(A) Treatment with anti–murine EphA2 antibody diminished EphA2 protein expression in tumor cells derived from MMTV-Neu and MMTV–PyV-mT mice. Tumor cells were treated with control IgG (10 μg/ml) or increasing concentrations of anti-EphA2 antibody for 48 hours. Uniform loading was confirmed by immunoblot for actin. Blots were stripped and reprobed with anti-EphA4 antibodies as a control for antibody specificity. (B) Cells derived from EphA2^+/+ MMTV-Neu mice were orthotopically transplanted into the cleared fat pads of female FVB recipient mice. At 2 weeks following transplantation, mice were injected intraperitoneally with anti-EphA2 antibody or control IgG (10 mg/kg) twice weekly for 3 weeks. We observed a significant reduction in tumor volume in anti-EphA2–treated animals relative to control IgG–treated mice (P < 0.05; 2-tailed, paired Student’s t test). Data are mean ± SEM. (C) Tumor cell proliferation was significantly impaired in anti-EphA2–treated animals relative to controls (P < 0.05; single-factor ANOVA; arrowheads indicate PCNA⁺ nuclei). Scale bar: 50 μm. (D) EphA2 expression was significantly diminished in anti-EphA2–treated tumors relative to IgG controls, as assessed by immunohistochemistry and immunoblot. Blots were stripped and reprobed for actin expression to verify uniform loading. Scale bar: 50 μm. (E) We observed significantly reduced (P < 0.05; 2-tailed, paired Student’s t test) microvascular density in tumors isolated from anti-EphA2–treated mice relative to controls (arrowheads indicate vWF⁺ blood vessels). Scale bar: 100 μm. (F) Cells derived from MMTV–PyV-mT mice were orthotopically transplanted in the cleared fat pad of FVB female recipient mice and were treated with anti-EphA2 antibody or control IgG as described above. We observed no change in tumor volume between animals treated with anti-EphA2 antibody relative to control IgG-treated mice.