ABSTRACT
The role of the gut microbiome in the development and progression of liver cancer has long been recognized. However, the presence of microbes in tumors that were previously considered sterile has only recently been discovered. The intratumor microbiome in liver cancer likely originates from various sources, including the gut, hematogenous spread from other mucosal locations, adjacent non-cancerous tissues, and co-metastasis with the tumor cells. As a newly discovered component of the tumor microenvironment, it regulates host immune responses, promotes chronic inflammation, modulates metabolic pathways, and exerts other influences in liver cancer. These unique features offer potential new biomarkers for liver cancer prognosis and treatment response. Exploring the complex interactions between intratumor microbiome and the host to modulate or target the intratumor microbiome may provide new avenues for liver cancer treatment. This article provides a comprehensive review of our current understanding regarding the potential origins of the intratumor microbiome in liver cancer, its unique characteristics, and the underlying mechanisms by which it affects liver cancer. Furthermore, we discuss the promising clinical implications and potential challenges that remain before this knowledge can be fully integrated into clinical practice.
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Keywords: Liver cancer; Microbiome; Clinical significance; Biomarkers; Therapeutic effects
INTRODUCTION
The liver stands as one of the most susceptible organs to both primary and metastatic tumors. The unique “gut-liver axis” mechanism fully demonstrates the complex bidirectional connections and interactions between the intestine and the liver [
1,
2]. With the rapid advancement of microbiomics, numerous studies have confirmed the significant influence of the gut microbiota on the occurrence and progression of primary and metastatic liver cancer [
3]. Recently, several groundbreaking studies have demonstrated that various tumors, once considered sterile, harbor bacteria and other microorganisms [
4-
6]. These pivotal findings have sparked widespread interest in the intratumor microbiome, stimulating rapid progress in research. Although the liver is the extraintestinal organ most closely associated with the intestine, potentially exposed to the gut microbiota through the portal vein and biliary tract systems, our current understanding of the intratumor microbiome in liver cancer still lacks depth.
Advancements in high-throughput sequencing technology have provided valuable opportunities to study intratumor microbiomes. Preliminary research has indicated that the main pathways through which the intratumor microbiome affects liver cancer include immunity, inflammation, and metabolism. However, further in vitro and in vivo experiments are required to comprehensively elucidate the interactions between the intratumor microbiome and liver cancer. While the diagnostic value of the intratumor microbiome in liver cancer may be limited, it shows potential in predicting patient prognosis and treatment response, and could offer new intervention measures for liver cancer treatment in the future.
POTENTIAL ORIGINS OF THE INTRATUMOR MICROBIOME IN LIVER CANCER
The unique microenvironment of tumor tissue may create favorable conditions for microbial colonization. For example, necrosis within tumors provides abundant nutrients for microbes, hypoxia promotes the growth of anaerobic microorganisms, and an immunosuppressive microenvironment hinders the clearance of microbes from the host. Although the precise origins of the intratumor microbiome in liver cancer are still in the air, a mounting body of evidence indicated that it likely originates from a variety of sources (
Fig. 1).
It is plausible that the gut microbiota serves as an important source of the intratumor microbiome in liver cancer, given the close anatomical and functional connections between the gut and the liver. Sookoian et al. [
7] found that lipopolysaccharides in the liver tissues of patients with metabolic dysfunction-associated steatotic liver disease (MASLD) were mainly located near the portal tracts, which suggests their possible intestinal origins. Fecal microbiota transplantation in germ-free mice further revealed that the liver microbiome is partially derived from the gut microbiota in a selective manner [
8]. The portal vein system is crucial conduit for the translocation of gut microbes to liver tissues as well as liver cancer. Enteric bacteria, such as
Escherichia coli that possesses the virulence regulator VirF, translocate to the liver through the portal vein after disrupting the gut vascular barrier and then foster a pre-metastatic niche in liver for colorectal cancer (CRC) [
9]. In patients with chronic liver diseases and liver cancer, the impairment of the gut barrier is often present, leading to increased intestinal permeability and subsequent dissemination of gut microbes, microorganism-derived metabolites, and pathogen-associated molecular patterns to the liver [
1]. An observational study found that compared to patients with MASLD, those with cirrhosis and hepatocellular carcinoma (HCC) had higher levels of gut microbiota deoxyribonucleic acid (DNA) enrichment in the liver tissues [
10], underscoring the notion that alterations of intestinal permeability in patients with cirrhosis and HCC facilitate the translocation of gut microbiota. The biliary tract provides an alternative route for the gut microbes to inhabit the liver cancer. It has been established that gut microbiota can colonize pancreatic cancer through retrograde migration via the pancreatic duct [
11]. The presence of microorganisms in the biliary tract lends credence to the hypothesis that retrograde translocation of intestinal microbiota is a contributor to the intratumor microbiome in liver cancer, though additional evidence is required to solidify this hypothesis.
Microbes originating from other mucosal sites external to the gut, including the oral cavity, the upper respiratory tract, genitourinary tract, can also traverse the bloodstream to reach the tumors or their precursors [
12,
13]. These mucosal sites are exposed to external environment and harbor commensal microbiota. When the mucosal barrier is damaged by pathological factors, mucosal microorganisms can gain access to peripheral blood vessels and travel through the bloodstream. Compared with healthy controls, the bacterial load is significantly increased in the peripheral blood of patients with cirrhosis, liver failure, and HCC, along with characteristic changes in microbial composition [
10,
14,
15]. Studies have also revealed that microbial communities exist in the peripheral blood of patients diagnosed with other can-cers [
16,
17]. The microbes circulating within the bloodstream exhibit a tropism for tumors, seemingly attracted by the chemotactic gradient of necrotic cellular debris emitted by the tumor [
18]. The disordered formation of neovascularization within the tumor and the incomplete structure of blood vessel walls provide favorable conditions that facilitate the dissemination of blood-borne microbes into distant tumor tissues [
19]. Abed et al. [
12] revealed that
Fusobacterium nucleatum adopted a hematogenous route to localize CRC and its metastases via the interaction between the bacterial Fap2 and host epithelial Gal-GalNAc overexpressed in CRC. Such molecular interactions, which offer a navigation for microbes during hematogenous invasion, might also exist in primary liver cancer as well as other tumor types, and remain an area of ongoing exploration.
Adjacent non-cancerous liver tissue is another potential reservoir for the intratumor microbiome of liver cancer. The liver has an intrahepatic microbiome [
20], and to some extent, the intratumor microbiome in liver cancer displays similarities with the microbiome present in the adjacent non-cancerous tissues [
21-
23]. The observation that microbial communities exhibit notable similarities between tumor tissues and adjacent tissues across various types of cancer further implies microbial invasion from the adjacent tissues [
4]. A comprehensive study involving different metastatic lesions, including lymph node, liver, and lung metastases, revealed that the organs in which metastases develop are the major determinant of the microbial composition within metastatic lesions, rather than the primary tumors from which they originate [
24]. This reflects the significant influence of adjacent non-cancerous tissue on the intratumor microbiome of liver cancer.
In metastatic liver cancer, the intratumor microbiome may also originate from the primary tumors through a co-metastasis route along with the tumor cells. Studies have revealed that the majority of intratumor bacteria are located within the tumor cells or immune cells [
4,
25]. The intracellular microbes present in breast cancer can be transported by the tumor cells and thus enable their migration to distant metastatic sites. Furthermore, these intracellular bacteria enhance the survival of circulating tumor cells by boosting their resistance to fluid shear stress within the bloodstream [
26]. Bullman et al. [
27] demonstrated that the intratumor microbiome of liver metastases from CRC exhibits a similarity to that of the corresponding primary tumors, with
F. nucleatum and its associated microbiota persisting in the distant liver metastases. They further confirmed the presence of identical viable strains of
F. nucleatum and
Fusobacterium necrophorum in matched primary tumors and liver metastases of CRC. This finding supports the hypothesis that some intratumor microorganisms migrate along with tumor cells to distant sites, aiding in the colonization of liver metastatic lesions [
27].
To further accurately clarify the origins of intratumor microbiome in liver cancer, tracing techniques such as GFP labeling would be very helpful. Considering the multifaceted origins of the microbiome in liver cancer, future studies should delve into the interconnections among microbiomes from diverse anatomical compartments through comprehensive multisite sampling. This will facilitate a deeper understanding of the changes and roles of the human commensal microbiota in the occurrence and progression of liver cancer, ultimately paving the way for innovative interventional strategies that leverage microbiota modulation for the treatment of liver cancer.
FEATURES OF INTRATUMOR MICROBIOME IN LIVER CANCER
The presence of bacteria in liver cancer was observed two decades ago. A study reported the presence of
Helicobacter in the liver tissues of patients with HCC [
28,
29]. Later studies suggested that certain species of the genus
Helicobacter, such as
Helicobacter pylori and
Helicobacter hepaticus, may be associated with hepatitis and liver cancer [
30,
31]. With advances in next-generation sequencing, proteomics, and metabolomics (
Table 1), the intratumor microbiome can now be detected in a high-throughput manner, without relying on microbial culture. Recent studies have depicted the characteristics of the intratumor microbiome within liver cancer (
Table 2), revealing differences in the abundance of some microorganisms within liver cancer and adjacent/normal tissues [
21-
23,
32-
37]. For example, He et al. [
33] discovered that microbes such as
Lactobacillus,
Fusobacterium, and
Neisseria were significantly enriched in HCC, while
Faecalibacterium and
Pseudomonas were decreased. Huang et al. [
23] found that Bacilli, Acdobacteriae, Parcubacteria, Saccharimonadia and Gammaproteobacteria are the most important differential taxa at the class level when comparing the microbiome between HCC and normal liver tissues. Based on these taxa, they proposed a classifier to distinguish HCC from normal liver tissues with considerable accuracy. Although studies have indicated that liver cancer possesses a unique intratumor microbiome that may distinguish it from adjacent and normal liver tissues to varying degrees, it seems that no microbe has been identified as the “specific marker” for liver cancer.
Despite recent progress in the research of the intratumor microbiome in liver cancer, individual studies have yielded conflicting reports on the features of intratumor microbiome (
Table 2). In addition to the differences in microbial diversity and the differential taxa associated with liver cancer among these studies, they have also revealed varying associations between the tumor microbiome and the clinical characteristics of patients. For example, while some studies have suggested that hepatitis viruses, alcohol consumption, and cirrhosis lead to significant changes in the intratumor microbiome of liver cancer [
23,
32,
33], others have not observed notable effects [
35,
37]. Given the differing principles underlying different high-throughput technologies, each possesses unique advantages and limitations (
Table 1), potentially leading to biases among the data they generate. Notwithstanding their sophistication, these advanced techniques remain vulnerable to potential contamination arising from multiple stages of the experimental process. Formalin-fixed paraffin-embedded (FFPE) tissues offer vast and practical resources for investigating the intratumor microbiome. Whereas the non-sterile procedures involved in sample processing and extensive DNA degradation tend to introduce additional external contamination and detection biases, rendering FFPE samples less optimal compared to fresh frozen ones. While methods like electron microscopy, immunohistochemical staining, immunofluorescence, and bacterial culture are indeed useful in determining the presence of the low-biomass tumor microbiome, their direct value in minimizing potential contamination in highthroughput detection remains limited. Therefore, standardized protocols and methodologies for generating and analyzing high-throughput data are crucial for ensuring conclusive findings in research concerning the features of intratumor microbiome within liver cancer.
INFLUENCES OF INTRATUMOR MICROBIOME IN LIVER CANCER
Emerging evidence has demonstrated that intratumor microbiome present within liver cancer exerts its influences by regulation of immune responses, promotion of chronic inflammation, alteration of metabolic pathways, and potentially other mechanisms. However, research in this area is still in its nascent stages, and further studies are required to delve into the causal relationships underlying the hostmicrobe crosstalk.
Intratumor microbiome in primary liver cancer
HCC is the most prevalent type of primary liver cancer and commonly arises from chronic liver inflammation caused by hepatitis viruses, MASLD, or alcohol abuse [
38]. Chronic infection with hepatitis viruses is one of the key contributors to HCC. Since the discovery of hepatitis B virus (HBV) and hepatitis C virus, extensive research has been conducted on their carcinogenic mechanisms [
39,
40]. Intrahepatic cholangiocarcinoma (ICC) originates from the epithelial cells of the secondary bile ducts and their branches, accounting for approximately 10–15% of primary liver cancer cases [
41]. It has the second highest incidence after HCC and has shown a significant global increase in recent years [
42]. Inflammation arising from viral hepatitis and bile duct stones is an important etiological factor of ICC [
43,
44]. Although it is acknowledged that microorganisms within hepatobiliary system are associated with primary liver cancer, recent research on its intratumor microbiome has introduced a new kid on the block.
The microbiota profoundly shapes the immune network of the host [
45]. The intrahepatic microbiome also plays a crucial role in maintaining normal immune function in the liver. Bacteria within the liver, especially Bacteroidetes, activate natural killer T cells by producing glycolipids, upregulating the CCL5 pathway, and enhancing the recruitment and activation of immune cells in the liver [
8]. Clearance of intrahepatic bacteria by antibiotics significantly reduces the number of immune cells in the liver, accompanied by decreased antigen presentation and adaptive immune function [
8]. In pathophysiological situation, dissemination of certain gut pathogens to the liver or other tissues can provoke autoimmune reactions in susceptible individuals [
46]. Increased levels of bacteria that produce aryl hydrocarbon receptor agonists, such as
Lactobacillus reuteri in liver tissue, can promote autoimmune hepatitis via INFγ-producing CD8+ T cells [
47].
The intratumor microbiome in liver cancer contributes to the local regulation of immune responses, altering the tumor immune microenvironment with immunosuppressive or immunostimulatory effects. Chakladar et al. [
48] found that some microbes present in HCC were relevant to cytokine signaling pathways, involving CCL28, CCL26, CSF3, IL6, and IL10. They also observed that the presence of microbes related to HBV or alcohol consumption in HCC showed a positive association with M1 and M2 macrophages, whereas it exhibited an inverse relationship with M0 macrophages [
48]. Li et al. [
49] revealed a significant role of the intratumor microbiome in shaping the immune microenvironment of HCC. They analyzed the intratumor microbiome of 29 patients with HBV-related HCC, categorizing them into virus-dominant and bacteria-dominant subtypes. The bacteria-dominant HCC displayed a different immune landscape compared with the virus-dominant subtype, with a remarkable increased infiltration of M2 macrophage. Since the M2-polarized tumor-associated macrophages promote tumor progression in multiple ways, including angiogenesis, stromal remodeling, and immunosuppression [
50], patients with HBV-related HCC classified as the bacteria-dominant subtype exhibited poorer clinical features.
Microorganisms can be recognized by pattern recognition receptors to trigger the innate immune system, which then prompts immune and inflammatory reactions. It has been shown that increased intestinal permeability allows bacterial lipopolysaccharides to spread from the gut to the liver, activating inflammation via Toll-like receptors (TLR) and subsequently promoting the progression of chronic liver diseases and hepatocarcinogenesis [
51,
52]. For instance, Suppli et al. [
53] proposed that the bacterial DNA load within the liver tissue may be a risk factor for MASLD based on the discovery that obese individuals exhibit a higher bacterial DNA load with abundant Proteobacteria and lower microbial diversity in the liver tissues as compared to healthy individuals. Furthermore, Proteobacteria in the liver tissue was associated with lobular and portal inflammation scores in patients with MASLD [
7]. The close relationships between microbiome within liver and the inflammatory reactions of the host provide novel insights into the development of liver cancer.
Persistent inflammation is a typical milieu that contributes to tumor initiation and progression. While viral hepatitis and cholangitis accompanied by dysbiosis in the biliary tract are significant risk factors for primary liver cancer, their pivotal roles driving carcinogenesis and cancer progression are not the focus of this review. Whereas inflammation in the tumor microenvironment (TME) fostered by the intratumor microbiome cannot be ignored. The translocation of the gut pathogen
Klebsiella pneumonia to liver exacerbates liver inflammation, fibrotic damage and hepatocarcinogenesis. Specifically, the penicillin-binding protein 1B located on the surface of
K. pneumonia binds to TLR4 on HCC tumor cells, and subsequently triggers pro-inflammatory and oncogenic signaling (
Fig. 2) [
54]. Similarly, Liu et al. [
22] discovered enrichment of
Stenotrophomonas maltophilia in the liver tissue of patients with HCC.
S. maltophilia, a gram-negative opportunistic pathogen, triggers the activation of the nuclear factor kappa-B (NF-κB) pathway through recognition by TLR4 in liver tissue, subsequently inducing a senescence-associated secretory phenotype in hepatic stellate cells. This process further triggers the formation and activation of the NLRP3 inflammasome (
Fig. 2), accelerating the progression of liver cirrhosis to HCC [
22].
Tumors undergo distinct metabolic reprogramming that is instrumental in their initiation and progression [
55]. The microbiotas present within tumors are able to generate a diverse range of functional metabolites, which can exert specific local effects. It has been shown that metabolic functions of the intratumor microbiome are associated with patients’ clinical characteristics across different tumor types [
4]. He et al. [
33] uncovered that specific functional processes of the intratumor microbiome, namely fatty acid and lipid synthesis, were markedly elevated in HCC tissues. This finding suggests that these microbial processes may have an important influence on liver cancer, given that fatty acids serve as crucial energy sources and secondary messengers during cancer progression [
56]. A study uncovered that infection with liver fluke promoted enrichment of enteric microbes, such as
Bifidobacteriaceae,
Enterobacteriaceae, and
Enterococcaceae, in tumor and adjacent tissues of patients with cholangiocarcinoma [
57]. The microbiome associated with liver fluke exhibited metabolic distortions, which were characterized by increased levels of bile salt hydrolase and an elevated capacity to produce bile acids and ammonia, potentially contributing to carcinogenesis (
Fig. 2) [
57]. This discovery indicates that infection with liver fluke is accompanied by abnormal shifts in the hepatobiliary microbiome, hinting at a potentially novel mechanism underpinning ICC, which involves the intersections between parasites and bacteria.
Xue et al. [
58] discovered that the metabolic profile of HCC was associated with the intratumor microbiome. Subsequently, they developed a mouse model of HCC to further validate that the intratumor microbiome indeed influences the host metabolic status [
59]. Chai et al. [
60] observed that
Paraburkholderia fungorum was more abundant in adjacent liver tissues compared to ICC tumor tissues. The intratumoral
P. fungorum not only correlated with the levels of carbohydrate antigen 199 in the peripheral blood, but also inhibited the proliferation and migration of ICC tumor cells in vitro. Furthermore, experiments conducted in a mouse model burdened with subcutaneous transplantation tumors demonstrated that this bacterium exerts antitumor effects by affecting amino acid metabolism (
Fig. 2) [
60]. Emerging research indicates that the microbiome present within tumor tissues plays a role in modulating metabolism of liver cancer. However, these associations remain incompletely analyzed due to the constraint of limited sample sizes, and more intensive investigation is required to elucidate the precise mechanisms by which intratumor microbiome of liver cancer exerts its influence through metabolic regulation.
As a component of the TME, the intratumor microbiome exerts influences on cancer development and progression across various types of malignancies through a series of mechanisms [
61-
65], which include: (1) inducing DNA damage [
66,
67]; (2) activating oncogenic pathways [
68-
70]; (3) promoting chronic inflammation [
71,
72]; (4) modulating anti-tumor immune responses [
73-
75]; (5) altering epigenetic modifications [
58,
76]; and (6) regulating metabolism in tumor [
59,
77].
Research suggests that the intratumor microbiome is a multi-faceted player in the TME of liver cancer. It may also impact liver cancer through dysregulation of signaling pathways. A study utilizing ribonucleic acid (RNA) sequencing data from the TCGA database revealed that HBV and alcohol consumption alter the abundance of certain microbes in HCC tumor tissues, and these microbial alterations are associated with oncogenic pathways, such as the WNT, PTEN, and AKT pathways (
Fig. 2) [
48]. Epigenetic reprogramming is also involved in the interaction of intratumor microbiome and liver cancer. Previous investigations have demonstrated that specific intratumor microbial species, such as
F. nucleatum and
H. pylori, induce abnormal DNA methylation in gastrointestinal carcinomas [
63]. It is interesting that the levels of DNA methylation in liver tissues of patients with MASLD were significantly correlated with intrahepatic Bacteroidetes, Gammaproteobacteria, Acidobacteria, and Actinobacteria. Additionally, Clostridiales and Sphingomonadales present within the liver affect steatosis by regulating histone acetyltransferase activity [
78]. The association between intratumor microbiome and epigenetic profiles of HCC was preliminarily reported by Xue et al. [
58]; however, their findings were constrained by a small sample size and lacked validation.
There exists intricate crosstalk among the various mechanisms by which intratumor microbiome plays a role in primary liver cancer. For instance, transcriptional differences between the aforementioned virus-dominant and bacteriadominant HCC subtypes are primarily associated with the immune and metabolic pathways. Tumor tissues of the bacteria-dominant subtype, which were infiltrated with higher levels of M2 macrophage, exhibited enhanced activity in the amino acid, carbohydrate, lipid, and energy metabolism pathways [
49]. Particularly, amino acid metabolism was positively correlated with macrophage infiltration, suggesting that the intratumor microbiome may influence the tumor immune microenvironment through metabolic regulation (
Fig. 2). Considering the intricate interplay between the intratumor microbiome and liver cancer, our current understanding of the intratumor microbiome may only be scratching the surface. Unraveling the complex network between the intratumor microbiome and liver cancer will provide novel perspectives on this deadly disease.
The liver is a common target organ for metastatic malignant tumors, especially those originating from the digestive tract, such as CRC, gastric cancer, and pancreatic cancer [
79]. Komiyama et al. [
32] conducted a preliminary analysis of 19 cases of liver metastases and found that their intratumor microbiome was mainly composed of Proteobacteria, Bacteroidetes, Firmicutes, and Verrucomicrobia, showing significant differences from the microbiota of adjacent normal liver tissue. Another study found that the α and β diversities of intratumor microbiomes in metastatic cancer are associated with key immune features such as immune cell infiltration and PD-L1 expression levels. Additionally, patients with lower α diversity metastatic lesions tend to have poorer prognosis [
24]. While this study revealed the potential clinical significance of the microbiome in metastatic cancers, it included a heterogeneous group of patients with various types of metastatic lesions, such as lymph node, liver, and lung metastases. Therefore, the specific relationship between the intratumor microbiome of liver metastases, immune infiltration, and patient prognosis requires further clarification.
During the entire course of CRC, liver metastasis occurs in 40–50% of cases, with simultaneous liver metastasis presenting in approximately 15–25% [
80]. The intratumor microbiome within the primary CRC lesions has been shown to promote metastasis to the liver (
Fig. 3).
F. nucleatum strains ATCC 10953 and ATCC 25586 activate the NF-κB pathway via the pattern recognition receptor ALPK1 on the surface of CRC tumor cells, upregulating the expression of adhesion molecule ICAM1 and enhancing adhesion between tumor and vascular endothelial cells, thereby promoting tumor cell extravasation and metastasis [
81]. CRC tumor cells infected with
F. nucleatum secrete exosomes containing enriched miR-1246/92b-3p/27a-3p and CXCL16/RhoA/IL-8, which are delivered to uninfected tumor cells, enhancing their migration by activating the β-catenin pathway [
82].
The impact of the intrahepatic microbiome on liver metastasis should also be highlighted (
Fig. 3). Harmful bacteria from the intestine that spread to the liver facilitate liver metastasis of CRC.
E. coli C17 carrying the virulence regulator VirF disrupts the gut vascular barrier, leading to increased intestinal permeability and dissemination of intestinal bacteria to the liver through the portal vein system. Increased levels of bacteria in liver tissue upregulate the expression of inflammatory cytokines, recruit macrophages and neutrophils, and promote extracellular matrix deposition, forming a pre-metastatic niche [
9]. Furthermore, bacteria residing within CRC liver metastases promote the lactylation of retinoic acid-inducible gene 1 by enhancing lactate production in tumor tissues, which suppresses the NF-κB signaling pathway and promotes M2 macrophage polarization [
83]. This inhibits the activation of CD8+ T cells and upregulates PD-L1 expression in regulatory T cells, ultimately establishing a more pronounced immunosuppressive microenvironment.
INTRATUMOR MICROBIOME AS A NOVEL BIOMARKER
Due to differences in the microbiomes between tumor tissues and adjacent normal tissues and the type-specific nature of intratumor microbiomes across different cancers [
4-
6], some researchers have suggested that intratumor microbiomes may have potential value in the auxiliary diagnosis of liver cancer [
23,
36]. However, for some types of cancer, including liver cancer, this value is quite limited. This is because intratumor microbiome detection still requires obtaining corresponding tissue specimens, which restricts its feasibility as a noninvasive diagnostic method. Nevertheless, intratumor microbiomes have potential applications in predicting survival outcomes and treatment responses in patients with liver cancer.
An accurate prognostic assessment of tumors holds paramount importance for clinical decision-making. A growing body of research has highlighted the significant prognostic value of the intratumor microbiome. Riquelme et al. [
73] demonstrated the significant impact of intratumor microbiomes on the prognosis of patients with pancreatic cancer. Data from two medical centers indicated that long-surviving patients had significantly higher tumor microbial diversity than short-term survivors.
Pseudoxanthomona,
Saccharopolyspora, and
Streptomyces have shown considerable value in predicting the prognosis of patients with pancreatic cancer. Similarly, Qu et al. [
34] conducted a preliminary study with a small sample size to explore the relationship between intratumor microbiome and prognosis in patients with primary liver cancer. They divided 28 patients with primary liver cancer into long- and short-term survivors according to their 5-year overall survival (OS) after surgery. The composition of the intratumor microbiota differed significantly between the two groups.
Pseudomonas was more enriched in patients with OS longer than 5 years, showing a strong positive correlation between its relative abundance and patient prognosis. However, owing to the small sample size and the inclusion of three pathologically distinct types (HCC, ICC, and combined liver cancer), the findings may be limited. Later, a subsequent study revealed that a specific intratumor microbiome signature, comprising
Intestinimonas,
Brachybacterium, and
Rothia, was a predictive indicator for post-surgery OS in a cohort of 172 patients diagnosed with HCC. Intriguingly, the functional analysis suggested that the intratumor microbiome in short-term survivors was associated with the activation of NOD-like receptor signaling, as well as the modulation of the PI3KAKT and AMPK signaling pathways [
21].
Another study classified HCC into two subtypes, referred to as “hepatotypes,” based on the composition of intratumor microbiome at the phylum level [
35]. Significant differences were observed in OS and recurrence-free survival between these two subtypes, with hepatotype B, characterized by higher diversity in intratumor microbiome, showing a better prognosis. Further analysis revealed the enrichment of
Methylobacterium and
Akkermansia in hepatotype B, which suggested a more favorable prognosis [
35]. Song et al. [
84] integrated multi-omics data from the TCGA database to provide important insights into the prognostic value of the intratumor microbiome in HCC. Their study identified a microbial signature comprising 27 intratumoral microbes that served as predictors of OS. Subsequent multi-omics analyses illuminated the pathways through which the intratumor microbiome influences the prognosis of HCC, including the modulation of immune responses and alterations in tumor cell stemness [
84].
Alpha diversity of the intratumor microbiome has been shown to be significantly associated with lymph node metastasis and CA19-9 levels in patients with ICC. In patients with higher alpha diversity, the infiltration of Foxp3+ regulatory T cells within the tumor is more pronounced [
36]. These patients have significantly shorter OS and recurrence-free survival than those with lower alpha diversity, with approximately twice the risk of recurrence and death [
36]. Based on 11 bacterial genera associated with prognosis, Xin et al. [
36] developed a microbe-based risk scoring system with internal validation demonstrating its role as an independent prognostic indicator for ICC.
Commensal microbiomes have a notable influence on the therapeutic outcomes of cancer treatment. With our expanding knowledge of the intratumor microbiome, there is growing optimism that its features will facilitate more accurate predictions of antitumor treatment efficacy in the foreseeable future.
Hermida et al. [
85] explored the value of the intratumor microbiome in predicting the efficacy of chemotherapy in various types of cancers. Compared to the clinical characteristics of patients, the intratumor microbiome demonstrated superior effectiveness in predicting treatment responses. When combined with gene expression profiles, the predictive performance of the models was further enhanced [
85]. Drug inactivation and autophagy modulation are important mechanisms by which the intratumor microbiome induces chemoresistance. Geller et al. [
11] found that Gammaproteobacteria can produce a specific subtype of cytidine deaminase that inactivates gemcitabine, thereby conferring resistance to gemcitabine in pancreatic cancer and other tumors.
F. nucleatum regulates tumor cell autophagy through LC3 and ATG7, leading to resistance of esophageal squamous cell carcinoma to 5-fluorouracil, cisplatin, and docetaxel [
86].
The intratumor microbiome also affects the efficacy of chemoradiation therapy.
Lactobacillus iners in cervical cancer produces L-lactic acid, which alters tumor metabolism and reduces the sensitivity of cervical cancer to chemoradiation [
77]. Sun et al. [
87] found that the intratumor microbiome can predict the efficacy of neoadjuvant chemoradiation therapy in locally advanced rectal cancer, and alter it through interactions with cancer-associated fibroblasts.
The advent of immunotherapy has changed the landscape of cancer treatment, with immune checkpoint inhibitors (ICIs) established as important therapies for various cancers, including liver cancer. Virus-associated tumors show greater sensitivity to ICIs, reflecting the influence of viruses on tumor immunogenicity [
88]. For instance, patients with HBV-associated HCC exhibit favorable responses to ICIs, whereas those with NASH (nonalcoholic steatohepatitis) -associated HCC rarely benefit from immunotherapy [
89]. Compared with human papillomavirus (HPV)-positive head and neck squamous cell carcinoma patients, HPV-negative patients exhibit more pronounced immunosuppressive TME and therefore respond poorly to ICI treatment [
90]. Recently, bacteria and fungi within tumor tissues have also been found to predict patient responses to immunotherapy [
4,
6,
91]. Pushalkar et al. discovered that eliminating microbes from pancreatic cancer tissues using antibiotics not only reshapes the tumor immune microenvironment but also upregulates PD-1 expression, thereby enhancing the effectiveness of ICIs [
74]. In esophageal squamous cell carcinoma,
F. nucleatum inhibits T cell proliferation and cytokine secretion, thereby weakening the therapeutic effects of ICIs [
92], whereas Streptococcus promotes CD8+ T cell infiltration, indicating a favorable response [
93]. Currently, ideal biomarkers for predicting the efficacy of ICIs in liver cancer are lacking [
94]. Given the prominent roles of viruses, bacteria, and fungi in immune modulation, exploring the potential of the intratumor microbiome as a biomarker for predicting the clinical outcomes of immunotherapy for liver cancer is an intriguing and promising research direction.
THERAPEUTIC INNOVATIONS WITH INTRATUMOR MICROBIOMES FOR LIVER CANCER
Despite significant advances in liver cancer treatment in recent years, the effectiveness of the current treatment methods requires further improvement. Promoting beneficial symbiotic relationships between the microbiota and hosts and integrating the application of the intratumor microbiome with existing cancer therapies could provide new opportunities to enhance interventions (
Fig. 4).
Some measures, such as modulating the intratumor microbiome, offer benefits in enhancing the effectiveness of current methods. For instance, fecal microbiota transplantation is a promising therapy for restoring microbiota imbalance. Animal models have demonstrated that fecal microbiota transplantation can significantly modulate the liver and intratumor microbiomes [
8,
73], suggesting its potential in improving chemotherapy and immunotherapy outcomes in cancer patients beyond the gut microbiota. Rational use of antibiotics is also an alternative approach. Animal experiments have shown that antibiotic treatment to eliminate intratumoral microbes can inhibit tumor growth and enhance the efficacy of immunotherapy [
27,
74]. However, antibiotics may inadvertently disrupt the beneficial microbiota and interfere with the gut microbiome, potentially compromising treatment outcomes. Therefore, their use requires optimal personalized strategies considering factors such as antibiotic type, dosage, administration route, and patient condition. Novel approaches like “cocktail” antibiotic formulations and nano-antibiotic particles show promising specificity in modulating the intratumor microbiome [
26,
95], thus warranting further exploration. Probiotic supplementation can promote beneficial gut microbiota and improve the treatment response in cancer patients [
96]. The oral probiotic
L. reuteri has been shown to migrate from the gut to extraintestinal tumor sites in mice, driving antitumor immune responses, enhancing the efficacy of ICIs treatment, and improving survival outcomes [
97]. Furthermore, bacteriophages, which can selectively invade bacteria without infecting human cells, offer a safe method for eliminating bacteria. Given their host specificity, bacteriophages offer a more targeted approach for clearing harmful bacteria from the human body, with studies exploring their feasibility in cancer therapy [
98]. Nevertheless, further research is needed to identify the specific microbes that promote tumor growth and metastasis and to develop corresponding bacteriophages for targeting them.
Other approaches do not target the intratumor microbiome for intervention, but instead directly utilize microbes to exert therapeutic effects on tumors. In the late 19th century, William B. Coley began injecting live or inactivated bacteria into patients with late-stage cancer in an attempt to save their lives [
99]. Today, engineered bacteria, made safer through methods including genetic modifications, can function therapeutically by enhancing anti-tumor immune responses, expressing prodrug-converting enzymes, producing cytotoxic substances, and modulating the TME [
100]. Naturally occurring or genetically modified oncolytic viruses selectively infect tumor cells, providing a novel cancer treatment method by directly lysing host cells, enhancing antitumor immune responses, and disrupting tumor vasculature. This approach has been approved for the clinical treatment of melanoma, glioblastoma, and nasopharyngeal carcinoma [
101]. Thus, innovative methods of using microbes directly for cancer therapy, with their unique antitumor mechanisms and acceptable safety profiles, show significant clinical potential and may provide new treatment options for liver cancer in the future.
FUTURE PERSPECTIVES
Approximately 13% of new cancer cases worldwide in 2018 were attributed to microbial infections, accounting for 2.2 million new cases [
102]. Despite the established connections between liver cancer and certain microbes, and extensive research on the mechanisms of the gut microbiota in liver diseases [
2,
103], the intratumor microbiome of liver cancer has only recently gained attention. Moreover, research on the intratumor microbiome in liver cancer is lacking.
Currently, our understanding of the intratumor microbiome in liver cancer remains primarily limited to the characterization of the microbial community and its association with patient clinical phenotypes and survival outcomes. However, the underlying mechanisms by which the intratumor microbiome influences the occurrence and progression of liver cancer require further clarification. Therefore, large-scale multi-omics studies are needed to fully analyze the interactions between intratumor microbes and their hosts. Mechanistic studies are crucial to elucidate the causal relationship between the intratumor microbiome and liver cancer. Many symbiotic microorganisms are difficult to isolate and culture using conventional techniques, which poses practical challenges for research [
104,
105]. Advances in novel technologies such as xenograft models, co-culture systems, organ-on-chips, and three-dimensional bioprinting have provided excellent in vitro and in vivo models for microbiome studies [
63,
106], aiding in a better understanding of the interplay between the microbiome and liver cancer.
Furthermore, current studies on this topic are cross-sectional in design; thus, they are unable to capture comprehensive changes of the intrahepatic or intratumor microbiome throughout the occurrence and progression of liver cancer. Changes in the evolution of the microbiome influence disease onset and prognosis [
107]. Therefore, conducting comprehensive longitudinal analyses of the intrahepatic microbiome during the progression of chronic hepatobiliary diseases, including dynamics in microbial composition, diversity, genetic variations, and functions, is crucial for a deeper understanding of pathogenesis. It also provides important clues for predicting disease progression and even the risk of carcinogenesis in the future, enabling early detection and warning.
We also need to understand the influence of the intratumor microbiome on liver cancer at higher spatial resolutions. Microbes are not uniformly distributed within tumor tissues [
108,
109], and the spatial heterogeneity of HCC intratumor microbiome influences immune cell infiltration [
109]. Given their close interactions with various components of the TME, intratumor microbiomes are likely to exert significant local effects on adjacent cells. Future research should integrate advanced imaging techniques, such as RNAscope multiplex fluorescence, single-cell sequencing, and spatial transcriptomics, to precisely elucidate the spatial localization and local effects of microbes within tumors.
Although the intratumor microbiome shows potential in predicting patient survival outcomes, rigorous multicenter external validation is necessary before it can effectively assist in clinical decision-making. This validation is crucial to ensure the generalizability and reliability of research findings. Future studies are needed to improve several aspects to overcome the barriers to clinical translation. The low biomass of the intratumor microbiome in liver cancer poses challenges for accurate detection using high-throughput sequencing. Establishing rigorous standardized experimental procedures and data analysis workflows is key to addressing this issue. Each step of the research process, including tissue sampling, nucleic acid extraction, amplification, library construction, and sequencing, requires standardized operations to minimize the introduction of exogenous microbial contamination. Developing effective decontamination algorithms is essential for the precise identification and removal of contaminants, thereby minimizing bias. Researchers should adhere to microbiome study standards such as the STORMS and RIDE checklists to facilitate comparative analysis and comprehensive summarization of research outcomes [
110,
111]. However, current research primarily relies on relative quantification methods in microbiome sequencing and analysis, which provide relative abundance information but cannot quantify the microbial load across samples. If the variation in microbial load is significant, accurately quantifying species composition and functional changes becomes difficult. This can also lead to biases when assessing the correlations between the microbiome and clinical characteristics [
112,
113]. Therefore, future clinical studies should adopt absolute quantification methods for intratumor microbiomes of patients with liver cancer. These methods, based on highthroughput sequencing combined with techniques such as flow cytometry, quantitative polymerase chain reaction, or microfluidics, enable more precise identification of the true association between the intratumor microbiome and liver cancer.
CONCLUSION
The rapidly advancing field of intratumor microbiome research has unveiled the coexistence of the intratumor microbiome with liver cancer. It is engaged in cancer initiation and progression through a series of interconnected mechanisms and is now considered a new dimension of the hallmark features of cancer [
114]. As emerging studies continue to delineate the unique features of the intratumor microbiome in liver cancer, it is imperative to conduct thorough assessments of its functional mechanisms within the TME and the complex interactions with liver cancer. The predictive value of the intratumor microbiome for prognosis and treatment response is becoming increasingly evident, and it holds promise for being harnessed in novel interventions tailored to liver cancer. Standardized methodologies and well-designed clinical trials are indispensable for unlocking the full potential of the intratumor microbiome in the context of the personalized management strategies for liver cancer in the future.
FOOTNOTES
-
Authors’ contribution
Xindi Ke, Shangze Jiang, and Qiaoxin Wei contributed equally to this work. Xindi Ke: conceptualization, investigation, methodology, writing - original draft; Shangze Jiang: conceptualization, investigation, visualization. Qiaoxin Wei: investigation, writing - review & editing, validation. Minghao Sun: investigation, validation. Hang Sun: validation. Mingchang Pang: methodology. Mei Liu: investigation. Lejia Sun: project administration, conceptualization. Huayu Yang: supervision, writing - review & editing. Yilei Mao: supervision, writing - review & editing, conceptualization.
-
Acknowledgements
This work was supported by the National High Level Hospital Clinical Research Funding (2022-PUMCH-B-034), Chinese Academy of Medical Sciences Innovation Fund for Medical Sciences (CIFMS) (No. 2021-I2M-1-058), China Postdoctoral Science Foundation (No. 2023T160328, No. 2023M731765), and University Science Research Project of Jiangsu Province (Natural Science) (No. 23KJB320002).
-
Conflicts of Interest
The authors have no conflicts to disclose.
Figure 1.Origins of the intratumor microbiome in liver cancer. The unique microenvironment in liver cancer provides favorable circumstances for intratumor microbiome. Given the intimate link between the gut and liver, the translocation of gut microbiota via the portal vein system and biliary tract constitutes a significant source of the intratumor microbiome in liver cancer. Additionally, microbiota originating from other mucosal sites can disseminate to liver tumors through the bloodstream. Furthermore, microorganisms present in adjacent liver tissues have the potential to colonize liver cancer. In the case of metastatic liver cancer, the intratumor microbes can be transported by tumor cells from the primary lesion through a co-metastasis route.
Figure 2.The role of intratumor microbiome in primary liver cancer. In hepatocellular carcinoma (HCC), hepatitis B virus and alcohol contribute to a pro-intratumor microbiome that correlates with oncogenic pathways and immune dysregulation. The bacteria-dominant intratumor microbiome in HCC influences metabolic pathways and immune landscape in tumor, leading to unfavorable clinical characteristics and a poor prognosis. Stenotrophomonas maltophilia induces NLRP3 inflammasome activation in hepatic satellite cells, thereby accelerating the progression from cirrhosis to HCC. Gut-liver translocation of Klebsiella pneumoniae promotes HCC by triggering pro-inflammatory responses via the PBP1B-TLR4 axis. Liver fluke infection alters the hepatobiliary microbiota, enhancing its capacity to produce bile acids and ammonia, which may contribute to the development of cholangiocarcinoma. Paraburkholderia fungorum within intrahepatic cholangiocarcinoma demonstrates anti-tumor potential by regulating amino acid metabolism. NF-κB, nuclear factor kappa-B; PBP1B, penicillin-binding protein 1B; TLR4, Toll-like receptor 4.
Figure 3.The role of intratumor microbiome in metastatic liver cancer. Intratumor microbes within the primary colorectal cancer stimulate liver metastases by upregulating the ICAM1 and modulating β-catenin signaling through exosomes. Disruption of the gut vascular barrier, induced by tumor-resident microbes, results in the dissemination of bacteria to the liver, thereby facilitating the establishment of a premetastatic niche through the recruitment of myeloid-derived cells and the deposition of extracellular matrix. Bacteria present in liver metastases promote tumor progression via lactylation of RIG-1, leading to subsequent immune modulation. ALPK1, alpha-kinase 1; ICAM1, intercellular cell adhesion molecule-1; NF-κB, nuclear factor kappa-B; PD-L1, programmed cell death ligand 1; PV-1, plasmalemmal vesicle associated protein-1; RIG-1, retinoic acid-inducible gene-1.
Figure 4.Innovative therapeutic approaches based on intratumor microbiome for the treatment of liver cancer. Some interventions, including fecal microbiota transplantation, antibiotics, prebiotics, and bacteriophages, can enhance the effectiveness of current treatments for liver cancer by modulating the intratumor microbiome. Other methods directly leverage microorganisms within the tumor, such as engineered bacteria and oncolytic viruses, to serve as therapeutic agents in the treatment of liver cancer.
Table 1.High-throughput technologies for detecting intratumor microbiome
Table 1.
|
Technology |
Principle |
Advantage |
Limitation |
|
Amplicon-based sequencing |
Sequencing of the hypervariable regions of 16S rRNA and 18S rRNA genes or the internal transcribed spacer in fugal rRNA gene |
Low cost; convenient; good accessibility |
Low resolution; bias induced by PCR |
|
Metagenomic shotgun sequencing |
Sequencing of DNA from all the organisms, including humans and microbiome |
Simultaneous multi-kingdom detection; high resolution; providing functional information |
Expensive; complex data processing; severe host genome contamination |
|
Metatranscriptomic shotgun sequencing |
Sequencing of RNA from all the organisms, including humans and microbiome |
Simultaneous multi-kingdom detection; high resolution; providing gene expression profiles |
Vulnerable to RNA instability; Host RNA contamination; complex data processing |
|
Data mining from DNA or RNA sequencing dataset of human samples |
Removal of human sequences followed by alignment with microbial genomic references |
Low cost; no new sample required; matched datasets for multi-omic analyses |
Complicated data interpretation; severe host genome contamination |
|
Metaproteomics |
Accessing human and microbial proteins in the samples |
Functional analysis at the protein level; able to evaluate the host-microbiome interplay |
Imcomplete reference database; difficulties in protein extraction |
|
Metabolimics |
Detection of global metabolites in the samples |
Direct reflection of physiological status and functional characteristics; able to display metabolic networks |
Unable to distinguish microbial or host-derived metabolites; imcomplete reference database |
Table 2.The characterization of intratumor microbiome in human liver cancer
Table 2.
|
Author |
Sample |
Sample type |
Sequencing method |
Removal of contamination |
α diversity |
β diversity |
Enriched taxa |
Decreased taxa |
|
Chakladar et al. [48] (2020) |
373 HCC and 50 adjacent tissues |
Not mentioned. |
RNA-sequencing from TCGA database |
Decontamination through correction based on dates, plates, and read counts |
Not mentioned. |
Not mentioned. |
Pantoea agglomerans, etc. |
Escherichia coli, etc. |
|
Komiyama et al. [32] (2021) |
47 HCC, 15 ICC, 19 metastatic LC samples, and some adjacent tissues |
Fresh frozen |
16S rRNA gene sequencing |
Sterile sampling procedures, but no removal of potential contaminations. |
Higher than peri-tumor samples. |
Different from peri-tumor samples. |
Bacteroides, Romboutsia, etc. |
Cutibacterium, Diaphorobacter, etc. |
|
Huang et al. [23] (2022) |
68 HCC samples, 71 adjacent tissues, and 29 samples of normal liver |
Fresh frozen |
16S rRNA gene sequencing |
None. |
Higher than normal liver tissues but comparable to peri-tumor samples. |
Different from normal liver tissues but similar to peri-tumor samples. |
Gammaproteobacteria, Saccharimonadia, Bacilli, etc. |
Acdobacteriae, Parcubacteria, etc. |
|
Liu et al. [22] (2022) |
46 HCC samples, 28 adjacent. tissues, and 33 samples from normal controls |
Fresh frozen and FFPE |
16S rRNA gene sequencing |
Decontamination through blank controls, and correction based on DNA extraction, PCR and sequencing batches |
Lower than normal liver tissues, but higher than peri-tumor samples. |
Different from normal liver tissues but similar to peri-tumor samples. |
Firmicutes, Streptococcus, Helicobacter, Bifidobacterium, Lactobacillus, Bacillus, etc. |
Proteobacteria, Acinetobacter, etc. |
|
Qu et al. [34] (2022) |
11 HCC, 8 ICC, 9 cHCC-ICC and 28 adjacent tissues |
FFPE |
16S rRNA gene sequencing |
None. |
Comparable to peri-tumor samples. |
Similar to peri-tumor samples. |
Rhizobiaceae and Agrobacterium
|
Pseudomonadaceae and Pseudomonas
|
|
Chai et al. [60] (2023) |
45 ICC and 49 adjacent tissues |
Not mentioned. |
16S rRNA gene sequencing and single-cell RNA-sequencing |
Decontamination through blank controls and the “decontam” algorithm |
Higher than peri-tumor samples. |
Different from normal liver tissues but similar to peri-tumor samples. |
Verrucomicrobia, Fusobacteria, Acidovorax, Staphylococcus, etc. |
Proteobacteria, Paraburkholderia fungorum, Pseudomonas azotoformans, etc. |
|
He et al. [33] (2023) |
99 HCC and adjacent tissues |
Not mentioned. |
16S rRNA gene sequencing |
None. |
Higher than peri-tumor samples. |
Different from peri-tumor samples. |
Fusobacteriota, Lactobacillus, Fusobacterium, Neisseria, etc. |
Actinobacteriota, Verrucomicrobiota, Faecalibacterium, Ruminococcaceae, Pseudomonas, etc. |
|
Li et al. [49] (2023) |
29 pairs of HBV-related HCC and adjacent tissues and 12 CHB liver tissues |
Fresh frozen |
Metagenomic sequencing |
Sterile sampling procedures, but no removal of potential contaminations. |
Lower than peri-tumor and CHB liver tissues. |
Different from normal liver tissues but similar to peri-tumor samples. |
Methylobacterium sp. XJLW. |
Klebsiella variicola
|
|
Sun et al. [35] (2023) |
HCC and adjacent tissues from 91 patients |
Fresh frozen |
16S rRNA gene sequencing |
Decontamination through blank controls and the “decontam” algorithm |
Comparable to peri-tumor samples. |
Different from peri-tumor samples. |
Actinobacteria, Nesterenkonia, Rubrobacter, Prauserella, etc. |
Deinococcus-Thmus, Anoxybacillus, Aeribacillus, etc. |
|
Xin et al. [36] (2024) |
121 ICC and 89 adjacent tissues |
Fresh frozen |
16S rRNA gene sequencing |
Decontamination through blank controls and the “decontam” algorithm |
Higher than peri-tumor samples. |
Different from peri-tumor samples. |
Acidobacteriota, Actinobacteria, Bacteroidetes, Firmicutes, etc. |
Proteobacteria, etc. |
|
Jiang et al. [21] (2025) |
172 pairs of HCC and adjacent tissues |
Not mentioned. |
16S rRNA gene sequencing |
Paraffin controls and a reference to literature |
Comparable to peri-tumor samples. |
Similar to peri-tumor samples. |
Not mentioned. |
Not mentioned. |
|
Li et al. [37] (2024) |
19 pairs of HCC and adjacent tissues |
Fresh frozen |
16S rRNA gene sequencing |
Sterile sampling procedures, but no removal of potential contaminations. |
Comparable to peri-tumor samples. |
Similar to peri-tumor samples. |
Lactobacillales, Veillonellaceae, Rhodobacter, Megasphaera, etc. |
Pseudochrobactrum, etc. |
Abbreviations
formalin-fixed paraffin-embedded
intercellular cell adhesion molecule-1
intrahepatic cholangiocarcinoma
immune checkpoint inhibitor
metabolic dysfunction-associated steatotic liver disease
penicillin-binding protein 1B
polymerase chain reaction
programmed cell death ligand 1
plasmalemmal vesicle associated protein-1
retinoic acid-inducible gene-1
ribosomal ribonucleic acid
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, Huayu Yang1
