A pre­clinical model combining cryopreservation technique with precision­cut slice culture method to assess the in vitro drug response of hepatocellular carcinoma.

Models considering hepatocellular carcinoma (HCC) complexity cannot be accurately replicated in routine cell lines or animal models. We aimed to evaluate the practicality of tissue slice culture by combining it with a cryopreservation technique. We prepared 0.3­mm­thick tissue slices by a microtome and maintained their cell viability using a cryopreservation technique. Slices were cultured individually in the presence or absence of regorafenib (REG) for 72 h. Alterations in morphology and gene expression were assessed by histological and genetic analysis. Overall viability was also analyzed in tissue slices by CCK­8 quantification assay and fluorescent staining. Tissue morphology and cell viability were evaluated to quantify drug effects. Histological and genetic analyses showed that no significant alterations in morphology and gene expression were induced by the vitrification­based cryopreservation method. The viability of warmed HCC tissues was up to 90% of the fresh tissues. The viability and proliferation could be retained for at least four days in the filter culture system. The positive drug responses in precision­cut slice culture in vitro were evaluated by tissue morphology and cell viability. In summary, the successful application of precision­cut HCC slice culture combined with a cryopreservation technique in a systematic drug screening demonstrates the feasibility and utility of slice culture method for assessing drug response.

Targeting mTORC2/HDAC3 Inhibits Stemness of Liver Cancer Cells Against Glutamine Starvation.

Cancer cells are addicted to glutamine. However, cancer cells often suffer from glutamine starvation, which largely results from the fast growth of cancer cells and the insufficient vascularization in the interior of cancer tissues. Herein, based on clinical samples, patient-derived cells (PDCs), and cell lines, it is found that liver cancer cells display stem-like characteristics upon glutamine shortage due to maintaining the stemness of tumor initiating cells (TICs) and even promoting transformation of non-TICs into stem-like cells by glutamine starvation. Increased expression of glutamine synthetase (GS) is essential for maintaining and promoting stem-like characteristics of liver cancer cells during glutamine starvation. Mechanistically, glutamine starvation activates Rictor/mTORC2 to induce HDAC3-mediated deacetylation and stabilization of GS. Rictor is significantly correlated with the expression of GS and stem marker OCT4 at tumor site, and closely correlates with poor prognosis of hepatocellular carcinomas. Inhibiting components of mTORC2-HDAC3-GS axis decrease TICs and promote xenografts regression upon glutamine-starvation therapy. Collectively, the data provides novel insights into the role of Rictor/mTORC2-HDAC3 in reprogramming glutamine metabolism to sustain stemness of cancer cells. Targeting Rictor/HDAC3 may enhance the efficacy of glutamine-starvation therapy and limit the rapid growth and malignant progression of tumors.

The combined induction of liver progenitor cells and the suppression of stellate cells by small molecules reverts chronic hepatic dysfunction.

Rationale: We developed a cocktail of soluble molecules mimicking the in vivo milieu supporting liver regeneration that could convert mature hepatocytes to expandable liver progenitor-like cells in vitro. This study aimed to induce endogenous liver progenitor cells by the administration of the soluble molecules to provide an alternative approach for the resolution of liver fibrosis. Methods:In vitro cultured hepatocyte-derived liver progenitor-like cells (HepLPCs) were transplanted into CCL4-treated mice to investigate the therapeutic effect against liver fibrosis. Next, we used HGF in combination with a cocktail of small molecules (Y-27632, A-83-01, and CHIR99021 (HACY)) to induce endogenous CD24+ liver progenitor cells and to inhibit the activation of hepatic stellate cells (HSCs) during CCL4-induced hepatic injury. RNA sequencing was performed to further clarify the features of HACY-induced CD24+ cells compared with CCL4-induced CD24+ cells and in vitro derived HepLPCs. Finally, we evaluated the expansion of HACY-induced CD24+ cells in human hepatocyte-spheroids from fibrotic liver tissues. Results: HepLPCs exhibited the capacity to alleviate liver fibrosis after transplantation into CCL4-treated mice. The in vivo administration of HACY not only induced the conversion of mature hepatocytes (MHs) to CD24+ progenitor cells but prevented the activation of HSCs, thus leading to enhanced improvement of liver fibrosis in CCL4-treated mice. Compared to CD24+ cells induced by CCL4 alone, HACY-induced CD24+ cells retained an enhanced level of hepatic function and could promote the restoration of liver function that exhibited comparable gene expression profiles with HepLPCs. CD24+ cells were also observed in human liver fibrotic tissues and were expanded in three-dimensional (3D) hepatic spheroids in the presence of HACY in vitro. Conclusions: Hepatocyte-derived liver progenitor-like cells are crucial for liver regeneration during chronic hepatic injuries. The administration of HACY, which allowed the induction of endogenous CD24+ progenitor cells and the inactivation of HSCs, exerts beneficial effects in the treatment of liver fibrosis by re-establishing a balance favoring liver regeneration while preventing fibrotic responses.

Cryopreserved biopsy tissues of rectal cancer liver metastasis for assessment of anticancer drug response in vitro and in vivo.

Living tumors are of great scientific value for clinical medicine and basic research, especially for drug testing. An increasing number of drug tests fail due to the use of imperfect models. The aim of the present study was to develop a novel method combining vitrification­based cryopreservation of tumor biopsies and precision­cut slice cultivation for the assessment of anticancer drug responses. Biological characteristics of rectal cancer liver metastasis biopsies could be retained by vitrification­based cryopreservation. The patient­derived xenograft models were successfully established using both fresh and warmed biopsy tissues. Precision­cut slicing provided a similar three­dimensional architecture and heterogeneity to the original tumor. The positive drug responses in the xenograft model were consistent with those in precision­cut slice cultures in vitro. The present study demonstrated that live tumor biopsies could be preserved using vitrification­based cryopreservation. The warmed tissues developed xenograft tumors, which were also useful for either in vivo or in vitro anticancer drug testing. Precision­cut slices derived from the warmed tissues provided an efficient tool to assess anticancer drug response in vitro.

Generation of hepatic spheroids using human hepatocyte-derived liver progenitor-like cells for hepatotoxicity screening.

Rationale: The idiosyncratic drug-induced liver injury (iDILI) is a major cause of acute liver injury and a key challenge in late-stage drug development. Individual heterogeneity is considered to be an essential factor of iDILI. However, few in vitro model can predict heterogeneity in iDILI. We have previously shown that mouse and human hepatocytes can be converted to expandable liver progenitor-like cells in vitro (HepLPCs). However, the limited proliferation potential of human HepLPCs confines its industrial application. Here, we reported the generation of a novel hepatocyte model not only to provide unlimited cell sources for human hepatocytes but also to establish a tool for studying iDILI in vitro. Methods: Human primary hepatocytes were isolated by modified two-step perfusion technique. The chemical reprogramming culture condition together with gene-transfer were then used to generate the immortalized HepLPC cell lines (iHepLPCs). Growth curve, doubling time, and karyotype were analyzed to evaluate the proliferation characteristics of iHepLPCs. Modified Hepatocyte Maturation Medium and 3D spheroid culture were applied to re-differentiate iHepLPCs. Results: iHepLPCs exhibited efficient expansion for at least 40 population doublings, with a stable proliferative ability. They could easily differentiate back into metabolically functional hepatocytes in vitro within 10 days. Furthermore, under three-dimensional culture conditions, the formed hepatic spheroids showed multiple liver functions and toxicity profiles close to those of primary human hepatocytes. Importantly, we established a hepatocyte bank by generating a specific number of such cell lines. Screening for population heterogeneity allowed us to analyze the in vitro heterogeneous responses to hepatotoxicity induced by molecular targeted drugs. Conclusions: In light of the proliferative capacity and the heterogeneity they represented, these iHepLPCs cell lines may offer assistance in studying xenobiotic metabolism as well as liver diseases in vitro.

Expansion and differentiation of human hepatocyte-derived liver progenitor-like cells and their use for the study of hepatotropic pathogens.

The study of pathophysiological mechanisms in human liver disease has been constrained by the inability to expand primary hepatocytes in vitro while maintaining proliferative capacity and metabolic function. We and others have previously shown that mouse mature hepatocytes can be converted to liver progenitor-like cells in vitro with defined chemical factors. Here we describe a protocol achieving efficient conversion of human primary hepatocytes into liver progenitor-like cells (HepLPCs) through delivery of developmentally relevant cues, including NAD + -dependent deacetylase SIRT1 signaling. These HepLPCs could be expanded significantly during in vitro passage. The expanded cells can readily be converted back into metabolically functional hepatocytes in vitro and upon transplantation in vivo. Under three-dimensional culture conditions, differentiated cells generated from HepLPCs regained the ability to support infection or reactivation of hepatitis B virus (HBV). Our work demonstrates the utility of the conversion between hepatocyte and liver progenitor-like cells for studying HBV biology and antiviral therapies. These findings will facilitate the study of liver diseases and regenerative medicine.

Inhibition of dipeptidyl peptidase IV prevents high fat diet-induced liver cancer angiogenesis by downregulating chemokine ligand 2.

Obesity is a major risk factor for hepatocellular carcinoma (HCC) and is typically accompanied by higher levels of serum dipeptidyl peptidase 4 (DPP4). However, the role of DPP4 in obesity-promoted HCC is unclear. Here, we found that consumption of a high-fat diet (HFD) promoted HCC cell proliferation and metastasis and led to poor survival in a carcinogen-induced model of HCC in rats. Notably, genetic ablation of DPP4 or treatment with a DPP4 inhibitor (vildagliptin) prevented HFD-induced HCC. Moreover, HFD-induced DPP4 activity facilitated angiogenesis and cancer cell metastasis in vitro and in vivo, and vildagliptin prevented tumor progression by mediating the pro-angiogenic role of chemokine ligand 2 (CCL2). Loss of DPP4 effectively reversed HFD-induced CCL2 production and angiogenesis, indicating that the DPP4/CCL2/angiogenesis cascade had key roles in HFD-associated HCC progression. Furthermore, concomitant changes in serum DPP4 and CCL2 were observed in 210 patients with HCC, and high serum DPP4 activity was associated with poor clinical prognosis. These results revealed a link between obesity-related high serum DPP4 activity and HCC progression. Inhibition of DPP4 may represent a novel therapeutic intervention for patients with HCC.

Maintaining viability and characteristics of cholangiocarcinoma tissue by vitrification-based cryopreservation.

Tumor tissue has great clinical and scientific value which relies highly on the proper preservation of primary materials. Conventional tumor tissue cryopreservation using slow-freezing method has yielded limited success, leading to significant cell loss and morphological damage. Here we report a standardized vitrification-based cryopreservation method, by which we have successfully vitrified and warmed 35 intrahepatic cholangiocarcinoma (ICC) tissues with up to 80% viability of the fresh tumor tissues. Cryopreserved ICC tissue could generate patient-derived xenografts (PDXs) with take rates of 68.2% compared to 72.7% using fresh tumor tissues. Histological and genetic analyses showed that no significant alterations in morphology and gene expression were introduced by this cryopreservation method. Our procedure may facilitate collection, long-time storage and propagation of cholangiocarcinoma or other tumor specimens for (pre)clinical studies of novel therapies or for basic research.

Aldehyde dehydrogenase-2 (ALDH2) opposes hepatocellular carcinoma progression by regulating AMP-activated protein kinase signaling in mice.

Potential biomarkers that can be used to determine prognosis and perform targeted therapies are urgently needed to treat patients with hepatocellular carcinoma (HCC). To meet this need, we performed a screen to identify functional genes associated with hepatocellular carcinogenesis and its progression at the transcriptome and proteome levels. We identified aldehyde dedydrogenase-2 (ALDH2) as a gene of interest for further study. ALDH2 levels were significantly lower at the mRNA and protein level in tumor tissues than in normal tissues, and they were even lower in tissues that exhibited increased migratory capacity. A study of clinical associations showed that ALDH2 is correlated with survival and multiple migration-associated clinicopathological traits, including the presence of metastasis and portal vein tumor thrombus. The result of overexpressing or knocking down ALDH2 showed that this gene inhibited migration and invasion both in vivo and in vitro. We also found that ALDH2 altered the redox status of cells by regulating acetaldehyde levels and that it further activated the AMP-activated protein kinase (AMPK) signaling pathway. CONCLUSION: Decreased levels of ALDH2 may indicate a poor prognosis in HCC patients, while forcing the expression of ALDH2 in HCC cells inhibited their aggressive behavior in vitro and in mice largely by modulating the activity of the ALDH2-acetaldehyde-redox-AMPK axis. Therefore, identifying ALDH2 expression levels in HCC might be a useful strategy for classifying HCC patients and for developing potential therapeutic strategies that specifically target metastatic HCC. (Hepatology 2017;65:1628-1644).

Blocking preferential glucose uptake sensitizes liver tumor-initiating cells to glucose restriction and sorafenib treatment.

Cancer cells display altered metabolic phenotypes characterized by a high level of glycolysis, even under normoxic conditions. Because of a high rate of glycolytic flux and inadequate vascularization, tumor cells often suffer from nutrient deficiency and require metabolic adaptations to address such stresses. Although tumor-initiating cells (T-ICs) have been identified in various malignancies, the cells' metabolic phenotypes remain elusive. In this study, we observed that liver T-ICs preferentially survived under restricted glucose treatment. These cell populations compete successfully for glucose uptake by preferentially expressing glucose transporters (GLUT1 and GLUT3), whereas inhibition of GLUT1 or GLUT3 abolished the survival advantage and suppressed the tumorigenic potential of liver T-ICs. Among signaling pathways related to T-ICs, IL-6/STAT3 was identified to be responsible for the elevation of glucose uptake in liver T-ICs under glucose limitation. Further investigation revealed that IL-6 stimulation upregulated GLUT1 and GLUT3 expressions in CD133+ cells, particularly during glucose deprivation. More importantly, inhibition of glucose uptake sensitized liver T-ICs to sorafenib treatment and enhanced the therapeutic efficacy in vivo. Our findings suggest that blocking IL-6/STAT3-mediated preferential glucose uptake might be exploited for novel therapeutic targets during hepatocellular carcinoma (HCC) progression.

Inhibition of SIRPα in dendritic cells potentiates potent antitumor immunity.

Despite their central function in tumor immunity, dendritic cells (DCs) can respond to inhibitory signals and become tolerogenic, curtailing T cell responses in vivo. Here, we provide the evidence for an inhibitory function of signal regulatory protein (SIRP) α in DC survival and activation. In tumors from human liver cancer patients, infiltrative DCs expressed elevated levels of SIRPα, which is correlated with the induction of immune tolerance within the tumors. Silencing of SIRPα resulted in a significant increase in the longevity of antigen-pulsed DCs in the draining lymph nodes. In addition, SIRPα controls the activation and output of DCs. Silencing of DC-expressed SIRPα induced spontaneous and enhanced production of IL12 and costimulatory molecules, resulting in more potent cytotoxic T lymphocyte responses, including the eradication of previously established solid tumors. SIRPα exerted such effects, at least in part, via the association and sequestration of p85 subunit of PI3K. Thus, SIRPα is a critical regulator of DC lifespan and activity, and its inhibition might improve the clinical efficacy of DC-based tumor vaccines.

Genomic and oncogenic preference of HBV integration in hepatocellular carcinoma.

Hepatitis B virus (HBV) can integrate into the human genome, contributing to genomic instability and hepatocarcinogenesis. Here by conducting high-throughput viral integration detection and RNA sequencing, we identify 4,225 HBV integration events in tumour and adjacent non-tumour samples from 426 patients with HCC. We show that HBV is prone to integrate into rare fragile sites and functional genomic regions including CpG islands. We observe a distinct pattern in the preferential sites of HBV integration between tumour and non-tumour tissues. HBV insertional sites are significantly enriched in the proximity of telomeres in tumours. Recurrent HBV target genes are identified with few that overlap. The overall HBV integration frequency is much higher in tumour genomes of males than in females, with a significant enrichment of integration into chromosome 17. Furthermore, a cirrhosis-dependent HBV integration pattern is observed, affecting distinct targeted genes. Our data suggest that HBV integration has a high potential to drive oncogenic transformation.

HBx regulates fatty acid oxidation to promote hepatocellular carcinoma survival during metabolic stress.

Due to a high rate of nutrient consumption and inadequate vascularization, hepatocellular carcinoma (HCC) cells constantly undergo metabolic stress during tumor development. Hepatitis B virus (HBV) X protein (HBx) has been implicated in the pathogenesis of HBV-induced HCC. In this study, we investigated the functional roles of HBx in HCC adaptation to metabolic stress. Up-regulation of HBx increased the intracellular ATP and NADPH generation, and induced the resistance to glucose deprivation, whereas depletion of HBx via siRNA abolished these effects and conferred HCC cells sensitive to glucose restriction. Though HBx did not affect the glycolysis and oxidative phosphorylation capacity of HCC cells under normal culture conditions, it facilitated fatty acid oxidation (FAO) in the absence of glucose, which maintained NADPH and ATP levels. Further investigation showed that HBx expression, under glucose deprivation, stimulated phosphorylation of AMP-activated protein kinase (AMPK) and acetyl-CoA carboxylase (ACC) via a calcium/CaMKK-dependent pathway, which was required for the activation of FAO. Conversely, inhibition of FAO by etomoxir (ETO) restored the sensitivity of HBx-expressing cells to glucose deficiency in vitro and retarded xenograft tumor formation in vivo. Finally, HBx-induced activation of the AMPK and FAO pathways were also observed in xenograft tumors and HBV-associated HCC specimens. Our data suggest that HBx plays a key role in the maintenance of redox and energy homeostasis by activating FAO, which is critical for HCC cell survival under conditions of metabolic stress and might be exploited for therapeutic benefit.

Acetyl-coenzyme A carboxylase alpha promotion of glucose-mediated fatty acid synthesis enhances survival of hepatocellular carcinoma in mice and patients.

UNLABELLED: Solid tumors often suffer from suboptimal oxygen and nutrient supplies. This stress underlies the requirement for metabolic adaptation. Aberrantly activated de novo lipogenesis is critical for development and progression of human hepatocellular carcinoma (HCC). However, whether de novo lipogenesis influences biological behaviors of HCCs under conditions of metabolic stress are still poorly understood. Here, we show that HCCs display distinct levels of glucose-derived de novo lipogenesis, which are positively correlated with their survival responses to glucose limitation. The enhanced lipogenesis in HCCs is characterized by an increased expression of rate-limiting enzyme acetyl-coenzyme A carboxylase alpha (ACCα). ACCα-mediated fatty acid (FA) synthesis determines the intracellular lipid content that is required to maintain energy hemostasis and inhibit cell death by means of FA oxidation (FAO) during metabolic stress. In accord, overexpression of ACCα facilitates tumor growth. ACCα forms a complex with carnitine palmitoyltransferase 1A (CPT1A) and prevents its mitochondria distribution under nutrient-sufficient conditions. During metabolic stress, phosphorylation of ACCα leads to dissociation of the complex and mitochondria localization of CPT1A, thus promoting FAO-mediated cell survival. Therefore, ACCα could provide both the substrate and enzyme storage for FAO during glucose deficiency. Up-regulation of ACCα is also significantly correlated with poorer overall survival and disease recurrence postsurgery. Multivariate Cox's regression analysis identified ACCα as an effective predictor of poor prognosis. CONCLUSION: These results present novel mechanistic insight into a pivotal role of ACCα in maintaining HCC survival under metabolic stress. It could be exploited as a novel diagnostic marker and therapeutic target.

Platelets promote tumour metastasis via interaction between TLR4 and tumour cell-released high-mobility group box1 protein.

Increasing evidence suggests that TLR4 expression by tumour cells promotes tumour progression, but it is unclear whether TLR4 is involved in metastasis. Here we show that TLR4 deficiency significantly diminishes experimental lung metastasis without affecting primary tumour growth. Bone marrow transplantation experiment and application of antiplatelet agents in mice demonstrate that TLR4 on platelets plays an important role in metastasis. TLR4 is critical for platelet-tumour cell interaction in vitro. Furthermore, high-mobility group box1 (HMGB1) neutralization attenuates platelet-tumour cell interaction in vitro and metastasis in vivo in a TLR4-dependent manner, indicating that tumour cell-released HMGB1 is the key factor that interacts with TLR4 on platelets and mediates platelet-tumour cell interaction, which promotes metastasis. These findings demonstrate a mechanism by which platelets promote tumour cell metastasis and suggest TLR4, and its endogenous ligand HMGB1 as targets for antimetastatic therapies.

p53 promotes inflammation-associated hepatocarcinogenesis by inducing HMGB1 release.

BACKGROUND & AIMS: Hepatocellular carcinoma (HCC) develops in response to chronic hepatic injury. Although induced cell death is regarded as the major component of p53 tumor-suppressive activity, we recently found that sustained p53 activation subsequent to DNA damage promotes inflammation-associated hepatocarcinogenesis. Here we aim at exploring the mechanism linking p53 activation and hepatic inflammation during hepatocarcinogenesis. METHODS: p53(-/-) hepatocytes expressing inducible p53 and primary wild type hepatocytes were treated to induce p53 expression. The supernatants were collected and analyzed for the presence of released inflammatory cytokines. Ethyl pyruvate was used in a rat model of carcinogen-induced hepatocarcinogenesis to examine its effect on p53-dependent chronic hepatic injury, inflammation, and tumorigenesis. RESULTS: Here we show that cytoplasmic translocation and circulating levels of potent inflammatory molecule high-mobility group protein 1 (HMGB1) were greater in wild type rats than in p53(+/-) rats following carcinogen administration. Restoration of p53 expression in p53-null hepatocytes or induction of endogenous p53 in wild type hepatocytes gives rise to the release of HMGB1. Administration of the HMGB1 release inhibitor ethyl pyruvate, which does not affect p53-mediated hepatic apoptosis, substantially prevented carcinogen-induced cirrhosis and tumorigenesis in rat livers. CONCLUSIONS: These results suggest that although p53 is usually regarded as a tumor suppressor, its constant activation can promote pro-tumorigenic inflammation, at least in part, via inducing HMGB1 release. Application of HMGB1 inhibitors when restoring p53 in cancer therapy might protect against pro-tumorigenic effects while leaving p53-mediated clearance of malignant cells intact.

Role of ß-Catenin in regulating the balance between TNF-α- and Fas-induced acute liver injury.

ß-Catenin plays many critical roles during various liver physiological and pathological processes. However, the role of ß-Catenin in acute liver failure remains unclear. Using hepatocyte specific ß-Catenin knockout mice, we found that loss of ß-Catenin in hepatocyte significantly reduced GalN/LPS-induced liver damage and hepatocyte apoptosis, but exacerbated Jo2-mediated liver injury. Mechanistically, the dual effects of ß-Catenin attributes on its function of inhibiting NF-κB signaling, which aggravates oxidative stress but decreases Fas expression under injury conditions. In conclusion, ß-Catenin plays an important role in regulating the balance between TNF-α and Fas-induced liver injury via its effect on NF-κB.

Rats deficient for p53 are susceptible to spontaneous and carcinogen-induced tumorigenesis.

The p53 tumor suppressor gene is highly mutated in human cancers. Individuals who inherit one p53 mutant allele are susceptible to a wide range of tumor types, including breast cancer and sarcoma. We recently generated p53 knockout rats through gene targeting in embryonic stem cells. Here we show that rats homozygous for the null allele are prone to early onset spontaneous sarcomas and lymphoma with high incidence of metastases. Heterozygous rats are also highly predisposed to cancer, but with a delayed onset and a wider spectrum of tumor types compared with homozygotes. Importantly, up to 20% of female heterozygotes developed breast cancer and about 70% of the tumors were positive for estrogen receptor. Exposing p53-deficient rats to a low dose of the carcinogen diethylnitrosamine dramatically decreased the latency for sarcoma development and survival time compared with equivalently treated wild-type rats. These unique features make this knockout line a valuable model for investigating human malignancy and in vivo carcinogenicity of chemicals and therapeutic compounds.

Profound impact of gut homeostasis on chemically-induced pro-tumorigenic inflammation and hepatocarcinogenesis in rats.

BACKGROUND & AIMS: Due to its anatomic connection, the liver is constantly exposed to gut-derived bacterial products or metabolites. Disruption of gut homeostasis is associated with many human diseases. The aim of this study was to determine the role of gut homeostasis in initiation and progression of hepatocellular carcinoma (HCC). METHODS: Disruption of intestinal homeostasis by penicillin or dextran sulfate sodium (DSS) and its restoration by probiotics were applied in a diethylnitrosamine (DEN) model of rat hepatocarcinogenesis. RESULTS: Patients with liver cirrhosis and HCC had significantly increased serum endotoxin levels. Chronic DEN treatment of rats was associated with an imbalance of subpopulations of the gut microflora including a significant suppression of Lactobacillus species, Bifidobacterium species and Enterococcus species as well as intestinal inflammation. Induction of enteric dysbacteriosis or intestinal inflammation by penicillin or DSS, respectively, significantly promoted tumor formation. Administration of probiotics dramatically mitigated enteric dysbacteriosis, ameliorated intestinal inflammation, and most importantly, decreased liver tumor growth and multiplicity. Interestingly, probiotics not only inhibited the translocation of endotoxin, which bears pathogen-associated molecular patterns (PAMPs) but also the activation of damage-associated molecular patterns (DAMPs) such as high-mobility group box 1 (HMGB1). As a result, the production of pro- and anti-inflammatory cytokines was skewed in favor of a reduced tumorigenic inflammation in the liver. CONCLUSIONS: The data highlights the importance of gut homeostasis in the pathogenesis of HCC. Modulation of the gut microbiota by probiotics may represent a new avenue for therapeutic intervention to treat or prevent HCC development.