Human astrocyte

The human glioblastoma cell lines (LN229, U87, A172, U118, and U251) were purchased from the American Type Culture Collection (ATCC, Manassas, USA). All of these cells were cultured in DMEM/F12 medium (Invitrogen, Waltham, USA) supplemented with 2 nM L-Glutamine, 10% fetal bovine serum (Gibco, Waltham, USA), 100 U/mL penicillin and 0.1 mg/mL streptomycin. 293FT cell line was purchased from Thermo Fisher Scientific (New York, USA), was cultured in DMEM(Invitrogen, Waltham, USA) supplemented with 10% fetal bovine serum, 1% P/S, 0.1 nM Non-Essential Amino Acids, 0.5 mg/mL G418, 4mL L-Glutamine, and 1 mM MEM sodium pyruvate. Human astrocytes (HA) and growth medium were from ScienCell Research Laboratories (Carlsbad, CA). All of these cells were maintained in 5% CO₂ at 37 °C.

Four GBM cell lines were employed in several combinations in order to simulate cell population heterogeneity as one might encounter in GBM tumors. GBM cell lines U-373 MG (ATCC^® HTB-16^TM) U-87 MG (ATCC^® HTB-14^TM), U-87 EGFRvIII cell line (gifted by Dr. Webster Cavenee from Ludwig Cancer Research Institute, San Diego) and A172 (ATCC^® CRL-1620^TM) (obtained from ATCC, Manassas, VA) were employed. All the cancer cell lines were cultured in Dulbecco’s Modified Eagle Medium (DMEM) - high glucose with 10% fetal bovine serum (FBS), 1% L-glutamine, and 1% penicillin/streptomycin in a tissue culture incubator at 37°C with 5% CO₂. In addition, human astrocytes (Sciencell Research Laboratories, Inc., Carlsbad, CA) were cultured in Astrocyte Medium containing 2% FBS, 1% astrocyte growth supplement, and 1% penicillin/streptomycin at 37°C with 5% CO₂. Cells were recovered from tissue culture plastic for subsequent studies using Trypsin/EDTA (Thermo Fisher, Waltham, MA).

Human cervical carcinoma cell (HeLa, catalog no. CRM-CCL-2^TM) and mouse mononuclear macrophages (J774A.1, catalog no. TIB-67^TM) were obtained from the American Type Culture Collection (ATCC). The human brain endothelial cell (hCMEC/D3, catalog no. 337728) was purchased from BeNa Culture Collection. The luciferase-transfected glioblastoma cell lines (U251-luc, catalog no. CBP30207L and GL261-luc, catalog no. iCell-0059a) were maintained in our laboratory (Shenzhen Second People’s Hospital, Shenzhen, China). Human astrocytes (catalog no. 1800) and brain vascular pericytes (catalog no. 1200) were purchased from Sciencell. The cells have been confirmed without mycoplasma contamination by mycoplasma detection kit (Beijing Solarbio Science & Technology Co., Ltd., CA1080). All types of cells were cultured in DMEM and supplemented with 10% FBS and 1% (v/v) penicillin-streptomycin. The normoxic cells were incubated in a humidified atmosphere with 5% CO₂ and the hypoxia cells were maintained in an incubator with 1% O₂, 5% CO₂, and 94% N₂. Red blood cells (RBC) were obtained from the peripheral blood of mice.

The GBM cell lines U87, A172, and H4 were obtained from American Type Culture Collection (ATCC). Cell lines SF295, SF268, SF539, SNB75, SNB19, and U251 were provided by National Cancer Institute Division of Cancer Treatment and Diagnosis (NCI, DCTD). Cell lines were cultured and maintained in Dulbecco’s Modified Eagle’s medium (DMEM, Corning Inc., Corning, NY, USA) supplemented with 10% heat-inactivated fetal bovine serum (Hyclone FBS, Thermo Fisher Scientific, Waltham, MA, USA), 1% Penicillin-Streptomycin (Thermo Fisher Scientific), and 0.1% Plasmocin Prophylactic (InvivoGen, San Diego, CA, USA). All cells were grown at 37 °C in a humidified incubator with 5% CO₂. Human astrocytes were purchased from ScienCell Research Laboratories (Carlsbad, CA, USA) and cultivated in accordance with the manufacturer’s protocol, using Astrocyte Medium (ScienCell) on poly-L-lysine-coated cell culture flasks and dishes. Cells were used for only two subcultures to prevent the analysis of senescent cells.