Unveiling hidden players: the role of intratumoral microbiota in gastrointestinal cancer dynamics.

Introduction

The expanding concept of the tumour microbiome
Over the past decade, the study of cancer-associated microorganisms has undergone a paradigm shift from an exclusive focus on the gut microbiome to the direct investigation of the intratumoral microbiota. In 2020, Nejman et al. analysed 1,526 tumour tissues and matched adjacent normal samples across seven cancer types and identified characteristic intratumoral microbiota that predominantly resided within both tumour and immune cells (Nejman et al. 2020). Initially recognized as commensals confined to the gastrointestinal lumen, microbial communities are now known to be translocated via portal-venous, biliary, and haematogenous routes, ultimately colonizing primary and metastatic digestive tumours. Within the tumour microenvironment (TME), these microorganisms, including bacteria, fungi, viruses, and their bioactive metabolites, constitute a discrete biological compartment that intersects with malignant, stromal, and immune cells. By engaging pattern recognition receptors, remodelling metabolic circuits, and modulating epigenetic landscapes, microbial components actively rewrite oncogenic signalling and immune checkpoints. Consequently, the “tumour microbiome” has evolved from a contaminant to be eliminated into an integral dimension of TME complexity, necessitating its integration into contemporary oncologic frameworks for precision diagnosis and therapy.

Why digestive organs are uniquely predisposed to intratumoral microbes
Digestive organs represent an unrivaled ecological gateway for intratumoral colonization, a predisposition rooted in their intimate anatomical, physiological, and immunological interfaces with the microbial world. First, the luminal surface of the gastrointestinal tract harbours the body’s densest microbial biomass, estimated at 3 × 1013 organisms, whose metabolites, endotoxins, and extracellular vesicles are continuously shuttled across a single epithelial layer (Sender et al. 2016). Second, portal venous and biliary drainage systems act as low-resistance conduits, allowing gut-derived bacteria such as Fusobacterium nucleatum (F. nucleatum) and Escherichia coli to traverse directly into the hepatic parenchyma and pancreatic ducts, where they seed primary tumours or foster premetastatic niches (Bullman et al. 2017; Bertocchi et al. 2021). Third, digestive tumours themselves exhibit microenvironmental hallmarks, such as hypoxia zones, necrotic cores, and immunosuppressive cytokine milieus, that preferentially sustain anaerobic and facultative anaerobic taxa (Nejman et al. 2020). Finally, mucosal barrier dysfunction, a common consequence of chronic inflammation, viral hepatitis, or alcohol abuse, amplifies microbial translocation and enables persistent intratumoral residence (Ke et al. 2025). Collectively, these features render digestive malignancies uniquely susceptible to microbial integration, thereby redefining the tumour microenvironment as a tripartite ecosystem of host cells, malignant clones, and microbial constituents.

Anatomical-physiological nexus: entry and retention of the intratumoral microbiota in digestive cancers

Gut-tumour translocation via the portal vein and biliary tree
The gut‒liver axis constitutes the most direct conduit for microbial translocation into hepatic and peri-ampullary malignancies. Under physiological conditions, portal venous blood carries ≤ 103 colony-forming units (CFUs) per millilitre; however, barrier dysfunction in chronic liver disease or colorectal neoplasia increases this load by > 100-fold (Sookoian et al. 2020). Escherichia coli expressing the virulence regulator VirF disrupts the gut vascular barrier, allowing bacterial and luminal metabolites to seed the hepatic parenchyma and establish a premetastatic niche enriched in TLR4 ligands (Bertocchi et al. 2021). Similarly, biliary reflux in patients with primary sclerosing cholangitis has been shown to transport gut-derived Fusobacterium and Bacteroides species directly into intrahepatic cholangiocarcinoma lesions, as confirmed by fluorescence in situ hybridization (Chng et al. 2016).

Oral-oesophageal seeding and swallowing-to-tumour routes
The oropharyngeal cavity harbours > 700 bacterial taxa that can be aspirated or swallowed into the upper digestive tract. In oesophageal squamous cell carcinoma, P. gingivalis DNA is localized within the tumour epithelium according to RNAscope, suggesting that repeated microaspiration seeds the lower oesophagus with periodontopathic organisms (Tsay et al. 2018). In vivo murine models further demonstrated that oral gavage with F. nucleatum results in sustained colonization of oesophageal xenografts, promoting IL-6/STAT3-mediated tumorigenesis (Abed et al. 2016).

Haematogenous dissemination from distant mucosal reservoirs
Beyond local mucosal surfaces, systemic circulation serves as a pathway for microbial dissemination. Metagenomic sequencing of peripheral blood from cirrhotic patients with hepatocellular carcinoma revealed a distinct blood microbiome signature enriched in Proteobacteria and Enterococcaceae (Cho et al. 2019). These circulating microbes exhibit tropism for tumour-associated neovasculature characterized by fenestrated endothelium and reduced pericyte coverage, facilitating extravasation into gastric and pancreatic tumours (Fu et al. 2022).

Cometastasis of microorganisms with circulating tumour cells
Recent single-cell sequencing analyses have demonstrated that intracellular bacteria can hitchhike within circulating tumour cells (CTCs). In a mouse model of colorectal cancer liver metastasis, F. nucleatum was detected within EpCAM-positive CTC clusters, enhancing their resistance to shear stress and promoting metastatic outgrowth (Zhang et al. 2022). Metastatic lesions retain microbial strains identical to those of primary tumours, indicating that microorganisms migrate alongside malignant cells rather than independently (Sender et al. 2016).

Methodological landscape for microbiota detection
The continuous advancement of sequencing technology has laid the foundation for modern microbiome research, with next-generation sequencing playing a pivotal role. The composition and function of the intratumoral microbiome can now be elucidated through an increasing array of methodologies. Current mainstream microbiome sequencing techniques, particularly 16S rRNA gene sequencing and shotgun metagenomic sequencing, have been widely applied in the study of the intratumoral microbiota (Fig. 1). However, each of these conventional approaches presents distinct advantages and limitations when applied to low-microbial-biomass tumour samples. In response to these challenges, several emerging techniques and analytical strategies specifically tailored for investigations of such low-biomass intratumoral microbiomes have been developed.

Immunohistochemistry (IHC)
Immunohistochemistry (IHC) serves as a valuable and complementary tool for visualizing the intratumoral microbiota within the tissue microenvironment. Unlike sequencing-based methodologies that characterize microbial composition and functional potential, IHC utilizes pathogen-specific antibodies to detect and localize entire microorganisms or specific microbial antigens directly in formalin-fixed, paraffin-embedded (FFPE) tissue sections. This approach provides a unique spatial context, allowing researchers to precisely identify whether bacteria are located within cancer cells, in the stroma, in areas of necrosis, or associated with specific immune cell populations.
The primary advantage of IHC lies in its ability to offer high-resolution spatial information, which is crucial for distinguishing true intratumoral residents from mere contaminants, adventitia, or commensals from adjacent normal tissue. This technique also facilitates the direct correlation of microbial presence with histopathological features, such as tumour grade, immune infiltration, and metastatic foci. However, a significant limitation of IHC is its inherent reliance on predefined targets; it can only detect microorganisms for which specific and validated antibodies are available, making it a hypothesis-driven rather than a discovery-based tool. Furthermore, issues of antibody cross-reactivity with host tissues can lead to false positives, and the technique is generally considered less sensitive than nucleic acid amplification-based methods for low-abundance microbes, potentially leading to false-negatives.
IHC is most effectively employed in scenarios where a specific, suspected pathogen is implicated in tumorigenesis. A classic and widely accepted application is the detection of Helicobacter pylori in gastric adenocarcinoma and gastric mucosa-associated lymphoid tissue (MALT) lymphoma. For this specific bacterium, IHC is highly sensitive and specific and is often considered the diagnostic gold standard in pathology practice, allowing direct visualization of the curved bacilli on the gastric epithelium. In addition to H. pylori, IHC has been explored for the detection of other intratumoral microbes, such as F. nucleatum in colorectal cancer and certain bacteria, such as Escherichia coli, associated with colorectal neoplasia.

16S rRNA gene amplicon sequencing
16S ribosomal RNA (rRNA) gene amplicon sequencing is a cornerstone technique for the taxonomic profiling of the intratumoral microbiota. It permits the identification of bacterial communities to the genus level and can reveal dysbiosis associated with different GI cancer subtypes. High-throughput 16S rRNA sequencing was used to analyse the variable region sequences of bacterial 16S rRNA genes to determine the taxonomic characteristics of each bacterium (Xue et al. 2023; Zhang et al. 2023); however, its resolution is limited at the species level, and it is highly susceptible to contamination from environmental DNA and reagents, which is a critical concern in low-biomass samples such as tissue. In addition, microorganisms other than bacteria cannot be identified through 16S rRNA sequencing. To improve microbial coverage and resolution, more 16S rDNA sequencing methods, such as 5R 16S rDNA sequencing, have been developed (Nejman et al. 2020).

Macrogenomic sequencing
The development of metagenomics has advanced the study of the tumour microbiome. The term “macrogenome” refers to the sum of the genomes of all microorganisms in the environment. Unlike 16S rRNA sequencing, metagenomics not only targets a specific microbial community (fungi, bacteria, or viruses) for targeted sequencing but also performs sequence analysis on the sum of all microbial genomes. Therefore, this technique has advantages in terms of precise species identification and can be used to infer the functional characteristics of the microbial community; its shortcomings lie in the fact that the assembly and alignment of sequences are limited by the reference sequence entries in microbial sequence databases, and the vast majority of genetic information extracted from tumour tissues is human DNA, with limited data on low-content microorganisms.

Fluorescence in situ hybridization (FISH)
FISH plays an important role in detecting the gut microbiota. The novel FISH technique called RNAscope-FISH can reduce background noise, increase signals, and observe molecules within individual cells. This technology has been pivotal in linking the spatial localization of F. nucleatum with poor prognosis and immune cell exclusion in CRC (Wang et al. 2012; Galeano Niño et al. 2022).

Single-cell sequencing (scRNA-seq)
The application of scRNA-seq to GI tumours represents a transformative approach for delineating the complex ecosystem of host cells and resident microorganisms at unprecedented resolution (Galeano Niño et al. 2022). While conventional bulk sequencing methods have revealed associations between specific pathogens and cancer progression, they obscure the critical cellular heterogeneity of both the host and the microbiome. Recent pioneering studies have begun to overcome significant technical challenges—including low microbial biomass, high host-to-microbe RNA ratios, and potential contaminants—inherent in single-cell analyses of the intratumoral microbiota. A key advancement is the development of sophisticated computational pipelines designed to mine microbial reads from host-derived scRNA-seq libraries. For instance, studies in CRC have leveraged these approaches not only to profile the transcriptional states of tumour-infiltrating immune and stromal cells but also to detect intracellular bacteria within specific epithelial cell subpopulations (Dohlman et al. 2022); this has provided direct evidence of microbial tropism for particular tumour cell types. Furthermore, the integration of scRNA-seq with other single-cell modalities, such as cellular indexing of transcriptomes and epitopes (CITE-seq), allows for the correlation of bacterial presence with detailed host cell surface protein expression, offering a more holistic view of host‒microbe interactions in the tumour microenvironment (TME)(Zhao et al. 2022a). The emerging field of single-cell metatranscriptomics holds immense potential to move beyond taxonomic identification and characterize the in situ metabolic activity and gene expression of intratumoral microbes. Such work could reveal how bacterial virulence pathways are activated in direct response to specific niches within a tumour. However, rigorous standardization and the implementation of robust negative controls remain paramount for distinguishing true microbial signals from artefacts (Gihawi et al. 2023). As these technologies mature, they are poised to elucidate the fundamental rules governing which host cells harbour microbes and how these interactions directly influence carcinogenesis, immune evasion, and the response to therapy in GI cancers.

Spatial transcriptomics
The emergence of spatial omics technologies has revolutionized our understanding of the intratumoral microbiota in gastrointestinal (GI) cancers by preserving the critical anatomical context of host‒microbe interactions (Galeano Niño et al. 2022; Zhao et al. 2022b; Erickson et al. 2022). Moving beyond bulk sequencing, which homogenizes tissue architecture, these methods enable precise mapping of microbial localization within specific tumour niches and their correlation with distinct cellular and molecular features of the TME. A pivotal advancement is the application of highly multiplexed in situ hybridization techniques. For instance, the development of platforms capable of simultaneously targeting bacterial 16S rRNA and host mRNA transcripts has enabled the comapping of numerous microbial taxa alongside tumour and immune cell populations at subcellular resolution. Zhao et al. developed slide DNA seq, a method for capturing spatially resolved DNA sequences from complete tissue sections (Zhao et al. 2022b). This method accurately preserves the local tumour structure and can rediscover different tumour clones and their copy number changes. Applying slide DNA-seq to transfer mouse models and primary human cancers can reveal the limitations of clone populations to different spatial regions. In addition, by integration with spatial transcriptomics, they discovered different gene sets associated with clone-specific genetic abnormalities, local tumour microenvironments, or both. Galeano Niño JL et al. used GeoMx digital spatial profiling in CRC and reported that compared with bacteria-negative tumour regions, bacterial communities populating microniches were less vascularized and highly immunosuppressive and associated with malignant cells with lower levels of Ki-67(Galeano Niño et al. 2022).
These spatial profiling efforts have revealed the fundamental principles of microbial ecology within GI tumours. They demonstrated that the intratumoral microbiome is spatially structured, forming microniches shaped by gradients of nutrients, hypoxia, and immune activity. Furthermore, the integration of spatial data with bulk sequencing has been instrumental in distinguishing true tissue-resident microbes from potential contaminants introduced during sampling or processing, thereby refining our definition of the cancer-associated microbiome (Dohlman et al. 2021). Despite this progress, challenges remain, including the sensitivity for detecting low-biomass communities and the development of robust bioinformatic pipelines for integrated analysis. As these technologies mature and become more widely adopted, spatial omics is poised to definitively establish the causal roles of specific host–microbe interactions in tumour initiation, progression, and therapeutic resistance, ultimately guiding novel spatially informed diagnostic and therapeutic strategies.

Microbial community profiles across digestive cancer types

Oesophageal carcinoma
The intratumoral microbiota has emerged as a significant contributor to the pathogenesis and progression of oesophageal carcinoma, which can be histologically categorized into oesophageal squamous cell carcinoma (ESCC) and oesophageal adenocarcinoma (EAC). Distinct microbial signatures are associated with each subtype and are influenced by anatomical location and aetiological factors. In ESCC, which is often linked to oral dysbiosis, enrichment of periodontal pathogens such as Porphyromonas gingivalis and F. nucleatum is frequently observed. Conversely, EAC, which typically arises from Barrett's oesophagus in the context of gastroesophageal reflux, often demonstrates a predominance of gram-negative bacteria of enteric origin (Li et al. 2023; Gillespie et al. 2021). These microbes are not only passive but also actively participate in tumorigenesis through multiple mechanisms, including the induction of chronic inflammation, the production of genotoxins, and the modulation of host cell proliferation and survival pathways.
The functional impact of these microbes on EC progression is profound. F. nucleatum, for instance, promotes tumour cell proliferation, invasion, and resistance to apoptosis through interactions mediated by its FadA adhesin, which activates β-catenin signalling and inflammatory cascades via the TLR4/NF-κB pathway (Yamamura et al. 2016). This creates a protumorigenic microenvironment conducive to cancer growth and metastasis. Critically, the intratumoral microbiota also substantially influences therapeutic outcomes. A high intratumoral burden of F. nucleatum has been correlated with poor response to neoadjuvant chemotherapy in ESCC patients, suggesting that it plays a role in mediating chemoresistance (Yamamura et al. 2019). In contrast, emerging evidence points to a potential beneficial role for other microbial constituents. Specifically, enrichment of intratumoral Streptococcus has been associated with a favourable response to neoadjuvant chemoimmunotherapy (NACI) in ESCC. This effect is mechanistically linked to enhanced CD8+ T-cell infiltration and activation within the tumour microenvironment, thereby sensitizing tumours to immune-mediated attack (Wu et al. 2023a). These findings suggest that the composition of the intratumoral microbiome could serve as a predictive biomarker for treatment selection.
In summary, the intratumoral microbiota in oesophageal cancer is a key determinant of disease biology and therapeutic efficacy. The balance between “harmful” microbes such as F. nucleatum, which drive aggressiveness and resistance, and “beneficial” microbes such as Streptococcus, which may potentiate immunotherapy, opens up novel avenues for prognostic stratification and combination therapies. Future research focusing on modulating the tumour microbiome, perhaps via targeted antimicrobial or microbial consortia transplantation, holds promise for improving the management of this deadly malignancy.

Gastric cancer
Compared with normal gastric mucosa, the gastric intratumoral microbiota is significantly altered and characterized by decreased microbial diversity and enrichment of specific bacterial taxa. Helicobacter pylori (H. pylori) is the most significant biological carcinogen in gastric carcinogenesis and employs sophisticated molecular strategies to initiate and promote malignant transformation. The pathogenicity of the bacterium is largely determined by the presence of the cag pathogenicity island (cagPAI), which encodes a type IV secretion system (T4SS). Through this apparatus, H. pylori translocates the cytotoxin-associated gene A (CagA) protein into gastric epithelial cells, where it is phosphorylated by Src and Abl kinases at EPIYA motifs. Phosphorylated CagA aberrantly activates multiple signalling pathways, including the MAPK/ERK, PI3K/Akt, and, notably, Wnt/β-catenin pathways, leading to the transcriptional upregulation of genes involved in cell proliferation and survival (Hatakeyama 2014). Simultaneously, CagA disrupts apical-junctional complexes by binding to tight junction proteins, including ZO-1 and JAM, resulting in loss of cell polarity and enhanced epithelial permeability—early events in gastric carcinogenesis (Amieva and Peek 2016). Vacuolating cytotoxin A (VacA) contributes to gastric pathology through multiple mechanisms. VacA forms anion-selective channels in mitochondrial membranes, inducing cytochrome c release and apoptosis while simultaneously inhibiting T-cell proliferation and activation, thereby compromising host immune surveillance (Cover and Blanke 2005). Recent studies have revealed that specific VacA genotypes (particularly s1/m1 strains) demonstrate enhanced virulence and are strongly associated with gastric cancer risk. Chronic inflammation represents a cornerstone of H. pylori-induced gastric carcinogenesis. The bacterium activates nuclear factor-kappa B (NF-κB) through multiple pattern recognition receptors, including Toll-like receptors (TLRs) and NOD-like receptors, leading to sustained production of proinflammatory cytokines such as IL-1β, IL-6, IL-8, and TNF-α (Polk and Peek 2010). This inflammatory milieu generates a mutagenic environment through reactive oxygen species (ROS) and reactive nitrogen species (RNS), causing DNA damage and genomic instability in gastric epithelial cells. H. pylori infection induces comprehensive epigenetic alterations that contribute to gastric tumorigenesis. Bacteria promote DNA hypermethylation of tumour suppressor gene promoters, including those of CDKN2A (p16), MLH1, and E-cadherin (CDH1), through the induction of DNA methyltransferases (DNMTs) and inflammation-mediated epigenetic changes (Maeda et al. 2017). Additionally, H. pylori alters histone acetylation and methylation patterns, further altering the transcriptional landscape of gastric epithelial cells. These epigenetic modifications facilitate the gradual transition from normal gastric mucosa to chronic gastritis, atrophy, intestinal metaplasia, dysplasia, and, ultimately, invasive carcinoma. The bacterium extensively remodels the gastric microenvironment to support carcinogenesis. H. pylori infection induces the expression of activation-induced cytidine deaminase (AID), which promotes somatic mutations in the TP53 tumour suppressor gene and other cancer-related genes (Matsumoto et al. 2007). Furthermore, the bacterium manipulates gastric stem cell dynamics, expanding a population of cells with increased susceptibility to malignant transformation.
H. pylori contributes to gastric cancer progression through multiple interconnected pathways. The bacterium enhances epithelial‒mesenchymal transition (EMT) by upregulating the expression of transcription factors, including Snail, Slug, and ZEB1, through CagA-dependent and inflammation-mediated mechanisms (Bessède et al. 2014). This induction of EMT increases cancer cell invasiveness and facilitates metastatic dissemination. Additionally, H. pylori infection promotes angiogenesis by upregulating vascular endothelial growth factor (VEGF) and hypoxia-inducible factor-1α (HIF-1α), creating a vascular network that supports tumour growth and metastasis.
Recent evidence has indicated that H. pylori can establish persistent intracellular populations within gastric epithelial cells, evading host immune responses and antibiotic treatments. These intracellular reservoirs serve as continuous sources of carcinogenic factors and inflammatory stimuli, contributing to disease progression and treatment failure (Zhang et al. 2020). The effect of H. pylori eradication on gastric cancer prevention is well established. Large-scale intervention studies have demonstrated that H. pylori eradication significantly reduces the incidence of gastric cancer, particularly in high-risk populations (Lee et al. 2016). However, the timing of eradication is critical, with maximal benefit observed before the development of preneoplastic lesions such as intestinal metaplasia and atrophy. With respect to cancer therapy, H. pylori infection influences treatment responses through multiple mechanisms. H. pylori modulates host immunity in GC patients treated with S-1 adjuvant chemotherapy (Koizumi et al. 2022). Conversely, H. pylori-derived CagA enhances glucose anabolism in gastric cancer cells and amplifies receptor tyrosine kinase signalling, thereby conferring chemoresistance (Gao et al. 2020; Chichirau et al. 2019). Recent emerging retrospective evidence has indicated that Helicobacter pylori infection attenuates the therapeutic efficacy of immune checkpoint inhibitors in patients with gastric malignancies (Gong et al. 2023).
The management of H. pylori infection faces challenges, including increasing antibiotic resistance rates, particularly to clarithromycin and metronidazole. Molecular detection of resistance mutations and tailored eradication regimens based on antimicrobial susceptibility testing are becoming increasingly important for successful eradication (Malfertheiner et al. 2022). Furthermore, the development of vaccines against H. pylori represents a promising strategy for the primary prevention of H. pylori-associated gastric cancer, although its clinical efficacy remains to be demonstrated in large-scale trials.
While Helicobacter pylori remains the most well-established bacterial carcinogen in gastric cancer (GC), recent high-throughput sequencing studies have revealed a complex microbial landscape beyond H. pylori. Comprehensive analyses of the gastric mucosal microbiota revealed substantial microbial dysbiosis in gastric cancer patients, characterized by decreased alpha diversity and distinct enrichment patterns of specific bacterial taxa. The non-H. pylori intratumoral microbiota in GC tissues shows distinct compositional patterns across molecular subtypes, with notable enrichment of genera such as Streptococcus, Pseudomonas, Staphylococcus, and Methylobacterium (Abate et al. 2022; Peng et al. 2022). The spatial distribution of these microbes within tumours is heterogeneous, with certain bacteria demonstrating preferential localization in specific tumour regions, potentially influenced by local microenvironmental conditions, including pH, oxygen tension, and nutrient availability.
The mechanisms through which these non-H. pylori bacteria contribute to gastric carcinogenesis through multiple interconnected pathways. Streptococcus species can induce DNA damage through ROS production and promote cellular proliferation via the activation of cyclin D1 and c-Myc expression. Furthermore, specific strains of Lactobacillus and Veillonella enhance inflammatory responses through the activation of Toll-like receptors (TLRs), particularly TLR2 and TLR4, leading to sustained NF-κB signalling and the production of proinflammatory cytokines, including IL-6, IL-8, and TNF-α (Ferreira et al. 2018). This chronic inflammatory microenvironment facilitates epithelial damage and accelerates the carcinogenic cascade from chronic gastritis to intestinal metaplasia and adenocarcinoma. F. nucleatum (F. nucleatum) has been shown to have particularly potent carcinogenic effects on gastric tissue (Hsieh et al. 2022, 2021). It may increase tumour mutational burden by inducing DNA damage and simultaneously inhibiting the DNA mismatch repair (MMR) pathway (Hsieh et al. 2022; Okita et al. 2020; Liu et al. 2023). F. nucleatum accelerates gastric cancer progression by activating the IL-17/NF-κB axis, remodelling the tumour microenvironment, recruiting tumour-associated neutrophils, and thereby upregulating PD-L1 expression to facilitate immune evasion (Sorino et al. 2025; Brennan et al. 2021; Zhou et al. 2022). Additionally, the bacterium increases tumour aggressiveness through cytoskeletal reorganization, and infection-elicited extracellular vesicles promote neoplastic proliferation, invasion, and metastatic spread (Meng et al. 2024).
The non-H. pylori gastric microbiome holds promise as a source of novel diagnostic biomarkers. Specific microbial signatures, including increased Streptococcus abundance and decreased Rothia abundance, demonstrate potential for early detection of gastric cancer and its precursor lesions (Liu et al. 2019). Furthermore, monitoring dynamic changes in gastric microbiota composition during treatment may provide valuable insights into treatment response and disease progression. Despite significant advances, several challenges remain in understanding the complex role of non-H. pylori microbiota in gastric cancer. The dynamic nature of microbial communities and their spatial heterogeneity within the stomach necessitate more sophisticated sampling and analysis approaches.

Colorectal cancer
The intratumoral microbiota of colorectal cancer originates primarily from the gut lumen, oral cavity, and adjacent normal mucosa; the compromise of the intestinal barrier and haematogenous dissemination collectively facilitate their enrichment within the tumour niche (Bertocchi et al. 2021). Studies have revealed that the composition of the intratumoral microbiota differs significantly among the molecular subtypes of colorectal cancer. Dmitri Mouradov et al. profiled the intratumoral microbiome of colorectal cancer (CRC) patients by 16S rRNA sequencing of paired tumour and adjacent normal tissues (Mouradov et al. 2023). They delineated three oncomicrobial community subtypes (OCSs)—OCS1, OCS2 and OCS3—that mirror distinct molecular and anatomical features of CRC. OCS1 (21% of cases) is enriched in F. nucleatum (F. nucleatum) and other oral pathogens, exhibits a proteolytic metabolic signature, and is tightly linked to right-sided, high-grade tumours that are MSI-high, CIMP-positive, and CMS1 and frequently harbour BRAF V600E and FBXW7 mutations. OCS2 (44%) is dominated by Firmicutes/Bacteroidetes and displays a saccharolytic phenotype, whereas OCS3 (35%) is characterized by Escherichia/Pseudescherichia/Shigella and active fatty acid β-oxidation; both OCS2 and OCS3 occur predominantly in left-sided tumours with chromosomal instability (CIN). Notably, OCS1 tumours exhibit a clear MSI-related mutational signature, whereas OCS2 and OCS3 tumours present mutation patterns that are indicative of reactive-oxygen-species-induced DNA damage (Mouradov et al. 2023).
Tumours of the left and right colon differ markedly in terms of epidemiology, clinical presentation, molecular profile, therapeutic response and prognosis. Recent work further revealed that their intratumoral microbial composition and abundance are distinctly different between right and left hemicolon tumours (Mouradov et al. 2023; Kolisnik et al. 2023; Oliveira Alves et al. 2024). Mouradov et al. demonstrated that right-sided tumours are preferentially colonized by oral-derived pathogens—most notably Fusobacteria—whereas left-sided lesions are dominated by Firmicutes, Bacteroidetes, Escherichia or Shigella (Mouradov et al. 2023). Complementing these findings, Younginger B.S. et al. documented a pronounced enrichment of Fusobacterium in association with right-sided tumours, high microsatellite instability (MSI-H), and BRAF mutation, reinforcing the notion that microbiota partitioning between the left and right colon is reproducible (Younginger et al. 2023).
Intratumoral microbiota act as double-edged swords, either accelerating or restraining colorectal tumorigenesis through distinct biological circuits. Collectively, they shape the fate of intestinal epithelial cells, neoplastic cells, and the entire tumour microenvironment; their mechanistic repertoire includes the induction of DNA damage, the modulation of apoptosis, and the orchestration of epithelial–mesenchymal transition, thereby tipping the balance between tumour suppression and progression (Cao et al. 2021; Dejea et al. 2018; Mu et al. 2020; Yu et al. 2017; Tang et al. 2023).
F. nucleatum has emerged as a key microbial driver in CRC pathogenesis, employing multiple sophisticated mechanisms to promote tumour development and progression. F. nucleatum, a gram-negative anaerobic bacterium extensively implicated in CRC, sustains chronic inflammation, suppresses host immune cell activity, and promotes tumorigenesis. The bacterium adheres to and invades human epithelial and endothelial cells primarily via the adhesins FadA and Fap2 (Fardini et al. 2011; Groeger et al. 2022). FadA binds E-cadherin on epithelial cells and VE-cadherin on endothelial cells, disrupting intercellular junctions (Fardini et al. 2011), whereas Fap2 facilitates adhesion through the recognition of tumour-associated Gal-GalNAc overexpression (Abed et al. 2023; Schöpf et al. 2025). FadA binding activates the WNT/β-catenin pathway, driving tumour growth and metastasis (Song et al. 2024; Sun et al. 2019; Dadgar-Zankbar et al. 2024), and disrupts the E-cadherin/β-catenin complex, inducing an epithelial-to-mesenchymal-like transition (Dadgar-Zankbar et al. 2024). Targeted interaction of the RadD protein with CD147 stimulates the PI3K-AKT-NF-κB-MMP9 axis, releasing matrix metalloproteinases (MMPs) to increase CRC proliferation (Maharati and Moghbeli 2023; Jia and Chen 2024; Galaski et al. 2024; Pezeshkian et al. 2021). F. nucleatum further induces genomic instability by inhibiting the glycosylase NEIL2 and impairing Chk2-mediated DNA damage repair, leading to DNA double-strand breaks and microsatellite instability (MSI)(Sayed et al. 2020). Epigenetically, it enhances DNA methyltransferase activity, promoting a CpG island methylator phenotype (CIMP) and the hypermethylation of tumour suppressor genes (TSGs), thereby contributing to high-level microsatellite instability (MSI-H) and the silencing of mismatch repair genes such as MLH1(Ranjbar et al. 2021; Li et al. 2024).
F. nucleatum also orchestrates significant immunosuppression within the tumour microenvironment. Activation of TLR4 signalling promotes myeloid differentiation primary response 88 (Myd88), leading to NF-κB activation; this upregulates the expression of miR-21, which suppresses RASA1 expression, thereby enhancing CRC cell proliferation and invasion (Yang et al. 2017). The Fap2 protein binds the inhibitory receptor TIGIT on immune cells, diminishing natural killer (NK) cell cytotoxicity and inducing T-a apoptosis to facilitate immune evasion (Gur et al. 2015). Concurrent activation of the inhibitory receptor CEACAM1 promotes T-cell exhaustion. Furthermore, F. nucleatum drives tumour-associated neutrophils (TANs) towards a protumorigenic N2 phenotype via TGF-β signalling; these N2 TANs generate ROS, causing DNA damage and accelerating tumour progression (Wu et al. 2023b; Fridlender et al. 2009). TLR4-mediated macrophage polarization towards an M2-like phenotype and a reduction in intratumoral CD4+ T-cell density collectively establish an immunosuppressive milieu conducive to tumour progression (Huang et al. 2024; Kim et al. 2023).
Metabolically, F. nucleatum reprograms cancer cells towards a more aggressive phenotype. Recent studies have demonstrated that F. nucleatum infection upregulates the long noncoding RNA ENO1-IT1, which enhances glycolytic flux and promotes oncogenesis through metabolic reprogramming (Hong et al. 2021a). This metabolic shift not only provides energy for rapid proliferation but also creates an acidic microenvironment that further supports tumour progression and immune suppression.
The impact of F. nucleatum extends to therapeutic resistance, presenting a significant clinical challenge. In chemotherapy resistance, F. nucleatum activates autophagy through TLR4 and Myd88 innate immune signalling, effectively protecting cancer cells from oxaliplatin-induced apoptosis (Yu et al. 2017). This autophagy-mediated chemoprotection has been consistently observed in both in vitro and in vivo models, providing a mechanistic explanation for the poor response to oxaliplatin-based chemotherapy in F. nucleatum-enriched CRC patients. F. nucleatum can also confer chemoresistance by upregulating the expression of the inhibitor of apoptosis protein BIRC3 or the chloride channel anoctamin-1 (ANO1)(Alon-Maimon et al. 2022). F. nucleatum-derived succinate suppresses the cGAS–IFN-β axis, thereby restricting CD8⁺ T-cell trafficking into the tumour microenvironment and driving resistance to anti-PD-1 monoclonal antibody therapy in colorectal cancer (Jiang et al. 2023). F. nucleatum further dampens antitumour immunity through Fap2-mediated engagement of the human TIGIT inhibitory receptor and concomitant activation of the additional immune checkpoint receptor CEACAM1 (Gur et al. 2019, 2019).
In addition to F. nucleatum, multiple bacterial taxa within the tumour microenvironment drive CRC progression through distinct oncogenic mechanisms. Enterotoxigenic Bacteroides fragilis (ETBF) has emerged as a significant contributor to CRC pathogenesis, primarily through the production of Bacteroides fragilis toxin (BFT). This 44.5 kDa zinc-dependent metalloprotease specifically cleaves the extracellular domain of E-cadherin, disrupting adherens junctions and activating β-catenin signalling. This cleavage initiates a cascade of cellular events, including increased epithelial permeability, IL-17 and IL-8 secretion, and sustained STAT3 activation, ultimately driving colonic epithelial cell proliferation and promoting malignant transformation (Kim et al. 2025; Allen et al. 2019; Chung et al. 2018; Wu et al. 2009; Yang et al. 2024). Furthermore, BFT induces spermine oxidase (SMO) expression, generating ROS that cause DNA damage in colonic epithelial cells (Goodwin et al. 2011). Additionally, via METTL14-mediated m6A RNA methylation, ETBF downregulates miR-149-3p to promote colorectal cancer cell proliferation (Cao et al. 2021).
Colibactin-producing Escherichia coli (CoPEC) strains harbour a polyketide synthase (pks) genomic island encoding the synthesis of colibactin, a genotoxin that induces DNA double-strand breaks and chromosomal instability. Mechanistically, colibactin alkylates DNA, forming interstrand cross-links that lead to characteristic mutational signatures in human colorectal tumours, including tandem duplications and specific single-base substitutions (Pleguezuelos-Manzano et al. 2020). In addition to direct genotoxicity, CoPEC infection promotes tumour progression by remodelling the tumour microenvironment (Lopès et al. 2020). Recent studies have demonstrated that CoPEC-infected tumours exhibit altered lipid metabolism characterized by glycerophospholipid accumulation, reduced CD8+ T lymphocyte infiltration, and enhanced chemoresistance through lipid droplet accumulation and phosphatidylcholine remodelling (Oliveira Alves et al. 2024).
Streptococcus gallolyticus (S. gallolyticus) promotes colorectal carcinogenesis through multiple mechanisms, including enhanced adhesion to collagen-rich extracellular matrix components via specific bacterial adhesins. S. gallolyticus infection activates β-catenin signalling and upregulates proinflammatory cytokines, including COX-2 and IL-8, through TLR2 and TLR4 recognition (Kumar et al. 2017). Additionally, this pathogen promotes angiogenesis by increasing VEGF production and facilitates metastatic spread through the induction of the expression of MMPs. Enterococcus faecalis (E. faecalis) contributes to colorectal carcinogenesis through the production of extracellular superoxide, which induces chromosomal instability through bystander effects. This bacterium generates persistent macrophage-dependent inflammation and promotes the formation of aneuploidy in colonic epithelial cells (Wang and Huycke 2007). Furthermore, E. faecalis produces collagenolytic enzymes that disrupt the mucosal barrier and facilitate bacterial translocation, exacerbating chronic inflammation and epithelial damage.

Hepatocellular carcinoma
The hepatic intratumoral microbiome is thought to originate primarily from the gut—the body’s largest bacterial reservoir (Komiyama et al. 2021; Berg 1999). Shifts in the gut microbial composition, compromised intestinal mucosal barrier, and host immunodeficiency are considered the main drivers of bacterial translocation to the liver. In addition, gut bacteria can reach the hepatic parenchyma via portal circulation, and circulating tumour cells may further disseminate these microorganisms to liver metastases (Komiyama et al. 2021; Ponziani et al. 2019). Compared with that in adjacent nontumour tissues, the intratumoral microbiome in hepatocellular carcinoma exhibits distinct alterations. Metagenomic analyses revealed a characteristic enrichment of Lactobacillus, Fusobacterium, and Neisseria in hepatocellular carcinoma (HCC), with concomitant depletion of Faecalibacterium and Pseudomonas (He et al. 2023a). Huang et al. demonstrated that Bacilli, Acidobacteriae, Saccharimonadia, Parcubacteria, Saccharimonadia, and Gammaproteobacteria represent the differential microbiota distinguishing HCC from normal liver tissues (Huang et al. 2022).
The intratumoral microbiota in hepatocellular carcinoma is influenced through the modulation of immune responses, the fostering of chronic inflammation, the reprogramming of metabolic pathways, and potentially other mechanisms. Chakladar et al. reported that in HBV-positive/nondrinker cohorts, the microbiota, including Staphylococcus epidermidis, Methylorubrum populi, and Acinetobacter calcoaceticus, significantly correlated with upregulation of ATF2, AKT, and PIGF; downregulation of TP53; and poor clinical prognosis (Chakladar et al. 2020a). This cohort also exhibited increased activity of the Wnt and PTEN/AKT signalling pathways concomitant with substantial microbial enrichment. Conversely, in HBV-positive patients who consumed alcohol, the hepatic microbial presence positively correlated with M1 and M2 macrophage populations but negatively correlated with M0 macrophages, suggesting that the HBV-associated microbiota may drive macrophage polarization (Chakladar et al. 2020a). K. pneumoniae is translocated from the gut to the liver, exacerbating chronic hepatic inflammation and hepatocarcinogenesis (Wang et al. 2025). Penicillin-binding protein 1B (PBP1B) on the surface of K. pneumoniae binds to TLR4 on HCC tumour cells, subsequently triggering proinflammatory and oncogenic signalling cascades (Wang et al. 2025). In liver tissue, Stenotrophomonas maltophilia activates NF-κB through TLR4 recognition, further inducing NLRP3 inflammasome formation and activation, thereby driving the progression from cirrhosis to HCC (Liu et al. 2022). Lipopolysaccharide (LPS) released during bacterial lysis engages TLR4 and subsequently activates NF-κB and STAT3 signalling, leading to elevated expression of the transcription factor Snail and ultimately exacerbating epithelial-to-mesenchymal transition and hepatocellular carcinoma metastasis (Jing et al. 2012). Microbial metabolites, including secondary bile acids and short-chain fatty acids, can directly promote hepatocarcinogenesis by inducing DNA damage and oxidative stress through ROS generation (Vital et al. 2019; Asarat et al. 2016). Microbial conversion of primary bile acids (BAs) into secondary BAs activates the JAK–STAT3 and phosphatidylinositol 3-kinase (PI3K) pathways, thereby fostering a protumorigenic microenvironment. Concomitantly, secondary BAs suppress the farnesoid X receptor (FXR) while stimulating Wnt/β-catenin signalling, collectively accelerating hepatocarcinogenesis (Chen et al. 2023; Song et al. 2023; Pallozzi et al. 2024).

Pancreatic ductal adenocarcinoma: oral–pancreatic cross talk
For the first time, Geller et al. detected bacterial DNA within PDAC tissues in 2017, and metagenomic sequencing revealed the overrepresentation of Acinetobacter, Pseudomonas and Sphingopyxis. Gene set enrichment analysis revealed that the abundance of these genera correlated with DNA replication, EMT, KRAS activation and MAPK signalling—pathways central to pancreatic carcinogenesis (Geller et al. 2017; Guo et al. 2021a). Oral and intestinal microbiota are considered the most common sources of tumour-associated microbiota in PDAC (Guan et al. 2023). Leng J et al. analysed 144 samples of pancreatic ductal adenocarcinoma from The Cancer Genome Atlas (TCGA) and reported that the most abundant phyla in pancreatic ductal adenocarcinoma were Firmicutes, Proteobacteria, and Actinobacteria, with Bacillus and Lactobacillus being the most common bacterial species (Leng et al. 2024). In the tumour microbiota of pancreatic ductal adenocarcinoma, Stenotrophomonas is associated with poor survival rates and is positively correlated with proinflammatory and innate immune-related genes but negatively correlated with adaptive immune genes (Leng et al. 2024). Jaideep et al. reported that the abundance of Proteobacteria, especially β-Proteobacteria and γ-Proteobacteria, is positively correlated with PDAC metastasis (Guo et al. 2021b; Sepich-Poore et al. 2022). Pseudomonas gingivalis is associated with the prognosis of pancreatic cancer patients. It promotes the inflammatory response by activating TLR signal transduction and promotes the progression of pancreatic cancer by increasing the secretion of neutrophil chemokines and neutrophil elastase (Michaud et al. 2013; Ahn et al. 2012; Hayashi et al. 2012; Mitsuhashi et al. 2015). Analysis of TCGA data revealed that A. ebreus, C. freundii, Pseudomonadales and S. sonnei were associated with poor prognosis and may be related to tumour immunity (Chakladar et al. 2020b). F. nucleatum also leads to poor survival in patients with pancreatic cancer, which is achieved through a variety of mechanisms (Mitsuhashi et al. 2015; Hayashi et al. 2023). It increases the secretion of GM-CSF, IL8, CXCL1, and MIP3 α to promote the proliferation and invasion of pancreatic cancer cells and recruits myeloid-derived suppressor cells (MDSCs) to inhibit CD8+ T cells (Hayashi et al. 2023; Udayasuryan et al. 2022). Assisted by E-cadherin, the FadA adhesin of F. nucleatum activates the Wnt/β-catenin pathway, regulating cancer cell proliferation and differentiation (Rubinstein et al. 2013; Pushalkar et al. 2018). It also delivers proteins and miRNAs to cells and acts on TLR4 to upregulate small extracellular vesicle (sEV) expression and promote pancreatic tumour metastasis (Niland et al. 2021).

Oncogenic mechanisms at the molecular and cellular levels
The intratumoral microbiota actively contributes to tumorigenesis and progression rather than acting as a passive bystander. The underlying mechanisms are multifaceted and include but are not limited to the induction of chronic inflammation, modulation of oncogenic signalling pathways to alter cellular proliferation and apoptosis, production of carcinogenic metabolites, induction of DNA damage, and profound remodelling of the local immune landscape and host metabolism (Fig. 2). These processes collectively shape the tumour microenvironment and drive malignant cell behaviour.

Role of intratumoral microbiota-induced chronic inflammation in malignant progression
The intratumoral microbiota has emerged as a critical regulator of tumour progression through the induction and maintenance of chronic inflammation within the tumour microenvironment. This inflammatory state is established and perpetuated through several interconnected molecular mechanisms that collectively promote carcinogenesis, immune evasion, and metastatic dissemination.
The primary mechanism involves the persistent activation of pattern recognition receptors (PRRs) on host cells by microbial components (Kawai and Akira 2010). Bacterial products such as LPS from gram-negative bacteria, including F. nucleatum, engage TLRs, particularly TLR4, leading to NF-κB and mitogen-activated protein kinase (MAPK) pathway activation (Pal et al. 2014; Xing et al. 2016). This results in the sustained production of proinflammatory cytokines, including tumour necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1β), IL-6, and IL-8. Similarly, Helicobacter pylori utilizes its cytotoxin-associated gene A (CagA) protein and LPS to activate these pathways in the gastric epithelium, establishing a chronic inflammatory state that drives the progression from normal mucosa to adenocarcinoma through continuous epithelial damage and compensatory proliferation. LPS engages TLR2/4/5/9, triggering the transcription factors NF-κB, AP-1 and CREB, which release IL-8, COX-2 and iNOS, thereby fuelling the GERD–Barrett's oesophagus–oesophageal adenocarcinoma sequence (Gillespie et al. 2021).
Another crucial mechanism involves the activation of inflammasome complexes, particularly the NLRP3 inflammasome (Barchi et al. 2025). Pathogens such as Porphyromonas gingivalis and specific strains of F. nucleatum activate this multiprotein complex, leading to caspase-1-dependent cleavage and maturation of IL-1β and IL-18. Concurrently, inflammasome activation induces pyroptosis, a highly inflammatory form of programmed cell death mediated by gasdermin D (GSDMD) cleavage (Xu and Núñez 2023). This process releases additional damage-associated molecular patterns (DAMPs) and proinflammatory cytokines, creating a self-amplifying inflammatory loop that sustains tumour-promoting signalling within the microenvironment.
Beyond inflammation, certain intratumoral microbes directly promote genomic instability through genotoxic mechanisms. CoPEC strains harbouring the pks genomic island generate colibactin, which induces DNA double-strand breaks and interstrand cross-links, resulting in characteristic mutational signatures in colorectal cancer (Pleguezuelos-Manzano et al. 2020). Additionally, the chronic inflammatory milieu generated by various intratumoral bacteria produces ROS/RNS that cause oxidative DNA damage, including 8-oxoguanine lesions, while simultaneously inhibiting DNA repair mechanisms. This combination of direct genotoxicity and inflammation-mediated DNA damage accelerates the accumulation of oncogenic mutations.
Microbiota-mediated metabolic reprogramming contributes significantly to chronic inflammation and immune suppression. Bacterial processing of host and dietary compounds generates metabolites with potent immunomodulatory properties. Specific gut bacteria convert primary bile acids to secondary bile acids such as deoxycholic acid, which function as DAMPs that induce epithelial ROS production and NF-κB activation (Jia et al. 2018). Similarly, microbial metabolism of tryptophan yields kynurenine derivatives that activate the aryl hydrocarbon receptor, promoting regulatory T-cell differentiation and suppressing antitumour immunity. These metabolic activities establish an immunosuppressive inflammatory environment conducive to tumour progression.
In summary, the intratumoral microbiota promotes malignant progression through integrated mechanisms involving PRR activation, inflammasome signalling, genotoxicity, and metabolic reprogramming. These pathways collectively establish a self-sustaining cycle of chronic inflammation that drives tumour initiation, progression, and therapeutic resistance. Understanding these mechanisms provides opportunities for novel therapeutic interventions targeting the interface between microbes, inflammation, and cancer.

Epigenetic reprogramming: methylation, acetylation, and noncoding RNAs
He intratumoral microbiota has emerged as a key regulator of tumour epigenetics, modulating gene expression patterns through multiple interconnected mechanisms without altering the DNA sequence itself. These epigenetic modifications represent a crucial interface between microbial presence and host cell transformation, contributing significantly to tumour initiation, progression, and therapeutic resistance.
Intratumoral microorganisms extensively influence DNA methylation patterns through both direct enzymatic activities and indirect inflammatory signalling. Specific bacterial species, including F. nucleatum and Helicobacter pylori, induce hypermethylation of tumour suppressor gene promoters by upregulating DNA methyltransferases (DNMTs). H. pylori infection in gastric carcinoma activates DNMT1 expression through NF-κB-mediated transcriptional upregulation, leading to the silencing of critical tumour suppressors, including CDKN2A and MLH1(Maeda et al. 2017). This methylation-mediated silencing facilitates uncontrolled cellular proliferation and genomic instability. Conversely, microbial metabolites such as short-chain fatty acids produced by commensal bacteria can induce hypomethylation (Hangul et al. 2025). Butyrate, for instance, not only inhibits histone deacetylases but also impairs DNMT activity, resulting in DNA hypomethylation and potential reactivation of normally silenced oncogenes (Cho et al. 2014). This bidirectional modulation of DNA methylation states represents a fundamental mechanism through which the intratumoral microbiota shapes the tumour epigenetic landscape.
Microbial communities within tumours significantly impact histone modification patterns, particularly acetylation and methylation marks. Butyrate-producing bacteria, including certain Clostridium and Bacteroides species, generate SCFAs that function as potent histone deacetylase inhibitors (HDACis). Butyrate-mediated HDAC inhibition results in histone hyperacetylation, predominantly at H4K18 residues, leading to chromatin relaxation and transcriptional activation of tumour-suppressive pathways (Fellows et al. 2018). This chromatin remodelling enhances the expression of genes involved in cellular differentiation and apoptosis while suppressing proinflammatory pathways. Additionally, microbiota-induced inflammatory cytokines, particularly IL-6 and TNF-α, activate histone acetyltransferases (HATs), including p300/CBP, which acetylate histones at the promoters of genes involved in EMT and cell survival. Beyond acetylation, intratumoral microbes influence histone methylation through metabolic intermediates. Microorganisms and their metabolites not only promote an increase in histone acetylation levels, a strong increase in H4 acetylation, and an increase in diacetylation H3 K9ac + K14ac and H3K18ac + K23ac but also promote an increase in methylation H3 K27me3 levels (Krautkramer et al. 2016).
The intratumoral microbiota extensively modulates the expression and function of noncoding RNAs, particularly microRNAs (miRNAs) and long noncoding RNAs (lncRNAs). F. nucleatum infection in colorectal cancer upregulates oncogenic miR-21 expression (Yang et al. 2017). Alterations in miR-21 target critical signalling pathways. Furthermore, bacteria-derived components such as LPS can modulate the expression of miRNAs—including miR-21, miR-140 and miR-223—to promote tumour cell invasion (Liu et al. 2014; Zhu et al. 2020; Hamza et al. 2024). In addition to miRNAs, intratumoral microbes significantly influence lncRNA expression. F. nucleatum upregulates the expression of the lncRNA ENO1-IT1, which interacts with glycolytic enzymes and enhances aerobic glycolysis in colorectal cancer cells (Hong et al. 2021b). This metabolic reprogramming supports tumour growth under nutrient-limited conditions. Similarly, microbial metabolites, including butyrate, modulate the expression of lncRNAs involved in chromatin remodelling complexes, thereby influencing broader epigenetic states in tumour cells.
These epigenetic mechanisms do not operate in isolation but form an integrated network that reinforces tumour-promoting gene expression programs. Changes in microbiota-induced DNA methylation involve the recruitment of histone-modifying enzymes that further stabilize repressed chromatin states, whereas noncoding RNAs serve as both effectors and regulators of these epigenetic modifications. The interconnected nature of these pathways creates a self-reinforcing epigenetic landscape that maintains cellular transformation. Therapeutically, the microbiota‒epigenome axis presents promising intervention opportunities. Probiotic administration, selective antimicrobial therapies, and microbiota-targeted dietary interventions represent strategies to reverse deleterious epigenetic modifications. Combining epigenetic therapies such as DNMTs or HDACis with microbiota modulation may synergistically restore normal epigenetic regulation and suppress tumour growth (Wang et al. 2024). Understanding the precise mechanisms of microbiota-mediated epigenetic reprogramming will enable the development of novel precision medicine approaches for cancer prevention and treatment.

Metabolic influences of the intratumoral microbiota in gastrointestinal cancers
The intratumoral microbiota plays an important role in reprogramming tumour metabolism within the GI tumour microenvironment, significantly affecting amino acid, glucose, and lipid metabolic pathways. These microbial-driven metabolic alterations not only support cancer cell proliferation and survival but also contribute to therapeutic resistance and immune evasion.

Amino acid metabolism
Intratumoral microbiota profoundly influence amino acid metabolism, which is crucial for protein synthesis and cellular signalling. Specific microbial species modulate the availability of key amino acids such as glutamine and arginine within the TME. For instance, certain bacteria can deplete arginine, impairing T-cell function and promoting immune evasion (Wang et al. 2023). Additionally, microbial dysbiosis can increase the production of immunosuppressive metabolites such as kynurenine via the tryptophan degradation pathway, which suppresses antitumour immunity and facilitates tumour progression (Pacheco and Elizondo 2023; Dehhaghi et al. 2020; Canavese et al. 2016). These modifications in amino acid metabolism highlight the role of the microbiota in shaping a tumour-permissive metabolic environment.

Glucose metabolism
The Warburg effect, characterized by enhanced glycolysis even under aerobic conditions, is a hallmark of metabolic reprogramming in cancer. Intratumoral microbiota contributes to this phenomenon by promoting glycolytic flux in cancer cells. F. nucleatum, for example, upregulates the expression of glucose transporters and glycolytic enzymes in CRC cells, leading to increased lactate production and acidification of the TME (Hong et al. 2021b; Ou et al. 2022). This acidic milieu not only fuels tumour growth but also inhibits immune cell function. In EAC, a shift to a gram-negative microbiota promotes glycolysis through Toll-like receptor activation and inflammatory signalling, further driving carcinogenesis (Gillespie et al. 2021).

Lipid metabolism
Lipid metabolic reprogramming is another critical aspect influenced by the intratumoral microbiota. Microbial dysbiosis enhances de novo lipogenesis and lipid accumulation in cancer cells. F. nucleatum has been shown to induce the expression of fatty acid synthase (FASN) in CRC cells, facilitating lipid biosynthesis and supporting membrane formation and energy storage (Liu et al. 2022). Such alterations in lipid metabolism are associated with increased tumour aggressiveness and chemoresistance. In HCC, intratumoral microbes, including Fusobacterium and Enterobacteriaceae, are linked to enhanced fatty acid and lipid synthesis pathways, contributing to disease progression (Xue et al. 2024; He et al. 2023b).

Bile acid biotransformation
Intratumoral microbiota play a crucial role in bile acid metabolism through biotransformation processes that generate oncogenic metabolites. Primary bile acids, such as cholic acid and chenodeoxycholic acid, are converted by microbial enzymes—particularly bile salt hydrolases (BSH) and 7α-dehydroxylase—into secondary bile acids such as deoxycholic acid (DCA) and lithocholic acid (LCA). These secondary bile acids promote DNA damage, oxidative stress, and inflammatory signalling, thereby fostering a tumorigenic environment (Xiang et al. 2023; Yang and Qian 2022). In CRC, high levels of DCA are associated with increased proliferation and invasion of cancer cells via activation of the farnesoid X receptor (FXR) and constitutive androstane receptor (CAR) signalling pathways. Additionally, secondary bile acids can suppress antitumour immunity by inhibiting dendritic cell maturation and promoting the expansion of regulatory T cells (Tregs), further facilitating immune evasion (Xiang et al. 2023; Yang and Qian 2022; Sheflin et al. 2014).

Impact of the intratumoral microbiota on immune evasion in gastrointestinal cancers
The intratumoral microbiota plays a critical role in promoting immune evasion within the gastrointestinal tumour microenvironment by modulating innate and adaptive immune responses. Specific microbial species, such as F. nucleatum and Bacteroides fragilis, contribute to immunosuppression through mechanisms such as M2 macrophage polarization and T-cell exhaustion. F. nucleatum has been shown to recruit MDSCs and induce M2 polarization of tumour-associated macrophages (TAMs) via activation of TLR4 and NF-κB signalling, resulting in the production of anti-inflammatory cytokines such as IL-10 and TGF-β, which suppress cytotoxic T-cell activity and promote a protumorigenic environment (Sakamoto et al. 2021; Lee et al. 2021). Concurrently, microbial metabolites, including short-chain fatty acids and secondary bile acids, can inhibit T-cell function by promoting the expression of checkpoint receptors (e.g., PD-1 and CTLA-4) and inducing epigenetic modifications that lead to T-cell exhaustion (Yang and Qian 2022; Yang et al. 2023). This dysregulated immune milieu facilitates tumour progression and resistance to immunotherapy.
Moreover, recent studies have shown that intratumoral microbes influence immune cell trafficking and function through metabolic competition. For instance, microbiota-derived lactate and other oncometabolites acidify the tumour microenvironment, further impairing T-cell receptor signalling and cytokine production (Ou et al. 2022; Sheflin et al. 2014). In colorectal cancer, F. nucleatum enrichment is correlated with decreased CD8+ T-cell infiltration and increased Tregs, highlighting its role in creating an immunosuppressive niche (Lee et al. 2021; Zepeda-Rivera et al. 2024). Therapeutic strategies targeting these microbiota–immune interactions, such as selective antimicrobial agents or probiotics, are emerging as promising adjuvants to increase the efficacy of immunotherapies in GI cancers.

Future perspectives in intratumoral microbiota research for GI cancers
The burgeoning field of intratumoral microbiota research holds transformative potential for understanding and treating GI cancers. Future investigations are poised to leverage multiomics technologies—including spatially resolved metagenomics, metatranscriptomics, and metabolomics—to decipher the functional dynamics of microbial communities within tumours at unprecedented resolution. These approaches elucidate not only which microorganisms are present but also their metabolic activity, spatial distribution, and immunomodulatory effects on the TME (Riquelme et al. 2019). A key goal lies in exploring microbial heterogeneity across different cancer subtypes, stages, and patient populations, which may reveal novel biomarkers for early detection and personalized prognosis. Furthermore, the integration of artificial intelligence and machine learning with microbial data offers promising avenues for identifying complex microbiome-based signatures that are predictive of treatment response and survival outcomes.
Harnessing the intratumoral microbiota for personalized therapy represents a paradigm shift in oncology. Future efforts will focus on developing microbiota-targeting interventions, such as engineered probiotics, selective antimicrobials, or faecal microbiota transplantation (FMT), tailored to modulate tumour-associated microbes and enhance the efficacy of conventional therapies and immunotherapies (Yang et al. 2023; Arıkan and Muth 2023). For instance, combining immune checkpoint inhibitors with microbial modulators may reverse immunosuppressive TME conditions and overcome therapeutic resistance. The success of these strategies will rely on robust translational studies and well-designed clinical trials that validate microbial targets and their mechanistic roles in tumour progression. Ultimately, multidisciplinary collaboration among microbiologists, oncologists, immunologists, bioinformaticians, and clinical trial experts is essential to advance this rapidly evolving field. Such integrative efforts will accelerate the development of microbiome-based diagnostics and therapeutics, paving the way for precision medicine interventions that improve outcomes for patients with GI cancers.