NG2 expression in glioblastoma identifies an actively proliferating population with an aggressive molecular signature

Abstract

Glioblastoma multiforme (GBM) is the most common type of primary brain tumor and a highly malignant and heterogeneous cancer. Current conventional therapies fail to eradicate or curb GBM cell growth. Hence, exploring the cellular and molecular basis of GBM cell growth is vital to develop novel therapeutic approaches. Neuroglia (NG)-2 is a transmembrane proteoglycan expressed by NG2+ progenitors and is strongly linked to cell proliferation in the normal brain. By using NG2 as a biomarker we identify a GBM cell population (GBM NG2+ cells) with robust proliferative, clonogenic, and tumorigenic capacity. We show that a significant proportion (mean 83%) of cells proliferating in the tumor mass express NG2 and that over 50% of GBM NG2+ cells are proliferating. Compared with the GBM NG2- cells from the same tumor, the GBM of NG2+ cells overexpress genes associated with aggressive tumorigenicity, including overexpression of Mitosis and Cell Cycling Module genes (e.g., MELK, CDC, MCM, E2F), which have been previously shown to correlate with poor survival in GBM. We also show that the coexpression pattern of NG2 with other glial progenitor markers in GBM does not recapitulate that described in the normal brain. The expression of NG2 by such an aggressive and actively cycling GBM population combined with its location on the cell surface identifies this cell population as a potential therapeutic target in a subset of patients with GBM.

Fig. 1.

Glioblastoma multiforme (GBM) neuroglia (NG)-2+ cells show higher levels of cell proliferation compared with the GBM NG2− cells from the same tumor. Analysis of 8 GBM showed proliferative dominance by NG2+ cells (Supplementary Material, Table S1). Illustrative example from 1 cell line: (A) Normalized data from MTS assay shows higher signal in the GBM NG2+ cell cultures, indicating more robust cell growth (3 repeats per cell line). (B) Higher number of total cells in the GBM NG2+ cultures (3 repeats per cell line). (C) Ki67 staining of the fluorescent-activated cell–sorted GBM NG2+ and GBM NG2− cells (scale bars: 100 µm). (D) Quantification of the number of Ki67+ cells showing higher number in the GBM NG2+ cultures (3 repeats for each of 3 cell lines). (E) Quantification of the proportion of Ki67 cells showing higher proportion in the GBM NG2+ cultures. Note: Comparisons were made between NG2+ and NG2− populations within each tumor. Comparison between tumors was not made owing to heterogeneity between patients.

Fig. 2.

The proliferative advantage of the glioblastoma multiforme (GBM) neuroglia (NG)-2+ cells is cell autonomous. MTS assay of NG2+ and NG2− fractions from 3 cell lines cultured under (A) basic culture conditions, (B) serum-free culture conditions, (C) Using conditioned media, and (D) Under differentiation (fetal calf serum) culture conditions. Higher absorbance values are observed in the GBM NG2+ cultures under all different culture conditions. Limiting dilution assay analysis of (E) GBM NG2+ and (F) GBM NG2− cells shows that the GBM NG2+ cells contain a higher number of clonogenic cells as indicated by u values. On the y axis r is the number of nonresponding wells and n is the total number of wells used. The dotted lines in E and F represent the 95% confidence intervals. All differences in A, B, C and D are statistically significant (Kruskal-Wallis ANOVA on ranks; P< .001).

Fig. 3.

The proliferative pdvantage of the glioblastoma multiforme (GBM) neuroglia (NG)-2+ cells can be observed in clinical samples. (A) Flow cytometry analysis of the coexpression of Ki67 and NG2 in freshly dissociated and fixed GBM tumor samples. (B) Confocal micrograph of a GBM tumor showing cells coexpressing NG2 (green) and Ki67 (red). (C) Higher magnification of the highlighted area in panel B showing the coexpression of NG2 (green) and Ki67 (red) with Z-stack. (D) Table of quantitative data from 10 GBM tumor samples showing the proportions of coexpression of NG2 and the proliferative marker Ki67. Scale bars: 50 µm in B and 10 µm in C.

Fig. 4.

Glioblastoma multiforme (GBM) neuroglia (NG)-2+ cells exhibit higher tumorigenic capability compared with the GBM NG2− cells. (A) Subcutaneous mass developed in the right hindlimb after the implantation of GBM NG2+ cells. (B) Small subcutaneous mass developed in the left hindlimb after the implantation of the GBM NG2− cells. (C) Subcutaneous tumors excised from A and B. (D) Intracerebral tumor masses can be observed in mice after orthotopic implantation of the GBM NG2+ cells as shown by human nuclei staining. (E) Orthotopic implantations of the GBM NG2− cells do not form tumors. (F) Occasionally isolated HN+ cells can be observed in brains of animals implanted with the GBM NG2− cells. (G) Comparative genomic hybridization (CGH) of the GBM NG2+ cells. (H) CGH of the GBM NG2− cells. In G and H, black arrows indicate gain of chromosome 7 and loss of chromosome 10 (black arrows), which are typical cytogenetic abnormalities in GBM. Green arrows show abnormalities that are more pronounced in the GBM NG2+ cells, whereas red arrows indicate abnormalities that only exist in the GBM NG2+ cells. (I) Example of a heat map of genes differentially expressed by the GBM-NG2+ and GBM-NG2− cells. Scale bars: 100 µm in D, E, and F; 10 mm in C.

Fig. 5.

The neuroglia (NG)-2 proteoglycan is expressed by glioblastoma multiforme (GBM) tumors. (A) Round-shaped cells expressing NG2 are observed in GBM. (B) The GBM-NG2+ cells have a close relationship to the GBM blood vessels. (C) The majority of GBM NG2+ cells do not express the endothelial marker von Willebrand factor (vWF). (D) Confocal microscopy confirms that the majority of GBM NG2+ cells (green) are negative for vWF (red). (E) Few GBM NG2+ cells express the pericyte marker PDGFRβ. (F) Confocal micrograph showing that some cells coexpress NG2 (green) and PDGFRβ (red) but most GBM NG2+ cells are NG2+/PDGFRβ−. Scale bars: 50 µm in A and D; 100 µm in B, C, and E; and 25 µm in F.

Fig. 6.

The phenotypic identity of the glioblastoma multiforme (GBM) neuroglia (NG)-2+ cells is distinct from nonmalignant NG2+ progenitors. Patterns of coexpression are variable: (A) 1% of GBM cells coexpress NG2+ cells (green) and Olig2 (red), (B) 1% are NG2−/Olig2+, and (C) 47% of GBM cells are NG2+/Olig2−. Similarly, coexpression of NG2 (green) and platelet derived growth factor receptor alpha (PDGFRα; red) can be observed in GBM tumors (D). Also, GBM PDGFRα+ (red) cells can be negative for NG2 (green; E) and some GBM NG2+ cells (green) do not express PDGFRα (red; F). Scale bars: 25 µm in A, B, C, D, and E; 50 µm in F.