0 × 105 cells/μl. U87ΔEGFR cells (5 μl) were injected into athymic rats (F344/N-rnu/rnu; CLEA Japan, Inc, Tokyo, Japan), and U87ΔEGFR cells (2 μl) were injected into athymic mice (BALB/c-nu/nu; CLEA Japan, Inc). The animals were anesthetized and placed in stereotactic frames (Narishige, Tokyo, Japan) with their skulls exposed. Tumor cells were injected with a Hamilton syringe (Hamilton, Reno, NV) into the right frontal lobe (in the athymic rats: 4 mm lateral and 1 mm anterior to the bregma at a depth of 4 mm; in the athymic mice: 3 mm lateral and 1 mm anterior to the bregma at a depth of 3 mm),

and the syringe was withdrawn slowly after 5 minutes to prevent reflux. The skulls were then cleaned and the incision was sutured. PBS, bevacizumab (for the athymic mice and rats: 6 mg/kg), cilengitide (for the athymic mice OSI-906 and rats: 10 mg/kg), or a combination of bevacizumab and cilengitide of the same amount was administered three times per week intraperitoneally, starting on day 5 after tumor

cell implantation. Athymic rats harboring U87ΔEGFR brain tumors were killed at 18 days after tumor implantation and six times administration of PBS, bevacizumab, cilengitide, or the combination of bevacizumab and cilengitide. The brains were removed and fixed BTK inhibitor in vivo immediately by perfusion of 2% glutaraldehyde. After fixation in 2% osmium tetroxide, the samples were dehydrated and embedded in Spurr’s resin. Thin sections poststained with salts of uranium and lead were cut to approximately 60 nm using an ultramicrotome (Leica EM UC6; Leica,

Wetzlar, Germany). The samples were observed under a transmission electron microscope (Hitachi H-7650 TEM; Hitachi, Tokyo, Japan). For histopathologic analysis, athymic rats harboring U87ΔEGFR brain tumors were killed at 18 days after tumor implantation. Athymic rats before were anesthetized, killed by cardiac puncture, perfused with 100 ml of PBS, and fixed with 50 ml of 4% paraformaldehyde (PFA). The brains were removed and stored in 4% PFA for 12 to 24 hours. Hematoxylin and eosin (HE) staining was performed as described previously [13]. For immunohistochemistry of PFA perfusion-fixed frozen sections, snap-frozen tissue samples were embedded in optimal cutting temperature compound for cryosectioning, and 16-μm-thick sections were processed for indirect immunofluorescence. After blocking non-specific binding with 10% normal goat serum, the slides were incubated overnight at 4°C with primary antibodies, including those targeting rat endothelial cell antigen 1 (RECA-1; 1:20, mouse monoclonal; Abcam, Inc, Cambridge, United Kingdom), von Willebrand factor (1:250, rabbit polyclonal; Abcam, Inc), integrin αvβ3 (1:100, mouse monoclonal; Abcam, Inc), and integrin αvβ5 (1:75, mouse monoclonal; Abcam, Inc). After three washes with PBS containing 0.