Abstract 6041: Perfusion-based bioreactor preserves microenvironment of hepatocellular carcinoma ex vivo
Authors: Hess Gabriel Fridolin , Muraro Manuele Giuseppe , Coto-Llerena Mairene , Däster Silvio , Ercan Caner , Muenst Simone , Kollmar Otto , Piscuoglio Salvatore , Soysal Savas Deniz 
Published: June 2022

Abstract
Background: Hepatocellular carcinoma (HCC) accounts for 75-85% of all primary liver malignancies. HCC represents an attractive target for immune checkpoint inhibitors (ICI) and tumor microenvironment (TME) agents. The development and success of ICI therapies requires a comprehensive model to study interactions of immune networks in the TME. Patient-derived organoids (PDO) have emerged as promising individualized models for ex vivo drug testing. While PDO are superior to previous models, due to their inherent heterogeneity and their enhanced capacity to capture tumor characteristics, the lack of mesenchymal and immune components limit their use for testing drug classes such as ICI or study tumor-TME interaction. Here, we developed and characterized a perfusion-based cultivated ex vivo HCC tissue model that preserves the tumor tissue architecture and maintains the pathophysiological microenvironment of human tissue for testing of TME-related treatments.
Materials and methods: Fresh tumor tissue was obtained from surgical specimens of six patients undergoing liver resection for HCC. Tissue fragments of 2x2x2 mm were installed between two discs of collagen type 1 porous scaffold and placed in a perfusion-based bioreactor (U-shaped Culture Under Perfusion; U-CUP) for tissue engineering or cultured in static condition. At different time points, cultured tissue fragments were formalin-fixed and paraffin-embedded for downstream analysis. Cell viability and morphology was monitored on day 0, 5 and 7 by using hematoxylin and eosin staining. The changes in TME composition were monitored by immunohistochemistry using cell-type specific antibodies like CD34 for endothelial cells, CD3 for T-cells, alpha-smooth muscle actin (a-SMA) for fibroblasts and arginase-L for urea cycle.
Results: Microscopic analysis revealed that, compare to static culture, the morphology and the viability of the cells were preserved during the culture period in the U-CUP. Tissue histology as well as the immunophenotypic pattern observed in the original tissue was maintained. Similarly, the presence of endothelial cells (CD34+) and fibroblasts (a-SMA+) were detected in the U-CUP cultured samples in a similar proportion to the original tumor samples. The presence of T-cells (CD3+) was stable at all time points. Compared to the samples cultured under static conditions, the U-CUP tended to show less necrosis during the seven-day course.
Conclusion: Compared to PDO, U-CUP bioreactor tumor culture seems to preserve mesenchymal and immune components. This enables further investigation and assessment of TME-related treatments, such as ICI.
Citation Format: Gabriel Fridolin Hess, Manuele Giuseppe Muraro, Mairene Coto-Llerena, Silvio Däster, Caner Ercan, Simone Muenst, Otto Kollmar, Salvatore Piscuoglio, Savas Deniz Soysal. Perfusion-based bioreactor preserves microenvironment of hepatocellular carcinoma ex vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6041.