DETECTION OF INDIVIDUAL HYPOXIC CELLS IN MULTICELLULAR SPHEROIDS BY FLOW-CYTOMETRY USING THE 2-NITROIMLDAZOLE, EF5, AND MONOCLONAL-ANTIBODIES

Purpose: The purpose of this work was to evaluate EF5, a 2-nitroimidaz ole compound, and anti-EF5 antibodies as a method to quantify radiobio logically hypoxic cells. Methods and Materials: Multicellular spheroid s of EMT6 mammary sarcoma cells were used as a model to identify hypox ic cells that were resistant to radiation damage, This was accomplishe d by incubating the spheroids with the 2-nitroimidazole (EF5), which f orms hypoxia-dependent adducts with cellular macromolecules that are d etected by fluorescent monoclonal antibodies. Results: Cells from sphe roids grown for 2 days in sealed flasks had an increased surviving fra ction 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 cryo sections with an anti-EF5 fluorochrome conjugated monoclonal antibody allowed for the identification of EF5-adduct containing cells, Spheroi ds grown under hypoxic conditions in the presence of EF5 showed limite d staining of the peripheral cell layers, intense staining of the inte rior, and an absence of staining within the necrotic center, In contra st, there was minimal staining in reoxygenated spheroids and no staini ng in control spheroids incubated in the absence of EP5, Flow cytometr ic analysis of single cells dissociated from spheroids allowed for the calculation of the percentage of stained cells, as well as the intens ity of staining, A comparison of the intensity of staining of EF5 trea ted hypoxic spheroids with the intensity of staining of single cells i ncubated with EF5 under controlled oxygen concentrations was used to e stimate the oxygen concentration range within spheroids, Selective dis sociation of spheroids provided a direct demonstration that the cells containing the highest level of EP5 binding were also the cells with i ncreased radiation resistance. Conclusion: This technique provides an excellent means of detecting and quantifying hypoxia, which should be directly applicable in tumors.