Profiling Paediatric Gliomas Figure 4. Expression profiling of paediatric and adult glioblastoma cell lines. (A) Heatmap demonstrating hierarchical clustering of 93 differentially expressed genes between paediatric (SF188, KNS42, UW479) and adult (LN229, A172, U118MG, U87MG, SF268) high grade glioma cell lines. (B) Quantitative real-time (TaqMan) RT-PCR confirming differential expression of CRKL, LYN, EPHA6 and AXL. Expression values are plotted relative to Universal Human Reference RNA. (C) Gene Set Enrichment Analysis highlighting co-ordinated differential expression of gene sets defined a priori. Enriched in paediatric high grade glioma cell lines - MORF_MSH2, GNF2_MLH1, GCM_RAD21, DNA_replication_reactome; enriched in adult lines IL6_SCAR_FIBRO_UP, CROONQUIST_IL6_RAS_UP, TGFBETA_C1_UP. Nominal p value,0.001. doi:10.1371/journal.pone.0005209.g004 confounding factors of grade. Despite the small number of lines profiled, we were able to identify 93 genes significantly differentially expressed between paediatric and adult high grade glioma cell lines (Figure 4A). The differentially expressed genes included several kinases which were validated by quantitative real- time RT-PCR (Figure 4B). These included members of the Src family kinases - LYN and the adaptor molecule CRKL, highly expressed in paediatric lines, along with SRC itself and also YES1. Also upregulated in the paediatric vs adult lines were several early response genes including FOS and FOSB. Highly expressed in the adult lines were the receptor tyrosine kinases EPHA6 and AXL, and also CDK9 and ILK. In addition, gene set enrichment analysis (GSEA) highlighted co-ordinated differentially high expression of several gene lists associated with maintenance of DNA replication and repair in paediatric vs adult cell lines (MORF_MSH2, GNF2_MLH1, GCM_RAD21, DNA_replication_reactome) (Figure 4C). Further- more, gene sets representing co-ordinated regulation of extracel- lular matrix protein in response to cytokines were found enriched in the adult compared to the paediatric lines (IL6_SCAR_- FIBRO_UP, CROONQUIST_IL6_RAS_UP, TGFBETA_- C1_UP). Although the nominal p values of these analyses was less than 0.001, q values for estimates of the false discovery rate were all greater than 0.9, presumably due to the small sample size. Discussion Understanding and exploiting the similarities and differences between adult and paediatric gliomas is an important strategy for the development of new targeted therapies. The intrinsic drug resistance of high grade gliomas represents a major challenge in the clinical management of children with the disease, and efforts to PLoS ONE | www.plosone.org 7 April 2009 | Volume 4 | Issue 4 | e5209