Isolation and Characterization of Glioblastoma Stem Cells

GSCs were isolated from patient-derived surgical specimens at the University of Texas MD Anderson Cancer Center, as previously described,^{27 (link)} and approved by the institutional review board (protocol #LAB04-0001). The GSCs were grown and maintained in suspension culture in Dulbecco’s modified Eagle medium (DMEM, Corning, NY, USA), supplemented with 2% B-27 (Thermo Fisher Scientific, Waltham, MA, USA), epidermal growth factor (EGF), basic fibroblast growth factor (FGF), and antibiotics at 37°C in a 5% CO₂ atmosphere, as described previously.^{28 (link)} Short tandem repeats using the Applied Biosystems AmpFISTR identifier kit (Foster City, CA, USA) were used to authenticate cells. All the GSCs lines used in this study are isocitrate dehydrogenase wild-type (IDHwt) except GSC5-22. The EGFR amplification in GSCs lines was characterized as described in the previous study.^{29 (link)} All MEK inhibitors (GDC-0623, MEK162, RO5126766 [RO5], and trametinib) were purchased from (Selleckchem, Houston, TX, USA). All cell lines tested negative for mycoplasma contamination using the MycoAlert Detection Kit (Lonza, Houston, TX, USA).