Methodology: preclinical models that will be usedto study drug delivery and efficacy in CLDs
In addition to the large collection of patient-derived human hepatoma cell lines provided by the ITN partners, genetargeted mouse models have been developed to study TGF-β signalling in liver disease. In particular, IT-LIVER’s partners have immediate access to mouse models which are needed for further studies within the network. These models include: (i) conditional liver-specific knock outs of Smad7 and Snai1; (ii)models of hepaticfibrosis such as Mdr2-/- or Mdr-/-/p19ARF-/- or Mdr-/-/Stat3-/- mice, or (iii) knock-out models of BMP9. To investigate liver fibrosis, we will also use cultured hepatocytes and hepatic stellate cells(HSC), as well as mouse models of chemicallyinduced liver fibrosis(CCl4, BDL). 3-dimensional (3D) cultures of hepatocytes, referred to as hepatospheres, provide versatile tools to investigate liver carcinoma development. 3D hepatosphere models will be available in the ITN network to analyze TGF-βsignalling and EMT. In particular, mouse hepatocytes generated from p19ARF deficient mice as well as human neoplastic hepatocytes established from HCC patients are suitable to generate hepatospheres with HCC cells before EMT and those induced to EMT. These 3D models further provide the opportunity to grow HCC cells as multicellular spheroids by co-cultivation with e.g. hepatic myofibroblasts or Kupffer cells as well as with liver endothelial cells. Hence, this in vitromodel closely mimics the architecture of human HCC allowing to reliably characterize tumour-stroma interactions at the molecular level. The modular setup, which recapitulates the tumour heterogeneity observed in vivo, enables to identify target molecules that mediate paracrine interactions in the tumour microenvironment and to validate the impact of these molecular targets on tumour growth and invasion in the supporting stroma.