title: HER-2/neu overexpression increases the viable hypoxic cell population within solid tumors without causing changes in tumor vascularization.
abstract: The effects of HER-2/neu overexpression on the tumor microenvironment in an aggressive breast cancer xenograft model were investigated. These studies focused on tumors derived following the subcutaneous injection of MDA-MB-435/LCC6 cells transfected with human c-erbB2 (LCC6(HER-2)) into SCID-Rag2M mice. LCC6(HER-2) tumors were more viable (H&E-stained tumor sections) than isogenic vector control tumors (LCC6(Vector)). Correspondingly, a 2.7-fold increase in trypan blue-excluding cells (P = 0.00056) and a 4.8-fold increase in clonogenic cells (P = 0.00146) were noted in cell suspensions derived from disaggregated LCC6(HER-2) versus LCC6(Vector) tumors. Tumor sections stained with the antibody detecting 2-(2-nitro-1H-imidazol-1-yl)-N-(2,2,3,3,3-pentafluoropropyl)-acetamide (EF5), a marker of hypoxia, showed a greater fraction of hypoxic tissue in LCC6(HER-2) tumors compared with control tumors. Flow cytometric analyses based on viable tumor cells (DNA content >/= 2N) in cell suspensions from disaggregated tumors confirmed that there were significantly more EF5-positive cells (i.e., hypoxic) in LCC6(HER-2) than in LCC6(Vector) tumors (16.41 +/- 8.1% and 5.96 +/- 4.1%, respectively; P = 0.0015). Protein levels of phosphorylated (Ser(536)) nuclear factor-kappaB p65 were significantly elevated in LCC6(HER-2) tumors (P = 0.00048), and a trend in increased hypoxia-inducible factor-1alpha protein levels was observed in LCC6(HER-2) compared with LCC6(Vector) tumors. Despite the substantial viable hypoxic cell fraction and a 1.7-fold increase of vascular endothelial growth factor protein (P = 0.05) in LCC6(HER-2) tumors, no significant differences were found (P > 0.05) between LCC6(HER-2) and LCC6(Vector) vasculature (CD31 staining and Hoechst 33342 perfusion). These results suggest that HER-2/neu overexpression may be linked with overall increased tumor viability and a significant increase in the population of viable hypoxic cells, which is not due to differences in tumor vascularization.
authors.affiliation: Department of Advanced Therapeutics, British Columbia Cancer Agency, 600 West 10th Avenue, Vancouver, British Columbia, Canada V5Z 4E6. vdragows@bccrc.ca.
authors.forename: Wieslawa H
authors.initials: WH
authors.lastname: Dragowska
authors.forename: Corinna
authors.initials: C
authors.lastname: Warburton
authors.forename: Donald T T
authors.initials: DT
authors.lastname: Yapp
authors.forename: Andrew I
authors.initials: AI
authors.lastname: Minchinton
authors.forename: Yanping
authors.initials: Y
authors.lastname: Hu
authors.forename: Dawn N
authors.initials: DN
authors.lastname: Waterhouse
authors.forename: Karen
authors.initials: K
authors.lastname: Gelmon
authors.forename: Kirsten
authors.initials: K
authors.lastname: Skov
authors.forename: Janet
authors.initials: J
authors.lastname: Woo
authors.forename: Dana
authors.initials: D
authors.lastname: Masin
authors.forename: Lynsey A
authors.initials: LA
authors.lastname: Huxham
authors.forename: Alastair H
authors.initials: AH
authors.lastname: Kyle
authors.forename: Marcel B
authors.initials: MB
authors.lastname: Bally
mesh_terms: D000222:Adaptation, Physiological / Q000235:genetics; D000818:Animals; D015415:Biomarkers; D001943:Breast Neoplasms / Q000098:blood supply / Q000235:genetics / Q000378:metabolism*; D002277:Carcinoma / Q000098:blood supply / Q000235:genetics / Q000378:metabolism*; D015687:Cell Hypoxia / Q000235:genetics; D045744:Cell Line, Tumor / Q000637:transplantation; D049109:Cell Proliferation; D019069:Cell Respiration / Q000235:genetics; D002470:Cell Survival / Q000235:genetics; D002999:Clone Cells / Q000378:metabolism; D019008:Drug Resistance, Neoplasm / Q000235:genetics; D017341:Etanidazole / Q000031:analogs & derivatives*; D005260:Female; D006085:Graft Survival / Q000502:physiology; D006801:Humans; D006845:Hydrocarbons, Fluorinated; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D051379:Mice; D016513:Mice, SCID; D016328:NF-kappa B / Q000378:metabolism; D009389:Neovascularization, Pathologic / Q000378:metabolism*; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000235:genetics* / Q000378:metabolism*; D051996:Transcription Factor RelA; D014157:Transcription Factors / Q000378:metabolism; D014183:Transplantation, Heterologous; D015854:Up-Regulation / Q000235:genetics; D042461:Vascular Endothelial Growth Factor A / Q000378:metabolism; D005466:Fluorocarbons
chemical_list: D015415:Biomarkers; D017341:Etanidazole; D006845:Hydrocarbons, Fluorinated; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D016328:NF-kappa B; D018719:Erb-b2 Receptor Tyrosine Kinases; D051996:Transcription Factor RelA; D014157:Transcription Factors; D042461:Vascular Endothelial Growth Factor A; C497442:HIF1A protein, human; C096011:2-(2-nitro-1H-imidazol-1-yl)-N-(2,2,3,3,3-pentafluoropropyl)acetamide; D005466:Fluorocarbons