Cell lines and tumor samples of astrocytoma and glioblastoma multiforme
All studies were performed after written informed consent was obtained under the auspices of a human subjects institutional review board (IRB) protocol approved by the Partners Human Research Committee. Primary frozen tissue from 78 human glioma specimens (including 15 low grade astrocytomas, 15 low grade oligodendrogliomas, 10 low grade gangliogliomas, 7 anaplastic astrocytomas and 31 glioblastomas (GBMs) were obtained from the Brain Tumor Tissue Bank in the Department of Neurosurgery at Brigham and Women's Hospital. Four human glioblastoma cell lines (U87, U251, U343, and T98) were obtained from the American Tissue Type Culture Collection. The D566 human glioblastoma cell line was a gift from D. Bigner, Duke University. All cell lines were cultured in DMEM (Invitrogen, Carlsbad, CA) supplemented with 10% FBS, and were maintained in a 5% CO2 incubator at 37°C.
mRNA expression profiling
Total RNA was isolated from 20 fresh frozen human glioma samples and from 7 non-tumor brain samples. In some experiments, RNA was isolated from U87 human glioblastoma cells after transduction with a SNAI2/Slug lentivirus or a control virus. The mRNA was reverse-transcribed to generate cDNA, which was then biotinylated and hybridized to Affymetrix HG-U133A expression arrays prior to scanning for quantitation. For data from primary glioma specimens, expression heatmaps were constructed using expression data from the non-tumor brain specimens as a reference. Statistical comparisons between histologic subgroups were performed using the t-test.
Taqman Real-time PCR
Total RNA was extracted from cell lines with TRIzol (Invitrogen, Carlsbad, CA), according to the manufacturer's protocol. Randomly primed cDNA was prepared using 1 μg of total RNA from each sample and the AMV 1st Strand cDNA Synthesis Kit (Roche Applied Science, Indianapolis, IN). Six ng of each cDNA were then used for real-time PCR analysis in a final reaction volume of 20 μl. Probes for β-actin (Hs99999903_m1) and human SNAI2/Slug (Hs00161904_m1) were purchased from Applied Biosystems (Foster City, CA). Samples were analyzed in triplicate using the ABI 7300 software system (Applied Biosystems, Foster City, CA) with DDCt quantification. Statistical analysis was performed using the t-test.
Lentivirus production and establishment of stable glioma lines
The full length human SNAI2 gene was cloned by RT-PCR from U87 cells using the forward primer 5'- CAC CAT GCC GCG CTC CTT CCT GGT C-3' and the reverse primer 5'-TCA GTG TAC ACA GCA GCC AGA-3', and was subsequently transferred into the pLenti6-IRES-EGFP vector. The correct SNAI2 sequence was confirmed by direct DNA sequencing. A lentiviral shRNA vector for Slug and an appropriate empty control vector were purchased from Open Biosystems (TRCN0000015389, Huntsville, AL). The lentiviral vectors were packaged in 293FT cells using the ViraPower Lentiviral Expression System (Invitrogen, Carlsbad, CA) according to the manufacturer's protocol.
Human U251 and U87 glioblastoma cells were transduced with the appropriate lentiviruses, and stable cell lines (U251-IRES-GFP and U251-Slug-IRES-GFP) were selected using 8 μg/ml blasticidin. U87-pLKO.1 and U87-shSlug stable cell lines were selected using puromycin.
Growth and proliferation assays
To measure cell growth, 1 × 103 glioma cells were plated into 96 well culture plates in triplicate, and cell growth was determined from day 0 to day 6 using a tetrazolium salt-based colorimetric assay (Cell Counting Kit-8, Dojindo Molecular Technologies, Gaithersburg, MD) according to the manufacturer's protocol.
To measure cell proliferation, 1 × 103 human glioma cells were plated into 96 well culture plates in triplicate and cultured for 6 hours. The cells were then incubated in BrdU overnight at 37°C. BrdU incorporation into DNA was quantitated by ELISA assay (Roche Applied Science, Indianapolis, IN) according to the manufacturer's protocol.
Total protein was extracted using RIPA buffer supplemented with proteinase inhibitors. Protein extracts were then separated by gel electrophoresis using 4-20% SDS-PAGE - Tris-HCl gels (Bio-Rad, Hercules, CA). The protein was transferred to nitrocellulose membranes and detected using a specific anti-Slug antibody (G-18, Santa Cruz Biotechnologies, Santa Cruz, CA) at a 1:1000 dilution. After washing and incubation in the appropriate secondary antibody, immunoreactive bands were visualized using the enhanced chemiluminescence system.
Animals containing subcutaneous tumors were anesthetized, and the subcutaneous tumors were then harvested, fixed with 4% paraformaldehyde at pH 7.4, immersed in 30% sucrose, embedded in OCT and cryostat-sectioned at 6 μm at -20°C. Hematoxylin and eosin (H&E) staining was performed, and immunohistochemistry was performed using the Vectastain Elite ABC kit (Vector Laboratories, Burlingame, CA). A specific anti-CD31 primary antibody (1:100; BD Biosciences PharMingen) was used to visualize blood vessels.
In vitro cell migration and invasion assays
Cell migration was measured using the in vitro
scratch wound healing assay [15
] and transwell assay [16
]. For the wound healing assay, U251-IRES-GFP and U251-Slug-IRES-GFP cells were cultured at 37°C in DMEM supplemented with 10% FBS until confluence was achieved. A cell-free area was then created by scratching the monolayer with a pipette tip, and the culture was photographed at time 0 and after 24 hours. The distance migrated from the edge of the scratch was measured using an automated imaging software program (SPOT, Diagnostic Instruments, Sterling Heights, MI).
For the transwell assay, 10 mm tissue culture inserts with polycarbonate membranes (8 μm pore, Nalge Nunc International, Rochester, NY) were used according to the manufacturer's protocol. Briefly, 1 × 105 U251-IRES-GFP or U251-Slug-IRES-GFP cells were suspended in DMEM supplemented with 1% FBS and plated into the 10 mm tissue culture inserts. DMEM supplemented with 10% FBS was used in the lower chamber as a chemoattractant, and the cells were allowed to migrate overnight. The cells that failed to migrate (located on the upper surface of the membrane) were removed using a cotton swab. The membranes were then stained with the 3 Step Stain Set (eosin-Y, azure A and methylene blue, Richard-Allen Scientific, Kalamazoo, MI), dried and mounted using Cytoseal 60. The cell number was determined by calculating the mean number of cells from five separate 40 × fields. The same protocol was used for the migration of U87-pLKO.1 and U87-shSlug cells, except that the migration time was decreased to 5 hours.
Invasion by glioblastoma cells in vitro
was measured using the Matrigel invasion assay [17
]. The BD BioCoat Matrigel invasion chamber (8 μm pore, BD Biosciences, San Jose, CA) was used according to the manufacturer's protocol. Briefly, 1 × 105
U251-IRES-GFP and U251-Slug-IRES-GFP cells were suspended in DMEM with 1% FBS and plated onto 10 mm tissue culture inserts. DMEM containing 10% FBS was placed into the lower chamber, and the cells were allowed to invade through the matrix overnight. All incubations were conducted at 37°C with 5% CO2
. The cells that failed to migrate through the membrane were removed with a cotton swab, and the membranes were stained with the 3 Step Stain Set for blood smear (Richard-Allen Scientific, Kalamazoo, MI). The cell number was determined by taking the average from five separate 40 × fields. Statistical significance was determined using the t-test.
Intracranial and subcutaneous tumor growth and survival assays
All animal experimental procedures were carried out in the animal facility at Brigham and Women's Hospital under the auspices of an approved protocol and in accordance with federal, local and institutional guidelines. For the subcutaneous tumor growth assay, approximately 5 × 105
U251-IRES-GFP or U251-Slug-IRES-GFP cells were subcutaneously injected into the flanks of 5-week-old nude mice. The tumor volumes were measured each week beginning with the second week after injection, and the tumors were allowed to grow for an additional 6 weeks. At the termination of the study, the mice were euthanized and the tumors were surgically removed and processed for histochemistry. A total of 4 mice were studied in each group. Tumor volume was calculated from the length (a) and width (b) using the formula V = 4/3π(ab2
]. Statistical significance was determined using the t-test.
For the intracranial orthotopic human glioma model, approximately 5 × 104 U87-pLKO.1 or U87-shSlug glioblastoma cells were injected intracranially into the frontal cortex of 5-week-old male nude mice. Six mice in the control group and 5 mice in the U87-shSlug group received intracranial human glioblastoma cell transplants. The animals were then followed until they showed signs of neurologic dysfunction or distress, at which time they were sacrificed. Kaplan-Meier survival analysis was then performed, and statistical significance was determined using the Logrank test.