Expression and clinical significance of EGFL7 in malignant glioma

Abstract

Purpose: Tumor angiogenesis is an important factor for the continuous growth of human malignancies and can be used to predict the prognosis for patients. In the current study, we examined the expression of EGF-like domain 7 (EGFL7), an endothelial cell-derived secreted factor, in malignant gliomas and explored its clinical significance.

Methods: We determined the steady-state mRNA levels of EGFL7 from 36 fresh glioma samples by semi-quantitative RT-PCR and the protein levels from 45 paraffin-embedded glioma samples by immunohistochemistry, respectively. Normal brain tissues from 10 patients with brain trauma were used as control. We also analyzed the correlations between the expression levels of EGFL7 and various clinical parameters, including patient gender, age, tumor grade, tumor proliferation marker Ki-67, and microvessel density (MVD).

Results: We found that EGFL7 was not detectable in normal brain tissues, but was up-regulated in both tumor cells and vascular endothelial cells within malignant glioma. The expression level of EGFL7 in malignant glioma significantly correlated with the tumor grade, Ki-67 expression and MVD (P < 0.01).

Conclusions: Our data suggest that EGFL7 expression is a novel predictive factor for the clinical progression of malignant glioma, and may constitute a therapeutic target for anti-angiogenesis therapy in patients with the disease.

Fig. 1

The EGFL7 expression level significantly correlated with tumor grade. a The relative mRNA levels of EGFL7 (as a ratio to the β-actin level) in 36 glioma samples and 10 samples of normal brain tissue (control) were determined by semi-quantitative RT–PCR and compared between samples with different histological grades as defined by WHO standards. b The protein levels of EGFL7 from 45 brain glioma and 10 normal brain tissues (control) were examined by immunohistochemistry, quantified by the IRS method, and compared between samples with different histological grades. *P < 0.01 as compared to the control group; ^# P < 0.01 as compared to samples of Grade I–II

Fig. 2

EGFL7 was expressed in tumor (the 2nd row) and vascular endothelial cells (the 3rd row), and its expression level, as well as that for Ki-67 and Factor VIII, increased with tumor grade. a Normal brain tissue and gliomas were analyzed by Hemotoxylin and Eosin (HE) staining or immunohistochemistry using the indicated antibodies (yellow or brown signals). Brain tissue areas with abundant tumor cells (the 2nd row) or with enriched vascular endothelial cells (the 3rd row) were chosen to present the expressions of EGFL7 in these two compartments, respectively. For immunohistochemistry, the sections were counter-stained with hemotoxylin (blue signals). b The expression of EGFL7 was examined in human glioma cell line U251 by immunocytochemistry (right panel). The isotype control IgG was used as negative control (left panel)

Fig. 3

Ki-67 and microvessel density levels significantly correlated with tumor grade. Normal brain (control) and glioma tissue samples were stained with antibodies against either Ki-67 or Factor VIII and examined by immunohistochemistry. The levels of positive signals were quantified as Ki-67 labeling index (Ki-67 LI) (a) and microvessel density (MVD) (b) as detailed in the “Materials and methods” section. *P < 0.01 compared to the control group; ^# P < 0.01 compared to Grade I–II samples

Fig. 4

EGFL7 expression, Ki-67 LI and MVD were positively and significantly correlated with each other. Based on the quantifications of immunohistochemistry signals from the 45 glioma samples, scatter plots were used to depict the correlations between Ki-67 LI and MVD (a) (r ² = 0.787, P < 0.01), Ki-67 LI and EGFL7 (b) (r ² = 0.793, P < 0.01) and MVD and EGFL7 (c) (r ² = 0.684, P < 0. 01)