Cell culture:

GL261 cells were obtained from the National Cancer Institute (NCI) and were grown at 37°C in RPMI 1640 medium containing 10% heat-inactivated fetal bovine serum, 10 mM HEPES, 100 U/ml penicillin, and 100 ng/ml streptomycin (all from Corning Inc. New York, NY, USA). U-87MG cells were from American Tissue Culture Collection and were grown in DMEM medium with 4.5g/l glucose and L-glutamine without sodium pyruvate. The medium was supplemented with 10% heat-inactivated fetal bovine serum, 100 U/ml penicillin, and 100 ng/ml streptomycin.

To generate spheroids from glioma cells, adherent U-87MG cells were enzymatically lifted with trypsin-EDTA, pelleted by centrifugation, counted, and resuspended in NeuroCult^™ NS-A Basal Medium with human recombinant epidermal growth factor (20ng/ml), basal fibroblast growth factor (10 ng/ml), heparin solution (2 ng/ml) (all from StemCell Technologies, Vancouver, Canada), and StemPro^™ neural supplement (1% v/v, Thermo Fischer) to a concentration of 2000 cells/μl. The cell suspension was mixed with cold Matrigel^® (Corning, Tewksbury, MA) at a 1:4 ratio. The Matrigel-cell suspension was added to the indentations created in the parafilm molds (20 μl) and polymerized for one hour at 37°C. After one hour, the spheroids were manually dislodged from the parafilm molds into DMEM and cultured for 72 hours at standard conditions (37°C, 5% CO2, 95% humidity). After 72 hours, the spheroids were placed on an orbital shaker within an incubator, where they remained for 11 additional days. On day 14, the Matrigel was removed using Cell Recovery Solution (Corning) per the manufacturer’s protocol. Briefly, spheroids were washed with cold PBS and gently resuspended in 1 ml of cold Cell Recovery Solution for 5 min. After 5 min, spheroids were washed with cold PBS three additional times and placed back into DMEM for seven days. After 21 days of culture, spheroids were ready for experimental use.

Tumor organoids were generated from GBM6 and GL261 orthotopic tumors as previously described [56]. Briefly, GBM6 and GL261 were implanted intracranially in NSG and C57BL/6 mice, respectively. Following tumor engraftment and development, mice were sacrificed, and tumors were dissected from brains. Tumors were subsequently diced into approximately 0.5 mm³ fragments with a sterile blade, washed twice with PBS, and cultured in DMEM supplemented with 10% FBS on an orbital shaker within a tissue culture incubator. After 72 hours of culture, tumor explants were ready for experimental use.