Gut Microbiota Interactions With Antitumor Immunity In Colorectal Cancer (CRC) PDF

Biomedicine & Pharmacotherapy 165 (2023) 115040 Contents lists available at ScienceDirect Biomedicine & Pharmacotherapy journal homepage: www.elsevier.com/locate/biopha Gut microbiota interactions with antitumor immunity in colorectal cancer: From understanding to application Yu-Pei Zhuang a, b, 1, Hong-Li Zhou c, 1, Hai-Bin Chen a, 1, Ming-Yue Zheng a, b, Yu-Wei Liang a, b, Yu-Tian Gu a, b, Wen-Ting Li a, b, *, 2, Wen-Li Qiu d, *, 2, Hong-Guang Zhou a, b, **, 2 a Department of Oncology, Affiliated Hospital of Nanjing University of Chinese Medicine, Nanjing, China Jiangsu Collaborative Innovation Center of Traditional Chinese Medicine in Prevention and Treatment of Tumor, The First Clinical College of Nanjing University of Chinese Medicine, Nanjing, China c College of Pharmacy, Nanjing University of Chinese Medicine, Nanjing, China d Department of Radiology, Affiliated Hospital of Nanjing University of Chinese Medicine, Nanjing, China b A R T I C L E I N F O A B S T R A C T Keywords: Colorectal cancer Gut microbiota Tumor immunity Modulation strategies Colorectal cancer (CRC) is one of highly prevalent cancer. Immunotherapy with immune checkpoint inhibitors (ICIs) has dramatically changed the landscape of treatment for many advanced cancers, but CRC still exhibits suboptimal response to immunotherapy. The gut microbiota can affect both anti-tumor and pro-tumor immune responses, and further modulate the efficacy of cancer immunotherapy, particularly in the context of therapy with ICIs. Therefore, a deeper understanding of how the gut microbiota modulates immune responses is crucial to improve the outcomes of CRC patients receiving immunotherapy and to overcome resistance in nonresponders. The present review aims to describe the relationship between the gut microbiota, CRC, and antitumor immune responses, with a particular focus on key studies and recent findings on the effect of the gut microbiota on the antitumor immune activity. We also discuss the potential mechanisms by which the gut microbiota influences host antitumor immune responses as well as the prospective role of intestinal flora in CRC treatment. Further more, the therapeutic potential and limitations of different modulation strategies for the gut microbiota are also discussed. These insights may facilitate to better comprehend the interplay between the gut microbiota and the antitumor immune responses of CRC patients and provide new research pathways to enhance immunotherapy efficacy and expand the patient population that could be benefited by immunotherapy. Abbreviations: AEs, adverse events; A2aR, Adenosine 2a receptor; B. fragilis, Bacteroides fragilis; BFT, Bacteroides fragilis toxin; B. producta, Blautia Producta; BSA, broad-spectrum antibiotics; CARD9, caspase recruitment domain-containing protein 9; ccRCC, Clear cell renal cell carcinoma; CLRs, C-type lectin-like receptors; CRC, colorectal cancer; cSCC, cutaneous squamous cell carcinoma; CTLA-4, anti-cytotoxic T-lymphocyte antigen-4; C. tropicalis, candida tropicalis; CXCL, C-X-C motif chemokine ligand; CXCR, C-X-C chemokine receptor; DCs, Dendritic cells; DFS, disease-free survival; EO-CRC, early-onset colorectal cancer; FMT, fecal microbiota transplantation; F. nucleatum, Fusobacterium nucleatum; GEJ, gastric/gastroesophageal junction; GVHD, graft-versus-host disease; GRFS, graft-versus-host disease and relapse-free survival; HCC, hepatocellular carcinoma; HDAC, histone deacetylase; HNSCC, head and neck squamous cell carcinoma; ICIs, immune checkpoint in hibitors; ID2, inhibitor of DNA binding 2; IFN-γ, interferon-gamma; IGF-1, insulin-like growth factor-1; IL, interleukin; LPS, lipopolysaccharides; MAMPs, microbialassociated molecular patterns; MDSCs, myeloid-derived suppressor cells; MHC, major histocompatibility complex class; MLMs, monocyte-like macrophages; MSI-H, high microsatellite instability; NADPH, nicotinamide adenine dinucleotide phosphate; NKs, natural killer cells; NLRP3, NOD-like receptor thermal protein domain associated protein 3; NSCLC, non-small cell lung cancer; ORR, Objective response rate; PCWBR2, putative cell wall binding repeat 2; PD-1, programmed cell death-1; PD-L1, programmed cell death ligand-1; P. anaerobius, Peptostreptococcus anaerobius; PI3K, phosphatidylinositol-3-kinase; PKB/AKT, protein kinase B; PKM2, py ruvate kinase M2; P. micra, Parvimonas micra; PRRs, pattern recognition receptors; PSF, Progression free survival; Pks+ E. coli, Pks+ Escherichia coli; PSMB4, pro teasome beta subunit 4; R. gnavus, Ruminococcus gnavus; RCC, renal cell carcinoma; STAT3, signal transducer and activator of transcription 3; STING, interferon genes; SYK, spleen tyrosine kinase; Th, T helper; TILs, tumor-infiltrating lymphocytes; TIME, tumor immune microenvironment; TLRs, toll-like receptors, TMP, tail sheath protein; TNF-a, tumor necrosis factor-a; Tregs, regulatory T cells; 5-FU, 5-fluorouracil. * Corresponding authors at: Department of Oncology, Affiliated Hospital of Nanjing University of Chinese Medicine, Nanjing, China. ** Corresponding author. E-mail addresses: [email protected] (W.-T. Li), [email protected] (W.-L. Qiu), [email protected] (H.-G. Zhou). 1 Yu-Pei Zhuang, Hong-Li Zhou and Hai-Bin Chen contributed equally to this work as co-first authors. 2 These authors have contributed equally to this work and share corresponding authorship. https://doi.org/10.1016/j.biopha.2023.115040 Received 17 April 2023; Received in revised form 17 June 2023; Accepted 20 June 2023 Available online 24 June 2023 0753-3322/© 2023 The Author(s). Published by Elsevier Masson SAS. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). Y.-P. Zhuang et al. Biomedicine & Pharmacotherapy 165 (2023) 115040 dimethylhydrazine exerted a significant carcinogenic effect on conven tional rats, but it did not exert such an effect on germ-free rats , this finding indicated the influence of the gut microbiota on susceptibility to CRC. A subsequent study of fecal cultures revealed disparities in gut microbiota composition between high-risk and low-risk CRC pop ulations. As shown in Table 1, following the development of next-generation sequencing technology, numerous studies have described the characteristics of the intestinal microbiota in CRC patients by conducting sequencing analysis of stool or intestinal mucosa samples [37–54]. In general, Fusobacterium nucleatum (F. nucleatum), Pks+ Escherichia coli (Pks+ E. coli), enterotoxigenic B. fragilis, Parvimonas, Peptostreptococcus, Gemella, Porphyromonas, Solobacterium, Streptococcus, Prevotella, and Clostridium have been repeatedly found to be enriched in CRC patients (Table 1). Notably, alterations in this microbial composi tion have also been found in the early stages of CRC [42,54,55]. To date, only a few studies have endeavored to characterize the dysbiosis of gut fungal or viral community in CRC. The elevated ratios of Ascomycota/Basidiomycota and the abundance of Trichosporon and Malassezia were the most prominent features of the gut fungal commu nity in CRC patients [44,49,50]. Inovirus, Tunalikevirus, and phages belonging to the order Caudovirales and Siphoviridae and Myoviridae families were reported to be closely associated with CRC occurrence [46, 47]. Besides, Jun Yu et al. reported the characteristics of archaea in the intestinal microbiota of CRC patients by performing shotgun meta genomic analyses of fecal samples from 585 participants (184 patients with CRC, 197 patients with adenomas, and 204 healthy individuals); the microbiota was characterized by the abundance of halophilic archaea and depletion of methanogenic archaea. Notably, compared to control samples, the feces of CRC patients showed a higher co-occurrence correlation among fungi as well as a higher co-exclusion correlation between fungi and bacteria; this finding indicated the po tential involvement of intra-fungal synergistic relationships and bacterial-fungal antagonistic associations in CRC development. Another two recent studies reported interactions between phages and oral bacterial in CRC [46,47], suggesting that interspecies interactions among different microbial species might influence CRC development. Nonetheless, the current research pertinent to this subject remains relatively scarce, with a majority being confined to studies with small sample sizes, thereby drawing clear and compelling conclusions is challenging. More comprehensive research is required to elucidate the intricate relationship between the gut microbiota and CRC. 1. Introduction Colorectal cancer (CRC) is a highly prevalent malignant tumor, it ranks third in global incidence and second in mortality rates. In recent years, the incidence of CRC has been on the rise, particularly among individuals under 50 years of age. In 2020, there were over 1.9 million new cases of CRC and 930,000 deaths due to CRC. Moreover, it is estimated that by 2040, the burden of CRC will increase to 3.2 million new cases and 1.6 million deaths , thereby exerting considerable pressure on public health and financial systems. Over the past decade, the application of immune checkpoint inhibitors (ICIs) including anti-cytotoxic T-lymphocyte antigen-4 (CTLA-4), anti-programmed cell death-1 (PD-1), and anti-programmed cell death ligand-1 (PD-L1) antibodies has dramatically changed the landscape of treatment for many advanced cancers [4–9]. However, CRC is one of the malignant tumors with a poor response to immunotherapy, with only a small proportion of CRC patients with DNA mismatch repair deficiency or high microsatellite instability (MSI-H) showing good response to ICIs therapy. The efficacy of ICIs therapy is limited for the majority of patients with high mismatch repair deficiency or microsatellite-stable CRC [11–15]. This situation presents an opportunity to identify inno vative strategies to augment immunotherapy response in patients with CRC. In recent years, burgeoning evidence has accentuated the profound implications of gut microbiota in both the ontogenesis of cancer and the potential modulation of its immunotherapy treatment. The studies of the differences in gut microbiota between responders and non responders have shown that the abundance of Bacteroides fragilis , A. muciniphila [18,19], Bifidobacterium spp [20–22], Eubacterium limo sum , Faecalibacterium spp , and Lactobacillus was posi tively correlated with the response to immunotherapy. Meanwhile, the diminished responses to anti-CTLA4 and/or anti-PD1 antibodies was observed in antibiotic-treated and germ-free murine models of solid tumors [17,18,26]. Further evidence for the direct link between microbiota composition and antitumor immunity was obtained by transplanting the microbiota from responder and nonresponder cancer patients into germ-free mice, which was found to elicit similar responses to ICIs [18,20,27]. It is evident that a healthy gut microbiota can ensure that T cells identify tumor antigens and activate cytotoxic CD8+ T cells to attack tumor cells, thereby enhancing the antitumor immune response and the efficacy of immunotherapy [28–30]. These findings suggests that the gut microbiota may exert far-reaching, systemic effects on the immune system, with a consequent impact on therapeutic out comes in the context of cancer. Therefore, a deeper understanding of how the gut microbiota modulates immune responses is crucial to improve the outcomes of CRC patients receiving immunotherapy and to overcome resistance in nonresponders. The present review summarizes the relationship between the gut microbiota, CRC, and antitumor immune responses, with particular emphasis on the influence of both pathogenic and protective microor ganisms on antitumor immunity. Concurrently, we discuss the potential mechanisms through which the gut microbiota influences host immune responses as well as the prospective role of intestinal flora in CRC treatment are discussed. Furthermore, the therapeutic potential and limitations of the various modulation strategies for the gut microbiota are also discussed. These insights could offer novel perspectives for improving immunotherapy efficacy and broadening the population of CRC patients benefited from immunotherapy. 3. Effect of the gut microbiota on antitumor immunity in CRC The process of CRC development has a complex association with host immune dysfunction [56–58]. The immune system primarily comprises innate immune cells such as neutrophils, macrophages, dendritic cells (DCs), and natural killer cells (NKs) as well as adaptive immune cells, including T and B lymphocytes , these cells exhibit pro- or anti tumor immune functions. The gut microbiota not only participates in the metabolism of host nutrients and maintenance of intestinal mucosal integrity but also exhibits characteristics that promote immune cell maturation; thus, the gut microbiota contributes to immune re sponses and plays a pivotal role in the development and maintenance of the host’s immune system [61,62]. Studies involving germ-free mice have shown that the gut microbiota influences the differentiation and activation of immune cells [63–66]. Colon tissue-resident commensal bacterial species such as Ruminococcus gnavus and Blautia producta can promote the activation of CD8+ T cells and enhance the antitumor im mune response by degrading tumor metabolites such as hemolytic glycerophospholipids, thus exerting a suppressive effect on CRC. Microbial dysbiosis promotes chronic inflammation and early T-cell exhaustion by excessive stimulation of CD8+ T cells, which consequently suppresses antitumor immunity. Another study showed that gut microbiota dysbiosis led to a deficiency of beneficial bacteria with urea-degrading functions (such as Bifidobacterium); this resulted in the 2. Dysbiosis in CRC The gut microbiota is a sophisticated microbial community comprising bacteria, fungi, viruses, and other microorganisms. Bacteria are the predominant organisms in the gut microbiota, with approximately 98% of the species belonging to the Bacteroidetes and Firmicutes phyla [32–34]. An early study in 1967 demonstrated that 2 Biomedicine & Pharmacotherapy 165 (2023) 115040 Y.-P. Zhuang et al. entry of urea into macrophages, thereby decreasing the binding effi ciency of phosphorylated signal transducer and activator of transcrip tion 1 (p-STAT1) to the promoter region of spermidine/spermine N1-acetyltransferase 1 (SAT1) and further promoting the polarization of macrophages toward a pro-tumor phenotype characterized by poly amine accumulation. Besides, the abundance of specific bacteria, such as Alloprevotella, Treponema, and Desulfovibrio, was also associated with the high expression of chemokines, which induced the infiltration of different T-cell subpopulations in the tumor immune microenviron ment (TIME) [29,30]. Thus, a multifaceted relationship exists between the gut microbiome and the host immune response in CRC [70–72]. To date, some beneficial or deleterious bacterial species have been reported to promote or interfere with antitumor immunity (Fig. 1; Table 2), thereby affecting CRC progression and treatment. 3.1. F. nucleatum F. nucleatum has been extensively studied in CRC. The abundance of F. nucleatum is associated with the malignant transformation of ade nomas to carcinomas [54,73]; furthermore, a high level of F. nucleatum in the TIME is correlated with the lower survival rate of CRC patients [74,75]. F. nucleatum has the potential to induce a pro-tumor immune microenvironment. In CRC tissue samples, the level of F. nucleatum was correlated with the expression pattern of a pro-inflammatory gene profile [70,76]. This finding has also been corroborated in CRC mouse model, where F. nucleatum enhanced the expression of proinflammatory cytokines and generated a proinflammatory microenvironment favor able for colorectal neoplasia progression by promoting tumor-infiltrating immune cell recruitment. F. nucleatum may also promote colorectal carcinogenesis through its suppressive effect on T Table 1 Correlation between Gut Microbiota and CRC. Study Sample size Study platform Sample types CRC-related microbiota Ref. Liu li et al. 2022 Fengzhu Sun et al. 2022 Jiachao Zhang et al. 2021 Caroline Young et al. 2021 Jun Yu et al. 2020 Takuji Yamada et al. 2019 Nicola Segata et al. 2019 Jun Yu et al. 2019 30 CRC, 29 adenomas, and 35 controls 462 CRC and 449 controls shotgun metagenomic sequencing shotgun metagenomic sequencing stool Peptostreptococcus stomatis, Clostridium symbiosum, Hungatella hathewayi, Parvimonas micra, and Gemella morbillorum Podoviridae, Siphoviridae, Myoviridae, Drexlerviridae, Inoviridae, and Herelleviridae 160 CRC and 157 controls shotgun metagenomic sequencing stool 400 CRC and 666 controls 16 S rRNA sequencing stool Peptacetobacter hiranonis Phage, Fusobacterium nucleatum animalis 7_1 Phage, Fusobacterium nucleatum polymorphum Phage, Fusobacterium nucleatum animalis 4_8 Phage, Parvimonas micra Phage F. nucleatum, Parvimonas, Peptostreptococcus, Gemella, Pks+ E. coli, Porphyromonas, Solobacterium, Bilophila, Atopobium, Dorea, and Alistipes 84 CRC, 197 adenomas, and 204 controls 616 CRC and 251 controls shotgun metagenomic sequencing shotgun metagenomic sequencing stool Halorubrum, Natrinema, and Halococcus stool F. nucleatum, Parvimonas, Solobacterium, Peptostreptococcus, Parvimonas, Porphyromonas, Gemella, Atopobium, Dorea, Bilophila, Clostridium, and Streptococcus 313CRC, 143 adenomas, and 308 controls shotgun metagenomic sequencing stool F. nucleatum, Solobacterium, Porphyromonas, Peptostreptococcus, Parvimonas, Clostridium, Gemella, and Streptococcus 184 CRC, 197 adenomas, and 204 controls 285 CRC and 290 controls shotgun metagenomic sequencing stool Malassezia, Moniliophthtora, Rhodotorula, Acremonium, Thielaviopsis, and Pisolithus shotgun metagenomic sequencing stool F. nucleatum, Pks+ E. coli, Porphyromonas, Parvimonas, Peptostreptococcus, Gemella, Prevotella, Solobacterium, and Clostridium Georg Zeller et al. 2019 stool Study Sample size Study platform Jun Yu et al. 2018 74 CRC and 92 controls shotgun metagenomic sequencing Patrick D. Schloss et al. 2018 Jun Yu et al. 2018 30 CRC and 30 controls shotgun metagenomic sequencing stool 255 CRC and 271 controls shotgun metagenomic sequencing stool H.Qin et al. 2017 74 CRC, 29 colon-polys and 28 controls 7 CAC, 10 sporadic cancer and 10 controls 120 CRC, 198 adenomas, and 172 controls 59 CRC, 21 polyps and 56 controls qPCR 74 CRC and 54 controls Harry Sokol et al. 2017 Patrick D. Schloss et al. 2016 Paul W O’Toole et al. 2016 Jun Wang et al. 2015 Joseph J.Y. Sung et al. 2015 52 CRC, 47 adenomas, and 61 controls Sample types stool CRC-related microbiota Ref. Orthobunyavirus, Inovirus, Tunalikevirus, Phikzlikevirus, Betabaculovirus, Sp6likevirus, Sfi21dtunalikevirus, Punalikevirus, Lambdalikevirus, C2likevirus, and Mulikevirus Siphoviridae and Myoviridae stool Enterotoxigenic B. fragilis, F. nucleatum, Porphyromonas asaccharolytica, Parvimonas micra, Prevotella intermedia, Alistipes finegoldii, and Thermanaerovibrio acidaminovorans Malassezia, Talaromyces, and Trametes ITS2 sequencing 16 S rRNA sequencing mucosal Basidiomycota, Malassezia, and Debaryomyces stool F. nucleatum, Peptostreptococcus, Pophyromonas, Gemella, and Parvimonas 16 S rRNA sequencing mucosal Porphyromonas, Peptostreptococcus, Parvimonas, Fusobacterium, Bacteroides, Roseburia, Ruminococcus, Oscillibacter, and Clostridium shotgun metagenomic sequencing 16 S rRNA sequencing stool F. nucleatum, Arvimonas micra, Solobacterium, and Peptostreptococcus mucosal F. nucleatum, Pks+ E. coli, Enterotoxigenic B. fragilis, Gemella, Parvimonas, Peptostreptococcus, and Granulicatella 3 Y.-P. Zhuang et al. Biomedicine & Pharmacotherapy 165 (2023) 115040 Fig. 1. Effect of the gut microbiota on antitumor immunity in CRC. cells-mediated antitumor immune response. Mima et al. analyzed tumor tissues from 598 CRC patients and revealed that F. nucleatum abundance was inversely correlated with CD3+ T cells infiltration in the tumor microenvironment (TME). Another study reported that F. nucleatum abundance was negatively correlated with the proportion of CD3+ T cells, particularly CD3+CD4+CD45RO+ T cells. Conversely, the density of CD45RO+ T cells was positively correlated with the longer survival of patients with CRC. Moreover, in CRC mouse model, F. nucleatum enrichment was positively correlated with the infiltration of CD11b+ bone marrow cells, particularly myeloid-derived suppressor cells (MDSCs) , which contribute to tumor growth by suppressing T-cell function [80–82]. Because of mismatch repair deficiencies, CRC patients with MSI-H show numerous mutations and the presence of immunogenic peptides, which increase the levels of tumor-infiltrating lymphocytes (TILs) and subsequently promote antitumor immune re sponses [83–86]. Notably, a study involving 1041 CRC patients demonstrated that the association between F. nucleatum and TILs varied according to the tumor MSI status. The presence of F. nucleatum was negatively associated with TILs in MSI-H tumors but positively associ ated with TILs in non-MSI-H tumors. Mechanistically, F. nucleatum may promote MDSC recruitment in the TIME and directly induce the death of T cells through Fap2 and RadD proteins, thereby suppressing host antitumor immune responses [88–90]. F. nucleatum can also pro mote immune evasion in CRC. F. nucleatum binds to the inhibitory re ceptor TIGIT (T cell immunoglobulin and ITIM domain) on immune cells through its Fap2 protein; this binding inhibits the activation of T cells and tumor cell clearance by NKs, thereby protecting F. nucleatum and nearby tumor cells from being killed by these immune cells [91,92]. F. nucleatum can also promote macrophage infiltration through acti vating tumor-derived chemokine ligand (CCL)− 20 and inducing M2 macrophage polarization to enhance CRC metastasis. Interestingly, a recent study revealed that F. nucleatum might contribute to the improvement of the effectiveness of CRC immuno therapy. Gao et al. discovered that feces and tumor tissues of CRC patients who respond to immunotherapy showed abundance of F. nucleatum; this might be associated with the improvement in response to the PD-1 blockade therapy. Mechanistically, F. nucleatum can upre gulate cyclic GMP-AMP synthase expression through p65 phosphoryla tion, activate stimulator of interferon genes (STING) signaling to induce PD-L1 expression by tumor cells, and induce the infiltration of interferon-gamma (IFN-γ)+ CD8+ TILs, thus increasing the sensitivity of tumor cells to the PD-L1 blockade therapy. Inversely, a recent study conducted metagenomic sequencing on fecal samples of metastatic CRC patients receiving treatment with anti-PD-1 inhibitors and Regorafenib, discovering an abundant presence of F. nucleatum in the feces of non responders and the presence of F. nucleatum showed a negative corre lation with the progression-free survival (PFS) of patients. Mechanistically, F. nucleatum-derived succinic acid suppresses the cyclic GMP-AMP synthase (cGAS)-interferon-β (IFN-β) pathway, which in turn reduced the levels of the T helper (Th) 1-type CCL5 and C-X-C motif chemokine ligand 10 (CXCL10) in tumors and consequently dampened the antitumor response by limiting the trafficking of CD8+ T cells to the TIME. Thus, for cancer patients undergoing immunotherapy, the potential influence of deleterious gut bacteria on treatment outcomes, either positive or negative, necessitates further research for substantiation. 3.2. Pks+ E. coli Pks+ E. coli can produce a colibactin toxin encoded by the pks genomic island. This bacterial species is abundant in the colonic mucosa of patients with CRC [40,45,54], and can promote tumorigenesis in mouse models [96,97]. Mathilde Bonnet et al. showed that Pks+ E. coli colonization in the intestine of CRC patients was associated with a reduced proportion of tumor-infiltrating CD3+ T cells. Pks+ E. coli infection in mice decreased the proportion of CD3+CD8+ T cells and led to a poor response to anti-PD-1 immunotherapy. Pks+ E. coli can also increase the expression of interleukin (IL)− 17 C by elevating the expression of BCL-2 and BCL-XL proteins through the Toll-like receptors (TLRs)-MyD88-dependent signaling pathway, suppress intestinal epithelial cell apoptosis , and induce macrophages to express cyclooxygenase-2 , thereby promoting CRC occurrence. 3.3. Enterotoxigenic B. fragilis B. fragilis is an obligate anaerobic, gram-negative commensal bac terium that is ubiquitously present in the human gastrointestinal tract. Enterotoxigenic B. fragilis (ETBF), a subtype of B. fragilis, is distinguished by the secretion of a specific B. fragilis toxin. ETBF was found to be enriched in CRC patient. In a mouse model, ETBF promoted distal colon tumorigenesis through a multistep proinflammatory immune response [98, 101–103]. Shaoguang Wu et al. revealed that persistent colonization by ETBF significantly expedited colonic adenoma formation. The tumorigenic 4 Y.-P. Zhuang et al. Biomedicine & Pharmacotherapy 165 (2023) 115040 Table 2 Effect of the Gut Microbiota on Antitumor Immunity in CRC. Microorganism Impact on antitumor immunity Underlying Mechanism F. nucleatum Inhibition 1. Promotes the infiltration of specific proinflammatory myeloid cell subsets into tumors. 2. Inhibits the function of NK cells and T cells through a direct interaction with TIGIT via the Fap2 protein. 3. Promotes macrophage infiltration through the activation of tumorderived CCL20 and induces M2 macrophage polarization. 4. suppresses the cGAS - IFN-β pathway which in turn reduced the levels of the Th1-type CCL5 and CXCL10 in tumors and conse quently dampened the antitumor response by limiting the trafficking of CD8 + T cells to the TIME. Induces PD-L1 expression by activating the STING signaling pathway and promotes the accumulation of IFN-γ+ CD8+ tumorinfiltrating lymphocytes. 1. Impairs antitumor T-cell response by decreasing CD3+ and CD8+ Tcells and increasing colonic inflammation. 2. Elevates the expression of IL-17 C by enhancing the expression of BCL-2 and BCL-XL through the MyD88-dependent pathway and increases IL-6 and TNF-α levels. 3. Stimulates macrophages to express cyclooxygenase-2. Enhances IL-17 secretion by Th17 cells and further activates STAT3 and NF-κB signaling pathway[101,103], thereby inducing a proximal to distal mucosal gradient of C-X-C chemokines that mediate the recruitment of CXCR2-expressing polymorphonuclear immature myeloid cells and promote the differentiation of pro-tumoral monocytic MDSCs. Promotes the activation of the PI3KAKT pathway in CRC cells through phospho-focal adhesion kinase to increase the activation of the NF-κB signaling pathway, thereby increasing the levels of proinflammatory cytokines such as IL-10 and IFN-γ and expanding the population of myeloidderived suppressor cells, tumorassociated macrophages, and granulocytic tumor-associated neutrophils. Enhances Th17 immune cell infiltration and stimulates the secretion of cytokines such as IL-17, IL22, and IL-23 in the colon. Stimulates the differentiation of MDSCs and activates their immunosuppressive activity. Enhances the immunosuppressive function of MDSCs and activates PKM2 to increase the expression of nitric oxide synthase, cyclooxygenase 2 and NADPH oxidase 2, with increase in the production of nitric oxide and reactive oxygen species. Enhancement Psk+ E. coli Inhibition Enterotoxigenic B. fragilis Inhibition P. anaerobius Inhibition P. micra Inhibition C. tropicalis Inhibition Table 2 (continued ) Microorganism Impact on antitumor immunity Underlying Mechanism Candida albicans Inhibition Ruminococcus gnavus and Blautia producta A. muciniphila Enhancement Lactobacillus plantarum Enhancement NS8 lactobacillis Enhancement Promotes the glycolysis of macrophages and IL-7 secretion, thereby inducing IL-22 production in group 3 innate lymphoid cells through the aryl hydrocarbon receptor and the STAT3 pathway. Promotes CD8+ T cell activation by degrading tumor metabolites such as hemolytic glycerophospholipids. 1. Facilitates the enrichment of M1like macrophages through the NLRP3-dependent pathway. 2. Increases the number of cytotoxic T lymphocytes, upregulates TNF-α expression, and inhibits PD-1 expression. Promotes IL-18 production suppresses inflammation and apoptosis, and elevates IgA secretion. Suppresses the activation of NF-κB and decreases the level of antiinflammatory cytokines IL-10- and IL17-producing T cells Enhancement effect of ETBF is probably mediated through the activation of the STAT3 pathway and mucosal IL-17 response. In mice infected with ETBF, IL-17 produced by Th 17 cells selectively activates the STAT3/nuclear factor kappa light-chain enhancer of activated B cells (NF-κB) pathway in distal colonic epithelial cells, thereby inducing the synthesis of proin flammatory cytokines such as CXCL1, CXCL2, and CXCL5. These cyto kines subsequently recruit C-X-C chemokine receptor (CXCR) 2+ myeloid progenitor cells and stimulate their differentiation into MDSCs, which culminates into MDSC infiltration and tumorigenesis, predomi nantly in the distal colon [102–104]. Christine M Dejea et al. also showed that ETBF could colocalize with Pks+ E. coli within poly microbial biofilms on polyps in patients with familial adenomatous polyposis. The concurrent presence of ETBF and Pks+ E. coli promoted their tumorigenic potential in murine models. Intriguingly, ETBF enhanced the pro-carcinogenic effect of Pks+ E. coli through IL-17-mediated inflammation and disruption of the mucosal barrier. Regulatory T cells (Tregs), which exert anti-inflammatory effects, usually play a contributing role in maintaining immune homeostasis and tolerance. In the context of ETBF-induced carcinogenesis, Tregs promote CRC development. Treg cells can suppress IL-2 activity within the TIME, attenuate T cell differentiation into Th1 cells in ETBF-infected mice, and conversely, enhance their differentiation into Th17 cells, thereby promoting tumor growth. ETBF-infected mice with Treg deficiency showed an amplified inflammatory response and diminished tumor formation, which corresponded to enhanced IFN-γ secretion by Th1 cells and reduced IL-2 secretion by Th17 cells. 3.4. Other bacteria Peptostreptococcus anaerobius (P. anaerobius), a gram-positive anaer obic bacterium, predominantly resides in the oral cavity and gastroin testinal tract. P. anaerobius is markedly enriched in the feces and mucosa of CRC patients [40,45] and is involved in promoting CRC development and regulating antitumor immunity. Jun Yu et al. confirmed that P. anaerobius activated the phosphatidylinositol-3-kinase (PI3K)-protein kinase B (PKB/AKT) signaling pathway in CRC cells by interacting with integrin α2/β1 through its surface protein putative cell wall binding repeat 2 (PCWBR2), thereby promoting cell proliferation and activating NF-κB pathway. Consequently, NF-κB initiated a proin flammatory response and elevated the levels of cytokines such as IL-10 5 Y.-P. Zhuang et al. Biomedicine & Pharmacotherapy 165 (2023) 115040 and IFN-γ. Additionally, in Apcmin/+ mice model, P. anaerobius admin istration through oral gavage expanded the population of MDSCs, tumor-associated macrophages, and tumor-associated neutrophils, thereby inducing a pro-tumor immune response in the TIME. Parvimonas micra (P. micra), another prevalent gram-positive, anaerobic pathogenic bacterial species in the human oral cavity, showed a high abundance in the feces and colon tissues of CRC patients, and this high abundance was correlated with poor prognosis [48,109, 110]. Mechanistically, the oral administration of P. micra in Apcmin/+ mice enhances Th17 cells infiltration and stimulates the secretion of cytokines such as IL-17, IL-22, and IL-23, thereby inducing a proin flammatory milieu that contributes to CRC development. Streptococcus gallolyticus can localize within polyps harboring APC mutation ; this induces proinflammatory cytokine secretion and attracts bone marrow cells, particularly CD11b⁺TLR4⁺ cells, thereby eliciting a pro-tumor immune microenvironment. In two separate clinical datasets, Akkermansia muciniphila (A. muciniphila) was significantly decreased in the feces of CRC patients as compared to that in healthy individuals. Furthermore, the abundance of A. muciniphila was positively correlated with NLRP3/TLR2 expression and M1-like macrophage infiltration in CRC patients. With regard to mechanisms, A. muciniphila induced the enrichment of M1-like tumor-associated macrophages in the CRC microenvironment through the NLRP3-dependent pathways, thereby inhibiting tumor occurrence. In another study, the oral administration of A. muciniphila increased the number of cytotoxic T cells and upregulated tumor ne crosis factor-α (TNF-α) expression while inhibiting PD-1 expression, thus enhancing antitumor immunity in the colon and mesenteric lymph nodes of CRC mice. and generated IFN-γ-producing CD8+ T cells, thereby suppressing colon cancer development. These studies highlight the involvement of gut fungi in tumor development, with CARD9 and its associated signaling pathways playing a pivotal role in regulating antifungal immunity. Some of these seemingly contradictory phenomena suggest that CARD9 may play a dual role in cancer [115,119,120], thus emphasizing the need to elucidate the more intricate mechanisms underlying the relationship between gut microbiota and host antitumor immune response. Tingting Wang et al. also reported that C. tropicalis enhanced the immu nosuppressive function of MDSCs and promoted CRC development by activating pyruvate kinase M2 (PKM2), which subsequently increased the expression of nitric oxide synthase, cyclooxygenase 2, and nicotin amide adenine dinucleotide phosphate (NADPH) oxidase 2 and the production of nitric oxide and reactive oxygen species. Moreover, in a mouse model lacking Dectin-3 (a c-type lectin), chemical induction led to tumor development and accumulation of Candida albicans. The enrichment of Candida albicans stimulated macrophage glycolysis and IL-7 secretion. Subsequently, IL-7 induced type 3 innate lymphoid cells to produce IL-22 through the aryl hydrocarbon receptor and STAT3, thereby increasing the incidence of intestinal inflammation and tumor formation. These studies suggest that fungal microbiota is emerging as a potential contributor to the development of CRC by exerting both positive and negative effects on antitumor immune re sponses, thus making them a potential target for CRC prevention and treatment. 3.6. Bacteriophage Several investigations conducted on the prevalence of bacterio phages in CRC have revealed intriguing insights into the impact of these phages on the disease [47,124]. Primarily, bacteriophages appear to modulate CRC by modifying the bacterial community, thus affecting both pro- and antitumorigenic bacterial species [125,126]. A seminal study by Lasha Gogokhia et al. demonstrated that Caudovirales phages not only selectively eradicate pro-carcinogenic bacteria, but also incite immune cells to generate copious amounts of IFN-γ through a TLR9-dependent mechanism, consequently bolstering host anti-tumor immunity. These emerging revelations pertaining to the role of phages in CRC not only clarify their involvement in carcinogenesis but also indicate the potential for phage-based therapeutic strategies targeting specific bacterial species involved in CRC. 3.5. Gut fungi To date, most research has primarily focused on the role of bacterial microbiota in antitumor immunity, while the role of viruses and fungi in antitumor immunity is poorly understood. Recently, two studies that used Caspase Recruitment Domain-containing protein 9 (CARD9)-defi cient mice have increased our knowledge of how intestinal fungi regu late host immunity and tumor development [115,116]. CARD9, an adaptor protein expressed in myeloid lineage cells, serves as a down stream molecule of C-type lectin-like receptors (CLRs). It recognizes not only bacteria, fungi, and viruses but also endogenous danger signals, subsequently forming the CARD9-adaptor protein BCL10-protease MALT1 signal complex, which is an upstream molecule of inflamma tory signaling pathways such as NF-κB and mitogen-activated protein kinase, thereby promoting the production and release of inflammatory cytokines by macrophages [117,118]. Tingting Wang et al. discovered that the proportion of Candida tropicalis (C. tropicalis) was notably higher in CRC patients than in healthy individuals. Under normal conditions, C. tropicalis is translocated to the intestinal lamina propria and eliminated by host macrophages through the CARD9 molecule. However, under CARD9 deficiency condition, the increased levels of C. tropicalis can stimulate the differentiation of MDSCs and induce their immunosuppressive activity, thereby promoting colon tumorigenesis. Tingting Wang et al. further demonstrated that CARD9 expression in the colon of CRC patients with low fungal loads was significantly higher than in those with relatively high fungal loads; moreover, fecal fungal load was positively correlated with the propor tion of MDSCs in blood and colon tissues. This finding suggested that excessive intestinal fungal accumulation might inhibit CARD9 expres sion, subsequently inducing MDSC differentiation and facilitating CRC development. However, another study conducted concurrently by Thirumala-Devi Kanneganti et al. revealed that in an azox ymethane/dextran sodium sulfate-induced mouse model of CRC, gut commensal fungi triggered inflammasome activation and downstream IL-18 maturation through the spleen tyrosine kinase (SYK)-CARD9 signaling axis. This promoted intestinal epithelial barrier restoration 4. The underlying mechanism through which gut microbiota influences antitumor immunity Cross-reactivity of antigens constitutes a crucial mechanism through which microbiota can influence antitumor immunity (Fig. 2A). Micro bial antigens can elicit cross-reactive T lymphocyte responses that may interact with tumor-associated antigens, thereby inducing specific antitumor immune reactions [128–130]. A prerequisite for T cells to initiate antitumor immunity in response to gut microbiota requires the presence of an epitope shared between tumor cells and bacteria. The antigen SVY present in Bifidobacterium breve is homologous to the novel antigen SIY in tumor cells; thus, it stimulates cross-reactive T cell responses against tumor cells , thereby implying that microbial “mimicry antigens” can modulate T cells and enhance antitumor im munity. Furthermore, circulating or tumor-infiltrating T cells can recognize major histocompatibility complex (MHC) class I- or II-restricted peptides derived from various microorganisms and tumor antigens. A recent study revealed that the tail sheath protein (TMP) of the prophage of Enterococcus hirae contained a MHC-I-binding epitope TSLARFANI (referred to as TMP1), which exhibited high similarity to the tumor antigen proteasome beta subunit 4 (PSMB4) and could bind to MHC-I molecules as an antigenic epitope, thereby inducing CD8+ T cell responses. These specific CD8+ T cells can cross-react with the tumor antigenic epitope PSMB4, thereby triggering antitumor effects. Mice 6 Y.-P. Zhuang et al. Biomedicine & Pharmacotherapy 165 (2023) 115040 Fig. 2. The underlying mechanism through which the gut microbiota influences antitumor immunity. (A) The gut microbiota influences antitumor immunity through cross-reactivity of antigens. (B) The gut microbiota modulates antitumor immunity by activating pattern recognition receptors. (C) The gut microbiota modulates antitumor immunity through their derived metabolites. harboring Enterococcus hirae in their gut exhibited rapid activation of CD8+ T lymphocyte responses when subjected to anti-PD-1 immuno therapy, thus enhancing the efficacy of the treatment. Addition ally, outer membrane vesicles derived from the gut microbiome can promote tumor immunity by inducing the generation of cross-reactive T cells. The activation of pattern recognition receptors (PRRs) represents another critical pathway through which gut microbiota modulates antitumor immunity (Fig. 2B). PRRs comprise a diverse group of re ceptors, which include both cell surface-bound and intracellular re ceptors such as TLRs, Nod-like receptors (NLRs), and CLRs. These receptors recognize microbial-associated molecular patterns (MAMPs) from bacteria, fungi, viruses, and protozoa and subsequently activate inflammatory cytokine signaling pathways to eradicate the infectious agent [132–135]. MAMPs include an array of bacterial motility proteins, lipopolysaccharides (LPS), and microbial nucleic acid structures present in the gut microbiota. Intestinal bacteria interact with TLRs on immune cells through MAMPs, thereby inducing cytokine release and activating the immune system with an inflammatory response. LPS derived from the gut microbiota can stimulate TLR4 on epithelial cells, thereby upregulating CCL2 expression and promoting the infil tration of colonic monocyte-like macrophages (MLMs) in the TIME; this further stimulates interleukin-1β production from MLMs, resulting in the activation of Th17 cells that promote inflammation. Commensal gut fungi can bind to CLRs in myeloid cells through MAMPs and sub sequently trigger inflammasome activation through the SYK-CARD9 signaling axis; this promotes the production of IFN-γ by CD8+ T cells. Resident commensal bacteria specifically activated the trans formation of native macrophages into M1-like macrophages through the TLR2/NOD-like receptor thermal protein domain-associated protein 3 (NLRP3) pathway, which released IL-1β in the TIME and suppressed CRC progression. Microbiota can also indirectly modulate immunity through their derived metabolites (Fig. 2C). For example, the microbiota-derived metabolite butyrate, a histone deacetylase (HDAC) inhibitor, promotes IFN-γ production by CD8+ T cells through the inhibitor of DNA binding 2 (ID2)-dependent mechanism, thereby enhancing antitumor immune responses. Bifidobacterium pseudolongum can increase the level of CD8+ T cells in tumors by secreting adenosine that binds to the T-cell adenosine receptor and subsequently induces the differentiation and activation of Th1 cells and increases IFN-γ production by Th1 cells. The bile salt hydrolase-producing non-enterotoxigenic Bacter oides can promote the accumulation of bile acids in the colon; this further activates the Wnt/β catenin signaling pathway and the G protein-coupled bile acid receptor to enhance CCL28 expression, which subsequently induces the accumulation of immunosuppressive Treg cells and exacerbates CRC progression. 5. Role of gut microbiota in CRC treatment The success of cancer treatment may depend on the interplay be tween the immune system and the microbiota. In patients with epithelial tumors such as melanoma, NSCLC, and renal cell carcinoma, a high abundance of A. muciniphila is associated with an increase in CCR9+CXCR3+CD4+ T cells and demonstrates a better clinical response to anti-PD-1 immunotherapy [18,19]. Given that these immune-related changes are the most significant ones in the local intestinal mucosa and regional mesenteric lymph nodes, this finding is particularly intriguing in CRC context. As shown in Table 3, preclinical studies have found that the specific gut microbiota can affect the antitumor immunity of CRC mice and the efficacy of immunotherapy [113, 114, 139, 141–147]. B. pseudolongum and secretes adenosine that binds to the T cell adenosine receptor, which promotes the differentiation and activation of Th1 cells, thereby leading to an increase in CD8+ T cell levels in tumors and enhancing the effec tiveness of efficacy of anti-PD-L1 and anti-CTLA-4 in CRC mice (including AOM/DSS induced colitis-associated cancer and MC38 tumor-bearing models). Besides, Li-Shun Wang et al. iso lated a novel strain of Lactobacillus paracasei sh2020 from healthy do nors and demonstrated through in vitro and in vivo studies that it upregulated CXCL10 in tumors, induced CD8+ T cell recruitment, and augmented the efficacy of anti-PD-1 therapy. In another investigation, Dr. Kenya Honda et al. discovered that 11 strains of the gut microbiota (Parabacteroides distasonis, Parabacteroides gordonii, Alistipes senegalensis, Parabacteroides johnsonii, Paraprevotella xylaniphila, Bacter oides dorei, Bacteroides uniformis JCM 5828, Eubacterium limosum, 7 Y.-P. Zhuang et al. Biomedicine & Pharmacotherapy 165 (2023) 115040 pursue more extensive, human-focused studies to provide a deeper, more substantial validation of these initial observations. Clinical studies have also substantiated the association between gut microbiota and the reactions to ICIs therapy in oncological patients [18–20, 24, 25, 149–157], intimating that specific gut microbiome compositions could potentially serve as predictors for the immuno therapeutic responses of cancer patients (Table 4). Remarkably, a sequencing study of fecal samples from 74 patients with advanced gastrointestinal cancers (including CRC) who were treated with ICIs, revealed a positive correlation between the treatment response and the presence of Prevotellaceae, Ruminococcaceae, and Lachnospiraceae in various types of gastrointestinal cancers. Besides, Cyanobacteria, Fusobacteria, Ruminococcaceae, and Veillonella also displayed a positive correlation with the better immunotherapeutic efficacy in CRC patients, whereas Bacteroides and Faecalibacterium were found to be enriched in CRC patients with poor responses. The microbial community also participates in mediating the thera peutic response to cancer chemotherapy. Elimination of gut bacteria by previous antibiotic treatment reduces the response of myeloid and Th17 cells, consequently diminishing the efficacy of oxaliplatin and cyclo phosphamide [158,159]; this indicates that the intestinal microbiota is essential for acquiring effective antitumor immunity after chemo therapy. Moreover, gut microbiota can regulate the therapeutic effect of 5-fluorouracil (5-FU) through the metabolism of bacterial vitamins B6, B9, and ribonucleotides, while the inhibition of bacterial deoxyribonu cleotide metabolism can enhance the efficacy of 5-FU. A high abundance of F. nucleatum is also associated with resistance to oxali platin and 5-FU in CRC patients [161,162]. Mechanistically, F. nucleatum activates the TLR4/MYD88-dependent pathway to stimu late innate immune responses, leading to microRNA loss that causes a shift in CRC cell fate from apoptosis to autophagy. This shift enhances the survival of CRC cells during chemotherapy and contributes to che moresistance development in CRC patients. Therefore, targeting F. nucleatum to reduce the autophagy level of CRC cells may potentially enhance the chemosensitivity of CRC. The response of tumors to radiation therapy may also be regulated by the microbiota. Interestingly, Stephen L. Shiao et al. found that both bacteria and fungi in the gut microbiota could affect post-radiotherapy antitumor immunity and exert opposing effects. The absence of symbiotic bacteria can lead to the increased proliferation of symbiotic fungi and decreased antitumor immunity after tumor radio therapy. Targeting the inhibition of symbiotic fungi can enhance radiation-induced antitumor immune response by reducing macrophage-mediated immune suppression. These findings emphasize the importance of comprehensively evaluating the diversity of the gut microbiota in understanding its impact on cancer treatment mecha nisms; moreover, such evaluation can help propose new microbiota-based therapeutic targets to improve cancer treatment. Gut microbiome may also influence anastomotic leaking after CRC surgery. Manuela M Santos et al. showed that anastomotic leakage in CRC patients and mice was correlated with the relative abundance of Alistipes onderdonkii and Parabacteroides goldsteinii. Oral supplementa tion with Alistipes onderdonkii increased anastomotic leakage in mice, while the administration of Parabacteroides goldsteinii through gavage improved anastomotic healing by exerting anti-inflammatory effects. Probiotic therapy may also contribute to reduce postoperative compli cations in CRC patients. A systematic review of 21 randomized controlled trials conducted between 2007 and 2017, which included 1831 CRC patients who underwent surgical treatment, found that mi crobial preparations significantly increased Actinobacteria abundance in patients’ fecal microbiota and altered the microbial composition in tumor tissues. This alteration in microbial composition reduced the levels of inflammatory factors, side effects of chemotherapy, post operative complications, and duration of antibiotic treatment in patients after surgery. Thus, the gut microbiota is a promising target for CRC treatment to enhance treatment efficacy and improve patient-related Table 3 Effects of Gut Microbiota in CRC Immunotherapy. Microorganism Underlying Mechanism Effect on immunotherapy of CRC Ref. B. pseudolongum and A. muciniphila Promotes the differentiation and activation of Th1 cells through the production of the metabolite inosine induces the expression of CXCL10 in the tumors and subsequently enhances CD8+ T cell recruitment Promote intratumoral infiltration and activation of CD8+ T cells Improves the efficacy of antiPD-L1 and antiCTLA-4 Improves the efficacy of antiPD-1 Improves the efficacy of antiPD-1 Augment the crosspriming capacity of dendritic cells through the activation of the STING-type I interferon signaling pathway, which subsequently stimulates CD8+ T cell-mediated immune responses Activates immune responses by increasing CD8+ T cell and effector memory T cells, decreasing Treg and M2 macrophages in the tumor microenvironment Induces the stable production of IFN-γ by CD8+ T cells in the intestine Improves the efficacy of antiCD47 Improves the efficacy of antiCTLA-4 Improves the efficacy of antiPD-1 and antiCTLA-4 Lacticaseibacillus paracasei sh2020 Commensal Clostridiales strains (Roseburia intestinalis, Eubacterium hallii, Faecalibacterium prausnitzii, and Anaerostipes caccae) Bifidobacterium species (B. bifidum, B. longum, B. lactis, and B. breve) Lactobacillus acidophilus Commensal bacterial strains (Parabacteroides distasonis, Parabacteroides gordonii, Alistipes senegalensis, Parabacteroides johnsonii, Paraprevotella xylaniphila, Bacteroides dorei, Bacteroides uniformis JCM 5828, Eubacterium limosum, Ruminococcaceae bacterium cv2, Phascolarctobacterium faecium, and Fusobacterium ulcerans) Ruminococcaceae bacterium cv2, Phascolarctobacterium faecium, and Fusobacterium ulcerans) isolated from healthy volunteers could induce the stable production of IFN-γ by CD8+ T cells in the intestine, thereby enhancing the antitumor immune response and exhibiting antitumor effects. Remarkably, the effect of these strains in impeding tumor pro gression is comparable to that of ICIs, and their combination with anti-PD-1 therapy can effectively inhibit tumor growth. These preclini cal data support the use of bacteria or a mixture of bacterial species as therapeutic agents for CRC, particularly as adjuvants in immune checkpoint blockade (ICB) therapy. Nevertheless, it should be noted that these interventional studies have been primarily conducted in animal models, which may not fully encapsulate the complexity of human biological systems. Consequently, these findings should be considered with a degree of circumspection. It becomes paramount, therefore, to 8 Y.-P. Zhuang et al. Biomedicine & Pharmacotherapy 165 (2023) 115040 Table 4 Relationship between Gut Microbiota and Immunotherapy. Cancer types n Intervention Study platform Responder Nonresponder Ref. NSCLC and RCC 160 Anti-PD-1 ↑A. muciniphila, Enterococcus hirae - NSCLC 338 ICIs Metagenomic Shotgun Sequencing Metagenomic Shotgun Sequencing 16 S rRNA Sequencing; Metagenomic Shotgun Sequencing 16 S rRNA Sequencing 16 S rRNA Sequencing; Metagenomic Shotgun Sequencing Metagenomic Shotgun Sequencing 16 S rRNA sequencing ↑A. muciniphila - ↑Bifidobacterium longum, Collinsella aerofaciens, and Enterococcus faecium ↑Ruminococcus obeum, Roseburia intestinalis ↑Faecalibacterium - ↑Prevotellaceae, Ruminococcaceae, and Lachnospiraceae； ↓Bacteroidacea, Prevotella/Bacteroides ratio ↑Lachnospiraceae spp. - ↑Streptococcaceae spp. ↑Faecalibacterium prausnitzii, Butyricicoccus pullicaecorum and A. muciniphilia ↑Akkermansiaceae, Enterococcaceae, Enterobacteriaceae, Carnobacteriaceae, and Clostridiales Family XI ↑A. muciniphila, B. longum, Faecalibacterium prausnitzii - - ↑Propionibacterium acnes, Veillonella, Staphylococcus aureus, Peptostreptococcus, Ruminococcus bromii, Dialister, and Sutterella ↑Proteobacteria ↑Lactobacillus, Clostridium, and Syntrophococcus ↑Alistipes putredini, Prevotella copri, B. longum, Lachnobacterium spp, Lachnospiraceae, and Shigella ↑ Alpha diversity ↑Faecalibacterium ↑Bilophila, Sutterella, and Parabacteroides ↑Ruminococcus_unclassified ↑Bacteroidales Ipilimumab, nivolumab, ipilimumab and nivolumab treatment: ↑Faecalibacterium prausnitzii, Bacteroides thetaiotamicron, and Holdemania filiformis; Pembrolizumab treatment: ↑Dorea formicigenerans; ICIs treatment: ↑Bacteroides caccae and Streptococcus parasanguinis - Metastatic melanoma 42 Anti-PD1 Melanoma 38 Anti-CTLA-4 Advanced-stage gastrointestinal cancer 74 Anti-PD-1 /Anti-CTLA-4 Melanoma 94 Anti-PD-1 Melanoma 103 Advanced thoracic carcinoma 42 AntiPD-1/AntiCTLA-4 Anti-PD-1 NSCLC 11 Anti-PD-1 16 S rRNA sequencing, 8 Anti-PD-1 Metagenomic Shotgun Sequencing 16 S rRNA sequencing 16 S rRNA sequencing, Hepato-cellular carcinoma NSCLC 17 ICIs NSCLC 37 Anti-PD-1 Melanoma 112 Anti-PD1 Melanoma 39 ICIs 16 S rRNA sequencing Metagenomic Shotgun Sequencing Metagenomic Shotgun Sequencing Higher taxa richness and more gene counts outcomes. microbiota [167,168] (Fig. 3). Disturbance of gut microbial homeostasis mediated by unhealthy dietary habits is closely linked with CRC occurrence and progression. The Western-style diet, which is characterized by high intake of fat, red meat, and processed meat products, is currently recognized as one of the risk factors for CRC. A study conducted on 217 healthy volunteers who underwent low-fat, moderate-fat, and high-fat dietary interventions for 6 months revealed that the low-fat diet group had the highest diversity of the gut micro biota, while the high-fat diet group had the lowest diversity, accompa nied by an increase in bacteria belonging to the genera Bacteroides and Prevotella and a decrease in bacteria belonging to the phylum Firmicutes. Targeted dietary changes (from high-fat/low-fiber diets to low-fat/high-fiber diets and vice versa) have led to significant alter ations in mucosal biomarkers related to cancer risk. In the low-fat/high-fiber diet group, glycation fermentation and butyric acid increased, while secondary bile acid synthesis was inhibited. Conversely, a high intake of total dietary fiber or whole grains is 6. Modulation strategies for the gut microbiota As previously noted, the gut microbiota exerts substantial influence on host antitumor immunity through diverse mechanisms (Figs. 1–2), hence strategizing its manipulation could potentially be beneficial for cancer immunotherapy. Several clinical trials are underway attempting to manipulate the microbiota in various ways, such as dietary in terventions, fecal microbiota transplantation (FMT), and probiotic intake (Table 5), which to assess the clinical benefits and safety of modulating gut microbiota in combination with immunotherapy for patients with various types of cancers. 6.1. Diet and exercise Diet significantly affects the structure and function of the gut 9 Y.-P. Zhuang et al. Biomedicine & Pharmacotherapy 165 (2023) 115040 Table 5 Clinical Trials on Gut Microbiota Modulation and Antitumor Immunotherapy. NCT number Cancer types Dietary intervention NCT05384873 NSCLC n Intervention Primary Outcome measures Status Ref. 180 Immunonutrition + Immunotherapy; Control dietary intervention + Immunotherapy High fiber, Plant based diet + Exercise prescription with Acceptance and Commitment Training sessions + Anti PD1/PD-L1 monotherapy; Standard diet and exercise recommendations + Anti PD-1/ PD-L1 monotherapy Prolonged nightly fasting + immunotherapy; Regular eating pattern + immunotherapy PFS Not yet recruiting Recruiting - Rates of prolonged nightly fasting compliance and change in gut microbiome and microbial metabolites ORR Active, not recruiting - Recruiting - Change in the gut microbiome Recruiting - Compliance, AEs of ICIs therapy and unanticipated AEs Quality of Life of Patients Completed - Completed - - NCT04866810 Melanoma 80 NCT05083416 Head and neck cancer 52 NCT0511901 RCC 60 NCT04645680 Melanoma 42 NCT04552418 Solid tumor 12 NCT04009122 NSCLC FMT NCT05750030 HCC 12 FMT + Atezolizumab + Bevacizumab Treatment safety 30 30 20 FMT + Sintilimab + Fruquintinib FMT FMT ORR Change in the intestinal microbiota FMT-related AEs, ORR NCT05251389 CRC Malignancies Melanoma, HNSCC, cSCC, ccRCC, and NSCLC Melanoma Not yet recruiting Recruiting Recruiting Recruiting 24 Clinical benefit Recruiting - NCT04988841 Melanoma 60 Treatment safety Recruiting - NCT04009122 NSCLC No-responding donor FMT; Responding donor FMT FMT + Ipilimumab + Nivolumab; Placebo + Ipilimumab + Nivolumab Standard treatment + nutritional support + IGEN-0206 (Dietary Supplement); Standard treatment + nutritional support + placebo; Standard treatment Quality of Life of Patients Completed - NCT number Cancer types n Intervention NCT04975217 NCT04951583 NCT04935684 Pancreatic cancer Melanoma and NSCLC Haematologic malignancy 10 70 150 NCT04924374 Lung Cancer 20 NCT04758507 RCC 50 NCT04729322 CRC 15 NCT04521075 NSCLC 42 FMT FMT + ICIs therapy FMT; No intervention FMT + anti PD1 therapy; Anti PD1 therapy Donor FMT; Placebo FMT FMT + Nivolumab; FMT + Pembrolizumab FMT + Nivolumab NCT04577729 Melanoma 60 NCT04264975 NCT04116775 NCT04056026 NCT04040712 Solid Tumors Prostate Cancer Mesothelioma RCC 60 32 1 20 NCT04038619 Genitourinary, melanoma, l ung, ovarian, uterine, cervical, and breast malignancies Melanoma, lung and genitourinary cancer 40 Allogenic FMT; Autologous FMT FMT FMT + Pembrolizumab FMT Donor FMT; Sham FMT FMT 800 FMT + prednisone, infliximab, or vedolizumab 30 20 40 NCT05279677 NCT05273255 NCT05286294 NCT03819296 NCT03812705 280 280 Continuous ketogenic diet + Nivolumab + Ipilimumab; Discontinuous ketogenic diet + Nivolumab + Ipilimumab; Beta-hydroxybutyrate supplementation + Nivolumab + Ipilimumab Isocaloric high-fiber diet + Immunotherapy; Isocaloric diet + Immunotherapy Potato starch (Resistant starch) + Dual ICIs Standard treatment + nutritional support + IGEN-0206 (Dietary supplement); Standard treatment + nutritional support + placebo; Standard treatment Hematopoietic and lymphoid cell neoplasm NCT03341143 Melanoma NCT03353402 Melanoma Probiotics and bacteria consortia Feasibility and compliance - - Primary Outcome measures FMT-related AEs ORR GRFS rate Status Ref. Recruiting Recruiting Recruiting - Treatment safety and responses Free from tumor progression rate ORR Recruiting - Recruiting - Recruiting - FMT-related AEs and Overall response rate PSF Recruiting - Recruiting - Overall response rate Anti-cancer effect PSF Diarrhea resolution rate Recruiting Recruiting Compeleted Compeleted FMT-related AEs and ICIsrelated diarrhea/colitis Recruiting - Recruiting - FMT Change in the gut microbiota and FMT-related AEs Treatment response rate Recruiting - FMT + Pembrolizumab FMT + ICIs therapy ORR FMT-related AEs Compeleted Compeleted (continued on next page) 10 Y.-P. Zhuang et al. Biomedicine & Pharmacotherapy 165 (2023) 115040 Table 5 (continued ) NCT05220124 Bladder urothelial carcinoma 190 Live Combined (Bifidobacterium, actobacillus and Enterococcus Capsules) + Immunotherapy; Immunotherapy Probiotic-V9 (Lactobacillus Bifidobacterium) + PD-1 inhibitor + platinum; Placebo + PD-1 inhibitor + platinum Probiotic-M9 (Lactobacillus rhamnosus) + PD-1 inhibitor; Placebo + PD-1 inhibitor Fermented soybean extract MicrSoy-20 + Pembrolizumab; Placebo + Pembrolizumab VE800 (11 distinct nonpathogenic, nontoxigenic, commensal bacterial strains) + Nivolumab + Vancomycin NCT05094167 NSCLC 46 NCT05032014 Liver cancer 46 NCT04909034 NSCLC 30 NCT04208958 Melanoma, GEJ adenocarcinoma, and Microsatellite stable CRC- 111 NCT number NCT03829111 Cancer types RCC n 30 NCT03686202 Solid tumor 65 Intervention Clostridium butyricum CBM 588 probiotic strain + Nivolumab + Ipilimumab; Nivolumab + Ipilimumab Microbial ecosystem therapeutics (MET-4) NCT03775850 Microsatellite stable CRC, triple negative breast cancer, NSCLC, bladder cancer, gastroesophageal cancer, and RCC 120 EDP1503 + pembrolizumab PFS Recruiting - ORR Recruiting - ORR Recruiting - The incidence of AEs Recruiting - Safety and tolerability of VE800, ORR Recruiting - Primary Outcome measures Change in Bifidobacterium composition of stool Status Active, not recruiting Ref. The abundance of immunotherapyresponsiveness associated species and treatment safety Safety and tolerability of EDP1503 alone and in combination with pembrolizumab Active, not recruiting - Completed Fig. 3. Gut microbiota modulation strategies. (A) Diet and exercise. (B) FMT. (C) Probiotics. (D) Antibiotics and bacteriophages. 11 Y.-P. Zhuang et al. Biomedicine & Pharmacotherapy 165 (2023) 115040 associated with a reduced incidence and mortality rate of CRC patients, with even greater benefits observed at higher levels of intake [173–175]. Notably, a high-fiber diet can promote the enrichment of Rumino coccaceae in melanoma patients receiving neoadjuvant therapy com bined with ICIs, thereby enhancing the optimal antitumor immune response and minimizing the risk of immune-related adverse events (AEs) during immunotherapy [150,176]. Similarly, a high-fiber diet is associated with enrichment of butyrate-producing Bifidobacterium spe cies and better clinical outcomes in metastatic NSCLC patients receiving ICIs therapy. These findings suggest that high-fiber diet inter vention combined with ICIs therapy may improve clinical outcomes of cancer patients (Fig. 4). Fasting, ketogenic diets, and other dietary interventions may also aid cancer treatment by regulating the gut microbiota and hormone levels, altering systemic immune status, and enhancing the effects of drug treatment. Daniel Ajona et al. conducted studies in various lung tumor models and confirmed that short-term fasting reduces insulin-like growth factor-1 (IGF-1) levels and downstream signaling; furthermore, the inhibition of IGF-1 increases cancer cell immunoge nicity, promotes CD8+ T cell antitumor response, and synergizes with PD-1 inhibitors to jointly inhibit lung cancer progression and metastasis. Ketone bodies induced by the ketogenic diet can regulate gut micro biota, thereby reducing the proportion of proinflammatory Th17 cells in the gut lamina propria. Additionally, the ketone body 3-hydroxy butyrate produced by the ketogenic diet can enhance the efficacy of ICIs therapy in a mice model. However, the ketogenic diet may ulti mately engender long-lasting adverse effects on an individual’s health. The ketogenic diet amplifies the predisposition to a multitude of afflictions, such as cardiovascular diseases, diabetes mellitus, and Alz heimer’s disease, eclipsing the prospective merits it might impart. Meanwhile, several clinical trials are currently evaluating the potential influence of diet intervention on cancer patients undergoing immuno therapy (Table 5). It should be noted that the microbiome’s response to diet is highly individualized, therefore, dietary interventions to modu late the gut microbiome should specifically tailored to each individual. Thus, the treatment modalities of dietary intervention should be Fig. 4. Manipulating the microbiota to improve immunotherapy efficacy. Different gut microbiome modulation strategies can be used to improve the efficiency of cancer immunotherapy by decreasing pathogenic bacteria and inflammation, increasing the microbiome diversity and function, and enhancing anti-tumor immunity. 12 Y.-P. Zhuang et al. Biomedicine & Pharmacotherapy 165 (2023) 115040 constructed based on personalized parameters such as age, gender, ge netic background, and clinical parameters to achieve precision therapy. Physical exercise and activity can also positively alter the composi tion of microbiota, thereby benefiting the host’s health. Individuals on a long-term exercise schedule have significantly increased diversity of intestinal bacteria, Akkermansia abundance, and the levels of microbial produced butyrate as compared to sedentary subjects. A recent prospective study reported that sports activities could significantly prolong disease-free survival (DFS) in patients with stage III CRC. The study recruited 1696 patients with resected stage III CRC to deter mine the effect of physical activity on 3-year DFS and found that a large amount of recreational physical activity as well as prolonged mild to moderate aerobic activity or any intense aerobic exercise significantly increased DFS in CRC patients. Although there is currently no strictly proven diet or exercise program for treating cancer, it is confirmed that maintaining moderate physical activity and appropriately adjusting the dietary structure, such as consuming more amounts of fruits, vegetables and reducing the intake of excessive fat, red meat, and processed meat products, can effectively reduce the risk of CRC. This is also the most cost-effective approach to prevent CRC. Obesity is recognized as a crucial risk factor of CRC and considered a significant element affecting gut microbiota homeostasis [187,188]. Clinical studies have demonstrated that diversity within the gut microbiota of obese individuals markedly decreases, with a signifi cant increase in Firmicutes abundance and the Firmicutes/Bacteroidetes ratio, and a considerable decline in Bacteroidetes abundance [189,190]. Moreover, certain pathogens, such as F. nucleatum, have been detected as overly enriched in the gut of both obese and CRC patients. Mechanistically, obesity induced by a high-fat diet could promote CRC incidence by disrupting gut microbiota and metabolic equilibrium [192, 193]. Moreover, obesity could impair the cytotoxic function of CD8+ T cells within the tumor microenvironment, thereby accelerating tumor progression. Consequently, weight control in obese individuals could potentially exert profound effects on gut microbiota, thereby influencing the progression of colorectal cancer. Exploring how obesity impacts gut microbiota and CRC may provide new insights for the pre vention and treatment of obesity-associated CRC. However, the specific obesity-associated microbiota related to CRC development remains un defined. Further studies were required to delve into the intricate relationships between obesity, gut microbiota, and CRC, as well as how to utilize this knowledge to enhance CRC prevention and treatment strategies. immunotherapy for patients with CRC. Apart from CRC, two recent clinical trials have confirmed that FMT combined with ICIs therapy can overcome resistance to immunotherapy in some refractory melanoma patients. Diwakar Davar et al. demonstrated the efficacy of FMT in converting 15 immunotherapy-resistant advanced melanoma patients into responders (NCT03341143). These patients were initially unre sponsive to therapy with ICIs; after they underwent FMT from re sponders, 6 patients exhibited tumor shrinkage or long-term disease stabilization after treatment with pembrolizumab or nivolumab. Notably, one patient demonstrated a sustained partial response for over 2 years. Further analysis revealed that the patients’ microbiota shifted toward the donor profile, with an increase in the abundance of Bifido bacterium longum and Faecalibacterium prausnitzii, an increase in tumor-infiltrating activated CD8+ T cells, and a decrease in immuno suppressive IL-8+ myeloid cells (Fig. 4). Erez N Baruch et al. treated 10 ICIs-unresponsive melanoma patients with FMT. Colonoscopy-based FMT was performed in 10 patients with metastatic melanoma who were unresponsive to anti-PD-1 therapy, along with repeated oral fecal capsules, followed by five cycles of combined therapy with nivolumab (NCT03353402). The results showed that three patients achieved complete/partial response (with the same donor’s stool), and the progression-free survival was more than 6 months, thus confirming the safety of the phase I clinical trial. The number of immune cells infiltrating the tumor in patients who responded to anti-PD-1 therapy was significantly increased, thus indicating that FMT successfully acti vated the patient’s immune response. These findings highlight the effect of FMT on cancer patients’ response to immunotherapy and represent a milestone. FMT also has the potential to mitigate immune-related colitis associated with immunotherapy , but this report is based on the data from a limited number of patients. As an emerging therapeutic approach, the safety of FMT remains debatable. A recent systematic review of 26 studies (including 1149 FMT recipients) found that severe adverse effects, such as death and viral infections, appear to be rare. However, it cannot be ignored that pathogenic bacteria, parasites, bacteriophages, and multidrug-resistant bacteria can be transferred from donor feces to FMT recipients, thereby leading to severe infection [200,201]. Additionally, the poten tial dissemination risk of certain pathogenic genes warrants attention. Preclinical studies have demonstrated that the transplantation of human feces from obese individuals into germ-free mice fed a low-fat diet can induce obesity and obesity-related metabolic phenotypes. A case report cautioned that a woman developed obesity after receiving FMT intervention from a healthy but overweight donor. In line with this finding, a randomized double-blind controlled trial confirmed that the metabolic characteristics of FMT donors could be partially transferred to recipients. This result suggests that during the FMT process, unknown pathogenic genes from donors may be transferred to recipients, thereby potentially inducing the onset of related diseases. Although current clinical evidence supports the rela tively good safety of FMT for treating cancer patients [21,197,206], it is still essential to strengthen donor screening and testing; additionally, more high-quality clinical data are required to further investigate whether FMT can be considered a safe cancer treatment option. The determination of an “ideal donor” remains a largely unexplored area. The identification of an “ideal donor” is based on optimal com ponents or a comprehensive health status of respondents or healthy volunteers, which requires further investigation [207–209]. Various studies have identified multiple microorganisms associated with response to ICIs therapy (Table 4). However, the current data are pri marily derived from comparative analyses between clinical efficacy and blood immune cell detection following gut microbiome profile. These conclusions should be interpreted cautiously as they pertain only to treatment efficacy; moreover, the majority of causal relationships remain unestablished. Additionally, the α/β diversity of microbiota, dominant strains, results of linear discriminant analysis effect size, Kyoto Encyclopedia of Genes and Genomes functions, and microbiota 6.2. FMT FMT combined with tumor immunotherapy is currently the major focus area of clinical translational research. FMT represents a direct modality for reshaping the recipient’s gut microbiota towards achieving compositional normalization. Typically, donors selected for FMT are sourced from individuals who have exhibited favorable responses to ICIs or healthy individuals (Fig. 3; Table 5). In CRC mice model, concurrently amplifying microbial diversity, FMT also has the capacity to alleviate inflammation, such as reducing the levels of pro-inflammatory cytokines and augmenting anti-inflammatory cytokine concentrations, thereby diminishing tumor formation [71,196]. However, to date, there is no clinical evidence that demonstrates the effectiveness and safety of FMT for treating CRC patients. Currently, a series of clinical trials are being conducted to evaluate the clinical benefits and safety of FMT in conjunction with immunotherapy in treating various types of cancer patients (Table 5). Notably, two ongoing early-phase clinical trials aim to evaluate the efficacy of FMT in combination with anti-PD-1 therapy for CRC patients [NCT05279677, current status: recruiting; NCT04729322, current status: recruiting]. If the initial results from these two clinical trials are positive, they would lay the groundwork for the design of larger, more in-depth studies, with the aim to further assess the clinical benefits and safety of FMT in combination with 13 Y.-P. Zhuang et al. Biomedicine & Pharmacotherapy 165 (2023) 115040 prediction signals are not completely consistent among the different studies , This inconsistency could be attributed to individual dif ferences in human gut microbiome composition and the influence of environmental factors such as diet and geographic distribution [211, 212]. Further basic and clinical research is necessary to determine the association between microbiota and the effectiveness of cancer immu notherapy, with an aim to identify the optimal microbiota composition that enhances antitumor response. elimination of pathogenic bacteria is also crucial in microbiome inter vention, such as antibiotics treatment (Fig. 3). Treatment of CRC animal models with antibiotics eradicates carcinogenic bacteria such as F. nucleatum, thereby leading to a reduction in the bacterial load within the tumors and subsequently impeding tumor growth and invasion [221, 222]. However, numerous clinical studies have reported conflicting re sults, wherein the use of antibiotics was found to be closely associated with an increased risk of CRC incidence [223–226]. This may be attributable to another type of dysbiosis induced by antibiotics. Anti biotics can effectively eliminate pathogenic or harmful bacteria; how ever, their nonselective antibacterial effects may exterminate symbiotic microbiota, thereby leading to an alternative form of dysbiosis , which disrupts normal immune function and precipitates the onset of chronic inflammation; this situation has been demonstrated to cause CRC occurrence. However, to date, the role of antibiotics in cancer treatment also remains contentious. In patients with hepatocellular carcinoma who used antibiotics within 30 days before and after initiating ICIs therapy, there was no significant change in the median overall survival period, objective response rate, and disease control rate, while the median progression-free survival period was significantly increased. In contrast, in patients with advanced cancer, the use of antibiotics within 30 days before the commencement of ICIs treatment significantly reduced the overall survival of the patients. Recent antibiotic usage significantly altered the gut microbiota composition of renal cell carcinoma patients receiving anti-PD-1 therapy, wherein the enrichment of Clostridium hathewayi was promoted and patients’ objective response rate (ORR) was markedly reduced. This finding agrees with the results of another study which found that in lung and renal cancer pa tients receiving anti-PD-1 therapy, patients administered broad-spectrum antibiotics (BSA) had significantly lower median sur vival period than non-BSA patients. A recent systematic review and meta-analysis of 48 studies also indicated that the use of antibiotics has a detrimental effect on overall survival, progression-free survival, treat ment response rate, and disease progression. These findings sug gest that cancer patients should avoid unnecessary use of antibiotics and postpone the initiation of immunotherapy following antibiotic administration. 6.3. Probiotics and bacteria consortia Probiotics are live microorganisms or their combinations that, when ingested in adequate amounts, confer health benefits to the host. A large prospective cohort study demonstrated that Lactobacillus and Streptococcus thermophilus could prevent CRC through the adenoma-to-carcinoma sequence and serrated pathway , thus indicating the prophylactic effect of probiotics on CRC. Moreover, certain probiotic strains exhibit antibacterial activity against F. nucleatum or modulate the microbiota associated with CRC, thus playing a protective role in the intestine (Fig. 3). In a prospective controlled trial , Peptostreptococcus and Comamonas abundance significantly decreased in CRC patients after the consumption of a mixture of B. longum, Lactobacillus acidophilus, and Enterococcus faecium. Notably, the quantity of F. nucleatum decreased 5-fold, which is believed to contribute to CRC development and progression. Another study revealed that Lactobacillus and Bifidobacterium significantly reduced the levels of TNF-α, IL-6, IL-10, IL-12, IL-17A, IL-17 C, and IL-22 in CRC patients , thus suggesting that probiotics could help modify the intestinal microenvironment by decreasing the production of proin flammatory cytokines (Fig. 3). Utilizing probiotics to enhance immunotherapy responses is another strategy currently warranting attention in cancer treatment research (Fig. 4). Notably, several clinical trials are presently assessing the safety and efficacy of combining probiotics with ICIs treatment (Table 5). One clinical trial (NCT03775850) has demonstrated that the combination of Bifidobacterium animalis lactis (EDP1503) and pembrolizumab is safe and well-tolerated. Biomarker research has revealed that EDP1503 may exert anti-tumor effects by increasing the ratio of CD8+ T cells to Tregs. Besides, one open-label, single-center study (NCT03829111) found that oral administration of CBM588 (a probiotic containing Butyricicoccus) significantly improved the intestinal microbiota stability of patients with metastatic renal cell carcinoma and markedly enhanced their immunotherapy response. The above-mentioned findings provide robust clinical evidence to formulate beneficial bacteria or bacteria with immunostimulatory effects as adjuvants for immuno therapy to improve its outcomes (Fig. 4). Preclinical studies indicates that the use of probiotics shows promising potential for CRC treatment (Table 3). However, due to unpredictable interactions with the host microbiota and colonization challenges within the intricate human gut microbiota , their anticipated efficacy might not be fully achieved in CRC patients. Currently, one can only speculate about the influence of these probiotic strains on CRC patients based on clinical evidence link ing probiotic strains to immunotherapy efficacy in other types of cancer. Thus, in the context of CRC, the probiotic strains correlated with patient clinical benefits remain to be defined. Notably, VB800 (a probiotic cocktail comprising a total of 11 strains) is currently being tested in combination with nivolumab in a phase I/II study involving patients with microsatellite stable CRC (NCT04208958). On the other hand, considering the significant inter-individual variations in mucosal colo nization by probiotics [219,220], we may need to further develop personalized approaches, rather than universal ones, to modify these probiotics. 6.5. Bacteriophages Bacteriophages are viruses that can infect and eliminate bacteria. They exhibit a high degree of specificity when invading bacteria, and they do not destroy bacterial species other than their targets; this avoids the development of dysbiosis caused by broad-spectrum antibiotic. As an antibacterial treatment, bacteriophage therapy can be used to regulate gut microbiota and eradicate multidrug-resistant bacteria (Fig. 3). Clinical studies have demonstrated that bacteriophage cocktails can effectively combat multidrug-resistant bacterial infections [234, 235]. Furthermore, bacteriophages can serve as a novel intervention measure by selectively eliminating potentially carcinogenic bacteria and thus impeding tumor progression. Xian-Zheng Zheng et al. developed a bacteriophage-guided nanomedicine consisting of F. nucleatum-specific bacteriophages isolated from human saliva linked to dextran nanoparticles loaded with the CRC chemotherapeutic drug irinotecan. This nanomedicine specifically targete F. nucleatum, thereby reducing CRC resistance to chemotherapy and significantly prolonging the survival of CRC mice. In another study, the researchers used a similar approach to assemble antibacterial silver nanoparticles on the surface of bacteriophages; these bacteriophages could specifically accumulate at tumor sites colonized by F. nucleatum and further exerted the bacteri cidal effect of silver nanoparticles on F. nucleatum. By inhibiting the proliferation of F. nucleatum, the recruitment of immunosuppressive cells in the colon TME could be effectively suppressed. This study also demonstrated that M13 bacteriophages, as a type of bacterial virus, could effectively induce an immune response in the host through their 6.4. Antibiotics Apart from maintaining a healthy symbiotic environment, the 14 Y.-P. Zhuang et al. Biomedicine & Pharmacotherapy 165 (2023) 115040 self-coat proteins, thereby significantly activating antigen-presenting cells, promoting the maturation of DCs, and reversing the suppressive immune microenvironment by polarizing M2 macrophages. In subse quent in vivo experiments, the authors found that by eliminating F. nucleatum with antibacterial silver nanoparticles, M13 bacteriophages could delay tumor growth; moreover, when combined with first-line chemotherapeutic drugs for colon cancer or ICIs, these bacteriophages could further significantly enhance the therapeutic effects of the drugs and thereby increase the survival rate of mice. These studies have pro vided new research avenues for bacteriophages to play a positive role in antitumor immunotherapy (Fig. 4). Additionally, the application of novel technologies, such as clustered regularly interspaced short palin dromic repeats (CRISPR-Cas9) with specific antimicrobial agents deliv ered by bacteriophages [239,240], could target specific bacteria at the microbiome-cancer interface, which could minimize the disruption of the commensal microbiome , and ensure effective cancer treat ment. Current research suggests that bacteriophage therapy has immense potential in cancer treatment; however, there are still several challenges to overcome. Issues such as bacteriophage dose, delivery method, and the emergence of bacteriophage-resistant strains remain to be investigated [242,243]. susceptible to various factors including age [253,254], sex , body mass index , dietary habits [167,183], medication usage [256, 257], gene [258–260], and geography [261,262]. Notably, these factors do not act independently but rather interact with each other, leading to a complex interplay that shapes the gut microbiota composition. If these factors are not adequately controlled, the outcome could poten tially yield false-positive results. Consequently, during the project planning phase, it is imperative to fully consider and control these fac tors influencing the gut microbiome. Simultaneously, methodological challenges persist. Owing to differing filters and algorithms used by gut microbiota sequencing platforms and reference databases [264–266], resulting the same data analyzed across different platforms may yield disparate results. Simultaneously, the reliance on databases for classifying or annotating sequencing data may result in uncategorizable or annotatable sequences representing unknown bacteria [268,269], sequences signifying unknown bacteria might not attain explicit classi fication or functional annotation, thus becoming unsuitable for subse quent analyses. Such data handling could potentially lead to key bacteria being overlooked or distortions in microbial marker represen tation. With advancements in third-generation sequencing technologies and single-cell microbial sequencing, scientists anticipate genomic analysis of microbial strains at a granular level, such as interpreting unknown microbial genomes through de novo genome assembly. thereby enriching our understanding of gut microbiota diversity. Additionally, we deliberate on the prospective role of gut microbiota in CRC treatment. Preclinical studies have unveiled the potential roles of individual bacteria or bacterial consortia in CRC treatment, particularly as adjuvants for ICIs treatment, thereby augmenting the effectiveness of immunotherapy (Table 3). Notably, these interventional studies are primarily conducted in animal models so that they do not fully encap sulate the complexity of human biology, although they offer valuable insights into the interplay between the microbiome and immunothera peutic drugs. Thus, these findings necessitate further validation through clinical trial data. Beyond bacterial components, other constituents of the microbiome such as gut fungi, viruses, and bacteriophages, also in fluence cancer progression and treatment but have yet to be thoroughly investigated. Clinical studies have also revealed a correlation between gut microbiota composition and responsiveness to immunotherapy (Table 4), specifically indicating that gut microbial compositions may aid in predicting the response of cancer patients to immunotherapy. However, these findings have not been consistent. Such incongruity in results may stem from the considerable influence factors, such as pa tient’s dietary patterns. Moreover, clinical heterogeneity and geographical factors among patients may also be tied to variations in microbiome research findings [149,150]. Hence, the ability to predict immunotherapeutic responses based on single-microbe signals is limited. Investigating whether a comprehensive and stable biomarker evaluation system can be constructed by integrating large-scale multi- sequence datasets, such as microbiomes, metabolomes, transcriptomes, and single-cell RNA sequencing , in conjunction with multiple biomarkers to enhance the capability of evaluating and predicting the prognosis of cancer patients remains a significant direction in current research. Manipulating the gut microbiota (such as through FMT, probiotic, and antibiotics) may represent a promising strategy to enhance the ef ficacy of immunotherapy in cancer patients (Fig. 4; Table 5), but the optimal approach remains incompletely elucidated. Studies conducted by Diwakar Davar and Erez N Baruch respectively have confirmed the efficacy of combining FMT with ICIs treatment in mela noma that is resistant to immunotherapy. These findings are exhila rating, yet the relevant clinical evidence remains considerably limited, consisting primarily of small-sample studies. Further high-quality clin ical trials are necessary to assess the clinical benefits and safety of combining FMT with immunotherapy for malignant tumors including CRC. Additionally, the clinical application of FMT encounters numerous 7. Discussion As our comprehension of ICIs continues to evolve, therapeutic stra tegies for cancer are undergoing a significant transformation. ICIs, encompassing monoclonal antibodies against PD-1, PD-L1, and CTLA-4, have become instrumental in the treatment of a multitude of cancer types. These pharmaceuticals reactivate the immune system’s capacity to counteract tumor cells by targeting the PD1/PD-L1 and CTLA-4 pathways. ICIs have demonstrated efficacy in improving the prognosis of various tumor types, including NSCLC, RCC, and mel anoma. Consequently, understanding and leveraging these inno vative therapeutic strategies will offer new avenues to enhance treatment outcomes and the quality of life for individuals diagnosed with cancer. However, the application of ICIs still poses numerous challenges. Some cancers, such as CRC displaying suboptimal responses to ICIs , along with drug tolerance issues and side effects. Therefore, it is imperative to explore novel therapeutic approaches aimed at enhancing the clinical efficacy of immunotherapy. Given the intricate interplay between gut microbiota and the host’s immune response, researchers had found that enriching understanding of gut microbe-anti-immunity interactions may provide invaluable assistance in devising innovative strategies to enhance the efficacy of CRC immunotherapy [248–250]. However, a myriad of intrinsic mech anisms warrants further exploration. Comprehensive identification of immune-stimulating and immune-suppressive related bacterial strains may assist in the discovery of more targeted intervention points, potentially ameliorating the clinical efficacy of immunotherapy in CRC patients. For instance, there is a hopeful expectation to discover mi crobial strains that can elicit sensitivity of CRC towards ICIs. Thus, we further synthesized existing knowledge regarding the impact of poten tial pathogenic and protective microbes on tumor immunity in the context of CRC (Fig. 1; Table 2), and emphasized potential mechanisms through which gut microbiota modulate anti-tumor immune activity (Fig. 2). Future studies unraveling whether these gut microbes act independently or synergistically with other microbes to influence anti-tumor immunity may provide novel therapeutic targets to augment the efficacy of immunotherapeutic strategies in CRC treatment. On another note, numerous clinical studies have analyzed and summarized the correlation between gut microbiota and CRC [250, 251]. However, the identified signature microbiota of CRC was not entirely consistent across disparate research cohorts (Table 1). The au thors suggest that this inconsistency may be attributed to the interfer ence of confounding factors. Primarily, the individual gut microbiomes display a significant degree of personal heterogeneity , and is 15 Y.-P. Zhuang et al. Biomedicine & Pharmacotherapy 165 (2023) 115040 challenges. Primely, FMT also harbors substantial drawbacks, encom passing risks of infection [200–202, 205] and a relative dearth of long-term safety data. Hence, stringent donor screening and testing are imperative to ensure the safe utilization of FMT in the context of cancer. Furthermore, for cancer patients, the definition of the ideal donor ne cessitates further investigation. Due to significant variations in the composition of the gut microbiota among different populations , therapeutic interventions based on the gut microbiota may also yield individual-specific outcomes. Clinical studies also indicated that the predominant microbial composition observed when administering different drugs for immunotherapy varies among different tumor pa tients (Table 4), suggesting that the ideal donor may require a person alized approach. Simultaneously, the gut microbiota is susceptible to various factors, such as dietary habits [167,183], age [253,254], gene [258–260], and geography [261,262]. Understanding this diversity may contribute to the design of more effective personalized treatments. For example, intestinal microbiota exhibits rapid responses to dietary modifications. Research indicated that following alterations in dietary structure, the composition of intestinal microbiota undergoes swift changes within a span of one to two days. Meanwhile, given the individualistic nature of gut microbiota, specific dietary modifications may yield highly variable impacts across different individuals. Based on these factors, a more intricate classification and metabolic analysis of the donor and recipient microbiota may facilitate personal ized matching between donors and recipients , thereby further optimizing treatment outcomes. Clinical studies have also demonstrated that probiotics or bacteria consortia with immune-stimulating proper ties can serve as adjuvants for CRC immunotherapy, enhancing its therapeutic efficacy [217,218]. However, in the context of CRC, further determination of the beneficial microbes associated with clinical bene fits for patients is still warranted. Although antibiotics can be utilized to eliminate potential harmful bacteria, it is important to note that many antibiotics lack specificity and may exacerbate disturbances in the gut ecosystem [227,275]. Lastly, selectively removing potential carcino genic microbes through bacteriophages may represent an ideal approach for cancer prevention or treatment; however, current clinical evidence supporting this approach is lacking. Despite the immense poten tial of gut microbiota interventions in cancer immunotherapy treatment (Fig. 4), further clinical and preclinical research is still required to determine the optimal strategies for microbial intervention. In general, the interplay between gut microbiota and anti-tumor immunity in CRC has been extensively explored, but the underlying mechanisms remain enigmatic and require validation through compre hensive preclinical models and trials. Given the role of gut microbiota in modulating anti-tumor immune responses, therapeutic approaches tar geting the gut microbiota may holds promise for CRC patients. However, the optimal strategy is yet to be fully elucidated. If suitable methodol ogies can be found to precisely manipulate the gut microbiota, it could offer vast possibilities and prospects for the prevention or deceleration of CRC onset, and the enhancement of the efficacy of immunotherapies. Funding This work was supported by the National Natural Science Foundation of China under Grant number 81973737, 82001883, and 81804058. CRediT authorship contribution statement Yu-Pei Zhuang: Investigation, Writing – original draft, Writing – review & editing. Hong-Li Zhou: Investigation, Writing – original draft. Hai-Bin Chen: Investigation, Writing – original draft. Ming-Yue Zheng, Yu-Wei Liang and Yu-Tian Gu: Investigation. Wen-Ting Li, Wen-Li Qiu and Hong-Guang Zhou: Methodology, Writing – review & edit ing, Supervision, Conceptualization. All authors approved the submitted version. Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Data Availability No data was used for the research described in the article. Acknowledgments We thank BioRender (www.BioRender.com) and Figdraw (www. figdraw.com) for their help in creating the figures. References H. Sung, J. Ferlay, R.L. Siegel, M. Laversanne, I. Soerjomataram, A. Jemal, F. Bray, Global cancer statistics 2020: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries, CA: Cancer J. Clin. 71 (3) (2021) 209–249, https://doi.org/10.3322/caac.21660. R.L. Siegel, N.S. Wagle, A. Cercek, R.A. Smith, A. Jemal, Colorectal cancer statistics, 2023, CA: Cancer J. Clin. (2023), https://doi.org/10.3322/caac.21772. E. Morgan, M. Arnold, A. Gini, V. Lorenzoni, C.J. Cabasag, M. Laversanne, J. Vignat, J. Ferlay, N. Murphy, F. 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