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1 Supporting Information Sasportas et al /pnas SI Methods Lentiviral Transduction. MSC and glioma cells were transduced with LVs at varying multiplicity of infection (moi) by incubating virions in a culture medium containing 4 g/ml protamine sulfate (Sigma) and cells were visualized for fluorescent protein expression by fluorescence microscopy. Following expansion in culture, both MSC and glioma cells were sorted by fluorescence activated cell sorting or flow cytometry (FACSAria Cell-Sorting System, BD Biosciences). Karyotyping and Genomic Profiling of MSC. Transduced and nontransduced MSC were plated to 80% confluency. Cultures were incubated at 37 C with 1 g/ml colcemid (Invitrogen) for 20 minutes and harvested, and metaphase spreads were prepared following routine protocol. Giemsa banded karyotypes were prepared using Applied Imaging CytoVision software (Genetix Limited). Genomic DNA was extracted from GFP-transduced and nontransduced MSC using routine protocol. acgh was performed using Agilent Human 105K oligonucleotide microarrays following the manufacturer s instructions. Genomic coordinates for this array are based on the NCBI build 36, March 2006, freeze of the assembled human genome (UCSC hg18), available through the UCSC Genome Browser. Microarray images were analyzed and data points were generated using the Feature Extraction software (version 9.1, Agilent) with linear normalization (protocol-v4 91). Data were subsequently imported into CGH Analytics software (version , Agilent). Detection of gains and losses were based on the z-score algorithm (threshold 2.5) and visual inspection of the log2 ratios. In general, log2 ratios 0.4 in at least 5 consecutive probes were considered as a reliable copy number alteration. Assaying Bystander Effect of MSC. To assess gap junction communication between glioma cells and MSC, Gli36-EGFRvIII cells were stained with the membrane-binding red fluorescent dye DI1 (5 L/mL cell suspension) (Molecular Probes), and MSC were stained with calcein-am (0.75 M) (Molecular Probes) as described (1). Cells were washed with phosphate-buffered saline, mixed at a ratio of 1:1, and seeded and incubated for 5 min and 3 hrs at 37 C. Calcein-AM transfer from MSC to glioma cells was assessed using a flow cytometer (BD Biosciences). Western Blot Analysis and ELISA. MSC and glioma cells were lysed and centrifuged at 30,000 g for 30 min at 4 C. Equal amounts of total cell protein (30 g) were denatured, separated by SDS/PAGE, and transferred to nitrocellulose membrane, blocked, and incubated for 1hatroom temperature with rabbit polyclonal antibodies to proteins: (i) Human Flt3L (Cell Science) and (ii) DR4 (Abcam); (iii) AKT (Cell Signaling); (iv) phospho-akt (Cell Signaling); (v) human PTEN (zcomsanta Cruz Biotechnology). Blots were developed using enhanced chemiluminescence reagents (Amersham). Membranes were then exposed to film for 30 s to 30 min. Eighty percent confluent 10-cm dishes of MSC expressing S-TRAIL or GFP were washed with PBS and incubated in 10 ml OptiMEM (Gibco) for 24 h. ELISA on MSC conditioned was done as described (2). Cell Viability and Caspase3/7 Assays. Transduced and nontransduced MSC were plated at 3x10 3 cells per well and cell viability at different time points was measured by quantitative luminescence assays using an ATP-dependent luminescent reagent (CellTiter-Glo, Promega). Assays were performed according to the manufacturer s instructions, and plates were read in a luminometer at 1 sec per plate. For in vitro S-TRAIL experiments, glioma cells were incubated with different concentrations of S-TRAIL ( ng/ml) obtained from conditioned medium quantified for S-TRAIL conc. or control conditioned medium and incubated for 24 hr, after which caspase-3/7 activity was determined with a caged, caspase 3/7-activatable DEVDaminoluciferin (Caspase-Glo 3/7, Promega). Cell viability was determined as described above. Immunocytochemistry. Gli36-EGFRvIII or GBM8 cells were incubated in a conditioned culture medium containing S-TRAIL (100 ng/ml). Eighteen hr later, cells were fixed, permeabilized and incubated with anti-cleaved caspase-3 antibody (Molecular Probes) and detected using goat anti-mouse Alexa dye 496 nm conjugated secondary antibody (Molecular Probes). GBM8 cells plated onto poly-l-ornithine/laminin-precoated coverslips were fixed, permeabilized, and incubated with rabbit anti-nestin (provided by Dr. Ron McKay, Bethesda, MD) or rabbit anti-gfap (Sigma) and detected using Cy3-conjugated goat anti-rabbit IgG (Jackson ImmunoResearch). To quantify CD133 stem cell marker expression on GBM8, cells were stained either with Phycoerythrin (PE)-conjugated anti-cd133/2 antibody (Miltenyi) or PE-conjugated isotype control (BD Biosciences), subjected to a flow cytometer (FACSCalibur, BD) and analyzed using FlowJo (Tree Star). In Vitro Bioluminescence Assays. To determine the correlation between the number of MSC-GFP-Fluc or Gli36-EGFRvIII-FD and the bioluminescence signal, cells were seeded in different concentrations and Fluc activity was measured as described. Each experiment was performed in triplicate. For dual-luciferase imaging of MSC progression and Gluc-S-TRAIL secretion, MSC were cotransduced with LV-Gluc-S-TRAIL and LV-GFP-Fluc at moi 4. Transduced MSC were washed with PBS and incubated in OPTIMEM. Eighteen hr later, different concentrations of transduced cells (ranging from to ) and the conditioned medium from cultured MSC was collected and both MSC and medium were imaged for Fluc and Gluc activity, respectively. Gaussia luciferase (Gluc) activity by incubating the medium with 1 g/ml coelenterazine and imaged as described (3). Intracranial Cell Implantation and Imaging in Vivo. To follow survival of MSC in the presence and absence of glioma cells, MSC-GFP- Fluc and Gli36-EGFRvIII-FD were harvested at 80% confluency and a mix of MSC-GFP-Fluc ( ) Gli36-EGFRvIII-FD ( ) or MSC-GFP-Fluc ( ) alone were implanted stereotactically (from bregma, AP: -2 mm, ML: 1.5 mm V (from dura): 2 mm) (n 4 in each case). Mice were injected intraperitoneally with 4.5 mg/mouse of D-luciferin on days 2, 6, 10, and 14 and imaged as described. To follow the effect of MSC on glioma growth, mice were implanted with either Gli36- EGFRvIII-FD ( ) or a mix of Gli36-EGFRvIII-FD ( ) and MSC-GFP ( ) stereotactically (from bregma, AP: -2 mm, ML: 1.5 mm V (from dura): 2 mm) (n 6 in each case). Mice were injected intraperitoneally with 2.0 mg/mouse of D-luciferin on days 2, 6, 12, and 16 and imaged as described. On day 16, mice were killed, and tumors were resected for histopathological analysis. To follow the effect of MSC secreted S-TRAIL on malignant glioma cell progression MSC-S-TRAIL or MSC-GFP were harvested at 80% confluency and a mix of 1of5

2 glioma cells ( ) and transduced MSC ( )(n 4 in each group) was implanted stereotactically (from bregma, AP: -2 mm, ML: 1.5 mm V (from dura): 2 mm) (n 4 in each case). Mice were injected intraperitoneally with D-luciferin and imaged as described above. Mice were killed, and brains were sectioned for histopathological analysis. To follow the effect of MSC secreting S-TRAIL on growth of established gliomas in the brain, CD133 ve primary glioma cells, GBM8 expressing GFP- Fluc were dissociated into single cell suspension and implanted into right frontal lobe of SCID mice brains ( /mouse, in 4 L PBS, n 10) (from bregma, AP: 0 mm, ML: 2 mm V (from dura): 2.5 mm). MSC expressing S-TRAIL or DsRed2 only were harvested and implanted stereotactically ( in 5 L PBS) into established tumors on days 7 and 14 after implantation. Mice were imaged for Fluc activity on days 7, 21, and 35 of implantation. They were then followed for survival and kiled when neurological symptoms became apparent. All in vivo procedures were approved by the Subcommittee on Research Animal Care at Massachusetts General Hospital. In Vivo Dual Bioluminescence Imaging. SCID mice (SCID; 3 weeks of age; Charles River Laboratories) were implanted with a mix of MSC-S-TRAIL-Gluc /GFP-Fluc and Gli36- EGFR-vIII subcutaneously (n 4). For dual-luciferase imaging 6 and 13 days after implantation, mice were injected with 100 g of coelenterazine/mouse via tail vein and imaged for Gluc activity as described. Eighteen hrs later, when there was no residual coelenterazine/gluc activity, mice were injected with 3 mg D-luciferin/mouse intraperitoneally and imaged for Fluc activity after 5 min as described above. Post processing and visualization were performed as described previously (2). Intravital Microscopy in Vivo. For intravital microscopy, mice were implanted stereotactically (from bregma, AP: -2 mm, ML: 2.5 mm, V (from dura): 2 mm) (n 4 in each case) with Gli36- EGFRvIII-GFP-Fluc ( ). Four days later, a small circular portion of the cortical surface was exposed and tdtomato expressing MSC ( ) were implanted stereotactically (from bregma, AP: -2 mm, ML: 1.5 mm, V (from dura): 2 mm) (n 3 in each case). Intravital microscopy was performed as described (4). Tissue Processing and Immunohistochemistry. Immediately following the imaging session, mice were perfused and brains were removed and sectioned. Brain sections on slides were washed in PBS and mounted for microscopy to be visualized for fluorescence on a confocal microscope (BioRad). For Nestin, GFAP, MAP-2, Ki67 and cleaved-caspase-3 staining, sections were incubated for 1 hr in a blocking solution (0.3% BSA, 8% goat serum and 0.3% Triton-X100) at room temperature (RT), followed by incubation at 4 C overnight with following primary antibodies diluted in blocking solution: (i) anti-human nestin (clone 10C2; Chemicon), (ii) anti-human GFAP (Chemicon), (iii) anti-ki67 (clone MIB-1; Dako), (iv) anti-map-2 (Chemicon), and (v) anti-cleaved-caspase-3 (Cell Signaling). Sections were washed three times with PBS, incubated in appropriate secondary antibody, and visualized using confocal microscope (LSM Pascal, Zeiss). Brain sections of GBM8 gliomas were incubated for 30 min in 10% goat serum at RT, followed by incubation with either anti-human nuclei (Chemicon), anticleaved-caspase-3 () or anti-ki67 at 4 C overnight. Sections were washed 3 times with PBS, incubated in Alexa Fluor 649 goat anti-mouse or anti-rabbit secondary antibody (Invitrogen), and visualized as described above. The number of Ki67 and anticleaved caspase-3 positive cells was calculated by counting the positive cells in randomly selected fields of view under the microscope. 1. Czyz J, Irmer U, Schulz G, Mindermann A Hulser DF (2000) Gap-junctional coupling measured by flow cytometry. Exp Cell Res 255: Shah K, et al. (2005) Glioma therapy and real-time imaging of neural precursor cell migration and tumor regression. Ann Neurol 57: Shah K, Tang Y, Breakefield X, Weissleder R (2003) Real-time imaging of TRAIL-induced apoptosis of glioma tumors in vivo. Oncogene 22: Shah K, et al. (2008) Bimodal viral vectors and in vivo imaging reveal the fate of human neural stem cells in experimental glioma model. J Neurosci 28: of5

3 Fig. S1. Characteristics of modified MSC and glioma cells. (A) Growth of transduced and non-transduced MSC in culture compared over 16 days. (B and C) In vitro correlation between Gli36-EGFRvIII-FD cells (B) or MSC-GFP-Fluc cells and bioluminescent signal intensity (C). (D) In vivo correlation of number of MSC-GFP-Fluc and Fluc activity. 3of5

4 Fig. S2. MSC are resistant to TRAIL mediated apoptosis. (A) Western blot analysis on cultured human Gli36-EGFRvlll and MSC showing the absence of death receptor (DR)4 in MSC. (B) Cell viability assay on MSC and glioma cells incubated with different concentrations of S-TRAIL for 24 hr. (C D) Photomicrograph showing GFP-positive (C) and FACS-sorted (D) MSC-S-TRAIL. (E) ELISA showing TRAIL protein concentration/ml in the conditioned medium of S-TRAIL and GFP expressing MSC. (F G) Photomicrographs of Gli36-EGFRvlll glioma cells incubated with conditioned medium from MSC-S-TRAIL (F) and MSC-GFP (G) for 18 hr and stained with anti-cleaved caspase-3 antibody. (H) Western blot analysis showing the expression of caspase-8 and PARP cleavage product in untreated and S-TRAIL-treated glioma cells. (I J) Cell viability and activated caspase-3/7 expression in human glioma cells incubated with different concentrations of S-TRAIL from MSC-S-TRAIL conditioned medium (I) and MSC-GFP conditioned medium (J). Original magnification 20 (C); 10 (F, G). 4of5

5 Fig. S3. Characteristics of modified GBM-8 cells. GBM8 cells were transduced with LV-GFP-Fluc. (A) FACS analysis on GBM8-GFP-Fluc cells stained with Phycoerythrin (PE)-conjugated anti-cd133/2 antibody and isotype control (B) In vitro correlation between GBM8-GFP-Fluc and bioluminescent signal intensity in culture. 5of5