Detection of individual hypoxic cells in multicellular spheroids by flow cytometry using the 2-nitroimidazole, EF5, and monoclonal antibodies.
OBJECTIVE
The purpose of this work was to evaluate EF5, a 2-nitroimidazole compound, and anti-EF5 antibodies as a method to quantify radiobiologically hypoxic cells.


METHODS
Multicellular spheroids of EMT6 mammary sarcoma cells were used as a model to identify hypoxic cells that were resistant to radiation damage. This was accomplished by incubating the spheroids with the 2-nitroimidazole (EF5), which forms hypoxia-dependent adducts with cellular macromolecules that are detected by fluorescent monoclonal antibodies.


RESULTS
Cells from spheroids grown for 2 days in sealed flasks had an increased surviving fraction following radiation as compared to fully reoxygenated spheroids, indicating the presence of radiobiological hypoxia. Treatment of the spheroids with EF5 and subsequent immunohistochemical staining of cryosections with an anti-EF5 fluorochrome conjugated monoclonal antibody allowed for the identification of EF5-adduct containing cells. Spheroids grown under hypoxic conditions in the presence of EF5 showed limited staining of the peripheral cell layers, intense staining of the interior, and an absence of staining within the necrotic center. In contrast, there was minimal staining in reoxygenated spheroids and no staining in control spheroids incubated in the absence of EF5. Flow cytometric analysis of single cells dissociated from spheroids allowed for the calculation of the percentage of stained cells, as well as the intensity of staining. A comparison of the intensity of staining of EF5 treated hypoxic spheroids with the intensity of staining of single cells incubated with EF5 under controlled oxygen concentrations was used to estimate the oxygen concentration range within spheroids. Selective dissociation of spheroids provided a direct demonstration that the cells containing the highest level of EF5 binding were also the cells with increased radiation resistance.


CONCLUSIONS
This technique provides an excellent means of detecting and quantifying hypoxia, which should be directly applicable in tumors.
Department of Microbiology and Immunology, University of Rochester School of Medicine and Dentistry, Rochester, NY 14642, USA.
M L
ML
Woods
C J
CJ
Koch
E M
EM
Lord
D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism*; D015687:Cell Hypoxia / Q000187:drug effects / Q000502:physiology / Q000528:radiation effects; D002470:Cell Survival / Q000187:drug effects / Q000528:radiation effects; D017341:Etanidazole / Q000031:analogs & derivatives / Q000276:immunology / Q000378:metabolism*; D005434:Flow Cytometry / Q000379:methods*; D006845:Hydrocarbons, Fluorinated / Q000276:immunology / Q000378:metabolism*; D007202:Indicators and Reagents / Q000378:metabolism*; D015674:Mammary Neoplasms, Animal / Q000378:metabolism; D051379:Mice; D008807:Mice, Inbred BALB C; D011853:Radiobiology; D018874:Spheroids, Cellular / Q000187:drug effects / Q000378:metabolism* / Q000528:radiation effects; D014407:Tumor Cells, Cultured; D005466:Fluorocarbons
D000911:Antibodies, Monoclonal; D017341:Etanidazole; D006845:Hydrocarbons, Fluorinated; D007202:Indicators and Reagents; C096011:2-(2-nitro-1H-imidazol-1-yl)-N-(2,2,3,3,3-pentafluoropropyl)acetamide; D005466:Fluorocarbons