Three-dimensional Organotypic Culture Models of Human Hepatocellular Carcinoma.

Atsushi Takai,Zhipeng Yu,Valerie Fako,Hien Dang,Anuradha Budhu,Xin Wei Wang,Marshonna Forgues

doi:10.1038/srep21174

Abstract

Three-dimensional cell culture methods are viable in vitro approaches that facilitate the examination of biological features cancer cells present in vivo. In this study, we demonstrate that hepatocellular carcinoma (HCC) cells in porous alginate scaffolds can generate organoid-like spheroids that mimic numerous features of glandular epithelium in vivo, such as acinar morphogenesis and apical expression patterns of EpCAM, a hepatic stem/progenitor cell marker highly expressed in a subset of HCC with stemness features. We show that the activation of Wnt/β-catenin signaling, an essential pathway for maintaining HCC stemness, is required for EpCAM+ HCC spheroid formation as well as the maintenance of the acinous structure. Furthermore, we demonstrate that EpCAM+ HCC cells cultured as spheroids are more sensitive to TGF/β-induced epithelial-mesenchymal transition with highly tumorigenic and metastatic potential in vivo compared to conventional two-dimensional (2D) culture. In addition, HCC cells in EpCAM+ spheroids are more resistant to chemotherapeutic agents than 2D-cultured cells. The alginate scaffold-based organotypic culture system is a promising, reliable, and easy system that can be configured into a high throughput fashion for the identification of critical signaling pathways and screening of molecular drug targets specific for HCC.

Highlights

Hepatocellular carcinoma (HCC), a major histological subtype of liver cancer, represents a lethal form of solid tumors with at least 600,000 deaths in the world annually[1] and a reported 5-year survival rate of < 20% in the US2
We demonstrate that certain EpCAM+ hepatocellular carcinoma (HCC) cells can generate organoid-like spheroids that recapitulate numerous features of the glandular epithelium in vivo, such as formation of acini and apical expression patterns of stem cell-associated proteins
We show that β -catenin signaling, including EpCAM that is essential for maintaining HCC stemness[16,17,18], is required for the formation of EpCAM+ HCC organoids

Summary

Introduction

Hepatocellular carcinoma (HCC), a major histological subtype of liver cancer, represents a lethal form of solid tumors with at least 600,000 deaths in the world annually[1] and a reported 5-year survival rate of < 20% in the US2. The main reason that many patients are refractory to treatment and have dismal outcome is the extended biological heterogeneity observed in HCC3 Such tumor heterogeneity makes it difficult to identify specific druggable cancer driver genes whose functions are essential for the fitness of cancer cells. We describe the characterization of an AlgiMatrix-based 3D culture method to support HCC organoid formation Using this method, we demonstrate that certain EpCAM+ HCC cells can generate organoid-like spheroids that recapitulate numerous features of the glandular epithelium in vivo, such as formation of acini and apical expression patterns of stem cell-associated proteins. We suggest that the AlgiMatrix-based HCC organoid culture system represents a reliable and efficient in vitro model for investigating candidate HCC driver genes and molecularly-targeted drug screening

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Results

Conclusion