Integrin inhibitor suppresses bevacizumab-induced glioma invasion

Abstract

Glioblastoma is known to secrete high levels of vascular endothelial growth factor (VEGF), and clinical studies with bevacizumab, a monoclonal antibody to VEGF, have demonstrated convincing therapeutic benefits in glioblastoma patients. However, its induction of invasive proliferation has also been reported. We examined the effects of treatment with cilengitide, an integrin inhibitor, on bevacizumab-induced invasive changes in glioma. U87ΔEGFR cells were stereotactically injected into the brain of nude mice or rats. Five days after tumor implantation, cilengitide and bevacizumab were administered intraperitoneally three times a week. At 18 days after tumor implantation, the brains were removed and observed histopathologically. Next, the bevacizumab and cilengitide combination group was compared to the bevacizumab monotherapy group using microarray analysis. Bevacizumab treatment led to increased cell invasion in spite of decreased angiogenesis. When the rats were treated with a combination of bevacizumab and cilengitide, the depth of tumor invasion was significantly less than with only bevacizumab. Pathway analysis demonstrated the inhibition of invasion-associated genes such as the integrin-mediated cell adhesion pathway in the combination group. This study showed that the combination of bevacizumab with cilengitide exerted its anti-invasive effect. The elucidation of this mechanism might contribute to the treatment of bevacizumab-refractory glioma.

Figure 1

Antiangiogenic effect of bevacizumab. (A) Tumor vessels identified with RECA-1. Untreated U87ΔEGFR orthotopic tumor was observed with angiogenic growth. (B) The reduction of tumor vessels with bevacizumab therapy. Quantitative assessment of tumor vessels between no treatment and bevacizumab therapy was shown in following graphs. (C) Bevacizumab decreased vessel density of tumors significantly. (D) There was no significant difference between vessel short diameters of tumors. (E) Intensity of vessel area stained with RECA-1 was significantly reduced in bevacizumab-treated tumors. Scale bar, 100 μm. *P < .01. HPF, high power field.

Figure 2

Microstructure of tumor vessels in the border area with bevacizumab treatment. (A) Contralateral normal brain. There was no space between the basal lamina and AE (black arrowheads). (B) There was a distance of more than 250 nm around the tumor microvessels; the fuzzy basal lamina and tumor cytoplasm (white arrowheads and double-headed black arrows) were separated in the border area of untreated U87ΔEGFR tumors. Conversely, a tight junction (white arrows) was maintained between the endothelial cells. (C) There was less space between the basal lamina and AE in the border area of bevacizumab-treated tumors compared to untreated tumors. (D) The distance between the endothelial cells and tumor was significantly narrowed in bevacizumab-treated tumors (Bev) compared to PBS-treated tumors (PBS). L, lumen of vessels. Scale bar, 1 μm. *P < .01.

Figure 3

Antiangiogenic therapy induces invasion in the orthotopic glioma model. (A) Well-defined borders toward the brain tissue in an untreated U87ΔEGFR orthotopic rat tumor. (B) Tumor cell invasion with vascular co-option in a bevacizumab-treated tumor. Scale bar, 100 μm.

Figure 4

Expression of integrins αvβ3 and αvβ5 in the glioma cell line. U87ΔEGFR cells expressed (A) αvβ3 and (B) αvβ5 integrins at a high level, especially αvβ3, in immunocytochemistry. (C) Integrins αvβ3 and (D) αvβ5 were expressed at a high level in tumor vessels stained with von Willebrand factor and surrounding tumor cells in U87ΔEGFR rat orthotopic tumors. Scale bar, 50 μm.

Figure 5

The effect of cilengitide on bevacizumab-induced invasion. (A) Hematoxylin and eosin staining of an untreated U87ΔEGFR rat orthotopic tumor demonstrated the expansion of the tumor with well-defined borders. (B) Following treatment with bevacizumab (Bev), the tumor border was irregular with tumor invasion. (C) Tumor invasiveness was reduced at the tumor border following combination therapy with bevacizumab and cilengitide (Bev + Cile) compared to bevacizumab monotherapy. (D) The depth of tumor invasion following treatment with bevacizumab and cilengitide was remarkably decreased compared to bevacizumab monotherapy. Scale bar, 200 μm. *P < .01.

Figure 6

Effects of combination treatment with bevacizumab and cilengitide on tumor vessel structure. (A) The tumor vessels treated with bevacizumab and cilengitide were inhibited more strongly than with bevacizumab treatment. Scale bar, 100 μm. (B, C) Tumor vessels around the tumor border area retained an orderly structured basal lamina. L, lumen of vessels. Scale bar, 5 μm. (D) The tumor vessel density following combination therapy (Bev + Cile) was inhibited to the same extent as with bevacizumab treatment (Bev), and the tumor vessel diameter was significantly smaller with combination therapy than with bevacizumab treatment (E). (F) Intensity of RECA-1 following combination therapy (Bev + Cile) was significantly less than that of bevacizumab-treated tumors. *P < .05. HPF, high power field.

Figure 7

Microarray analysis of the effect of combination treatment compared to bevacizumab monotherapy. (A) There were 947 differentially expressed genes between the bevacizumab monotherapy group (Bev) and the group with bevacizumab and cilengitide combination therapy (Bev + Cile), with 486 upregulated genes and 461 downregulated genes. (B, C) Caveolin 3 (CAV3) and c-src tyrosine kinase (CSK) were significantly decreased by combination therapy compared to monotherapy (*P < .05; mean ± SE, n = 3). RQ, relative quantification.

Figure W1

Microstructure of perivascular space in the center area of a U87ΔEGFR rat brain tumor. (A) The fuzzy basal lamina and loose ECM were observed at perivascular space in the center area of an untreated U87ΔEGFR tumor. (B) Numerous collagen fibers were increased at perivascular space in the center area of a bevacizumab-treated U87ΔEGFR tumor. (C) Cluttered and dense ECM around endothelial cells following combination therapy was observed. Scale bar, 500 nm.