Plgf/Vegfr-1-Dependent Activation Of Dll4/Notch4 Pathway Contributes To Liver Vessel Abnormalities And Tumor Growth In Hepatocellular Carcinoma

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: Annemilai 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.