title: Enhancement of tumor growth and vascular density by transfection of vascular endothelial cell growth factor into MCF-7 human breast carcinoma cells.
abstract: BACKGROUND
  Vascular endothelial growth factor (VEGF) is a secreted endothelial-specific growth factor that is angiogenic in vivo. It is commonly expressed in a range of carcinomas.
  
  
  OBJECTIVE
  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 (VEGF121) was stably transfected into MCF-7 cells. Transfected clones were screened for VEGF121 messenger RNA (mRNA) expression by ribonuclease protection analysis and for secretion of VEGF121 protein by Western blot analysis. Secretion of biologically active VEGF121 by 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 wild-type or 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-endothelial marker CD31.
  
  
  RESULTS
  Stable VEGF121-overexpressing MCF-7 cells 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 growing tumors than did wild-type cells (P < .01) when xenografted subcutaneously into nude mice with either 3T3 fibroblasts or MDA-435S cells. Tumors formed from V12 cells were more vascular (P < .01) and showed a heterogeneous distribution of vessels when compared with the homogeneous distribution seen in tumors formed from 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 advantage 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 VEGF121.
  
  
  CONCLUSIONS
  Tumor formation by V12 cells could provide a useful model for the assessment of anti-angiogenic drugs.
authors.affiliation: Imperial Cancer Research Fund, Institute of Molecular Medicine, University of Oxford, John Radcliffe Hospital, England.
authors.forename: H T
authors.initials: HT
authors.lastname: Zhang
authors.forename: P
authors.initials: P
authors.lastname: Craft
authors.forename: P A
authors.initials: PA
authors.lastname: Scott
authors.forename: M
authors.initials: M
authors.lastname: Ziche
authors.forename: H A
authors.initials: HA
authors.lastname: Weich
authors.forename: A L
authors.initials: AL
authors.lastname: Harris
authors.forename: R
authors.initials: R
authors.lastname: Bicknell
mesh_terms: D016475:3T3 Cells; D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D001943:Breast Neoplasms / Q000098:blood supply / Q000473:pathology*; D018076:DNA, Complementary; D016228:Endothelial Growth Factors / Q000096:biosynthesis / Q000502:physiology*; D004730:Endothelium, Vascular; D006801:Humans; D008222:Lymphokines / Q000096:biosynthesis / Q000502:physiology*; D051379:Mice; D008807:Mice, Inbred BALB C; D008819:Mice, Nude; D008969:Molecular Sequence Data; D009368:Neoplasm Transplantation; D009389:Neovascularization, Pathologic / Q000503:physiopathology; D011817:Rabbits; D011994:Recombinant Proteins / Q000096:biosynthesis; D014162:Transfection; D014407:Tumor Cells, Cultured; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors
chemical_list: D018076:DNA, Complementary; D016228:Endothelial Growth Factors; D008222:Lymphokines; D011994:Recombinant Proteins; C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors