Figure 3.

NSC-sTRAIL in combination with Lan C induce apoptosis and inhibit neurosphere recovery in primary glioblastoma stem-like cells. (A): 10³ GBM8 cells (~20 small neurospheres) expressing Fluc and mCherry fluorescent protein were seeded in each lower well of 8 μm Transwell plate in NBM E/F20. 10³ NSC-Vluc or NSC-sTRAIL cells expressing Gluc and GFP were seeded in the upper well. After 12 hours (Day 1), GBM cells were treated with DMSO or 0.1 μM Lan C. The lower wells were imaged on day 3 for mCherry and GFP expression using fluorescence microscopy. At day 5, treatment was stopped and at day 7, sphere recovery was imaged. Neurospheres were then dissociated to single cells at day 10 and replated to monitor secondary neurosphere formation at day 15. Shown are representative overlay images of GBM8 cells (red), and NSC-Vluc or NSC-sTRAIL cells (green). The side length of a square = 500 μm. (B): 10³ GBM8 cells were treated with conditioned medium-containing sTRAIL, Lan C, or both. Three days later, cells were fixed, stained with PI, and analyzed by flow cytometry. Fractions of cells in G1 (green), S (brown), and G2/M (blue) phases are determined using Flowjo; the percentage of cells in G2/M phase is shown. (C): GBM8 cells were treated with Lan C, sTRAIL, or both. Seventy-two hours later, cells were stained with Cy5-conjugated Annexin-V and PI and analyzed using flow cytometry. Numbers in the right upper quadrants indicate the percentage of advanced apoptotic/necrotic cells. (D): 3 × 10³ GBM8 cells were cocultured with 300 NSC-Vluc or NSC-sTRAIL cells and/or treated with 0.1% DMSO or 0.1 μM Lan C. Activities of caspase 3/7 and 8 were measured 48 hours later. Caspase activities in cells cocultured with NSC-Vluc and treated with 0.1% DMSO were arbitrarily set at 100 (**, p <.01 vs. control). Data shown in (A)–(C) are representative of three independent experiments. Abbreviations: DMSO, dimethyl sulfoxide; NSC, neural stem cell; TRAIL, tumor necrosis factor-related apoptosis-inducing ligand.