title: Normal human fibroblasts enable melanoma cells to induce angiogenesis in type I collagen.
abstract: BACKGROUND
  We previously reported that fibroblasts induce human microvascular endothelial cells (HMVECs) to differentiate from monolayer to capillarylike morphology. We now test the hypothesis that fibroblasts modulate angiogenesis in melanoma cells.
  
  
  METHODS
  We tested 12 human melanoma lines (2 radial growth phase (RGP), 3 vertical growth phase (VGP), and 7 metastatic (MM)) for ability to induce HMVECs to invade/migrate into collagen and form capillarylike networks. HMVEC monolayers were overlaid with 3-dimensional collagen gels embedded with melanoma cells alone (M), fibroblasts alone (F), or a 1:1 mixture of the 2 cells (M+F). After 5 days, gels were removed, fixed, and HMVEC networks were quantified by von Willebrand's factor (vWF) immunofluorescence. The influence of soluble factors on HMVEC invasion/migration into collagen was assessed with the use of acellular 3-D collagen gels overlaid on HMVEC monolayers, cultured with conditioned media (CM) derived from monolayers of M, F, or M+F. Angiogenic growth factors involved in the observed invasion/migration were identified with the use of a RayBio Cytokine Antibody Array (RayBiotech, Norcross, Ga).
  
  
  RESULTS
  Cell line-specific variability in melanoma-supported angiogenesis was observed only when in combination with fibroblasts (analysis of variance [ANOVA], P < .01). Melanoma plus fibroblasts uniformly resulted in a significantly higher angiogenic response than melanoma alone (P < .05). One vertical growth phase and one metastatic melanoma line, while weakly angiogenic alone, induced significantly higher angiogenesis than either fibroblast or melanoma alone (P < .05) when combined with fibroblasts. CM from M or M+F induced significantly less HMVEC invasion/migration into collagen than CM from fibroblasts alone. Interleukin 8, monocyte chemotactic protein-1, and tissue inhibitor of metalloproteinase-2 were identified as significantly elevated in the media derived from M+F cultures, compared with either cell type alone.
  
  
  CONCLUSIONS
  To our knowledge, this is the first report demonstrating that melanoma-supported angiogenesis in collagen is more significantly influenced by normal skin-derived fibroblasts than by the intrinsic biology of the melanoma cell type. Interleukin 8, monocyte chemotactic protein-1, and tissue inhibitor of metalloproteinase-2 are implicated as potential paracrine factors regulating this observed effect.
authors.affiliation: Hospital of the University of Pennsylvania, Philadelphia 19104, USA.
authors.forename: Lee J
authors.initials: LJ
authors.lastname: Goldstein
authors.forename: Haiying
authors.initials: H
authors.lastname: Chen
authors.forename: Richard J
authors.initials: RJ
authors.lastname: Bauer
authors.forename: Stephen M
authors.initials: SM
authors.lastname: Bauer
authors.forename: Omaida C
authors.initials: OC
authors.lastname: Velazquez
mesh_terms: D042501:Angiogenic Proteins / Q000032:analysis*; D000077214:Becaplermin; D045744:Cell Line, Tumor; D002478:Cells, Cultured; D003094:Collagen; D017077:Culture Media, Conditioned; D004730:Endothelium, Vascular; D004815:Epidermal Growth Factor / Q000032:analysis; D005109:Extracellular Matrix; D005347:Fibroblasts / Q000502:physiology*; D006801:Humans; D008545:Melanoma / Q000098:blood supply*; D009389:Neovascularization, Pathologic; D018919:Neovascularization, Physiologic; D010982:Platelet-Derived Growth Factor / Q000032:analysis; D020574:Proto-Oncogene Proteins c-sis; D016212:Transforming Growth Factor beta / Q000032:analysis; D014471:Umbilical Veins
chemical_list: D042501:Angiogenic Proteins; D017077:Culture Media, Conditioned; D010982:Platelet-Derived Growth Factor; D020574:Proto-Oncogene Proteins c-sis; D016212:Transforming Growth Factor beta; D000077214:Becaplermin; D004815:Epidermal Growth Factor; D003094:Collagen