understand and overcome this are hampered by a lack of mechanistic data in paediatric glioma models. We have sought to address this by collecting and carrying out a detailed molecular and phenotypic characterisation of the few available paediatric glioma cell lines, with a view to incorporating them into ongoing drug development programmes. There is an acute clinical need to develop better treatment strategies for the refractory high grade gliomas in childhood, and having lines representative of paediatric glioblastoma (WHO grade IV – SF188 and KNS42) and anaplastic astrocytoma (WHO grade III – UW479), would be extremely useful in preclinical and mechanistic studies of the disease. These lines all grew as either stellate or bipolar cells, with some polygonal features, in monolayers, and the immunophenotypes were representative of high grade glial tumours, including expression GFAP, S100 and vimentin. There were slight discrepancies with a previous publication referring to some of these lines [19], with a greater degree of S100 staining in UW479, and less GFAP in Res259 in our study, however it unclear whether this is reflective of methodological differences, culture conditions or changes in gene expression over time. It is of considerable interest to note the relative high levels of CD133 positive cells in both SF188 (7%) and KNS42 (4%). Intrinsic drug resistance in the proportion of clinical glioblastomas comprised of tumour stem cells has been suggested to play a key role in drug resistance in this disease, and the high CD133-positive content of these cells grown as monolayers may make them particularly relevant in preclinical drug screening programmes. All three high grade lines had highly complex genomic profiles and harboured amplifications and deletions at several known cancer genes dysregulated in paediatric glioma, including CCND1, MYC, CDK4, PIK3CA, CDKN2A/B, and RB1 [2,20–23]. Although there are no molecular alterations yet described that are diagnostic for paediatric high grade glioma, these genes cover a broad spectrum of reported genomic aberrations. In addition, large scale chromosomal imbalances such as gain of chromosomes 1q and 7, and losses of 10q and 13q, reported at high frequencies in primary tumours [24], are present within these cell lines. There were contrasting genomic profiles in the two paediatric glioblastoma lines. SF188 harboured an amplifier phenotype, with coordinated amplification of CCND1 and CDK4 evidence of a lack of cell cycle control by virtue of dysregulation of the RB1 pathway. By contrast, KNS42 cells achieved a similar end through direct deletion of RB1 itself. No RB1 hypermethylation was detected. Simultaneous abrogation of core signalling pathways in glioblas- tomas, as recently reported for primary tumours [25,26], are in evidence in both these cell lines, with further disruption to the p53 pathway (through point mutations of TP53 in both lines), and the RTK/PI3K/AKT pathway via NF1 deletion in SF188 and PIK3CA copy number gain in KNS42. Interestingly, UW479 cells, as well as harbouring numerous high level amplifications, also had extensive promoter hyper- methylation at a variety of epigenetically regulated genes, including APC, CASP8, CD44, CDH13, CHFR, ESR1, GSTP1, IGSF4, MGMT, PAX5A, PAX6, and RARB. They also had, in common with all the lines tested, methylated RASSF1A and TMS1 genes. RASSF1A promoter methylation is one of the most common molecular changes in cancer, and may modulate multiple apoptotic and cell cycle checkpoint pathways [27]. TMS1/ASC is an intracellular signaling molecule with proposed roles in the regulation of apoptosis, nuclear factor-kB activation, and cytokine maturation, and has been found to be hypermethylated in approximately 40% of adult glioblastoma cell lines and tumours [28]. Profiling Paediatric Gliomas Of potential significance in preclinical drug screening was the observation that UW479 and Res186 cells to harbour methylated GSTP1. Glutathione-S-transferase p acts to enzymatically conju- gate glutathione to the reactive metabolites of alkylating agents, and up-regulation in glioblastoma tumour specimens has been correlated with resistance to BCNU [29]. Of further direct translational relevance was the observation that KNS42 cells were found to have methylation in the MGMT promoter, but counter to the expectation in glioblastoma cells, this did not confer a sensitivity to in vitro treatment with the alkylating agent temozolomide [30]. Two of the cell lines (Res259 and Res186) were reported to be diffuse astrocytoma (WHO grade II) and pilocytic astrocytoma (WHO grade I), which would make them extremely valuable in vitro models. Due to the difficulty in culturing these lesions successfully, and the relatively stable genomes reported in the human disease [31–33], it is likely that these lines may have acquired genetic alterations in culture. Interestingly, both lines harboured genetic alterations reminiscent of high grade gliomas in humans - including the archetypal 4q12 amplification spanning PDGFRA and KIT in Res259, and PTEN deletion in Res186. Despite this, their immunophenotypes more closely resembled low grade lesions, with an absence of synaptophysin expression, in contrast to the occasional positive cells seen in the high grade lines, and higher grade tumours in humans [34]. Whether or not they are truly reflective of the lesion from which they were derived, they are likely to make excellent models for the study of specific pathway modulation, i.e. that of the receptor tyrosine kinase and PI3K/PTEN systems. This is of particular importance, as the paediatric high grade lines were all PTEN wild-type, and there was significantly lower constitutive levels of phosphorylated Akt in these cells than a selection of the commonly used adult glioblastoma lines. Similarly, constitutive activation of additional key signalling pathways would further increase the usefulness of the cell line panel for drug development. The MAPK pathway is represented in the KNS42 glioblastoma cells, which otherwise has little constitutive activation of Akt. High levels of phosphorylated S6 in both KNS42 and Res259 cells shows a significant activation of the mTOR pathway, potentially independent of upstream PI3K/Akt signalling. mTOR is emerging as a therapeutic target in human gliomas [35], and extending this to the paediatric setting will be facilitated by the use of these models for screening mTOR inhibitors, alone and in combination strategies. Global measures of gene expression also served to highlight the differing genetic background of the paediatric lines. The differential expression of diverse kinases including LYN, CRKL, EPHA6 and AXL between the high grade glioma cells derived from patients of different ages suggests a number of potential new drug targets, and provides impetus for molecular pathology investiga- tions to confirm their presence in vivo. Two such observations are the overrepresentation of genes associated with DNA integrity in the paediatric cells, and underrepresentation of those associated with the immune response. As both of these areas are under intense research for the development of novel therapeutic agents in gliomas [36–40], measurement of the specific elements responsible for their modulation is needed in order to ensure the most appropriate model is utilised for mechanistic and preclinical assessment. The paediatric lines have already proven their usefulness in vitro with studies identifying differential drug sensitivity and mecha- nisms of action/resistance [19,30,41,42]. As an adjunct to this, we are currently establishing and profiling them as in vitro neurosphere cultures, and in vivo as xenografts in immunodeprived mice. It is PLoS ONE | www.plosone.org 8 April 2009 | Volume 4 | Issue 4 | e5209