Chondroitin Sulphate Proteoglycan 4 (NG2/CSPG4) Localization in Low- and High-Grade Gliomas

Abstract

Background: Neuron glial antigen 2 or chondroitin sulphate proteoglycan 4 (NG2/CSPG4) is expressed by immature precursors/progenitor cells and is possibly involved in malignant cell transformation. The aim of this study was to investigate its role on the progression and survival of sixty-one adult gliomas and nine glioblastoma (GB)-derived cell lines.

Methods: NG2/CSPG4 protein expression was assessed by immunohistochemistry and immunofluorescence. Genetic and epigenetic alterations were detected by molecular genetic techniques.

Results: NG2/CSPG4 was frequently expressed in IDH-mutant/1p19q-codel oligodendrogliomas (59.1%) and IDH-wild type GBs (40%) and rarely expressed in IDH-mutant or IDH-wild type astrocytomas (14.3%). Besides tumor cells, NG2/CSPG4 immunoreactivity was found in the cytoplasm and/or cell membranes of reactive astrocytes and vascular pericytes/endothelial cells. In GB-derived neurospheres, it was variably detected according to the number of passages of the in vitro culture. In GB-derived adherent cells, a diffuse positivity was found in most cells. NG2/CSPG4 expression was significantly associated with EGFR gene amplification (p = 0.0005) and poor prognosis (p = 0.016) in astrocytic tumors.

Conclusion: The immunoreactivity of NG2/CSPG4 provides information on the timing of the neoplastic transformation and could have prognostic and therapeutic relevance as a promising tumor-associated antigen for antibody-based immunotherapy in patients with malignant gliomas.

Keywords: CAR-Ts; NG2/CSPG4; gliomagenesis; immunotherapy; neo-angiogenesis; prognosis.

Figure 1

NG2/CSPG4 immunohistochemistry in oligodendroglial tumors. (A) WHO grade II oligodendroglioma. Isolated tumor cells (arrow) with NG2/CSPG4-positive cell membranes; DAB, original magnification (OM) ×400. (B) WHO grade III oligodendroglioma. NG2/CSPG4-positive area with honeycomb appearance; DAB, OM ×400. (C) Id. Isolated CNPase-positive tumor cells in infiltration area; DAB, OM ×400. (D) Id. Sox10-positive tumour cells; DAB, OM ×200. (E) Id. NG2/CSPG4-positive reactive astrocytes inside the tumor; DAB, OM ×200. (F) Id. GFAP-positive reactive astrocytes (arrow) intermingled with GFAP-negative tumor cells; DAB, OM ×400. (G) Id. GFAP-positive (alkaline phosphatase red) and mIDH1^R132H-negative reactive astrocytes (arrow) intermingled with mIDH1^R132H-positive (DAB) and GFAP-negative tumor oligodendrocytes (arrow); OM ×400. WHO, World Health Organization; DAB, 3,3′-Diaminobenzidine.

Figure 2

NG2/CSPG4 immunohistochemistry in astrocytic tumors. (A) WHO grade II astrocytoma. NG2/CSPG4-negative tumor cells and -positive reactive astrocytes; DAB, original magnification (OM) ×200. (B) IDH-wild type glioblastoma (GB). Area of NG2/CSPG4-positive cells; DAB, OM ×200. (C) Id. NG2/CSPG4-positive tumor cells and reactive astrocytes oriented on vessels; DAB, OM ×200. (D) Id. NG2/CSPG4-negative area of necrosis; DAB, OM ×200. (E) Id. NG2/CSPG4-positive vascular pericytes in microvascular proliferations; DAB, OM ×200. (F) Id. NG2/CSPG4-positive pericytes in a glomerulus with sprout; DAB, OM ×200. (G) Id. α-SMA-positive pericytes in infiltration area; DAB, OM ×200. (H) Id. PDGFRβ-positive pericytes in hyperproliferative area; DAB, OM ×200. WHO, World Health Organization; IDH, isocitrate dehydrogenase 1/2; DAB, 3,3’-Diaminobenzidine.

Figure 3

Immunofluorescence (IF) analysis of NG2/CSPG4 expression in glioblastoma (GB)-derived human cell lines. IF of a GB-derived neurosphere cell line for (A) NG2/CSPG4 (green), original magnification (OM) ×200; (B) Nestin (red), OM ×200; (C) MSel1 (red), OM ×200; (D) Notch-2 (green), OM ×200. IF staining of an adherent cell line for (E) NG2/CSPG4 (green), OM ×400; (F) GFAP (green), OM ×400; (G) β-III Tubulin (red), OM ×200; (H) Galactocerebroside C (red), OM ×200. Nuclei were counterstained with DAPI (blue). DAPI, 4′,6-diamidino-2-phenylindole.

Figure 4

Relationship between NG2/CSPG4 expression and survival in glioma patients. Kaplan–Meier survival curves for overall survival (OS) with respect to NG2/CSPG4 immunoreactivity after patient stratification for the histologic grade in 24 patients with astrocytic gliomas.

Figure 5

Scheme of NG2/CSPG4 expression in CNS cytogenesis and gliomagenesis. (A) Scheme representing the expression of the NG2 marker during normal neurogenesis and the different cell fates of NG2-glia. Canonically, NG2-glia (NG2+ glial restricted progenitors) has the capability to proliferate and differentiate into oligodendrocyte precursor cells (OPCs), then giving rise to oligodendrocytes in the immature and mature brain. However, NG2-glia can also differentiate into astrocytes. Additionally, in vitro, NG2-glia can differentiate into type-2 astrocytes through the O2A progenitor cells. (B) NG2+ cells can also be considered as potential cells for the origin of malignant glioma. During tumorigenesis, the glioma stem cell is believed to derive from the transformation of neural stem cells or from the dedifferentiation and transformation of NG2+ glial restricted progenitors, OPCs, or mature cells (astrocytes and oligodendrocytes). The arising tumor cells show potential for self-renewal and express markers associated with both stem and progenitor cell types [85].