The plate was examined and photographed for the formation of capillary-like endotubes under a phase-contrast microscopy at 3 h, 6 h and 22 h. In vivo angiogenesis (matrigel plug) assay The method of this assay was described in detail in previous publication [15]. In brief, 4 groups of mice with H226-containing matrigel plugs were treated with IgG (control), bevacizumab alone (1 mg/kg intraperitoneally), radiation alone (2 Gy/fraction), or combination treatment in which bevacizumab was administered immediately following radiation, PCI-34051 mw twice a week for 2 weeks. At the end of week 2, mice were injected with FITC-Dextran solution. The plugs were removed and examined for

the perfused blood vessels. The intensity of fluorescence in captured images was quantified by Adobe Photoshop software. Growth inhibition assay in tumor xenograft models

A series of in vivo experiments in athymic mice bearing SCC1 and H226 xenografts were conducted to examine the anti-tumor activity of bevacizumab, radiation and combined therapy in concurrent and sequential fashion. Design and treatment schedule of those experiments are described in the Results Section. Details on xenografts, animal care, tumor measurement and radiation delivery were described in previous publication [15]. Statistical Crenolanib analysis Analysis of variance (ANOVA) was performed to compare tumor volume in groups of mice treated with bevacizumab and/or radiation LY3023414 supplier with the control group. Treatment interaction and linear contrasts were used to evaluate the synergistic effect of the bevacizumab and radiation therapy combination.

Tumor volume was log-transformed to meet the assumption of normality. Effects of bevacizumab and radiation on tumor growth in mice bearing SCC1 and H226 xenografts were analyzed using ANOVA and linear mixed-effects models. An autoregressive correlation structure was assumed to account for correlations between repeated measurements within an experimental unit. Tukey’s HSD method was used to control the type 1 error for the pairwise comparisons between treatment groups. All p values were two sided and considered significant when ≤0.05. Statistical analyses were performed with SAS statistical software (version 8.2; SAS Institute, Gefitinib chemical structure Cary, NC). Results Bevacizumab inhibits HUVEC proliferation in vitro and tumor growth in vivo In the crystal violet assay, bevacizumab induced a modest inhibition of HUVEC growth at a concentration as low as 0.001 μM (Figure 1). This inhibition was observed across 5 logs of bevacizumab dose ranging from 0.001Â μM – 10Â μM with approximately 30-40% HUVEC growth inhibition. No clear dose response effect was observed suggesting saturation of VEGF blockade in the higher dose range. Figure 1 Inhibitory effect of bevacizumab on proliferation of HUVEC.