Glioblastoma Cell Culture and Drug Treatment

U87 (alias U87-MG) and A172 were purchased from ATCC and cultured in DMEM 4.5 g/L glucose (Thermo Fisher Scientific, Waltham, MA, USA), supplemented with 10% fetal bovine serum (Thermo Fisher Scientific). GSCs were isolated from patients’ surgical samples at the Institute of Neurosurgery, Catholic University of Rome as previously described [16 (link)] and grown in suspension in DMEM/F12 serum-free medium (Thermo Fisher Scientific) containing 2 mM L-glutamine, 0.6% glucose, 9.6 mg/mL putrescine, 6.3 ng/mL progesterone, 5.2 ng/mL sodium selenite, 0.025 mg/mL insulin, 0.1 mg/mL transferrin sodium salt (Sigma Aldrich, St Louis, MO, USA), EGF (20 ng/mL; Peprotech, London, UK), bFGF (10 ng/mL; Peprotech), and heparin (2 μg/mL; Sigma Aldrich) at 37 °C, 5% CO₂. All cell lines were regularly checked to exclude mycoplasma contamination by Mycoalert Detection Kit (Lonza, Basel, Switzerland).
Regorafenib (Bay73-4506) was purchased from Selleckem (Houston, TX, USA) and resuspended in DMSO.