The role of microbiota in the development and treatment of gastric cancer

The stomach was once considered a sterile organ until the discovery of Helicobacter pylori (HP). With the application of high-throughput sequencing technology and macrogenomics, researchers have identified fungi and fivemajor bacterial phyla within the stomachs of healthy individuals. These microbial communities exert regulatory influence over various physiological functions, including energy metabolism and immune responses. HP is a well-recognized risk factor for gastric cancer, significantly altering the stomach’s native microecology. Currently, numerous studies are centered on the mechanisms by which HP contributes to gastric cancer development, primarily involving the CagA oncoprotein. However, aside from exogenous infections such as HP and EBV, certain endogenous dysbiosis can also lead to gastric cancer through multiple mechanisms. Additionally, gut microbiota and its metabolites significantly impact the development of gastric cancer. The role of microbial therapies, including diet, phages, probiotics and fecal microbiota transplantation, in treating gastric cancer should not be underestimated. This review aims to study the mechanisms involved in the roles of exogenous pathogen infection and endogenous microbiota dysbiosis in the development of gastric cancer. Also, we describe the application of microbiota therapy in the treatment and prognosis of gastric cancer.


Introduction
Gastric cancer is the fifth most common cancer and the third leading cause of cancerrelated deaths worldwide (1).Gastric cancer rarely detects in its early stage, and the lack of effective biomarkers can accurately diagnose early gastric cancer and monitor prognosis during the adjuvant stage, which results in higher mortality rates, with only one in five patients surviving more than five years after being diagnosed with gastric cancer (2).Consequently, the development of more accurate and accessible biomarkers is necessary to aid in the early-stage diagnosis of gastric cancer and to monitor relapse.
The microbiota in the human body comprises viruses, fungi and bacteria.High-fat diets, antibiotics, exogenous microbial infections and host genetics can lead to dysbiosis (3)(4)(5).The definition of dysbiosis is a change in the composition and function of the microbiota (5).A healthy and stable microbiota inhibits cancer development, whereas a dysbiotic microbiota has a limited protective effect on the host and promotes cancer development.Gastric cancer is the result of the interaction between the dysbiosis of the gastric microbiota and the host gastric epithelial cells (6).Moreover, as gastric cancer progresses, damage to the gastric mucosal barrier and persistent chronic inflammation will further promote dysbiosis (7).
Helicobacter pylori (HP) infection is one of the riskiest factors for gastric cancer (8).The majority of intestinal-type gastric cancers are associated with HP.HP infection accelerates the progression from atrophic gastritis (AG) to intestinal metaplasia (IM) and eventually to gastric cancer.Furthermore, individuals with HP infection have a 3-6 fold higher risk of developing gastric cancer than those without HP infection (2,9).In recent years, viruses such as Epstein-Barr virus (EBV), Hepatitis B virus (HBV), and John Cunningham virus (JCV) have also attracted attention for their role in gastric cancer carcinogenesis.In addition to exogenous pathogen infections, some dysbiotic endogenous microbiota such as Lactobacillus, F. nucleatum, and Candida albicans can also be involved in developing gastric cancer through various mechanisms.Metabolites from the gut microbiota influence gastric cancer progression and improve anticancer therapy for gastric cancer patients.Microbiota therapy will be an essential target for future cancer treatment.In this review, the direct and indirect mechanisms by which exogenous infections and endogenous dysbiosis contribute to gastric cancer development are examined first.Next, the effects of oral and gut microbiota on gastric cancer are explored.Finally, the application of microbiota therapy in treating gastric cancer, including diet, bacteriophages, probiotics, and fecal microbiota transplantation (FMT), is discussed.Our goal is to investigate the impact of microbiota on gastric cancer, which could provide new insights for early screening and subsequent treatment of gastric cancer.

Gastric microbiota Composition of the gastric microbiota in healthy individuals
Due to its high acidity, the stomach was once regarded as a sterile organ and unsuitable for microbial colonization.However, the discovery of HP led to the realization that some microbes could indeed colonize the stomach (2).Advancements in high-throughput sequencing technologies in microbiology have facilitated the discovery of an increasing number of exogenous and endogenous microbiota.The normal human stomach harbors a diverse microbiota, with over 130 lineages representing 7 to 13 bacterial phyla, of which 5 are the most dominant, including Proteobacteria, Firmicutes, Bacteroidetes, Actinobacteria, and Fusobacteria (10, 11).Fungi, a common yet easily overlooked component of gastric microbiota, have also been identified.Investigations of the gastric environment in healthy individuals, patients with chronic gastritis (CG), and gastric ulcers have detected hundreds of fungal strains in the stomach.Fungi may possess an effective acid tolerance mechanism, enabling them to proliferate more efficiently in a gastric environment with a pH of only 1.4 compared to bacteria.Researchers have detected fungal species, such as Candida tropicales and Candida lusitanae, in addition to the most common Candida albicans.Fungi usually colonize areas like the gastric mucosa and stomach contents (12,13).
Both bacteria and fungi in the human stomach play crucial roles in various physiological activities, including energy metabolism, nutrient absorption, immune response, and nervous system regulation (14).However, some studies have observed differences in the flora of the gastric mucosa and gastric juice (8,10,11,15,16).Due to swallowing in the oral cavity and reflux in the duodenum, the gastric juice may contain more bacteria than the gastric mucosa (17).Although most current gastric flora studies have sampled the gastric mucosa, bacteria in the gastric juice should not be overlooked.These bacteria can contribute to the proper functioning of the gastrointestinal system but may also cause some adverse reactions.

Composition of the gastric microbiota in gastric cancer patients
The results are diverse for changes in the diversity and abundance of microbiota in gastric cancer.Some studies showed that the abundance and diversity of the gastric microbiota did not change gradually with the development of gastric cancer (18,19).Other studies have taken the opposite view, suggesting that the abundance and diversity of the microbiota decrease as gastric cancer progresses, which is what most studies have concluded (20)(21)(22).Another study has shown a significant increase in the abundance and diversity of the microbiota in cancerous tissues compared to paracancerous tissues.They also found that oral bacteria (such as Peptostreptococcus, Streptococcus, and Fusobacterium) dominated the cancerous tissues, and lactic acid- producing bacteria (such as Lactococcus lactis and Lactobacillus brevis) were enriched in the paracancerous tissues (23).However, Liu et al. found that Prevotella melanogenic, Streptococcus anginosus and Propionibacterium acnes were enriched in cancerous tissues compared to normal and paracancerous tissues.In contrast, the abundance of HP, Prevotella copri and Bacteroides uniformis was significantly reduced in cancerous tissues (24).Many current studies exist on the microbiota of gastric cancer patients and controls.However, their results varied widely, and some were even contradictory, possibly due to different sample types, study populations, sequencing technologies and analysis methods (Table 1).However, it is undeniable that there is a significant difference in the microbial composition of gastric cancer patients compared to controls.In general, the microbiota of gastric cancer patients shows a decreased abundance of HP, a significant enrichment of the oral bacteria, and reduced diversity and abundance of the microbiota.
Effect of HP-positive and HP-negative gastric cancer on the gastric microbiota HP is a gram-negative bacterium that parasitizes the human stomach.The colonization of HP can lead to dysbiosis.The diversity and abundance of the gastric microbiota decrease after infection with HP (27).HP-positive gastric cancers demonstrate an The diversity of GM was significantly lower than that of GF.As the disease progressed, the differences between GM and GF in GC were significantly reduced compared with SG and IM.
• Enriched phyla: GF: Bacteroidetes, Fusobacteria and Proteobacteria GM: Firmicutes There was no significant difference in the diversity and richness of gastric microbiota between SG and GC.The richness and diversity of the microbiota showed a clear downward trend from normal to para-cancerous to cancer tissues.abundance of Lactobacillus, Achromobacter, Citrobacter, Clostridium, and Rhodococcus but lack HP and Neisseria compared to other gastric diseases (26).A recent meta-analysis suggests that eradicating HP can restore the diversity of the gastric microbiota and reduce the risk of gastric cancer (28).However, some studies contradict this result by suggesting that eradicating HP does not entirely prevent the development of gastric cancer, which indicates that other microorganisms in the stomach besides HP are also associated with gastric cancer carcinogenesis (29).
Lertpiriyapong et al. demonstrated this hypothesis with INS-GAS mice.They found that HP mice colonized by complex or restricted microbiota were more susceptible to HP-induced gastric cancer than germ-free mice (30).This result suggests that after HP infection of the gastric epithelium causes dysbiosis, HP acts synergistically with the dysbiotic microbiota to promote gastric carcinogenesis.
Although HP is recognized as a type 1 carcinogen by the World Health Organization (WHO), not all gastric cancers are caused by HP infection.HP-negative gastric cancers also account for a percentage ranging from 0.7% to 47.8% of gastric cancer patients (31).The role of microbiota in the development of HP-negative gastric cancer has attracted increasing attention.In HPnegative gastric cancer, Kim et al. found there was a significant increase in the abundance and diversity of Lactobacillaceae, Streptococcaceae, and Prevotellaceae and a decrease in the numbers of Burkholderiaceae, Haemophilus, and Campylobacter (32) (Figure 1).These findings are inconsistent in related studies, but the increased abundance of Lactobacillaceae was detected in almost all patients with gastric cancer.Lactobacillaceae is a nitrosating bacterium, and Haemophilus is a nitrate-reducing bacterium.An increase in the abundance of Lactobacillaceae and a decrease in the abundance of Haemophilus can increase the formation of N-nitroso compounds, which can cause persistent inflammation and promote the development of gastric cancer (32).Ding et al. also concluded that HP-negative gastric cancer occurs due to disruption of the normal microbiota, with elevated levels of some bacteria with pro-cancer activity and decreased levels of cancer-suppressing bacteria (33).In other words, it is not a certain kind of bacteria that causes gastric cancer alone.Instead, the microbiota in the stomach undergoes dysbiosis, and the various dysbiotic microbiota work together to participate in the development of gastric cancer.

Gastric carcinogenesis
Numerous studies have found a connection between the development of gastric cancer and exogenous infections, such as HP and EBV, as well as endogenous dysbiosis.A shared mechanism among these factors is the induction of chronic inflammation, which heightens the risk of cancer (34).Chronic inflammation is vital in all three stages of gastric cancer development, from AG to IM and finally to gastric cancer.Chronic inflammation leads to the accumulation of mutations and the destabilization of the host genome by accelerating DNA replication rates and diminishing DNA damage repair capabilities.The result is pro-oncogenes' activation and tumor suppressor genes' (TSG) inactivation, ultimately leading to gastric cancer development (35).

Exogenous pathogens
HP and its potential mechanism in gastric carcinogenesis HP, a representative exogenous bacteria in the stomach's microecology, can be transmitted through saliva, vomit, and feces.The carcinogenic mechanism of HP is closely linked to its virulence factors, which include cytotoxin-associated gene A (CagA), CagA, an oncoprotein, interacts with various signaling pathways upon entering the cytoplasm of gastric epithelial cells, disrupts intercellular junctions, and activates pro-inflammatory and oncogenic signaling pathways.This process leads to sustained inflammatory responses and uncontrollable cell proliferation.CagA can be transfected into the cytoplasm of gastric epithelial cells via the type 4 secretion system (T4SS) encoded by the Cag pathogenicity island (CagPAI) (2).CagA can regulate signal transduction through both phosphorylated and nonphosphorylated pathways.Phosphorylated CagA can directly bind or recruit the phosphatase SHP2 to activate the MAPK/ ERK pathway.SHP2 is a key protein in CagA pathogenesis and can regulate ERK signaling in a non-Ras-dependent manner (36).Non-phosphorylated CagA, when combined with E-cadherin, decreases E-cadherin expression and promotes epithelialmesenchymal transition (EMT), accelerating gastric cancer progression (37).
VacA possesses selective anion channel properties that facilitate the release of bicarbonate and organic anions into the cytoplasm, contributing to HP colonization in gastric epithelial cells and leading to apoptosis (36).Moreover, HP infection can induce gastric carcinogenesis through hypermethylation silencing of multiple TSGs, such as miR-124a-1, miR-124a-2, and miR-124a-3 (60).Abnormal alterations in the methylation levels of the CpG island of TSG promoters following HP infection affect the transcription of downstream genes, causing irreversible TSG inactivation (61).
NAP is the main pro-inflammatory factor in HP infection.The protein was initially named for its ability to activate neutrophils to produce Reactive Oxygen Species (ROS) and promote neutrophils' adhesion to gastric epithelial cells (62).In addition to neutrophils, NAP activates monocytes and mast cells to release a variety of proinflammatory chemokines such as IL-8, IL-6, TNF-a, MIP-1a, MIP-1b, and b-hexosaminidase (63).The release of these factors causes damage to the gastric mucosa and accelerates the process of chronic gastritis.In addition, NAP has been found to play an essential role in bacterial protection.NAP is a DNA-binding protein belonging to the Dps family and has structural similarity to bacterial ferritin.Therefore, NAP can isolate free iron and bind DNA, which can protect HP DNA from oxidative damage and promote the growth and colonization of HP (40).
The most studied adhesins among HP outer membrane proteins are blood group antigen-binding adhesin (BabA) and sialic acidbinding adhesion (SabA).They can bind to receptors on the surface of gastric epithelial cells, and this binding contributes to HP adhesion and colonization, increases HP pathogenicity, and promotes persistent infection (64).BabA binds to Leb and enhances CagA translocation by promoting T4SS activity.CagA induces the production of massive pro-inflammatory factors, leading to IM and precancerous lesions (49).SabA binds to the sialyl-Lex antigen and promotes neutrophil activation and infiltration, inducing oxidative damage and causing a persistent inflammatory response (51).The expression of BabA and SabA is closely associated with the development and prognosis of various gastrointestinal diseases, such as chronic gastritis and gastric cancer (50, 65).Therefore, they may be potential targets for preventing and treating HP-related diseases.
Additionally, HP infection may significantly diminish the effectiveness of immunotherapy for gastric cancer.Firstly, HP can decrease the activity of CD4+ T cells, dendritic cells (DCs), macrophages, and NKT cells, while also increasing the activity of myeloid-derived suppressor cells (MDSCs) and regulatory T cells (Treg cells) (66).Secondly, HP elevates the expression of PD-L1 in gastric epithelial cells and induces CD4+T cell apoptosis (67) (Figure 2).This finding implies that HP infection leads to nonspecific suppression of T cells and reduced immune checkpoint inhibitors (ICIs) efficacy.Finally, Oster et al. discovered that HP infection could reduce the effectiveness of anti-CTLA-4 and anti-PD-L1 therapy, decrease the potency of cancer vaccines, and inhibit in situ tumor immunotherapy (66,68).

EBV and its potential mechanism in gastric carcinogenesis
EBV, first isolated from Burkitt's lymphoma cell line in 1964, was among the initial viruses identified to be linked to human malignancy.EBV infection also elevates the risk of developing gastric cancer by more than 18-fold, with EBV-associated gastric cancer constituting approximately 10% of all gastric cancers (69,70).
Following EBV infection, viral genes such as EBERs, EBNA-1, and BARTs are expressed and can interact with host proteins to promote cell proliferation and inhibit apoptosis.The DNA damage and cell proliferation resulting from this process contribute to gastric cancer development.Additionally, EBV can disrupt the host's epigenetic machinery and induce malignant transformation of the epigenome.EBV upregulates host DNA Methyltransferases (DNMTs) through the expression of Latent Membrane Protein 1 (LMP1) and Latent Membrane Protein 2A (LMP2A) viral oncoproteins (13,71).Consequently, most EBV-associated gastric cancers exhibit CpG island hypermethylation at TSGs, such as PTEN, leading to the inactivation of TSGs and immune escape.Plasma EBV DNA load is a valid marker for assessing the efficacy and prognosis of EBV-associated gastric cancers (72).It has been discovered that EBV and HP may synergistically impact the development of gastric cancer.HP-positive patients have a higher EBV DNA load.EBV infection can enhance the oncogenic potential of CagA.Gastric cancers co-infected with EBV and HP display numerous methylated genes.EBV and HP can synergistically promote chronic inflammation and increase tissue damage during the early stages of the gastric carcinogenesis process (2, 73).Immunotherapy is typically employed to treat patients with EBVassociated gastric cancer because EBV subtypes are positively associated with PD-L1 overexpression (35).Su et al. observed an increased cellular immune response and cytotoxicity in EBVassociated gastric cancer xenograft model mice through CRISPR/ Cas9 system-mediated disruption of PD-1 on T cells (74).
Several studies have suggested that other viruses, such as JCV and HBV, may also play a crucial role in gastric cancer development (75).The oncogenic effect of JCV is primarily achieved through its encoded oncoprotein T-Ag.T-Ag can interact with tumor suppressor proteins like p53 and pRB and participate in gastric carcinogenesis through critical signaling pathways, such as Wnt/b-Catenin (76).HBV infection results in a 1.29-fold increased risk of gastric cancer, with the hepatitis B virus X protein (HBx) potentially playing a vital role in this process (77).Cui et al. discovered that HBV could induce gastric epithelial cell carcinogenesis through HBx (78).Moreover, chronic inflammation caused by HBV infection heightens the risk of gastric carcinogenesis (79).

Lactobacillus and its potential mechanism in gastric carcinogenesis
Although Lactobacillus is widely regarded as a probiotic in the intestine, it is often abundantly enriched in gastric cancer, particularly in advanced gastric cancer (32,80).Several factors contribute to Lactobacillus causing gastric cancer.First, lactate, produced by Lactobacillus, plays a massive role in the Warburg effect of tumors (81).Second, Lactobacillus can upregulate proinflammatory genes, such as Ptger4 and Tgf-b, and oncogenic genes, such as Nos2, Tnf-a, Cxcl1 (Kc), and Ccl2 (Mcp-1) to induce gastric carcinogenesis (30).Third, Lactobacillus can reduce nitrate to nitrites and eventually form nitrosamines (82).Furthermore, Lactobacillus is a potent ROS inducer, leading to DNA damage (83).Lastly, Lactobacillus can convert human fibroblasts into multipotent cells, directly promoting gastric cancer development (84).These factors may jointly contribute to gastric cancer development, but the exact mechanism requires further investigation.

F. nucleatum and its potential mechanism in gastric carcinogenesis
Fusobacterium nucleatum (F.nucleatum) is most widely known for its carcinogenic effect on colorectal cancer.However, in recent years, F. nucleatum has also been recognized as a significant cause of gastric cancer development.F. nucleatum is more abundant in gastric cancer than normal tissues, and F. nucleatum positivity is associated with a worse prognosis and shorter overall survival in patients with diffuse-type gastric cancer (85, 86).F. nucleatum binds to E-cadherin in gastric epithelial cells via the characteristic virulence factor FadA, increasing endothelial permeability, promoting self-colonization, propagation, and immune escape (87), and activating the Wnt/b-catenin signaling pathway to enhance tumor cell proliferation (88).Familial adenomatous polyposis 2 (Fap2) is another virulence factor of F. nucleatum.Fap2, through binding and interaction with T cell immunoreceptors with IG and ITIM domains (TIGIT), can inhibit the killing of NK cells and lymphocytes to tumors, thereby protecting tumors and promoting the formation of an inflammatory microenvironment (89,90).F. nucleatum also produces an induced inflammatory response through NF-kB-mediated cytokines such as IL-6, IL-1b, IL-17, IFN-g, and TNF-a (87,91).

Candida albicans and its potential mechanism in gastric carcinogenesis
Zhong et al. conducted ITS rDNA gene analysis on cancerous and non-cancerous tissues from 45 gastric cancer patients and discovered an imbalance of fungal communities in gastric cancer.After statistical analysis, they determined that Candida albicans were abundantly enriched in gastric cancer.Ultimately, it was demonstrated that Candida albicans could serve as a fungal biomarker for diagnosing gastric cancer.Candida albicans may promote gastric cancer development by reducing the diversity and abundance of fungi in the stomach, such as Candida glabrata, Aspergillus montevidensis, Saitozyma podzolica, and Penicillium arenicola (92).
Candida albicans may possess multiple carcinogenic mechanisms, including damage to mucosal epithelial cells, induction of chronic inflammation, and production of carcinogens leading to cancer.
Upon invasion of the mucosal epithelium, Candida albicans cause apoptosis and necrosis, which disrupts the immune barrier of the epithelium and leads to structural changes in the epithelium (93).Long-term colonization and infestation of Candida albicans in the mucosal epithelium can cause chronic inflammation.During this process, the expression of the inflammatory factor prostaglandin E2 (PGE2) increases.PGE2 downregulates macrophage cytokines, inhibits DC and NK cell activity and function, blocks cytotoxic T cell activation, and promotes maturation and suppressive activity of Treg cells, thereby suppressing tumor immunity.PGE2 aids in the migration of MDSCs to the tumor microenvironment, proliferation of malignant cells, and inhibition of apoptosis, directly promoting tumor progression.PGE2 can also promote tumorigenesis through ROS production, activation of oncogenic transcription factors, and promotion of angiogenesis (94).
Candida albicans-produced nitrosamines and acetaldehyde can contribute to cancer development to some extent.Acetaldehyde impacts DNA replication and can cause point mutations of genes and chromosomal abnormalities.Meanwhile, acetaldehyde interferes with enzymes involved in cytosine methylation and DNA damage repair, resulting in proto-oncogene activation and cell cycle abnormalities, thereby promoting cancer development.Additionally, acetaldehyde can cause mitochondrial damage and induce apoptosis (93).
In conclusion, Candida albicans dysbiosis can increase the likelihood of cancer occurrence and promote cancer progression.

Microbiota from other habitats and gastric cancer Oral microbiota and gastric cancer
The oral and gastric environments are linked through swallowed saliva and ingested food.The oral microbiota is a major source of gastric microbiota (95).Many studies have found that dysbiosis of the oral microbiota is associated with gastric carcinogenesis.Oral bacteria such as Parvimonas, Eikenella and Prevotella-2 were significantly enriched in gastric cancer compared to other precancerous stages (18).The abundance of oral bacteria such as Peptostreptococcus, Streptococcus, and Fusobacterium was higher in cancerous tissues than in paracancerous tissues (23).Network analysis revealed that oral microorganisms such as Peptostreptococcus stomatitis, Slackia exigua, Parvimonas micra, Streptococcus anginosus, and Dialister pneumosintes might play critical roles in the development of gastric cancer (25).Under normal conditions, most of the oral microbiota entering the stomach is either destroyed by gastric acid or protected by the mucus-bicarbonate barrier from invading the gastric epithelium.However, certain disease states, such as gastric ulcers and gastric cancer, can damage the gastric mucosa and neutralize gastric acid, allowing oral bacteria to colonize the gastric mucosa from gastric juices (17).Recently, some studies have also been on the interaction between oral microbiota and HP.On the one hand, HP infection can disrupt the homeostasis of the oral microbiota (96).On the other hand, ectopic colonization on the gastric mucosa by the oral microbiota may, in turn, be necessary for HP-induced ecological dysbiosis (95).

Gut microbiota and gastric cancer
Since the pH in the intestine is more conducive to microbial colonization, intestinal bacteria are nearly 10 7 times more abundant than those in the stomach (16,97).Approximately 99% of the 10 14 microorganisms that comprise the human microbiome reside in the intestine (98).Numerous studies have demonstrated that intestinal flora can directly and indirectly affect the development, treatment, and prognosis of gastric cancer.
In a study conducted in Shanxi province, researchers investigated changes in the gut microbiota of 116 patients with gastric cancer and 88 healthy controls.They observed an increase in the abundance of intestinal flora, a decrease in butyrate-producing bacteria, and a significant enrichment of Lactobacillus, Escherichia, and Klebsiella in patients with gastric cancer (99).According to Sarhadi et al., Enterobacteriaceae were abundant in stool samples from patients with all types of gastric cancer (100).
Enterobacteriaceae may serve as a potential biomarker in the diagnosis of gastric cancer.
Certain metabolites of intestinal bacteria, such as acetic acid and butyrate, also influence the development of gastric cancer.Acetic acid and butyrate are the major short-chain fatty acids, and intestinal bacteria such as Eubacterium, Clostridium, Ruminococcus, and Coprococcus can produce butyrate (35).Increasingly, research has found that butyrate plays a crucial role in inhibiting gastric cancer development.First, butyrate inhibits the Warburg effect of gastric cancer by binding pyruvate kinase M2 (PKM2), increasing the content of glucose intermediates in mitochondria, preventing the conversion of tricarboxylic acid cycle intermediates to ATP, and ultimately depriving tumor growth of sufficient energy, thus inhibiting gastric cancer development.Second, butyrate can directly interfere with the mitochondria of gastric cancer cells, upregulate oxidative stress, and significantly increase the level of ROS.Moreover, butyrate can promote caspase 9 production and inhibit BCL-2 synthesis, leading to the apoptosis of gastric cancer cells (101).Lastly, a recent study discovered that butyrate could inhibit the growth, migration, and invasion of gastric cancer cells and aerobic glycolysis by blocking the Wnt/b-catenin/c-Myc signaling pathway (102).

Microbiota modification and immunotherapy for gastric cancer
Alterations in the microbiota may directly impact gastric cancer immunotherapy (103, 104).Matson et al. discovered that higher proportions of Bifidobacterium longum, Collinsella aerofaciens, and Enterococcus faecium led to more effective anti-PD-L1 therapy (105).A meta-analysis revealed that Bacteroides caccae was enriched in all types of immune checkpoint inhibitor therapies.Faecalibacterium prausnitzii, Bacteroides thetaiotamicron, and Holdemania filiformis were enriched in responders to anti-CTLA-4 and anti-PD-1 ICIs treatments (106).Based on these findings, researchers have proposed microbiota modification as a means to enhance the efficacy of immunotherapy for gastric cancer.Current approaches to microbiota modification include the use of probiotics and FMT.

Probiotics
Probiotics, such as Bifidobacterium, Lactobacillus, and Saccharomyces, are active microbes that benefit the host's health (107, 108).There is growing evidence that probiotics may inhibit the growth of gastric cancer to some extent.Probiotics can significantly reduce inflammatory responses, enhance the immune system, promote tumor cell apoptosis, restore gut microbiota homeostasis, and inhibit cancer signaling pathways (35,109,110).Lactobacillus can inhibit the development of gastric cancer by reducing the expression of NF-kB and the phosphorylation of the PI3K/Akt/mTOR signaling pathway (111).Bifidobacterium can directly induce DC maturation and cytokine (IFNg, TNFa, IL-10, IL-17) production, thus promoting anti-tumor immunity and anti-PD-L1 efficacy and almost inhibiting tumor progression after combination therapy with PD-L1 monoclonal antibody (mAb) (104).Bacteroides thetaotaomicron and Bacteroides fragilis (B.fragilis) are also potential probiotics.Vetizou et al. found that the antitumor and immunotherapeutic effects of CTLA-4 blockade were associated with Bacteroides thetaotaomicron and B. fragilis.Oral administration of Bacteroides thetaotaomicron or B. fragilis to germ-free mice enhances the antitumor effects of CTLA-4 blockers (112).B. fragilis also increase macrophage phagocytosis, prompting them to polarize towards an M1 phenotype (113).
Probiotics have become a prominent research topic due to their beneficial effects on gastric cancer.Immunotherapeutic probiotics could be developed to improve the efficacy of immunotherapy (114).However, contrary to the aforementioned findings, it is relatively easy to identify inconsistencies.The role of Lactobacillus in gastric cancer remains unclear.In addition to Lactobacillus, there are inconsistent findings on the effects of other probiotics on gastric cancer.Based on human microbiome studies and animal models, researchers have cautioned against the direct use of Lactobacillus in treating patients with gastric cancer (81).

FMT
FMT is transplanting fecal material from a healthy donor into a recipient's intestine, ultimately altering the recipient's intestinal flora to match the donor's microbial profile (115).Several methods of fecal microbiota transplantation exist, including oral, nasal, and rectal administration.Rectal administration is preferred, as nasal administration may cause pulmonary or gastrointestinal complications, and although oral administration is convenient, careful avoidance of first-pass effects is necessary (116,117).Given FMT's numerous benefits, there is growing interest in its potential for enhancing cancer immunotherapy.
Routy et al. discovered that the aberrant composition of intestinal flora results in primary resistance to immune checkpoint inhibitors (ICIs) in patients with late-stage cancer and that antibiotics interfere with the therapeutic efficacy of ICIs in patients.Their findings revealed that the anti-tumor effects of PD-1 inhibitors are more effective in germ-free mice or those not treated with antibiotics after FMT from cancer patients who respond to ICIs.However, this was not the case in the FMT of patients who did not respond to ICIs.They also analyzed the metagenomics of fecal material from patients and found a correlation between the efficacy of ICIs and the abundance of Akkermansia muciniphila (A.muciniphila).After FMT, patients who did not respond were given A. muciniphila can restore the efficacy of PD-1 inhibitors (103).Fecal material from immunotherapy responders was transplanted into germ-free mice, resulting in slower tumor growth and increased immunotherapy efficacy (105).In summary, FMT can enhance patient responses to immunotherapy.
Since FMT has a more substantial impact on intestinal flora than gastric flora, most current research focuses on the effect of FMT on intestinal diseases.However, researchers should also consider FMT as a treatment for gastric cancer and precancerous lesions in the stomach.As a safe and effective therapy, FMT may treat gastric cancer in the future by interfering with the composition of the intestinal flora.However, we need further studies to evaluate FMT's safety and reduce the risk of side effects.

Other interventions and therapeutic strategies for gastric cancer treatment
In addition to the FMT and probiotics mentioned above, many potential microbial interventions have been used to treat gastric cancer, including diet, prebiotic, and bacteriophage-based strategies, all pointing to the promising future of microbial therapies.

Bacteriophage-based strategies
Bacteriophages are viruses that selectively infect bacteria (118) and are more than 100 times more abundant in the gut than in bacteria and human cells (119).Given that most bacteriophages are particular to specific pathogenic bacteria but do not disrupt the homeostasis of normal flora (120), many studies have constructed bacteriophage-based biotic-abiotic hybrid nanosystems to accurately remove tumor microorganisms (121).
In order to precisely regulate intestinal flora to treat colorectal cancer, Zheng et al. loaded irinotecan inside dextran nanoparticles (122).And through bioorthogonal reaction, it was covalently linked with azide-modified phages that could specifically lyse F. nucleatum, which could not only effectively inhibit the proliferation of F. nucleatum, but also reduce the toxic side effects caused by the non-targeted release of conventional chemotherapeutic drugs.Moreover, phages can also remodel TME.F. nucleatum selectively amplifies MDSCs, thereby blocking the body's anti-tumor immune response.Through phage display technology, Dong et al. screened M13 phage that can specifically bind F. nucleatum (123).They further assembled silver nanoparticles on the surface capsid protein of the M13 phage (M13@Ag).Subsequently, it was also demonstrated that M13@Ag could specifically clear F. nucleatum and inhibit the proliferation of MDSCs, reversing the immunosuppressive TME.In addition, the M13 phage utilizes its coat protein to activate antigen-presenting cells, which further activates the host immune response and inhibits CRC.Animal experiments demonstrated that M13@Ag combined with immune checkpoint inhibitors or chemotherapeutic agents significantly prolonged the survival of CRC mice.A recent study on phage strategies targeting HP may open new avenues for treating HP-positive gastric cancer (124).

Diet and prebiotic strategies
Diet has an important influence on the composition of the gut microbiota.Different dietary habits can lead to differences in the composition of the gut microbiota (125).Compared to omnivores, the intestinal flora of strict vegetarians was characterized by a higher abundance of Bacteroidetes and a lower abundance of Firmicutes (126).However, the opposite alteration of the intestine flora was observed in people on chronic high-fat diets (127).Therefore, the composition of the intestinal flora can be adjusted by dietary interventions, thereby creating a more favourable microecological environment for the host.Dietary interventions such as fasting and calorie restriction have been used in clinical trials for various cancers, and the results have shown that dietary interventions can alter the systemic metabolism of cancer patients, improve antitumor immunity (128), and inhibit tumor growth.For example, in an ongoing clinical trial (NCT01642953), researchers restrict patients' diets after gastric cancer surgery to assess whether fasting promotes recovery and reduces mortality and adverse events (129).Dietary interventions can also inhibit inflammation, improve anti-cancer immune surveillance (130) and prevent chemotherapy-induced toxic side effects (131).
WHO defines the concept of prebiotics as "an inactive food ingredient that promotes the health of the organism by modulating the composition of the microbiota (107)."Common prebiotics are fructooligosaccharides (FOS), galactooligosaccharides (GOS) and inulin (132).Prebiotics can help the body resist pathogenic infections, maintain stable gastrointestinal function, promote the production of short-chain fatty acids by beneficial bacteria, regulate immunity and energy metabolism, and increase the absorption of micronutrients, thus improving the efficacy of cancer treatment (132-134).To demonstrate the role of prebiotics in cancer treatment, many preclinical studies and clinical trials are underway or have been completed.One clinical trial in the United States (NCT04682665) is investigating eicosapentaenoic acid's role in treating liver metastases from colorectal cancer (135).Furthermore, It has been shown that a high intake of Ganoderma spp (136).Polysaccharides and raffinose (137) potentially reduce the risk of gastric cancer, but more extensive independent studies and clinical trials are needed to confirm this.

Conclusions and perspective
The microbiota has dual effects on gastric cancer.On the one hand, pathogenic bacterial infections and dysbiosis can promote the development of gastric cancer and is detrimental to the treatment and prognosis of gastric cancer.On the other hand, microbial therapies that optimize the microbiota through dietary changes, the use of bacteriophages and probiotics, and even transplantation of the fecal microbiota hold promise as potential treatments for gastric cancer.Furthermore, specific microorganisms and metabolites in gastric and intestinal flora could potentially serve as markers for diagnosing and monitoring the prognosis of gastric cancer, thereby improving early detection and treatment.Studying the microbiota helps us further understand the mechanisms of gastric cancer development and may provide new ideas for targeting microbiota interventions to treat cancer.However, there are still some problems in the current research on the microbiota of gastric cancer.Firstly, the mechanism by which some microbes promote gastric cancer has not been conclusively demonstrated and requires further investigation.Second, due to the differences in sequencing technology, analysis method or study population, the results obtained by many studies are quite different, and the interference of these factors should be excluded as far as possible.Third, there is currently a lack of more definitive biomarkers for gastric cancer other than prevention or eradication of HP infection, which hinders the early diagnosis of gastric cancer.In the future, more convenient and efficient biomarkers should be explored in larger sample sizes.
In conclusion, further exploration is needed to modify the microbiota and ultimately improve the prognosis and effectiveness of gastric cancer treatment.

FIGURE 1 Factors
FIGURE 1 Factors affecting gastric microecological dysbiosis and potential mechanisms underlying microecological dysbiosis causing gastric carcinogenesis.A multitude of microorganisms resides in the stomach, creating normal gastric microecology.Normal microecology plays a vital role in various physiological activities such as energy metabolism, nutrient absorption, immune response, and nervous system regulation.High-fat diet, antibiotics, genetic variation, and exogenous infections can all dysregulate normal gastric microecology, turning the normal microenvironment into the tumor microenvironment and eventually leading to gastric cancer development.Exploring the mechanism of gastric cancer progression from the perspective of gastric microecology is essential for early diagnosis, treatment, and prognosis of gastric cancer.HP, Helicobacter pylori; EBV, Epstein-Barr virus; HBV, Hepatitis B virus; JCV, John Cunningham virus, F. nucleatum, Fusobacterium nucleatum; Th, helper T cell; CTL, Cytotoxic T lymphocyte.Created with BioRender.com.(accessed on 17 May 2023).

FIGURE 2
FIGURE 2 Pathogens and probiotics have effects on anti-tumor immunity.Pathogens correlated with gastric carcinogenesis comprise Helicobacter pylori, F. nucleatum, and Candida albicans, all of which can suppress immune cell function, stimulate tumor cell proliferation and hinder apoptosis, thereby fostering tumor advancement.Helicobacter pylori upregulate PD-L1 expression in gastric epithelial cells, consequently inducing T cell apoptosis.Helicobacter pylori additionally activate MDSCs, reducing DCs and macrophage function while enhancing Treg cell activity.The F. nucleatum virulence factor Fap2 obstructs tumor elimination by immune cells via binding and interacting with TIGIT on NK cells or lymphocytes.Candida albicans infection leads to heightened expression of the inflammatory factor PGE2. PGE2 diminishes cytokine levels such as IL-1, IL-12, and TNF-a, which restrain DC and NK cell activity and function, obstruct CTL activation, and promote Treg cell maturation, ultimately suppressing tumor immunity.Furthermore, PGE2 facilitates MDSC migration to TME, thus promoting tumor immune escape.Probiotics like Bifidobacterium and B. fragilis elicit robust immune responses, enhance anti-tumor immunity, and impede tumor progression.Bifidobacterium directly stimulates DC maturation and fosters immune response production by T cells.B. fragilis induces macrophage polarization toward the M1 phenotype and enhances macrophage phagocytic capacity.In summary, microbial pathogens associated with gastric carcinogenesis suppress anti-tumor immunity.However, probiotics can potentially enhance anti-tumor immunity, which has significant implications for the treatment and prognosis of gastric cancer.F. nucleatum, Fusobacterium nucleatum; B. fragilis, Bacteroides fragilis; MDSCs, myeloid-derived suppressor cells; Treg cells, regulatory T cells; TIGIT, T cell immunoreceptors with IG and ITIM domains; DC, Dendritic cell; TME, Tumor microenvironment.Created with BioRender.com.(accessed on 17 May 2023).

TABLE 1
Studies on the difference in gastric microbiota composition between patients with gastric cancer and controls.

TABLE 2
Virulence factors closely associated with the carcinogenic mechanism of HP.
• Selective anion channel properties • Targets different organelles • Promotes HP colonization • Induces apoptosis and necrosis • Causes cell vacuolization • Enhances autophagy NAP (40-43) • Overactivation of ERK and p38-MAPK pathways • DNA-binding protein • Structurally similar to bacterial ferritin • Activates neutrophils, monocytes, and mast cells • Produces ROS • Promotes neutrophils' adhesion to gastric epithelial cells • Promote the release of IL-8, IL-6, TNF-a, MIP-1a, MIP-1b, and bhexosaminidase • Damages the gastric mucosa • Accelerates the process of chronic gastritis • Protects HP DNA from oxidative damage • Promotes the growth and colonization of HP

Table 2 )
. These factors damage DNA in gastric epithelial cells and cause the destabilization of the host genome.