sDll4 inhibits the tumor growth in a murine tumor xenograft model. (A) Mice (n = 6/group) were implanted with 1 × 106 HT29 cells in a Matrigel preparation with PBS or sDll4-Fc or sDll4-His (5 μg/mL) and tumor volumes (mean ± SEM) were measured after 2 weeks; tumors were then harvested and analyzed. Tumor volumes were significantly smaller in the sDll4 arm. *P < .05 compared to control. The experiment was repeated twice. (B) In assessing the effect of endogenous expression of sDll4, HT29 cells were transfected with expression vector with Dll4-FL, sDll4-Fc, sDll4-His, or vector alone. Coexpression of truncated CD4 was done to allow sorting of the transfected cells. Equal numbers of the transfected cells were implanted in mice (n = 6/group). Tumor volumes (mean ± SEM) were assessed. Tumor volumes were significantly smaller in the sDll4 groups. *P < .05 compared to vehicle. (C) Microvasculature was assessed by PECAM immunostaining, and the blood vessel volume was quantitated as described in “Materials and methods.” Mean ± SEM. *P < .05 compared to vehicle. (D) Hypoxy probe was infused prior to tumor harvest; tumor sections were then probed with MAb and fluorescent-labeled secondary antibody as described in “Materials and methods.” Hypoxic areas were quantitated (mean ± SEM) using ImageJ as described in “Materials and methods.” All values are expressed as mean ± SEM. *P < .01. Photomicrographs were taken with a Nikon Eclipse 80 microscope with a Nikon Plan Fluor ∞, 0.17 10×/0.3 NA dry objective mounted with a Photometrics CoolSNAP camera and processed with Metamorph V 6.3r2. (E) Vascular perfusion was determined by injecting fluorescent-labeled lectin 10 to 15 minutes prior to killing mice and harvesting tumors. Lectin was localized to perfused areas, while blood vessels were delineated with PECAM staining. Lectin and PECAM colocalized in control group, while sDll4 group showed marked deficiency of perfusion. (F) Localization of α-SMA in tumor vessel. Control group showed colocalization of α-SMA and PECAM, while sDll4 group had paucity of α-SMA–positive cells in the microvessels.
