UK) and a complete mini protease inhibitor cocktail (Roche Diagnostics, Mannheim, Germany). Cells were then centrifuged at 11,000 rpm at 4uC for 15 min, and protein concentration determined (Pierce BCA assay, Rockford, IL, USA). Total protein extracts (30 mg/lane) were separated electrophoretically in 4–20% SDS-polyacrylamide gel and transferred to nitrocel- lulose membranes (Invitrogen Ltd, Paisley, UK). Immunodetec- tion was performed using antibodies against cRAF (1:500, Santa Cruz Biotechnologies, Santa Cruz, CA, USA), phosphorylated and total Erk1/2, Akt, GSK3b and S6 (all 1:1000, Cell Signaling), and GAPDH (1:2000, Chemicon, Hampshire, UK). Blots were revealed with peroxidase-conjugated secondary anti- rabbit or anti-mouse antibodies (GE Healthcare, Amersham, UK) followed by ECL chemiluminescence solution (GE Healthcare). mRNA expression profiling 10 mg of total RNA was used to generate labelled cRNA and hybridised to U133Plus 2.0 oligonucleotide arrays (Affymetrix, Santa Clara, CA, USA) containing more than 40,000 transcripts by the UCL Institute of Child Health Gene Microarray Centre according to standard manufacturer protocols. Probe intensity data for each array was normalized to a baseline array with median signal intensity using the ‘‘invariant set’’ model. Model- based expression was performed using the perfect-match/ mismatch (PM/MM) model to summarize signal intensities for each probe set. All data have been submitted according to MIAME guidelines [14] to the public data repository ArrayEx- press (http://www.ebi.ac.uk/arrayexpress/) with accession num- ber E-TABM-579. For the supervised identification of genes exhibiting differential expression between paediatric and adult high grade cell lines, expression values were subjected to a variation filter to exclude probe sets with minimal variation across the samples, and an absolute signal to noise metric of greater than 1.5 was subsequently used to identify marker probe sets using GenePattern software (http://www.broad.mit.edu/ cancer/software/genepattern/);. Differentially expressed co-ordi- nate sets of genes were identified using Gene Set Enrichment Analysis (GSEA, www.broad.mit.edu/gsea/) with a nominal p value cut-off of 0.001. Real-time quantitative RT-PCR cDNA was prepared from 1 mg of tumour or reference RNA by random primed reverse transcription using Superscript II (Invitrogen, Paisley, UK). TaqManH Gene Expression Assays were obtained from Applied Biosystems (Warrington, UK) for Profiling Paediatric Gliomas LYN (Hs00176719), CRKL (Hs00178304), AXL (Hs00242357) and EPHA6 (Hs00297133). PCRs were performed in a 10 ml reaction volume containing 5 ml 26 buffer/enzyme mix, 0.5 ml 206assay mix, 0.5 ml 206GAPDH endogenous control assay mix (Hs99999905) and 1 ml input cDNA. Assays were run on an Applied Biosystems 7900 Sequence Detection System and results analysed by the standard curve method. Data were normalised to Universal Human Reference RNA (Stratagene, La Jolla, Califor- nia, USA). Results Paediatric glioma cell lines grown as monolayers in vitro express astrocytic and stem cell markers All paediatric glioma cell lines were derived from astrocytomas of differing grades arising in patients aged 3–16 years old. They were established in vitro as monolayer cultures, with doubling times of between 24–48 hours, and demonstrated a mixture of stellate and bipolar morphologies, with some cells of polygonal, cuboidal or flattened appearance (Table 1). The astrocytic nature of the cells was confirmed in culture by immunohistochemistry with a range of glial markers (Figure 1). All cells showed some degree of GFAP-positivity, although SF188 and Res259 were only weakly expressing. All cells were strongly positive for S100 and vimentin, with the exception of UW479, which was vimentin negative. There was little synaptophysin expression, save for the occasional isolated cell in SF188 and KNS42 cultures. There was a surprisingly high level of expression of stem cell markers in the paediatric glioblastoma lines, with both SF188 and KNS42 strongly positive for nestin, and containing 7% and 4% CD133- positive cells respectively. Genomic profiling reveals numerous chromosomal abnormalities and focal amplifications/deletions Affymetrix 500K SNP array analysis revealed all paediatric glioma cell lines to display complex genomes with numerous gross chromosomal copy number abnormalities and rearrangements. After exclusion of known copy number variants, this high- resolution copy number profiling additionally highlighted a number of focal copy number aberrations, which were confirmed by FISH on metaphase preparations (Figure 2). The glioblastoma cell line SF188 harboured high-level amplifications of several oncogenes known to be present in paediatric glioblastoma tumour samples. These included MYC at 8q24, CCND1 at 11q13, CDK4 at 12q14, all of which were due to extrachromosomal double minute formation, and overexpression of the corresponding proteins was Table 1. Characteristics and immunophenotype of paediatric glioma cells grown as monolayers. Cell line WHO grade Diagnosis Age Sex Morphology Doubling time Immunohistochemistry Synapto- GFAP S100 Vimentin physin SF188 KNS42 IV IV UW479 III Res259 II Res186 I Glioblastoma multiforme 8 yrs male stellate, polygonal, bipolar 26 hrs Glioblastoma multiforme 16 yrs male cuboidal, polygonal, bipolar 48 hrs Anaplastic astrocytoma 13 yrs female polygonal, stellate 28 hrs Diffuse astrocytoma Pilocytic astrocytoma 4 yrs 3 yrs female polygonal, cuboidal, bipolar 24 hrs female stellate, flattened, bipolar 46 hrs ++ +++ +++ + +++ ++ +++ +++ ++ ++ +++ +++ 2 +++ +++ ++ ++ + 2 2 The astrocytic nature of the cells was confirmed in culture by immunohistochemistry with a range of glial and stem cell markers. (+++) strongly positive; (++) moderately positive; (+) weakly positive; (2) negative. doi:10.1371/journal.pone.0005209.t001 Nestin CD133 7% 4% 1% 0% 0.1% PLoS ONE | www.plosone.org 3 April 2009 | Volume 4 | Issue 4 | e5209