Accepted Article

DR. PURU RATTAN (Orcid ID : 0000-0001-7410-488X)

DR. VINOD RUSTGI (Orcid ID : 0000-0002-4105-5783)

Article type : Review Articles

TITLE PAGE
Title: The Microbiome and Hepatocellular Carcinoma
Short Title: Microbiome and HCC

Authors:
Puru Rattan¹,², email: pr447@rwjms.rutgers.edu
Carlos D. Minacapelli¹,², email: minacacd@rwjms.rutgers.edu
Vinod Rustgi¹,², email: vinod.rustgi@rutgers.edu
1 Division of Gastroenterology and Hepatology, Rutgers Robert Wood Johnson Medical School, New
Brunswick, New Jersey
2 Center for Liver Diseases and Masses, Rutgers Robert Wood Johnson Medical School, New
Brunswick, New Jersey

Keywords:
Hepatocarcinogenesis; Dysbiosis; Chronic Liver Disease

This article has been accepted for publication and undergone full peer review but has not been
through the copyediting, typesetting, pagination and proofreading process, which may lead to
differences between this version and the Version of Record. Please cite this article as doi:
10.1002/LT.25828
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 FOOTNOTE PAGE
Accepted Article
Abbreviations:
BA, bile acid; CLD, chronic liver disease; DCA, deoxycholic acid; DEN, diethyl-nitrosamine; ESLD,
end-stage liver disease; FXR, farnesoid X receptor; HBV, hepatitis B virus; HCC, hepatocellular
carcinoma; HCV, hepatitis C virus; HSC, hepatic stellate cell; LPS, lipopolysaccharide; MAMPs,
microbe-associated molecular pattern; miRNA, microRNA; mTOR, mammalian target of rapamycin;
NAFL, non-alcoholic fatty liver; NAFLD, non-alcoholic fatty liver disease; NASH, non-alcoholic
steatohepatitis; NBNC, non-hepatitis B, non-hepatitis C; NF-κB, nuclear factor κB; NOD, nucleotide-
binding oligomerization domain; OCA, obeticholic acid; PBC, primary biliary cholangitis; PRR, pattern
recognition receptor; rRNA, ribosomal RNA; SASP, senescence-associated secretory phenotype ;
SCFA, short-chain fatty acids; TLR, toll-like receptor; TNF, tumor necrosis factor

Corresponding Author:
Vinod Rustgi, MD, MBA
Rutgers Robert Wood Johnson School of Medicine
One Robert Wood Johnson Place
Medical Education Building, Rm # 466
New Brunswick, New Jersey 08901
Email: vinod.rustgi@rutgers.edu
Phone: 301-801-5814
Fax: 723-235-5537

Funding: This work received no external funding

Disclosures: No conflicts of interest, financial or otherwise, are declared by the authors.

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 Abstract
Accepted Article The human microbiome is a vast and complex system encompassing all the microbes and
their genes that occupy the environmentally exposed surfaces of the human body. The gut
microbiota and its associated microbiome play an integral role in mammalian metabolism, immune
tolerance as well as immunocompetence. Disruptions in the human gut microbiome are associated
with a cycle of hepatocyte injury and regeneration characteristic of chronic liver disease (CLD). The
persistence of this inflammation has been shown to induce the accumulation of genetic and
epigenetic changes leading to hepatocellular carcinoma (HCC). Therefore, the importance and
prognostic influence of the gut microbiome on hepatocarcinogenesis has been increasingly studied in
recent years. This review discusses the mechanisms by which imbalances in the gut microbiome
disturb the gut-liver axis to impact hepatocarcinogenesis, including disruption of the intestinal barrier,
changes in bile acid metabolism and reduction in tumor-suppressing miRNA. Furthermore, this
review summarizes recent advances in potential microbiome-based therapeutic opportunities in
HCC.

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 Introduction
Accepted Article The liver is now the second-most commonly transplanted organ with both acute and chronic
liver diseases accounting for at least 2 million deaths worldwide every year (1). Primary liver cancer,
a majority being hepatocellular carcinoma (HCC), contributes significantly to this burden of liver
disease. With over 840,000 new cases and approximately 780,000 deaths attributed to it in 2018,
liver cancer is the sixth most common cancer diagnosis and the fourth most common cause of
malignancy-related mortality in the world (2,3). Additionally, the 5-year survival rate of liver cancer is
18%, placing it second only to pancreatic cancer as the malignancy with the worst prognosis. In
2020, an estimated 42,810 adults (30,170 males and 12,640 females) in the United States will be
diagnosed with primary liver cancer (4).
Curative treatment for HCC is effective only in early-stage tumors and involves invasive
modalities such as ablation, surgical resection or liver transplant (5). A low surveillance rate, often
below 20% in high-risk populations (6,7), as well as a lack of options for chemoprevention contribute
to the high mortality of HCC. While numerous agents, including non-steroidal anti-inflammatory drugs
(NSAIDs), statins, and nutritional supplements such as coffee have been studied, none have
demonstrated a clear benefit in HCC chemoprevention (8). A scarcity of non-invasive therapeutic
options has been attributed to the heterogeneity of HCC genetics and etiology (8,9).
This review explores the mechanisms by which gut dysbiosis, an imbalance in the
microbiome, has been discovered to impact hepatocarcinogenesis. Furthermore, it summarizes the
potential for microbiome-based therapeutic opportunities for HCC.
The Microbiome
The emergence of microbiome-related discoveries over the last two decades can be related
directly to technological advances in gene sequencing and bioinformatics. The human microbiota is
present on all surfaces of the human body that interact with the environment including the skin, the
aerodigestive tract, the vaginal cavity in females and the entirety of the gastrointestinal lumen. Each
of these surfaces house a specific niche of the microbiome forming their own unique signature (10).
The gut microbiota, and its associated microbiome, has been recognized to play an integral role in
mammalian metabolism, immune tolerance as well as immunocompetence (11).
Composed mainly of bacteria, with significant contributions from viruses, fungi and archaea,
the gut microbiota is thought to number over 100 trillion cells (12). The gut microbiome is comprised
of all the genetic information contained within these organisms. Consisting of over 9 million genes,
the gut microbiome outnumbers the human genome by 500-fold (13,14). Each individual’s

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 microbiome begins to form during birth and goes through myriad changes through development
Accepted Article
influenced by environmental and behavioral factors (15,16). Additionally, disease states such as
inflammatory bowel disease, liver cirrhosis and even Parkinson’s disease have been associated with
alterations in the gut microbiota with this effect being bi-directional (17–19). The liver is known to play
an essential part in the mammalian metabolic and immune systems via the gut-liver axis, which
constitutes its unique connection with the gastrointestinal tract through the portal circulation (20,21).
With increased microbiome-related study in the natural history of chronic liver disease (CLD), the
significance of the gut microbiome as an intermediary to form the gut-microbiota-liver axis is being
realized.
High-throughput sequencing of 16S ribosomal RNA (rRNA) has allowed the detailed profiling
of the human microbiome (22). 16S rRNA is a component of the small 30S subunit of bacterial
ribosomes and is a reliable phylogenetic marker (10,22). Samples from humans can be analyzed for
bacterial genetic material and, if detected, can undergo further PCR amplification of 16S rRNA (23).
These rRNA sequences are then matched against databases of known microbial genetic material to
generate a profile of the microorganisms which contributed to the sample (10,23). The raw rRNA
sequences can identify organisms down to the level of genus but further metagenomic analyses of
specific gene products can give additional insights to the species level (23) (See Figure 1). Shotgun
HTS techniques such as pyrosequencing allow this methodology to be simultaneously applied to
hundreds of microbial communities (22). Leveraging these technologies has led to the creation of
large databases of human-associated microbial communities such as the Human Microbiome Project
created by the US National Institutes of Health (24) and MetaHIT in Europe (10).
These large databases have shown that every anatomical niche has a distinct pattern of
microbiome composition even after adjustment for significant interpersonal variation (10).
Importantly, the unique microbiome signature of these anatomical sites plays an integral part in
various stages of human life. For example, the vaginal microbiome, composed mainly of the genus
Lactobacillus, determines the initial microbial colonies that are established on the skin and
gastrointestinal tract of a newborn human after vaginal birth (10). Later in life, the cutaneous
microbiome has also been postulated to influence the presence of acne vulgaris due to the amount
of Propionibacterium acnes on the skin, as well as immune-mediated dermatological conditions such
as psoriasis which is associated with an increased abundance of organisms within the Firmicutes
phylum and a decreased presence of the Actinobacteria phylum (10). However, the role that the
microbes residing in the GI tract play in human health and disease have been the most thoroughly

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 studied.
Accepted Article
The Gut Microbiota and Liver Disease
The course of CLD is defined by a chronic inflammatory insult to the hepatic parenchyma
which over time results in worsening fibrosis with eventual progression to end-stage liver disease
(ESLD) (See Figure 2). This cycle of hepatocyte injury and regeneration not only leads to persistent
inflammation but also disrupts the regulation of an intricately balanced relationship between the
gastrointestinal tract and the liver. The gut microbiome mediates this bi-directional interaction
through the unique hepatic portal system, the enterohepatic circulation of bile acids and the systemic
circulation. Homeostasis within the gut-liver axis is further preserved via extensive crosstalk between
the gut microbiome and the immune, metabolic, and neuroendocrine systems. As critical
intermediaries in the gut-liver axis, the composition and function of the gut microbiome have co-
evolved with their hosts (10). As such, at baseline the majority of human gut microbiota is composed
of organisms within the phylum Firmicutes with contributions from the Actinobacteria and
Verrucomicrobia phyla (23). These commensal organisms maintain this balance by preventing the
overgrowth of pathogenic organisms such as certain families in the Bacteroidetes and the
Proteobacteria phyla. Additionally, a healthy microbiome functions as a whole to preserve the
integrity of the intestinal epithelium, modify bile acids to be potent signals in the gut-liver axis and
immune system, and metabolize dietary contents into short-chain fatty acids (SCFA) as
intermediaries to the metabolic system (23).
An intact gut epithelium, the natural barrier formed by multiple layers and tight junctions
between enterocytes, has an integral role in modulating the gut-liver axis. Disruption of this barrier
leading to increased intestinal permeability can be due to overgrowth of pathogenic bacteria, CLD-
related systemic inflammation, as well as direct effects of certain CLD etiologies such as excess
ethanol (25). This “leaky gut” exposes the liver parenchyma to various microbiota-associated
molecular patterns (MAMPs) and metabolites via the portal circulation. MAMPs tend to be essential
microbial structures, such as lipopolysaccharide (LPS) a cell wall component in all gram-negative
bacteria, whose effects are mediated by evolutionary conserved receptors known as the pattern-
recognition receptors (PRRs) (25,26). Several families of PRRs, including toll-like receptors (TLRs)
and the nucleotide-binding oligomerization domain (NOD)-like receptors, have been described. The
cell type expressing the PRR determines the nature of the innate immune response; however, these
share common signaling pathways which eventually lead to the activation of pro-inflammatory
transcription factors such as NF-κB and interferon regulatory factor 3 (26). The liver contains

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 numerous PRRs to identify and sequester blood-borne pathogens in the portal circulation.
Accepted Article
Specifically the resident macrophages of the liver, Kuppfer cells, are known to express TLR4 which
specifically recognizes LPS and release inflammatory cytokines such as interleukin-1, interleukin-6
and tumor necrosis factor in response (25,27). The activation of hepatic TLR4 via LPS has been
demonstrated to promote chronic hepatic inflammation in the presence of increased intestinal
permeability (28–30).
The enterohepatic circulation of bile acids serves an equally important role in preserving the
balance of the gut-liver axis. Primary conjugated bile acids produced in the liver are metabolized in
the gut by commensal organisms, especially certain restricted clusters within the order Clostridiales,
into secondary conjugated bile acids through 7α-dehydroxylation (31). These bile acids are then
sensed by the farnesoid X receptor (FXR) and epidermal growth factor receptors which direct
feedback to the liver as well as conducting local regulation of the gut epithelium and microbiota
composition (25,31,32).
The presence of dysbiosis in ESLD has been studied and reviewed in great detail (18,33–37).
Specifically, the microbial composition in patients with ESLD tend to have increased pathogenic
bacteria from the Enterobacteriaceae and decreased commensal organisms from the Firmicutes
phylum. The reduced enterohepatic circulation of bile acids, impaired gut motility, and small intestinal
bacterial overgrowth in ESLD have been implicated in the development of these disruptions in the
microbiome (35,38). Additionally, the presence of portal hypertension related complications in ESLD
such as ascites with spontaneous bacterial peritonitis (34) and hepatic encephalopathy (36,38–40)
have also been shown to contribute to the altered gut microbiome. Importantly, a combination of
these increased pathogenic organisms with ESLD-mediated leaky gut can further potentiate the
development of systemic infections in ESLD due to bacterial translocation. These infections can lead
to an increased incidence of decompensation and acute-on-chronic liver failure which, with their poor
prognosis, add to the burden of liver disease (41).
The physiological changes that lead to dysbiosis in ESLD precede the development of overt
cirrhosis and are increasingly present in CLD as that disease process advances. The specific
etiologies underlying CLD have been shown to have distinct changes in their gut microbiome profile
(See Table 1). Specifically, alcohol (42–44), non-alcoholic fatty liver disease (NAFLD) (45,46), and
chronic hepatitis B (HBV) (43,47,48) and hepatitis C (HCV) (47) infections have been studied in the
most detail. The mechanisms underlying dysbiosis in these specific liver diseases have been
reviewed extensively by Shen et al. (35). Computational analysis with machine-based learning

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 utilizing 16S rRNA databases has reaffirmed these observations of compositional imbalance in liver
Accepted Article
disease (49). This tool allows assessment of the predictive power of microbiome composition by
large scale shotgun metagenomics and provides an accurate and practical use for the archived
microbiome profile.
The study of the human gut microbiome and liver disease has shown some heterogeneity
and conflicting results in terms of the abundance of specific organisms (See Table 2). However, the
compositional change in CLD is generally characterized by a loss in microbial diversity with loss of
beneficial microorganisms and an increased presence of pathogenic microbes. Interestingly, these
changes tend to be most pronounced between healthy individuals and patients with advanced
fibrosis or cirrhosis (36).
The Gut Microbiota and Hepatocarcinogenesis
HCC is the end-product of a multifaceted and complex process. Most HCC develops in the
setting of advanced fibrosis in ESLD but can also occur prior to significant fibrosis in the setting of
chronic HBV infection or NAFLD (50). The persistent inflammation and hepatic fibrosis associated
with CLD described above also tends to favor the accumulation of genetic and epigenetic changes,
such as mutations in the TERT promoter, TP53 or WNT signaling genes, that lead to HCC
development (9). However, due to the remarkable genetic heterogeneity of HCC lesions (9,51), and
the rising prevalence of non-cirrhotic HCC in NAFLD (50), there has been an increasing focus on the
contribution of the chronic inflammatory milieu in which HCC develops (52). This tumor
microenvironment is composed of pre-cancerous hepatocytes surrounded by fibrotic tissue and the
vast quantity of innate and adaptive immune cells native to the liver. These cells include Kuppfer
cells, hepatic stellate cells (HSCs), natural killer cells, natural killer T cells, and other innate
lymphocytes. As the liver continually processes foreign material via the portal circulation, a mildly
immunosuppressive environment is maintained by these cells with a balanced release of pro-
inflammatory and anti-inflammatory cytokines to prevent over activation of the immune system (53).
It is theorized that with persistent chronic inflammation and advancing fibrosis this balance is
disrupted resulting in reduced immune surveillance and resultant hepatocarcinogenesis (27,53). The
importance and prognostic influence of the gut microbiota on this process has been increasingly
described in recent years.
The evidence of the relationship between the gut-microbiota-liver axis relationship and HCC
formation has been primarily derived from pre-clinical animal model studies and based on the
theories that CLD-related inflammation causes dysbiosis and intestinal barrier dysfunction (See

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 Figure 2). Yu et al. showed that elevated levels of plasma LPS were found in rats that developed
Accepted Article
HCC after diethyl-nitrosamine (DEN) exposure (28). This group also demonstrated that the effect of
LPS is in fact mediated by TLR4 using knock-out models. The TLR4 receptor knock-out animals
showed a much lower incidence of HCC as well as lower levels of inflammatory cytokines such as IL-
6 and TNF-alpha. Further studies have linked this effect to dysbiosis by demonstrating DEN-exposed
animals having an increased incidence of LPS-containing Gram-negative bacteria (Escherichia coli,
Atopobium, Collinsella, Eggerthella, and Coriobacterium). The use of high-dose probiotics in these
animals not only reversed the dysbiosis to a degree but also reduced the number and size of HCC
lesions (29,30).
In addition to the LPS-TLR4 mediated effects, CLD-related dysbiosis can also cause
suppression of the immune system (54,55) resulting in further propagation of hepatocarcinogenesis.
The induction of HSCs in the tumor microenvironment into a senescence-associated secretory
phenotype (SASP) has been associated with the suppression of antitumor immunity (56,57) (See
Figure 2). Yoshimoto et al. demonstrated the development of the SASP HSCs in obese animal
models did not appear to be mediated by the “leaky gut” pathway or TLRs, but instead was
associated with increased levels of deoxycholic acid (DCA), a secondary bile acid with increased
prevalence in obesity-induced dysbiosis (56). More recent studies have delineated that the gut
microbiota in NAFLD-related HCC animal models has increased Bifidobacterium and a group of
organisms in the Clostridiales order, formerly known as the “Clostridium cluster XVIII”. Both of these
groups are involved in bile acid modification and implicated in the conversion of primary bile acids to
secondary bile acids, such as DCA (58,59).
The relationship between bile acids and the gut microbiome goes far beyond obesity-induced
changes. CLD, especially when it has progressed to ESLD, impacts bile flow resulting in lower
secretion of conjugated bile acids into the small intestine. This process has marked effects on the gut
microbiome as the pool of conjugated bile acids can directly influence the composition of organisms
via their intrinsic anti-microbial properties (31). Kakiyama et al. postulated that the reduced bile
production in cirrhosis leads to overgrowth of potentially pathogenic bacteria such as the
Enterobacteriaceae family (31). Notably, the enteric FXR, which is classically involved in bile acid
transport and production, plays a significant role in this bile acid and gut microbiome interplay.
Conjugated bile acids activate FXR to release anti-microbial peptides modulating the microbiome
and enhancing enteric epithelial cell integrity to preserve the intestinal barrier (32). Furthermore, this
interaction between the bile acid pool and the gut microbiota also appears to influence the activity of

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 the FXR (60). The downregulation of intestinal FXR leads to the accumulation of bile acids in the liver
Accepted Article
and has been associated with hepatocarcinogenesis (61,62).
The mechanistic details of the links between FXR activity and hepatocarcinogenesis appear
to be multifactorial. Yamada et al. showed that a NASH-related HCC animal model, created by the
administration of a steatohepatitis-inducing high-fat diet (STHD-01), had microbiota-dependent
increase in DCA. However, the authors noted that the mTOR pathway, a known oncogenesis
cascade, was activated in these animals and attributed it to secondary bile acid associated activation
of hepatocyte FXR (59). Others have identified FXR as a critical mediator in the relationship between
the gut microbiome and tumor-suppressive miRNA expression (63,64).
A pre-clinical study by Xie et al. showed that sex-based disparity in liver carcinogenesis is
associated with gut microbiota, bile acids and miRNAs (64) in male and female mice treated with
streptozotocin high-fat diet. Short non-coding RNA molecules (18-22 nucleotides), miRNAs regulate
many biological processes and have critical roles in cell proliferation, differentiation, and cell death
(65,66). Specific miRNAs can regulate the translation of specific genes by pairing with target
messenger RNAs (mRNA) at the 3’-untranslated region and blocking their translation (65). In this
study, Xie et al. demonstrate that dysbiosis was achieved by the loss of microbiota diversity, resulting
in increased aerobic and pro-inflammatory Enterococcus, Erysipelatrichales, and Enterobacteriales,
and reduced beneficial anti-inflammatory Lactobacillus and Lactobacillaceae. This microbiota
composition prevented the conversion of primary to secondary bile acids as well as de-conjugation
into free bile acids, thus leading to significant accumulation in the liver. The presence of bile acids in
the liver lowered the expression of tumor-suppressive microRNA’s miR26a, miR26a-1, miR-192,
miR122, miR22, and miR125b, whereas tumor-promoting mRNAs miR-10b and miR99b were
upregulated in the male mice. These miRNA patterns proceed from significant decreases in FXR
expression which facilitates bile acid transport (63,64,67). On the other hand, high levels of FXR
expression were observed in female mice correlating with increased expression of miR26a, miR26a-
1, and miR122, all suppressors of liver cancer. These observations indicate a relationship between
microbiota composition, bile acids, and miRNAs in the progression of liver disease and provide novel
therapeutic targets of disease prevention such as FXR.
The influence of dysbiosis-related functional changes in the microbiome, such as changes in
bile acids and other downstream bacterial metabolites, on HCC development continues to be
explored. SCFAs, such as acetate, propionate and butyrate, are formed as a result of carbohydrate
metabolism by the gut microbiota. Butyrate and propionate are generally considered beneficial

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 SCFAs as they provide nutrition to colonocytes as well as strengthen enteric tight junctions to
Accepted Article
maintain intestinal barrier integrity (68). Interestingly, Singh et al. fed mice lacking TLR5, a PPR for
the bacterial protein flagellin, a diet enriched with fermentable fibers to increase their intraluminal
SCFA content. These knock-out mice tend to develop metabolic syndrome at baseline, and while the
increased butyrate content did reduce inflammation associated with metabolic syndrome, it seemed
to also lead to intrahepatic cholestasis and development of HCC in these animals (69). This
unexpected finding again illustrates the importance of balanced interactions within the gut-
microbiota-liver axis. Though the underlying mechanism currently remains unclear, this study does
show that a surfeit of certain SCFAs can potentially lead to increased hepatocarcinogenesis.
Animal studies have increased our understanding of the mechanisms underlying dysbiosis-
related hepatocarcinogenesis; however, data from humans have been relatively scarce (See Table
2). Grąt et al. did show, in a small sample of cirrhotic patients, that higher fecal counts of E.coli were
associated with HCC (70). Ponziani et al. recently analyzed the biomarker and gut microbiota profile
of 20 cirrhotic patients with NAFLD and early HCC (71). This group noted that all patients with
cirrhosis showed an increase in markers of intestinal permeability and inflammation, such as serum
LPS and fecal calprotectin respectively, when compared to healthy controls. However, only the fecal
calprotectin and other markers of intestinal inflammation differentiated the patients with HCC. All
cirrhotic patients also showed reduced Akkermansia with increased Enterobacteriaceae while the
HCC subset showed a further increase in Bacteroides and Ruminococcus abundance with reduced
Bifidobacterium. These patterns were found to be consistent with increased inflammation when a
correlation network was created. This network showed an inverse relationship between Akkermansia
and fecal calprotectin while Bacteroides was associated with increased pro-inflammatory cytokines,
namely IL-8 and IL-13 (71). Animal models have demonstrated similar findings with both
Akkermansia and Bifidobacterium linked to reduced inflammation and attenuation of liver injury
(72,73), while numerous models have shown increased Bacteroides in NASH animal models (23,45).
Future Therapies and Directions
Although human studies have not provided a direct correlation with the mechanisms identified
in animal studies, they appear to show dysbiosis patterns associated with increased inflammation,
disruption of the intestinal barrier and possible effects on the immune system. Therefore, directing
future therapeutics towards maintaining a healthy microbiome, preventing dysbiosis and the
impacting downstream effector mechanisms related to HCC development, remains a promising
pursuit. With increasing data becoming available regarding the gut-microbiota-liver axis and

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 hepatocarcinogenesis, several approaches to prevent HCC based on these mechanisms have been
Accepted Article
proposed (Table 3).
A common theme illustrated in several animal HCC models studying the effects of dysbiosis
on hepatocarcinogenesis is that administration of a combination of broad-spectrum antibiotics results
in sterilization of the gut and a reduction in tumor burden with prevention of HCC development
(56,74). These observations might be mediated through mTOR activation and might initiate other key
mechanisms in the development of HCC (59). Although continuous administration of broad-spectrum
antibiotics is not a realistic therapy for clinical studies, there has been investigation into various
antibiotics that can modulate the gut microbiome into a more favorable profile and present fewer side
effects. Rifaximin, a non-absorbable antibiotic, has been shown to induce the overgrowth of
“beneficial” microbes such as Bifidobacterium, Faecalibacterium, and Lactobacillus without overly
altering the microbiome profile (75). Though not widely used in HCC animal model studies for gut
decontamination, there has been at least one study that showed a moderate reduction in HCC
development in DEN-exposed animals treated with Rifaximin (74). With the known efficacy of
Rifaximin for the treatment of hepatic encephalopathy in patients with ESLD, further study into its
effect on the human microbiome and HCC development is warranted.
Probiotics have also been used in murine HCC models to oppose dysbiosis and have shown
a reduction in HCC development. Zhang et al. administered VSL#3 to DEN-exposed rats with a
reduction in HCC development by restoring the gut homeostasis and ameliorating intestinal and
hepatic inflammation (29). VSL#3 is a widely used probiotic that contains eight different bacterial
species such as Streptococcus thermophiles, Bifidobacterium breve, Bifidobacterium longum,
Bifidobacterium infantis, Lactobacillus acidophilus, Lactobacillus plantarum, Lactobacillus paracasei,
and Lactobacillus delbrueckii subsp. bulgaricus. Prohep, which is composed of Lactobacillus
rhamnosus GG, viable Escherichia coli Nissle 1917, and heat-inactivated VSL#3, has also been
shown to reduce tumor burden by 40% in HCC mouse models (30) by reducing pro-inflammatory
mediators such as IL-17 and inhibiting angiogenesis. These studies suggest that probiotics promote
the establishment of non-inflammatory, beneficial microbes that shift the composition of a dysbiotic
microbiome resulting in reduced HCC development. There are limited data on the efficacy of
probiotics to modulate the human gut microbiome especially in the setting of ESLD (76). If certain
probiotics can show a viable and persistent shift in the human microbiome, they could be a possible
therapeutic option for HCC prevention in the future.
Fecal microbiota transplantation has become a viable way to alter the human gut microbiome

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 in certain conditions such as Clostridioides difficile infection (77) or after stem cell transplant (78).
Accepted Article
Although preliminary studies have shown some efficacy in beneficial outcomes in liver disease such
as metabolic syndrome (79), severe acute alcoholic hepatitis (80) or improvement in hepatic
encephalopathy (40), the longevity of these effects and the optimal mixture of flora needed to impact
ESLD and HCC continue to be investigated.
As detailed above, the increase in gut permeability, with a resultant increase in inflammatory
cytokine release, forms the basis of numerous mechanisms studied in HCC animal models. The
effects of the LPS-TLR4 pathway and the FXR-related modulation of the gut-microbiota-liver axis are
central to a number of these mechanisms (See Figure 3). Therapeutic targeting of these pathways
provides another avenue of study in HCC prevention. There has been extensive study of various
TLR antagonists especially in the treatment of sepsis (81) but they have yet to be studied in CLD or
HCC development. Due to the systemic ubiquity of TLRs and their important role in human
immunocompetence, there are valid concerns about the long-term antagonism of such receptors,
especially in an immunocompromised ESLD population. These concerns need to be addressed
before such agents can be considered for study in HCC prevention. Alternatively, FXR agonists
including obeticholic acid (OCA), have already been approved for use in CLD such as primary biliary
cholangitis (PBC) (82) and have shown promising effects on fibrosis associated with NASH (83,84).
Given the vital role FXR plays in the modulation of the gut microbiome as well as the integrity of the
intestinal epithelial barrier, it will be important to determine if there are any effects on HCC
development independent of effects on PBC and NASH.
Conclusion
The advances in techniques to study the human microbiome are allowing the exploration of
the intricate links between two complex systems, specifically the HCC tumor microenvironment and
the gut microbiome. Although there are clear associations between dysbiosis-related inflammation
and HCC in animal models, this needs further exploration in human studies. The significance of the
microbial constitution of the human microbiome is confounded by numerous factors such as
antibiotics, comorbidities and diet. Studies on non-antibiotic therapies to modulate the composition
such as FMT, and drugs that affect functional targets of the microbiome such as FXR, warrant further
investigation. New technologies such as miRNA analysis and artificial intelligence-based techniques
to analyze the metabolomic, genetic, and overall functional profile show further promise in
delineating this relationship.

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 FIGURES AND TABLES
Accepted Article
See attached file

Figure 1. Schematic illustration of gut microbiota processing and bioinformatics analysis of 16s
rRNA.

See attached file

Figure 2. Mechanisms by which the Gut microbiome influences hepatocarcinogenesis.

BA- bile Acid. CLD- Chronic liver disease. ESLD- End-stage liver disease. NAFLD- Non-alcoholic
fatty liver disease. Amps- Microbiota-associated molecular patterns. SCFAs- short-chain fatty acids,

See attached file

Figure 3. Mechanisms linking gut microbiota to HCC. Dysbiosis and leaky gut increase hepatic
exposure to bacterial metabolites and MAMPs. MAMPs and bacterial metabolites such as BA and
SCFA promote hepatic inflammation (via IL-1, IL-6, TNF), fibrosis, proliferation, and immune
suppression via miRNA dysregulation and mTOR. These cancer promoting signaling pathways can
be interrupted at several levels. Probiotics can be used to restore normal microbiota composition.
Antibiotics can be used to eliminate disease-promoting bacteria and decrease release of MAMPs
and metabolites from a leaky gut. TLR antagonists can block the propagation of downstream
cytokine release. FXR agonists can modulate various downstream immune-related pathways.
BA- Bile acids. CLD- Chronic liver disease. FXR- farnesoid X receptor. HCC- Hepatocellular
carcinoma. HSCs- hepatic stellate cells. IL-1- Interleukin 1. IL-6- Interleukin 6. MAMPs- Microbiota-
associated molecular patterns. miRNA- micro RNA. SASP- senescence associated secretory
phenotype. SCFAs- short-chain fatty acids. TLR- Toll-like receptor. TNF- Tumor necrosis factor.

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Accepted Article
TABLE 1 – Studies of the Gut Microbiome in Chronic Liver Disease

Source Model Organism

NASH/NAFLD
Boursier et al (45) Human study ↑ Bacteroides
57 pts
↑ Ruminococcus
↓ Prevotella
Loomba et al. (46) Human study ↑ E.coli
86 pts
↑ Bacteroides
Vulgatus

ALD Mutlu et al. (42) Human study ↑ Enterobacteriaceae

48 pts
Chen et al. (43) Human study ↓ Bacteroidetes
36 pts
↓ Lactobacillus
Yang et al. (44) Human/mouse ↑ Candida
studies

CIRRHOSIS Qin et al. (36) Human study ↓ Bacteroides

98 pts
↑ Prevotella
↑ Clostridium
↑ Streptococcus
↑ Veillonella

HBV + Alcohol Chen et al. (43) Human study ↓ Bacteroidetes

36 pts
↑ Proteobacteria

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 ↓ Fusobacteria
Accepted Article ↑ Streptococcaceae
↑ Veillonellaceae

HBV Milosevic et al. (47) Review ↓ Bifidobacteria

↓ Lactobacillus
↑ Enterococcus
↑ Enterobacteriaceae
Wei et al. (48) Human study ↓ Bacteroidetes
120 pts
↑ Proteobacteria

HCV Milosevic et al. (47) Review ↓ Acinetobacter

↓ Veillonella
↓ Phascolarctobactriu
m
↑ Prevotella

TABLE 2 – Studies Of The Gut Microbiome Involving Hepatocellular Carcinoma

Source Notes/Mech Organism

Grat et al. (70) Fecal counts ↑ E.coli

Ponziani et al. (71) Cirrhosis ↓ Akkermansia
Cirrhosis ↑ Enterobacteriaceae
Cirrhosis + HCC ↑ Bacteroides
Cirrhosis + HCC ↑ Ruminococcus
Cirrhosis + HCC ↓ Bifidobacterium
Ren et al. (85) HBV Cirrhosis + HCC ↑ Actinobacteria
↓ Verrucomicrobia
Liu et al. (86) NBNC Cirrhosis + HCC ↑ Escherichia
NBNC Cirrhosis + HCC ↑ Enterococcus

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 NBNC Cirrhosis + HCC ↓ Faecalibacterium
Accepted Article NBNC Cirrhosis + HCC ↓ Ruminococcus
NBNC Cirrhosis + HCC ↓ Ruminoclostridium
HCC: Hepatocellular Carcinoma; NBNC: Non-Hepatitis B, Non-Hepatitis C

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Accepted Article
TABLE 3 – Therapeutics involving Gut Microbiome to prevent liver disease

Source Model Experiment Findings

Vrieze et al. Human study Infusion of intestinal ↑ Insulin sensitivity

(79) microbiota
↑ butyrate-producing intestinal
microbiota

Philips et al. Human study Fecal microbiota Improvement in liver function and
(80) transplantation survival

Bajaj et al. Human study Fecal microbiota ↑ Cognition improvement

(40) transplantation
↑ diversity and beneficial taxa

Dapito et al. Animal study/ Rifaximin ↓ HCC development

(74) murine mice

Ponziani et Animal and Rifaximin ↑ Bifidobacterium

al. (75) Human studies
↑ Faecalibacterium
↑ Lactobacillus

Zhang et al. Animal Study/ Probiotic VSL#3 ↓ HCC

(29) DEN model of
RAT HCC
↑ Gut homeostasis
↓Hepatic inflammation

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 Li et al. (30) Animal study/ Prohep ↓ HCC burden by 40%
Accepted Article mouse model
↑ Prevotella
↑ Oscillibacter
Yoshimoto Animal study/ Blocking DCA production ↓ HCC
et al. (56) obese mice or reducing gut bacteria

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