There is evidence that specific dietary components can help increase the efficacy or reduce the toxicity of cancer therapeutics.

Review article

Gut microbiota contributes towards immunomodulation against cancer: New frontiers in precision cancer therapeutics

Author links open overlay panelKentaroInamura

The gut microbiota contributes to anti-cancer immunomodulation: a new frontier in precision cancer therapy

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The microbiota influences human health and the development of several diseases, including cancer. Microorganisms can influence the occurrence and progression of tumors in a positive or negative way. In addition, the composition of the gut microbiota influences the efficacy and toxicity of cancer treatments as well as treatment resistance. The significant impact of the microbiota on tumorigenesis and cancer treatment provides compelling evidence to support the idea that manipulating microbial networks is a promising strategy for treating and preventing cancer. Specific microorganisms or microbial ecosystems can be modified by a variety of processes, and therapeutics and approaches are always evolving. Microbial manipulation can be used as an adjunct to traditional cancer therapies such as chemotherapy and immunotherapy. In addition, this approach has shown great promise as a stand-alone therapy after the failure of standard therapies. In addition, patients benefit from such strategies by avoiding side effects that can lead to treatment discontinuation. microbiome, which will help expand the frontiers of precision cancer treatment to improve patient care. This review discusses the role of the microbiota in tumorigenesis and cancer treatment, with a focus on efforts to harness the microbiota to fight cancer.

There is growing evidence of the impact of the microbiota on human health and disease. The microbiota has been widely thought to influence a variety of physiological processes, such as metabolism, neurotransmission, circulation, and immunity. Specifically, the microbiota shapes the host's immune system by regulating local and systemic immune responses. Microorganisms exert a role in promoting or inhibiting tumors and affect the efficacy and toxicity of cancer treatment. For example, beneficial gut microbes promote the antitumor activity of immunotherapy drugs through immunomodulation, including immune checkpoint inhibitors (ICIs). Given the close relationship between the microbiota and cancer, microbial manipulation is being heavily utilized as a promising strategy for the treatment and prevention of cancer. The manipulation of the microbiota can be used as an adjunct to traditional cancer therapies (e.g., chemotherapy, immunotherapy). In addition, this approach can be used as a stand-alone therapy after failure of standard therapy. In addition, such a strategy can benefit patients by avoiding side effects that can lead to discontinuation of treatment.

However, the use of the microbiota in cancer treatment is still in its infancy. Specific microorganisms or microbial ecosystems can be modified through a variety of processes, and treatments and technologies are constantly evolving. These include standard methods (e.g., dietary modification, prebiotics, probiotics, antibiotics, fecal microbiota transplantation [FMT]) and new techniques (e.g., bacteriophages, nanotechnology, bacterial mixtures to increase CD8+ T cell counts). Understanding the role of the host-microbial ecosystem in cancer and the development of microbial manipulation methods will help expand the frontiers of precision cancer therapy to improve patient care. This review aims to provide a concise discussion of the existing knowledge about the role of the microbiota in tumorigenesis and cancer treatment, with a focus on efforts to harness the microbiota in the fight against cancer.

2 . Immunomodulation through host-microbiota interactions

The microbiota, especially the gut microbiota, shapes the host's immune system by regulating local and systemic immune responses. The intestinal mucosa is the internal compartment of the intestinal wall that includes several types of intestinal epithelial cells (IECs), including goblet cells and Paneth cells, as well as the underlying lamina propria (Figure 1). Goblet cells produce a mucus layer that hinders microbial binding to IEC through steric hindrance and acts as a releasable decoy for microbial adhesion. Instead, microorganisms are a prerequisite for mucus production, as illustrated by the absence of a mucus layer in germ-free (GF) mice [35]. In addition, Paneth cells block microbial invasion by releasing antimicrobial peptides that maintain the sterility of the internal mucus layer. The lamina propria beneath the IEC contains a variety of immune cells, including antigen-presenting cells (e.g., dendritic cells [DCs]), T cells, and B cells.

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Figure 1. The structure of the gut, cells, and microorganisms. The intestinal mucosa contains a single layer of intestinal epithelial cells (IECs), including goblet cells and Paneth cells, and the underlying lamina propria. Goblet cells produce a mucus layer that prevents the organism from binding to the IEC. Paneth cells stop microbial invasion by releasing antimicrobial peptides. The lamina propria beneath the IEC contains several types of immune cells, including antigen-presenting cells (e.g., dendritic cells), T cells, and B cells.

Microorganisms trigger local immune responses by interacting with immune cells expressing pattern recognition receptors (PRRs) (e.g., Toll-like receptors [TLRs]). Microorganisms or microbial-derived elements (e.g., components, products, metabolites) activate local DCs through interaction with PRR. These encounters result in the transfer of activated DCs from the gastrointestinal tract to mesenteric lymph nodes (mLNs), where they present microbial-derived antigens that subsequently induce naïve T cell differentiation into effector T cells, specifically regulatory T cells (Tregs) and T helper 17 (Th17) cells [40]. A subset of these effector T cells migrate back into the gastrointestinal tract and affect the local immune response. The remaining population enters the systemic circulation and affects systemic immunity. Tregs mediate the transition of the immune system from a pro-inflammatory state to an anti-inflammatory state by releasing anti-inflammatory cytokines (e.g., IL-10, TGF-β) or by participating in DCs [41]. In contrast, Th17 cells mediate the transition of the immune system to a pro-inflammatory state by secreting immunostimulatory cytokines (e.g., IL-17) or by activating and recruiting neutrophils [36]. The lamina propria of GF mice lacks these pro-inflammatory Th17 cells; However, their generation is restored by a specific subgroup of bacteria called segmented filamentous bacteria. This intriguing relationship strongly suggests an important role for microorganisms in Th17 cell activation [42].

3 . The influence of microorganisms on the occurrence and progression of cancer

Microorganisms promote or inhibit tumors in a tissue-specific manner. Some microorganisms directly promote the occurrence and progression of tumors by producing toxic or tumorigenic products, and indirectly promote the occurrence and progression of tumors by producing pro-inflammatory or anti-inflammatory microenvironments. Conversely, other microbes exert tumor suppressive effects in the gastrointestinal tract or other organs by enhancing the host's anti-tumor immune response. Specific microbial-derived products (e.g., short-chain fatty acids [SCFAs]) create a tumor-inhibiting microenvironment [46]. For example, microbial-derived SCFA butyric acid molecules are released in the gut by microbial fermentation fibers, inhibiting tumor growth by inactivating histone deacetylase in tumor cells and transforming the immune system into an anti-inflammatory state. This section presents examples of the effects of microorganisms on tumorigenesis and progression, with a focus on Helicobacter pylori and Fusobacterium nucleatus, both of which are closely related to tumorigenesis.

3.1 . Helicobacter pylori

According to an analysis of the GLOBOCAN 2018 Cancer Incidence and Mortality Database, approximately 2.2 million cases of cancer worldwide can be attributed to infectious agents, of which 810,000 cases are caused by Helicobacter pylori. H. pylori promotes the entry of cytotoxin-associated gene A into gastric epithelial cells through type IV secretion-mediated pathogens. This microorganism can induce stomach cancer; On the contrary, its elimination from the host effectively reduces the risk of gastric cancer. Chronic infection with Helicobacter pylori causes multi-step carcinogenesis, i.e., progression from chronic gastritis, tissue atrophy, intestinal metaplasia, and benign tumors to cancer.

There is evidence that Helicobacter pylori is the only bacterium that causes stomach cancer. However, high-throughput techniques have recently identified other bacterial species as potential drivers of gastric cancer (eg, oral digestive streptococcus, streptococcus angina, micromonas, Slackia exigua, Dialister pneumosintes) [59]. The carcinogenic potential of these newly discovered microorganisms remains to be evaluated [60].

3.2 . F. nucleatum

F. nucleatum contributes to the development and progression of colorectal cancer (CRC). F. nucleatum is significantly more abundant in human CRC tissues than in paired normal tissues. It creates a tumor-promoting immunosuppressive environment by interacting with the host's immune cells. Specifically, this microorganism stimulates anti-inflammatory bone marrow cells, damages NK and T cells by activating TIGIT and CEACAM1 inhibitory receptors, and induces the WNT/β-catenin (CTNNB1) modulator annexin A1 (ANXA1) [70]. F. nucleatum-positive CRC is characterized by right-sided anatomical position, BRAF mutations, and a high state of microsatellite instability (MSI). Evidence of these features in serrated tumors suggests that Clostridium sclerotioides promote tumorigenesis and progression through a serrated pathway. F. nucleatum-positive CRC is classified as a common molecular subtype 1 [76], which is typically characterized by MSI-high status and activated immune-related pathways [77]. In humans and mice, this organism is present in primary and metastatic lesions with Fusobacterium nucleatus-positive CRC, suggesting its persistence during metastasis [78]. Population-based studies have shown that cautionary or inflammatory dietary patterns reduce or increase the risk of F. nucleatum-positive CRC, respectively, as a key factor in preventing F. nucleatum. Nucleatum-positive CRC provides a potential strategy.

4 . Effect of microbiota on chemotherapy efficacy and toxicity

The action of chemotherapy drugs is closely related to the immune response of the host. The composition of the microbiome influences this response and inevitably affects the efficacy and toxicity of chemotherapy as well as chemoresistance. Chemotherapy requires certain bacteria to exert their antitumor effects, while others induce chemoresistance . Similarly, specific bacteria can cause or inhibit adverse events caused by chemotherapy.

The microbiota regulates chemotherapy efficacy and toxicity through multiple mechanisms collectively known as the TIMER mechanistic framework (translocation, immunomodulation, metabolism, enzymatic degradation, and reduction of diversity) [83]. When chemotherapy drugs disrupt the intestinal barrier, bacteria can move from the intestine to secondary lymphoid organs (e.g., mLNs, spleen). In turn, gastrointestinal microbes influence the efficacy and toxicity of chemotherapy through immunomodulation. For example, cyclophosphamide (CTX) increases intestinal permeability and induces translocation of certain gut microbes to mLN and spleen, thereby stimulating antitumor activity by modulating local and systemic immunity to inhibit CTX [84]. The pharmacological fate, as well as the beneficial and side effects of chemotherapy drugs, may be directly determined by the gastrointestinal microbiota. In fact, a high-throughput study systematically identified a range of drug-metabolizing microbial enzymes [85]. These microbiome-specific enzymes directly affect intestinal and systemic drug metabolism and explain the drug-metabolizing role of gut microbes [85]. As an example of the effects of microorganisms through enzymatic degradation, Mycoplasma hyorhinis deaminations gemcitabine to reduce its beneficial effects [86]. In addition, certain chemotherapy drugs can reduce microbial diversity , which can cause adverse effects. This section focuses on the impact of the microbiota on the efficacy and toxicity of chemotherapy.

4.1 . Oxaliplatin

Oxaliplatin is a platinum-based chemotherapy drug whose anticancer effects are enhanced by the intestinal microbiota [87, 88]. Specifically, gut microbes stimulate bone marrow cells to produce reactive oxygen species (ROS), through which oxaliplatin exerts its anticancer activity. Thus, bacterial ablation abolishes the cytotoxicity of oxaliplatin in mice by reducing the production of microbiota-dependent ROS, suggesting that oxaliplatin is unable to kill cancer cells without the involvement of gut microbes [87]. In contrast, the beneficial effects of oxaliplatin and 5-fluorouracil (5-FU) are attenuated by Clostridium sclerotioculate. By coordinating TLR activation, microRNA expression, and autophagy networks, Clostridium nucleatum promotes resistance to these drugs [88].

The gut microbiota has been implicated in oxaliplatin toxicity (eg, mechanical hyperalgesia) [89]. Oxaliplatin side effects were reduced in GF and antibiotic-treated mice; Conversely, the restoration of the microbiota eliminates this protection [89]. Immunomodulation of the gut microbiota through the TLR4-lipopolysaccharide (LPS) pathway causes oxaliplatin toxicity [89].

4.2 . Cisplatin

Cisplatin disrupts the integrity of the epithelial barrier of the gastrointestinal tract. This, in turn, promotes the transfer of microorganisms into the bloodstream and leads to systemic inflammation [87]. Ruminococcus gnavus, a bacterium selectively depleted by cisplatin, partially alleviates this systemic inflammation [90]. In contrast, reconstitution of the entire gut microbiota by fecal gavage can cure cisplatin-induced intestinal injury and completely eliminate systemic inflammation [90]. Therefore, FMT represents a potential strategy for the prevention of cisplatin-induced adverse events [90]. In addition, D-methionine prevents cisplatin-induced intestinal damage by modulating the gut microbiota [91]. In rats, D-methionine attenuates cisplatin-induced intestinal mucositis by increasing the abundance of beneficial microorganisms, such as lactobacilli, and attenuating oxidative stress and pro-inflammatory immune responses [91].

4.3 . CTX

The efficacy of the antitumor immunomodulator CTX is influenced by the gut microbiota [83, 92]. CTX disrupts the mucosal barrier of the small intestine and promotes the transfer of a group of gram-positive microorganisms (e.g., Enterococcus hela, Lactobacillus josli, Lactobacillus m. munitii) from the gut to mLN or spleen, where they promote a Th17-mediated pro-inflammatory immune response [84]. In tumor-bearing mice, bacterial ablation reduces this pro-inflammatory immune response by eliminating gram-positive bacteria [84]. Mice that are depleted of these bacteria then develop CTX resistance. In contrast, Th17 cell metastasis restored the efficacy of CTX [84], suggesting that the gut microbiota enables CTX to exert its antitumor activity by enhancing Th17-mediated pro-inflammatory immunity.

E. hirae and Barnesiella intestinihominis synergistically enhanced the anticancer effects of CTX [93]. E. hirae metastasized from the small intestine to secondary lymphoid organs and increased intratumoral CD8/Treg ratio; Conversely, B. intestinihominis accumulates in the colon and increases the number of INF-γ-producing γδ T cells in the tumor bed [93]. These microbes synergistically enhance the efficacy of CTX by transforming the immune system into a pro-inflammatory state, albeit through different mechanisms].

CTX-induced toxicity is susceptible to dietary influences. For example, polysaccharides from squid ink altered the composition of the gut microbiota after CTX treatment in mice [94]. Specifically, these molecules prevent CTX-induced toxicity by preventing this drug-induced gut microbial overgrowth [94]. Similarly, polysaccharides from Camellia sinensis L. ameliorate CTX-induced intestinal damage [95].

4.4 . Gemcitabine

Gammaproteobacteria aboligate the anticancer activity of the nucleoside analogue 2',2'-difluoro-2'-deoxycytidine (gemcitabine) by producing a long isoform of the bacterial enzyme cytidine deaminase [86]. In mice with colon cancer, intratumoral gammaproteobacterial species induce gemcitabine resistance by producing this enzyme, while concomitant treatment with the antibiotic ciprofloxacin can restore gemcitabine efficacy, suggesting that gammaproteobacteria are involved in gemcitabine resistance [86]. These microorganisms are rich in pancreatic cancer. Therefore, combination therapy with gemcitabine and antibiotics may be more effective for pancreatic cancer than gemcitabine alone.

4.5 . 5-FU

Manipulating the microbiome increases the efficacy of 5-FU while reducing its toxicity. For example, ginseng metabolites produced by the microbiota enhance the anticancer activity of 5-FU [[96], [97], [98], [99]. Similarly, urolithin A is a microbial-induced ellagic acid metabolite that increases the efficacy of 5-FU [100]. In an in vitro model of CRC, the Lactobacillus plantarum supernatant increases the cytotoxicity of 5-FU by inducing apoptosis in cancer cells and reducing the number of stem cell-like cancer cells [101]. The effect of the probiotic Lactobacillus fermentum and prebiotics on the effect of fructooligosaccharides on 5-FU-induced intestinal mucositis was investigated using a rat model [102]: Although probiotics improved 5-FU-induced toxicity, co-treatment with prebiotics was no better than probiotics alone [102]. In addition, in mice transplanted with syngeneic CT26 colorectal adenocarcinoma cells, FMT reduced 5-FU-induced intestinal injury by attenuating intestinal mucositis and enhancing the intestinal barrier [103].

4.6 . Yasushi Itachi

Microbial enzymes cause side effects of irinotecan (CPT-11). The prodrug is converted to its active form (SN-38) by carboxylesterase and inactivated by hepatic UDP-glucuronosyltransferase (SN-38G). SN-38 G is secreted through the bile ducts into the intestine, where it is reactivated by the bacterial β-glucuronidase. SN-38 can damage the intestinal lining and cause diarrhea and other adverse events [104, 105]. Irinotecan-induced toxicity can be mitigated by bacterial ablation or administration of bacterial β-glucuronidase inhibitors [104]. Given that irinotecan-induced toxicity is caused by bacterial enzymes and that the gut microbiota regulates the integrity of the gut barrier, manipulation of gut bacteria or their components may improve the adverse effects caused by irinotecan. In fact, the utility of probiotics in reducing irinotecan-induced toxicity was demonstrated in a mouse model study, in which administration of Escherichia coli Nissle 1917 (EcN) reduced the side effects of irinotecan treatment by strengthening the intestinal barrier [107].

5 . Effect of microbiota on efficacy and toxicity of immunotherapy

The ability of cancer cells to survive in the host is achieved through the creation of an immunosuppressive microenvironment. The beneficial effects of enhancing host immunity against cancer provide important information to facilitate the development of effective immunotherapy strategies. This approach dramatically changes the therapeutic landscape of malignancy, especially when it utilizes immune checkpoint blockade [110]. Unfortunately, we lack biomarkers that can accurately predict clinical response to immunotherapy, and our understanding of the mechanisms that determine its efficacy and toxicity is inadequate, and it is clear that it must be expanded. Emerging evidence suggests that the microbiota influences response to immunotherapy by modulating the host's local and systemic immune responses. Therefore, the utilization of microorganisms, their components, or both may help identify biomarkers and therapeutic targets that may aid in the development of cancer immunotherapy. These efforts are briefly described in this section.

5.1 . Adoptive cell transfer (ACT) therapy

The anticancer effect of ACT therapy after total body irradiation (TBI) is determined by the gut microbiota, as shown by the loss of treatment effect when the bacterial group is ablated [24, 116, 117]. TBI promotes the translocation of gut microbes to secondary lymphoid organs, increases circulating levels of microbial-derived LPS, and stimulates the expression of pro-inflammatory cytokines by DCs [116]. Conversely, bacterial ablation inhibits microbial translocation, reduces the number of activated DCs, and reduces anti-tumor immunity, thereby eliminating the efficacy of ACT therapy [118]. The gut microbiota influences the anticancer activity of ACT through the binding of LPS to TLR4. Thus, TLR4-deficient tumor-bearing mice did not respond to ACT treatment, whereas removal of LPS reduced the beneficial effect of TBI on tumor reduction [118]. In addition, the gut microbiota affects the efficacy of ACT treatment by increasing systemic CD8α+ DC count and IL-12 levels.

5.2 . CpG 寡脱氧核苷酸 (ODN) 和抗 IL-10 治疗

When the immunosuppressive activity of IL-10 is inhibited, CpG ODN, which mimics bacterial DNA, exerts its anticancer effects. CpG ODN induces the release of immunostimulatory cytokines (e.g., TNF, IL-12) from bone marrow cells. These cytokines then induce a transition to a pro-inflammatory state in tumor-associated macrophages (TAMs) and DCs, thereby inhibiting tumor growth.

The beneficial effects of CpG ODNs and anti-IL-10 therapy require microbial-driven pro-inflammatory immunity. As a result, the efficacy of this treatment was canceled in GF and antibiotic-treated mice, with reduced levels of TNF and IL-12. However, in contrast, Alistipes shaii enhances the therapeutic effect of CpG ODN by activating bone marrow cells to produce immunostimulatory cytokines (e.g., TNF) through the TLR4 signaling pathway. In addition, with A. SHAVII gavage restores the therapeutic effect of CpG ODNs in microbiota-depleted mice. In addition, administration of TLR4 agonists restored TNF expression in tumors of microbiota-depleted mice, but not TNF expression in TLR4-deficient mice.

5.3 . IC

The gut microbiota has a significant impact on the efficacy of ICI treatment in human and mouse models. In general, those enriched with gut microbiota in patients who respond to ICI treatment are considered "favorable", while those enriched with gut microbiota in patients who are not responding are considered "unfavorable". FMT or oral administration of beneficial microorganisms often enhances the efficacy of ICIs in mouse models. In addition, high microbial diversity in the gastrointestinal tract is associated with increased efficacy in ICI treatment.

The effect of antibiotics on the antitumor activity of ICIs varies depending on the tumor type and treatment strategy. For example, the efficacy of cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) blockade was significantly reduced in antibiotic-treated mice with melanoma, colon cancer, or sarcoma [125]. Bacterial ablation reduced the effectiveness of programmed cell death 1 (PD-1)/programmed cell death 1 ligand 1 (PD-L1) blockade on non-small cell lung cancer (NSCLC), renal cell carcinoma (RCC), and urothelial carcinoma [123]. Consistent with these findings, patients with NSCLC or RCC who received antibiotics within one month of initiation of ICI therapy had a shorter survival time than those who did not take antibiotics [127]. On the contrary, bacterial ablation is beneficial for pancreatic cancer patients, in which the abundance of intratumoral microorganisms creates an immunosuppressive tumor microenvironment. PD-1 blockers and antibiotics work synergistically to produce anti-tumor effects in patients with pancreatic cancer.

The gut microbiota modulates local and systemic anti-tumor immunity through complex interactions between the microbiota and the host, thereby influencing the efficacy of ICIs. Specifically, administration of beneficial microorganisms to mice can enhance the efficacy of CTLA-4 blockade by inducing a Th1-dependent immune response in tumor draining lymph nodes and transforming DCs into a pro-inflammatory state [125]. Similarly, favorable microorganisms enhance the efficacy of PD-1/PD-L1 blockade by activating DCs and tumor-specific CD8+ T cells [129]. Patients who are beneficial to CTLA-4 blockade of responsive microorganisms have fewer circulating Tregs than non-responders [130].

In 2018, three groups reported on the effect of gut microbiota on the efficacy of ICIs in humans and mice. Specifically, favorable microorganisms increase the number of CD8+ T cells in the tumor bed and decrease the number of FOXP3+ Tregs [131]. In addition, favorable microbiota induces the secretion of immunostimulatory IL-12 by DCs, which in turn recruits CCR9+CXCR3+CD4+ T cells into the tumor bed [123]. In addition, patients who are beneficial to PD-1 blockade of reactive microorganisms are rich in circulating Th17 cells, while unresponsive patients who are unfavorable to microbes are characterized by large amounts of activated Treg and myeloid-derived suppressor cells (MDSCs) [124]. Experimental data suggest that favorable microorganisms activate antigen processing and presentation, and enhance the function of Th17 cells present in circulating and tumor beds [124]. In contrast, anti-tumor immunity in nonresponders is abrogated by impaired Th17 cell function, activation of Tregs and MSDCs, limited infiltration of pro-inflammatory myeloid cells into the tumor bed, and reduced antigen processing and presentation [27, 124].

The gut microbiota influences the toxicity of ICIs, which often leads to treatment discontinuation. Certain microorganisms increase ICI-induced toxicity, while others reduce the risk of immune-mediated adverse events. However, the identity of specific microorganisms that have protection or adverse effects on ICI-induced toxicity is unknown. The abundance of Bacteroides species is associated with a reduced risk of CTLA-4-induced colitis in melanoma patients. The protective effects of Bacteroides species are explained by their immunosuppressive effects, as these microorganisms enhance anti-inflammatory immunity by promoting Treg differentiation. A recent study on the use of FMT for the treatment of ICI-induced adverse events demonstrates another example of the important role of Treg in the treatment of ICI. Specifically, FMT cures refractory ICI-induced colitis by promoting the reconstitution of the gut microbiota and increasing the number of anti-inflammatory Tregs in the colonic mucosa.

Current immunotherapies, especially ICI therapy, exert anti-tumor effects mainly by activating T cell-mediated anti-tumor immunity. As a result, many studies have examined the effects of T cells on cancer treatment. In contrast, B cells have not been extensively studied to date. In 2020, three independent research groups have demonstrated that B cells and tertiary lymphoid structures (TLS) are key determinants of ICI efficacy. In cancer patients, the invasion of B cells and the presence of TLS in tumor tissues are positively correlated with increased response to ICIs. In addition, patients who respond to ICIs generally have more abundant B-cell and TLS features compared to non-responders. Given the significant impact of B cells and TLS on ICI responses, therapies that enhance B cell responses and activate TLS are promising as complements to T cell-mediated immunotherapy. As mentioned above, the microbiota has a huge impact on the host's immunity; Therefore, manipulation of microorganisms and/or their components that affect B cells and TLS is a potential strategy to enhance the efficacy of immunotherapy.

6 . Microbial intervention as cancer treatment

The significant impact of microbiota on tumorigenesis and cancer treatment provides compelling evidence that the manipulation of microbial networks represents a promising cancer treatment strategy. Specific microorganisms or microbial ecosystems can be modified through many processes, and therapeutic approaches and pathways are rapidly evolving (Figure 2). This section discusses emerging and traditional strategies for using the microbiota to treat and prevent cancer.

1. Figure 2. Microbial intervention as cancer treatment. A high-fiber diet increases the number of beneficial microorganisms that produce short-chain fatty acids (SCFAs). Prebiotics, such as oligosaccharides, are inactive substances that promote the growth or activity of specific bacteria. Probiotics (e.g., lactobacillus , bifidobacteria ) include individuals or combinations of bacteria. Synbiotics are a mixture of prebiotics and probiotics. Postbiotics, such as SCFAs, are microbial-derived soluble products and metabolites. Antibiotics can eliminate unfavorable microorganisms, but due to insufficient specificity, they may cause dysbiosis by reducing the diversity of commensal microorganisms. Bacteriophages can specifically destroy the target bacteria. Fecal microbiota transplantation (FMT) transfers the entire gut microbial ecosystem to the recipient. Attenuated Salmonella (oncolytic bacteria) can spread to tumor tissue, survive in an oxygen-deficient environment, and release specific molecules in the field. Nanotechnology is being applied to treat and prevent cancer through microbial manipulation. A mixture of bacteria containing a specific mixture of bacteria can increase the number of specific anti-tumor immune cells (e.g., CD8+ INF-γ+ T cells).

6.1 . diet

Gut microbes metabolize dietary products that are not metabolized by the host [146]. Conversely, intense dietary modifications can rapidly alter the composition of the microbial community. For example, reducing the intake of animal fats reduces the abundance of harmful bacteroides, while a high-fiber diet increases the number of SCFA-producing microorganisms. As a result, there is growing interest in the use of diet for its effects on the microbiota and its role in pathogenesis.

Fiber plays an important role in strengthening the gastrointestinal barrier function. A low-fiber diet weakens the ability to fight off pathogens. When dietary fiber is deficient, gut microbes use mucus glycoproteins as a source of nutrients, leading to erosion of the gut mucus barrier [106]. The collapse of the mucus layer disrupts its barrier function, allowing mucosal pathogens (eg, Citrobacterium) to induce the development of fatal colitis [106]. The Western-style diet is characterized by being high in fat, low in calcium, and low in vitamin D, and by regulating the gut microbiota to reduce the integrity and growth rate of the mucus layer. However, the consumption of fiber or the application of specific microorganisms can prevent these adverse effects. Specifically, inulin consumption prevents mucus penetration, while Bifidobacterium longum restores mucus production [154]. Cross-sectional studies have reported that high-fiber diets increase SCFA levels as well as the abundance of SCFA-producing microorganisms (e.g., Eubacterium rectal, Roche spp., Faecobacterium prazi) [156, 157]. In addition, randomized controlled trials have found that fiber supplementation or related prebiotics can increase the number of beneficial Lactobacillus species. and Bifidobacterium spp. [ 75、158 ] 。 _ These bacteria produce lactic acid and acetic acid and increase butyrate levels by interacting with butyrate-producing microorganisms (e.g., Escherichia coli) [75].

The beneficial effects of dietary fiber on CRC risk are related to gut microbiota [152, 159]. Microbial production of SCFAs reduces the risk of CRC by regulating the immune system and metabolism [159]. Patients with CRC had lower numbers of Lactobacillus spp., Bifidobacterium spp., and other SFCA-producing microorganisms compared to the control group. In addition, patients with CRC had lower numbers of butyrate-producing microorganisms and fecal butyrate concentrations compared to controls, which supported the inhibitory effect of butyrate on CRC [75, 162].

High intake of whole grains is associated with increased numbers of SCFA-producing microorganisms (e.g., Roseburia, Lachnospira), decreased numbers of pro-inflammatory microorganisms (e.g., Enterobacteriaceae), and high concentrations of fecal SCFAs. The polyphenols and flavonoids in whole grains are key regulators of the gut microbiota. Whole-grain wheat in the diet increases the levels of ferulic acid (the most abundant phenolic compound in whole grains) in the feces and the concentration of dihydroferulic acid (a metabolite produced by lactobacilli and bifidobacteria from ferulic acid). Peripheral circulation. A variety of bacteria produce SCFA and dihydroferulic acid, suggesting a synergistic mechanism that promotes the synthesis of beneficial metabolites. These mechanisms may explain the superior effect of whole grains on CRC risk relative to other sources of dietary fiber. For example, a population-based study showed that a cautious diet that included whole grains reduced F. Nucleatum positivity, but does not reduce the risk of F. Nucleatum-negative CRC.

As a clinical trial comparing the effects of whole grain and refined grain diets on gut microbiota and insulin sensitivity has shown, different types of whole grains may have different effects on individual microbial species]. Surprisingly, the whole grain diet did not significantly alter gut microbiota or insulin sensitivity compared to the refined grain diet, although the whole grain diet was associated with weight loss and systemic inflammatory markers (e.g., CRP, IL-6) [164]. The limited effect of a whole grain diet on the gut microbiota may be explained by the composition of different grains (e.g., wheat, rye, oats), which may have different effects on individual microbial species. Therefore, future studies need to investigate the effects of specific grains on the gut microbiota.

There is evidence that specific dietary components can help increase the efficacy or reduce the toxicity of cancer therapeutics. For example, a preclinical study showed that the popular herb ginseng indirectly enhanced the efficacy of 5-FU through its microbiota-derived metabolites. Gastrointestinal microbes metabolize ellagic acid , a polyphenol found in pomegranates, nuts and certain berries to produce urolithin [165]. Urolithin A inhibits the proliferation of human CRC cell lines [166]. In addition, the co-administration of urolithin A with 5-FU enhances the therapeutic effect of the latter. In addition, recent studies in mouse models have found that ellagic acid attenuates cisplatin-induced hepatotoxicity [167], while polysaccharides extracted from squid ink can prevent chemotherapy-induced toxicity by altering the composition of the microbial community [94]. Specifically, these molecules derived from squid ink ameliorate CTX-induced intestinal injury by increasing the number of Bifidobacteria and Bacteroidetes spp. After CTX treatment. Similarly, polysaccharides in camellia ameliorate CTX-induced toxicity [95]. Specifically, the polysaccharides in Camellia sinensis L. attenuate CTX-induced intestinal damage and enhance intestinal barrier function by modulating gastrointestinal microbes and stimulating SCFA production [95].

6.2 . Prebiotics

Prebiotics are inactive substances that promote the growth or activity of specific bacterial species, resulting in health benefits for the host. Prebiotics include fiber (e.g., soy fiber), fructans (e.g., inulin), resistant starch (RS), and oligosaccharides.

In the large intestine, fiber or related prebiotics are fermented by commensal microorganisms to produce SCFAs, which lower the intestinal pH and thus maintain the growth of beneficial gut microbes (e.g., lactobacilli, bifidobacteria). RS induces the proliferation of butyrate-producing microorganisms. Current evidence suggests that these prebiotics can be used to treat cancer. For example, a study using a mouse model of pancreatic cancer found that RS reduced tumor growth by modulating the composition and metabolism of the gut microbiota [170]. In addition, other studies have demonstrated the beneficial effects of prebiotics (e.g., soy fiber, oat fiber, pectin) on methotrexate-induced toxicity.

Prebiotics often increase the amount of specific microorganisms that are already present in the host or ingested for a long time. Therefore, when prebiotics are used alone, heterogeneity of the natural gut microbiota among patients may hinder the beneficial effects of prebiotics. Synbiotics are a combination of prebiotics and probiotics that represent a pattern of potentially greater benefits than prebiotics alone [28].

6.3 . Probiotics

Probiotics are individuals or combinations of microorganisms that provide health benefits when consumed in the right amounts. Probiotics are often administered as supplements or consumed in fermented foods, such as yogurt, making them readily available.

Probiotics are mainly represented by lactic acid-producing microorganisms, particularly Lactobacillus and Bifidobacterium [169]. Administration of probiotics containing strains of Lactobacillus acidophilus and Bifidobacterium lactis to patients with CRC increases the abundance of butyrate-producing bacteria (e.g., Faecalibacterium, Clostridium) in mucosal and fecal samples and decreases the abundance of CRC-related genera (e.g., Fusobacterium, Streptococcus pepticus) [29, 178].

Efforts have been made to identify beneficial probiotics and to assess their impact on cancer initiation and progression. For example, several studies have investigated the efficacy of probiotic supplementation in patients with bladder cancer. Specifically, the use of probiotics (e.g., Lactobacillus casei) improved clinical outcomes in patients with early-stage bladder cancer compared with untreated controls [179, 180]. In mouse models, probiotic supplementation containing Lactobacillus helveticus prevents colitis-associated colorectal neoplasia by modulating the tumor immune microenvironment and microbial homeostasis [182]. Specifically, this probiotic inhibits colitis-associated hyperplasia and tumor formation by inactivating nuclear factor kappa B (NFκB), upregulating anti-inflammatory IL-10 activity, downregulating IL-17-producing pro-inflammatory T cells, and influencing the composition of beneficial components. Gut microbiota. In a mouse model of breast cancer, Lactobacillus acidophilus administration reduces tumor size by altering the levels of certain cytokines (e.g., INF-γ, IL-4, TGF-β) and increasing the number of tumor-infiltrating lymphocytes [183]. In melanoma-bearing mice, nebulized Lactobacillus rhamnosus induces an immune response against lung metastases and represents another beneficial probiotic delivery strategy that modifies the microbiome [184]. A recent study in a mouse model showed that administration of EcN reduced irinotecan-induced toxicity. Specifically, EcN ameliorates irinotecan-induced weight loss, diarrhea, and dysbiosis by modulating gut microbes and strengthening intestinal barrier function.

Probiotics vary depending on the kind, dosage, and type of preparation. These variables affect the survival of the species in the presence of gastric acid and its ability to colonize the intestinal mucosa. In addition, human probiotic colonization with diverse natural host-microbiota ecosystems presents a daunting challenge. Although antibiotics can be used to establish an ideal gastrointestinal environment that promotes microbial colonization, probiotics may hinder the re-establishment of multiple host-microbiota ecosystems after antibiotic administration. Therefore, these methods require careful study in humans, such as the recently published personalized probiotic approach [30].

6.4 . Biostime

Synbiotics are expected to provide greater benefits to the host than prebiotics or probiotics. In synbiotic formulations, prebiotics are expected to selectively promote the growth or activity of probiotics to achieve a synergistic effect .

However, there is little information on the effects of synbiotics on the gut microbiota and their efficacy as cancer therapeutics. Contrary to expectations, some evidence suggests that synbiotics do not confer additional benefits compared to prebiotics or probiotics [169]. For example, a rat model study examined the effects of probiotics (Lactobacillus fermentum) and prebiotics (fructooligosaccharides) alone or in combination on 5-FU-induced intestinal mucositis [102]. The results suggest that although probiotics improved chemotherapy-induced toxicity, the synbiotic combination did not further enhance the efficacy of probiotics [102, 169]. In contrast, another study showed a beneficial effect of synbiotics in attenuating chemotherapy-induced toxicity [190]. Specifically, the combination of the probiotics Lactobacillus casei and Bifidobacterium breve with the prebiotic galacto-oligosaccharides inhibits chemotherapy-related adverse events (eg, lymphopenia, diarrhea) in patients with esophageal cancer [190]. In patients treated with synbiotics, the number of harmful and beneficial bacteria decreased and increased, respectively, compared to the control group [190]. The reduced toxicity may be attributed to synbiotic-induced changes in the microbiota. A rat model study found that the synbiotic combination of Taiwan quinoa (Djulis; prebiotics) and Lactobacillus acidophilus (probiotics) inhibit tumorigenesis more strongly than any one ingredient alone through their effects on proliferative, inflammatory, and apoptotic pathways [191].

6.5 . Postbiotics

Postbiotics are microbial-derived soluble products and metabolites that benefit the health of the host. The best-studied postbiotics are SCFAs (e.g., butyrate, acetate, propionate), which are produced by microbial fermentation of dietary fiber [8]. Culture supernatants of microorganisms can be used as a safe alternative to live microbial administration [169]. For example, supernatants prepared from cultures of Lactobacillus plantarum enhance the efficacy of 5-FU by increasing apoptosis and hindering stemness of CRC cell lines [101], implying that postbiotics can be used as adjuvants in the treatment of patients with chemoresistant cancers [188].

6.6 . antibiotic

Antibiotics have a significant impact on clinical outcomes in cancer patients by altering the microbiota [8]. Antibiotic therapy increases or decreases the response to treatment depending on the type of cancer and drug type [8]. This treatment eliminates unfavorable microorganisms, which increases the effectiveness of the treatment. However, without sufficient specificity, antibiotics may induce dysbiosis by reducing the diversity of gut microbes. For example, the use of broad-spectrum antibiotics often worsens clinical outcomes.

The anti-tumor activity of chemotherapy is due to a strong immune response. Therefore, the disruption of the microbial network by antibiotics may undermine the therapeutic efficacy. For example, in cisplatin-treated lung cancer mice, antibiotics promote tumor growth and worsening survival by upregulating VEGFA expression and downregulating BAX and CDKN1B expression [192]. In the clinical setting, antibiotics can adversely affect the anticancer activity of CTX and cisplatin [193]. Specifically, anti-gram-positive antibiotics reduce chemotherapy efficacy and worsen clinical outcomes in patients with leukemia treated with CTX, as well as in patients with lymphoma treated with cisplatin versus those who do not receive antibiotics [193]. In both patient populations, antibiotic use was independently associated with poor clinical outcomes [193].

As shown in human and mouse studies, antibiotics may also impair the efficacy of immunotherapy (e.g., ICI therapy) in cancer patients, although the effect depends on the cancer and the drug [123, 125]. For example, a study in tumor-bearing mice found that bacterial ablation significantly reduced the therapeutic efficacy of CTLA-4 blockers [125]. In addition, another study found that antibiotic-induced dysbiosis was associated with reduced PD-1 blockade and poor clinical outcomes [123]. In addition, antibiotic therapy is associated with resistance to anti-PD-1 therapy, independent of standard prognostic factors, highlighting the impact of microbial network disruption on the efficacy of ICIs [123]. In addition, a recent clinical study showed that the use of antibiotics immediately before or immediately after initiation of ICI therapy had an adverse effect on clinical outcomes in patients with metastatic NSCLC or RCC [127].

In contrast, antibiotics can improve the efficacy of ICI therapy. Pancreatic cancer contains more bacteria than normal pancreatic tissue, and intratumoral microorganisms create an immunosuppressive microenvironment by activating anti-inflammatory immune cells [194]. Bacterial ablation reduces tumor burden by reducing intratumoral microbial load [126]. In addition, oral antibiotics reduce the number of MDSCs and activate antitumor M1 macrophages, thereby promoting Th1 differentiation of CD4+ T cells and activation of CD8+ T cells [126]. Antibiotics also increase the efficacy of ICIs by upregulating PD-1 expression [126].

Similar to the effects of immunotherapy, antibiotic therapy can also promote the effects of chemotherapy in cancer patients. In tumor beds, pancreatic cancer contains a large number of gammaproteobacteria, which eliminate the antitumor activity of gemcitabine by deamination [86]. This resistance caused by gammaproteobacteria is reduced by co-treatment with an antibiotic (ciprofloxacin) [126]; Therefore, gemcitabine in combination with antibiotics may be effective against gemcitabine-resistant pancreatic cancer [126]. A study in mice with metastatic melanoma in the lungs showed the potential use of antibiotic nebulization [184], in which nebulized antibiotics reduce microbial load and inhibit the growth of metastatic tumors in the lungs by inactivating Treg and activating T and NK cells. In addition, antibiotic nebulization increased the efficacy of dacarbazine [184].

Insufficient specificity of antibiotics may alter the innate microbial network or lead to dysbiosis. Therefore, individualized antibiotic therapy that selectively eliminates specific microorganisms will potentially improve the treatment of cancer patients [8].

6.7 . Fungus

Antibiotics can eliminate beneficial bacteria and disrupt the commensal microbial network. In contrast, the host range of lysed phages is highly specific. For example, in vitro, bacteriophages are as effective as ciprofloxacin in killing specific bacterial species (e.g., E. coli) while having a much more modest effect on the commensal microbiota [195]. Bacteriophages enhance the efficacy of FMT in the treatment of refractory Clostridium difficile infection (CDI) in humans by modulating the gut microbiota [198]. In order to take full advantage of bacteriophages as cancer therapeutics, it is necessary to comprehensively study the mechanisms by which these viruses regulate the gut microbiota, immunity, and tumor microenvironment [8, 199].

6.8 . FMT

FMT is the most straightforward method of manipulating the gut microbiome with a remarkably high response rate and is currently widely used to treat refractory CDI. In addition, FMT is effective in treating ulcerative colitis without increasing adverse events. The knowledge gained from the use of FMT in the treatment of these malnourished gastrointestinal diseases will inform further efforts to adopt FMT and design clinical trials for cancer treatments [30].

A recent report describes the efficacy of FMT in mitigating the side effects of ICI therapy in cancer patients [140]. Specifically, FMT cures ICI-associated colitis by restoring the gut microbiota and increasing Treg's infiltration of the colonic mucosa [140]. In addition, FMT suppresses the side effects of 5-FU-based chemotherapy through immunomodulation [103]. Specifically, FMT alleviates chemotherapy-induced diarrhea and intestinal mucositis by modulating the TLR-MyD88-NFκB innate immune signaling pathway in CRC mice.

FMT transfers the entire gut microbial ecosystem to the recipient, which may be more advantageous than providing a single microbial species. In microbial ecosystems, multiple microorganisms cooperate with each other for the benefit of the host [30]. FMT provides not only presumed beneficial microorganisms, but also the diversity needed to contribute to the homeostasis of the microbiome ecosystem.

There are some risks associated with FMT. A recent report described bacteremia associated with antibiotic-resistant E. coli in two patients after receiving FMT capsules from the same donor [205]. It is important that transplanted stool is rigorously screened for pathogens to minimize the risk of infection. In addition, clinicians need to be aware that certain microorganisms in the stool may induce chronic inflammation and inflammation-induced cancer. For example, FMT in CRC patients yielded adenoma and adenocarcinoma in GF mice, whereas tumorigenesis was not observed in mice receiving feces from healthy donors.

The clinical benefits of FMT in the treatment of cancer need to be validated. Ongoing clinical trials are investigating the effects of FMT on cancer patients treated with ICIs (e.g., NCT03353402, NCT03341143).

6.9 . Lytic bacterium

Advanced bioengineering techniques have allowed us to create genetically modified oncolytic bacteria for cancer therapy [209, 210]. Bioengineered anaerobes can enter tumor tissue, survive in an oxygen-deficient tumor environment, and release anti-tumor or immunomodulatory molecules in situ. For example, anaerobes are bioengineered strains of Salmonella enterica subsp. Enterica Serovar Typhimurium has been extensively studied as an oncolytic agent. Attenuated Salmonella strains, engineered to secrete Vibrio vulnificus flagellin B (FlaB) in tumor beds, inhibits tumor growth and metastasis by stimulating anti-tumor immune responses with TLR4 signaling [211]. Engineered Salmonella strains create a pro-inflammatory microenvironment with abundant tumor-infiltrating immune cells (e.g., monocytes/macrophages, neutrophils). Activation of M1 type TAM by secretion of FlaB by colonizing Salmonella and attenuation of M2-like immunosuppressive activity of TAM [211]. When another strain of Salmonella is injected intravenously into tumor-bearing mice, tumor growth is inhibited by inducing the expression of various chemokines (e.g., CXCL9) [212]. A phase I clinical study found that intravenously administered attenuated Salmonella spread to tumor sites in melanoma patients without exerting any beneficial effects [213]. Currently, the beneficial effects of Salmonella-mediated cancer therapy observed in animal models have not been replicated in clinical studies. Therefore, attention should be paid to developing safe and effective strategies for the use of bioengineered oncolytic bacteria to fight cancer.

6.10 . Nanotechnology

Nanotechnology has been widely used for diagnostic and therapeutic purposes [31]. Examples of the application of nanotechnology to cancer include the accumulation of nanoparticles at the tumor site through strong permeability and retention effects [214, 215], liposome-based delivery of breast cancer therapeutics [216], and controlled release of encapsulated drugs. The application of nanotechnology to the treatment or prevention of cancer through microbiome-targeted interventions is being studied [31].

LPS is a cell wall component of gram-negative bacteria that promotes tumorigenesis by activating the TLR4, NFκB, and STAT3 signaling pathways. Therefore, LPS is a potential target for cancer therapy. For example, a nanoparticle system that captures LPS selectively blocks LPS within tumors by expressing a protein that captures LPS (LPS-Trap protein) [218]. This LPS capture system increases the efficacy of PD-L1 blockade by enhancing anti-tumor T cell-mediated immunity and reversing the immunosuppressive tumor microenvironment [218].

Helicobacter pylori is a potential target for the prevention of gastric cancer, as described in subsection 3.1. An engineered nanoparticle encapsulated with an antibiotic and coated with a gastric membrane selectively binds to H. pylori and releases a soluble antibiotic in situ [222], highlighting a prophylactic strategy that applies nanotechnology to selectively kill carcinogenic microorganisms before tumors form.

F. nucleatum is associated with progression and chemoresistance to CRC, as described in subsection 3.2, . Although bacterial ablation kills this harmful microorganism, this treatment may also eliminate beneficial microorganisms. A multifaceted nanotechnology-based approach uses a bacteriophage isolated from human saliva that selectively eliminates Clostridium nucleotica without affecting Clostridium butyricum, thereby reducing the viability of CRC cells by producing SCFAs. In addition, dextran-based nanoparticles act as prebiotics by providing metabolites that promote the growth of Clostridium butyricum. This approach enables nanoparticles to increase the abundance of Clostridium butyricum at tumor sites [223]. In addition, irinotecan, encapsulated by dextran-based nanoparticles, kills CRC cells through a unique mechanism [223]. Future research necessitates the customization of this phage-guided nanotechnology to modulate specific microorganisms and provide other drugs to treat cancer ].

Local delivery of ICI molecules to the tumor site can alleviate immune-mediated side effects. For example, an engineered probiotic system for controlling nanobody production and intratumoral release targets the immune checkpoint molecules PD-L1 and CTLA-4 [226]. After injection into tumors, these bacteria ablate primary tumors and distant metastases by enhancing systemic anti-tumor immunity. These probiotics have been engineered to release immune-stimulating cytokines (granulocyte-macrophage colony-stimulating factor) to further stimulate responses to hypoimmunogenic tumors [226].

6.11 . The bacterial mixture expands the number of CD8+ T cells

Cytotoxic CD8+ T cells eliminate infected cells and tumor cells. ICIs eliminate the activity of immunosuppressive immune checkpoint molecules and reactivate cytotoxic CD8+ T cells. A recent study found that a mixture of 11 bacterial strains promoted the proliferation of CD8+ INF-γ+ T cells and improved their ability to recognize and kill infected or malignant cells. These T cells specifically recognize bacterial antigens present in 11 bacterial strains, work in tandem with antigen-presenting DCs, and enhance protective immunity against pathogens such as Listeria monocytogenes. In addition, the transfer of these bacterial strains into tumor-bearing mice enhances the efficacy of ICIs by increasing the number of CD8+ INF-γ+ T cells in the tumor bed. These tumor-infiltrating T cells do not originate in the colon, and the 11 strains of bacteria remain in the intestine. These findings have led to the hypothesis that certain molecules produced by these microorganisms may circulate at distant tumor sites and increase the abundance of CD8+ INF-γ+ T cells [229].

To identify the gut microbes that influence the efficacy of ICIs, previous studies have focused on the different levels of microbes that exist between ICI responders and non-responders. For the efficacy of ICIs, highly abundant microorganisms in responders and non-responders are considered favorable and unfavorable, respectively. In contrast, this group of beneficial bacterial strains has been identified to specifically increase CD8+ INF-γ+ T cell counts [229]. However, most healthy individuals do not contain these beneficial microorganisms, and in the few who do contain them, they are not abundant [229]. These findings may explain why previous studies have not identified these microbes as key effectors required to respond to ICI treatment.

In order to take advantage of these findings in cancer treatment, certain challenges must be overcome. For example, further research must determine the abundance required for the beneficial effects of these microorganisms. Colonization of these microorganisms in the gastrointestinal tract may require bacterial ablation, which can lead to microbial pathogen infection. Therefore, adverse events due to antibiotics must be considered. In addition, ICI treatment is often associated with immune-mediated adverse events. Although ICI-induced toxicity was not severe in mice treated with 11 strains [229], future studies are necessary to evaluate ICI-induced toxicity in humans treated with these microorganisms. These factors require further research to develop a safe and effective strategy to apply these promising data to the clinic.

As described in subsection 5.3, recent studies have revealed a significant impact of B cells and TLS on the efficacy of ICI therapy and provide a promising target for immunotherapy. Microorganisms that increase the number of B cells and activate TLS may enhance the efficacy of immunotherapy. Further research is needed to identify the specific microorganisms that activate B cells and TLS, and to determine whether these microorganisms enhance the efficacy of immunotherapy.

7 . Conclusion and future directions

A growing body of evidence reveals the important role of the gut microbiota in tumorigenesis and how cancer cells respond to treatment. The significant impact of the microbiota on cancer has prompted efforts to manipulate the microbiota to prevent and treat cancer. Microbial manipulation can be used as an adjunct to conventional cancer therapy or as a stand-alone therapy after failure of standard therapy. In order to apply these promising strategies, a lot of effort should be put into delineating complex microbial networks. Knowledge about the entire range of microorganisms and the specific functions of each microorganism in a particular cancer type is still limited. In microbiome-related research, researchers must recognize that mouse models may not accurately mimic human host-microbiota ecosystems. For example, the gut microbiota of laboratory mice is significantly different from that of wild mice. Reconstitution of the microbiota of wild mice in laboratory mice restores protection against tumorigenesis, which highlights the functional differences between wild and laboratory microbiota. In addition, tumor cells transplanted into mice do not undergo multistep tumorigenesis and do not interact closely with the microenvironment [232]. In addition, tumor transplantation procedures can alter tumor characteristics and anti-tumor immunity, and differences in composition between human and mouse microbiota significantly confound the results [232]. Therefore, efforts should be made to develop a model that faithfully mimics the human host-microbiota ecosystem. In addition, microorganisms that are highly abundant in certain ecological niches are not necessarily functionally important. In contrast, specific microorganisms with experimentally proven functionality should be targeted for cancer treatment. To gain insight into complex host-microbiota interactions, molecular pathological epidemiology (MPE) approaches that combine microbiology, molecular pathology, and epidemiology may be promising for their multi-level and multidimensional design. Increased understanding of the cancer host-microbiota ecosystem, as well as evolving approaches, will help expand the frontiers of precision cancer therapy to improve patient care.