Pathology - Theses

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Start date: 2015 × End date: 2015 × Type: PhD thesis × Author: Brown, Daniel ×

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    Investigating stemness and plasticity models as sources of intra-tumoural heterogeneity in Glioblastoma multiforme
    Brown, Daniel ( 2015-09-09)
    Glioblastoma mutiforme (GBM) is a heterogeneous tumour of the brain with a poor prognosis. Genome-wide profiling has revealed four molecular subtypes, yet there is no significant difference in long-term survival between subtypes. Recurrence and resistance to GBM therapy is believed to be due to an underlying Glioma Stem Cell (GSC) subpopulation. To identify gene expression differences with the ability to predict patient survival, the cancer stem cell subpopulation of Patient Derived Glioma Cells (PDGCs) was isolated based on the expression of the putative stem cell marker CD133. RNA-seq libraries were prepared from six different PDGCs with the identification of 37 differentially expressed genes. Downstream characterisation of the cellular phenotypes exhibited by CD133+ and CD133- cells indicated that CD133 does not enrich for stem and malignant phenotypes. Genes coexpressed with GSCs markers were used to build a gene signature that classified patients based on a CD133 coexpression module signature (CD133-M) or a CD44 coexpression module signature (CD44-M) subtype. CD133-M tumours were enriched for the Proneural GBM subtype and correspondingly CD44-M tumours were enriched for the Mesenchymal subtype. Gene set enrichment identified proliferative pathways as activated in CD133-M and invasion/ migration pathways as enriched in CD44-M. CD133-M patients benefitted most from radiotherapy while CD44-M classified patients responded equally well to temozolomide or radiotherapy. In different PDGCs there was a culture specific equilibrium of distinct PN and MES subpopulations, potentially due to the genetic background of the original patient. The distribution of Proneural and Mesenchymal cells in the same tumour was measured in GBM sections using the expression of Olig2 and CD44 proteins as markers. Heterogeneous expression of these markers in tumours was observed, consistent with the heterogeneity observed in cell cultures. The influence of oxygen tension and chemoradiotherapy on the intra-tumoural equilibrium of PN and MES cells in PDGCs was investigated, with hypoxia inducing a MES to PN shift and chemoradiotherapy inducing a PN to MES shift respectively. The results of this study favour a cellular plasticity model over a hierarchical cancer stem cell model and is in agreement with accumulating evidence that CD133 and CD44 expression are markers of PN and MES molecular subtypes respectively. Both PN and MES subtypes coexist in the same tumours while rare cells that transiently express both CD44 and CD133 (having PN and MES properties) may be cancer stem cells. The results of this thesis suggest the tumour specific proportion of these states is determined by a combination of genetic and environmental factors. Surveillance and modulation of intra-tumoural heterogeneity could be of benefit in the clinical management of GBM.