Histidine-rich glycoprotein can prevent development of mouse experimental glioblastoma

Abstract

Extensive angiogenesis, formation of new capillaries from pre-existing blood vessels, is an important feature of malignant glioma. Several antiangiogenic drugs targeting vascular endothelial growth factor (VEGF) or its receptors are currently in clinical trials as therapy for high-grade glioma and bevacizumab was recently approved by the FDA for treatment of recurrent glioblastoma. However, the modest efficacy of these drugs and emerging problems with anti-VEGF treatment resistance welcome the development of alternative antiangiogenic therapies. One potential candidate is histidine-rich glycoprotein (HRG), a plasma protein with antiangiogenic properties that can inhibit endothelial cell adhesion and migration. We have used the RCAS/TV-A mouse model for gliomas to investigate the effect of HRG on brain tumor development. Tumors were induced with platelet-derived growth factor-B (PDGF-B), in the presence or absence of HRG. We found that HRG had little effect on tumor incidence but could significantly inhibit the development of malignant glioma and completely prevent the occurrence of grade IV tumors (glioblastoma).

Figure 1. RCAS-HRG infected DF-1 cells could produce functional HRG.

(A) Viral produced HRG protein could be detected by western blot in conditioned media from DF-1 cells infected with RCAS-HRG but not from RCAS-PDGFB or RCAS-eGFP infected cells. Purified HRG was used as a positive control (+). (B) Viral produced HRG could significantly inhibit migration of HUVEC cells towards VEGF. t-test, ** p<0.01, *** p<0.001.

Figure 2. HRG had no effect on primary glial cell proliferation.

(A) Primary glial cells were infected with RCAS-eGFP, RCAS-HRG or RCAS-PDGFB-HA and expression of the viral transduced proteins was analyzed with immunocytochemistry. The infection efficiency was similar in all conditions. (B) Proliferation assay on infected cells showed no effect of HRG compared to control cells on glial cell proliferation at day 7. Curves show the mean (±SEM) from three independent experiments for HRG and eGFP, and two independent experiments for PDGF-B.

Figure 3. Presence and expression of viral transduced PDGF-B and HRG in tumors.

(A) Insertion of the viral transduced human PDGF-B and HRG cDNA in genomic DNA prepared from PDGF-B+X (P+X) and PDGF-B+HRG (P+H) induced tumors. The tumor grade is given above each sample. Genomic DNA from U-706MG-a cells was used as positive control (+), and genomic DNA from an untreated mouse was used as negative control (−). (B) Expression of human PDGF-B and HRG mRNA in P+X and P+H tumors. RNA extracted from U-343MG and DF-1 RCAS-HRG cells were used as positive control for PDGF-B and HRG, respectively (+), and RNA from an untreated mouse brain was used as negative control (−).

Figure 4. Tumor histopathology and vessel morphology.

(A) Histopathology (H&E) of representative grade II–IV tumors induced with PDGF-B+X (P+X) and corresponding immunostainings for CD31. Arrows indicate mitoses. (B) Histopathology (H&E) of representative grade II–III PDGF-B+HRG (P+H) tumors and corresponding CD31 stainings. Bar = 100 µm.

Figure 5. Distribution of tumor malignancy grades.

(A) Distribution of tumor grades (II–IV) in PDGF-B+X (P+X) and PDGF-B+HRG (P+H) injected Ntv-a Arf-/- mice. * p<0,05 (B) Distribution of low grade (II) versus malignant (III+IV) glioma. *p<0,05.