Fusobacterium nucleatum: Unraveling its potential role in gastric carcinogenesis

Abstract

Fusobacterium nucleatum (F. nucleatum) is a Gram-negative anaerobic bacterium that plays a key role in the development of oral inflammation, such as periodontitis and gingivitis. In the last 10 years, F. nucleatum has been identified as a prevalent bacterium associated with colorectal adenocarcinoma and has also been linked to cancer progression, metastasis and poor disease outcome. While the role of F. nucleatum in colon carcinogenesis has been intensively studied, its role in gastric carcinogenesis is still poorly understood. Although Helicobacter pylori infection has historically been recognized as the strongest risk factor for the development of gastric cancer (GC), with recent advances in DNA sequencing technology, other members of the gastric microbial community, and F. nucleatum in particular, have received increasing attention. In this review, we summarize the existing knowledge on the involvement of F. nucleatum in gastric carcinogenesis and address the potential translational and clinical significance of F. nucleatum in GC.

Key Words: Fusobacterium nucleatum; Gastric microbiota; Gastric cancer; Preneoplastic changes; Prognosis

Core Tip: Fusobacterium nucleatum (F. nucleatum) plays a key role in the development of oral inflammatory diseases and is a member of the oral microbiota. Recently, however, F. nucleatum has been associated with colorectal cancer and several other malignancies, including gastric cancer (GC). In particular, F. nucleatum has been associated with patient outcomes, suggesting its role in clinical practice. In this review, we summarize the current knowledge on the potential role of F. nucleatum in the development and progression of GC from a basic and translational perspective and highlight its potential therapeutic relevance.

INTRODUCTION

Gastric cancer (GC) is not only one of the most common and deadliest tumor types worldwide, but is probably also one of the most representative cancers related to the microbiome and microenvironment[1]. Historically, GC has been recognized as a multifactorial disease caused by the interaction of host, microbial and environmental factors[2,3]. The strongest known risk factor for the disease is chronic infection with Helicobacter pylori (H. pylori), which favors the development of premalignant lesions such as atrophic gastritis (AG) and intestinal metaplasia (IM), which in turn can lead to dysplasia and eventually GC[4-6]. H. pylori infection of the stomach is a complex disease that depends on bacterial virulence factors, host factors and probably other nutritional and environmental factors that can influence not only acid production but all physiological processes in the stomach. For example, H. pylori may contribute to increased acidity with a higher risk of peptic ulcer complications due to antrum-dominant gastritis, or lead to a decrease in pH due to corpus-dominant inflammation and subsequent progression of chronic gastritis (CG) to AG/IM[7]. H. pylori-dependent or -independent changes in the acidic gastric environment can therefore directly reduce the local defense of the highly proliferative gastric epithelial cells, but also the induction of local apoptosis and inflammation with accompanying changes can further contribute to changes in the gastric microbiota that favor the development of GC[8].

The gastric microbiota also has other diverse members besides H. pylori, but the acidic gastric environment and thick protective mucin layer strongly contribute to the limited growth of bacteria in the niches of the stomach. The microbiota of the stomach is therefore less rich than the microbiota of the intestine and oral cavity, but its potential effect on the mucosa is probably at least as great, if not more pronounced[9]. The latest research findings suggest that not only H. pylori but also the entire microbiota of the human stomach can have important effects on the development of GC[10-12].

The proximity to the oral microenvironment suggests that the oral microbiome may play a functionally relevant role in controlling local inflammation in the upper gastrointestinal (GI) tract and especially in the stomach. However, the alteration of the upper GI acid defense mechanism is also likely to affect the distal lower GI tract. Therefore, it is not surprising that the first reports analyzing the lower GI tumor microenvironment and stool samples from patients with colorectal carcinoma demonstrated an enrichment of Fusobacterium nucleatum (F. nucleatum)[13,14]. F. nucleatum is a widespread Gram-negative anaerobic bacterium that is common in the oral cavity and plays a key role in the development of inflammatory diseases in the mouth, such as periodontitis and gingivitis[15]. Over the past decade, there has been increasing evidence that F. nucleatum may accelerate the proliferation and metastasis of colorectal cancer (CRC) through several potential mechanisms[16,17]. In addition, the increased abundance of F. nucleatum has been reported in tissue samples from other cancers, including esophageal[18,19], pancreatic[20] and breast[21] and more recently, in GC[22,23]. To clarify the emerging data on this clinically highly relevant topic, in this review we systematically summarize the current knowledge on the potential role of F. nucleatum in the development of GC.

F. NUCLEATUM FROM CRC AND OTHER CANCERS

Before reviewing the existing data on F. nucleatum in GC, it is useful to summarize the evidence from other cancers and specifically from CRC. Initial studies, supported by subsequent reports, have convincingly shown that the level of F. nucleatum is significantly higher in CRC tissue than in adjacent normal tissue. A higher abundance of F. nucleatum has been found in stool samples from CRC patients compared to normal controls. The prevalence of F. nucleatum has been reported to be associated with CRC progression, metastasis and poor disease outcome[24,27-29].

From a molecular point of view, several mechanisms of the role of F. nucleatum in the pathogenesis of CRC have been explored[30]. F. nucleatum has been shown to accelerate cancer development by increasing cell proliferation via the FadA/E-cadherin/β-catenin pathway[31]. FadA binds to E-cadherin, activating β-catenin signaling, which increases the expression of inflammatory and oncogenic genes. This pathway also leads to the overexpression of chk2, a multifunctional enzyme involved in DNA damage[32]. F. nucleatum may promote CRC by regulating gene expression through noncoding RNAs. Yang et al[33] showed that F. nucleatum induces high expression of miR-21 in CRC, which promotes cell proliferation. F. nucleatum promotes the expression of several inflammatory genes and the release of inflammatory cytokines, particularly interleukin (IL)-8, IL-10 and tumor necrosis factor-α, which are associated with activation of the NF-κB pathway, thereby creating a tumor-promoting proinflammatory environment that stimulates colorectal tumor progression[34,35]. Fusobacterial Fap2 protein can inhibit immune cell activity and protect tumors from killing by natural killer cells and tumor-infiltrating T cells through activation of the TIGIT inhibitory receptor[36].

Bullman et al[37] successfully detected and cultured F. nucleatum not only from the primary CRC tumor, but also from the liver metastasis of the same patient. The average nucleotide similarity of F. nucleatum in tumor and metastasis was mostly > 90%. The discovery of nearly identical viable Fusobacterium strains in matched primary and metastatic CRCs confirms the persistence of viable Fusobacterium species during the metastatic process and suggests that Fusobacterium could potentially migrate with CRC cells to the metastatic site.

Abed et al[38] found that CRC-specific recognition by Fusobacteria is mediated by the fusobacterial Fap2 lectin, which specifically recognizes and binds tumor-derived D-galactose-b(1-3)-N-acetyl-D-galactosamine (Gal-GalNAc). Gal-GalNAc is overexpressed in colorectal tumors. High levels of Gal-GalNAc have also been found in adenocarcinomas of the prostate, ovary, colon, uterus, pancreas, breast, lung, esophagus and stomach[39].

POTENTIAL ROLE OF MICROBIOTA IN GASTRIC CARCINOGENESIS

It is now well established that the microbiota plays an important role in human gastric health, and changes in the composition of the microbiota may be associated with various diseases[12,40]. The microbiota of the human digestive tract varies in richness and diversity, with the highest numbers of bacteria in the oral cavity and gut, and lower numbers in the esophagus and stomach. Because of its acidic environment, the stomach[41-43] was thought to be a sterile organ until the discovery of H. pylori; however, recent advances in molecular techniques have shown that other microorganisms besides H. pylori can colonize the gastric epithelium[44]. The composition of the gastric microbiota varies considerably between individuals with different conditions, particularly in relation to H. pylori infection, as has been repeatedly shown[45-47]. Although the gastric microbiome can be strongly modified by medication use, dietary habits, age, gender and other factors, the key factor responsible for the gastric microbiome pattern is H. pylori infection[48-51]. Overall, the five most abundant phyla (Proteobacteria, Firmicutes, Actinobacteria, Bacteroidetes and Fusobacteria) were identified using culture dependent, mass spectrometry and sequencing techniques[8,9,52]. H. pylori colonization significantly reduces the overall diversity of the gastric microbiota[46,53]. For example, Maldonado-Contreras et al showed that four phyla (Proteobacteria, Firmicutes, Actinobacteria and Bacteroidetes) dominated in the stomach; however, H. pylori infection increased abundance of non-H. pylori-Proteobacteria, Spirochaete, and Acidobacteria, while decreasing the abundance of Actinobacteria, Bacteroidetes and Firmicutes[54]. Klymiuk et al[55] found that the genera Actinomyces, Granulicatella, Veillonella, Fusobacterium, Neisseria, Helicobacter, Streptococcus and Prevotella were significantly different between the H. pylori-positive and H. pylori-negative individuals[55].

Although H. pylori infection is the strongest risk factor for developing GC, other members of the gastric microbiota may influence disease progression. H. pylori infection plays a crucial role in the initial steps of carcinogenesis by causing inflammation of the gastric mucosa, leading to hypochlorhydria and mucosal atrophy[5,6]. A decrease in gastric acidity favors bacterial growth, which contributes to malignant transformation by maintaining inflammation and converting nitrates into N-nitrosamines[56]. Some studies have shown significant differences in the gastric microbiota of patients with gastritis, IM and GC, suggesting that changes in the microbiota are associated with the progression of gastric carcinogenesis[57-59]. Studies of the composition, richness and biodiversity of the microbiota in patients with GC have shown mixed results. Due to the large variation between study cohorts and associated factors, data on the predominant bacteria in the cancer-associated gastric microbiota vary widely.

Wang et al[60] found that five types of bacteria (Lactobacillus, Escherichia–Shigella, Nitrospirae, Burkholderia fungorum and Lachnospiraceae) were enriched in GC[60]. Ferreira et al[61] found that the GC microbiota had an over-representation of Actinobacteria and Firmicutes, and lower abundance of Bacteroidetes and Fusobacteria[61]. Some studies have shown the enrichment of Fusobacteria in GC compared to other disease stages[22,23,62]. Among the various bacteria reported above, Fusobacteria are among the most common associated with the gastric microbiome and are gaining further attention, particularly due to the concurrent association data from CRC and increasing understanding of molecular interactions.

FUSOBACTERIA IN THE STOMACH

As previously reported, Fusobacteria are one of the five major phyla that dominate a diverse gastric bacterial community, while having the lowest relative abundance of 5%–10%[9,52,53,55,63]. Bik et al[9] found 56 clones and 10 phylotypes of Fusobacteria in the stomach. However, the presence of Fusobacteria is influenced by the highly dominant presence of H. pylori in the stomach, which shows a lower abundance of Fusobacteria[53,55,64,65]. Klymiuk et al[55] showed that the relative abundance of Fusobacteria was significantly lower in H. pylori positive individuals infected with CagA-positive strains. The frequency of Fusobacteria in all samples examined was 2.53%, while it was 3.79% in the H. pylori-negative group. Schulz et al[53] also found a lower relative abundance of all four major phyla, including Fusobacteria, in gastric biopsy samples from H. pylori positive individuals. It has also been shown that operational taxonomic units with the annotation Fusobacterium are significantly reduced in patients treated with proton pump inhibitors[66]. Overall, based on the summarized evidence, Fusobacteria are among the most common phyla reproducibly identified in the gastric environment.

GASTRIC MICROBIOME, FUSOBACTERIA AND F. NUCLEATUM IN GC

Despite the growing evidence, there are still few studies that have investigated the microbial composition in relation to gastric carcinogenesis. In Table 1, we have summarized the most relevant studies and their characteristics related to the Fusobacteria and F. nucleatum in the stomach and GC. While during the earlier years the focus of GC was rather on other entities, it is obvious that the strong and consistent data on the potential role of F. nucleatum in gastric carcinogenesis is gaining substantial attention. As shown in Figure 1 using a time trend, significant progress has been made in understanding the potential role of F. nucleatum in GC.

Figure 1 Timeline on scientific progress on Fusobacterium nucleatum research. CRC: Colorectal cancer; GC: Gastric cancer; H. pylori: Helicobacter pylori; F. nucleatum: Fusobacterium nucleatum.

Table 1 Summary of the studies with focus on Fusobacteria and Fusobacterium nucleatum in stomach and gastric cancer.

Ref.	Year	Taxonomic rank	Methods	Sample type	Sample origin	No. of subjects	No. of controls	No. of preneoplastic conditions	No. of GC	GC	Major findings	Clinical value	Enriched in GC tissue
Bik et al[9]	2006	Fusobacteria	16S rRNA seq	Gastric biopsy	United States	23	11 H. pylori-negative, 12 H. pylori- positive			No	Frequent phyla: Proteobacteria, Firmicutes, Actinobacteria, Bacteroidetes, and Fusobacteria
Dicksved et al[63]	2009	Fusobacteria	16S rRNA seq	Gastric biopsy	Sweden	15	5 controls		10 GC	Yes	GC microbiota not significantly different from controls, with the phyla Proteobacteria, Firmicutes, Actinobacteria, Bacteroidetes, and Fusobacteria
Maldonado-Contreras et al[54]	2011	Fusobacteria	16S rRNA gene microarray	Gastric biopsy	Venezuela	12	12 healthy			No	Inverse correlation of Fusobacteria with Helicobacter
Aviles-Jimenez et al[59]	2014	Fusobacteria	16S rRNA gene microarray	Gastric biopsy	Mexico	15	5 gastritis	5 IM	5 GC	Yes	Fusobacteria among the most prevalent phyla
Parsons et al[66]	2017	Fusobacterium	16S rRNA seq	Gastric biopsy	UK	95	20 healthy, 19 PPI treated, 22 gastritis	34 AG		No	Fusobacterium lower in PPI treated patients
Yamamura et al[19]	2017	F. nucleatum	qPCR	Gastric tissue in paraffin	Japan	40	20 controls		20 GC	Yes	F. nucleatum detected in GC
Klymiuk et al[55]	2017	Fusobacterium	16S rRNA seq	Gastric biopsy	Austria	30	30 gastritis			No	Inverse correlation of Fusobacterium with H. pylori
Castaño-Rodríguez et al[22]	2017	Fusobacterium	16S rRNA seq	Gastric biopsy	Malaysia, Singapore	32	20 functional dyspepsia		12 GC	Yes	Fusobacterium enriched in GC		↑
Li et al[58]	2017	Fusobacterium	16S rRNA seq	Gastric biopsy	Hong Kong (China)	33	8 healthy, 9 gastritis	9 IM	7 GC	Yes	Fusobacterium is enriched after H. pylori eradication		↑
Coker et al[62]	2018	Fusobacterium	16S rRNA seq	Gastric biopsy	China	207	77 gastritis	74 AG; 17 IM	39 GC	Yes	Fusobacterium enriched in GC		↑
Schulz et al[53]	2018	Fusobacterium	16S rRNA seq	Saliva, gastric juice, duodenal juice, gastric biopsy, duodenal biopsy	Germany	24	8 H. pylori- positive, 16 H. pylori-negative			No	Inverse correlation of Fusobacterium with H. pylori
Hsieh et al[23]	2018	F. nucleatum	16S rRNA seq	Gastric biopsy	Taiwan	27	9 gastritis	7 IM	11 GC	Yes	Fusobacterium enriched in GC		↑
Ferreira et al[61]	2018	Fusobacteria	16S rRNA seq	Gastric biopsy	Portugal	135	81 gastritis		54 GC	Yes	Fusobacteria lower in GC; correlation with of Helicobacter		↓
Chen et al[69]	2019	Fusobacterium	16S rRNA seq	Gastric tissue	China	62			62 GC + Adj	Yes	Fusobacterium enriched in GC		↑
Shao et al[70]	2019	Fusobacteria	16S rRNA seq	Gastric tissue	China	103			36 GC + Adj	Yes	Fusobacteria enriched in GC		↑
Gantuya et al[64]	2019	Fusobacterium	16S rRNA seq	Gastric biopsy	Mongolia	75	24 controls; 11 H. pylori- positive, 40 H. pylori-negative			No	Inverse correlation of Fusobacteria with H. pylori
Guo et al[65]	2020	Fusobacterium	16S rRNA seq	Gastric biopsy	China	164	49 healthy; 115 H. pylori gastritis			No	Inverse correlation of Fusobacterium with H. pylori only in advanced gastric lesion patients
Rodriguez et al[91]	2020	F. nucleatum	Exome sequencing	Gastric tissue	United States	85			85 GC	Yes	F. nucleatum detected in GC
Gantuya et al[74]	2020	Fusobacterium	16S rRNA seq	Gastric biopsy	Mongolia	168	20 gastritis	40 IM, 40 AG	48 GC	Yes	Fusobacterium enriched in GC regardless of H. pylori infection		↑
Boehm et al[84]	2020	F. nucleatum	qPCR	Gastric tissue	Lithuania Germany	125	18 controls; 17 gastritis	9 AG/IM	81 GC + Adj	Yes	F. nucleatum-positivity associated with prognosis; lower than in CRC; no difference in AG/IM	Prognosis
Ravegnini et al[78]	2020	Fusobacteria	16S rRNA seq	Gastric tissue in paraffin	Italy	20			20 GC	Yes	signet-ring cell GCs were significantly enriched in the phyla Fusobacteria
Zhang et al[72]	2021	Fusobacteria	16S rRNA seq	Stool samples	China	146	61 healthy		85 GC	Yes	Fusobacteria enriched in GC; Fusobacteria reduced following chemotherapy		↑
Hsieh et al[86]	2021	F. nucleatum	Nested PCR	Gastric tissues	Taiwan	60			60 GC	Yes	F. nucleatum+ and H. pylori+ associated with worse prognosis	Prognosis
Nascimento et al[82]	2021	F. nucleatum	qPCR	Gastric biopsies in paraffin	Brazil	120	40 controls		80 GC	Yes	No difference to controls; F. nucleatum associated with aging
Xu et al[80]	2021	Fusobacterium	16S rRNA seq	Tongues coating	China	293	112 controls		181 GC	Yes	Fusobacterium shows diagnostic value	Diagnosis	↑
Nie et al[88]	2021	Fusobacterium	16S rRNA seq	Gastric tissue	China	61			61 GC	Yes	Fusobacteria enriched in GC and common in elderly. Fusobacterium positive GC patients more likely to have tumour-infiltrating lymphocytes
Gunathilake et al[47]	2021	F. nucleatum	16S rRNA seq	Gastric biopsy	South Korea	556	288 controls		268 GC	Yes	Fusobacteria enriched in controls		↓
Conti et al[92]	2021	Fusobacteria	16S rRNA seq	Gastric biopsy	Italy	55	32 controls	23 AG		No	Fusobacteria enriched in controls
Hsieh et al[87]	2022	F. nucleatum	TruSight Oncology 500 panel	Gastric tissue	Taiwan	36			36 GC	Yes	F. nucleatum and high tumour mutation burden are associated with poor prognosis	Prognosis
Chen et al[93]	2022	F. nucleatum	qPCR	Saliva	China	232	20 normal controls; 35 gastritis; 26 gastric polyps	31 AG	120 GC	Yes	F. nucleatum in saliva as biomarker for GC	Diagnosis	↑
Liu et al[94]	2022	Fusobacterium	16S rRNA seq	Gastric biopsy	Columbia, China, Italy, Mexico, South Korea	1270	109 healthy controls, 183 gastritis	135 AG; 124 IM, 94 IN	344 GC; 281 GC adj	Yes	Fusobacterium enriched in GC		↑
Aziz et al[85]	2022	F. nucleatum	Mass spectrometry	Gastric biopsy	Pakistan	75	12 normal controls, 38 gastritis, 12 ulcers		13 GC	Yes	Protein of F. nucleatum detectable in GC and gastritis
Zhang et al[81]	2022	Fusobacterium	16S rRNA seq	Saliva, stool, GC tissues	China	146	70 healthy controls		76 GC	Yes	Fusobacterium enriched in GC stool and saliva sample		↑
Li et al[95]	2022	Fusobacteria	16S rRNA seq	Saliva, esophageal swab, gastric juice, gastric biopsy, stool sample	China	40	22 healthy controls, 18 esophagitis			No	Fusobacteria in ~3% in all regions except faeces
Lehr et al[83]	2023	F. nucleatum	16S rRNA seq	Gastric tissue	Lithuania	64			64 GC + Adj	Yes	Higher abundance of F. nucleatum in GC tumour tissue associated with poorer survival; microbial network	Prognosis
Liu et al[89]	2023	F. nucleatum	qPCR	Gastric tissue	China	91			91 GC	Yes	Fusobacterium nucleatum conditions megakaryocytes by induction of extracellular vesicles
Wei et al[73]	2023	Fusobacterium	16S rRNA seq	Gastric juice	China	139	61 healthy controls		78 GC	Yes	Fusobacterium enriched in advanced GC		↑
Nikitina et al[46]	2023	Fusobacterium	16S rRNA seq	Gastric biopsy	Lithuania	105	29 healthy controls		76 GC	Yes	Fusobacterium abundance increased in tumour DNA versus RNA
Jeong et al[96]	2024	Fusobacteria	Whole metagenome shotgun sequencing	Stool sample	Taiwan	65	33 gastritis		32 GC	Yes	Fusobacteria enriched in GC		↑
Meng et al[90]	2024	F. nucleatum	16S rRNA seq	Gastric tissue	China	30			30 GC + Adj	Yes	F. nucleatum is more abundant in tumor than in adjacent samples and extracellular vesicles of F. nucleatum promote the growth of GC tumors and metastasis in the liver

GC: Gastric cancer; IM: Intestinal metaplasia; AG: Atrophic gastritis; PPI: Proton pump inhibitors; CRC: Colorectal cancer; H. pylori: Helicobacter pylori; F. nucleatum: Fusobacterium nucleatum.

As it has already been mentioned, there is a considerable variation in the results, which can be explained, at least in part, by the multiple factors, including the sample size, the origin of the patients, the H. pylori status, as well as the methodological factors. Therefore, taking into account the heterogeneity, it remains to be determined what role does the microbiome play in gastric carcinogenesis and whether the interaction may include H. pylori or may even go beyond directing carcinogenesis[67,68].

Several studies have been conducted to evaluate the alteration of the microbiome in GC. One of the earliest studies found that the microbiota did not differ significantly between controls and GC patients[63]. However, several studies later found a variety of differences, for example a group from Taiwan analyzed the gastric microbiota in patients with gastritis, IM and GC[23]. The results showed that the prevalence of H. pylori was significantly lower in the gastric microbiota of patients with GC compared to patients with noncancerous gastric diseases, while the abundance of Fusobacterium, Clostridium and Lactobacillus species was higher in patients with GC. Similar results have been reported by other groups. For instance, Chen et al[69] and Shao et al[70] reported that Fusobacterium was more abundant in tumor compared to nontumor tissue.

Wang et al[71] analyzed changes in the gastric microbiome at different stages of neoplastic progression. Bacterial diversity decreased progressively from nonatrophic CG to GC[71-80]. Actinobacteria, Bacteriodes, Firmicutes, Fusobacteria, SR1 and TM7 were more abundant in intraepithelial neoplasia and GC compared to other histological types. Zhang et al[81] reported that Fusobacteria are also enriched in GC, but also in patients who have undergone chemotherapy. In addition, Wei et al[73] found an increased abundance of F. nucleatum in the gastric juice of patients with advanced GC.

The study by Castaño-Rodríguez et al[76] conducted in ethnic Chinese subjects, compared the gastric microbiomes of GC patients and controls with functional dyspepsia. They found that the gastric microbiome was significantly different in GC, and these differences were independent of the contribution of H. pylori to relative abundance, which was also reported by Gantuya et al[74]. Bacterial genera commonly found in the oral cavity including Fusobacterium, Veillonella, Leptotrichia, Haemophilus and Campylobacter were enriched in GC patients. The authors discussed that Fusobacteria may induce inflammation, with Toll-like receptor (TLR)4 and autophagy playing an important role, while polymorphisms in TLR4 and autophagy have been shown to increase the risk of developing GC in Chinese individuals[75,76]. In addition, Fusobacteria have been shown to coaggregate with other bacteria to form biofilms, which play an important role in colorectal adenocarcinoma, and Veillonella species could be a plausible pro-oncogenic partner, as enrichment of these bacteria has been found in other types of cancer[77].

Ravegnini et al[78] identified potential microbial biomarkers associated with GC subtypes. In signet-ring cell carcinoma, the phyla Fusobacteria, Bacteroidetes and Patescibacteria were significantly enriched, whereas, in the adenocarcinoma type, Proteobacteria and Acidobacteria were found.

Based on the observation of an increased abundance of Fusobacteria or F. nucleatum in GC, efforts have been made to evaluate the potential value of the microbiome as a diagnostic biomarker. For example, the combination of F. nucleatum and Clostridium colicanis in tissue has been proposed as a diagnostic biomarker for GC with a sensitivity of 73% and specificity of 100%[23]. While the tumor microbiome may be of limited translational and clinical value for diagnostic purposes, the potential differences in the microbiome are still helpful in identifying potential species in saliva, blood or faces. In particular, the vicinity of the oral and gastric niches may be most exciting in terms of identifying non-invasive microbiome-based biomarkers for GC.

The over-representation of the members of the oral microbiome in GC was reported by Coker et al[62] in a Chinese population. They observed that 14 of the 21 bacterial taxa enriched in GC were bacteria commonly found in the oral cavity, such as Parvimonas micra, Peptostreptococcus stomatis, Gemella and F. nucleatum. After adjusting for potential confounders, including age, sex, tissue location and H. pylori status, the differences between GC and all precancerous stages remained statistically significant. The diversity and richness of the microbiota was increased in tumor tissue. The enriched bacterial taxa were represented by oral bacteria such as Peptostreptococcus, Streptococcus and Fusobacterium, while lactic acid producing bacteria such as Lactococcus lactis and Lactobacillus brevis were more abundant in adjacent nontumor tissue. The association between periodontal disease caused by oral microbial dysbiosis and GC was confirmed in a meta-analysis of observational studies[79].

Going one further Xu et al[80] and also Zhang et al[81] investigated the differences of the microbiome and specifically F. nucleatum in oral specimens from GC patients. They showed that both tongue coating and saliva samples can be used as potential biomarkers for GC patients.

Although several studies have reported the higher abundance of Fusobacterium in GC patients, there are also studies that have not been able to confirm the data. Specifically, Nascimento et al[82] found no differences in the abundance of F. nucleatum between GC patients and noncancer controls, but an association of F. nucleatum with age has been reported. Similarly, Ferreira et al[61] even reported a decrease in the relative abundance of Fusobacteria in patients with GC compared to patients with gastritis, 0.5% and 1.8%, respectively. The enrichment of other bacterial genera, mostly represented by intestinal commensals, was found. Two other studies reported a higher abundance of Fusobacterium in GC tissues compared to adjacent tissues, but the data did not reach significance using either quantitative qPCR analysis or 16S rRNA sequencing[83,84]. In addition, the authors also included the subcohort of subjects with normal mucosa, CG including H. pylori and preneoplastic conditions such as AG and IM, but although the variation in GC samples was much higher than in the non-neoplastic cohort, the differences did not reach quantitatively significant differences[84]. It is worth mentioning that all these studies did not include Asian population but were from Brazil, Portugal, Lithuania and Germany, suggesting that many factors may be attributed to the variation of microbiome and Fusobacteria in gastric mucosa and GC.

Most detections of F. nucleatum in the stomach are based on sequencing techniques, but these results do not guarantee that the bacteria detected are transcriptionally active. Recently, Aziz et al[85] found F. nucleatum proteins in gastric biopsies from patients with GC, demonstrating that these bacteria are metabolically active in this biological niche. This supports the hypothesis that F. nucleatum plays a mechanistic role in GC. Furthermore, Nikitina et al[46] have also reported differences between 16S rRNA data depending on the samples used, either DNA or RNA, suggesting that this variable may also contribute to the differences between studies.

F. NUCLEATUM AND PROGNOSIS OF GC PATIENTS

Analysis of the microbiota in gastric tumors not only provides evidence of its potential role in cancer initiation, but may also lead to the identification of its potential impact on prognosis and prediction of treatment response. In particular, the first evidence has been reproducibly reported for CRC showing an association between F. nucleatum and worse prognosis of CRC patients, including poor overall survival[29].

Boehm et al[84] performed the first analysis to investigate the prognostic role of Fusobacterium and specifically F. nucleatum in GC patients. Using a qPCR-based quantitative targeted approach, the authors found that F. nucleatum positivity was associated with significantly worse overall survival in patients with GC, specifically Lauren's diffuse type GC. These findings were further supported by Hsieh et al[86] in a GC patient cohort from Taiwan. The authors also showed that the combination of F. nucleatum infection and high tumor mutation burden is a highly effective biomarker for poor prognosis[87]. In these studies, F. nucleatum was detected by qPCR, but last year Lehr et al[83] showed in a 16S rRNA-based study that F. nucleatum in tumor tissue, but not in adjacent tissue, was associated with worse overall survival, independent of GC classification. In particular, phylogenetic tree analysis linked F. nucleatum and in some cases Fusobacterium necrophorum to GC, while Fusobacterium peridonticum was also frequently observed in healthy controls[83].

The mechanistic reason for this effect on survival has not been elucidated and mechanistic data are generally limited. Abed et al[39] found high levels of Gal-GalNAc in normal and cancerous gastric cells, suggesting that Fusobacteria can enter and accumulate in gastric cells and may be involved in carcinogenesis by inducing inflammation and inhibiting immune cell activity. It has also recently been reported that GC patients infected with Fusobacterium sp. are more likely to have tumor-infiltrating lymphocytes and p53 expression[88].

Liu et al[89] recently demonstrated that F. nucleatum colonization is associated with the occurrence of portal vein thrombosis in patients with GC. However, the underlying mechanism by which F. nucleatum promotes thrombosis remained unclear. They also found that F. nucleatum-positive patients had increased neutrophil extracellular traps and thrombocyte counts. Extracellular vesicles from F. nucleatum-positive patients were able to promote the differentiation and maturation of megakaryocytes and had upregulated 14-3-3 proteins, particularly 14-3-3ε. Upregulation of 14-3-3ε promotes megakaryocyte differentiation and maturation in vitro[89].

The extracellular vesicles of F. nucleatum appear to play a major role in other molecular mechanisms in GC. In vivo analyses have shown that they not only promote the growth of GC tumors and metastasis in the liver, but also the resistance of GC tumors to oxaliplatin[90]. Together with the results of Liu et al[89], this makes the extracellular vesicles of F. nucleatum a highly exciting field of research for the future.

Although limited evidence is available to elucidate the translational role of microbial deregulation and F. nucleatum positivity, the evidence from other cancers may help to explore the pathogenic mechanisms of F. nucleatum in GC are unknown.

CONCLUSION

GC remains a common malignancy worldwide, characterized by high incidence and poor prognosis. This review provides a comprehensive overview of the current knowledge on microbiome deregulation, with a special focus on Fusobacteria and F. nucleatum in GC. Despite accumulating evidence implicating Fusobacterium in GC pathogenesis, several research gaps remain. First, there is considerable heterogeneity in F. nucleatum positivity among reported GC data, which may be due to differences in the demographics of the cohorts studied (Asian vs non-Asian populations). Therefore, future studies should aim to provide more robust evidence on this issue. Second, although sample sizes in studies, particularly those investigating the diagnostic role of F. nucleatum, are generally adequate, further improvements are warranted. In particular, analysis of microbiome data at higher taxonomic levels may not provide superior value given the predominant association of F. nucleatum with GC. Thirdly, the mere identification of F. nucleatum in tumor tissue is not sufficient to establish its carcinogenic role, especially as samples with preneoplastic conditions do not show significant differences from controls. One of the most intriguing aspects of F. nucleatum in GC is its potential prognostic significance. Although not yet translated into clinical implications for GC patients, it suggests the emergence of novel therapeutic strategies. These may include specific targeted therapeutics or even bacteriophages designed to selectively target F. nucleatum. In summary, our understanding of the contribution of the microbiome to GC has advanced significantly. F. nucleatum is emerging as a reproducible and clinically relevant species, providing insights beyond H. pylori infection. Going forward, high-quality data are essential to comprehensively characterize microbial alterations and explore translational implications, including novel therapeutic strategies and mechanistic insights.