The Impact of the Microbiota on the Immune Response Modulation in Colorectal Cancer

The activation of the innate immune response represents the initial line of defense the body employs against pathogenic incursions. This process is critical for the recognition and response to harmful microorganisms [24]. Pathogenic bacteria can activate the innate immune system by interacting with pattern recognition receptors (PRRs) on immune and epithelial cells, triggering pro-inflammatory signaling pathways [25,26]. Some pathogenic bacteria release molecules such as lipopolysaccharides (LPS), flagellin, or peptidoglycans that bind to receptors on epithelial cells and immune cells (e.g., toll-like receptor 4—TLR4, toll-like receptor 5—TLR5) and trigger the activation of the nuclear factor-κB (NF-κB) and mitogen-activated protein kinase signaling pathways (MAPK) that lead to the increased synthesis and release of pro-inflammatory cytokines, such as interleukine-6 (IL-6), interleukine-1beta (IL-1β), and tumor necrosis factor alpha (TNF-α) [27]. Thus, the generated inflammatory process is sustained and contributes to DNA damage, epithelial barrier disruption, and tumor progression. The activation of nucleotide-binding and oligomerization domain (NOD)-like receptors (NLRs) in epithelial cells by pathogens such as ETBF or certain strains of E. coli leads to the formation of the inflammasome (e.g., the NLRP3 inflammasome) and the release of IL-1β and IL-18, which support chronic inflammation and tumorigenesis [28,29]. In addition, as a consequence of the increased synthesis of pro-inflammatory cytokines, there is a massive recruitment of myeloid cells, including macrophages and neutrophils, which produce reactive oxygen species (ROS) and reactive nitrogen species (RNS) in large quantities, leading to DNA damage in epithelial cells [30]. Consequently, chronic inflammation promotes angiogenesis by increasing the production of pro-angiogenic factors, such as vascular endothelial growth factor (VEGF), supporting tumor growth (Figure 1).

The suppression and evasion of adaptive immunity encompass the mechanisms employed by pathogens to evade or diminish the body’s specific immune response. This phenomenon typically occurs subsequent to the activation of the adaptive immune system. T cells, including CD4⁺ helper cells and CD8⁺ cytotoxic cells, as well as B cells, play a role in inhibiting the presentation of major histocompatibility complex (MHC) molecules. They also contribute to the suppression of the immune response through the action of regulatory T cells (Tregs). As a result of these interactions, pathogens can evade detection, creating conditions that allow for chronic infections [31]. Pathogenic bacteria like Fn can impair the function of anti-tumor immune cells. Fn interacts with immune cells through the fusobacterium adhesin A (FadA) adhesin, which binds to E-cadherin on tumor cells, activating Wnt/β-catenin signaling and promoting cell proliferation and invasion. Also, Fn expresses the fibroblast activation protein-2 (Fap2), which binds to the immune inhibitory receptor known as T cell immunoreceptor with Ig and ITIM domains (TIGIT) on T cells and natural killers (NK) cells, inhibiting their functions. The result of this process is reduced tumor cell killing and allowing cancer cells to evade immune surveillance. Pathogens may recruit or induce populations of immune cells that facilitate tumor growth and suppress anti-tumor immunity [32]. For example, Fn recruits myeloid-derived suppressor cells (MDSCs) and regulatory T cells (Tregs), both of which suppress cytotoxic T lymphocyte (CTL) functions, which are crucial for attacking tumor cells (Figure 1). Additionally, Fn secretes various factors that impair antigen-presenting cells (APCs), like dendritic cells, preventing an effective anti-tumor immune response (Table 1).

Pathogenic bacteria produce metabolites that interfere with immune functions. Bacterial toxins like colibactin from E. coli not only damage DNA but also lead to chronic inflammation which promotes immunosuppressive cell recruitment, making the immune system less effective at targeting tumor cells. These bacterial toxins contribute to promoting immune evasion in CRC (Table 1). Metabolites produced by certain bacteria, like secondary bile acids (e.g., deoxycholic acid), promote oxidative stress and DNA damage [40]. In addition, hydrogen sulfide (H2S) generated by sulfur-reducing bacteria damages epithelial DNA and impairs mitochondrial function. These toxins and metabolites create a pro-carcinogenic environment and enhance tumor evolution [41]. Pathogenic bacteria can suppress anti-tumor immune responses, allowing tumors to grow unchecked and spread to distant sites.

Pathogenic bacteria alter the balance between pro-inflammatory and anti-inflammatory cytokines in the tumor microenvironment. Many bacteria trigger the release of IL-10 and TGF-β, which suppress effector T cells and promote immune tolerance. At the same time, they inhibit interferon-gamma (IFN-γ) and TNF-α, which are crucial for the activation of macrophages, dendritic cells, and T cells that attack tumors. Reduced levels of IFN-γ and IL-12 impair the activation of CTLs and NK cells. Persistent inflammation can exhaust effector T cells, promote Treg activity, and create a tumor-promoting cytokine milieu [42,43]. This shift in cytokine balance makes the tumor microenvironment immunosuppressive, allowing cancer cells to evade immune attack.

Elucidating how the gut microbiota drives ROS generation reveals that these microbes shape colorectal carcinogenesis by stimulating pro-tumor inflammation, reprogramming immune cells toward a suppressive phenotype, and facilitating tumor immune evasion [44]. Some of the pathogenic bacteria that significantly influence the production of ROS in CRC initiation and progression, and several interconnected mechanisms are presented in Table 2. These bacteria contribute either directly through toxins or genotoxins or indirectly modulating host cells to produce them by inducing inflammation that promotes ROS release by immune cells [45].

Bacteroides fragilis (particularly enterotoxigenic B. fragilis) releases BFT, which activates nicotine adenine dinucleotide phosphate oxidase (NOX) in colon epithelial cells, leading to ROS production. E. coli strains with the pks+ island produce colibactin, a genotoxin that induces DNA double-strand breaks, partly through ROS-mediated mechanisms. These ROS can cause mutagenesis (DNA damage), lipid peroxidation, and protein oxidation that contribute to genomic instability, a hallmark of cancer initiation. In early CRC, microbial ROS activation may break the tolerance of the gut immune system leading to the over-activation of innate immunity [51].

Immune system interactions with microbial imbalance lead to chronic inflammation, and pathogenic bacteria facilitate the infiltration of inflammatory cells like neutrophils and macrophages in the colon mucosa and release high levels of ROS. Chronic exposure to this oxidative environment promotes the oncogenic mutations and activation of pro-cancer signaling pathways (e.g., NF-κB, or Signal Transducer and Activator of Transcription 3-STAT3) [52]. In late-stage CRC, some pathogen bacteria increase oxidative stress which suppresses anti-tumor immunity, for example, Fn inhibits NK and T cell activity. However, ROS can suppress APCs and impair dendritic cell (DC) function [53].

In addition, ROS alters immune cell function and polarization. Low ROS levels activate immune responses, including antigen presentation and pro-inflammatory cytokine release. High levels of ROS induce T cell exhaustion, impair CTLs, promote differentiation of Tregs and Th17 cells (linked to tumor growth) or lead to M2-like macrophage polarization (pro-tumor), rather than M1 (anti-tumor) [54,55]. In consequence, ROS reprogram the immune landscape to support immune evasion and tumor tolerance (Table 2).

Some pathogenic bacteria act to induce antioxidant pathway suppression, downregulating host antioxidant enzymes like glutathione peroxidase (GPx) and superoxide dismutase (SOD). This increases intracellular ROS, pushing epithelial cells toward transformation. Fusobacterium nucleatum, E. coli, and B. fragilis together create a biofilm and facilitate a hypoxic, inflammatory microenvironment that promote sustained ROS production at the mucosal surface. This further encourages epithelial-to-mesenchymal transition (EMT), tumor growth, and invasion. ROS can contribute to more microbial translocation and dysbiosis but when ROS is locally higher concentrated ROS feedback appears to alter the microbiome and form a vicious cycle responsible for CRC progression [56].

Understanding these relationships between the microbiome, ROS production, and the immune response can help pave the way for innovative therapeutic strategies and enhance our approach to prevention and treatment.

Bacterial pathogens can contribute to tumorigenesis by disrupting tight junctions, increasing intestinal permeability, and triggering inflammation. BFT produced by Bacteroides fragilis cleaves E-cadherin, disrupts tight junction proteins (such as ZO-1, occludin, and claudins), and increases epithelial permeability, facilitating tumor development [59,60].

Fusobacterium nucleatum can activate inflammatory pathways, including NF-κB and β-catenin, which lead to the downregulation of tight junction proteins and a weakening of barrier integrity [61]. Furthermore, F. nucleatum enhances bacterial invasion and colorectal cancer progression through the adhesin FadA, which binds to E-cadherin and disrupts tight junctions. Additionally, Escherichia coli (pks+ strains) secrete colibactin, which can modify epithelial adhesion molecules and impact both DNA integrity and epithelial barriers [62].

The mucus in the intestinal epithelium contains a highly glycosylated polymeric protein called mucin, which reduces the exposure of epithelial cells to bacteria and viruses from the human microbiome. Recent studies have provided compelling evidence for the significant role of the intestinal microbiota at both the metabolic and immunological levels [63]. Akkermansia muciniphila contributes under dysbiotic conditions to the degradation of mucins, thinning the protective mucus layer and thus allowing pathogens to reach the epithelial surface. Some species of Bacteroides spp. can act to degrade mucins, exposing epithelial cells to bacterial toxins and inflammatory signals. S. gallolyticus also produces virulence factors that promote bacterial adhesion and disrupt epithelial integrity [64].

The intestinal barrier, composed of the intestinal mucosa, epithelial cells, and a protective mucus layer, plays a critical role in regulating the passage of substances between the intestine and the bloodstream while preventing the entry of pathogens and endotoxins. The degradation of this barrier by pathogenic bacteria can significantly influence the immune response in CRC. Both systemic and local immune activation, driven by various molecular and cellular mechanisms, are associated with the disruption of the intestinal barrier [65]. In CRC, it has been demonstrated that specific pathogens, including Fn, B. fragilis, and E. coli, facilitate the translocation of bacteria across the mucosa, thereby initiating a local inflammatory response. This process compromises the integrity of the intestinal barrier and is linked to intestinal dysbiosis, which may contribute to tumor progression [66].

When pathogenic bacteria, such as CD, E. coli, and B. fragilis, compromise the intestinal barrier, they can create epithelial lesions that enable the penetration of endotoxins and bacteria. This process can initiate a systemic inflammatory response, highlighting the importance of maintaining intestinal health. Key to this response are pattern recognition receptors (PRRs), including TLR4 (which recognizes LPS), NOD1/NOD2 (which detect peptidoglycan), STING, and cGAS pathway (which respond to bacterial DNA). The activation of these receptors triggers significant intracellular pathways, such as NF-κB and IRF3, ultimately leading to the production of inflammatory cytokines. For example, the bacterium Fn activates the NF-κB pathway through TLR4, resulting in the increased secretion of IL-6 and TNF-α, which can negatively impact epithelial integrity. Moreover, bacterial components like LPS, flagellin, and BFT engage TLR4/NF-κB signaling, further contributing to the release of pro-inflammatory cytokines (including IL-6, TNF-α, and IL-1β) that can disrupt the intestinal barrier [67,68]. In response to these challenges, macrophages and dendritic cells act as APCs. The cells capture bacteria or their components, undergo a process of activation, and subsequently migrate to the lymph nodes, where they present antigens to T lymphocytes. This process triggers the activation of CD4⁺ (helper) and CD8⁺ (cytotoxic) T lymphocytes, facilitating a robust adaptive immune response. By comprehending these mechanisms, we can develop strategies that promote intestinal health and enhance immune function [69,70,71] (Figure 1).

Also, dysbiosis accompanied by inflammation generates a polarization of T cells, translated by favoring Th17 cells involved in tumor angiogenesis and Th1 cells responsible for the secretion of pro-inflammatory cytokines associated with the reduction in the Treg population. This immune imbalance process favors the establishment of chronic inflammation and the suppression of immunological tolerance [72,73]. For example, ETBF and Fn activate the IL-17/Th17 pathway, which is responsible for the increase in IL-17 secretion. This cytokine is responsible for promoting chronic inflammation and intestinal barrier damage, thus creating a microenvironment favorable to tumor development [74]. E. coli pks+, capable of secreting colibactin, intervenes in the activation of the Wnt/β-catenin pathway and TGF-β that affects EMT and induces intestinal barrier dysfunction, thus promoting CRC progression [75,76]. At the same time, systemic inflammation contributes to the repeated stimulation of the immune system, which can result in T cell exhaustion and reduced effective anti-tumor response, favoring tumor progression [77].

When the intestinal flora’s homeostasis is disrupted, many harmful bacteria can induce the oxidative stress-related pathway in the host cell, leading to complex interactions, such as the abnormal differentiation and apoptosis of intestinal cells, the disruption of the epithelial barrier, and inflammatory response (Table 2). Pathogenic bacteria such as Bacteroides fragilis ETBF, E. coli (pks+), and Fn invade the intestine and release toxins (e.g., BFT, colibactin), triggering inflammation. This process causes increased ROS production by host epithelial cells (via the activation of NOX enzymes) or by activated immune cells such as macrophages and neutrophils. Increased ROS production causes oxidative damage to DNA, proteins, and lipids and promotes genomic instability. In addition, high levels of ROS induce epithelial stress and apoptosis, contributing to the weakening of the intestinal barrier and the amplification of the process [78]. The disruption of the intestinal barrier is accompanied by the loss of tight junction proteins (e.g., ZO-1, E-cadherin), a thinning of the mucus layer, and epithelial cell death. As a consequence of these processes, microbial translocation into the submucosa occurs, and immune cells are exposed to bacterial antigens, which results in an amplification of the immune response followed by the increased secretion of TNF-α and IL-6, but also an increase in ROS production, which will generate more tissue damage. Consequently, a weakened barrier allows more pathogenic bacteria to infiltrate and colonize; thus, the cycle repeats and intensifies. These processes facilitate the accumulation of DNA mutations, result in immune suppression, and contribute to the progression of cancer [79].

The detrimental cycle involving pathogenic bacteria, the production of ROS, and the disruption of the intestinal barrier in CRC represents the central loop to tumor initiation, chronic inflammation, and progression (Figure 3).

Clearly, these mechanisms collectively weaken the intestinal barrier, promote chronic inflammation, and create a microenvironment favorable for tumor development. Understanding and combating these mechanisms is a crucial task for our audience in the fields of microbiology, oncology, and gastroenterology.

Beneficial bacteria, such as Bifidobacterium and Lactobacillus, produce SCFAs like butyrate, propionate, and acetate through the fermentation of dietary fiber. These SCFAs and other bacterial metabolites suppress NF-κB activation and reduce inflammation by suppressing pro-inflammatory cytokines, such as IL-6 and IL-1β, while simultaneously promoting the production of anti-inflammatory cytokines such as IL-10 [96]. Moreover, SCFAs have a significant impact on the differentiation, recruitment, and activation of immune cells, including neutrophils, DCs, macrophages, and T lymphocytes [97]. Furthermore, SCFAs facilitate the differentiation of regulatory Tregs, which are essential for the modulation of chronic inflammation. They also play a crucial role in the differentiation of regulatory Tregs, which are important for controlling chronic inflammation. Additionally, SCFAs are involved in epigenetic regulation by inhibiting histone deacetylases (HDACs) [98], which enhances the expression of anticancer genes and helps to slow the progression of CRC [99,100]. Beneficial bacteria play a vital role in repairing and regenerating damaged epithelial cells. SCFAs enhance the expression of antimicrobial peptides on the external surface of epithelial cells and modulate their production of immune mediators, including IL-18, which is critical for the maintenance and restoration of epithelial integrity [101]. Notably, butyrate, a type of SCFA, activates the Wnt signaling pathways that promote the proliferation and differentiation of epithelial cells. This process is essential for the continuous renewal of the epithelial layer, which helps maintain barrier integrity in CRC [102].

Some bacteria directly activate anti-tumor immune responses. Beneficial bacteria can promote immune tolerance by stimulating regulatory Tregs or encouraging a balanced immune response between Th1 and Th2 cells, which is important for anti-tumor effects [103]. Furthermore, CTLs can directly activate anti-tumor immune responses. This activation can be enhanced by improving antigen presentation, such as with Bifidobacterium, or by increasing the cytotoxic activity of NK cells, both of which help destroy tumor cells [104]. Additionally, these beneficial bacteria can influence the regulation of innate immunity, helping to maintain immune homeostasis. They do this by encouraging macrophage polarization towards the anti-inflammatory M2 phenotype and by modulating neutrophil activity to prevent excessive tissue damage and inflammation [105,106].

Immune system cells receive signals from various bacterial substances, which integrate and initiate the activation of appropriate immune response mechanisms. DCs exhibit a broad spectrum of receptors that facilitate the recognition of various bacterial constituents. For instance, specific free fatty acid receptors (FFARs) are capable of detecting bacterial metabolites classified as SCFAs. The activation of these receptors serves as an indication of the bacteria’s viability and demonstrates how their metabolites interact with the immune system to promote anti-inflammatory functions [107]. Furthermore, the diverse array of receptors expressed by DCs enables them to activate multiple signaling cascades concurrently. This ability to integrate several signals simultaneously ensures the generation of an appropriate response to both antigenic and tissue-related stimuli [108]. The modulation of dendritic cell function by beneficial bacteria influences DCs to act as antigen-presenting cells for T cells. Consequently, beneficial bacteria promote the development of tolerogenic DCs, which enhance the induction of Treg cells or stimulate DCs to produce anti-inflammatory cytokines and prevent the over-activation of the immune system [109]. Furthermore, these bacteria can stimulate dendritic cells to produce anti-inflammatory cytokines, such as IL-10, thereby contributing to the balance of immune responses and preventing the excessive activation of the immune system, particularly in the context of CRC [110].

The intestinal barrier is a crucial barrier for an organism against invasion by foreign pathogens, including mechanical, chemical, immune, and microbial barriers. Beneficial bacteria play a crucial role in strengthening the intestinal barrier in CRC by maintaining intestinal homeostasis and reducing inflammation (Figure 4). Lactobacillus, Bifidobacterium, and Akkermansia muciniphila act beneficially and promote mucin secretion by goblet cells, forming a protective barrier that prevents the entry of pathogens and minimizes the activation of the immune system, thus promoting overall gut health [111].

Several beneficial bacteria (Lactobacillus rhamnosus GG, Lactobacillus plantarum, Lactobacillus reuteri, Bifidobacterium breve, Bifidobacterium longum, Bifidobacterium bifidum, Akkermansia muciniphila, and Faecalibacterium prausnitzii) play a significant role in improving the integrity of the intestinal epithelial barrier by upregulating the expression of tight junction proteins (occludin, claudins, and zonula occludens) and preventing CRC progression (Figure 4).

Beneficial bacteria are instrumental in safeguarding the epithelial barrier by scavenging ROS and mitigating oxidative stress. For example, species within the Lactobacillus genus are known to synthesize antioxidants and detoxify harmful metabolites, thereby preventing oxidative damage to epithelial cells [112]. Furthermore, specific bacterial metabolites can diminish epithelial inflammation and reinforce the integrity of the barrier [113]. Indole derivatives resulting from tryptophan metabolism are known to activate the aryl hydrocarbon receptor (AhR) in epithelial cells, facilitating both barrier integrity and immune regulation. Moreover, certain intestinal bacteria possess the capacity to degrade toxic compounds, such as bile acids and polyamines, which are implicated in the progression of CRC [114]. Additionally, beneficial bacteria occupy competing niches, effectively outcompeting harmful bacteria for the occupation of adhesion niches on the mucosa by secreting bacteriocins and organic acids that directly inhibit pathobionts, maintaining a local pH unfavorable to pathogenic species, and preferentially consuming essential nutrients, thus preventing pathogenic bacterial colonization that could compromise the intestinal barrier (Figure 4) [115].

Collectively, these mechanisms underscore the significance of beneficial bacteria in fortifying tight junctions, reducing intestinal permeability, and providing protection against the progression of CRC. These mechanisms emphasize the crucial role of beneficial bacteria in enhancing the integrity of tight junctions, which are vital barriers within the intestinal lining. Furthermore, their protective effects play a significant role in reducing the risk of developing CRC, offering an important defense in the battle against this disease [116].

Chemotherapy remains the standard first-line treatment in CRC, with an essential role in controlling the disease, and preparing the patient for combination treatments or surgery, where possible. For localized stages (local disease), oncological resection remains the foundation, complemented by chemotherapy (FOLFOX/CAPOX) and, for rectal tumors, preoperative radio- or chemoradio-therapy (“short course” (5 × 5 Gy) or long chemoradiotherapy (50 Gy + capecitabine), to reduce tumor volume and preserve sphincter function) [118]. At the advanced or metastatic stages, chemotherapy is often combined with other types of therapies, depending on the stage of the disease and the molecular profile of the tumor.

Microbiome bacteria can modulate the activation or deactivation of chemotherapy drugs before their delivery to tumors, thereby influencing their efficacy and toxicity. Certain microbial species produce enzymes, such as β-glucuronidases, which can convert inactive drug metabolites back into their active and toxic forms, resulting in adverse side effects. A pertinent example is Irinotecan (CPT-11), which is metabolized in the liver to an inactive form (SN-38G) and subsequently reactivated by bacterial β-glucuronidase, leading to inflammatory responses [119]. Furthermore, the microbiome exerts a considerable influence on immune cell recruitment and the inflammatory pathways that are critical to chemotherapy-induced tumor cell death. Empirical studies have demonstrated that the composition of the microbiota can impact immune responses to treatments such as 5-Fluorouracil (5-FU), thereby modulating both the effectiveness and side effects of the treatment oxaliplatin, another chemotherapeutic agent, which induces tumor cell death through the generation of ROS and enhances the anti-tumor immune response by activating mechanisms of innate immunity [120]. Additionally, specific bacteria, including Alistipes and Lactobacillus, have been identified as enhancers of the immune-mediated anti-tumor effects associated with oxaliplatin. Conversely, other bacteria may promote chemoresistance through mechanisms such as inflammation, the disruption of the mucosal barrier, or immune system suppression. For instance, Fn has been implicated in promoting chemoresistance via pathways that inhibit apoptosis and facilitate autophagy [121].

In metastatic disease, treatment is based on the genetic profile of the tumor (RAS, BRAF, HER2, or MSI/dMMR) and the extent of the disease [122]. Programmed cell death 1 (PD-1) immunotherapy (pembrolizumab, nivolumab) has become the standard in patients with microsatellite instability-high (MSI-H) or mismatch repair deficiency (dMMR) tumors, conferring durable responses, and current research is exploring bimodal synergies (ICI and anti VEGF/EGFR, radiotherapy, or microbiota manipulation) for refractory microsatellite stable (MSS) tumors. This multimodal and biomarker-targeted approach transforms CRC from a uniformly treated disease to one managed by sequential strategies tailored to each patient. Anti-PD-1 immunotherapy has decisively changed the treatment paradigm for the subgroup of CRC with MSI-H or dMMR [123]. MSI H/dMMR tumors accumulate thousands of “frameshift” mutations that generate abnormal peptides, neoantigens, which are processed by the proteasome, loaded on major histocompatibility complex-1 (MHC I) molecules, and presented on the surface of tumor cells. Due to their uniqueness, they are recognized as non-self by naive T lymphocytes in the mesenteric nodes, inducing intense proliferation and differentiation into effector cells in CTLs. The large number and diversity of these neoantigens leads to the polyclonal clonal expansion of distinct T cells, each with a unique specificity, from which some dominant clones are selected and multiplied that exert a strong immune pressure on the tumor, secreting IFNγ and TNFα and releasing granzymes/perforin that induce the apoptosis of malignant cells [124]. In the absence of PD-1 blockade, the response is rapidly exhausted by tumor evasion mechanisms, and the therapeutic inhibition of PD-1 re-energizes these expanded clones and prolongs their activity. PD-1 inhibition restructures the tumor microenvironment: re-energized CTLs secrete IFN-γ that polarizes macrophages towards an M1 phenotype and stimulates stromal cells to secrete the chemokines CXCL9/10, which additionally attract NK cells to the tumor [125]. Concomitantly, the density of suppressor cells, such as Treg and myeloid-derived suppressor cells (MDSCs), decreases [126], either through apoptosis induced by the pro-inflammatory environment or through unfavorable competitive chemotaxis, and MHC-I/II expression on tumor cells increases, enhancing antigen presentation. The synergy between high neoantigen load, clonal expansion, and PD-1 re-energization supports a robust polyclonal response, explaining the durable remissions observed in MSI-H/dMMR patients. For patients with MSI-H/dMMR tumors, the introduction of PD-1 inhibitors has transformed the prognosis from less-than-one-year survival to five-year survival rates exceeding 55% [127], validating the concept of “durable benefit” and establishing immunotherapy as the backbone of treatment in this molecular subset of CRC.

The efficacy of immunotherapy, particularly in relation to immune checkpoint inhibitors (ICIs), can be significantly impacted by the composition of the gut microbiota. Immunotherapy, particularly ICIs, shows great promise in treating CRC that is characterized by MSI-H or dMMR [128]. Conversely, it exhibits limited effectiveness in microsatellite stable (MSS) CRC, unless influenced by the microbiome. Certain beneficial microbes can potentially enhance the therapeutic response to ICIs by improving antigen presentation and promoting T cell activation. For instance, Akkermansia muciniphila has been shown to stimulate DCs, thereby augmenting the effectiveness of PD-1 blockade. Additionally, Bacteroides fragilis (non-toxigenic) can induce Th1 immunity, which increases the efficacy of anti-cytotoxic T lymphocyte antigen 4 (anti-CTLA-4) treatment. Furthermore, Faecalibacterium prausnitzii plays a crucial role in maintaining mucosal integrity and is also noteworthy for highlighting the potential of microbiome modulation in cancer therapies [81]. Moreover, while the use of antibiotics is often necessary, it is important to be aware that their administration before or during ICI therapy can lead to adverse effects on response rates due to gut dysbiosis [129]. Addressing this issue and understanding the implications on the gut microbiota, such as the impact of Fn related to immune evasion and therapy resistance, will be crucial in optimizing treatment strategies and improving patient outcomes [130]. By focusing on these microbial interactions, we can potentially enhance the effectiveness of immunotherapy in colorectal cancer patients.

Targeted therapies constitute an essential aspect of CRC management, particularly in cases of metastasis. This therapy involves the use of molecules that block angiogenesis and are known as anti-VEGF (Vascular Endothelial Growth Factor) inhibitors, such as Bevacizumab (Avastin), Ziv-aflibercept (Zaltrap), and Ramucirumab (Cyramza). Furthermore, in the context of RAS wild-type and BRAF wild-type tumors, anti-EGFR (Epidermal Growth Factor Receptor) inhibitors are employed to obstruct signaling pathways that promote cell growth. These agents are marketed under various trade names, including Cetuximab (Erbitux) and Panitumumab (Vectibix). The microbiota may interact with the tumor microenvironment, influencing the responses to anti-EGFR or anti-angiogenic therapies; thus, in metastatic disease, chemotherapy is supplemented with targeted therapies against EGFR or VEGF, and newly approved combinations—encorafenib + cetuximab for BRAFV600E, adagrasib + cetuximab for KRASG12C, and trastuzumab deruxtecan for HER2 positive—expanding the options in advanced lines [131,132,133].

Emerging research on the therapeutic interplay between the gut microbiota and targeted treatments reveals a fascinating landscape where specific gut bacteria, like Akkermansia muciniphila and Bacteroides fragilis, play pivotal roles. These microorganisms possess the remarkable ability to invigorate immune responses and mitigate inflammation, which may significantly bolster the effectiveness of anti-EGFR therapies. Furthermore, Bifidobacterium emerges as a powerful ally, capable of enhancing the therapeutic impact of anti-VEGF treatments [134].

These studies emphasize the increasing interest in the integration of microbiome modulation into treatment strategies for CRC by synergistically blending traditional therapies with innovative interventions designed to influence the gut microbiota. The aspiration is to significantly elevate treatment effectiveness, surmount resistance barriers, and alleviate the burden of adverse effects, ultimately paving the way for more holistic and patient-centered care.

The introduction of microecological agents such as probiotics, prebiotics, synbiotics, and postbiotics, in CRC management hold the potential to modulate gut microbiota. These agents contribute to both the prevention and treatment of CRC by restoring microbial balance, enhancing immune responses, reducing inflammation, and suppressing tumor-promoting pathways.

Probiotics, living microorganisms which are specific non-pathogenic strains of bacteria or yeast, are beneficial to human health when consumed in adequate amounts, helping to support a balanced and healthy gut microbiota. They belong to the genus Lactobacillus and Bifidobacterium but also include Bacillus, Pediococcus, or yeasts and are found in fermented food and drinks (yogurt, kefir) or as dietary supplements [135]. Certain probiotic strains from the genera Lactobacillus and Bifidobacterium (especially Lactobacillus rhamnosus GG, Lactobacillus casei, and Bifidobacterium longum) have demonstrated the ability to reduce the activity of pro-carcinogenic bacterial enzymes such as β-glucuronidase, azoreductase, and nitro-reductase, which are involved in the conversion of food or bile pro-carcinogens into active metabolites. Moreover, these probiotics can sequester mutagens (e.g., heterocyclic amines from processed meat), reducing their bioavailability. Prebiotics, indigestible fibers or substances found in specific foods that nourish the beneficial bacteria in the gut, support the growth and activity of good bacteria, helping to maintain a healthy digestive system. Inulin [136], fructo-oligosaccharides (FOS), galacto-oligosaccharides (GOS) [137], and pectin [138], are among the most investigated prebiotics, with the aim of evaluating their potential role in CRC prevention and treatment [139].

A recent study underlines that, in contrast to the conventional methods of vaccination that stimulate systemic immune response, the administration of probiotics as oral vaccines holds several advantages, such as the ability to target both systemic and mucosal immunity, convenient storage and transport without the need for refrigeration or cold chain systems, and inherent acid and bile tolerance, which aids survival in the gut environment. Unlike attenuated vaccines, probiotics eliminate the risk of reversion to virulence. Advanced strategies that employ CRISPR-Cas editing, the insertion of heterologous genes into bacterial chromosomes, and Phage-Mu transposition improve the genetic stability of probiotics. Moreover, the association of probiotics with prebiotics, encapsulation, and enhancing acid tolerance leads to improved host colonization, thereby optimizing probiotic vaccine performance [140].

Synbiotics combine probiotics and prebiotics to promote a healthy gut microbiome. The synergy of probiotics with prebiotic fibers enhances the production of SCFA and contributes to the restoration of the intestinal barrier integrity (by increasing the expression of zonulin and occludins), reducing bacterial translocation and systemic inflammation, which are critical factors in the progression of CRC. Thus, their administration can modulate the intestinal metabolic profile in a protective sense [141]. In colorectal oncology, postbiotics, structural or metabolic products of viable bacteria that exert host benefits even in the absence of the living microorganism, offer the advantage of controllable bioavailability, eliminating the risk of bacterial translocation in immunocompromised patients. Compared with probiotics, postbiotics have been shown to have a safer and more stable chemical structure. Postbiotics include SCFAs (butyrate, acetate, and propionate), enzymes, cell wall fragments (peptidoglycan), bacterial metabolites, exopolysaccharides, bacteriocins, vitamins, and amino acids [142]. A recent article explores the potential of bacteriocins—specifically Lactacin B, Lactacin F, and Doderlin—produced by lactic acid bacteria (LAB) as safe and effective therapeutic agents for colorectal cancer (CRC). The study utilized in silico analysis to identify strong binding affinities between these compounds and key CRC-associated genes, such as CTNNB1 and LRP5. This indicates their potential to modulate the Wnt signaling pathway and to provide a less toxic therapeutic option compared with current CRC treatments [143].

Used both as prevention and adjuvant therapy in CRC treatment, postbiotics exert their anticancer role through multiple mechanisms including the modulation of the intestinal microbiota, the enhancement of the intestinal mucosal barrier, the regulation of the immune response, and their influence on systemic metabolism [144,145].

The incorporation of beneficial microbes or compounds into supplementation strategies serves to promote their growth. This approach not only aids in reducing the inflammation associated with colorectal cancer (CRC) but also enhances the mucosal barrier function and improves tolerance to chemotherapy.

FMT is an innovative medical procedure in which fecal matter from a healthy donor is transferred into the intestine of a person with dysbiosis to restore the balance of the intestinal microbiota [146]. FMT contains the bacteria and viruses naturally present in healthy stools. FMT is currently widely accepted and recommended as an effective therapeutic strategy for the management of recurrent Clostridioides difficile infection [147], and its use in experimental studies for other intestinal diseases such as inflammatory bowel disease (IBD), constipation, short bowel syndrome (SBS), and irritable bowel syndrome (IBS) showed promising results. In obesity, type 2 diabetes, and metabolic syndrome, FMT is being investigated for its potential to modulate the metabolism and systemic inflammation by intervening in the composition of the intestinal microbiota [148,149]. A growing number of experimental studies are currently exploring the potential of FMT to inhibit the progression of CRC. Using a chemically induced murine model of CRC with intestinal microbial dysbiosis, a recent study [150] investigated the effects of introducing healthy gut bacteria through FMT. This strategy restored the composition and diversity of the gut microbiota in CRC mice, facilitated the recruitment of intestinal immune cells, improved host immune activity targeting CRC, modulated cytokine expression within the CRC microenvironment, and thereby inhibited tumor growth and prolonged survival.

Immunotherapy can enhance the immune system’s ability to recognize and attack cancer cells, while also potentially disrupting tumor growth and metastasis. Combining FMT with pembrolizumab or nivolumab may offer a synergistic effect, improving disease control. A phase II clinical trial, conducted by the MD Anderson Cancer Centre (NCT04729322↗), is currently exploring the efficacy of FMT in combination with the reintroduction of anti-PD-1 therapy (pembrolizumab or nivolumab) in patients with advanced metastatic CRC who have failed anti-PD-1 therapy [151].

The interaction between the microbiota and radiotherapy is a complex process. Bidirectional anticancer treatments can alter the microbiome, leading to dysbiosis, and these microbial changes, in turn, can affect both the efficacy of treatments and the severity of treatment-related gastrointestinal toxicities. In this context, FMT may be associated with radiotherapy and could restore the diversity of the intestinal microbiota, consequently improving the response to radiation and reducing side effects [152]. These data support the beneficial effect of transferring a healthy microbiome to a patient, enhancing the efficacy of immunotherapy and reducing dysbiosis induced by chemotherapy, radiotherapy, or antibiotics.

Bacteriophages, commonly referred to as phages, are specific viruses that infect bacteria and play a vital role in regulating bacterial populations. These phages can be modified to deliver therapeutic agents. Preliminary research and preclinical studies have explored the use of lytic phages targeting Fusobacterium nucleatum, a bacterium linked to enhanced tumor cell proliferation and the promotion of chemoresistance by shielding tumors from immune cell attacks. Furthermore, the increased abundance and prevalence of Fusobacterium nucleatum correlate with more advanced tumor stages and a poorer prognosis in patients diagnosed with CRC [153,154]. These phage therapies have shown potential in reducing tumor growth in CRC xenograft mouse models achieved through a decrease in bacterial load, a reduction in inflammation, and an increase in T cell infiltration [155].

Innovations derived from the CRISPR-Cas (clustered regularly interspaced short palindromic repeats) immune system of prokaryotes have facilitated the application of CRISPR-Cas tools for human gene editing. These tools may also be employed to target the pathogenic and commensal bacteria that colonize the human body, thereby providing novel avenues for treating infections and modulating the microbiome [156].

Phages can be engineered to selectively delete virulence genes or to eliminate antibiotic-resistant bacteria associated with CRC by utilizing CRISPR-Cas3 or CRISPR-Cas9 technologies. CRISPR-Cas tools possess the capability to eliminate or modify specific bacteria through DNA and RNA targeting strategies, effectively disarming them by deleting, altering, or silencing particular genes [157,158]. Additionally, the delivery of CRISPR-Cas tools via bacteriophages or conjugation highlights promising therapeutic opportunities for treating infectious diseases and modifying the microbiome, underscoring both the advancements achieved and the challenges that remain in translating these methodologies into clinical practice.

One notable advantage of phage therapy is its high specificity, which ensures the targeted elimination of pathogens while preserving the diversity of the gut microbiome, thus offering a potential strategy to address antibiotic resistance. Therapeutic bacteriophage approaches in CRC are an emerging and promising area of research that targets pro-tumor bacteria in the gut, modulates the tumor microenvironment, and potentially enhances the efficacy of existing cancer therapies [159,160].