Sorafenib

sorafenib tosylate (Cat. No. S-8502) was purchased from LC Laboratories, dissolved in DMSO at 10 mM aliquots, and stored at −20°C. Tumor organoids were plated at a density of 5 × 10³ cells in 15 μL BME2 droplets in order to form organoids. At day 6, sorafenib was added to the medium, and cell viability was measured after 6 days using CellTiter-Glo 3D reagent (Promega). Luminescence was measured on a Synergy H1 Multi-Mode Reader (BioTek Instruments). Results were normalized to vehicle (=100% DMSO). Curve fitting was performed using Prism (GraphPad) software and the nonlinear regression equation. All experiments were performed at least two times in duplicate. Results are shown as mean ± SEM.

HA22T⁄VGH undifferentiated cells, kindly provided by N. D'Alessandro (University of Palermo, Palermo, Italy), were maintained in RPMI-1640 (Life Technologies) supplemented with 10% fetal bovine serum at 37°C in a 5% CO₂ incubator. SKHep1Clone3 (SKHep1C3), selected from human HCC-derived cells (SKHep1: ATCC HTB-52), was maintained in Earle's MEM (Life Technologies) supplemented with 10% fetal bovine serum (Life Technologies) at 37°C in a 5% CO₂ incubator. sorafenib (Nexavar^®, BAY43-9006) was synthesized at Bayer Corporation and was dissolved in 100% DMSO (Sigma-Aldrich) and diluted in RPMI-1640 (Life Technologies) to the required concentration with a final DMSO concentration of 0.1%. Cells were cultured in 10 cm Ø plates up to almost confluence and were treated with sorafenib (5 µM) or solvent (0.1% DMSO) for 24 h. Genomic DNA and total RNA were extracted from treated and untreated cells using Wizard Genomic DNA purification kit (Promega) and TRIzol reagent (Life Technologies), respectively, according to the manufacturer's instructions, and the nucleic acid quantifications were performed using NanoDrop (Thermo Fisher Scientific).