Abstract 237: Establishment of an ex vivo tissue culture model as a potential preclinical drug testing platform for hepatocellular carcinoma
Authors: Feng Huiche , Cieri Miriam , Esposito Cinzia , Guja-Jarosz Karolina , Liberati Daniela , Bubendorf Lukas , Soysal Savas Deniz , Coto-Llerena Mairene , Terracciano Luigi Maria , Kollmar Otto , Piscuoglio Salvatore 
Published: March 2024

Abstract
Background: Hepatocellular carcinoma (HCC) is a leading cause of cancer-related deaths worldwide, and there is a critical need for more effective treatments. The development of new therapies, especially those aiming to modulate the tumor microenvironment (TME), is limited by the lack of in vitro and in vivo models that recapitulate the complexity of the TME and spatial architecture of HCC. Here, we aimed to establish an HCC model to assess the treatment response and hepatotoxicity of selected drugs by ex vivo culturing fresh human liver tumor tissue and adjacent normal tissue, respectively.
Materials and Methods: Fresh human liver tissues, including 5 malignant and 20 adjacent-normal, were obtained from surgical resections. PDX tumors were generated from patient-derived organoids. Tissue resections were cut into ultra-thin slices (from 150µm to 350µm) using a vibratome. Related culture conditions such as high oxygen partial pressure, air-liquid interface (ALI) culture, artificial extracellular matrix (AEM), and delay duration were evaluated to optimize the protocol. As a readout, cell viability was quantified by an expert pathologist using Hematoxylin/Eosin staining. The preservation and complexity of the TME were evaluated by immunohistochemistry or flow cytometry using specific antibodies for each cell population (T cells, anti-CD3; stroma cells, a-SMA; endothelial cells, CD34; Kupffer cells, CD68). Liver cell function was tested by measuring ALT/AST activity, albumin release, and CYP340 activity.
Results: The highest tissue viability was observed in normal and tumor tissue with a thickness of 150µm and 250µm, respectively. Compared to standard conditions, the combination of ALI and high-oxygen partial pressure conditions dramatically improved tissue viability, with most of the samples having 70% and 50% cell viability at days 3 and 5 of ex vivo culture, respectively (p&lt;0.05). AEM could prevent the efflux of cellular components (p=0.016). The impact of the time delay between the liver resection and the start of slicing was minimal when the slicing occurred within two hours after hepatectomy (p&lt;0.05). Key cellular components including hepatocytes, T cells, fibroblasts, and Kupffer cells also remained better preserved throughout the 5-day culture period in optimized conditions.
Conclusion: Our study has successfully optimized the ex vivo tissue culture model of liver tissue, capable of maintaining the tissue for several days while preserving the unique TME. These results highlight the potential of using the ex vivo tissue culture model in the preclinical setting to further investigate and evaluate TME-related treatments and their hepatotoxicity in HCC tissues.
Citation Format: Huiche Feng, Miriam Cieri, Cinzia Esposito, Karolina Guja-Jarosz, Daniela Liberati, Lukas Bubendorf, Savas Deniz Soysal, Mairene Coto-Llerena, Luigi Maria Terracciano, Otto Kollmar, Salvatore Piscuoglio. Establishment of an ex vivo tissue culture model as a potential preclinical drug testing platform for hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 237.