Fibrotic liver extracellular matrix induces cancerous phenotype in biomimetic micro-tissues of hepatocellular carcinoma model

Abstract

Background: Despite considerable advancements in identifying factors contributing to the development of hepatocellular carcinoma (HCC), the pathogenesis of HCC remains unclear. In many cases, HCC is a consequence of prolonged liver fibrosis, resulting in the formation of an intricate premalignant microenvironment. The accumulation of extracellular matrix (ECM) is a hallmark of premalignant microenvironment. Given the critical role of different matrix components in regulating cell phenotype and function, this study aimed to elucidate the interplay between the fibrotic matrix and malignant features in HCC.

Methods: Liver tissues from both control (normal) and carbon tetrachloride (CCl₄)-induced fibrotic rats were decellularized using sodium dodecyl sulfate (SDS) and Triton X-100. The resulting hydrogel from decellularized ECM was processed into micro-particles via the water-in-oil emulsion method. Micro-particles were subsequently incorporated into three-dimensional liver biomimetic micro-tissues (MTs) comprising Huh-7 cells, human umbilical vein endothelial cells (HUVECs), and LX-2 cells. The MTs were evaluated using the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay at day 11, immunofluorescence staining, immunoblotting, and spheroid migration assay at day 14 after co-culture.

Results: Fibrotic matrix from CCl₄-treated rat livers significantly enhanced the growth rate of the MTs and their expression of CCND1 as compared to the normal one. Fibrotic matrix, also induced the expression of epithelial-to-mesenchymal transition (EMT)-associated genes such as TWIST1, ACTA2, MMP9, CDH2, and VIMENTIN in the MTs as compared to the normal matrix. Conversely, the expression of CDH1 and hepatic maturation genes HNF4A, ALB, CYP3A4 was decreased in the MTs when the fibrotic matrix was used. Furthermore, the fibrotic matrix increased the migration of the MTs and their secretion of alpha-fetoprotein.

Conclusions: Our findings suggest a regulatory role for the fibrotic matrix in promoting cancerous phenotype, which could potentially accelerate the progression of malignancy in the liver.

Keywords: Cell differentiation; Decellularized extracellular matrix; Epithelial-to-mesenchymal transition; Hepatocellular carcinoma; Liver fibrosis; Spheroid.