LEGENDS TO SUPPLEMENTARY TABLES, FIGURES AND VIDEOS

Supplementary Table 1: List of different GBM and NSC lines generated with engineered viruses

Supplementary Figure 1: Generation of GBM-FmC and neural stem cell lines and GBM-NSC co-cultures: (a) Representative fluorescent images of glioma cells transduced with Fluc-mCherry. (b) Representative fluorescent images of NSCs transduced with LV-GFP or LV-TRAIL-IRES-GFP growing as neurosphere or as adherent cultures. (c) Representative images from GBM-NSC co-cultures (Top:Gli36-EvIII-FmC cells; Middle: U251-FmC cells; Bottom: LN229-FmC cells) at 120 hours of co-culturing with NSC-GFP or NSC-TRAIL.

Supplementary Figure 2: Generation of GBM-IMSRP cells and monitoring of mitochondrial outer membrane permeabilization in GBM-NSC co-cultures: (a) Representative fluorescent images of GBM cells transduced with MOMP reporter, IMS-RP. (b) Live-cell imaging of MOMP in Gli36-EVIII-IMSRP cells (red) co-cultured with NSC-TRAIL (green). White arrows point to the switch from punctate to diffuse fluorescence distribution representative of cells undergoing MOMP and apoptosis. (c) Live-cell imaging of MOMP in U251-IMSRP cells (red) co-cultured with NSC-TRAIL (green). White arrows point to the cells undergoing MOMP and blue arrows show NSCs migrating across GBM cells. Insets from time points between 6hr and 8hr show the close association between NSCs and GBM cells undergoing MOMP. (d) The mean time-to-MOMP in U251-IMSRP cells co-cultured with NSC-TRAIL and controls (NSC-GFP). N/D: not detected

Supplementary Figure 3: Comparison of systemically delivered TRAIL and NSC-delivered TRAIL in vivo: (a) Description of the experimental plan.Gli36-EvIII-FmC cells were implanted subcutaneously in nude mice and established tumors were treated either by systemically delivered TRAIL (i.p. injections of 10 mg/kg daily for 4 days) or intratumorally injected NSC-TRAIL. Fluc imaging was performed to monitor tumor volumes. (b) Top: Representative BLI images of Fluc signal on days 0 and 4 for each group. Bottom: Quantitation of Fluc activity (n=9 control; n=7 systemic TRAIL; n=2 NSC-TRAIL). Data presented as percent of day 0.

Supplementary Figure 4: Combined effect of MS-275 and TRAIL on GBM cell viability (a-c) CellTiterGlo assays showing thecombined effect of MS-275 and TRAIL treatment on GBM cells with MS-275 (5 M) and TRAIL (0-1000 ng/ml).

Supplementary Figure 5: Detection of AnnexinV positive cells in response to MS-275 and TRAIL treatment: (a-b) AnnexinV/PI analysis of apoptosis in Gli36-FmC cells (a) or LN229 cells (b) treated with MS-275 (2.5 M), TRAIL (100 ng/ml) or MS-275 and TRAIL for 16 hours. The percent AnnexinV positive cells are indicated on the histogram graphs on the right. The dot plots of PI and AnnexinV positivity are shown on the left.

Supplementary Figure 6: Generation of GBM-ECRP cell lines and monitoring of effector caspase activation in real time: (a) Representative fluorescent images of GBM cells transduced with ECRP. (b) Representative snapshots of the CFP and YFP channels and FRET trajectory of U251-ECRP cells undergoing ECRP cleavage in response to 20 ng/ml TRAIL. Data presented as CFP/YFP at each time point measured at 4min intervals for 17 hours (n=256 time points). (c) Plot showing the mean time of cell death (hr) of U251-ECRP cells with 20 (n=3), 100 (n=15) or 250 ng/ml (n=39) TRAIL. (d) Plot showing the percentage of U251-ECRP cell survival with TRAIL.

Supplementary Figure 7:Live-cell imaging with apoptosis reporters reveals the dynamics of GBM cell apoptosis induced by stem cell-delivered TRAIL. (a) Representative fluorescent images of NSCs transduced with LV-mCherry or LV-TRAIL-IRES-mCherry growing as neurosphere or as adherent cultures. (b) Representative snapshots of the U251-ECRP GBM cells (green and cyan) and NSC-mCh or NSC-TRAIL cells (red) in co-cultures over 24 hours. Arrows point to the cells undergoing apoptosis. (c) Representative FRET trajectory of U251-ECRP cells co-cultured with NSC-mCh or NSC-TRAIL. Data presented as CFP/YFP ratio of ECRP reporter at 9min intervals for 27 hours (n=175 time points). (d-e) Plot showing the percentage of U251-ECRP cell survival (d), and mean time of death of U251-ECRP cells (e) co-cultured with NSC-mCh or NSC-TRAIL. N/D: not detected

Supplementary Figure 8: The effect of MS-275 on NSCs, alone, or in GBM co-cultures: (a) Cell viability showing the effect of MS-275 (0-5 M) treatment for 24 hours on NSCs as measured by CellTiterGlo assay. (b) Plot showing the NSC viability as measured by Rluc activity of NSC-GFP-Rluc cells co-cultured with Gli36-EvIII-FmC in the presence of MS-275 (5 M) for 48 or 96 hours.

Supplementary Figure 9: The effect of MS-275 on the surface expression of DR4 and DR5 in TRAIL-resistant cells: (a-b) Histograms showing cell surface levels of DR4 (a) and DR5 (b) in LN229 cells treated with MS-275 (5 M) and measured using PE-conjugated DR4 or DR5 antibodies.

Supplementary Figure 10: The effect of MS-275 administration on the DR5 promoter activity in TRAIL-sensitive cells in vivo: (a) Imaging of DR5 activity in Gli36-EvIII-pDR5-Fl-CMV-RlD cells in vivo. Representative BLI images of Fluc and Rluc signal on before and after MS-275 (10 mg/kg i.p. daily) treatment are shown. (b) Quantitation of Fluc/Rluc activity shown in a (n=4 tumors/group). Data presented as Fluc/Rluc ratio as fold of day 0. (c) Imaging of DR5 activity in U251-pDR5-Fl-CMV-RlD cells in vivo as shown in A. (d) Quantitation of Fluc/Rluc activity of U251-pDR5 activity shown in c.

SUPPLEMENTARY VIDEO LEGENDS

Supplementary Video 1: Migration of NSCs towards GBM cells in co-cultures: NSCs (GFP, green) moving towards GBM cells (Gli36-EvIII, mCherry, red) were imaged by time-lapse microscopy. Frames were taken at 9-minute intervals. A total of 360 frames were taken during 54 hours and video is played at 10 fps.

Supplementary Video 2: Apoptosis of GBM cells induced by NSCs secreting TRAIL: NSCs secreting TRAIL (green) killing the surrounding TRAIL-sensitive GBM cells (Gli36-EvIII, mCherry, red) were imaged by time-lapse microscopy. Frames were taken at 9-minute intervals. A total of 360 frames were taken during 54 hours and video is played at 10 fps.

Supplementary Video 3:Apoptosis of a mid-sensitive GBM cell line in response to TRAIL: U251-ECRP (CFP, cyan) cells treated with TRAIL (200 ng/ml), were imaged by time-lapse microscopy. Frames were taken at 4-minute intervals. A total of 241 frames were taken during 16 hours and video is played at 10 fps.

Supplementary Video 4: Apoptosis of a mid-sensitive GBM cell line in response to TRAIL and MS-275: U251-ECRP (CFP, cyan) cells treated with MS-275 (1.25 M) and TRAIL (200 ng/ml) were imaged by time-lapse microscopy. Frames were taken at 4-minute intervals. A total of 241 frames were taken during 16 hours and video is played at 10 fps.

Supplementary Video 5:Apoptotic resistance of a GBM cell line in response to NSCs secreting TRAIL: LN229-ECRP (YFP, green) cells co-cultured with NSC-TRAIL (mCherry red) were imaged by time-lapse microscopy. Frames were taken at 9-minute intervals. A total of 175 frames were taken during 26.25 hours and video is played at 10 fps.

Supplementary Video 6: Sensitization of TRAIL-resistant GBM cell line to NSC-TRAIL by MS-275: LN229-ECRP (YFP, green) cells co-cultured with NSC-TRAIL (mCherry red) and treated with MS-275 (2.5 M) were imaged by time-lapse microscopy. Frames were taken at 9-minute intervals. A total of 175 frames were taken during 26.25 hours and video is played at 10 fps.

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