Chronic inflammation is a key component in the development of virtually all types of primary liver cancers. However, how chronic inflammation potentiates or even may initiate liver parenchymal cell transformation remains unclear. Cancer stem cells (CSCs) represent an exciting target for novel anticancer therapeutic strategies in several types of cancers and were also described in primary liver cancers as tumor initiating cells. Recently, we reported a key role of Interleukin (IL)-17 in Liver Progenitor Cell (LPC) accumulation in preneoplastic cirrhotic livers. In this study, we evidenced in vitro, that long-term stimulation of LPCs with IL-17 led to their transformation into CSCs. Indeed, they acquired CSC-marker expression, and self-renewal properties, showed by their increased capacity to form spheroids. The miRNome analysis revealed that long-term IL-17 treatment of LPCs led to a 90% decrease in miR-122 expression. In a model using immunodeficient mice, ectopic engraftment of LPCs in an IL-17-enriched environment led to tumor occurrence with an aggressive phenotype. Contrastingly, in a murine model of hepatocellular carcinoma induced by a unique injection of diethyl-nitrosamine associated with chronic administration of carbon tetrachloride, IL-17-deficiency or anti-IL-17 therapy protected mice from liver tumor growth. In conclusion, we showed that a chronic exposure of LPCs to IL-17 cytokine promotes their transformation into CSCs. In addition, we demonstrated that IL-17-neutralizing strategies limit CSC occurrence and liver tumor progression through miR-122 restored-expression.

To assess the performance of 405 nm–induced autofluorescence for the characterization of primary liver nodules on ex vivo resected specimens. Forty resected liver specimens bearing 53 primary liver nodules were included in this IRB-approved prospective study. Intratissular spectroscopic measurements were performed using a 25-G fibered-needle on all ex vivo specimens: 5 autofluorescence measurements were performed in both nodules and adjacent parenchyma. The spectra derivatives of the 635 and 670 nm autofluorescence peaks observed in nodules and in adjacent liver parenchyma were compared (Kruskal–Wallis and Mann–Whitney when appropriate). A total of 42 potentially evolutive primary liver nodules—34 hepatocellular carcinomas, 4 intrahepatic cholangiocarcinomas, 4 hepatocellular adenomas—and 11 benign nodules—5 focal nodular hyperplasias, 6 regenerative nodules—were included. Both 635 and 670 nm Δderivatives were significantly higher in benign as compared to potentially evolutive (PEV) nodules (respectively 32.9 ± 4.5 vs 15.3 ± 1.4; p < 0.0001 and 5.7 ± 0.6 vs 2.5 ± 0.1; p < 0.0001) with respective sensitivity and specificity of 78% and 91% for distinguishing PEV from benign nodules. 405 nm–induced autofluorescence enables the discrimination of benign from PEV primary liver nodules, suggesting that autofluorescence imaging could be used to optimize US targeted liver biopsies. • 405 nm–induced autofluorescence can distinguish liver tumors from the adjacent liver parenchyma. • The analysis of autofluorescence imaging observed within primary liver tumors can discriminate benign tumors from those requiring follow-up or targeted liver biopsy. • In current practice, autofluorescence imaging could be embedded within biopsy needle, to enable, in addition to ultrasound guidance, optimal targeting of liver nodules which could optimize tissue sampling.

Background: Non-alcoholic fatty liver disease (NAFLD), especially non-alcoholic steatohepatitis (NASH) is a chronic liver disease commonly associated with hepatic fibrosis. NASH patients have an increased risk for hepatocellular carcinoma (HCC). Due to western way of life, NASH incidence is rising and is predicted to become the leading cause of HCC in the next decades. Therefore, there is an urgent need for robust animal models fully recapitulating the NASH-related HCC carcinogenesis. In this study, we develop and characterize specific diet-induced variants from our transgenic HCC mouse model, focusing on immune landscape.Methods: To mimic NASH, ASV-B, a transgenic mouse model (C57BL/6J) that spontaneously develops a reproducible stage-defined HCC (hyperplasia at week(W)8, nodular stage at W12, and diffuse carcinoma at W16-20) was exposed to 5 different diets. Ten ASV-B and 5 control mice were fed as follows: classic diet as control (yellow), or a high-fat diet (blue), a diet enriched with saturated fatty acids + 1.25% cholesterol (green), a diet containing 22% of vegetal oil + 0.2% cholesterol (orange), and a 1.25% cholesterol diet containing 21% of milkfat (red). All mice fed with special diets also received 30% fructose in the drink water. RNA was extracted from frozen livers at W20 for 40 immune markers analysis using qRT-PCR (LightCycler, Roche). Immune populations were assessed using automated immunohistochemistry (IHC) (Bond Max, Leica).Results: ASV-B model shows an increase in liver volume and angiogenesis, ASV-B livers harboring marked arterialization and capillarization as compared to control. Assessing immune markers on 7 evaluable tumor specimens, we observed an increase in CD8, Foxp3, INOS, CD11b, PD-1, PD-L1, IL1β, IFN-γ, TNF-α, IL17A and IL17F mRNA expression, as frequently observed in human inflammatory HCC. In addition, IHC staining showed intratumoral infiltration of lymphocytes (CD8+) and macrophages (F4/80+, a well-characterized and extensively referenced mouse macrophage marker). ASV-B mice receiving yellow, blue, and green regimens showed similar liver volumes and weight. By macroscopic analysis, we observed increased liver steatosis, and fibrosis in the red and orange regimen compared to others. Moreover, we observed a 40% mortality rate in the orange regimen, and a 20% mortality rate in the blue and the green regimens. At the conference, we will show the morphologic changes of the livers using HPS staining and the immune landscape in the livers of the diet-variants.Conclusion: ASV-B transgenic mouse model mimics several characteristics of human HCC developing on healthy liver including inflammatory reaction and immune cell infiltration. In the ASV-B model, we have been able to develop specific-diets variants aiming at mimicking NASH that could be used for drug testing.Citation Format: Annemilaï Tijeras-Raballand, Christian Hobeika, Benoit Rousseau, Patricia Hainaud, Philippe Bonnin, Aurélie Rodrigues, Fouad Ladfil, Marc Pocard, Valérie Paradis, Armand de Gramont, Eric Raymond, Evelyne Dupuy, Clarisse Eveno, Sandrine Faivre. Diet-variants and immune characterization of a stage-defined, transgenic immunocompetent mouse model of HCC (ASV-B) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 91.

Background & aims The recent approval of direct acting anti-virals (DAA) has dramatically changed the landscape of hepatitis C virus (HCV) therapy. Whether viral clearance could promote liver carcinogenesis is debated. It has been hypothesized that changes in intrahepatic immune surveillance following viral cure could favour tumour growth. This study aimed at characterizing the intrahepatic immune changes induced by HCV cure following DAA therapy. Methods Patients with compensated cirrhosis who underwent surgical resection for hepatocellular carcinoma (HCC) after sustained virological response (SVR) to DAA therapy were included. A control group of untreated HCV-infected patients with compensated cirrhosis was selected. RNA was extracted from tumoral and non-tumoral tissues and analysed using the Nanostring Immuno-Oncology-360 panel. Immune cells were quantified by immunohistochemistry. Results Twenty patients were included: 10 patients with a DAA-induced SVR and 10 untreated controls. All of them had a de novo BCLC 0/A HCC. Non-tumoral tissue profiling showed down-regulation of interferon-related genes (including MX1, ISG15 and IFIT1) after DAA therapy. No other differences in immune profiles/immune cell densities were identified between the two groups. The intra-tumoral immune profiles of HCCs that occurred after DAA therapy were not qualitatively or quantitatively different from those of tumours occurring in untreated patients. Conclusion In conclusion, removal of HCV infection after DAA-based therapy results only in a down-regulation of interferon-stimulated genes in non-tumoral tissues from patients with cirrhosis who develop HCC. These minor changes in the liver immune microenvironment are unlikely to favour HCC occurrence or recurrence after DAA-induced SVR.

Objective: Cancer stem cells (CSCs) have been the focus ofseveral studies because oftheir involvement in cancer initiationand progression. CSCs were identified in 28% to 50% ofhepatocellular carcinomas (HCCs). The origin of CSCs is stillunclear, but it has been recently suggested that CSCs couldoriginate from the transformation of liver progenitor cells (LPCs)during chronic liver inflammation. We previously reporteda correlation between increased expansion of LPCs and thenumber of interleukin (IL)-17-producing cells. In this study,we hypothesized that chronic IL-17 exposure may lead to LPCtransformation into CSCs. Methods: Preneoplastic liver samples from 33 patients wereimmunostained with specific antibodies to identify LPCs (anti-CK19), CSCs (anti-CD133), and IL-17-producing cells (anti-IL-17).IL-17 tumorigenic effects were assessed in vivo, inNOD/SCIDmice that were subcutaneously engrafted with a murine LPCline, “Bipotential Murine Oval Liver" (BMOL) transfected withluciferase and IL-17 genes or empty vector. Tumor growth wasfollowed over 12 weeks by bioluminescence imaging (IVIS Camerasystem). MicroRNA-regulated expression effect on IL-17-treatedBMOL cells over 40 days was determined by next-generationsequencing. Acquired self-renewal ability of transformed BMOLcells was assessed by spheroid formation capacity. Results: Semi-quantitative analysis of CK19+, CD133+, andIL-17+ cells in cirrhotic livers showed a positive correlationbetween the number of infiltrated IL-17+ cells and LPC andCSC accumulation. In vivo, when mice were engrafted withBMOL cells that constitutively producing IL-17, enhancedcell expansion was observed, in contrast to the conditionwhere BMOL cells with empty vector control were engrafted.Immunohistological and mRNA analyses of the tumors thatdeveloped at 12 weeks showed a significant increase in cancercell makers including CD133, α-fetoprotein, and glypican-3in the presence of IL-17. MiRNome analysis of BMOL treatedwith IL-17 revealed a 90% decrease in miR-122 expressionand this was accompanied by a higher self-renewal capacity.However, over expression of a miR-122 mimic in BMOL cellsabolished the IL-17-mediated self-renewal capacity. Conclusions: In preneoplastic liver samples, the correlationbetween IL-17+ cells and increased expression of CD133strongly suggests that IL-17 leads to LPC transformation intoCSCs. This was supported by aggressive tumor developmentin mice engrafted with LPCs in an IL-17-enrichedmicroenvironment. IL-17 mediates LPC transformationthrough miR-122 downregulation. Therefore, strategies aimingto neutralize IL-17 may contribute to CSC niche eradicationand could prevent HCC initiation. DOI: 10.20892/j.issn.2095-3941.2018.S124