be improved when compared with historical controls [8–11]. Similarly, the disappointing results obtained in early phase clinical trials by the use of inhibitors directed against EGFR [12] and PDGFR [13] are likely in part due to inadequate patient selection, although as yet there are no validated markers predictive of response in high grade gliomas, and it is unclear whether there are differences in the paediatric setting. The mechanisms of drug resistance in paediatric high grade glioma are poorly understood, in part due to the limited availability of suitable models of the disease. Nearly all preclinical and functional work is carried out in a relatively small panel of adult glioblastoma cell lines in vitro, and it is currently unclear how representative these are of the underlying biology of paediatric lesions. In order to begin to address these questions, we have carried out a detailed molecular and phenotypic characterisation of a series of paediatric high grade glioma cell lines in comparison to routinely used adult lines. Through an integrated profiling approach, we demonstrate that glioma cell lines derived from paediatric patients show key molecular differences to those from adults, some of which are well known, whilst others may provide novel targets for evaluation in primary tumours. We thus provide the rationale for using paediatric high grade glioma cell lines for preclinical and mechanistic studies. Materials and Methods Cell culture Adult glioblastoma cell lines, A172, LN229, SF268, U87MG, U118MG and U138MG were obtained from ATCC (LGC Promochem, Middlesex, UK). Paediatric glioblastoma KNS42 cells were obtained from the JCRB (Japan Cancer Research Resources) cell bank. Paediatric SF188 cells were kindly provided by Dr Daphne Haas-Kogan (UCSF, San Francisco, CA, USA), whilst UW479, Res259 and Res186 were kindly provided by Dr Michael Bobola (University of Washington, Seattle, WA, USA). They were grown as monolayers in DMEM/F12 Ham’s medium+10% FCS in 5% CO2. Immunohistochemistry Cells were grown to 70–80% confluency in chambered slides (Nunc, Rochester, NY, USA) fixed in either 4% paraformalde- hyde:0.5% TritonX-100 in PBS (GFAP, synaptophysin, vimentin, nestin and CD133) or ice cold methanol (S100). The cells underwent a peroxidase block (Dako, Ely, UK) followed by washes in 0.05%Tween in TBS prior to incubation with primary antibody – GFAP (clone 6F2, 1:50 dilution, Dako); synaptophysin (SY38, 1:10, Dako); vimentin (V9, 1:100, Dako), S100 (B32.1, 1:20, Abcam, Cambridge, UK), nestin (10C2, 1:400, Miltenyi Biotech, Bergisch Gladbach, Germany), or CD133 (W6B3C1), 1:40, Miltenyi Biotech). Staining was visualised by the Envision horseradish peroxidase system (Dako) according to the manufacturer’s instructions. Genomic analysis 500 ng of DNA was analysed on the Affymetrix GeneChip Human Mapping 250K Nsp and Sty arrays by the UCL Institute of Child Health Gene Microarray Centre, according to the manufacturer’s protocol. The raw data were processed and genotypes called using the BRLMM method of the Affymetrix GTYPE package. Copy number ratios were calculated by the Affymetrix CNAT 4 algorithm, using quantile normalisation and a Gaussian smoothing bandwidth of 0.1 Mb. Normal references were provided by public data from an unmatched panel of 30 Caucasian female DNA samples, the Centre d’Etude du Polymorphisme Humain (CEPH) trio mothers, analysed on the Profiling Paediatric Gliomas same platform by The International HapMap Consortium (International HapMap Consortium 2007, http://www.hapmap. org). The copy number data set was compared to the positions of known copy number variations (CNVs) in the HapMap popula- tions displayed on the UCSC genome browser and any copy number changes in the current data that overlapped with these CNVs were noted. LOH was inferred using the linkage disequilibrium Hidden Markov Model as implemented in dChip (http://www.dchip.org). False positives were reduced by excluding apparent LOH regions present in more than 10% of all 60 CEPH trio parents. LOH was taken as copy neutral if the copy number measured by dChip was close to diploid (1.7–2.3 copies). All data have been submitted according to MIAME guidelines [14] to the public data repository ArrayExpress (http://www.ebi.ac.uk/ arrayexpress/) with accession number E-TABM-579. Multiplex fluorescence in situ hybridisation (M-FISH) analysis was performed on metaphase spreads prepared from Res259 cells using a Vysis SpectraVysion probe (Abbot Molecular, Abbott Park, IL, USA) following the manufacturer’s instructions as previously described [15]. Images were captured using a Zeiss Axiophot microscope equipped for epifluorescence using cooled CCD-camera (Hamamatsu, Welwyn Garden City, UK). Image analysis was performed using SmartCapture X software (Digital Scientific, Cambridge, UK). Fluorescent in situ hybridisation (FISH) FISH analysis was carried out on metaphase chromosome preparations as previously described [16]. Probes directed against CDK4 (RP11-66N19,RP11-571M6), PDGFRA (RP11-231C18, RP11-626H4, RP11-117E8), and PTEN (RP11-105A10) were labelled with Cy5 (GE Healthcare, Amersham, UK), whilst commercial probes against RB1 (Abbot Molecular) and CDKN2A/B (Abbott Molecular) were SpectrumOrange-labelled. Appropriate centromeric probes were labelled with either fluorescein or SpectrumGreen. Hybridised preparations counter- stained with DAPI in antifade (Vector Laboratories Inc., Burlingame, CA, USA). Images were captured using a cooled charge-coupled device camera (Photometrics, Tuscon, AZ, USA). Methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) Promoter methylation was assessed by kits ME001, ME002 and ME011 according to manufacturer’s instructions (MRC-Holland, Amsterdam, Netherlands) [17]. Briefly, 100 ng of tumour DNA was denatured in 5 mL of Ultra-Pure Water at 98uC for 5 min and then incubated with the probe mix for 20 h at 60uC. After probe hybridization, each sample was divided into two different tubes. Half of the sample was ligated using a ligase enzyme and in the other half ligation was combined with HhaI digestion resulting in ligation of the methylated sequences only. The resulting products were amplified by PCR using a FAM-labeled primer, following manufacturer’s instructions. PCR products were analyzed on ABI Prism 310 single capillary genetic analyzer (Applied Biosystems) using the GeneScan 3.7 software (Applied Biosystems). Duplicate experiments were performed for methylation analysis and average ratios were calculated. We interpreted ratios as absence of hypermethylation (0.00–0.24), mild hypermethylation (0.25– 0.49), moderate hypermethylation (0.50–0.74), and extensive hypermethylation ($0.75), as previously described [18]. Western blot analysis Cells at 80% confluence were trypsinised, washed with PBS and lysed for 1 hr at 4uC in lysis buffer (Cell Signaling, Hertfordshire, PLoS ONE | www.plosone.org 2 April 2009 | Volume 4 | Issue 4 | e5209