Clinical Specimens and Immunohistochemistry
Snap-frozen primary glioma tissues were obtained from the Department of Pathology, Johns Hopkins University School of Medicine, with Institutional Review Board approval. RNA was extracted from gliomas using Trizol reagent (Invitrogen, Carlsbad, CA, http://www.invitrogen.com) and further purified using RNeasy columns (Qiagen, Valencia, CA, http://www1.qiagen.com) according to the manufacturers' instructions. The construction of the tissue microarray containing high-grade astrocytomas has been previously described . Immunohistochemistry was performed on freshly cut, deparaffinized tumor or tissue microarray sections essentially as previously described . The following antibodies were used for immunocytochemistry: Nestin, clone MAB5326 (1:2,500; Chemicon, Temecula, CA, http://www.chemicon.com); glial fibrillary acidic protein (GFAP), Z0334 (1:2,500; DAKO, Carpinteria, CA, http://www.dako.com). Statistical analyses were performed using GraphPad Prism4 (GraphPad Software, Inc., San Diego, http://www.graphpad.com).
The U87-MG, A172, U251, and SW1088 cell lines were obtained from American Type Culture Collection (ATCC) (Manassas, VA, http://www.atcc.org) and maintained in ATCC's recommended growth medium supplemented with 10% fetal bovine serum (FBS). The CJ-MG and BK-MG cell lines were a kind gift from Dr. Carol A. Kruse . JHH-GBM2 was derived as follows: fresh tumor was chopped to small pieces using sterile razor blade and then mechanically triturated through a 21-gauge needle and filtered through a 40 μm cell strainer (BD Falcon; BD Biosciences, Bedford, MA, http://www.bdbiosciences.com). Cells were concentrated by centrifugation and then seeded in Dulbecco's modified Eagle's medium (DMEM)/Ham's F-12 medium supplemented with 10% FBS. The glioblastoma-derived neurosphere line HSR-GBM1 was derived and propagated as previously described . 293T, 293T/SHH-N′, and Light2 cells were the kind gift of Dr. Phil Beachy.
The Gli reporter assay was performed in either U87-MG by transient transfection of the Gli firefly luciferase reporter construct or in NIH3T3 cells stably transfected with Gli firefly luciferase and Renilla luciferase reporter constructs (Light2), as previously described with minor modifications . Briefly, Light2 cells were plated in a 24 well plates at 7 × 104 cells per well in DMEM supplemented with 10% FBS. When confluent (normally the next day), the monolayers were washed once each with phosphate-buffered saline (PBS) and DMEM supplemented with 0.5% FBS. Cells were incubated overnight in a humidified incubator kept at 37°C, 5% CO2, and then growth medium was removed and monolayers were overlaid with conditioned medium from 293T, 293T/Shh-N′, or HSR-GBM1 and incubated for an additional 48 hours. Gli reporter activity was quantified using the dual-luciferase reporter assay system (Promega, Madison, WI, http://www.promega.com) and normalized to Renilla luciferase activity. In U87-MG, Gli reporter activity was quantified using the luciferase reporter assay system (Promega) and normalized to total protein.
For mRNA quantification and 3-(4,5-dimethylthiazol-2-yl)-5(3-carboxymethonyphenol)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assays, cells were plated in six-well plates (RNA) or 96-well plated (MTS) and incubated overnight, and the monolayers were washed once with PBS, followed by overlay with low-serum (0.5% fetal bovine serum) media. The following day (approximately 16 hours after serum withdrawal), medium was replaced with medium supplemented with 0.5% FBS and either ethanol or cyclopamine (5 or 10 μM). RNA was extracted at various time points after cyclopamine addition using Qiagen RNeasy kits. MTS assays were performed at various time points using the CellTiter96 assay (Promega) according to the manufacturer's instructions.
To assay neurosphere differentiation, HSR-GBM1 cells were triturated and plated at 2 × 104 cells per cm2 in 10-cm2 cell culture dishes precoated with Matrigel (BD Biosciences) diluted 1:100 in Neurocult medium (Stem Cell Technologies, Vancouver, BC, Canada, http://www.stemcell.com) without epidermal growth factor (EGF). After attachment of cells to the plate surface, medium was replaced with Neurocult medium lacking mitogens and heparin but supplemented with 1% bovine calf serum (HyClone, Logan, UT, http://www.hyclone.com). Cells were incubated for 5 days as before and then washed, and RNA was extracted as described above.
shRNA targeting Gli1 was purchased from Sigma-Aldrich (St. Louis, http://www.sigmaaldrich.com). pLKO.1-puro was a kind gift from Dr. Bob A. Weinberg and Dr. Carl D. Novina . U87-MG cells were plated at 2.5 × 105 cells per well or 7.5 × 105 cells per dish in six-well plates or 10-cm culture dishes, respectively. The next day, medium was replaced with 2 ml of transduction medium. Dishes were incubated overnight in humidified incubator at 37°C. RNA was extracted from cells grown in six-well plates 48 hours after infection. For cells grown in 10-cm dishes, medium was replaced 72 hours postinfection with fresh medium containing 10 μg/ml puromycin (Sigma-Aldrich). Cells were stained with 4,6-diamidino-2-phenylindole, and pictures of 10 random high-power fields were taken for each condition.
For Gli1 and Gli2 short interfering RNA (siRNA) experiments, 2 × 105 HSR-GBM1 cells were plated in duplicates in six-well plate format for RNA analysis and at 1.5 × 104 cells per well in a 48-well format for growth assays (MTS). Cells were incubated overnight and then transfected every other day according to the manufacturer's directions using RNAiFect (Qiagen) with 8 nM/well of a nonspecific siRNA (Dharmacon, Lafayette, CO, http://www.dharmacon.com) as negative control, Gli1 pooled siRNA, or a single Gli2 siRNA duplex (Dharmacon) with the following oligo sequences: Gli1 forward, 5′-GGAAAUGACUGGCAAUGCAUU-3′, and Gli1 reverse, 5′-PUGCAUUGCCAGUCAUUUCCUU-3′ for duplex 1; Gli1 forward, 5′-GCACUGGUCUGUCCACUCUUU-3′, and Gli1 reverse, 5′-PAGAGUGGACAGACCAGUGCUU-3′, for duplex 2; Gli1 forward, 5′-GUCCUCACUUGAACAUUAUU-3′, and Gli1 reverse, 5′-PUAAUGUUAAGUCGAGGACUU-3′, for duplex 3; Gli1 forward, 5′-AGGCUCAGCUUGUGUGUAAUU-3′, and Gli1 reverse, 5′-PUUACACACAAGCUGAGCCUUU-3′, for duplex 4; Gli2 forward, 5′-CGUCAACCCUGUCGCCAUUUU-3′, and Gli2 reverse, 5′-PAAUGGCGACAGGGUUGACGUU-3′.
Clonogenic assays were performed as previously described . Briefly, U87-MG cells plated at 3 × 103 cells per well. Cells were treated with either ethanol (vehicle [V]) or cyclopamine (5 and 10 μM). Each condition was tested in triplicate, and each assay was repeated twice with similar results. Colony number and size were scored with the ChemiDoc-XRS imager, using the QuantityOne software package (Bio-Rad, Hercules, CA, http://www.bio-rad.com).
HSR-GBM1 cells were seeded in T75 cm2 culture dishes at 1 × 105 cells per dish. Cells were treated with either ethanol or cyclopamine (10 μM) in a final volume of 20 ml of Neurocult medium (Stem Cell Technologies) supplemented with 0.002% heparin, 10 ng/ml human EGF, and 10 ng/ml human fibroblast growth factor-b (Peprotech, Rocky Hill, NJ, http://www.peprotech.com). Dishes were incubated upright for 7 days in a humidified incubator with 5% CO2 at 37°C. Cells were retreated with 2 ml of medium containing the indicated concentration of drug on days 2, 4, and 6. Seven days after treatment, spheres were triturated and replated in T25 cm2 at 2 × 103 cells per dish. Subsequent sphere formation was monitored and scored by light microscopy after 8 days. Ten random fields were photographed for both vehicle and cyclopamine-treated conditions, and the number of spheres over 50 μm in size was scored.
Xenograft and Side Population Assays
For side population analyses, U87-MG and C6 cells were treated for 7 days in low-serum (0.5% FBS) media containing either ethanol (V) or cyclopamine (5 or 10 μM), with medium changes every 2 days. HSR-GBM1 cells were treated in 10 ml of media, with 2 ml of additional medium containing drug or vehicle supplemented every 2 days. Side population analyses on treated cells were performed as previously described . For xenograft studies, HSR-GBM1 cells were treated as described above, and an aliquot collected from each flask following treatment was scored for viability by Guava-PCA flow cytometry system and ViaCount reagent, according to the manufacturer's instructions (Guava Technologies, Hayward, CA, http://www.guavatechnologies.com). Next, viable cells were diluted with fresh medium and injected over 10 minutes into the right striatum of athymic (nu/nu) mice (Harlan, Indianapolis, IN, http://www.harlan.com). Mice were monitored daily and sacrificed at the first indication of tumor development (ataxia, seizure, lethargy, or cachexia). Brains were surgically removed and fixed immediately in formalin before submission for histological analysis, as previously described .
HSR-GBM1 cells were plated at 5 × 103 cells per well (in triplicate) in 48-well culture plates and treated with either ethanol (0) or cyclopamine (5 or 10 μM). After 3 days of growth, the cultures were irradiated at either mock (0 Gy) or 10 Gy using a Gammacell 40 irradiator equipped with a 132cesium source (MDS Nordion, Ottawa, ON, Canada, http://www.mds.nordion.com). Cell mass was subsequently measured on day 7 using the MTS assay (Promega), and growth rate calculated from the first day of cyclopamine treatment.
For cotreatment with cyclopamine and radiation, HSR-GBM1 cells were plated at 1 × 106 cells per T75 culture flask and treated with either ethanol (0) or cyclopamine (5 or 10 μM) at plating and every other day thereafter. Cells were irradiated 4 days after plating, and percentages of side and aldehyde dehydrogenase (ALDH)-positive populations were determined on day 7.