title: Cross-talk between endothelial and breast cancer cells regulates reciprocal expression of angiogenic factors in vitro.
abstract: Reciprocal growth factor exchange between endothelial and malignant cells within the tumor microenvironment may directly stimulate neovascularization; however, the role of host vasculature in regulating tumor cell activity is not well understood. While previous studies have examined the angiogenic response of endothelial cells to tumor-secreted factors, few have explored tumor response to endothelial cells. Using an in vitro co-culture system, we investigated the influence of endothelial cells on the angiogenic phenotype of breast cancer cells. Specifically, VEGF, ANG1, and ANG2 gene and protein expression were assessed. When co-cultured with microvascular endothelial cells (HMEC-1), breast cancer cells (MDA-MB-231) significantly increased expression of ANG2 mRNA (20-fold relative to MDA-MB-231 monoculture). Moreover, MDA-MB-231/HMEC-1 co-cultures produced significantly increased levels of ANG2 (up to 580 pg/ml) and VEGF protein (up to 38,400 pg/ml) while ANG1 protein expression was decreased relative to MDA-MB-231 monocultures. Thus, the ratio of ANG1:ANG2 protein, a critical indicator of neovascularization, shifted in favor of ANG2, a phenomenon known to correlate with vessel destabilization and sprouting in vivo. This angiogenic response was not observed in nonmalignant breast epithelial cells (MCF-10A), where absolute protein levels of MCF-10A/HMEC-1 co-cultures were an order of magnitude less than that of the MDA-MB-231/HMEC-1 co-cultures. Results were further verified with a functional angiogenesis assay demonstrating well-defined microvascular endothelial cell (TIME) tube formation when cultured in media collected from MDA-MB-231/HMEC-1 co-cultures. This study demonstrates that the angiogenic activity of malignant mammary epithelial cells is significantly enhanced by the presence of endothelial cells.
authors.affiliation: Virginia Tech - Wake Forest University School of Biomedical Engineering and Sciences, Virginia Tech, Blacksburg, VA 24061, USA. cfbuchan@vt.edu
authors.forename: Cara F
authors.initials: CF
authors.lastname: Buchanan
authors.forename: Christopher S
authors.initials: CS
authors.lastname: Szot
authors.forename: Tia D
authors.initials: TD
authors.lastname: Wilson
authors.forename: Steven
authors.initials: S
authors.lastname: Akman
authors.forename: Linda J
authors.initials: LJ
authors.lastname: Metheny-Barlow
authors.forename: John L
authors.initials: JL
authors.lastname: Robertson
authors.forename: Joseph W
authors.initials: JW
authors.lastname: Freeman
authors.forename: Marissa Nichole
authors.initials: MN
authors.lastname: Rylander
mesh_terms: D042683:Angiopoietin-1 / Q000235:genetics / Q000378:metabolism*; D042702:Angiopoietin-2 / Q000235:genetics / Q000378:metabolism*; D001943:Breast Neoplasms / Q000378:metabolism / Q000473:pathology*; D002460:Cell Line; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D018920:Coculture Techniques; D004730:Endothelium, Vascular / Q000166:cytology* / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D006801:Humans; D012333:RNA, Messenger / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D042461:Vascular Endothelial Growth Factor A / Q000235:genetics / Q000378:metabolism*
chemical_list: C467630:ANGPT1 protein, human; D042683:Angiopoietin-1; D042702:Angiopoietin-2; D012333:RNA, Messenger; D042461:Vascular Endothelial Growth Factor A