Supplementary Methods
Generation of TSP-1 lentiviral vectors: For Ss-aaTSP-1 the cDNA sequence encoding amino
acid (a.a.) 412–499 of Thrombospondin-1 (TSP-1) (aaTSP-1) were amplified by PCR using
primers in which a His6-tag and an EcoRI site were introduced in the forward primer (5’ CG
GAATTTC ATG AAG AGA TTT AAA CAG 3’) and a Flag-tag and an XhoI site were introduced
in the reverse primer (5’CCG CTC GAG TCA ACG ACT ACGTTT CT 3’). The resulting 0.26 kb
EcoRI/XhoI fragment was ligated in frame with a 63 bp NheI/EcoRI cDNA fragment encoding
the human Flt3L secretion signaling sequence (Ss) (a.a. 1–21) or NheI/EcoRI digested 0.7 kb
cDNA fragment encoding the extracellular domain of human Flt-3L (a.a. 1–81) (HF) into the
NheI/XhoI digested pLV CSC-IG vector thus resulting in the LV Ss-His-aaTSP-1-Fg and LV-HFHis-aaTSP-1Fg construct respectively. To construct the aaTSP-1 vector without the His and the
FLAG tag, a 261 bp sequence of aaTSP-1 was amplified by PCR with a forward primer
introducing an EcoRI site and a reverse primer introducing an XhoI site. The resulting 0.26 kb
fragment was ligated in frame with NheI/EcoRI digested 0.7 kb cDNA fragment encoding the
extracellular domain of human Flt-3L (a.a. 1–81) or human Flt3L secretion signal sequence (a.a.
1–81) and ligated into the NheI/XhoI digested pLV-CSC-IG vector, resulting in the LV-HFaaTSP-1 and LV-Ss-aaTSP-1 construct. To construct the Ss-aaTSP-1-Gluc vector, the
Nhe1/EcorV aaTSP-1 fragment (generated by PCR) and EcorV/XhoI Gluc fragment (0.6 kb)
(obtained from LV-Gluc vector), were ligated into the NheI/XhoI digested pLV-CSC-IG vector
resulting finally in the LV-Ss-aaTSP1-Gluc construct. Bi-modal lentiviral vectors: LV-DsRed2,
LV-GFP-Fluc, LV-GFP-Rluc and LV-Fluc-DsRed2, generated as described previously (Corsten
& Shah, 2008), were also used to engineer different hNSC control lines and glioma tumor lines.
All lentiviral constructs were packaged as lentiviral (LV) vectors in 293T/17 cells using a helper
virus-free packaging system as described previously (Kock et al., 2007).
Endothelial Cell culturing: Human microvascular endothelial cells (HMVECS) and human
brain microvascular endothelial cells (HBMVECs) (Cambrex, East Rutherford, NJ) were grown
in EGM-2-MV medium (Cambrex) supplemented with hEGF, Hydrocortisone, GA-1000
(Gentamicin, Amphotericin-B), 5% FBS, VEGF, hFGF-B (w/ heparin), R3 -IGF-1 and ascorbic
Acid (Clonetics). Both endothelial cell types were passaged before they reached confluency (7080%) and not grown beyond seventh passage.
Western blot analysis: Cells were lysed 24 h after transduction in a buffer containing 50 mM
Tris (pH 8.0), 150 mM NaCl, and 1% NP40, supplemented with proteinase inhibitor (CompleteMini cocktail; Boehringer-Mannheim, Indianapolis, IN) and centrifuged at 40,000 g for 30 min at
4°C. Medium was prepared by adding 1/10th of normal sample buffer to medium sample and
incubating at 95°C to denature proteins. Equal amounts of total cell protein (30 g) were
denatured, separated by SDS-PAGE, and transferred to nitrocellulose membrane, blocked and
incubated for 1 h at room temperature with rabbit polyclonal antibodies to proteins: (a) Human
Flt3L (Cell Science, MA) and (b) biotinylated monoclonal anti Flag (Sigma, St Louis, MO, USA),
washed and further incubated for 1 h with goat anti-rabbit IgG peroxidase-conjugated secondary
antibody (Santa Cruz Biotechnology, Santa Cruz, CA) in TBS-T. Blots were developed using
enhanced chemiluminescence reagents (Amersham, Piscataway, NJ). Membranes were then
exposed to film for 30 s to 30 min.
Dot blot analysis and ELISA: Eighty percent confluent 10-cm dishes of transduced hNSC were
washed with PBS and incubated in 10 mL OptiMEM (Gibco) for 24 h. Different amounts of
medium were spotted on to nitrocellulose filter and dot blot was performed as described
previously (Shulga-Morskoy & Rich, 2005) using biotinylated monoclonal anti FLAG antibody.
Blots were developed as discussed above and subjected to quantification as described
previously (Arwert et al., 2007). For ELISA, Ni++ coated wells (Sigma, St Louis, MO, USA) were
incubated with His6 and FLAG tagged Ss-aaTSP-1 and HF-aaTSP-1 hNSC conditioned medium
for 2 hours at room temperature. The plates were washed with PBS and incubated for 1 hour
with biotinylated anti-FLAG antibody, with streptavidin-HRP for 30 minutes and developed with
Chromogen (Biosource, Camarillo, CA, USA). OD450 was determined and compared to a known
protein standard.
Pharmacokinetics: dual-bioluminescence assay in culture: hNSC were cultured as
described above. For dual-luciferase imaging of neural stem cell progression and aaTSP-1-Gluc
secretion, hNSC were co-transduced with LV-aaTSP-1-Gluc and LV-GFP-Fluc (Corsten &
Shah, 2008) Twenty four hrs after incubation of different concentrations of transduced cells
(ranging from 1.5x104 to 1.3x105), the culture medium of the cells was collected and cells and
medium were imaged for Fluc and Gluc activity respectively. Dual bioluminescence assays on
transduced
cells culture medium containing secreted aaTSP-1-Gluc was performed as
described previously (Sasportas et al., 2009)
Intravital fluorescence microscopy: A prototype multichannel upright laser scanning
fluorescence microscope (Olympus IV100) with a custom-designed stage and scanning unit for
intravital observations was used for intravital microscopy (Alencar et al., 2005). The stage was
equipped with a heating plate regulated by a thermostat (37°C) and gas anesthesia. Mice were
anesthetized as described above and images were acquired with Fluoview imaging software
(Olympus). Lasers used for excitation included a 488-nm argon laser, a 561-nm solid-state
yellow laser, and a 633-nm HeNe-R laser. Emission signal was filtered using 505-525 nm, 586615 nm and 660-730 nm band-pass filters, respectively.
Tissue Processing and Immunohistochemistry: The subcutaneous tumors and brain was
removed and incubated in cold 4%PFA for 4-6 hours. Subsequently, tumors and brain were
placed in 15% sucrose for 8-12 hours and 30% sucrose
overnight before it was embedded in
OTC prior to cryo-sectioning. Sections used for immunohistochemistry (IHC) were cut at 7
microns and deposited on Poly- L- Lysine coated slides. CD31 IHC was performed using goat
polyclonal IgG at 1:100 dilution (M-20; Santa Cruz Biotechnology Inc, Santa Cruz, CA). Slides
were counterstained using hematoxylin and visualized by confocal microscopy. For Nestin,
GFAP, MAP-2 and Ki67 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: 1) anti-human
nestin (clone 10C2; Chemicon), 2) anti-human GFAP (Chemicon), 3) anti-Ki67 (clone MIB-1;
DAKO) and anti-MAP-2 (Chemicon). Sections were washed three times with PBS, incubated in
appropriate secondary antibody. Photomicrographs of both IHC and H&E slides were taken
using the Nikon E400 light microscope (Nikon Instruments Inc, Melville, NY) attached to a SPOT
CCD digital camera (Diagnostics Instruments, Inc., Sterling Heights, MI) and also by confocal
microscope (LSM Pascal, Zeiss) using rhodamine, cy3.3 and cy5.5 channels.
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