Sorafenib

HepG2215 cells (HBV⁺/HBsAg⁺ human hepatoblastoma) were a gift from Dr. Jun-Jen Liu (Institute of Medical Biotechnology, Taipei Medical University, Taipei, Taiwan). Hep3B cells (HBV⁺ HBsAg⁺ human HCC, HB-8064) were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). Sorafenib-resistant cells (HepG2215_R and Hep3B_R) were generated by treating naïve cells (HepG2215 and Hep3B) with Sorafenib (Cell Signaling, Danvers, MA, USA) starting at a low concentration and proceeding to a high concentration. The Sorafenib-resistant cells were then maintained in 10 µM Sorafenib (Cell Signaling, Danvers, MA, USA). Dulbecco’s modified Eagle medium (DMEM, Gibco-BRL, Thermo Fisher Scientific, Waltham, MA, USA) plus 10% fetal bovine serum (FBS), 3.7 g/L sodium bicarbonate (Sigma-Aldrich, St. Louis, MO, USA), 1% penicillin-streptomycin (PS, Gibco, Grand Island, NY, USA), and 1% glutamate (Gibco, Grand Island, NY, USA) was used for culturing all cell lines.
HCC tissues were obtained from a patient who had received Sorafenib after resection of a large HCC and had a peritoneal lymph node excised 2 months after Sorafenib treatment. This study was approved by the Institutional Review Board of Chang Gung Medical Foundation (Approval number: 201800008B0C601).

Hep3B cells (HBV⁺/HBsAg⁺ human HCC, HB-8064) were purchased from the American Type Culture Collection (Manassas, VA, USA) and HepG2215 cells (HBV⁺/HBsAg⁺ human hepatoblastoma) were kindly provided by Dr. Jun-Jen Liu (Institute of Medical Biotechnology, Taipei Medical University, Taipei, Taiwan). These cell lines were maintained in Dulbecco’s modified Eagle medium (DMEM, Gibco-BRL, Thermo Fisher Scientific, Waltham, MA, USA) with 10% fetal bovine serum, 3.7 g/L sodium bicarbonate (Sigma-Aldrich, St. Louis, MO, USA), 1% penicillin–streptomycin (PS, Gibco, Grand Island, NY, USA), and 1% glutamate (Gibco) in a humidified incubator in 5% CO_2. Sorafenib-resistant cells (HepG2215_R and Hep3B_R) were generated by treating naïve cells (HepG2215 and Hep3B) with low to high concentrations of Sorafenib (Cell Signaling, Danvers, MA, USA) from low to high concentrations for continuing 3 months. And Sorafenib-resistant cells were maintained in 10 µM Sorafenib with the medium as mentioned above. The cell line protocol was the same as the published study by Yen-Hua Huang’s lab [23 (link)].

For proliferation assay of the 2D cells, the naïve or sorafenib-resistant cells were seeded in 96- well plates at 5000 cells/well and incubated at 37 °C in 5% CO₂ for 24 h or 48 h. For the drug sensitivity assay, the cells were seeded for 24 h and treated with various concentrations of sorafenib (#8705 Cell Signaling) and these cells were then incubated at 37 °C in 5% CO₂ for 48 h. The 3D organoids were cultivated in Matrigel (Sigma-Aldrich) by seeding a drop in a 48-well plate. A total of 10³ cells were mixed with 50 μL Matrigel. Organoids were cultivated for 14 days. Thereafter, a WST-1 assay (Roche) was used to detect cell proliferation according to the manufacturer’s instructions. Three experiments were performed for each experimental condition. Cell viability is expressed as the percentage of non-treated cells.