Abstract
BACKGROUND: Glioblastoma (GB) is a lethal malignancy whose clinical intransigence has been linked to extensive intra-clonal genetic and phenotypic diversity and the common emergence of therapeutic resistance. This interpretation embodies an implicit assumption that cancer stem cells or tumour propagating cells are themselves genetically and functionally diverse. Sub-clonal evolution and cancer stem cells (CSCs) have been considered as alternative mechanisms for disease progression but it can also be argued that they are part of the same process because clonal diversity is likely to be generated and sustained by genetically distinct CSCs, which provide the units for evolutionary selection. However, this has yet to be demonstrated in GB. METHODS: We screened primary GB tumours by SNP array to identify copy number alterations (minimum of three) that could be visualised in single cells by multi-colour FISH. Interrogation of neurosphere-derived cells (from four patients) and cells derived from secondary transplants of these same cells in NOD-SCID mice allowed us to infer the clonal and phylogenetic architectures. RESULTS: In all four cases studied by multicolour FISH there was genetic heterogeneity in the neurosphere cells, with each case showing a unique, branched phylogenetic architecture. In each case, more than one sub-clone was capable of propagating tumours in secondary transplanted mice. CONCLUSIONS: Our interrogation of sub-clonal genetic diversity of human GB has revealed for the first time that tumour-propagating cells in GB are genetically heterogeneous, with variable competitive capacity for tumour propagation in vivo. The link forged between genetic diversity, clonal architecture and propagating activity in vivo may facilitate the characterisation of mutational variants responsible for disease recurrence and therapeutic resistance in patients.