EGFRvIII-expressing, control MSCV, and PTEN knockdown (PTENi) astrocytes were generated by infection of SV40 large T-immortalized Stat3loxP/loxP
astrocytes with the EGFRvIII or control MSCV retrovirus or with a pSUPER-Puro retrovirus encoding shRNAs targeting PTEN as described (de la Iglesia et al., 2008a
). The Stat3
gene flanked by loxP sites was excised in vitro using adenovirus encoding the recombinase Cre (University of Iowa) to generate Stat3−/−
astrocytes. Primary astrocytes were cultured from postnatal day 2 (P2) B6 mice as described (Di Giorgio et al., 2007
; Nagai et al., 2007
Virus production and infection
Lentiviruses were based on the pLKO.1 vector carrying the blasticidin resistance gene and obtained from the RNAi Consortium at the Broad Institute. Cloning of recombinant lentiviruses coding for short hairpin RNAs directed against iNOS was performed by annealing and insertion of complementary oligonucleotides into stuffed-pLKO.1 plasmid using AgeI and EcoRI sites. Oligonucleotides were generated as follows: iNOSi1, forward: 5′-C CGG TCC ATG CAA AGA ACG TGT TTA
TAA ACA CGT TCT TTG CAT GGA TTTTTG -3′; iNOSi1, reverse: 5′-AATTCAAAAA TCC ATG CAA AGA ACG TGT TTA
TAA ACA CGT TCT TTG CAT GGA-3′; iNOSi2, forward: 5′-C CGG GAG CAG GTG GAA GAC TAT TTC
GAA ATA GTC TTC CAC CTG CTC TTTTTG -3′; iNOSi2, reverse: 5′-AATTCAAAAA GAG CAG GTG GAA GAC TAT TTC
GAA ATA GTC TTC CAC CTG CTC-3′. Hairpin structures containing the stem sequences and loops are indicated by underlining and in bold, respectively. Correct insertion of the desired oligonucleotides was confirmed by sequencing. pLKO.1-TRC026 containing the null-T sequence was used as the control.
Recombinant lentiviruses were made by transfecting human embryonic kidney 293T (HEK293T) cells with pCMV-dR8.91 (containing gag, pol, rev), pMD2.G (vesicular stomatitis virus glycoprotein), and the transfer plasmid (pLKO.1-TRC026, pLKO.1-iNOSi1 or pLKO.1-iNOSi2) via FuGENE 6 (Roche). Viral supernatants were collected and flash frozen.
EGFRvIII;Stat3loxP/loxP and MSCV;Stat3loxP/loxP astrocytes were infected in 3 cm dishes by incubating for 24 hrs with supernatant containing lentivirus. Cells were expanded into 10 cm plates and selected with blasticidin at a concentration of 5 μg/mL. Selection of uninfected astrocytes under the same conditions confirmed that blasticidin completely killed all cells at this concentration (data not shown). Blasticidin resistant control and iNOS knockdown astrocytes were expanded and frozen into cell stocks for use in biochemical and functional assays.
RNA was prepared from cells using Trizol extraction and purification. For gel-based RT-PCR analyses, purified RNA was quantified and reverse transcribed and amplified using the appropriate primers and SuperScript One-Step RT-PCR with Platinum Taq system (Invitrogen) according to manufacturer’s protocol. Amplified cDNA products were resolved using agarose gel electrophoresis and visualized using ethidium bromide. Primer sequences were as follows: iNOS, forward: 5′-GTG GTG ACA AGC ACA TTT GG-3′; iNOS, reverse: 5′-GGC TGG ACT TTT CAC TCT GC-3′; Hyaluronan Synthase, forward: 5′-AGT ATA CCT CGC GCT CCA GA-3′; Hyaluronan Synthase, reverse: AGC AGC AGT AGA GCC CAG AG-3′; Bcl-XL, forward: 5′-TGG TGG TCG ACT TTC TCT CC-3′; Bcl-XL, reverse: 5′-TGC AAT CCG ACT CAC CAA TA-3′; MMP3, forward: 5′-CAG GTG TGG TGT TCC TGA TG-3′; MMP3, reverse: 5′-GCC TTG GCT GAG TGG TAG AG-3′; Pim1, forward: 5′-CAT GGA AGT GGT CCT GTT GA-3′; Pim1, reverse: GAC CCG AAG TCG ATG AGT TT-3′; VEGF, forward: 5′-CTA CAG ATG TGG GGG TTG CT-3′; VEGF, reverse: 5′-CAC AGC GGC ATA CTT CTT CA-3′; TERT, forward: 5′-AGG GTA AGC TGG TGG AGG TT-3′; TERT, reverse: 5′-TGC TGA GGA AGG TTT TTG CT-3′; Survivin, forward: 5′-ATC GCC ACC TTC AAG AAC TG-3′; Survivin, reverse: 5′-CAG GGG AGT GCT TTC TAT GC-3′; Myc, forward: 5′-GCC CAG TGA GGA TAT CTG GA-3′; Myc, reverse: 5′-GAA TCG GAC GAG GTA CAG GA-3′; GAPDH, forward: 5′-ACC ACA GTC CAT GCC ATC AC-3′; GAPDH, reverse: 5′-TCC ACC ACC CTG TTG CTG TA-3′.
For quantitative RT-PCR analyses, cDNA was prepared using oligo(dT) primers and SuperScript III First-Strand cDNA synthesis system (Invitrogen) according to manufacturer’s protocol. Quantitative PCR was performed using the Lightcycler 480 SYBR Green 1 Master Kit on a Lightcycler 480 thermocycler (Roche). For all quantitative PCR experiments, gene expression was normalized to GAPDH levels. Specific amplification of target genes was confirmed by agarose gel electrophoresis and calculation of melting temperature of the amplified product. Primer sequences were as follows: STAT3, forward: 5′-ACC CTT AGG GAG CAG AGA TGT G-3′; STAT3, reverse: 5′-GTT CTT GGG GTT ATT GGT CAG C-3′; iNOS, forward: 5′-GGA TTG TCC TAC ACC ACA CCA A-3′; iNOS, reverse: 5′-ATC TCT GCC TAT CCG TCT CGT C-3′; GAPDH, forward: 5′-TGC TGG TGC TGA GTA TGT CG-3′; GAPDH, reverse: 5′-GCA TGT CAG ATC CAC AAC GG-3′.
Rabbit iNOS antibody for immunocytochemical analyses and mouse actin antibody (Santa Cruz Biotechnology), rabbit iNOS antibody for immunoblotting analyses (BD Biosciences), polyclonal rabbit Ki67 antibody (Vector Labs), mouse EGFR, rabbit STAT3, and rabbit CC3 antibodies (Cell Signaling Technology), mouse phospho-Tyr (clone 4G10) antibody (Millipore), mouse α-tubulin antibody (Covance), and mouse Flag antibody (Sigma) were purchased. Mouse GFP antibody was obtained through the NeuroMab antibody consortium in association with Antibodies Incorporated.
For visualization of iNOS and GFP proteins, astrocytes were fixed in methanol for ten minutes at −20°C and subjected to microwaving and immunocytochemical analyses with the indicated antibodies. For visualization of Ki67 and CC3, astrocytes were fixed in 4% paraformaldehyde for 20 minutes at room temperature and processed in a similar fashion. For BrdU analyses, astrocytes were treated with incubated with BrdU for two hours and then processed as per manufacturer’s protocol with BrdU Labeling and Detection Kit I (Roche). Primary mouse astrocytes were incubated with BrdU for four hours before fixing and staining.
The upstream 2 kb and 0.3 kb regions of the mouse iNOS transcriptional start site were cloned into the pGL2basic (Promega) vector to generate the iNOS-luciferase reporter genes (2 kb iNOS-pGL2b and 0.3 kb iNOS-pGL2b) using standard cloning techniques and the following primer sequences: 2 kb iNOS-pGL2b, forward: 5′-CCG CTC GAG GCC AAG CAC TCC AAT GTA AAA-3′, 0.3 kb iNOS-pGL2b, forward: 5′-CCG CTC GAG AAG CCA GCC TCC CTC CCT-3′, 2kb and 0.3 kb iNOS-pGL2b, reverse: 5′-CCC AAG CTT CCA AGG TGG CTG AGA AGT TT-3′. Astrocytes were plated at a density of 100,000 cells/well in a 24 well format. Following incubation overnight, astrocytes were transfected with the reporter plasmid and constructs of interest using FuGENE transfection reagent. Twenty four hours after transfection, astrocytes were subjected to dual-luciferase assays (Promega). In all experiments, astrocytes were transfected with a Renilla firefly reporter to control for transfection efficiency.
For manual counting experiments, EGFRvIII;Stat3loxP/loxP astrocytes were plated at a density of 20,000 cells per 6 cm plate. The small molecule 1400W or vehicle (water) was added to cells 12 hours after plating. At each time point analyzed, cells were washed, trypsinized, and counted in triplicate for each condition via hemocytometer.
For high-throughput proliferation assays, astrocytes (250 cells), primary mouse astrocytes (1000 cells), or U87 human glioblastoma cells (250 cells) in 50 μL of media were added to each well of a 96 well plate and incubated for 24 h at 37°C. Pharmacological agents or their corresponding vehicles were then added as follows: 1400W (500 μM), S-MIU (25 μM), c-PTIO (5 μM), and SNAP (1.25 μM). Cells were lysed 48, 96, and 144 hours after initial plating with Cell TiterGlo reagent (Promega), agitated for 2 min, and then incubated at 25°C for 10 min. Wells were then read via a SpectraMax luminometer (Molecular Devices). For all experiments, cells were also lysed two hours after plating to ensure that equal numbers were initially plated across conditions. Wells containing media only were used to determine background signal.
astrocytes were washed with PBS and cross linked with 1% formaldehyde in PBS for 10 minutes. A solution containing 0.125 M glycine in PBS was used for quenching for 5 minutes at room temperature. Cells were harvested and the pellet was dissolved in ChIP lysis buffer (40 mM Tris-HCl, pH 8.0; 1.0% Triton X100; 4 mM EDTA; 300 mM NaCl) containing protease inhibitors. Chromatin was fragmented by sonication in a water bath sonicator at 4°C to an average length of 500 base pairs. The lysates were spun at 14,000 rpm for 15 minutes and the supernatant was diluted to 1:1 in ChIP dilution buffer containing 40 mM Tris-HCl, pH 8.0 and 4 mM EDTA plus protease inhibitors. Immunoprecipitation was done using a ChIP grade STAT3 antibody (Cell Signaling Technologies) against the endogenous STAT3 protein. Antibody–protein–DNA complexes were collected, washed, and eluted, and cross links were reversed according to manufacturer’s instructions (Millipore) as previously described (Gillespie et al., 2009
). The following primer sequences were used: STAT3, forward: 5′-GAG CTA ACT TGC ACA CCC AAC-3′; STAT3, reverse: 5′-GTG GGG CCA GAG TCT CAG T-3′, iNOS, forward: GAA CAG ACA GAA AGC CAG AGA GC, iNOS, reverse: GAC ACT CCT AGT CTG TGT GCT TGA.
Matrigel invasion assays
Matrigel was diluted and coated onto invasion chambers (BD) with an 8 μm pore size membrane overnight. Polymerized gel was re-hydrated with serum free media and astrocytes (2.5 × 104) in 500 μL of media were added to each of the inserts and incubated for 22 h at 37°C. Astrocytes on the lower surface of the membrane that had migrated through the matrigel were fixed, stained with crystal violet, and counted. Noninvasive NIH3T3 cells were used as a negative control. Equivalent numbers of NIH3T3 failed to invade the matrigel. The effect of iNOS knockdown or pharmacological inhibition on invasiveness was not secondary to a change in cell proliferation, as the invasive potential of these cells was measured at a time (22 h after plating) prior to a significant decrease in cell number upon inhibition of iNOS.
Control vector transfected and iNOS knockdown EGFRvIII; Stat3loxP/loxP astrocytes (1 × 106) were resuspended in serum-free, antibiotic-free media and injected subcutaneously into 4–6 wk old male severe combined immunodeficiency (SCID) mice. For experiments with administration of 1400W, EGFRvIII; Stat3loxP/loxP astrocytes were injected subcutaneously, and 100 μL of vehicle or 1400W (500 μM) was injected locally three times per week. Four weeks after initial injection of cells, mice were sacrificed and the tumors were removed, measured, and fixed for histological analyses.
Tumors were fixed in 4% paraformaldehyde, embedded in Paraplast, and serially sectioned at 7 microns. Sections were deparaffinized through xylenes and graded ethanol and subsequently stained with hematoxylineosin for histological evaluation. Representative areas are shown in figures. For immunohistochemistry, antigen retrieval was performed via microwaving for 20 minutes in 10 mM Sodium Citrate (pH 6.0). Proliferating cells were stained with a rabbit polyclonal antibody to Ki67 (Vector Laboratories) and visualized the immunolabeling with the Vectastain Elite ABC Reagent and DAB Substrate Kit (Vector Laboratories). Sections were counterstained with hematoxylin.
All analyses were completed from a minimum of three independent experiments. Statistical analyses were performed with GraphPad Prism 4.0. All histograms are presented as mean + SEM unless otherwise noted. The Student’s t-test was utilized for comparisons in experiments with two sample groups. In experiments with more than two sample groups, analysis of variance (ANOVA) was performed followed by Bonferroni’s post-hoc test. For non-parametric analyses of more than two sample groups, Kruskal-Wallis was performed followed by Dunn’s post-hoc test.