title: Validation of a hypoxia-inducible factor-1 alpha specimen collection procedure and quantitative enzyme-linked immunosorbent assay in solid tumor tissues.
abstract: Hypoxia-inducible factor-1 alpha (HIF-1α) is an important marker of hypoxia in human tumors and has been implicated in tumor progression. Drugs targeting HIF-1α are being developed, but the ability to measure drug-induced changes in HIF-1α is limited by the lability of the protein in normoxia. Our goal was to devise methods for specimen collection and processing that preserve HIF-1α in solid tumor tissues and to develop and validate a two-site chemiluminescent quantitative enzyme-linked immunosorbent assay (ELISA) for HIF-1α. We tested various strategies for HIF-1α stabilization in solid tumors, including nitrogen gas-purged lysis buffer, the addition of proteasome inhibitors or the prolyl hydroxylase inhibitor 2-hydroxyglutarate, and bead homogenization. Degassing and the addition of 2-hydroxyglutarate to the collection buffer significantly increased HIF-1α recovery, whereas bead homogenization in sealed tubes improved HIF-1α recovery and reduced sample variability. Validation of the ELISA demonstrated intra- and inter-assay variability of less than 15% and accuracy of 99.8±8.3% as assessed by spike recovery. Inter-laboratory reproducibility was also demonstrated (R(2)=0.999). Careful sample handling techniques allow us to quantitatively detect HIF-1α in samples as small as 2.5μg of total protein extract, and this method is currently being applied to analyze tumor biopsy specimens in early-phase clinical trials.
authors.affiliation: Division of Cancer Treatment and Diagnosis, National Cancer Institute, Bethesda, MD 20892, USA.
authors.forename: Sook Ryun
authors.initials: SR
authors.lastname: Park
authors.affiliation: Laboratory of Human Toxicology and Pharmacology, Applied/Developmental Research Directorate, Leidos Biomedical Research, Frederick National Laboratory for Cancer Research, Frederick, MD 21702, USA. Electronic address: kindersr@mail.nih.gov.
authors.forename: Robert J
authors.initials: RJ
authors.lastname: Kinders
authors.affiliation: Laboratory of Human Toxicology and Pharmacology, Applied/Developmental Research Directorate, Leidos Biomedical Research, Frederick National Laboratory for Cancer Research, Frederick, MD 21702, USA.
authors.forename: Sonny
authors.initials: S
authors.lastname: Khin
authors.affiliation: Biological Testing Branch, Developmental Therapeutics Program, Frederick National Laboratory for Cancer Research, Frederick, MD 20892, USA.
authors.forename: Melinda
authors.initials: M
authors.lastname: Hollingshead
authors.affiliation: Division of Cancer Treatment and Diagnosis, National Cancer Institute, Bethesda, MD 20892, USA.
authors.forename: Smitha
authors.initials: S
authors.lastname: Antony
authors.affiliation: Laboratory of Human Toxicology and Pharmacology, Applied/Developmental Research Directorate, Leidos Biomedical Research, Frederick National Laboratory for Cancer Research, Frederick, MD 21702, USA.
authors.forename: Ralph E
authors.initials: RE
authors.lastname: Parchment
authors.affiliation: Division of Cancer Treatment and Diagnosis, National Cancer Institute, Bethesda, MD 20892, USA.
authors.forename: Joseph E
authors.initials: JE
authors.lastname: Tomaszewski
authors.affiliation: Division of Cancer Treatment and Diagnosis, National Cancer Institute, Bethesda, MD 20892, USA.
authors.forename: Shivaani
authors.initials: S
authors.lastname: Kummar
authors.affiliation: Division of Cancer Treatment and Diagnosis, National Cancer Institute, Bethesda, MD 20892, USA; Center for Cancer Research, National Cancer Institute, Bethesda, MD 20892, USA.
authors.forename: James H
authors.initials: JH
authors.lastname: Doroshow
mesh_terms: D000818:Animals; D014408:Biomarkers, Tumor / Q000378:metabolism; D045744:Cell Line, Tumor; D002471:Cell Transformation, Neoplastic; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D006801:Humans; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000378:metabolism*; D051379:Mice; D009369:Neoplasms / Q000473:pathology*; D013048:Specimen Handling / Q000379:methods*
chemical_list: D014408:Biomarkers, Tumor; D051795:Hypoxia-Inducible Factor 1, alpha Subunit
keywords: HIF-1α; Hypoxia-inducible factor; Pharmacodynamics; Quantitative ELISA; Solid tumors