B 27 no serum supplement

All patient-derived GSC cell lines and neural progenitor cells (NPCs) were kindly donated by Dr. Krishna P.L. Bhat (The University of Texas, M.D. Anderson Cancer Center, Houston, TX). GSC11, GSC8–11, GSC20, GSC267, GSC28 were established and widely applied in previous studies (6 (link), 29 (link), 30 (link)); their subtypes had already been identified according to the metagene score for PN or MES subtypes based on Philips and Verhaak gene set, respectively (3 (link), 5 (link)). All cell lines were cultured in medium prepared from DMEM/F12 (10565018; Gibco, USA), 2% B-27 no serum supplement (17,504,044; Gibco, USA), 20 ng/mL human recombinant EGF (236-EG; R&D Systems, USA), and 20 ng/mL human recombinant bFGF (233-FB; R&D Systems) using a 37°C, 5% CO2 environment. Accutase solution (A6964; Sigma–Aldrich, USA)-digested tumor spheres were used for passaging. All cell lines used in the experiments were free of mycoplasma contamination. Poly-L-ornithine solution (P4957; Sigma–Aldrich) and laminin (L4544; Sigma–Aldrich) were used to coat the plates to make the cells adhere to the wall for the experiment.

Human glioma cell lines U118MG, U87MG, LN229 and U251MG were purchased from the Chinese Academy of Sciences Cell Bank and cultured in DMEM medium (Gibco, USA) with 10% fetal bovine serum (FBS). The neural progenitor cell (NPC) and GBM patient-derived GSC cell lines and were kindly donated by Dr. Krishna P.L. Bhat (The University of Texas, M.D. Anderson Cancer Center, Houston, TX). GSC lines (GSC20, GSC267, GSC8–11, GSC11) have been used extensively in previous studies and the subtypes of GSCs have been clarified according to the Verhaak or Philips gene signatures, respectively. GSCs and NPC were cultured in DMEM/F12 (Gibco, USA) with 2% B-27 no serum supplement (Gibco, USA), 20 ng/mL human recombinant bFGF (R&D Systems) as well as 20 ng/mL human recombinant EGF (R&D Systems, USA). The GSC or NPC spheres were digested using accutase solution (Sigma-Aldrich, USA). All cell lines were cultured in a humid chamber at 37 °C and containing 5% carbon dioxide and 5% oxygen.