Characteristics of the Binding of Labeled Fluoromisonidazole in Cells in Vitro

Abstract

Radiolabeled fluoromisonidazole (FMISO) is being investigated as an imaging agent for hypoxia in tumors and nonmalignant tissues in myocardial infarct or stroke. In this study in vitro cell cultures were used to characterize the oxygen dependency of FMISO uptake and to examine other modifying factors. The uptake of [3H]FMISO was measured in four cell lines in vitro: V-79, EMT-6(UW), RIF-1, and CaOs-1. The modifying effects of different O2 levels as well as cell growth state and concentration of glucose and nonprotein sulfhydryls were examined. In these cell types an O2 level between 720 and 2300 ppm inhibited FMISO binding by 50%, relative to binding under anoxic conditions. These values bracket the O2 level which confers full radiobiologic hypoxia, about 1000 ppm. Some bound label was released from cells in the first 1 to 3 h after a 3-h anoxic labeling with [3H]FMISO, but this does not represent tritium loss from the parent molecule. Cells from unfed plateau-phase cultures took up less [3H]FMISO than did exponentially growing cells incubated at comparable O2 levels. Reducing glucose to 1/10 or 1/100 of the usual concentration in medium had little effect on binding of micromolar levels of FMISO, except in V-79 cells, where reduced glucose levels were associated with increased FMISO accumulation. Adding cysteamine to the culture medium moderately increased FMISO uptake. We conclude that cell growth state, glucose, and nonprotein sulfhydryl concentrations affect FMISO binding, albeit less than varying O2 levels: anoxic/oxic binding ratios vary from 12.6 to 28 for the four cell types examined. Nonetheless these factors must be considered in evaluating the oxygen-dependent binding of this nitroimidazole in tumors or tissues.

Hypoxia Imaging with 18F-FMISO PET for Brain Tumors

“…Lower-grade gliomas do not develop necrosis and may be expected to suffer from only a milder degree of hypoxia than that of glioblastomas [18,19]. FMISO accumulation requires severe hypoxic conditions (pO2 < 10 mmHg) [43,44], and the results here are consistent with this.…”

Resolution in PET hypoxia imaging: Voxel size matters

“…In support of that, we have observed a significant loss of decay-corrected radioactivity when previously anoxically FAZA loaded cells were transferred to tracer free medium (Â35% loss in one h, Busk et al, in press). Nonirreversible binding (or loss of radioactive label) has also been reported for other 2-nitro-imidazole tracers in vitro [16,26]. Finally, the high positron energy of 124 I leads per se to a loss in resolution of Â1mm compared to 18 F [27].…”

“…These tracers differ in their lipophilicity and resistance towards metabolism but a complete and objective ranking of their ability to identify and map hypoxia is not possible as only few studies have compared tracers directly. The absolute tracer uptake in animal tumor models shows considerably variability but tumor-to-reference tissue ratios, and possible intratumoral contrast, proved rather similar for FMISO, FETA and FETNIM [15,16]. FAZA, the tracer used in the present study, is less lipophilic, and thus easier to clear from non-hypoxic tissue areas than FMISO and has indeed generated considerably higher tumor-to-reference tissue ratios in tumor-bearing mice [17,18] but not in rats [19].…”

In vivo evaluation of [18F]fluoroetanidazole as a new marker for imaging tumour hypoxia with positron emission tomography

“…Of the numerous possibilities in nuclear medicine, appropriately labelled 2-nitroimidazole probes are particularly attractive for imaging tumour hypoxia (pO 2 o10 mmHg (Höckel et al, 1996;Nordsmark et al, 1996); radiobiological hypoxia, pO 2 o1 mmHg (Rasey et al, 1990)). A combination of pharmacological and physical properties that need to be considered as part of an ideal design goal include: (i) a nitro group with appropriate redox potential (E 1/7 of BÀ380 to À390 mV) for selective reduction and binding in hypoxic tumour cells; (ii) lipophilicity that is high enough to enable diffusion across cellular membranes to the site of metabolism (octanol -water partition coefficient of BX0.1 (Brown and Workman, 1980;Workman, 1982), but low enough (Bp2) to assure rapid systemic elimination and, hence, convenient imaging times (within 2 h); (iii) stability to hypoxiaindependent degradation; and (iv) high photon flux (and low energy) to assure high detection sensitivity and spatial image resolution.…”