Background: Vascular endothelia growth factor (VEGF) is a secreted endothelial-specific growth factor that is angiogenic in vivo. it is commonly expressed in a range of carcinomas. Purpose: The study was designed to investigate the effect of constitutive expression of VEGF on tumor formation by estrogen-dependent human MCF–7 breast carcinoma cells. Methods: A full-length complementary DNA encoding the shortest isoform of VEGF (VEGF₁₂₁was stably transfected into MCF–7 cells. transfected clones were screened for VEGF₁₂₁messenger RNA (mRNA) expression by ribonuclease protection analysis and for secretion of VEGF₁₂₁protein by Western blot analysis. secretion of biologically active VEGF₁₂₁by transfectants was confirmed by 1) a competitive radioreceptor-binding assay, 2) stimulation of the growth of microvascular endothelial cells in vitro, and 3) potent angiogenic activity in the rabbit corneal assay. tumor models were then established by subcutaneously implanting wildtype of VEGF₁₂₁-transfected MCF–7 cells, together with either mouse BALB/3T3 clone A31 fibroblasts or human MDA–435S breast carcinoma cells, into ovariectomized nude mice either with or without a separately implanted slow-release estrogen pellet. Tumor vascularity was quantitatively assessed by capillary vessel counting after staining with the pan-endothelia marker CD31. Results: Stable VEGF₁₂₁-overexpressing MCF–7cells were isolated and designated V12 cells. when implanted into the rabbit cornea, V12 cells elicited a strong directional outgrowth of capillaries. The growth rate of V12 cells in vitro was indistinguishable from that of MCF–7 wild—type cells. V12 cells formed faster growting tumors than did wild-type cells (P←.01) and showed a heterogeneous distribution of vessels when compared with the homogeneous distribution seen in tumors formed form wild-type cells. VEGF₁₂₁ overexpression had no effect on hormone dependence or tamoxifen sensitivity of tumor formation by MCF–7 cells in mice. No macroscopic evidence for metastasis from subcutaneous implants was obtained conclusions: VEGF₁₂₁ expression by breast carcinoma cells confers a growth advantange in vivo but not in vitro. Tumors formed by V12 transfectants were more vascular than those formed by wild-type MCF–7 cells, and we surmise that the growth advantage arises from increased tumor vascularization induced by VEGF_121.Implications: Tumor formation by V12 cells could provide a useful model for the assessment of anti-angiogenic drugs. [J Natl Cancer Inst 87: 213–219, 1995]