sEphB4 inhibits the tumor growth in a murine tumor xenograft model. (A) 1 × 104 MCF-7 and SCC-15 cells were grown in triplicate in the presence of increasing concentrations of sEphB4 for 72 hours. Cell viability was assessed by MTT assay. The experiment was repeated twice. (B) Mice (n = 6/group) were given implants with 5 × 106 SCC-15 or MCF-7 cells in a Matrigel preparation with PBS or sEphB4 (70 nM) and tumor volumes measured 3 times a week. Three weeks after implantation, tumors were harvested and weighed (right axis). Tumor volumes were significantly smaller in the sEphB4 arm, beginning on day 7 of measurement. The experiment was repeated twice. (C) Representative histology (hematoxylin and eosin) of SCC-15 tumors is shown. Tumor proliferation was assessed by immunohistochemical detection of Ki-67 protein and apoptosis by TUNEL. Microvasculature was assessed by CD31 immunohistochemistry. Number of cells staining positive was averaged over 5 random 40 × fields by a blinded observer and is shown at the bottom of each photomicrograph. (D) For assessing the effect of systemic administration of sEphB4, mice (n = 6/group) were given implants of tumor cells subcutaneously and treated with sEphB4 or CF2 (10 mg/kg/d) or an equal volume of PBS intravenously starting on day 4. Tumor volumes and weights were assessed as in panel B. Tumor volumes were significantly smaller in the sEphB4 arm compared to animals treated with PBS and CF2. (E) Histologic analysis of harvested MCF-7 tumors was performed as in panel C. All values are expressed as mean ± SEM. *P < .01 by Student t test. Bar in bottom right panel in E represents 100 μm in hematoxylin-eosin section, 60 μm in Ki-67 and apoptosis sections, and 75 μm in CD31 sections. Photomicrographs in panels C and E were taken using a Nikon Coolpix 5000 camera and a Nikon Eclipse E400 microscope with a 10 × eyepiece. Magnification was as follows: top row, 4 ×/0.13 NA; middle rows, 40 ×/0.75 NA; and bottom rows, 20 ×/0.5 NA objectives.
