Cell culture

Patient samples used for this research were provided by the Biobank Core Facility at St. Joseph’s Hospital and Medical Center and Barrow Neurological Institute (BNI), Phoenix, Arizona. The samples were de-identified and conformed to the Biobank Institutional Review Board (IRB) protocol. Patient-derived cell line GB3 was established from resected primary GBM tumor tissue at BNI. Briefly, tumor tissue was processed using the Gentle MACS Dissociator and Tumor Tissue Dissociation kit (Miltenyi Biotec Inc.). Cells were expanded as neurospheres in tissue culture dishes coated with poly-(2-hydroxyethyl methacrylate) (Sigma-Aldrich) or grown adherent on laminin (Fisher Science), in neural stem cell (NSC) medium consisting of DMEM and F12-Glutamax supplemented with B27 and N2 (Fisher Science), in the presence of 20 ng ml⁻¹ EGF and 20 ng ml⁻¹ FGF2 (EMD Millipore). To generate GB3-RFP cell line, GB3 cells were transduced with pre-made lentiviral particles (Amsbio) expressing RFP-Luc (GB3-RFP) and were selected using blasticidin (2 μg ml⁻¹). Human umbilical vein endothelial cells (HUVECs) were obtained from Lonza and cultured in EGM-2 (Endothelial Growth Medium, Lonza) and passaged at 70–80%.