Abstract
The novel fluorinated 2-nitroimidazole SR-4554 is undergoing preclinical development as a magnetic resonance spectroscopy and imaging probe for hypoxic tumour cells. We have used electron energy loss spectroscopic analysis (EELS) to show selective reduction and differential subcellular localisation of SR-4554 in human ovarian multicellular spheroids. SR-4554 was demonstrated to be metabolised by these A2780 cells under hypoxic but not under normal aerobic cell culture conditions. The EELS technique illustrated that the relative amount of drug within the cytoplasm of cells from both the inner region (150-160 microns from edge) and outer edge of the spheroid did not differ significantly after an initial 3 h incubation with drug. In contrast, an 8-fold differential between the amount of drug retained in the cytoplasm (primarily ribosomes and endoplasmic reticulum) of cells from the inner vs outer regions of the spheroids was observed following a subsequent 2 h 'chase' culture in drug-free medium. Within cells from the hypoxic region of the spheroid, SR-4554 was mainly associated with the endoplasmic reticulum, nucleus and the cytoplasmic side of intracellular vesicles and also to a lesser extent with the nuclear periphery. Interestingly, the drug was only weakly associated with the mitochondria and plasma membrane of the cells. The characteristics of cellular and subcellular distribution of SR-4554 are consistent with the hypothesis that 2-nitroimidazole compounds undergo hypoxia-mediated enzymatic reduction to reactive species. These reactive species are selectively retained in the cells in which they are metabolised through covalent association with subcellular components. These findings provide additional support for the clinical development of the drug as a non-invasive probe for tumour hypoxia and at the same time illustrate the utility of the EELS technique for examining the heterogeneity of drug distribution both between and within cells.
Highlights
Energy loss contributions of both fluorine and nitrogenCH2CONHCH2 CH were measured following background modelling for each pixel in median filtered energy loss image sequence
SR-4554 [N-(2-hydroxy-3,3,3 trifluoropropyl)-2-(2-nitro-1-imi- The spheroids were incubated with culture medium condazolyl) acetamide] was synthesised and supplied by SRI taining a non-toxic concentration of 1 mM SR-4554 at 37°C
The human ovarian A2780 cell line was used in this study since this cell line has previously been shown to express cytochrome P450 reductase, an enzyme involved in the reductive metabolism of 2-nitroimidazoles, by enzyme assay and PCR analysis
Summary
CH2CONHCH2 CH were measured following background modelling for each pixel in median filtered energy loss image sequence. Localisaflon of a 2-nitroimidazole hypoxia probe in human spheroids EO Aboagye et al eV portion of the post-ionisation edge for fluorine and nit- average grey level value representing energy loss for fluorine rogen were used to calculate elemental ratios. In this way, as a function of section area for each pixel. Using our in-house image analysis software, regions of interest (ROI) for each electron spectroscopic image sequence (ESIS) were defined by drawing on the
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