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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: Hepatocellular carcinoma (HCC) is a complex multistep malignancy in need of new therapeutic options often arising on underlying chronic liver disease. HCC animal models relevant to clinical situations are crucial to investigate new anticancer drugs, alone or in combination. Mice bearing xenografts and transgenic mice are the two main models used for preclinical drug development, but most of them fail to mimic the different step of HCC observed in patients. In this study, we aim to describe a stage-defined, transgenic immunocompetent HCC mouse model. Methods: ASV-B is a transgenic mouse model that spontaneously develops, upon SV40 T-Ag oncogene expression in hepatocytes, a reproducible stage-defined HCC. Hyperplasia at week(W)8 is followed by nodular stage at W12 (multiple well-delimited tumor nodules of about 1 mm, growing progressively up to 1 cm), then diffuse carcinoma stage at W16-20. HCC is restricted to male, backcrossed with C57BL/6J mice, the female littermates being used as controls. Results: Liver volume assessed by ultrasound at each step of carcinogenesis showed a 2.7-, 2.7-, 3.9-, and 5.8-fold increase (p Conclusion: ASV-B transgenic mouse model mimics several characteristics of human HCC developing on healthy liver including multinodular disease, vessel abnormalities, and dedifferentiation toward mesenchymal phenotype. Further steps will consist of characterizing the adjacent nontumor liver, and developing nonalcoolic steatohepatitis (NASH) on ASV-B model using specific diets. Citation Format: Annemilaï Tijeras-Raballand, Patricia Hainaud, Christian Hobeika, Clarisse Eveno, Marc Pocard, Philippe Bonnin, Valérie Paradis, Mohamed Bouattour, Eric Raymond, Armand de Gramont, Evelyne Dupuy, Sandrine Faivre. Stage-defined, transgenic immunocompetent mouse model (ASV-B) to investigate new drugs for hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4096.

Background: Hepatocellular carcinoma (HCC) is characterized by strong abnormalities of liver vasculature linked to proangiogenic pathways in the tumor and its microenvironment. Upregulation of Notch4 and its ligand Dll4 in endothelial cells (ECs), as well as PlGF is involved in liver tumor growth, arterialization and sinusoid remodeling. However, the specific role of PlGF and its receptor VEGFR-1 toward Dll4/Notch4 in ECs and tumor vascular remodeling remains to be characterized. Material and Methods: In vitro, we used primary culture of HUVECs, isolated in our laboratory from human umbilical cords. Active forms of Notch4 and Dll4 in HUVECs were assessed by Western blot. In vivo, differential expression of VEGF-A, PlGF and their receptors were evaluated by qRT-PCR and immunostaining in ASV-B transgenic mice developing stage-defined HCC. Finally, we assessed in vivo PlGF and Notch inhibition, using siRNA PlGF, DAPT and a Notch inhibitor, LY3039478, alone or in combination with sorafenib. Results: In vitro, we first investigated the relative contribution of VEGF-A, VEGF-E and PlGF on Notch4 pathway, showing that all 3 growth factors increased Dll4 and Notch4 expression in HUVECs, the effect being more pronounced with PlGF. Downregulation of VEGFR-1 by si RNA abrogated VEGF-A and PlGF stimulation on Dll4 expression and Notch4 activation, whereas VEGFR-2 siRNA had no effect. PlGF silencing significantly reduced Dll4 dependent expression and Notch4 activation in response to VEGF-E and VEGF-A. To confirm in vivo the prevalence of PlGF/VEGFR-1 pathway on vessel abnormalities, we evaluated the differential expression of PlGF, VEGF-A, VEGFR-1 and VEGFR-2 in our ASV-B HCC model. VEGFR-1 expression increased with HCC progression and was restricted to macrophages and sinusoidal endothelial cells. PlGF levels were maximal at the stage that coincides with the beginning of liver vessels abnormalities in HCC. Silencing in vivo PlGF or blocking Notch4 using DAPT delayed tumor growth, reduced arterial vessel length, and sinusoids abnormalities while decreasing Dll4 and active Notch4 expression. Using a clinically relevant Notch inhibitor, LY3039478, alone or in combination with sorafenib, liver size and tumor macronodules number were significantly lower in all treatment arms vs placebo, Y3039478/sorafenib combination showing increased tumor control. Angiogenesis measured by mean blood flow in the coeliac trunk (TCm) decreased in all treatment arms vs placebo, confirmed by CD31 staining. Conclusions: These data suggest that PlGF/VEGFR-1-Dll4/Notch4 pathways are involved in vessel remodeling and tumor growth. In vivo, silencing PlGF or inhibiting Notch4 prevent tumor vessels remodeling/arterialization and delay tumor growth, further warranting new antiangiogenic strategies for HCC treatment. Citation Format: Annemilaï Tijeras-Raballand, Christian Hobeika, Jean-Olivier Contreres, Patricia Hainaud, Matthieu Martinet, Philippe Bonnin, Clarisse Eveno, Evelyne Dupuy, Marc Pocard, Valerie Paradis, Eric Raymond, Sandrine Faivre, Armand de Gramont. PlGF/VEGFR-1-dependent activation of Dll4/Notch4 pathway contributes to liver vessel abnormalities and tumor growth in hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2043.

Angiotensinogen, a member of the serpin family, is involved in the suppression of tumor growth and metastasis. To investigate whether human angiotensinogen protects against tumor progression in vivo, we established an original bitransgenic model in which transgenic mice expressing human angiotensinogen (Hu-AGT-TG mice) were crossed with a transgenic mouse model of hepatocellular carcinoma (HCC-TG mice). Bitransgenic mice overexpressing human angiotensinogen (HCC/Hu-AGT-TG) had a significantly longer survival time than the HCC-TG mice and a reduction of both tumor growth and blood flow velocities in the liver. This antitumor effect of angiotensinogen is related to a reduced angiogenesis, impaired expression of endothelial arterial markers (active Notch4, Delta-like 4 ligand, and ephrin B2) with a decrease of arterial vessel density in HCC/Hu-AGT-TG mice liver. Overexpression of human angiotensinogen decreases angiogenesis, and prevents tumor sinusoids from remodeling and arterialization, thus delaying tumor progression in vivo. [Cancer Res 2009;69(7):2853–60]

Background: Hepatocellular carcinoma (HCC), is characterized by severe vessel anomalies with an intense arterial blood supply, and acquisition of a basal membrane rich in laminin by sinusoids. A role of the VEGF-A/Dll4-Notch4/ephrin B2 cascade in tumor vessel anomalies has been demonstrated, raising the question of the nature of the VEGF receptor pathway implicated in triggering this cascade. This study was undertaken to determinate the role of PlGF and its receptor VEGFR-1 in the development of vascular anomalies in HCC and to evaluate the role of the downstream Notch4 pathway. Material and Methods: In vitro, we used primary culture of HUVECs, isolated in our laboratory from human umbilical cords. HUVECs were cultured in EBM2 medium, with 20% of FBS and 2ng/ml of FGF-2. The expression of the active form of Notch4 and Dll4 in HUVECs were assessed by Western Blot. In vivo, the differential expression of VEGF-A, PlGF and their receptors were evaluated by qRT-PCR and immunostaining in transgenic mice developing stage-defined HCC. Results: In vitro, to determine which growth factor/receptor combination was predominant in activating the Notch4 pathway, we compared the effects of VEGF-A, VEGF-E and PlGF on Dll4 expression and Notch4 activation in HUVECs. Treatment with all 3 growth factors increased Dll4 and active Notch4 expressions whose levels were significantly greater in PlGF than VEGF-A and VEGF-E treated HUVECs. Moreover, downregulation of VEGFR-1 by si RNA, but not VEGFR-2, abrogated the effects of VEGF-A and PlGF stimulation on Dll4 expression and Notch4 activation. PlGF silencing significantly reduced Dll4 dependent expression and Notch4 activation in response to VEGF-E and VEGF-A. To confirm in vivo the prevalence of the PlGF/VEGFR-1 pathway in induction of vessel anomalies, we evaluated the differential expression of PlGF, VEGF-A, VEGFR-1 and VEGFR-2 in our transgenic HCC model at different stages. VEGFR-1 expression is increased with HCC progression and restricted in macrophages and sinusoidal endothelial cells. Moreover, PlGF levels were maximal at the stage that coincides with the beginning of liver vessels anomalies in HCC. Silencing PlGF or blocking Notch4 activation, using a γ-secretase inhibitor (DAPT), in vivo delayed tumor growth, reduced arterial vessel length, and sinusoids anomalies with a decrease in Dll4 and active Notch4 expression levels without affecting microvascular density or normal liver. Conclusions: Together, these data suggest that the activation of PlGF-VEGFR-1/ Dll4- Notch4/ephrin B2 cascades played a role in vessel remodeling and tumor growth. Inhibitor of γ-secretase or silencing PlGF in vivo reduced activation of Notch4, prevent tumor vessels remodelling and arterialization, leading to a delay in tumor growth. These results would argue that targeting PlGF or active Notch4 may be valuable new anti-angiogenic strategies in the treatment of HCC. Citation Format: Annemilai Tijeras-Raballand, Jean-Olivier Contreres, Patricia Hainaud-Hakim, Carole Le Hénaff, Marc Pocard, Evelyne Dupuy, Armand de Gramont. PlGF/VEGFR-1-dependent activation of the Dll4-Notch4/Ephrin B2 cascade contributes to liver vessel anomalies in hepatocellular carcinoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3564. doi:10.1158/1538-7445.AM2015-3564

Introduction: Hepatocellular carcinoma (HCC), is characterized by severe vessel anomalies with an intense arterial blood supply (arterialisation), and acquisition ofa basal membrane rich in laminin by sinusoids(sinusoid capillarisation). A role of the VEGF-A/Dll4-Notch4/ephrin B2 cascade in tumor vessel anomalies has been demonstrated, raising the question of the nature of theVEGF receptor pathway implicated in triggering this cascade. This study was aimed at determining whether PlGF/VEGFR-1 or VEGF-A/VEGFR-2 pathways were implicated in the vessel anomalies observed during HCC development. Material and Methods:In vitro, we used primary culture of HUVECs, isolated in our laboratory from human umbilical cords. HUVECs were cultures in EBM2 medium, complemented with 20% of FBS and 2ng/ml of FGF-2. To determine which pathway was predominant in the activation of the Dll4/Notch4 cascade, the effects of PlGF (a specific ligand of VEGFR-1), VEGF-E (a specific ligand of VEGFR-2) and VEGF-A (a ligand of both receptors) on the expression of the active form of Notch4 and Dll4 in HUVECs were compared by Western Blot. In vivo, () the differential expression of VEGF-A, PlGF and their receptors were evaluated by qRT-PCR and immunostainings in transgenic mice developing stage-defined HCC. Results: In vitro, HUVECs stimulation by either VEGF-A, VEGF-E or PlGF increased Dll4 expression and Notch 4 activation. However, PlGF had the most important effect with a 5-fold increase of Dll4 expression level and a 4-fold increase in Notch 4 activation, compared to control HUVECs. Moreover, downregulation of VEGFR-1 by small interfering RNA, but not VEGFR-2, abrogated the effects of VEGF-A and PlGF stimulation on Dll4 expression and Notch4 activation. To confirm in vivothe prevalence of the PlGF/VEGFR-1 pathway in induction of vessel anomalies, we first evaluated the differential expression of PlGF, VEGF-A, VEGFR-1 and VEGFR-2 in our transgenic HCC model at different stages of the disease. We showed that VEGFR-1 increased with HCC progression and was expressed by macrophages and sinusoidal endothelial cells. Moreover, PlGF levels were maximal at the stage that coincides with the beginning of liver vessels anomalies in HCC. Silencing PlGF in vivo delayed tumor growth (p Conclusions: We demonstrated that PlGF and VEGFR-1 played a key role in the development of vessel anomalies in HCC and that silencing PlGF prevents liver vessel anomalies and delays tumor growth. Our results bring a new insight in the mechanism responsible for vessel anomalies observed in HCC, giving a strong rationale for the development of new antiangiogenic strategies based on PlGF inhibition in HCC. Citation Format: Annemilai Tijeras-Raballand, Armand de Gramont, Patricia Hainaud, Jean-Olivier Contreres, Carole Le Hénaff, Marc Pocard, Evelyne Dupuy. PlGF/VEGFR-1 dependent activation of the Dll4-Notch4/Ephrin B2 cascade contributes to liver vessel anomalies in hepatocellular carcinoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 19. doi:10.1158/1538-7445.AM2014-19

To overcome the resistance to VEGF-inhibitor treatment, additional anti-angiogenic agents need to be valorized. Placental growth factor (PlGF), a VEGF homologue, stimulates endothelial, tumor and inflammatory cells. Studies in independently generated PlGF−/− mice by us and Regeneron identified a role for PlGF in inflammatory, ischemic and malignant disease, while the anti-PlGF mAb 5D11D4 blocks growth of implanted tumors. Several clinical studies also show that PlGF levels correlate with poor prognosis in diverse types of cancer, while the humanized anti-human PlGF mAb TB403 has been succesfully tested in a phase I trial in out-treated cancer patients. However, genetic evidence for a disease-candidate role of PlGF or an effect of the anti-PlGF mAb 5D11D4 in spontaneous cancer models has been lacking so far. Here, we show that loss of PlGF inhibited a carcinogen-induced skin tumor model papilloma formation and angiogenesis. Furthermore, silencing of PlGF in a transgenic hepatocellular carcinoma (HCC) model reduced tumor size and incidence. In a carcinogen-induced HCC model, most WT but only a minority of PlGF−/− mice died, while fewer tumor nodules developed in PlGF−/− mice. When WT mice with established HCC were treated with 5D11D4, more control IgG than 5D11D4 treated mice died. PlGF blockage inhibited the characteristic arterialization response in these tumors and blocked vessel abnormalization in HCC leading to reduced levels of hypoxia, known to stimulate HCC growth, however, without affecting microvascular density. These findings indicate that the anti-cancer activity of PlGF-blockage is not reflected by a change in vessel density alone. In the Rip1Tag2 model, no differences between WT and PlGF−/− mice or 5D11D4-treated mice in tumor burden or incidence were found, indicating that 5D11D4 does not have off-target effects. Resistance of this model is likely due to the lack of infiltration by Gr1+ neutrophils by PlGF blockage. These findings and previously published results of a Phase I trial with TB403 warrant furter analysis of anti-PlGF strategies for (non)-oncology indications. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-362.