Sorafenib reduces steatosis‐induced fibrogenesis in a human 3D co‐culture model of non‐alcoholic fatty liver disease

Abstract

Non‐alcoholic fatty liver disease (NAFLD) affects around 25% of the worldwide population. Non‐alcoholic steatohepatitis (NASH), the more progressive variant of NAFLD, is characterized by steatosis, cellular ballooning, lobular inflammation, and may culminate on hepatic stellate cell (HSC) activation, thus increasing the risk for fibrosis, cirrhosis, and HCC development. Conversely, the antifibrotic effects of sorafenib, an FDA‐approved drug for HCC treatment, have been demonstrated in 2D cell cultures and animal models, but its mechanisms in a NAFLD‐related microenvironment in vitro requires further investigation. Thus, a human 3D co‐culture model of fatty hepatocytes and HSC was established by culturing hepatoma C3A cells, pre‐treated with 1.32 mM oleic acid, with HSC LX‐2 cells. The fatty C3A/LX‐2 spheroids showed morphological and molecular hallmarks of altered lipid metabolism and steatosis‐induced fibrogenesis, similarly to the human disease. Sorafenib (15 μM) for 72 hours reduced fatty spheroid viability, and upregulated the expression of lipid oxidation‐ and hydrolysis‐related genes, CPT1 and LIPC, respectively. Sorafenib also inhibited steatosis‐induced fibrogenesis by downregulating COL1A1, TGFB1, PDGF, and TIMP1 and by decreasing protein levels of IL‐6, TGF‐β1, and TNF‐α in fatty spheroids. The demonstration of the antifibrotic properties of sorafenib on steatosis‐induced fibrogenesis in a 3D in vitro model of NAFLD supports its clinical use as a therapeutic agent for the treatment of NAFLD/NASH patients.

In Vitro Liver Toxicity Testing of Chemicals: A Pragmatic Approach

“…The absolute quantification of total and/or small fractions of lipids contained in the cell is a frequently used method for evaluation of lipid levels present in in vitro liver steatosis models [ 116 , 317 , 318 , 319 ]. Cellular lipid quantification is a multi-step procedure, which starts with a cellular lysis step followed by the extraction of the lipids, leading to their quantification in the last step.…”

Modelling metabolic diseases and drug response using stem cells and organoids

“…In contrast, magnesium treatment, which is a highly controversial clinical therapy for treating T2DM and liver metabolic discorders 144 , has proven ineffective in this 3D system 101 . Moreover, sorafenib was shown to reduce steatosis-induced fibrogenesis in a human 3D co-culture model of NAFLD 145 . Thus, liver organoids represent an innovative approach to the development of new treatments and the testing of drug responses ex vivo.…”

In vitro models for non-alcoholic fatty liver disease: Emerging platforms and their applications

“…They can provide new insights into complex physiological mechanisms and potential therapeutic targets, increasing our knowledge of NAFLD. Furthermore, they represent a suitable alternative to in vivo models at early stages of drug development ( Romualdo et al., 2021 ; Beisner et al., 2021 ; Eilenberger et al., 2020 ). The main 3D in vitro models used for NAFLD –spheroids, organoids, and microfluidic devices– are detailed in the following section.…”

“…Additionally, fibrogenic spheroids have been used to test the efficacy of putative drugs (e.g., sorafenib ( Romualdo et al., 2021 ), lanifibranor, elafibranor, or cenicriviroc ( Hurrell et al., 2020 )) in mediating resolution of NASH, thereby validating the role of spheroids for drug screening.…”