Abstract 5142: Detection of genomic changes in glioblastoma stem cell subpopulations by array comparative genomic hybridisation
Authors: Daniela Loetsch, Christine Pirker, Sabine Spiegl-Kreinecker, Michael Grusch, Johannes Fischer, Michael Micksche, Walter Berger
Journal: Cancer Research (2010)

Abstract Glioblastoma multiforme (GBM) is one of the most aggressive tumors characterised by bad prognosis and limited treatment response. Stem-cell like GBM subpopulations are believed to cause tumor initiation and treatment resistance. Aim of this study was to clarify whether the GBM stem-cell like subpopulations (GSCLS) present in GBM cell cultures are characterized by specific genomic alterations. Fourteen GBM primary cultures and 6 cell lines were analyzed for the presence of GSCLS compartments by investigating their ability to grow in serum-free neurobasal medium. Growth in neurospheres was compared to the expression of the stem cell markers CD133, nestin and SOX2. Nine out of 14 GBM primary cultures and 3 out of 7 GBM cell lines were able to grow as stable non-adherent neurospheres in NBE-medium for 3 up to 14 months. From those GBM samples, able to grow as neurospheres in NBE-medium, 33% expressed all 3 stem cell markers, 42% two markers and 25% one stem cell marker. The samples who grew as monolayers or died in NBE-medium, expressed only one (30%) or no stem cell marker (70%). Genomic changes in selected primary cultures (N=8) and a GBM cell line grown in parallel for 6 to 60 weeks in spheres and monolayer cultures were comparably analyzed by (array)CGH. In all cases analyzed, GSCLS grown as neurospheres contained widely the identical chromosomal gains/amplifications and losses as the respective parental cell cultures. Differences concerned random low-level gains/losses of large chromosomal regions or whole chromosome arms. However, on closer inspection by aCGH small but distinct amplifications/deletions could be found in the GSCLS grown in neurospheres mainly affecting single gene loci. Further changes at the SOX2 gene locus and SOX2 gene expression were analyzed in GBM cell lines and primary culture. An increase of SOX2 gene expression was observed in GSCLS grown as neurospheres compared to the respective monolayer cultures as determined by qRT PCR. Preliminary data show that the SOX2 expression is higher in GBM tumor samples compared to the respective cell lines indicating loss of stem cell features during in vitro cell culture. Consequently, the association between the expression of stem cell markers, like SOX2, and the survival time of GBM patients is currently established. In ongoing experiments genome-wide gene expression differences between GSCLS neurospheres and the respective monolayer cultures are established and compared with the aCGH data. In conclusion, we demonstrate that the majority of human GBM primary cell cultures/cell lines contain stem cell-like subpopulations. These sphere-forming cell clones harbor small distinct gene amplifications/deletions as compared to the respective monolayer cell cultures and the genes residing in the affected regions are currently characterized. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5142.