Basic Science Liver

Abstract

Journal of Gastroenterology and HepatologyVolume 32, Issue S2 p. 3-14 Supplement ArticleFree Access Basic Science Liver First published: 17 August 2017 https://doi.org/10.1111/jgh.13887Citations: 1AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat Subsets of innate lymphoid cells in acute liver injury SY Alabbas1,2, R Movva2,3, AS Purdon2, J Begun1,2, TH Florin1,2 and I Oancea1,2 1Faculty of Medicine, University of Queensland, Brisbane, Queensland, Australia; 2Chronic Disease Biology and Care, Mater Research Institute-University of Queensland, Brisbane, Queensland, Australia; 3School of Pharmacy, Griffith University, Gold Coast, Queensland, Australia Introduction: Innate lymphoid cells (ILCs) are a recently described immune cell population that has been shown to mirror the phenotype and function of T cells without the requirement for antigen specificity. Specifically, ILCs are classified into three main groups based on the dominant transcription factor and the cytokines that they secrete. Group 1 ILCs secrete IFNγ and TNF; group 2 ILCs secrete IL-5, IL-9 and IL-13; and group 3 ILCs secrete IL-17A and IL-22. They have been linked to tissue repair and remodeling, and early responses against pathogens, and are associated with a number of autoimmune disorders. Thus, ILCs are considered to have an important role in inflammatory responses due to their secretion of important cytokines and interaction with components of the adaptive immune system. Various studies have suggested that different cytokines play an anti- or pro-inflammatory role following acute liver injury, outlining a potential role for ILCs in liver injury. In the gut, it has been suggested that NKp46+ ILC3 (IL-1710w and IL-22high) cells may play a protective role, whereas NKp46-ILC3 (IL-17high and IL-2210w) cells may play a pathogenic role. We hypothesized that might be the case in liver pathology as well. Methods: Four- to five-week-old C57BI/6 (wild type [WT]) mice were treated with either intraperitoneal vehicle control (water) or 500 mg APAP, to model acute liver injury. Mice were fasted overnight and feed was re-allowed immediately after APAP administration. They were sacrificed 24 hours after treatment. Tissues like liver and lamina propria isolated from small and large intestine were collected, and changes in the frequencies of ILC subsets were measured via flow cytometry (FACS). We also evaluated IL-17 and IL-22 cytokine production and measured a number of gene expressions associated with hepatic injury. Results: APAP-treated WT mice had significant weight loss and hepatic inflammation at 24 hours, confirming the model of acute liver injury. FACS data from liver and lamina propria tissues revealed an expansion in ILC2 (about threefold) and ILC3 NKp46+ (about 15-fold) populations in APAP-treated mice at this time point. On the other hand, the frequency of ILC3 NKp46− (about threefold) was decreased following liver injury in the same tissues as above. Conclusion: At 24 hours after liver injury, a time point associated with a significant anti-inflammatory component, we have shown an expansion of anti-inflammatory and a decrease of pro-inflammatory ILC populations. This suggests an associated role of ILCs in hepatic injury. We are further exploring ILC functions with cytokine production in the absence of T cells and how this anti-/pro-inflammatory balance changes with time. Iron blocks the secretion of apolipoprotein E in cultured human adipocytes LJ Britton1,2,3,4, K Bridle1,2, LA Jaskowski1,2, J He4, C Ng5, JE Ruelcke6, J Reiling1,2,7, N Santrampurwala1, MM Hill8, JP Whitehead5, VN Subramaniam9 and DHG Crawford1,2 1Gallipoli Medical Research Institute, Brisbane, Queensland, Australia; 2School of Medicine, University of Queensland, Brisbane, Queensland, Australia; 3Department of Gastroenterology, Princess Alexandra Hospital, Brisbane, Queensland, Australia; 4Mater Research Institute, Brisbane, Queensland, Australia; 5School of Life Sciences, University of Lincoln, Lincoln, UK; 6Translational Research Institute, Brisbane, Queensland, Australia; 7Department of Surgery, NUTRIM School of Nutrition and Translational Research in Metabolism, Maastricht University, Maastricht, The Netherlands; 8QIMR Berghofer Medical Research Institute, Brisbane, Queensland, Australia; 9Institute of Health and Biomedical Innovation and School of Biomedical Sciences, Queensland University of Technology, Brisbane, Queensland, Australia Introduction: Adipose tissue dysfunction plays a central role in the pathogenesis of non-alcoholic steatohepatitis (NASH) through the dysregulation of adipokines and the generation of insulin resistance. Recent data support a role for iron in the regulation of adipose tissue function. The effects of iron on the secretome of adipocytes have not been established. Methods: We treated human adipocytes (Simpson–Golabi–Behmel syndrome adipocytes—differentiated in vitro) with 0, 25, 100, and 500 uM iron, as ferric ammonium citrate, for 48 hours. We found that treatment with 100 uM iron caused significant increases in intracellular iron concentration but did not alter proliferation or total protein secretion from these cells. To determine if iron modulated the secretion of specific proteins from the adipocyte secretome, we performed quantitative proteomics using SILAC (stable isotope labeling with amino acids in cell culture), followed by tandem mass spectrometry. Results: A total of 339 proteins were quantified from the adipocyte secretome, of which 59 were differentially secreted in response to iron, with a more than twofold change and P value < 0.05. We validated the iron-induced down-regulation of apolipoprotein E (ApoE) by western blotting, with a reduction of 67% (P = 0.001) and 76% (P = 0.007) by SILAC and western blot, respectively. Conversely, cell lysate samples demonstrated a greater than 11-fold increase in intracellular ApoE in response to iron (P = 0.0005), without a significant change in ApoE mRNA levels. The ratio of secreted ApoE to cell lysate ApoE was reduced by 98% by iron (P = 0.002). Conclusion: These findings indicate that cellular iron loading blocks ApoE secretion, causing ApoE to become sequestered within adipocytes. ApoE has been shown to have a protective role in promoting adipocyte hyperplasia over hypertrophy and also protects against diet-induced steatohepatitis in mice. The interaction between iron and ApoE in adipocytes may represent a novel therapeutic target for treating humans with NASH. Liver oxidative stress, apoptosis, and autophagy are increased in a mouse model of iron overload ACG Chua1, A Domenichini1, RD Delima1, C Elsegood1, JK Olynyk2 and D Trinder1 1School of Medicine, University of Western Australia, Perth, Western Australia, Australia; 2Department of Gastroenterology, Fiona Stanley Hospital, Perth, Western Australia, Australia Introduction: Hereditary hemochromatosis (HH) is a common primary iron overload disorder caused by mutations in HFE or TFR2 genes, which may cause liver iron overload and liver fibrosis, cirrhosis, and hepatocellular carcinoma. Secondary liver iron overload can also develop from excess dietary iron intake. The aim of this study was to examine the role of iron overload in the development of liver injury by examining oxidative stress, apoptosis, and autophagy in mouse models of primary (HH) and secondary (dietary iron supplementation) iron overload. Methods: HH mice with disrupted HFE and TFR2 genes and wild-type (WT) mice were fed a control iron diet (0.01%) until they reached 13 or 26 weeks of age. Other HH and WT mice were fed an iron-supplemented diet (2% carbonyl iron) from 10 weeks of age for 3 weeks. Liver iron concentration (LIC) was measured by inductively coupled plasma mass spectrometry, and liver injury was assessed by measurement of serum alanine transaminase activity. Oxidative stress was determined by measurement of lipid peroxidation (malondialdehyde) and antioxidant enzymes heme oxygenase 1, and copper and manganese superoxide dismutase protein expression. Apoptosis was evaluated by Tunel, and mRNA and protein expression of apoptosis and autophagy markers were determined by polymerase chain reaction and western blot, respectively. Results: LIC was increased in HH mice at 13 weeks (HH, 137 ± 6 vs WT, 20 ± 1 μmol/g; P < 0.001) and was further raised at 26 weeks of age (HH, 168 ± 12 vs WT, 27 ± 2 μmol/g; P < 0.001). Dietary iron supplementation increased LIC in WT mice to similar levels observed in HH mice fed a control iron diet (137 ± 8 μmol/g), and in HH mice, levels were elevated markedly (245 ± 5 μmol/g; P < 0.001). Serum alanine transaminase activity was raised in HH mice at both ages (P < 0.01), but levels did not change with dietary iron loading. Malondialdehyde levels were increased in HH (P < 0.01) and dietary iron-loaded WT mice (P < 0.05), but levels did not change significantly with age. Antioxidant enzyme heme oxygenase protein levels were increased in HH (P < 0.01) and dietary iron-loaded WT mice (P < 0.001) and were further augmented by 1.7-fold in dietary iron-loaded HH mice. In contrast, both copper and manganese superoxide dismutase protein levels were reduced in HH mice (P < 0.01) and dietary iron-loaded WT mice (P < 0.01), with an additional decline by 30%–50% in dietary iron-loaded HH mice. Apoptosis was evident in dietary iron-loaded HH mice by Tunel. Furthermore, expression of pro-apoptotic proteins Bid and Bak were significantly upregulated in HH (P < 0.01) and dietary iron-loaded HH mice (P < 0.05), while anti-apoptotic Bcl-xl mRNA expression was reduced in HH mice (P < 0.05) at 26 weeks. Protein expression of autophagy proteins LC3II and Atg3 were raised in HH mice at both ages (P < 0.05), but dietary iron loading did not enhance expression further in HH mice. Conclusions: Iron-induced liver injury was observed in mouse models of primary and secondary iron overload. There was hepatic oxidative stress with increased lipid peroxidation and changes in antioxidant enzyme expression in both models, while apoptosis and autophagy were both evident in HH mice, suggesting they contribute to liver injury processes in iron overload disease. Circulatory microRNA-365a-3p as a potential biomarker for the diagnosis of pediatric cystic fibrosis-associated liver disease DA Calvopina1, MA Coleman1, MA Fernandez-Rojo1, LF Wockner2, CJ McDonald3, PJ Lewindon1,4,5 and GA Ramm1,5 1Hepatic Fibrosis Group, QIMR Berghofer Medical Research Institute, Brisbane, Queensland, Australia; 2QIMR Berghofer Statistics Unit, QIMR Berghofer Medical Research Institute, Brisbane, Queensland, Australia; 3Membrane Transport Group, QIMR Berghofer Medical Research Institute, Brisbane, Queensland, Australia; 4Department of Gastroenterology and Hepatology, Lady Cilento Children's Hospital, Brisbane, Queensland, Australia; 5Faculty of Medicine and Biomedical Sciences, University of Queensland, Brisbane, Queensland, Australia Introduction: In Australia, cystic fibrosis (CF) affects one out of 2500 newborns, of whom 15–20% develop severe liver abnormalities and 5% die due to end-stage liver disease. CF is an autosomal recessive disorder that affects the CF transmembrane regulator channel expressed in cholangiocytes and the gall bladder. As a consequence of decreased bile flow, thickened secretions block the bile ducts and lead to hepatic and cholangiocyte injury, characteristics of CF-associated liver disease (CFLD). CFLD is one of the leading non-respiratory causes of morbidity and mortality in children with CF. Liver biopsy, an invasive procedure, remains the gold standard to assess the severity of liver damage in CFLD before the advent of cirrhosis and portal hypertension. We investigated circulatory microRNAs (miRNAs) as non-invasive biomarkers to diagnose and assess disease progression of pediatric CFLD. Methods: Our study assessed the circulating miRNA signature of 90 children allocated into three study cohorts based on clinical, biochemical, and imaging assessment, as follows: healthy controls (controls, n = 30); CF patients with no evidence of liver disease (CFnoLD, n = 30); and CFLD (n = 30; sub-divided according to liver biopsy fibrosis stages: F0, F1–2, and F3–4; n = 10 each). Serum miRNAs were analyzed using the Ion Torrent sequencing platform. Sequencing libraries were created by pooling 10 samples per group. Significant differentially expressed miRNAs were detected using normalized read counts and pair-wise comparisons. Selected miRNAs were further validated in 124 individual samples by quantitative real-time polymerase chain reaction and analyzed using anova (P < 0.05). Results: We identified let-7 g-5p, miR-34a-5p, miR-122–5p, miR-365a-3p, miR-18a-5p, miR-126–5p, and miR-142–3p to be significant differentially expressed between all three study groups. Post hoc analysis showed the upregulation of miR-34a-5p (P = 0.0042), miR-122–5p (P = 0.0015), and miR-365a-3p (P = 0.0002), while let-7 g-5p (P = 0.0034) and miR-142–3p (P = 0.0047) were downregulated in CFLD patients compared with CFnoLD patients. ROC curve analysis was used to determine the ability of these miRNAs to differentially diagnose liver disease in children with CF. miR-365a-3p showed the best predictive value, with an area under the curve (AUC) of 0.745 (P = 0.0001; sensitivity, 70.4%; specificity, 67.5%). Additionally, let-7 g-5p with an AUC of 0.708 (P = 0.0010; sensitivity, 70.4%; specificity, 65%), miR-34a-5p with an AUC of 0.707 (P = 0.0014; sensitivity, 71.4%; specificity, 64.1%), and miR-122–5p with an AUC of 0.708 (P = 0.0010; sensitivity, 70.4%; specificity, 62.5%) were also capable of differentiating between CFLD and CFnoLD. In contrast, miR-18a-5p was significantly upregulated in both CFnoLD and CFLD compared with controls (P < 0.0001), with a predictive value of AUC = 0.822 (P < 0.0001; sensitivity, 80%; specificity, 80%), suggesting an important role in CF. Conclusion: Although diagnostic panels using combinations of these miRNAs require validation in larger cohorts, our study has identified several serum miRNAs with potential to diagnose liver disease in children with CF. Alagebrium inhibits progression of non-alcoholic fatty liver disease to steatohepatitis and liver fibrosis by blocking AGE/RAGE pathway in mice HKDH Fernando1, DIG Rajapaksha1, PW Angus1,2 and CB Herath1 1Department of Medicine, University of Melbourne, Austin Health, Melbourne, Victoria, Australia; 2Department of Gastroenterology and Hepatology, Austin Health, Melbourne, Victoria, Australia Introduction: Non-alcoholic fatty liver disease (NAFLD) affects up to 30% of the adult population and is now a major cause of liver disease-related premature illness and death in Australia. There is currently no established drug therapy, and treatment is largely based on lifestyle modification, which is difficult to achieve in most patients. Advanced glycation end products (AGEs), formed as a result of non-enzymatic reaction between reducing-sugars and proteins, lipids or nucleic acids and which works through their receptor, RAGE (receptor for advanced glycation end products), have been implicated as a second hit that drives NAFLD to steatohepatitis and liver fibrosis. Therefore, in this study, we investigated the therapeutic potential of the AGE crosslink breaker alagebrium (Ala) in mice fed a high-fat, high-cholesterol (HFHC) diet or HFHC baked (HFb) diet to increase dietary AGE exposure. Methods: Six-week-old male C57Bl/6 mice were fed an HFHC diet for 40 weeks. A second group of mice was fed the HFb diet (baked 1 hour at 160 °C) to increase dietary AGE levels. A third group of mice, which was fed on the HFb diet, was treated with Ala (10 mg/kg body weight) by daily oral gavage for the last 10 weeks of the experiment. The content of N(6)-(carboxymethyl)lysine (CML), the best characterized AGE type, in HFHC and HFb, was measured by gas chromatography mass spectrometry. At 40 weeks, animals were sacrificed, and blood and liver tissue were harvested. Liver function tests were determined in plasma. Gene expression of RAGE, profibrotic and proinflammatory cytokines, and hepatic stellate cell (HSC) activation marker was determined by quantitative polymerase chain reaction. Hepatic fibrosis was quantified by picrosirius red staining, and therapeutic effects of Ala were tested in mice fed a high AGE diet. Results: CML levels were threefold higher in HFb than in HFHC pellets. Long-term consumption of the HFHC diet produced steatosis, steatohepatitis, and fibrosis after 40 weeks. Alanine aminotransferase (ALT) and alkaline phosphatase (ALP) levels were higher (P < 0.01) in mice fed the HFb diet compared with those of HFHC-fed mice. However, Ala treatment significantly reduced ALT (P < 0.001) and ALP (P < 0.01) in HFb-fed mice, suggesting that Ala improved hepatocellular damage. The increased liver RAGE expression (P < 0.001) in mice fed the HFb diet was abrogated (P < 0.001) by Ala treatment. Moreover, Ala significantly (P < 0.05) downregulated the gene expression of inflammatory cytokines, monocyte chemoattractant protein-1 and tumor necrosis factor-α and HSC activation marker, α-smooth muscle actin in mice fed the HFb diet. Furthermore, the HFb diet caused increased (P < 0.001) expression of profibrotic cytokine, transforming growth factor-β1 (TGF-β1) and extracellular matrix (ECM) gene collagen I in the liver. Moreover, Ala treatment reduced the increased expression of TGF-β1 and collagen I by more than 50% (P < 0.01) in the livers of HFb-fed mice. Gene expression changes of proinflammatory and profibrotic cytokines were accompanied by increased (P < 0.05) ECM deposition in mice fed the HFb diet, leading to liver fibrosis. Interestingly, Ala treatment strongly inhibited liver fibrosis in mice fed the HFb diet, restoring it to that seen in HFHC-fed mice. Conclusion: The progression of NAFLD to steatohepatitis and liver fibrosis is triggered by activation of RAGE with elevated AGE content in the diet. We conclude that activation of AGE/RAGE pathway may serve as a second hit that drives simple steatosis to steatohepatitis and fibrosis. Moreover, we show that the AGE crosslink breaker alagebrium inhibits the activation of the AGE/RAGE pathway in NAFLD and, thus, has potential to be used as a treatment for fibrosis associated with NAFLD. Evolution of the renin angiotensin system in a rat model of non-cirrhotic portal hypertension LS Gunarathne1, DIG Rajapaksha1, HKDH Fernando1, PW Angus1,2 and CB Herath1 1Department of Medicine, University of Melbourne, Austin Health, Melbourne, Victoria, Australia; 2Department of Gastroenterology and Hepatology, Austin Health, Melbourne, Victoria, Australia Aim: The rat model of partial portal vein ligation (PPVL) has been widely used in studies investigating pathophysiology of non-cirrhotic portal hypertension (PH). However, the contribution of the renin angiotensin system (RAS) in the development of PH following PPVL remains largely unknown. Therefore, this time course study investigated the evolution of RAS gene expression in three closely related vascular beds, namely, the mesenteric, gut and liver vasculatures, during the development of PH in PPVL rats. Methods: Six-week-old Sprague Dawley rats were randomly allocated into six groups (n = 6). In rats undergoing PPVL surgery, a calibrated stenosis in the portal vein was created using a 19G needle (OD, 1.067 ± 0.0005). Sham-operated rats served as controls. On Days 1, 2, 5, 10, and 14 after PPVL, the portal vein of anesthetized rats was cannulated through an ileocolic vein with a PE-10 catheter, which was connected to a highly sensitive pressure transducer for continuous monitoring of portal pressure (PP). PP in sham-operated rats was measured 14 days after surgery. Fifteen minutes after continuous PP recording, rats were sacrificed for sample collection. Gene expression of RAS components, including angiotensin converting enzyme (ACE), ACE2, Mas receptor (MasR), angiotensin II type 1 receptor (AT1R), and angiotensin II type 2 receptor (AT2R), was determined in second and third order mesenteric vessels, jejunum and liver tissues by quantitative polymerase chain reaction (qPCR). Results: PPVL significantly (P < 0.0005) increased PP from a mean (±SD) of 5.25 ± 0.91 mmHg in sham-operated rats to more than 12 mmHg (12.4 ± 0.99) in PPVL rats and was dropped to about 10 mmHg (10.6 ± 0.12) after Day 5. A drop in pressure is consistent with the establishment of collateral circulation in this model. qPCR analysis showed jejunal AT1R expression was downregulated (P < 0.005) by more than sixfold from Day 1 to about threefold at Day 14 after PPVL, whereas ACE2 expression tended to be increased. Consistent with AT1R expression in the gut, mesenteric vascular AT1R was also downregulated (P < 0.0005) by more than sixfold. We found that in this vasculature, both AT2R (P < 0.05) and ACE2 (P < 0.0005) were also downregulated in PPVL rats compared with sham-operated controls. In contrast, AT1R expression in the liver was upregulated from Day 2 after PPVL compared with the controls. Moreover, liver ACE, ACE2, and MasR were upregulated by more than 10-fold at Day 2 of PPVL rats compared with the controls. Conclusion: The gut is the first to respond to portal vein obstruction to the blood flow with decreased AT1R expression, leading to inhibition of angiotensin II-mediated vasoconstriction, which allows more blood to flow through the vasculature. This in turn may allow adequate gut perfusion. The mesenteric vasculature also responds by blocking vasoconstriction pathway mediated by angiotensin II, thus allowing adequate gut perfusion. A decrease in the expression of vasodilatory components of the RAS, ACE2, and AT2R, in the mesenteric vasculature may help in maintaining the gut perfusion without exacerbating the vasodilatation. Increased liver ACE2, which may increase vasodilatory Ang(1–7) production, and together with increased MasR, is likely to be a counter-regulatory mechanism in response to increased angiotensin II-mediated vasoconstriction. We conclude that development of non-cirrhotic PH may result from decreased angiotensin II-mediated vasoconstriction in the gut and mesenteric vascular beds. New murine model for hepatocellular carcinoma JM Henderson1, N Polak1, JB Chen1, GW McCaughan1, JG Kench1, B Roediger1, WW Bachovchin2 and MD Gorrell1 1Centenary Institute and Sydney Medical School, University of Sydney, Sydney, New South Wales, Australia; 2Tufts University, Boston, Massachusetts, USA Introduction: Hepatocellular carcinoma (HCC) is responsible for more than 70% of primary liver cancer and is the second leading cause of cancer-related deaths worldwide. Due to the ineffectiveness of current therapies, HCC patients have a 16% 5-year survival rate. HCC models typically involve an initial insult of N-nitrosodiethylamine (DEN), followed by the long-term administration of either a pro-fibrotic or pro-steatotic insult. The major drawback of this approach is the time taken for HCC to appear (8–12 months). Here, we show that combining pro-steatotic and pro-fibrotic insults can produce HCC in 6 months. Liver fibrosis is predominantly driven by activated hepatic stellate cells (HSCs). Following liver insult, HSCs undergo activation from a quiescent vitamin A-storing cell to a proliferative, myofibroblast-like, alpha smooth muscle actin (SMA)-expressing cell exhibiting upregulated collagen synthesis, as well as increased expression of fibroblast activation protein (FAP). FAP activity is very low in resting tissues but is highly induced during inflammation and upregulated in carcinoma-associated fibroblasts (CAFs) of most human tumors. Method: Mice were injected with DEN at 12 days of age and treated from weaning with thioacetamide (TAA) or both TAA and a high-fat diet (HFD). Histological measurement of inflammation, steatosis, and fibrosis used hematoxylin and eosin staining. Alpha-SMA and F4/80 were assessed by immunohistochemistry. Flow cytometry was carried out when tumors were large, at 36 weeks of age. Statistics used non-parametric t-test and anova. Quantitative real-time polymerase chain reaction was performed on 93 genes. Results: The incidence of HCC was greater in the DEN–TAA–HFD-treated mice (83%) than the DEN–TAA-treated mice (20%) at 24 weeks of age (n = 5–6). DEN–TAA–HFD treatment showed increased fibrosis and inflammatory cell aggregation compared with control, and increased steatosis compared with both DEN–TAA and control at 24 weeks (n = 5–6, t-test; P < 0.05, P < 0.001, respectively). The number of dysplastic lesions in DEN–TAA–HFD-treated mice (25 lesions) was greater than in DEN–TAA mice (14 lesions) (n = 5–6). Alpha-SMA (% positive area/total tissue area) expression increased in both the DEN–TAA–HFD livers and the DEN–TAA livers compared with control (n = 5–6, t-test: P < 0.05, P < 0.01, respectively). Flow cytometry analysis of tumors and surrounding liver tissue from DEN–TAA–HFD mice, compared with non-diseased liver showed a decrease in CD45+CD90+ and a decrease in CD45+CD19+ populations inside the tumors and surrounding tissue compared with control (P < 0.0001). By polymerase chain reaction, 34 transcripts were differentially expressed in DEN–TAA–HFD liver (P < 0.05), including upregulation of the macrophage-associated genes CD163, Nos2, CD47, CD64, CD68 and Keap1 and the cell growth-associated genes Bc12-L1 and IGF-1. CD136 mRNA was downregulated in DEN–TAA–HFD liver. Myofibroblasts in DEN–TAA–HFD-treated mice at 36 weeks of age were targeted with 3996, a FAP activated bortezomib-like prodrug. Flow cytometry using the fluorescent FAP inhibitor 4613b showed that the intrahepatic CD45-CD29 + FAP+ population of myofibroblasts decreased in 3996-treated mice. Conclusion: This new DEN–TAA–HFD model provides a more rapid means of examining HCC and potential therapies. Characterizing and combating the immune modulatory and epithelial-to-mesenchymal plasticity properties of hepatocellular carcinoma-derived cancer stem cells A Jayachandran1,2, R Shrestha1,2, H Wang2,3, P Prithviraj4, B Dhungel1,2, I-T Huang1,2 and JC Steel1,2 1Liver Cancer Unit, Gallipoli Medical Research Institute, Greenslopes Private Hospital, Brisbane, Queensland, Australia; 2Faculty of Medicine, University of Queensland, Brisbane, Queensland, Australia; 3Translational Research Institute, Brisbane, Queensland, Australia; 4Olivia Newton John Cancer Research Centre, Melbourne, Victoria, Australia Background: Hepatocellular carcinoma (HCC) is the fastest increasing cause of cancer mortality in Australia despite the development of various therapeutic strategies. The development of an effective immunotherapy for HCC has proven difficult, with the induction of anticancer-directed immune responses seldom resulting in complete tumor eradication. Understanding the mechanisms by which cancer cells are able to escape host immune responses is critical for the development of successful immunotherapeutic treatments. Cancer stem cells (CSCs) represent a specialized population of transformed cells distinct from the majority of “differentiated” tumor cells. These cells are responsible for tumor initiation, organization and maintenance and play a major role in the resistance to radiation and chemotherapy. Recently, epithelial-to-mesenchymal plasticity (EMP) that enables tumor metastasis has emerged as an important regulator of CSC immune modulation. Despite the importance of CSCs, very little is known about the sensitivity of these cells to immune surveillance and immune killing in HCC. Hypothesis and Aims: Our hypothesis is that, to successfully form and maintain tumors, CSCs must suppress or avoid host immune responses, and that EMP is closely associated with CSC immune modulation. The aim of this study is to enrich and purify CSCs from human and murine HCC cell lines and to examine the relationship between immune modulatory and EMP properties of CSCs. Methods and Results: In this study, using a modified sphere-forming serum-free culture system, we enriched stem-like cells from a panel of 20 human- and mouse-derived HCC cell lines. We assessed their stemness characteristics by evaluating the mRNA and protein levels of stemness genes and a cell surface stem cell marker. We imaged the CSCs in vivo with high-resolution multiphoton fluorescence lifetime imaging. Subsequently, we analyzed the immune-modulating characteristics of the CSCs using quantitative reverse transcription polymerase chain reaction (qRT-PCR), flow cytometry and ELISA. We identified immune modulatory pathways, including MHC-I down regulation and TGF-β and IL-10 upregulation, that CSCs may use to suppress immune recognition. Next, we show evidence that CSCs with immunosuppressive characteristics are closely linked with an EMP phenotype and function assessed by qRT-PCR, immunofluorescence staining and motility assays. To examine the clinical relevance of our findings, we analyzed HCC patient datasets available from the Cancer Genome Atlas and found that the expression of immune modulatory and EMP markers resulted in worse overall survival (hazard ratio [HR], 1.7; 95% CI, 1.24–2.44; P = 0.001) and recurrence-free survival (HR, 1.67; 95% CI, 1.16–2.41; P = 0.005). Conclusions: Our study demonstrated that the HCC CSCs exhibit distinctive immune modulatory and EMP markers that can be potentially used as biomarkers to stratify patients with HCC. The insights gained from this research