A non-hierarchical organisation of tumorigenic NG2 cells in glioblastoma promoted by EGFR

Background. Expression of neuron-glial antigen 2 (NG2) identifies an aggressive malignant phenotype in glioblastoma (GBM). Mouse models have implicated NG2 in the genesis, evolution, and maintenance of glial cancers and have highlighted potential interactions between NG2 and epidermal growth factor receptor (EGFR). However, it is unknown whether the lineage relationship of NG2+ and NG2? cells follows a hierarchical or stochastic mode of growth. Furthermore, the interaction between NG2 and EGFR signaling in human GBM is also unclear. Methods. Single GBM NG2+ and NG2? cells were studied longitudinally to assess lineage relationships. Short hairpin RNA knockdown of NG2 was used to assess the mechanistic role of NG2 in human GBM cells. NG2+ and NG2? cells and NG2 knockdown (NG2-KD) and wild type (NG2-WT) cells were analyzed for differential effects on EGFR signaling. Results. Expression of NG2 endows an aggressive phenotype both at single cell and population levels. Progeny derived from single GBM NG2? or GBM NG2+ cells consistently establish phenotypic equilibrium, indicating the absence of a cellular hierarchy. NG2 knockdown reduces proliferation, and mice grafted with NG2-KD survive longer than controls. Finally, NG2 promotes EGFR signaling and is associated with EGFR expression. Conclusions. These data support a dynamic evolution in which a bidirectional relationship exists between GBM NG2+ and GBM NG2? cells. Such findings have implications for understanding phenotypic heterogeneity, the emergence of resistant disease, and developing novel therapeutics.