New strategy for the analysis of phenotypic marker antigens in brain tumor–derived neurospheres in mice and humans

Abstract

OBJECT: Brain tumor stem cells (TSCs) hypothetically drive the malignant phenotype of glioblastoma multiforme (GBM), and evidence suggests that a better understanding of these TSCs will have profound implications for treating gliomas. When grown in vitro, putative TSCs grow as a solid sphere, making their subsequent characterization, particularly the cells within the center of the sphere, difficult. Therefore, the purpose of this study was to develop a new method to better understand the proteomic profile of the entire population of cells within a sphere. METHODS: Tumor specimens from patients with confirmed GBM and glioma models in mice were mechanically and enzymatically dissociated and grown in traditional stem cell medium to generate neurospheres. The neurospheres were then embedded in freezing medium, cryosectioned, and analyzed with immunofluorescence. RESULTS: By sectioning neurospheres as thinly as 5 mum, the authors overcame many of the problems associated with immunolabeling whole neurospheres, such as antibody penetration into the core of the sphere and intense background fluorescence that obscures the specificity of immunoreactivity. Moreover, the small quantity of material required and the speed with which this cryosectioning and immunolabeling technique can be performed make it an attractive tool for the rapid assessment of TSC character. CONCLUSIONS: This study is the first to show that cryosectioning of neurospheres derived from glioma models in mice and GBM in humans is a feasible method of better defining the stem cell profile of a glioma.