Vascular endothelial growth factor immunoneutralization plus Paclitaxel markedly reduces tumor burden and ascites in athymic mouse model of ovarian cancer

Abstract

Ovarian cancer is characterized by rapid growth of solid intraperitoneal tumors and production of large volumes of ascites. Our previous studies of intraperitoneal ovarian carcinoma in an athymic mouse model demonstrated that a monoclonal antibody (mAb) to human vascular endothelial growth factor (VEGF) could prevent ascites formation. Although ascites was almost completely inhibited, tumor burden was variably reduced. To develop more effective therapy, we assessed the combination of a human VEGF mAb plus paclitaxel. Four groups of female athymic nude mice were inoculated intraperitoneally with OVCAR3 cells. Two weeks after inoculation, one group was treated with a human VEGF mAb intraperitoneally twice weekly plus paclitaxel intraperitoneally three times weekly for 6 weeks. The second group was treated with VEGF mAb alone. The third group was treated with paclitaxel alone. The remaining group was treated with vehicle only. Tumor burden in the VEGF mAb plus paclitaxel and paclitaxel alone groups was reduced by 83.3% and 85.7% and 58.5% and 59.5%, respectively, in two separate experiments, compared to controls. VEGF mAb alone caused no significant decrease in tumor burden, nor did treatment of mice inoculated intraperitoneally with HEY-A8 cells, a non-VEGF-secreting ovarian cell line. Virtually no ascites developed in the combined treatment group or the group treated with VEGF mAb alone. Paclitaxel alone reduced ascites slightly, but not significantly. Morphological studies demonstrated that VEGF immunoneutralization enhanced paclitaxel-induced apoptosis in these human ovarian cancers. Thus, combination therapy with inhibitors of VEGF plus paclitaxel may be an effective way to markedly reduce tumor growth and ascites in ovarian carcinoma.

Figures

Figure 1.

Effects of VEGF mAb plus paclitaxel on tumor burden (A) and ascites formation (B) in mice inoculated with OVCAR3 cells. Experiment 1. Four groups of athymic immunodeficient mice were used. OVCAR3 cells (2 × 106) were injected as a bolus intraperitoneally in 5- to 7-week-old athymic immunodeficient mice. Treatment was initiated 2 weeks after inoculation. Treatment groups consisted of control (vehicle alone), VEGF mAb alone, VEGF mAb plus paclitaxel, and paclitaxel alone. The VEGF mAb (5 μg/g body weight) was administered intraperitoneally twice weekly for 6 weeks. Administration of paclitaxel (20 μg/g body weight) was twice weekly in the first week and increased to three times weekly for the last 5 weeks. At autopsy, ascites fluid was quantified and tumors were excised and weighed (n = 15). Data are expressed as mean ± SE **. P < 0.01 versus control; ++, P < 0.01 versus paclitaxel.

Figure 2.

Effects of VEGF mAb plus paclitaxel on tumor burden (A) and ascites formation (B) in mice inoculated with OVCAR3 cells. Experiment 2. Four groups of athymic immunodeficient mice were used. The OVCAR3 cells (2 × 106) were injected as a bolus intraperitoneally in 5- to 7-week-old athymic immunodeficient mice. Treatment was initiated 2 weeks after inoculation. Treatment groups consist of control (vehicle alone), VEGF mAb alone, VEGF antibody plus paclitaxel, and paclitaxel alone. The VEGF mAb (5 μg/g body weight) was administered intraperitoneally twice weekly for 6 weeks. Administration of paclitaxel (20 μg/g body weight) was three times weekly for 6 weeks. At autopsy, ascites fluid was quantified and tumors were excised and weighed (n = 49). Data are expressed as mean ± SE. **, P < 0.01 versus control; ++, P < 0.01 versus paclitaxel.

Figure 3.

Effects of VEGF mAb plus paclitaxel on tumor growth in mice inoculated with Hey-A8 cells. Four groups of athymic immunodeficient mice were used. Hey-A8 cells (2 × 106) were injected as a bolus intraperitoneally in 5- to 7-week-old athymic immunodeficient mice. Treatments were started 2 weeks after inoculation. Treatment groups consisted of control (vehicle alone), VEGF mAb alone, VEGF mAb plus paclitaxel, and paclitaxel alone. The VEGF mAb (5 μg/g body weight) was administered intraperitoneally twice weekly for 4 weeks. Paclitaxel (20 μg/g body weight) was administered three times weekly for 4 weeks. At autopsy, tumors were excised and weighed (n = 20). Data are expressed as mean ± SE. *, P < 0.05 versus control.

Figure 4.

Histological appearance and apoptosis in tumor tissue from OVCAR3-inoculated athymic mice with and without paclitaxel or VEGF mAb plus paclitaxel treatment. A: Representative section of tumor from control group. Tumor cells have large, atypical nuclei, prominent nucleoli, a moderate amount of cytoplasm, grow in sheets, and undergo mitosis. B: Section of tumor from VEGF mAb plus paclitaxel treatment group. Focus of necrosis and apoptosis with cytoplasmic debris and calcification. A few clusters of viable tumor cells surround this central focus (bottom right). Similar foci were scattered throughout tumors removed from the treated animals. The brown-stained cells (arrows) indicate apoptosis. C: Section of tumor from VEGF mAb-treated group shows tumor cells with large, atypical nuclei. Some cells are swollen, whereas in others the cytoplasm is dense and decreased in amount. No evidence of apoptosis was detected. D: Section of tumor from paclitaxel-treated group shows degenerative changes, including decreased nuclear size, hyperchromasia, and smudging of the nuclear chromatin. A few cells are swollen, whereas in others the cytoplasm is dense and decreased in amount. Arrows indicate apoptotic cells. Original magnifications, ×300.