Molecular and phenotypic characterisation of paediatric glioma cell lines as models for preclinical drug development

Abstract

Background: Although paediatric high grade gliomas resemble their adult counterparts in many ways, there appear to be distinct clinical and biological differences. One important factor hampering the development of new targeted therapies is the relative lack of cell lines derived from childhood glioma patients, as it is unclear whether the well-established adult lines commonly used are representative of the underlying molecular genetics of childhood tumours. 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.

Principal findings: All lines proliferate as adherent monolayers and express glial markers. Copy number profiling revealed complex genomes including amplification and deletions of genes known to be pivotal in core glioblastoma signalling pathways. Expression profiling identified 93 differentially expressed genes which were able to distinguish between the adult and paediatric high grade cell lines, including a number of kinases and co-ordinated sets of genes associated with DNA integrity and the immune response.

Significance: These data 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 and demonstrate the practicability of using paediatric glioma cell lines for preclinical and mechanistic studies.

Figure 1. Immunophenotyping of paediatric glioma cell lines.

All lines were grown as monolayers and stained with a variety of glial and stem cell markers including glial fibrillary acidic protein (GFAP), S100, vimentin, synaptophysin, nestin and CD133. H&E – haematoxylin and eosin. All images original magnification ×400.

Figure 2. Genomic profiling of paediatric glioma cell lines.

Copy number and loss of heterozygosity (LOH) profiles were generated by Affymetrix 500K SNP arrays. Log₂ ratios are plotted (y axis) for each probeset according to chromosomal location (x axis). Loss (blue) and retention (yellow) of heterozygosity is depicted in the lower portion of the plots. Fluorescent in situ hybridisation validation of selected copy number changes is represented with clones for CDK4, RB1, CDKN2A/B, PDGFRA and PTEN (lCy5/SpectrumOrange) and appropriate centromeres (fluorescein/SpectrumGreen).

Figure 3. Constitutive activation of key signalling pathways in glioma cell lines.

Western blots analysis of c-Raf, phospho/total Erk1/2, phospho/total Akt, phospho/total GSK3β, phospho/total S6 and GAPDH as loading control in adult (LN229, A172, U118MG, U138MG, U87MG, SF268) and paediatric (SF188, KNS42, UW479, Res259, Res186) glioma cell lines.

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.