Use of a high frequency ultrasound microscope to image the action of 2-nitroimidazoles in multicellular spheroids

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Abstract
A system was designed to allow imaging of control and drug treated multicellular spheroids with a high frequency backscatter ultrasound microscope. It allowed imaging of individual spheroids under good growth conditions. Since little data were available on cellular toxicity of ultrasound at these high frequencies (80 MHz), studies were undertaken to evaluate effects on cell survival, using a colony forming assay. No toxicity was observed on cell monolayers subjected to pulsed ultrasound at the intensities used for imaging experiments. Spheroids were also subjected to pulsed ultrasound and no growth delay was observed when exposed spheroids were compared with mock-exposed spheroids. Imaging studies were performed and pictures of untreated spheroids were obtained in which the necrotic and viable regions are clearly distinguishable. When the hypoxic cell cytotoxin 1-methyl-2-nitroimidazole (INO2) was added to the spheroid, dramatic changes were observed in the backscatter signal. The interior viable cells of the spheroid were selectively affected. Changes in the backscatter signal were also observed when the reduction product 1-methyl-2-nitrosoimidazole (INO) was added to spheroids. With INO however, the changes were located at the periphery of the spheroid, presumably due to the high reactivity of INO which limits diffusion of the drug into the spheroid. The present work demonstrates the potential usefulness of ultrasound backscatter microscopy in following the action of selected drugs in this in vitro tumour model.