The gut microbiota not only affects digestive system tumors but also impacts tumors in other sites. A specific study found that the gut microbiota is directly involved in the efficacy of trastuzumab treatment (75). Patients who achieved complete remission (CR) exhibited lower alpha diversity and abundance of Lachnospiraceae, Turicibacteraceae, Bifidobacteriaceae, and Prevotellaceae, similar to antibiotic-treated mice (75). Dubigeon et al. collected feces from breast cancer patients and healthy women for microbial comparisons and found that microbiota diversity was reduced in breast cancer patients, with a relative enrichment in Firmicutes and a depletion in Bacteroidetes compared to those of healthy women. A tendency towards a decreased relative abundance of Odoribacter spp., Butyricimonas spp., and Coprococcus spp. was observed (76). In addition, Zhu et al. found that in postmenopausal women with breast cancer, the intestinal macrogenome was enriched with genes encoding lipopolysaccharide biosynthesis, iron complex transporter system, PTS system, secretion system, and β-oxidation (77).

An imbalance of microbiota in the respiratory and intestinal tracts, known as ecological dysbiosis, is associated with alterations in immune responses and the development of lung disease (78). Fecal microbiota analysis showed that non-small cell lung cancer patients were associated with significant upregulation of Prevotella, Gemmiger, and Roseburia when gut microbiota was dysbiotic (79). By examining the role of gut microbiota in cachexia in 31 lung cancer patients, it was found that in patients with cachexia, the gut microbiota showed increased consumption of branched-chain amino acids (BCAAs) and methylhistamine, with alterations in microorganisms involved in functional pathways (80). Analysis of the gut microbiota in lung cancer by 16S rRNA gene sequencing revealed specific microbiota. Firmicutes and Actinobacteria were significantly reduced, whereas Aspergillus, Ruminococcus spp., uncharacteristic genera of Enterobacteriaceae, and uncharacteristic genera of Lactobacillaceae showed high abundance (81). In patients with advanced NSCLC treated with first-line ICIs, it was found that those with an objective response rate (ORR) and progression-free survival (PFS) > 6 months had an enrichment of Gastrocysticercaceae UCG-013 and Agathobacter, while patients with overall survival (OS) > 12 months were enriched in Gastrocysticercaceae UCG-013 (82).

The innate immune response plays a vital role in maintaining the homeostasis of the internal environment by clearing pathogenic bacteria and modulating adaptive responses to the microbiota. These effects are mediated through sIgA (93), TLR4 (94), autophagy (95), and inflammatory vesicles (95). sIgA, the primary immunoglobulin in the gut, targets polysaccharides and flagellin on the surface of bacteria, leading to the formation of sIgA-encapsulated bacteria. sIgA-encapsulated Lactobacillus rhamnosus contributes to the growth of Dendritic cells(DCs), particularly in Peyer’s patches. In addition, sIgA-encapsulated L. rhamnosus upregulates Toll-like receptor 2 (TLR2) expression in 3-week-old mice (96). LPS, a significant component of the outer membrane of Gram-negative bacteria, bind to the TLR4-MD2 complex to activate innate immunity (97). Pro-inflammatory hepatic macrophages produce ROS through phagocytosis of monomeric TLR4-MD2 complexes by NADPH oxidase 2. Palmitate-stimulated CD11b+F4/80low hepatic macrophages produce ROS through initiator protein-mediated endocytosis of TLR4 and NOX2, ROS production, and increased expression of IL-1β in macrophages (98). Inflammasomes, also known as inflammatory vesicles, are cytoplasmic protein complexes that play a crucial role in inflammation and cell death. Inflammasomes can be activated by a variety of pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs), and activation of inflammasome motifs by PAMPs and DAMPs leads to the activation of cysteinyl asparagine-2016, which cleaves the precursors of IL-1β and IL-1 in order to produce the mature forms of these cytokines (99, 100). In addition, NLRP12 plays an essential role in inflammatory vesicles, inducing inflammatory vesicle-dependent release of IL-1β and IL-18 during Yersinia pestis or Plasmodium falciparum infection. NLRP12 acts as a negative regulator of NFκB and MAPK signaling pathways during infection with Salmonella enterica serotype S. Typhimurium, vesicular stomatitis virus, Mycobacterium kansasii, or Mycobacterium tuberculosis, as well as colon tumorigenesis (101).

In homeostasis, myeloid cells represent the most abundant immune cell type in the digestive system, among which DCs and macrophages play crucial roles in maintaining epithelial barrier integrity and anti-tumor immunity. For instance, DCs expressing FFAR2 inhibit their expression of IL-27 to sustain murine intestinal mucosal barrier integrity, reduce tumor bacterial burden, and suppress CD8⁺ T cell exhaustion, thereby restraining tumor initiation (102). DCs from gut-associated lymphoid tissues, spleen, and tumor-draining lymph nodes sense and respond to specific bacteria, stimulating anti-tumor immunity by producing IL-12, thereby amplifying anti-tumor T cell responses (103)and/or IFN-mediated (104) signaling. Intestinal macrophages promote gut barrier integrity and restrict tumor development. In patients with inflammatory bowel disease and colorectal cancer associated with colitis, intestinal macrophages respond to dysbiotic microbiota and metabolites that disrupt the epithelium. For example, inherent FXR receptors in intestinal macrophages perceive aberrant bile acids, leading to the release of pro-inflammatory factors, thereby promoting intestinal stem cell proliferation (105). The gut microbiota stimulates monocyte-derived macrophages to produce IL-1β upon exposure to lipopolysaccharides, inducing T helper cell production of interleukin 17, thus creating an inflammatory environment conducive to tumor initiation (106).

Abnormal communication between the innate immune system and the gut microbiota may lead to complex diseases. A study identified that the gut microbiota plays an essential role in modulating peripheral cGAS-STING activation, thereby promoting systemic resistance to viral infections (101). Complete loss of the innate immune signaling molecule TAK1 in myeloid cells (Tak1ΔM/ΔM) leads to complete resistance to chemically induced colitis and CRC, a phenomenon associated with microbiome alterations that drive protective immunity (107). Analysis of MyD88/TRIF knockout mice with nuclear ATF6 expression in intestinal epithelial cells (nATF6IEC) showed that tumor development was dependent on the activation of STAT3 through MyD88/TRIF signaling (108). In a model of DSS-induced colitis, the PI3K/PTEN signaling pathway in DCs was found to enhance IL-6 production, and DC-specific PTEN knockout (PTENΔDC) resulted in heightened Th1 cell responses and increased mortality (109). Wang et al. performed RNA sequencing analyses to show that simulated microgravity (SMG) inhibits the production of inflammatory cytokines, including TNF-α and IL-6, and suppresses the activation of innate immune signaling in enteropathogenic Escherichia coli (EPEC)-infected macrophages through the p38MAPK and Erk1/2MAPK pathways (110).

Gut microbiota metabolites play a significant role in immunomodulation. SCFAs, produced by microbial fermentation of dietary fibers, have been shown to induce the differentiation of regulatory T (Treg) cells in vitro and in vivo, and mitigate colitis development induced by the transfer of CD4(+) CD45RB(hi) T cells in Rag1(-/-) mice (111). Specifically, propionate-derived SCFAs promote the expansion of colonic Tregs by inhibiting histone deacetylase (HDAC) activity and targeting forkhead box protein P3 (FOXP3), a key nuclear transcription factor of Tregs (111). SCFAs also upregulate the production of interleukin-22 (IL-22) by promoting the expression of the aromatic hydrocarbon receptor (AhR) and hypoxia-inducible factor 1α (HIF1α), which enhances intestinal homeostasis (112). Bile acids are also crucial in innate immunity. The agonist INT-747, a Farnesoid X receptor agonist, significantly reduces TNF-α secretion in activated human peripheral blood monocytes, purified CD14 monocytes and DCs, and monocytes from the lamina propria of inflammatory bowel disease (IBD) patients (113).

The mammalian gut microbiota plays a crucial role in the development and maturation of the host immune system, particularly during the adaptive immune response. Regulatory T (Treg) cells and helper T (Th17) cells are the most abundant subpopulations of lamina propria CD4⁺ T cells under steady-state conditions (114). Th17 cell cytokines are important activators of the innate immune system (115). Treg cells are vital for developing oral tolerance and suppressing hyperinflammatory responses to numerous resident commensal bacteria. The balance between Treg cells and Th17 cells is crucial for mucosal immune homeostasis. Certain Clostridium spp. clusters, such as IV and XIVa, promote the accumulation of Treg cells in the colonic mucosa (116). ETBF promotes the differentiation of preneoplastic mononuclear cells and myeloid-derived suppressor cells (MO-MDSCs) through the combined action of its toxins BFT and interleukin-17 (IL-17) on the colonic epithelium. ETBF selectively upregulates nitric oxide synthase 2 (NOS2) and arginase 1 (ARG1), leading to nitric oxide (NO) production and inhibition of T cell proliferation (117). Segmented filamentous bacteria (SFB) are symbionts that drive postnatal maturation of the intestinal adaptive immune response. SFB stimulate isolated lymphoid follicles and tertiary lymphoid tissues, acting as an alternative to Peyer’s patches for inducing intestinal Th17 cell responses (118). Analysis of the microbiota in immunodeficient Rag1(-/-) mice compared to wild-type mice using 16S rRNA gene sequencing revealed a high enrichment of the mucin-degrading bacterium Akkermansia muciniphila (A.muciniphila) in Rag1(-/-) mice (119).

Several studies have shown that specific gut microbiota can induce the presence of CD8⁺ T cells in the systemic circulation or tumor microenvironment (TME). Modulation of the gut microbiota, as observed in preclinical and preliminary clinical studies, is associated with increased infiltration of CD8⁺ effector T cells into tumors. This infiltration is typically accompanied by enhanced activity of type 1 helper T cells and dendritic cells within the tumor and a decrease in the density of immunosuppressive cells ( Figure 2 ) (121). Tanoue et al. identified 11 bacterial strains from healthy human feces that could strongly induce IFN-γ-producing CD8⁺ T cells in the gut. Additionally, colonization of mice with a mixture of these 11 strains enhanced the host’s resistance to Listeria infection and improved the therapeutic effects of ICIs (103).

An increase in CD8⁺IFNγ⁺ T cells has been associated with intestinal tumorigenesis. Dysbiosis of the intestinal flora can promote chronic inflammation and early T-cell depletion by overstimulating CD8⁺ T cells, which reduces anti-tumor immunity and increases susceptibility to colon tumors (122). In mice infected with the influenza virus, disruption of the intestinal microbiota induced by gentamicin leads to elevated levels of branched-chain amino acids (BCAAs). This inhibits the development of CD11b⁺Ly6G⁺ cells and results in overactive CD8⁺ T cell responses, thereby exacerbating the severity of viral infection (123).

SCFAs regulate colonic Treg cell homeostasis. Treatment of initial CD4⁺ T cells from healthy donors with SCFAs increased the frequency of CD4⁺CD25⁺Foxp3⁺ cells and, to a lesser extent, the proliferation of differentiated Treg cells (124). SCFAs also regulate the size and function of the colonic Treg pool and prevent colitis in mice in a Ffar2(GPR43)-dependent manner (125). During the active immune response, SCFAs promote the production of Th1 and Th17 cells. In T cells, inhibition of HDAC by SCFAs increases the acetylation of p70S6 kinase and phosphorylation of rS6, regulating the mTOR pathway required for the production of Th1, Th17, and IL-10(+) T cells (126). Liang et al. demonstrated differential regulation of Th1 and Th17 cell differentiation by butyrate, which also promoted IL-10 production to control T-cell-induced colitis when butyrate-treated CBirT cells were transferred into Rag1-/- mice (127). SCFAs profoundly affect the mTOR pathway in T cells and cellular metabolism. Oral administration of butyrate, an SCFA, improved humoral immunity by promoting Akt-mTOR-promoted plasma cell production in vancomycin-treated mice. Valeric acid lipids and butyrate lipids led to increased production of effector molecules, such as CD25, IFNγ, and TNFα, through metabolic and epigenetic reprogramming, significantly enhancing anti-tumor activity of specific CTL and transgenic (CAR) T cells in murine melanoma and pancreatic cancer models (128).

As a significant factor influencing the TME, the gut microbiota impacts tumor progression through its metabolites, genotoxins, and signaling pathways. In an APC ^Min/+ mice model of intestinal tumorigenesis, Clostridium nucleatum increased tumor diversity and selectively recruited myeloid-derived suppressor cells (MDSCs), thereby promoting tumor progression (129). An experiment analyzing the relationship between CD8⁺ T cells and melanoma found that the intestinal microbiota within the tumor modulates chemokine levels, influencing the infiltration of CD8⁺ T cells and impacting the survival of melanoma patients (130). In a mice model of primary gastric cancer established by Peng et al., methyl bacillus significantly reduced the diversity of tumor microbiota and the expression of transforming growth factor beta (TGFβ) and CD8⁺ tissue-resident memory (TRM) cells in the tumor microenvironment of gastric cancer (131). IL-25 induced alternate macrophage activation in the TME, promoting HCC cell migration, invasion, and tumorigenesis in vivo and in vitro. This induction increased the expression of waveform protein, Snail, and phosphorylated ERK, and decreased the expression of E-cadherin in HCC cells (132). In CRC, Bifidobacterium adolescentis induced a new subset of CD143+ cancer-associated fibroblasts (CAFs) to suppress tumorigenesis through the Wnt signaling-regulated gene, GAS1, as revealed by single-cell RNA sequencing (133). In antibiotic-treated or GF mice, tumor-infiltrating bone marrow-derived cells showed poor response to treatment, resulting in reduced cytokine production and tumor necrosis after CpG oligonucleotide treatment, and insufficient ROS and cytotoxicity production after chemotherapy (134). Additionally, single-cell RNA sequencing has shown that a lack of microbiota predisposes the TME to tumorigenic macrophages. Mechanistically, microbiota-derived stimulators of interferon genes (STING) agonists induce production of interferon I (IFN I) by monocytes within the tumor, modulating macrophage polarization and natural killer (NK) cell- DCs crosstalk (135). Researchers have found that the STING agonist cdAMP induces the production of IFN-I, which can increase the number and functionality of tumor-infiltrating NK cells and DCs, thereby enhancing the overall anti-tumor immune response. This indicates that the microbiota modulates DC-NK cell crosstalk within the TME through the STING-IFN-I signaling pathway, playing a crucial role in anti-tumor immunity (135). These biological targets offer new avenues for future interventions using the gut microbiota to manage tumor development, treatment, and prognosis.

Recently, numerous studies have highlighted the potential of the gut microbiota as a prognostic biomarker for predicting responses to ICIs. In patients with advanced gastrointestinal (GI) cancers treated with ICIs, analysis of fecal samples by Peng et al. revealed an elevated Prevotella/Bacteroides ratio post-treatment, correlating with a more favorable response to ICIs (144). Similarly, patients with unresectable hepatocellular carcinoma undergoing ICI therapy showed significant enrichment of Lachnoclostridium and Prevotella 9 in their fecal microbiota (145). For mCRC and NSCLC patients receiving Cetuximab + Avelumab, Agathobacter and Blautia strains were identified as potentially crucial factors associated with anti-tumor activity, indicating the potential of gut microbiota as a prognostic biomarker (146). In metastatic melanoma patients treated with avelumab, higher levels of Faecalibacterium and other butyrate-producing bacteria were linked not only to prolonged survival but also to increased avelumab-induced colitis (147). Additionally, intestinal A.muciniphila has emerged as a potential prognostic biomarker for PD-1 inhibitors in mNSCLC patients, showing associations with improved ORR and OS, and exhibiting a predictive value superior to PD-L1 expression in a multivariate analysis (148).

Pathogenic bacteria, such as F. nucleatum, have emerged as potential diagnostic or prognostic markers. Genomic data from cancer patients have demonstrated an enrichment of F. nucleatum in CRC tissues compared to normal tissues. F. nucleatum levels have been observed to increase with tumor malignancy and are associated with metastasis. Interestingly, higher levels of F. nucleatum have been linked to an improved response to PD-1 inhibitor therapy in CRC patients. Additionally, F. nucleatum has been shown to enhance the anti-tumor effects of PD-L1 inhibitors in CRC mice models, leading to prolonged survival. Mechanistically, F. nucleatum induces PD-L1 expression through activation of the STING signaling pathway, leading to increased accumulation of IFN-γ⁺CD8⁺ tumor-infiltrating lymphocytes (TILs) during PD-L1 blockade therapy, thus enhancing tumor sensitivity to PD-L1 blockade. Furthermore, patient-derived organoid models have corroborated these findings, showing that higher F. nucleatum levels are associated with an improved therapeutic response to PD-L1 blockade (149). However, the relationship between F. nucleatum and tumor methylation status, which is crucial for its role as a prognostic marker, requires further clarification.

Given that various members of the intestinal microbiota can produce microbial metabolites and structures associated with the efficacy of ICIs, analyzing the functional capacity of the gut microbiota offers a promising approach to identifying predictive markers of ICI responsiveness. For instance, certain phages such as A. muciniphila, as well as specific bacterial species like B. intestinihominis and B. thetaiotaomicron, have been mechanistically linked to improved therapeutic responses through immunomodulation, highlighting their potential translational significance (12, 150, 151). However, a major challenge lies in achieving consensus across studies regarding the characterized species, which hinders the establishment of a predictive therapeutic index for the gut microbiota. Meta-analytical data suggest a more consistent relationship between microbial gene content and responsiveness to ICIs compared to microbiome composition. Therefore, further functional studies and clinical trials are warranted to explore the translational implications of these findings.

Dysbiosis may contribute to the intestinal inflammation and reduced efficacy of ICIs observed with antibiotic therapy. In experimental colitis, different antibiotic regimens alter the gut microbiota’s ability to control intestinal inflammation by changing microbial community structure and metabolite profiles (152). Streptomycin- and vancomycin-treated microbiota failed to control inflammation, showing proliferation of pathogenic bacteria linked to IBD and metabolites associated with oxidative stress and monosaccharide metabolism. Conversely, metronidazole-treated microbiota maintained inflammation control, with enrichment of Lactobacillus and innate immune responses involving iNKT cells (152). Broad-spectrum antibiotics, and chronic overexposure in the presence of definite or latent infections, can dysregulate the gut microbiota and suppress the immune response. Routy et al. found that in patients receiving PD-1 immunotherapy for epithelial neoplasms, the use of antibiotics (β-lactams+/-inhibitors, fluoroquinolones, or macrolides) led to significantly shorter PFS and OS (12). This indicates that antibiotic therapy, even prior to ICI treatment, can impact treatment efficacy. PINATO et al. similarly reported that prior antibiotic therapy, rather than concurrent therapy, was associated with poorer treatment response and overall survival in patients receiving ICIs (153). Among patients with advanced RCC and NSCLC treated with ICIs, those receiving antibiotics within 30 days of ICI initiation had reduced clinical activity of ICIs compared to those who did not (154). This negative effect of antibiotics on ICIs was also observed in melanoma patients. Patients with advanced melanoma receiving systemic antibiotics along with eplerenone had shorter median overall survival [6.3 m vs. 15.4m, HR=1.88, 95% CI (1.46; 2.43), P=10^-6] (155). Additionally, one study found an increased risk of esophageal, gastric, and pancreatic cancers with increasing penicillin regimens (156). However, some studies suggest a beneficial effect of antibiotics on expanding VδVγ9 T cells in HCC immunotherapy. The efficacy of γδ T cells in tumor therapy in mice models was enhanced by antibiotic-induced gut microbiota dysbiosis (157). Sethi et al. investigated the effect of oral antibiotic depletion of the gut microbiota on tumor growth in models of subcutaneous and hepatic metastases of PDAC, CRC, and melanoma. They found that depletion of the gut microbiota reduced tumor burden in all tested models (158).

Overall, the use of antibiotics during or before the treatment of ICIs is fraught with controversy and is likely to alter the dynamic balance between probiotics and harmful bacteria in the gut. As mentioned above, the use of antibiotics may provide benefits in the treatment of ICIs, but usually, the widespread use of most antibiotics may lead to the development of resistance. Clinicians should consider the duration of antibiotic use to enhance the efficacy of the gut microbiota for ICIs.

The role of diet and dietary interventions in shaping the gut microbiota has garnered significant attention in recent decades. A one-year dietary intervention study conducted in five European countries revealed that adherence to the Mediterranean diet led to alterations in the gut microbiota composition, resulting in reduced frailty and improved health outcomes among older adults (159). This dietary intervention was associated with an increase in the abundance of microbial genes and improvements in clinical phenotypes (160). In another study involving 128 melanoma patients, dietary fiber intake was significantly associated with improved progression-free survival (161). A randomized controlled trial (NCT03275662) demonstrated that a high-fiber diet increased the activity of microbial-encoded glycan-degrading carbohydrate-activating enzymes (CAZymes), leading to a gradual enhancement of microbial diversity, reduced inflammatory markers, and modulation of the immune status (162). Additionally, Nie et al. observed selective increases in the activity of Mycobacterium avium spp., Prevotella spp., and Spirochaetes militaris following consumption of different dietary fiber beverages, along with reductions in the abundance of Clostridium perfringens and Anaplasma fragilis ( 163). Changes in dietary patterns, such as increased consumption of high-fat and high-salt foods, have been implicated in altering the gut microbiota composition and function, thereby promoting chronic inflammation and tumorigenesis. Studies using the azoxymethane (AOM) and APC^min/+ models of CRC have demonstrated that a high-fat diet can drive CRC development by inducing dysregulation of the gut microbiota, metabolomic alterations including elevated lysophosphatidic acid levels, and dysfunction of the intestinal barrier (164). Similarly, a mice model fed with a high-salt diet showed alterations in the composition and function of the fecal microbiota, reduced butyrate production by Lactobacillus, and disruption of intestinal immune homeostasis through cytokine modulation (165).

FMT was initially utilized as a treatment for recurrent Clostridioides difficile infections (166). FMT involves the transplantation of gut microbiota from a donor to a recipient. Patients with sustained remission after FMT treatment showed increased alpha diversity, higher abundance of Lactococcaceae and Lactobacillaceae, reduced levels of Enterobacteriaceae, more successful engraftment of donor microbiota, and restoration of gut microbiota ecological balance, compared to non-responders (167). A trial (NCT03568734) assessed the restoration of gut microbiota in infants born via cesarean section following postnatal oral administration of FMT (168). Furthermore, FMT has shown promise in overcoming resistance to ICIs in refractory melanoma ( Table 1 ). FMT, when combined with PD-1 inhibitors, has been effective in overcoming PD-1 resistance in refractory melanoma patients. Studies have demonstrated that FMT, in conjunction with PD-1 inhibitors, leads to expansion of activated CD56⁺CD8⁺ T cells, increased activation of CD8⁺ T cells and mucosal-associated invariant T (MAIT) cells in peripheral blood mononuclear cells (PBMCs) in responsive patients (169). Additionally, FMT combined with anti-PD-1 therapy counteracted bone marrow-induced immunosuppression, thereby enhancing CD8⁺ T cell activation in the TME of responsive patients (169). In a multicenter phase I trial (NCT03772899), post-FMT, patients in the responsive group showed immunogenic enrichment and reduction in harmful bacteria (170). Moreover, FMT has shown efficacy in treating immune-related adverse events induced by ICIs, such as ICIs-induced diarrhea and colitis (IMDC). Comparison of pre- and post-treatment fecal samples in patients with complete responses revealed increased α-diversity of the intestinal microbiota and higher abundance of Cyanobacteria and Bifidobacteria following FMT treatment (171).

Despite the promising results of FMT therapy in patients treated with ICIs, the long-term safety of FMT remains a concern due to its relatively recent adoption as atreatment modality. Two patients in separate clinical trials developed bacteremia caused by broad-spectrum β-lactamase (ESBL) Escherichia coli after receiving FMT from the same donor, resulting in one fatality (172). This severe outcome prompted the U.S. Food and Drug Administration to issue a safety bulletin advising caution and emphasizing the need to avoid FMT from donors carrying potentially pathogenic bacteria. Additionally, a retrospective cohort study revealed that healthy fecal donors could be colonized with multidrug-resistant organisms during donation campaigns. Among 66 donors, 6 (9%) tested positive for multidrug-resistant organisms, with 11 (17%) showing positivity at any given time (173). Regular screening of donor feces is essential to mitigate the risk of transmitting microorganisms that may cause adverse infectious events, particularly in immunocompromised patients. Further clinical investigations are necessary to elucidate the optimal donor selection criteria, transplantation protocols, and recipient characteristics that are critical for the successful implementation of ICI-FMT combination therapy.

Probiotics, defined as ‘live microorganisms that, when administered in adequate amounts, confer a health benefit on the host (174), have shown promise in CRC treatment. A randomized controlled trial investigating CRC and probiotics revealed that probiotic supplementation improved patients’ quality of life, increased gut microbiota diversity, reduced postoperative infectious complications, and suppressed the production of pro-inflammatory factors (175). Moreover, specific probiotic strains have been found to accumulate at CRC lesion sites after oral administration, where they are fermented by Bacillus butyricus, resulting in the production of SCFAs with known anticancer properties (176). Additionally, probiotic spore-dextrose has been shown to modulate the gut microbiota, enhancing the population of SCFA-producing bacteria and overall microbiota abundance (176). Probiotics have also demonstrated relevance in immunotherapy ( Table 2 ). Griffin et al. demonstrated in a murine tumor model that active Enterococcus faecalis expresses and secretes a direct homolog of the NlpC/p60 peptidoglycan hydrolase, SagA, leading to the production of immunologically active microparticles (177). Another pro-inflammatory strain, Bifidobacterium animalis lactis EDP1503, has shown promise as a novel anti-tumor immunotherapy. EDP1503 reduces the activation of immunosuppressive cells such as myeloid-derived suppressor cells (MDSCs) and regulatory T cells (Tregs), while increasing the number of activated, immune-stimulating DCs, IFN-γ-producing tumor-infiltrating lymphocytes, and cytotoxic CD8⁺ T cells. These effects contribute to the immunogenic remodeling of the TME (178).

Prebiotics, including oligofructose, oligogalactose, and inulin, are known to contain specific chemicals that selectively stimulate the growth of particular bacterial taxa and modify SCFA levels in the gut (179). These substances play a crucial role in maintaining a healthy gut microbiota ( Table 2 ). For example, a randomized, double-masked, crossover intervention study (NCT02548247) demonstrated that inulin-type fructans, a type of prebiotic, induced changes in the human intestinal microbiota, particularly affecting the relative abundance of anaerobic digestive bacteria and bifidobacteria (180). In mice with cefixime-induced alterations in the gut microbiota, administration of a prebiotic mixture increased the abundance of Parabacteroides Goldstein and reduced the abundance of Robinsoniella psoriasis and A.muciniphila. Furthermore, the prebiotic mixture modulated the levels of microbial metabolites, including unsaturated fatty acids and bile acids (181). Studies have also shown that prebiotic oligogalactans can attenuate acute graft-versus-host disease in mice (182). The antitumor effects of inulin have been linked to the activation of intestinal and tumor-infiltrating γδ T cells, which are critical for αβ T cell activation and subsequent control of tumor growth (183). Moreover, adding

inulin or mucin to the diet of C57BL/6 mice has been found to induce antitumor immune responses and inhibit the growth of BRAF mutant melanoma in a subcutaneously implanted mice model (184).

Novel probiotics and prebiotics are being isolated and characterized using various advanced tools, such as sequencing technologies and analytical pipelines. These tools have enabled the identification of new bacterial taxa associated with therapeutic responses. However, there is only modest overlap in the bacterial taxa identified in different studies. While single probiotic strains have shown efficacy in enhancing the effects of ICIs, a combination of multiple strains (flora) may be more effective in maintaining the ecological balance within the gut microbiota. For example, a consortium of 11 species has been shown to confer sustained resistance to Listeria monocytogenes infection in GF mice and to have an additive antitumor effect when combined with anti-PD-1 therapy (103). However, further trials are needed to validate these findings.

Tumor-targeted engineered probiotics have shown promise in the treatment of CRC. Synthetic probiotics have been engineered to significantly reduce CRC tumor size and inhibit tumor growth. These synthetic probiotics have also been shown to modulate the intestinal microbiota and attenuate chemically induced dysbiosis (185). Tang et al. also demonstrated the inhibition of CRC and modulation of mice intestinal microbial homeostasis, providing further support for this approach (186). Engineered bacteria have been developed to induce systemic antitumor immunity. Chowdhury et al. engineered a nonspecific E. coli strain to specifically lyse and release a nano-antagonist encoding CD47 (CD47nb) in the TME. Topical injection of CD47nb-expressing bacteria stimulated a systemic tumor-antigen-specific immune response and reduced the growth of untreated tumors (187). Another engineered bacterium, SYNB1891, targets STING to activate phagocytic antigen-presenting cells (APCs) in tumors and activate complementary innate immune pathways (188). Additionally, the probiotic E. coli Nissle1917 strain has been shown to colonize tumors and continuously convert ammonia to L-arginine. Increasing L-arginine levels has been associated with increased numbers of tumor-infiltrating T cells and significant synergistic effects in tumor clearance (189). In the CT26 model of microsatellite stable (MSS) CRC in mice, triple-engineered E. coli expressing PD-L1 and anti-CTLA antibodies, as well as granulocyte-macrophage colony-stimulating factor, were found to reduce tumor growth (190).

Engineered bacteria still have limitations. Firstly, they are complex and active agents, rather than precise ones. This complexity can lead to instability in the targeting effect of the bacteria, resulting in varying treatment efficacy among patients with different tumors. Additionally, engineered bacteria may exhibit different levels of immune expression. Low expression levels may result in poor efficacy, while high expression levels may increase the risk of autoimmune diseases. Furthermore, designing personalized treatments with engineered bacteria for different types and stages of cancer requires significant time and economic support, which hinders their widespread use.