Mapping of the vascular endothelial growth factor-producing hypoxic cells in multicellular tumor spheroids using a hypoxia-specific marker.
We have investigated the hypoxia inducibility of vascular endothelial growth factor (VEGF) in multicellular tumor spheroids of HT29 cells using a monoclonal antibody to a fluorinated bioreductive drug, EF5 [2-(2-nitro-1H-imidazol-1-yl)-N-(2,2,3,3,3-pentafluoropropyl)aceta mide], a chemical probe for hypoxia. We have shown that VEGF expression is predominantly localized in interior spheroid cells that are sufficiently hypoxic to bioreductively activate the 2-nitroimidazole and produce immunologically detectable adducts of the EF5 compound. Northern blotting analyses demonstrated that VEGF165 is the predominant form of VEGF produced by HT29 cells and that the phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate did not induce VEGF expression. This study demonstrates that VEGF expression is up-regulated in response to hypoxia and in the microenvironments found in human multicellular tumor spheroids. This investigation also illustrates the utility of the EF5 binding in multi-cellular tumor spheroids as a means of studying the expression and regulation of hypoxia-inducible genes.
Waleh
N S
NS
Cellular and Molecular Biology Laboratory, SRI International, Menlo Park, California 94025, USA.
Brody
M D
MD
Knapp
M A
MA
Mendonca
H L
HL
Lord
E M
EM
Koch
C J
CJ
Laderoute
K R
KR
Sutherland
R M
RM
D002277:Carcinoma / Q000098:blood supply*; D003110:Colonic Neoplasms / Q000098:blood supply*; D016228:Endothelial Growth Factors / Q000235:genetics*; D017341:Etanidazole / Q000031:analogs & derivatives; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D006845:Hydrocarbons, Fluorinated; D000860:Hypoxia / Q000378:metabolism; D017403:In Situ Hybridization; D007202:Indicators and Reagents; D008222:Lymphokines / Q000235:genetics*; D009389:Neovascularization, Pathologic; D009940:Organoids; D012333:RNA, Messenger / Q000235:genetics; D012334:RNA, Neoplasm / Q000235:genetics; D014407:Tumor Cells, Cultured; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors; D005466:Fluorocarbons
D016228:Endothelial Growth Factors; D017341:Etanidazole; D006845:Hydrocarbons, Fluorinated; D007202:Indicators and Reagents; D008222:Lymphokines; D012333:RNA, Messenger; D012334:RNA, Neoplasm; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors; C467484:VEGFA protein, human; C096011:2-(2-nitro-1H-imidazol-1-yl)-N-(2,2,3,3,3-pentafluoropropyl)acetamide; D005466:Fluorocarbons