Figure 2.

VEGF expression increases tumor growth and vascularization. A, tumor growth in athymic nude mice. C9V4 cells (10⁷), suspended in 200 µL of 50% phenol red–free Matrigel (BD Biosciences), were inoculated into the inguinal mammary fat pad of four groups of ovariectomized athymic nude mice on day 0. A slow-release 0.72-mg E₂ pellet (Innovative Research) was implanted into each mouse the day before the cell inoculation. The control group was provided with drinking water containing 2.5% sucrose only; three other groups were provided with 2.5% sucrose and 0.05 or 0.2 mg/mL of doxycycline (dox) starting the day before the cell inoculation. Doxycycline was removed from one of 0.2 mg/mL groups on day 11. There were six mice for each group (n = 6). Tumor sizes were measured twice a week. Symbols, mean tumor volume; bars, SE. B, relationship between tumor growth and VEGF levels. Cytosolic extracts were prepared from xenograft tumors as described in ref. 6, and the amount of VEGF present was determined by ELISA (R&D Systems) and normalized to total protein concentration. The mean tumor volume at the end of the study is indicated by the red columns and the average tumor VEGF level of each mouse group (n = 6) is indicated by the green columns. C, VEGF overexpression is associated with increased tumor microvessel density. Paraffin-embedded tumor samples prepared from the animal study were sectioned and immunostained with a rat monoclonal antibody against mouse PECAM-1 (BD Biosciences). Representative images of tumor microvessels from two tumors, one with 0.2 mg/mL doxycycline induction of VEGF expression and the other without. Quantitative analysis, as shown in the histogram, was done by counting the number of PECAM-1–positive blood vessels per microscopic field with five tumor samples for each condition and three microscopic fields per sample (×10 magnification).