Cellstar

3D-MCTS were established by the hanging-drop method as previously described [22 (link)]. Briefly, 5,000 GL261 cells (National Cancer Institute, Frederick, MD) were suspended in 20 μL of spheroid media, prepared as described in the Online Resource file, on a lid of a petri-dish. The 3D-MCTS were then grown upside down by covering a phosphate buffered saline (PBS)-filled/moisturized petri-dish with the cell-seeded lid for 2 days. We then transferred the 3D-MCTS onto a sterile U-bottom 96-well plate (CELLSTAR®; Sigma-Aldrich, St. Louis, MO) and added RPMI 1640 medium (ThermoFisher Scientific, Waltham, MA). The spheroids were then incubated at 37 °C for 48 hours to reach ~500 μm in diameters prior to use. Subsequently, 3D-MCTS were treated with various BPN formulations carrying plasmids encoding a green fluorescent reporter protein, ZsGreen, at 1 μg DNA/spheroid. After 48 hours of incubation, the 3D-MCTS were transferred onto poly-D-lysine coated 35 mm glass bottom dishes (MatTek Corp., Ashland, MA). We then captured Z-stack fluorescence images at a depth of ~200 μm using an LSM 710 confocal microscope (Carl Zeiss; Hertfordshire, UK) under 10X magnification and conducted a quantitative analysis of spheroid area-normalized mean fluorescence intensity using an ImageJ software (NIH, Bethesda, MD).

3D tumor spheroids were established by the hanging-drop method in a house-made spheroid media. The spheroid media was prepared by dissolving autoclaved methylcellulose (Sigma-Aldrich) at 2.4% (w/v) in preheated (60 °C), serum-free DMEM followed by dilution with an equivolume of DMEM supplemented with 10% FBS and an overnight stirring at 4 °C. The solution was then centrifuged at 5,000 xg for 2 hours at 4 °C to precipitate undissolved methylcellulose and the supernatant stored at 4 °C until use. To form each spheroid, we cultured 5,000 cells, either F98 glioma cells or F98 cells engineered to constitutively express a red fluorescent protein, mKate (F98-mKate; provided by Dr. Surojit Sur; Johns Hopkins University), in 20 μL spheroid media on the lid of a petri-dish. Spheroids were then grown upside down by covering a PBS-filled/moisturized petri-dish with the cell-seeded lid for 2 days. Finally, spheroids were transferred onto a sterile U-bottom 96-well plate (CELLSTAR®; Sigma-Aldrich) along with DMEM and incubated at 37⁰ C for 24 hours prior to their use for subsequent experiments.