Expression of integrin alphavbeta3 in gliomas correlates with tumor grade and is not restricted to tumor vasculature

Abstract

In malignant gliomas, the integrin adhesion receptors seem to play a key role for invasive growth and angiogenesis. However, there is still a controversy about the expression and the distribution of alpha(v)beta(3) integrin caused by malignancy. The aim of our study was to assess the extent and pattern of alpha(v)beta(3) integrin expression within primary glioblastomas (GBMs) compared with low-grade gliomas (LGGs). Tumor samples were immunostained for the detection of alpha(v)beta(3) integrin and quantified by an imaging software. The expression of alpha(v)beta(3) was found to be significantly higher in GBMs than in LGGs, whereby focal strong reactivity was restricted to GBMs only. Subsequent analysis revealed that not only endothelial cells but also, to a large extent, glial tumor cells contribute to the overall amount of alpha(v)beta(3) integrin in the tumors. To further analyze the integrin subunits, Western blots from histologic sections were performed, which demonstrated a significant difference in the expression of the beta(3) integrin subunit between GBMs and LGGs. The presented data lead to new insights in the pattern of alpha(v)beta(3) integrin in gliomas and are of relevance for the inhibition of alpha(v)beta(3) integrin with specific RGD peptides and interfering drugs to reduce angiogenesis and tumor growth.

Figure 1

Hematoxilin & Eosin (H&E) (A) and immunostain of integrin α_vβ₃ (C), CD31 (D) and fluorescent overlay (B) in samples of glioblastoma. Standard H&E staining (A) shows typical morphology of a malignant glioma. Immunohistochemical staining of α_vβ₃ (C) is intense in vascular structures (asterisk) as confirmed by a consecutive section stained for the endothelial cell marker CD31 (D). Rather, α_vβ₃ is clearly detectable throughout the whole section as expressed by the glial tumor cells (arrow). A fluorescent overlay picture of another area with dense tumor growth (B) demonstrates that the integrin α_vβ₃ (green) is not restricted to vascular structures (red, asterisk) but shows a ubiquitous distribution pattern (cell nuclei blue). Scale bars: 100 µm. Abbreviations: EP = endothelial proliferation; M = mitosis.

Figure 2

Detection of α_vβ₃ integrin in malignant (A) and a low‐grade (B) glioma and analysis by an imaging software (C,D). The malignant glioma (A) exhibit a much higher expression of α_vβ₃ integrin (red) than the low‐grade glioma (B), where only very few glial cells are immunopositive for the integrin. The detection of the staining intensity in the malignant glioma shows a large overall baseline (red) expression with certain strong (yellow) or even intense (white) expression in microvascular structures (C). In contrast, only some red dots can be detected in the low‐grade glioma (D). Scale bars: 100 µm

Figure 3

Statistical analysis of the α_vβ₃ integrin immunohistochemistry in patients with glioblastomas (GBMs) and low‐grade gliomas (LGGs) and non‐CNS tumors (non‐CNS). A. Mean staining intensity of non‐CNS tumors, GBMs and LGGs. B. Fraction of mild, moderate and strong stained tissue in GBMs and LGGs. C. Mean staining intensity of glial tissue only in GBMs and LGGs. D. Fraction of mild, moderate and strong stained tissue of glial tissue only in GBMs and LGGs. Significant differences (P < 0.05, Student's t‐test) are indicated with an asterisk.

Figure 4

Western blot analysis of subunits and degradation products of the α_vβ₃ integrin. The Western blot analysis of five different glioblastoma (GBM) tissue samples (A, left lanes) and densitometric analysis of integrin subunits (B). In all glial tumors, degradation products of the α_v subunit (α_v light chains) were detected as a double band at 25/27 kDa (▸). Expression was constantly present but heterogeneous between different GBMs. The β₃ subunit was detected at about 96 kDa (▸). Degradation products of α_v and β₃ subunit were found at 55 kDa (▸). The detection of CD31 at about 80 kDa (right ▸) and β‐actin at about 42 kDa (right ▸) is additionally demonstrated in the lane on the right. B. Densitometric analysis revealed significant differences for the β₃ subunit between malignant gliomas and low‐grade gliomas (LGGs) (P < 0.05; Student's t‐test; indicated with an asterisk). The α_v subunit (about 137 kDa) and its degradation products (α_v light chains, 25/27 kDa) showed no significant differences between high‐ and low‐grade gliomas.

Figure 5

PCR analysis of α_vβ₃ integrin subunits. PCR analysis of α_vβ₃ integrin subunits α_v (A,B) and β₃ (C,D) of a glioblastoma (GBM; A,C) and low‐grade glioma (LGG; B,D) after different recombination cycles. Lane 1: 24 cycles, lane 2: 27 cycles, lane 3: 30 cycles, lane 4: 33 cycles and lane 5: 36 recombination cycles. Standard PCR revealed that both subunits α_v (690 bp) and β₃ (510 bp) are synthesized in high as well as low‐grade gliomas indicating that both tumor entities are able to express a functionally active α_vβ₃ integrin receptor. Marker (M) was a 50 bp ladder (Novagen, Perfect DNA ladder).