided into three main areas: (1) Cell-cell interaction circuits: Intestinal epithelial-derived microvesicles deliver dual apoptotic signals to induce T cell death[25,26], while programmed death 1 (PD-1)-expressing epithelial cells subvert immunity through both CD28 co-stimulation blockade and Treg homeostasis maintenance[27,28]; (2) Metabolic cross-talk: The glycolytic switch in CRC cells elevates lactic acid to 10-30 mmol/L in TME[29]. Creating a paradoxical scenario where lactic acid simultaneously inhibits effector T cells/natural killer (NK) cells while fueling Treg immunosuppressive functions through HIF-1α-mediated metabolic adaptation[30]; and (3) Spatial reconfiguration: Recent spatial transcriptomics reveals M2-polarized tumor-associated macrophages (TAMs; CD163⁺, CD206⁺) preferentially accumulate at tumor-stroma interfaces, forming physical barriers that sequester CD8⁺ T cells from carcinoma nests[31-33]. The effects of immune cells from the TME indicated in Figure 2.

Figure 2 The effects of immune cells from the tumor microenvironment. Different types of immune cells have various effects on the tumor microenvironment: CD4⁺ T cells can differentiate into different subsets, such as Th1 cells, which release interferons and activate other immune cells to enhance anti-tumor immune responses. However, Tregs common in the tumor microenvironment can suppress immune responses, contributing to immune evasion by the tumor. Macrophages have the ability to engulf tumor cells, release inflammatory factors, recruit other immune cells, clear tumor cell debris, and promote anti-tumor immune responses. However, in certain cases, tumors can activate macrophages to produce inhibitory factors, thereby suppressing their anti-tumor effects. myeloid-derived suppressor cells are a type of immunosuppressive cells that inhibit T cell activity in the tumor microenvironment, suppressing immune responses. They hinder the function of immune cells by releasing inhibitory molecules like NO and H₂O₂, aiding in immune evasion by the tumor. Dendritic cells are antigen-presenting cells that can recognize tumor antigens and present them to T cells, activating T cell immune responses. The presence of dendritic cells can enhance anti-tumor immune effects, but certain factors in the tumor microenvironment may inhibit dendritic cell function. Natural killer (NK) cells can rapidly recognize and kill tumor cells without the need for prior immune memory. They induce tumor cell apoptosis by releasing perforin and other cytotoxic substances. The activity of NK cells may sometimes be suppressed in the tumor microenvironment. This Figure was drawn by Figdraw (Supplementary material).

The above examples are intended to illustrate that the antitumor immune response and the immunosuppressive response in CRC TME is a complex process, and that synergistic or antagonistic effects between various immune cells may have a greater impact than those produced by a single immune cell, for example, moving away from an immune cell-centered immunotherapy mindset alone to targeting the entire TME on the basis of metabolic alterations in immune cells may be the key to breaking through immunosuppression. Next, we will start from the metabolic changes of the main immune cells, and gradually reveal the active or passive synergism and antagonism between immune cells, while exploring the game between immune cells and tumor cells in TME, with a view to providing new ideas for CRC immunotherapy.

The metabolic characteristics of immune cells in CRC

T-cells & B-cells: The metabolic plasticity of tumor-infiltrating CD8⁺ T cells is hijacked during CRC progression through multiple co-opted pathways. In early tumor stages, activated CD8⁺ T cells undergo Warburg-like glycolytic shifts[34] (Figure 3), characterized by PDK1-mediated diversion of pyruvate to lactate production rather than mitochondrial CoA generation, and this process, amplified by PI3K/AKT-driven GLUT1 trafficking[35-37]. Consequently, should the glycolytic process in T cells be compromised, the functional efficacy of these cells would diminish. Thus, CRC cells actively suppress CD8⁺ T cell glycolysis by exploiting inhibitory checkpoints such as TIGIT, thereby impairing antitumor immunity[38], a preclinical study showed that in mouse models, inducing functional impairment—a vulnerability partially reversed by rhus chinensis-derived triterpenoids that reactivate glycolytic genes[39]. Intriguingly, CRC-associated regulatory T cells (Tregs) exhibit glycolytic hyperactivity too. Through suppression of the MondoA-TXNIP axis, Tregs upregulate GLUT1 to acquire a Th17-like phenotype in high-lactate niches, creating protumor inflammation via interleukin (IL)-17A/IL-23 feedforward loops[40].

Figure 3 Mechanisms of glucose metabolism and fatty acid metabolism in tumor cell. Glucose metabolism and fatty acid metabolism are two essential metabolic pathways in cells that provide the necessary energy and building blocks for various cellular processes. Here is a detailed description of these two metabolic processes, including the roles of key molecules: Glucose metabolism: Glycolysis is the process by which glucose is broken down into pyruvate and lactate, typically occurring in the cytoplasm. This pathway involves a series of reactions where glucose is converted into two three-carbon molecules, generating a small amount of ATP and NADH. PFK is the rate-limiting enzyme of glycolysis, and its activity increases in response to energy demands, driving glycolysis. PK promotes the conversion of pyruvate into lactate in the final step of glycolysis. Citric acid cycle (Krebs cycle) takes place in the mitochondria and involves the oxidation of pyruvate into carbon dioxide and ATP. NAD⁺ and FAD accept electrons during the citric acid cycle and ultimately drive ATP synthesis in the electron transport chain. The electron transport chain is located on the inner mitochondrial membrane and transfers electrons from NADH and FADH₂ to oxygen, resulting in the production of a significant amount of ATP. Cytochrome c responsible for electron transfer, ultimately driving ATP synthesis. Fatty acid metabolism: Fatty acid synthesis occurs in the cytoplasm, where acetyl-CoA is used to synthesize long-chain fatty acids through a series of enzymatic reactions. Acetyl-CoA is the initial substrate for fatty acid synthesis, often derived from glucose metabolism. Fatty acid synthase is a key enzyme responsible for synthesizing fatty acids. Fatty acid oxidation occurs in the mitochondria, where long-chain fatty acids are oxidized to generate acetyl-CoA, which is used to produce ATP. This Figure was drawn by Figdraw (Supplementary material). MCT: Monocarboxylate transporter; LDH: Lactate dehydrogenase; PDH: Pyruvate dehydrogenase complex; OAA: Oxaloacetate.

Next, when the main energy substance glucose is gradually depleted, T cells shift to OXPHOS and fatty acid (FA) catabolism, but CRC-induced lipid overload disrupts mitochondrial cristae ultrastructure, causing reactive oxygen species (ROS)-mediated dysfunction[41-43]. In addition, short-chain FAs (SCFAs) like butyrate exhibit dual roles: While beneficial microbes (e.g., Lactobacillus plantarum) epigenetically suppress Saa3 to reduce cholesterol toxicity[44]. Pathobionts remodel glycerophospholipid metabolism to impair CD8⁺ T cell surveillance[45].

By the discussion in the previous paragraph, we already know the main metabolic changes that occur in T cells in CRC TME, that is, one of the essences of the metabolic reprogramming phenomenon in CRC TME is the competition between CRC cells and immune cells for energy substances. This competition also exists among other nutrients that can provide energy, for example, amino acids. The metabolic competition between tumor-infiltrating lymphocytes (TILs) and CRC cells manifests as reciprocal fuel preferences: TILs predominantly utilize glycolysis, while CRC cells rely on glutamine metabolism—a dichotomy conserved across CRC subtypes[46]. Activated effector T cells require glutamine uptake to sustain proliferation, yet paradoxically, pharmacological glutaminase inhibition triggers compensatory glycolytic hyperactivation, compromising their long-term functionality[47]. While L-arginine supplementation enhances TIL cytotoxicity by forcing metabolic reprogramming from glycolysis to OXPHOS[48,49]. this therapeutic potential is subverted by myeloid-derived suppressor cells (MDSCs) through dual enzymatic sabotage: MDSC-derived arginase-1 depletes extracellular L-arginine, while inducible nitric oxide synthase generates peroxynitrite to nitrosylate TCR complexes, collectively blunting antigen recognition[50].

Importantly, B cells amplify this metabolic warfare—their reprogramming toward NADPH-generating PPP and immunoregulatory lipid metabolism reshapes the TME to favor CRC immune evasion[51]. B cells deploy context-dependent metabolic programs in CRC. Upon activation, B cells increase glycolysis and OXPHOS, fueled by PI3K/mTORC2-mediated glucose/glutamine co-utilization[52,53]. However, chronic antigen exposure drives a metabolic shift towards leucine dependency—LARS B cells utilize LARS2 to stabilize mitochondrial NADH dehydrogenases, sustaining proliferation against tumor-derived oxidative stress[54,55]. This phenomenon existing in B cells further supports our view that paying attention to changes in the entire TME may be more important than focusing on changes in a single immune cell.

Macrophages: Based on distinct functional characteristics, it is possible to further categorize the predominant macrophage population within TAMs into M1 and M2 subtypes. The presence of M1-type macrophages is considered to be associated with a favorable prognosis in CRC[56]. The former exhibits a predilection for OXPHOS in glucose metabolism, while the latter leans towards glycolysis and FA oxidation (FAO)[57]. In early-stage CRC, the pro-inflammatory subtype of TAMs undergoes a metabolic reprogramming that results in an increase in itaconate salts, promoting the progression of CRC, and this suggests the existence of a metabolic reprogramming in CRC-infiltrating macrophages, including but not limited to glucose metabolism[58].

When intestinal epithelial cells (IECs) are damaged, macrophages increase the metabolism of various amino acids, such as arginine, through the Tsc2/mTORC1 pathway to generate more energy, but accordingly, polyamine metabolites from amino acid metabolism further promote the proliferation of CRC cells[59], while the key transporter protein amino acid transporter LAT1 (also known as SLC7A5) promotes CRC progression by mediating increased leucine metabolism and inducing polarization of TAMs toward the M2 phenotype[60].

Regarding lipid metabolism reprogramming, the phenomenon of metabolic reprogramming in macrophages manifests in a more diverse manner. A clinical study involving 40 CRC patients demonstrated that GRP78, when incorporated into macrophage lipid droplets, enhances the protein stability of adipose triglyceride lipase by inhibiting ubiquitination. This process promotes the hydrolysis of triglycerides and the production of arachidonic acid and docosahexaenoic acid to maintain a tumor-immunosuppressive TME and also induce the polarization of macrophages towards the M2 phenotype, this phenomenon particularly pronounced in advanced stages of CRC[61]. In addition, contrary to T cells, the accumulation of cholesterol in CRC infiltrating macrophages facilitates their polarization towards the M1 phenotype[62]. This polarization enhances the activation of T cells by macrophages, thereby amplifying the anti-tumor effect. This reprogramming of cholesterol metabolism is mediated by 5-aza-2'-deoxycytidin[63]. Additionally, the ectopic expression of lipolytic co-activator ABHD5 in CRC-associated macrophages suppresses the production of spermidine ultimately induces the growth of CRC[64].

We have mentioned earlier that of equal interest to the altered metabolism of immune cells is the game between immune cells and tumor cells in the TME, and we would now like to add further to this idea by drawing on the discussion of the altered metabolism of macrophages in this subsection and this example to be mentioned next. Platinum-based drugs are an important component of cancer chemotherapy; however, macrophages treated with naphthylplatin trigger calcium release via Mcoln1, leading to activation of lysosomal metabolism. This in turn induces activation of p38 and nuclear factor-kappa B, promotes M2 polarization[65], and exacerbates immunosuppression in CRC, which is the exact opposite of the original intent of the treatment. So, is there more than a game of immune cells vs tumor cells behind those phenomena of gradual immunosuppression as the number of treatments increases in CRC patients? If so, how much influence do chemotherapeutic agents, often applied in combination with immunotherapy, have in this? How is this influence to be controlled and monitored? These questions urgently require an answer. Figure 4 show the roles of macrophages in cancer.

Figure 4 The roles of inflammatory macrophages and immunosuppressive macrophages in cancer. Role of inflammatory macrophages in tumors: Inflammatory macrophages typically participate in early anti-tumor immune responses. Inflammatory macrophages can recognize and phagocytose tumor cells, eliminating potential tumor cell threats. They release inflammatory cytokines such as tumor necrosis factor alpha, interleukin (IL)-1β, and IL-12 to stimulate an inflammatory response, recruit other immune cells like T cells and natural killer cells, and assist in clearing tumor cells. Inflammatory macrophages also influence tumor development through various molecules and signaling pathways, such as NF-κB signaling pathway, iNOS, IL-12 and IL-23, which promote T cell activation and enhance the immune response. Immunosuppressive macrophages, also known as M2-type macrophages, typically play an immunosuppressive role in the tumor microenvironment, helping to maintain immune balance and reduce inflammation. Immunosuppressive macrophages release anti-inflammatory cytokines like IL-10 and transforming growth factor beta (TGF-β) to suppress the activity of immune cells, especially T cells and influence the growth and survival of tumor cells in the tumor microenvironment through various molecules and signaling pathways, such as STAT3 signaling pathway which is activated in immunosuppressive macrophages, promoting the production of anti-inflammatory factors like TGF-β. Besides, the activity of ARG1 is increased in immunosuppressive macrophages, leading to the degradation of L-arginine and the suppression of T cell activity. This Figure was drawn by Figdraw (Supplementary material). FAS: Fatty acid synthase; FAO: Fatty acid oxidation; DAMP: Damage-associated molecular pattern; IDO: Indoleamine-2,3-dioxygenase; TNF: Tumor necrosis factor; IL: Interleukin; TGF-β: Transforming growth factor-beta.

MDSCs: MDSCs renowned for their immunosuppressive effects on T cells. Under physiological conditions, bone marrow-derived multipotent hematopoietic stem cells have the capacity to differentiate into macrophages, dendritic cells, and granulocytes[66]. Conversely, the TME can suppress MDSCs differentiation, leading to the accumulation of a significant population of immature pathological cells[67]. These pathological cells have been linked to adverse prognoses in CRC, immunosuppression, drug resistance, invasion, and metastasis[68].

During the differentiation and activation of MDSCs, its metabolic changes have many similarities to those mentioned earlier[69,70]. For example, activation of leucine-rich repeats, nucleotide-binding oligomeric structural domains, and pyrimidine-containing structural domain 3 inflammatory vesicles via the STAT1-NLRP3 axis, which in turn drives CRC progression[71,72]. On the contrary, CRC cells contribute to the generation of MDSCs. These MDSCs induced by CRC can inhibit T cell proliferation and facilitate the growth of CRC cells through cell-cell contact. However, by suppressing oxidative metabolism, including the production of NO and ROS, this suppression of T cells can be mitigated[72].

The upregulation of the scavenger receptor CD36 promotes the uptake of fats by MDSCs, shifting their primary energy source from glycolysis to FAO[73], in contrast, deletion of Mir4435-2hg leads to an increase in polymorphonuclear (PMN)-MDSCs and disrupts their FA metabolism to enhance their immunosuppressive capacity[74]. In light of this process, it is widely acknowledged in preclinical research that FA transport protein 2 acts as a regulatory factor in the immunosuppressive function of PMN-MDSCs, and inhibiting FA transport protein 2 can significantly enhance the efficacy of immunotherapy[75]. Taking another lipid as an example, retinoic acid inhibits glycolysis in myeloid cells to activate the AMPK pathway and weakens the immunosuppressive power of MDSCs, but in human CRC, alcohol dehydrogenase 1 mediates a decrease in retinol metabolism and impairs retinoic acid signaling leading to abnormal aggregation of PMN-MDSCs, resulting in a poor prognosis for CRC[76].

Finally, there are changes in amino acid metabolism. Amino acid metabolism is included as a separate segment because this metabolic alteration in MDSCs is often accompanied by suppression of T cells. The metabolism of arginine and nitric oxide involved here has been introduced earlier[77,78], but a deficiency in 5-lipoxygenase reduces the activity of arginase 1 in MDSCs, thereby diminishing their capacity to suppress T cell functionality, and this reduction leads to a decrease in the occurrence of colonic polyps and CRC[79]. Similarly, this mechanism of impairing T cell functionality by depleting essential amino acids in T cells also occurs with amino acids such as tryptophan and cysteine[80]. MDSCs diminish tryptophan levels in the TME by relying on indoleamine-2,3-dioxygenase (IDO)-dependent tryptophan metabolism, depleting T cell nutrients, thereby inhibiting their normal immune activity[81]. It is an interesting phenomenon that in addition to the game between immune cells and tumor cells, there is also a mutual game between immune cells, which, although also mentioned in the previous section, appears to be particularly obvious in MDSCs and T cells as two mutually antagonistic cells, which undoubtedly re-emphasizes the holistic nature of the TME.

NK cells: NK cells exhibit rapid cytotoxic responses against tumor and virus-infected cells through granzyme/perforin-mediated killing and IFN-γ production[82]. Competitive uptake of cystine/glutamine by CRC cells leads to polyamine depletion in NK cells, and subsequent inhibition of c-Myc expression downregulates key enzymes of glycolysis by approximately 60%, ultimately leading to a reduction in the efficiency of perforin-mediated lysis of tumor cells to 39% ± 6% of baseline levels[83,84]. In patients with liver metastases of CRC, there is a significant increase in lactate levels within the TME. NK cells unable to regulate their internal pH consequently experience mitochondrial metabolic dysfunction, then leads to an excessive accumulation of ROS, ultimately resulting in mitochondrial stress and cell apoptosis[85]. Additionally, NK cell FA metabolism is enhanced to ensure their energy supply too. However, if NK cells continue to be stimulated, such as IL-15, they are eventually induced to generate PPARγ-driven lipid overload and endoplasmic reticulum stress, resulting in NK cell exhaustion[86].

REGULATORY MECHANISMS OF GUT MICROBIOTA ON IMMUNE METABOLISM IN CRC

Direct effects of gut microbiota

Specific conserved molecules on the surface of gut microbiota can be recognized by pattern recognition receptors (PRRs, such as Toll-like receptors TLRs and NOD-like receptors NLRs) on immune cells, thereby activating downstream signaling pathways and precisely regulating immune responses. Studies have found that certain intestinal bacteria (such as CAG-977) promote the transformation of effector T cells into phenotypes with more anti-tumor effects by activating specific signaling pathways[87]. F. nucleatum adheres to, invades, and induces the occurrence of CRC through its unique FadA adhesin. It can also directly bind to the TIGIT receptor on immune cells, inhibiting the killing function of NK cells and cytotoxic T cells[88,89]. For example, in diarrhea caused by E. coli, some regulatory factors can induce microorganisms to express antigens that have structural similarities to host or tumor-associated antigens[90]. This cross-reactivity may activate T cells and other immune responses.

Metabolites of gut microbiota

SCFAs: SCFAs, primarily acetate, propionate, and butyrate, are produced by gut bacteria such as Bacteroides, Prevotella, and Clostridium. In CRC, SCFAs modulate immune cell metabolism to exert anti-tumor effects. For instance, SCFAs activate the GPR41 receptor on CD8⁺ T cells, enhancing both their OXPHOS and glycolytic capacities. This metabolic reprogramming promotes CD8⁺ T cell proliferation, differentiation, and strengthens their anti-tumor immune response[91,92]. Furthermore, SCFAs promote FA uptake in these cells. Utilizing FA catalysts and glutamine, the cells shift their glucose metabolism towards OXPHOS and differentiate into memory T cells, sustaining long-term immunity[93,94]. Additionally, butyrate acts as a histone deacetylase (HDAC) inhibitor to promote regulatory T cell (Treg) differentiation, suppress pro-inflammatory factors [e.g., IL-6, tumor necrosis factor alpha (TNF-α)], and enhance intestinal barrier function. Similarly, butyrate and valerate can upregulate immunoregulatory B10 cells via p38 MAPK activation[95,96]. Notably, the anti-inflammatory effects of butyrate are primarily observed in healthy intestinal tissue. In the CRC TME, butyrate can exert opposing, pro-tumorigenic effects. This context-dependent duality highlights the intricate interplay between the TME and SCFA-mediated metabolism. For example, within the TME, butyrate induces metabolic reprogramming in macrophages towards OXPHOS and lipid metabolism, promoting their M2 polarization and thereby accelerating CRC progression[97].

Tryptophan metabolites: Tryptophan is metabolized by gut microbiota, including Bacteroides, Clostridium, and E. coli, into indoles and kynurenine[98]. Alterations in gut microbiota-mediated tryptophan metabolism are closely related to intestinal barrier function and may be involved in the occurrence and development of CRC[99]. These metabolites exert divergent immunomodulatory effects via the aryl hydrocarbon receptor (AhR). Indole derivatives, such as indolepropionic acid, activate AhR to promote Treg differentiation and suppress Th17-driven inflammation. In contrast, kynurenine activates AhR to inhibit CD8⁺ T cell infiltration, thereby facilitating tumor immune escape. Inhibiting the key enzyme IDO, which drives kynurenine production, can reverse this immunosuppression and shows therapeutic potential in inflammatory CRC models[100].

Polyamines: Polyamines, including putrescine, cadaverine, and spermidine, are primarily produced by gut microbiota such as Bacteroides, Clostridium, and Streptococcus[101]. Mouse studies indicate that polyamines help maintain gut homeostasis. Spermine oxidase deficiency reduces α-defensin production, leading to microbial dysbiosis and promoting the transition from colitis to CRC. Conversely, spermidine supplementation can restore homeostasis and prevent CRC development[102]. Moreover, in the context of CRC, spermidine supplementation can reactivate NK cells by alleviating c-Myc suppression and restoring glycolytic energy production, thereby restoring their tumor-killing activity[83]. Beyond their role in immune cell function, polyamines can also directly induce apoptosis in CRC cells by mechanisms that include inhibiting MYC synthesis and promoting mitochondrial depolarization.

Secondary bile acids: Secondary bile acids mainly include deoxycholic acid (DCA), lithocholic acid (LCA), etc., and are mainly derived from Firmicutes (such as Clostridium scindens). Studies have shown that the metabolic level of secondary bile acids in the gut microbiota is significantly increased in inflammatory bowel disease and CRC, and DCA and LCA are more carcinogenic than other secondary bile acids due to their properties of causing DNA damage and inducing oxidative stress[103]. This process has been confirmed to be closely related to two bile acid-sensitive receptors: Farnesoid X receptor (FXR) and G protein-coupled bile acid receptor 1[104]. Among them, FXR has been proven to play a role in inhibiting the progression of CRC. For example, ginsenoside Rh4 can regulate gut microbiota -mediated bile acid metabolism, promote the production of UDCA and activate the FXR receptor, and regulate the TLR4-NF-κB signaling pathway, thereby inhibiting the development of CRC in a gut microbiota-dependent manner[22]. In addition, studies have found that DCA can promote tumor cell proliferation, induce epithelial-mesenchymal transition (EMT), increase vasculogenic mimicry formation, and activate VEGFR2, thereby leading to intestinal carcinogenesis[22].

Lipopolysaccharide: Lipopolysaccharides (LPS) are mainly derived from Gram-negative bacteria (such as E. coli, F. nucleatum), etc. LPS involved in blood circulation can promote systemic inflammation and affect the coagulation system, thereby leading to chronic inflammation and the occurrence of tumors[105]. The production of this inflammation is related to the negative regulation of glutathione peroxidase 4 by acetylated high mobility group box-1. Further studies have shown that LPS can regulate immune responses in the CRC TME, and this process is related to macrophage M2 polarization and inhibition of T cell responses[106]. In addition, LPS can also increase intestinal muscular inflammation by activating p38-MAPK, increasing the risk of intestinal obstruction in patients after CRC surgery[107]. It can thus be inferred that regulating inflammatory responses is also one of the pathways through which LPS promotes the development of CRC.

H₂S: Hydrogen sulfide mainly originates from sulfate-reducing bacteria (such as Desulfovibrio) and the like. Studies have found that H₂S derived from Desulfovibrio can inhibit mitochondrial respiration and induce the unfolded protein response in intestinal L cells, thereby hindering the secretion and gene expression of GLP-1 and further impairing the host's metabolism[108]. At the same time, hydrogen sulfide can also induce metabolic reprogramming of colon cells by disrupting mitochondrial bioenergy[109]. In addition, F. nucleatum in CRC can regulate the autophagy process and disrupt the normal gut microbiota by producing hydrogen sulfide[110]. It can be seen that disrupting intestinal barrier function and inducing metabolic disorders are the main ways by which hydrogen sulfide promotes the development of CRC.

Branched-chain amino acids: Branched-chain amino acids (BCAAs), namely leucine, isoleucine, and valine, are essential amino acids that serve as crucial nutrient signals and metabolic fuels in the CRC TME. While diet is the primary source, the gut microbiota also contributes to the host's BCAA pool through synthesis and degradation, thereby influencing systemic and local BCAA availability. Parabacteroides merdae can decompose BCAAs to produce three BCAA metabolites-isovaleric acid, 2-methylbutyric acid, and isobutyric acid. It can delay aging in mice through various health benefits such as reducing oxidative stress and inflammation, improving muscle capacity, reversing brain acetylcholine levels, and regulating blood glucose[111]. In addition, BCAA metabolites can also regulate metabolic disorders, thereby maintaining the homeostasis of gut microbiota and effectively preventing the occurrence of precancerous lesions such as inflammatory bowel disease[112].

REGULATION OF THE CRC TME BY GUT MICROBIOTA

Inducing pro-tumor inflammatory responses

Gut microbiota represented by F. nucleatum, enterotoxin-producing B. fragilis, and adherent-invasive E. coli can impair intestinal barrier function through various pathways. This causes a large number of bacteria and their metabolites to penetrate into the submucosa, which are then recognized by immune cells and continuously activate inflammatory pathways (such as the NF-κB signaling pathway)[113], and release pro-inflammatory factors, ultimately forming a chronic inflammatory environment that is conducive to the occurrence of CRC[114]. Preclinical studies have shown that F. nucleatum can release outer membrane vesicles, activate TLR4 and NF-κB, and promote the development of intestinal inflammation[115]. In CRC, F. nucleatum activates the TLR4/Myd88/NF-κB signaling pathway, transcriptionally activates the expression of miRNA-155-5p, thereby negatively regulating MSH6 and accelerating the malignant progression of CRC in mice[116]. In addition, enterotoxigenic B. fragilis disrupts the intestinal barrier by stimulating IECs to induce the activation of MMP-7 and transcription factors NF-κB and AP-1, and promoting the release of syndecan-2[117]. However, B. fragilis that can produce capsular polysaccharide A can promote the expression of anti-inflammatory IL-10, inhibit inflammation, and maintain the integrity of the intestinal barrier[118]. In patients with inflammatory bowel disease, the promoter of this type of capsular polysaccharide A is often in a closed state[119], which suggests the potential of targeting intestinal microorganisms for CRC treatment. Another type of microbiota that has been confirmed to play an important pro-inflammatory role is adherent-invasive E. coli. By virtue of its produced FimH adhesin and cell adhesion molecule 6, it activates the NF-κB pathway to promote the expression of a large number of pro-inflammatory cytokines such as TNF-α and chemokines such as IL-8, thereby inducing chronic inflammation. At the same time, it activates inflammasomes to induce the formation of IL-1β and IL-18, aggravating the inflammatory response[120,121], and ultimately promoting the occurrence and development of CRC.

Promote the formation of an immunosuppressive TME

First, the gut microbiota can directly suppress anti-tumor immunity. A representative mechanism is the inhibition of the killing ability of CD8⁺ T cells through the calcineurin-NFAT axis[122]. For example, the metabolite secondary bile acid inhibits this pathway by targeting the plasma membrane Ca²⁺ ATPase, thereby achieving immunosuppression[123]. Secondly, the gut microbiota can recruit myeloid-derived immunosuppressive cells or induce M2 polarization of macrophages to achieve immunosuppression. For example, the gut microbiota metabolite 4-hydroxyphenylacetic acid upregulates the transcription of CXCL3 by activating the JAK2/STAT3 pathway, thereby recruiting PMN-MDSCs into the CRC TME, inhibiting the anti-tumor responses of cells such as CD8⁺ T cells, and promoting the progression of CRC[124], In metastatic CRC, the intestinal microbiota induces the secretion of cathepsin K, and promotes the metastasis of CRC by mediating the polarization of TLR4-dependent M2 macrophages[125]. Third, the gut microbiota competes with immune cells for nutrients, directly or indirectly participating in the shaping of an immunosuppressive TME. For example, as mentioned earlier, the gut microbiota can consume tryptophan and produce kynurenine, which directly or indirectly inhibits T cell proliferation while promoting the function of Tregs cells. In addition, different microbial metabolites can also regulate the direction of T cell differentiation by inhibiting the activation of antigen-presenting cells (APCs). For instance, SCFAs and secondary bile acids inhibit APC activation and reduce the differentiation of inflammatory T cells (Th1/Th17); whereas trimethylamine N-oxide and others promote APC activation and enhance the anti-tumor immunity of T cells[126]. Finally, the gut microbiota can also affect the CRC TME by acting on stromal cells. For example, F. nucleatum attaches to and invades cancer-associated fibroblasts (CAFs) in CRC, causing them to exhibit an inflammatory CAF phenotype. This phenotype is characterized by the secretion of pro-inflammatory cytokines (such as CXCL1, IL-6, and IL-8), the production of ROS, and enhanced metabolic activity. This transformation makes CRC cells more prone to proliferation and metastasis[127]. In addition, in young CRC patients, researchers have observed that actinomycetes abnormally reside in CAFs, thereby activating the TLR2/NF-κB pathway and reducing the infiltration of CD8⁺ T lymphocytes in the CRC TME[128]. In the formation of the immunosuppressive TME, these mechanisms often coexist and act synergistically, jointly constructing a powerful immunosuppressive network, enabling tumors to evade the surveillance and attack of the host immune system. This part is shown in Figure 5.

Figure 5 The impact of gut microbiota metabolites on immunometabolism and the tumor microenvironment in colorectal cancer. This diagram systematically illustrates the impact of six major metabolites of the gut microbiota on immune cells and the tumor microenvironment. Among them, short-chain fatty acids can, on the one hand, promote the killing effect of T cells and inhibit Tregs cells, thereby suppressing the development of colorectal cancer (CRC); on the other hand, they can induce the polarization of M2 macrophages to promote the progression of CRC. Tryptophan metabolites represented by kynurenine can inhibit the activity of CD8⁺ T cells. Polyamine metabolites are related to the apoptosis of CRC cells, and their deficiency often indicates the formation of an immunosuppressive microenvironment. Secondary bile acids such as lithocholic acid and deoxycholic acid can induce the development of CRC by directly damaging DNA, inducing oxidative stress, and promoting epithelial-mesenchymal transition. Lipopolysaccharides promote the progression of CRC by inducing pro-inflammatory responses, while H₂S affects the metabolism of normal intestinal epithelial cells by influencing mitochondrial function, thereby affecting the development of CRC. This Figure was drawn by Figdraw (Supplementary material). SCFA: Short-chain fatty acid; OXPHOS: Oxidative phosphorylation; IL: Interleukin; TNF-α: Tumor necrosis factor alpha; CRC: Colorectal cancer; LPS: Lipopolysaccharides; AhR: Aryl hydrocarbon receptor; IPA: Indolepropionic acid; LCA: Lithocholic acid; DCA: Deoxycholic acid; EMT: Epithelial-mesenchymal transition; VM: Vasculogenic mimicry; UDCA: Ursodeoxycholic acid.

GUT MICROBIOTA EMPOWERS CRC TREATMENT

Biomarker

It has been confirmed that the enrichment of certain gut microbiota is significantly associated with the early diagnosis of CRC, treatment response, and prognostic levels. F. nucleatum is one of the representative microbiota[129]. A clinical study has shown that F. nucleatum, especially the F. nucleatum a C2 subgroup, dominates the TME of CRC, and its enrichment level is significantly correlated with the early diagnosis and prognosis of CRC[130]. An increase in the enrichment level often indicates a decrease in the response of CRC to treatment and a poor prognosis. However, in the immunotherapy of patients with microsatellite-stable CRC, researchers have observed the opposite phenomenon. The study found that butyrate, a metabolite of F. nucleatum, inhibited HDAC 3/8 in CD8⁺ T cells, induced H3K27 acetylation and expression of the Tbx21 promoter, and instead made MSS CRC more sensitive to anti-PD-1 therapy[131]. This indicates that the regulation of gut microbiota on CRC is not static; even the same microbiota can play different roles in different stages of CRC. Regarding the toxic and side effects after chemotherapy, clinical studies on the toxicity and side effects after chemotherapy have shown that Fusicatenibacte, Cetobacterium, and Paraeggerthella are significantly associated with post-chemotherapy leukopenia and may serve as biomarkers for predicting post-chemotherapy myelosuppression in CRC patients[132]. In addition, specific cyclomodulins produced by gut microbiota, such as colibactin, cytotoxic necrotizing factor, and cytolethal distending factor produced by E. coli, can also be used to improve the positive rate of early screening for CRC[133]. Moreover, this non-invasive biomarker screening method using specimens such as feces has the characteristics of high acceptance among the screening population and relatively low cost, which will also improve the efficiency of early screening for CRC.

Regulate the sensitivity to chemotherapy and radiotherapy

Although treatment methods such as immunotherapy and targeted therapy are developing rapidly, radiotherapy and chemotherapy, as traditional therapies, still occupy an important position in the treatment of CRC. This is particularly true for CRC patients who require surgery, where adjuvant radiotherapy and chemotherapy before and after surgery play an undeniable positive role in the surgery and postoperative expectations. Researchers conducted 16S rRNA sequencing on fecal samples from patients with locally advanced rectal cancer and found that the diversity of gut microbiota decreased after preoperative neoadjuvant radiotherapy and chemotherapy. Take Bacteroides vulgatus as an example: The nucleotide biosynthesis mediated by it reduces the sensitivity of CRC cells to 5-fluorouracil or radiotherapy[134]. In addition, the gut microbiota has also been found to be closely related to the abscopal effect in radiotherapy[135], and fecal microbiota transplantation technology has been confirmed by clinical research to reduce radiotherapy-related toxicity and improve its efficacy[136]. Other preclinical studies have shown that, by artificially intervening to regulate the gut microbiota, for example, using nanoparticles constructed with the prebioticxylan-stearic acid conjugate to delay the clearance of capecitabine in the blood, while promoting the proliferation of probiotics and the production of SCFAs, the anti-tumor immune response and chemotherapy response are ultimately enhanced, increasing the tumor inhibition rate from 5.29% to 71.78% and significantly prolonging the survival period of mice with CRC[137]. Similarly, the chemotherapy effect was enhanced after modifying 5-FU-loaded liposomes with the prebiotic xylan derivative Sxy and the active phospholipid homolog 1,2-dipalmitoylphosphatidylethanolamine of the probiotic Akkermansia muciniphila[138]. Moreover, artificially supplementing irinotecan-loaded nanoparticles covalently linked to azide-modified bacteriophages to inhibit the growth of F. nucleatum significantly improved the efficacy of chemotherapy drugs for CRC[139]. Although nanotechnology is not yet mature, these preliminary studies have provided us with new therapeutic ideas to try.

Improving immunotherapy

As mentioned earlier, researchers have confirmed that the efficacy of drugs such as anti-PD-1/programmed death ligand-1 (PD-L1) can be improved by altering the composition of the gut microbiota in CRC[140]. For example, studies have shown that in CRC patients, Prevotellaceae can regulate the immune TME, and its abundance is significantly positively correlated with the therapeutic effect of anti-PD-L1 therapy[141]. Another preclinical study found that the ileal microbiota determines the tolerance and immunogenic cell death of ileal IECs, as well as the accumulation of follicular helper T cells (TFH). Inhibiting the apoptosis of IECs impairs chemotherapy-induced immune surveillance against colon cancer. However, the colonization of B. fragilis and Erysipelotrichaceae in the ileum enables apoptotic ileal IECs to induce the production of PD-1⁺ TFH cells in an IL-1R1 and IL-12-dependent manner, thereby enhancing the efficacy of chemotherapy and PD-1 blockade therapy for CRC[142]. The improvement in this therapeutic effect has also been confirmed to be related to immune metabolism mediated by gut microbiota. Changes in the intestinal microbiome affect the glycerophospholipid metabolic pathway, thereby regulating the response to PD-1 antibodies in immunotherapy for patients with MSS-type CRC[143]. B. fragilis can enhance the anti-tumor immune capacity of CD3⁺ T cells, NK cells, and IFNγ⁺ CD8⁺ T cells in the CRC TME, and improve the immunosuppressive TME by downregulating isobutyric acid. In contrast, CRC with a high level of F. nucleatum is often accompanied by more succinate, a metabolite that inhibits the cGAS-interferon-β pathway, thereby restricting the trafficking of CD8⁺ T cells to the TME in vivo, suppressing anti-tumor responses, and inducing tumor resistance to immunotherapy[144]. Moreover, artificially supplementing probiotics, such as dietary Lactobacillus rhamnosus, can also improve the therapeutic efficacy of immune checkpoint inhibitors by regulating immune metabolism[145]. In addition, by combining clostridium butyricum spores (CBS) with drug-loaded chitosan (CS)-coated liposomes loaded with oxaliplatin and metronidazole, the constructed colon-targeted drug delivery system (CBS-CS@Lipo/Oxp/MTZ) can, through the synergistic regulation of gut microbiota by Clostridium butyricum, block the penetration of intestinal pathogenic bacteria from the lumen to the tumor, prevent their recruitment to immunosuppressive cells, and further synchronize with the tumor-associated antigens produced by immunogenic dead cells caused by metronidazole-induced dead F. nucleatum, collectively enhancing the tumor infiltration of CD8⁺ T cells, reactivating and reshaping the anti-tumor immune TME[146]. It is worth noting that most of these studies are still in the stage of basic research, and whether their clinical translation can meet expectations still needs further verification through clinical research.

Directly used as a therapeutic target

This treatment method includes two directions: Positive and negative. First, it promotes the stability of “good” gut microbiota. For example, ENPP2 inhibitors restore the intestinal barrier by increasing the expression of tight junction proteins claudin-1, occludin, and ZO-1, reduce the polarization of M2 TAMs and colonic inflammation, and alleviate gut microbiota imbalance in mice, thereby inhibiting the proliferation of CRC cells[147,148]. In addition, some clinical studies have confirmed that, gut microbiota imbalance has been confirmed to be associated with the development of colorectal tumors related to a high-fat diet, while berberine significantly improves intestinal barrier damage and inhibits colonic inflammation and related carcinogenic pathways, providing a promising microbiota-regulating therapeutic strategy for the clinical prevention and treatment of Western diet-related CRC. In contrast to the above, for example, as mentioned earlier, F. nucleatum causes immunotherapy resistance through its metabolite succinate. In such cases, the abundance of F. nucleatum can be reduced by directly using antibiotics such as metronidazole to improve immunotherapy[144], nanogels modified by nanotechnology can combat F. nucleatum-infected CRC through the cascade release of metronidazole and chemotherapeutic drugs, further enhancing the therapeutic effect[149]. In addition, neutrophil-mimicking nanomedicines loaded with metronidazole and oxaliplatin can simultaneously achieve antibacterial and anticancer effects. They can reverse the EMT in the process of F. nucleatum-induced tumor cell metastasis, reshape the immunosuppressive TME, thereby inhibiting the development of CRC and liver metastasis, and minimizing damage to the normal symbiotic microbiota[150]. It can be seen from this that the combination of nanotechnology and gut microbiota may become a new therapeutic approach for CRC. Similarly, antimicrobial peptides represent a new class of antibacterial drugs. The halogenated derivative Jelleine-I, Br- Jelleine-I shows significant anti-F. nucleatum activity. At the same time, Br-JI may target membrane-associated FadA to disrupt the F. nucleatum membrane. Compared with metronidazole, it has a stronger ability to kill F. nucleatum and is sensitive to the anticancer effect of the chemotherapy drug 5-fluorouracil, which may become a new strategy for the treatment of CRC[151]. This part is shown in Figure 6 and Table 1 respectively.

Figure 6 Clinical applications of gut microbiota in colorectal cancer. In terms of biomarkers, the enrichment level of gut microbiota such as Fusobacterium nucleatum can serve as a multi-dimensional marker for diagnosis, prediction of treatment response, and prediction of prognosis. In improving the sensitivity of traditional treatments, gut microbiota can directly or indirectly affect the blood clearance rate or response efficiency of chemotherapy drugs through their metabolites or by combining with technologies such as nanoparticles, thereby enhancing the therapeutic effect. In improving immunotherapy, gut microbiota can enhance the response efficiency of immunotherapy by regulating the activity of immune cells or controlling the apoptosis of intestinal epithelial cells. Finally, gut microbiota can directly be used as therapeutic targets. A representative example is the antibacterial effect of metronidazole on Fusobacterium nucleatum, which assists in enhancing the therapeutic effect of chemotherapy drugs. Meanwhile, emerging drugs such as antimicrobial peptides can also assist in anti-cancer treatment through their antibacterial properties. This Figure was drawn by Figdraw (Supplementary material). CRC: Colorectal cancer; HDAC: Histone deacetylase; MSI-H: High microsatellite instability; MSS: Microsatellite stable; PD-1: Programmed death 1; IL: Interleukin; NK: Natural killer; IEC: Intestinal epithelial cell; SCFA: Short-chain fatty acid; FMT: Fecal microbiota transplantation; Fn: Fusobacterium nucleatum; EMT: Epithelial-mesenchymal transition.

Table 1 The immune checkpoint, metabolic inhibitors and gut microbiota currently under clinical investigation for the treatment of colorectal cancer.

	Drug	Targets	Clinical phase	NCT	Sponsored by	Status	Purpose
ICIs	Relatlimab	LAG-3	Phase 2	NCT03642067	Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins	Completed	The purpose of this study is to evaluate the safety and clinical activity of nivolumab and relatlimab in patients with metastatic or locally advanced MSS colorectal cancer
	MK-4830	ILT-4	Phase 2	NCT04895722	Merck Sharp & Dohme LLC	Active, not recruiting	Evaluation of co-formulated pembrolizumab/quavonlimab (MK-1308A) vs other treatments in participants with MSI-H or dMMR stage IV CRC (MK-1308A-008/KEYSTEP-008)
	Tucatinib	HER2	Phase 3	NCT05253651	Seagen, a wholly owned subsidiary of Pfizer	Recruiting	A study of tucatinib with trastuzumab and mFOLFOX6 vs standard of care treatment in first-line HER2+ metastatic CRC (MOUNTAINEER-03)
	B1962	VEGF/PD-L1	Phase 2	NCT04296019	Tasly Biopharmaceuticals Co., Ltd.	Not yet recruiting	A multicenter, open phase IIb clinical study to evaluate the efficacy and safety of B1962 injection in the treatment of advanced CRC
	MGD007	GPA33	Phase 1	NCT02248805	MacroGenics	Completed	Phase 1 study of MGD007 in relapsed/refractory metastatic colorectal carcinoma
	Belzutifan	HIF-2α	Phase 2	NCT04976634	Merck Sharp & Dohme LLC	Active, not recruiting	Pembrolizumab plus lenvatinib in combination with belzutifan in solid tumors (MK-6482-016)
	PF-07062119	GUCY2c/CD3	Phase 1	NCT04171141	Pfizer	Terminated	Study to test the safety and tolerability of PF-07062119 in patients with selected advanced or metastatic gastrointestinal tumors
	BMS-986340	CCR8	Phase 1 and phase 2	NCT04895709	Bristol-Myers Squibb	Recruiting	A study of BMS-986340 as monotherapy and in combination with nivolumab or docetaxel in participants with advanced solid tumors
	Sintilimab	PD-1	Phase 3	NCT05236972	Sun Yat-sen University	Recruiting	PACE: PD-1 antibody for dMMR/MSI-H stage III CRC
	Avelumab	PD-L1	Phase 2	NCT03854799	Gruppo Oncologico del Nord-Ovest	Completed	Immunotherapy in locally advanced rectal cancer (AVANA)
	Tislelizumab	PD-1	Phase 1 and phase 2	NCT04577963	Hutchison Medipharma Limited	Terminated	A study of fruquintinib in combination with tislelizumab in advanced solid tumors
	Pembrolizumab	PD-1	Phase 2	NCT05131919	The Netherlands Cancer Institute	Active, not recruiting	Pembrolizumab for locally advanced, irresectable, non-metastatic dMMR CRCs (PUMA)
	Nivolumab	PD-1	Phase 2	NCT03642067	Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins	Completed	The purpose of this study is to evaluate the safety and clinical activity of nivolumab and relatlimab in patients with metastatic or locally advanced MSS CRC
	HFB200603	BTLA	Phase 1	NCT05789069	HiFiBiO Therapeutics	Recruiting	A study of HFB200603 as a single agent and in combination with tislelizumab in adult patients with advanced solid tumors
	MK0457	pan-Aurora	Phase 1	NCT00099346	Merck Sharp & Dohme LLC	Terminated	MK0457 (an aurora kinase inhibitor) study in patients with advanced CRC and other advanced solid tumors (0457-001)
	HRO761	WRN	Phase 1	NCT05838768	Novartis Pharmaceuticals	Recruiting	Study of HRO761 alone or in combination in cancer patients with specific DNA alterations called microsatellite instability or mismatch repair deficiency
Metabolic inhibitor	Epacadostat	IDO1	Phase 1 and phase 2	NCT03516708	Washington University School of Medicine	Recruiting	Epacadostat (INCB024360) added to preoperative chemoradiation in patients with locally advanced rectal cancer
	Ivosidenib	IDH1	Phase 2	NCT04195555	National Cancer Institute	Active, not recruiting	Ivosidenib in treating patients with advanced solid tumors, lymphoma, or histiocytic disorders with IDH1 mutations (a pediatric MATCH treatment trial)
	Metformin	Mitochondria	Phase 3	NCT05921942	Ain Shams University	Completed	The impact of metformin administration on the clinical outcome of stage IV colon cancer
	Telaglenastat	GLS	Phase 1 and phase 2	NCT02861300	David Bajor, MD	Completed	CB-839 + capecitabine in solid tumors and fluoropyrimidine resistant PIK3CA mutant CRC
Gut microbiota	Gut microbiota	/	Observational	NCT02845973	Shanghai Jiao Tong University School of Medicine	Completed	Study of fecal bacteria in early diagnosis of CRC
	Gut microbiota	/	Observational	NCT06738173	Gianluca Ianiro, Catholic University of the Sacred Heart (Responsible Party)	Recruiting	Microbiome-based diagnostic tool for the screening of CRC (GUILTI)
	Gut microbiota	/	Observational	NCT06588166	Fondazione Policlinico Universitario Agostino Gemelli Irccs	Recruiting	Microbiome testing for the screening of CRC (NI-GUILTI)
	Gut microbiota	/	Observational	NCT04291755	Persephone Biosciences (Responsible Party)	Recruiting	Development and analysis of a stool bank for cancer patients
	Gut microbiota		Observational (patient registry)	NCT07154173	Sixth Affiliated Hospital, Sun Yat-sen University	Recruiting	Comprehensive analysis of gut microbiota signatures in metastatic CRC
	Fusobacterium nucleatum		Interventional	NCT05945082	Malmö University	Recruiting	Fusobacterium nucleatum at CRC sites
	EPEC		Observational (patient registry)	NCT02373020	Mansoura University	Recruiting	EPEC: Does it have a role in colorectal tumourigenesis (EPEC)?

MSS: Microsatellite stable; MSI-H: High microsatellite instability; dMMR: Mismatch repair deficient; CRC: Colorectal cancer; EPEC: Enteropathogenic Escherichia coli; ICI: Immune checkpoint inhibitor; NCT: National clinical trial; VEGF: Vascular endothelial growth factor; PD-L1: Programmed death ligand-1; PACE: Pembrolizumab adjuvant colorectal evaluation; PD-1: Programmed death 1; PUMA: Pembrolizumab for unresectable high microsatellite instability/mismatch repair deficient advanced colorectal cancer; GUILTI: Gut microbiota-based diagnostic tool for the screening of colorectal cancer; NI-GUILTI: Non-invasive gut microbiota-based diagnostic tool for the screening of colorectal cancer.

LIMITATIONS OF THE STUDY

Although we have made every effort to conduct a systematic review on this topic, there are still several limitations. First, due to space constraints, many studies have inevitably not been included in the analysis, which may fail to fully reflect the complexity of human CRC. Second, although we have revealed the association between gut microbiota-mediated immunometabolism and the TME, many of their exact causal mechanisms remain to be elucidated. Finally, many of the possible clinical translation pathways mentioned in the article have not been further verified, and the efficiency and feasibility of their clinical translation still need further research and improvement.

CONCLUSION

In recent years, the relationship between the intestinal microbiota and CRC has attracted much attention. Studies have confirmed that the gut microbiota significantly regulates the immunometabolism of CRC through its metabolites, thereby affecting the TME and its progression. This finding provides a new perspective for us to understand the role of the gut microbiota in cancer development. Immunometabolic reprogramming is considered an important mechanism of tumor immune escape and therapeutic resistance, and the gut microbiota plays a key role in this process, which provides us with new targets. In addition, by understanding the characteristics of an individual’s gut microbiota, we can develop more targeted prevention and treatment strategies to improve patient prognosis. Research in this direction will promote the application of gut microbiota in cancer and ultimately achieve the goal of personalized medicine. However, there are still many complex issues. The role of specific microorganisms is often context-dependent, and the inconsistencies in human cohort studies - which may stem from dietary, geographical, and host genetic factors - highlight the challenges in identifying universal microbiota biomarkers. Furthermore, some preclinical studies have not been clinically validated, and the feasibility and efficiency of their clinical translation remain unknown. Emerging technologies represented by artificial intelligence and nanotechnology have provided many conveniences for our clinical treatment, but their technical limitations still need to be overcome, so there is still a long way to go before clinical application.

ACKNOWLEDGEMENTS

The authors would like to express their sincere gratitude to all colleagues and collaborators who contributed valuable insights and support throughout the development of this manuscript. Special appreciation is extended to the researchers whose studies laid the foundation for our discussion. Finally, we acknowledge the constructive feedback from reviewers, which greatly improved the quality of this paper.