Cirrhotic Human Liver Extracellular Matrix 3D Scaffolds Promote Smad-Dependent TGF-β1 Epithelial Mesenchymal Transition

• Published in: Cells (Basel, Switzerland) Vol. 9; no. 1; p. 83
• Main Authors: Mazza, Giuseppe, Telese, Andrea, Al-Akkad, Walid, Frenguelli, Luca, Levi, Ana, Marrali, Martina, Longato, Lisa, Thanapirom, Kessarin, Vilia, Maria Giovanna, Lombardi, Benedetta, Crowley, Claire, Crawford, Mark, Karsdal, Morten A, Leeming, Diana J, Marrone, Giusi, Bottcher, Katrin, Robinson, Benjamin, Del Rio Hernandez, Armando, Tamburrino, Domenico, Spoletini, Gabriele, Malago, Massimo, Hall, Andrew R, Godovac-Zimmermann, Jasminka, Luong, Tu Vinh, De Coppi, Paolo, Pinzani, Massimo, Rombouts, Krista
• Format: Journal Article
• Language: English
• Published: Switzerland MDPI AG 28.12.2019; MDPI
• Subjects: 3-dimensional (3d) platform; 3d ecm scaffolds; Biomechanics; Cell culture; Chronic illnesses; Collagen; decellularized extracellular matrix (decm); decellularized human liver; Disease; Enzyme inhibitors; Extracellular matrix; Fibronectin; Gene expression; Gene regulation; Hepatocellular carcinoma; hepatocellular carcinoma (hcc); hepatocellular carcinoma cells; Hepatocytes; Homeostasis; Kinases; Liver; Mechanical properties; Mesenchyme; Microenvironments; Phosphorylation; Proteins; proteomics; Scanning electron microscopy; Smad protein; Smad2 protein; Therapeutic targets; tissue engineering; transforming growth factor beta1 (tgf-β1); Transforming growth factor-b1; tumor microenvironment (tme); United Kingdom > UK; decellularized extracellular matrix (dECM); tissue engineering; tumor microenvironment (TME); proteomics; Transforming growth factor beta1 (TGF-β1); 3-dimensional (3D) platform; decellularized human liver; hepatocellular carcinoma (HCC); 3D ECM scaffolds; hepatocellular carcinoma cells
• ISSN: 2073-4409; 2073-4409
• DOI: 10.3390/cells9010083

An altered liver microenvironment characterized by a dysregulated extracellular matrix (ECM) supports the development and progression of hepatocellular carcinoma (HCC). The development of experimental platforms able to reproduce these physio-pathological conditions is essential in order to identify and validate new therapeutic targets for HCC. The aim of this work was to validate a new in vitro model based on engineering three-dimensional (3D) healthy and cirrhotic human liver scaffolds with HCC cells recreating the micro-environmental features favoring HCC. Healthy and cirrhotic human livers ECM scaffolds were developed using a high shear stress oscillation-decellularization procedure. The scaffolds bio-physical/bio-chemical properties were analyzed by qualitative and quantitative approaches. Cirrhotic 3D scaffolds were characterized by biomechanical properties and microarchitecture typical of the native cirrhotic tissue. Proteomic analysis was employed on decellularized 3D scaffolds and showed specific enriched proteins in cirrhotic ECM in comparison to healthy ECM proteins. Cell repopulation of cirrhotic scaffolds highlighted a unique up-regulation in genes related to epithelial to mesenchymal transition (EMT) and TGFβ signaling. This was also supported by the presence and release of higher concentration of endogenous TGFβ1 in cirrhotic scaffolds in comparison to healthy scaffolds. Fibronectin secretion was significantly upregulated in cells grown in cirrhotic scaffolds in comparison to cells engrafted in healthy scaffolds. TGFβ1 induced the phosphorylation of canonical proteins Smad2/3, which was ECM scaffold-dependent. Important, TGFβ1-induced phosphorylation of Smad2/3 was significantly reduced and ECM scaffold-independent when pre/simultaneously treated with the TGFβ-R1 kinase inhibitor Galunisertib. In conclusion, the inherent features of cirrhotic human liver ECM micro-environment were dissected and characterized for the first time as key pro-carcinogenic components in HCC development.