EPH Profiling of BTIC Populations in Glioblastoma Multiforme Using CyTOF

The ability to elucidate the phenotype of brain tumor initiating cell (BTIC) in the context of bulk tumor in glioblastoma multiforme (GBM) provides significant therapeutic benefits for therapeutic evaluation. For the identification of such an elusive and rare subpopulation of cells, a single cell analysis technology with deep profiling capabilities known as Mass Cytometry (CyTOF) can prove to be highly useful. CyTOF circumvents the spectral overlap limitations of traditional flow cytometry by replacing fluorophores with metal isotope tags, allowing the accurate detection of significantly more parameters at the same time. In this chapter, we demonstrate that synthetic antibodies can be conjugated with metal isotope tags for CyTOF analysis, resulting in the development of a highly tailored, custom multi-parameter panel. This toolset was used to stain patient-derived GBM cells, which was analyzed via CyTOF. Analysis software viSNE and SPADE were applied to study the co-expression patterns of the Eph Receptor (EphR) family and several putative BTIC markers in GBM, resulting in the identification of a distinct group of cells consistent with a BTIC subpopulation. This approach can be readily adapted to the detection of cancer stem-like cells in other cancer types.