Abstract PO008: Aggressive hepatocellular carcinoma and intrahepatic cholangiocarcinoma cell lines share similar response to proangiogenic priming
Authors: Milosa Fabiola , Romanzi Adriana , Marcelli Gemma , Critelli Rosina Maria , Lasagni Simone , Cadamuro Massimiliano , Fabris Luca , Villa Erica 
Published: September 2022

Abstract
Hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (iCCA) are the most common primary liver cancers. Traditionally, both neoplasms are considered distinct entities due to their different background. However, the comparison of the proliferative subgroup of iCCA with the subgroup of aggressive HCCs (overexpressing transcriptomic signature (TS) (10.1136/gutjnl-2014-308483)) showed that they share 95% of the overexpressed genes and that they hold similar severe prognosis. In TS-positive aggressive HCCs, the most expressed gene is Angiopoietin 2 (ANGPT2), which is associated with increased angiogenesis and proliferation, epithelial-mesenchymal transition (EMT), and PD1/PDL1 activation. We therefore, studied the effect of ANGPT2 and of its regulator VEGF on in vitro 3D models, with the aim of evaluating putative common carcinogenic pathways shared by aggressive HCCs and iCCA. We generated spheroids from HepG2 and HuCCT-1 cell (HCC and iCCA derived cell lines respectively), while EGI-1 cells, from extrahepatic cholangiocarcinoma (eCCA), served as control. Four days after seeding, well-shaped spheroids were stimulated with either 200 ng/mL ANGPT2 or VEGF, or with 100 ng/mL of ANGPT2/VEGF mix. After 3 and 48h, migration of adherent spheroids and expression of EMT markers (E-cadherin, N-cadherin, Vimentin, Beta-catenin) were evaluated by Western blot (WB) and immunofluorescence. Unstimulated spheroids served as negative controls. In both HepG2 and HuCCT-1, but not in EGI-1, treatment with ANGPT2, VEGF or their combination, stimulated the migration of the adherent spheroids. With respect to EMT biomarkers, in HepG2 E-cadherin expression dropped significantly starting from 3h and remaining low even at 48h. Notably, E-cadherin expression by untreated spheroids was lower at 48h than at 3h. In HuCCT-1, E-cadherin expression levels were not modified by the treatments either at 3h or at 48h. N-cadherin expression levels increased, regardless of treatment, in HepG2 from 3h, while in HUCCT-1 from 48h. Vimentin expression strikingly increased after 48h stimulation in both HepG2 and HuCCT-1, but not in controls. No differences were observed in any of the cell lines in Beta-catenin expression for any treatment. Expression of EMT markers remained unchanged in EGI-1 regardless of any treatment,. Immunofluorescence analysis of the adherent spheroids revealed a marked decrease in E-cadherin expression at the periphery of the migrating cells in both HepG2 and HuCCT-1, paralleled by an increase of N-cadherin and Vimentin expression. Taken together, our results demonstrate a similar response to proangiogenic stimulation of both HCC and iCCA cells, but not of EGI-1, thus underlying a) the substantial phenotypic differences between eCCA and iCCA, and b) the pro-invasive effects of angiogenic growth factors on both HCC or iCCA cells Supported by AIRC under IG 2020 - ID. 24858 project – P.I. Villa Erica
Citation Format: Fabiola Milosa, Adriana Romanzi, Gemma Marcelli, Rosina Maria Critelli, Simone Lasagni, Massimiliano Cadamuro, Luca Fabris, Erica Villa. Aggressive hepatocellular carcinoma and intrahepatic cholangiocarcinoma cell lines share similar response to proangiogenic priming [abstract]. In: Proceedings of the AACR Special Conference: Advances in the Pathogenesis and Molecular Therapies of Liver Cancer; 2022 May 5-8; Boston, MA. Philadelphia (PA): AACR; Clin Cancer Res 2022;28(17_Suppl):Abstract nr PO008.