Analysis of proliferation and tumor angiogenesis in MDA-MB-231/sFRP1 xenografts. (a) Mice bearing MDA-MB-231/secreted Frizzled-related protein 1 (sFRP1)-P1 or control-P1 tumors were injected with bromodeoxyuridine (BrdU) and sacrificed 2 hours later. Tumors of similar weight from each group were sectioned and stained with anti-BrdU antibody. Bar = 250 μm. (b) BrdU-positive nuclei and total nuclei were counted in nine randomly chosen areas from each section. Bar graph shows the quantification ± standard error of the mean (SEM). **P < 0.01. (c) Aniokis assays were performed by seeding cells in 1% FCS-containing medium on polyHema-coated plates to prevent adhesion. Cells were harvested 24 hours later, stained with propidium iodide and analyzed with a FACScalibur. Representative cell cycle distribution of three independent control and sFRP1-expressing MDA-MB-231 clones shown. (d) Total blood vessels in tumor sections visualized by staining for the endothelial cell marker CD31. y axis, ratio of CD31+ area/total area. Average calculated from five sFRP1-expressing and eight control tumors ± SEM. n.s., not significant. (e) Functional blood vessels in tumor-bearing mice visualized by injecting FITC-lectin into tail veins 5 minutes before sacrificing. The FITC+ and CD31+ areas were measured on tumor sections and the average was calculated on three sFRP1-expressing and five control tumors. y axis, FITC+ area/CD31+ area ratio ± SEM. n.s., not significant. (d), (e) Image analysis performed using IMARIS software (Bitplane).