Project Summary/Abstract Non-alcoholic steatohepatitis (NASH) is a major cause of HCC. It is clinically recognized that HCC in NASH often arises at a pre-cirrhotic stage, however the pathomechanism of HCC in non-cirrhotic livers is not well understood. As T2DM with poor glycemic control is an independent risk factor for HCC it is plausible that there are distinct pathways that create a pro- carcinogenic niche in non-cirrhotic T2DM/NASH. We showed that the accumulation of advanced glycation end products (AGEs) in patients with T2DM/NASH and in an animal model are key to necroinflammation and oxidative liver injury. Downregulation of the AGE clearance receptor AGER1 accelerated AGE deposition, and correcting AGER1 in vivo improved NASH. To study how high AGE environment creates permissive conditions for transformed cells, we modulated diet/AGE content prior to hydrodynamic injection of hMET/mutant β-catenin: 1) High AGE background induced an earlier and more invasive HCC, 2) AGE accumulation was linked to significant changes in matrix dynamics-with an increase in energy dissipation or loss and faster stress relaxation in response to a deformation - in an AGE and receptor for AGEs (RAGE)-dependent manner, 3) Inhibiting AGE production reversed changes in matrix viscoelasticity in vivo and lowered tumor burden. We will test the hypothesis that in non- cirrhotic T2DM/NASH accumulation of AGEs contribute to an increase in matrix viscoelasticity and matricellular changes creating a pro-invasive environment. Aim 1: We propose to investigate the link between diet/AGE content, matrix viscoelasticity, sex and HCC phenotypes and outcomes in a novel HCC model. In particular we will focus on how AGE-mediated collagen crosslinks on specific amino acids can alter intermolecular recognition and interaction with proteoglycans thereby affecting matrix dynamics. Aim 2: We will dissect the effects of RAGE and AGER1 signals and AGE accumulation on matrix viscoelastic parameters and HCC phenotypes. In Aim 3 we propose to develop a 3D hydrogel system with tunable viscoelasticity and the impact of AGEs-modified matrix on cell behavior will be studied. Based on the RNAseq data we will focus on the pathomechanism of how viscoelastic changes are sensed by cells, and the key matricellular signals that confer invasive and migratory properties. These studies will demonstrate the impact of AGEs on the liver matrix in non-cirrhotic NASH and outline the pathomechanism for a pro-invasive environment. Defining the key matricellular signals that drive invasion will enable us to pursue translational studied in the future.