Electron Paramagnetic Resonance (EPR) studies on tumor oxygenation

The aim of this review is to show how Electron Paramagnetic Resonance (EPR) and related methods such as dynamic nuclear polarisation (DNP) can be used to detect, localize and quantify oxygen concentration in biological tissues. Several applications will be described with special emphasis on tumor oxygenation. Introduction It is well established that the partial pressure of oxygen (pO2) plays important roles in the response of tumors to cytotoxic treatments such as chemotherapy, radiotherapy, and photodynamic therapy. Additionally, hypoxic microenvironments in tumors are known to promote processes driving malignant progression. The assessment of tissue oxygenation is therefore of great physiological and clinical interest, and numerous methods have been developed to measure this parameter. Here, we will focus on EPR-based methods that monitor the oxygen level in tissues. Comparison between NMR and EPR EPR (Electron Paramagnetic Resonance) or equivalently ESR (Electron Spin Resonance) detects only impaired electron species. The greatest difference between NMR and EPR arises because the gyromagnetic ratio of a free electron (28 GHz/T) is 659 times that of a proton (42.5 MHz/T). Standard EPR spectrometers operate at 9 GHz (0.34 Tesla, X-Band). At this frequency, non-resonant absorption of the electromagnetic radiation by the liquid water of biological samples presents a major problem. The lack of sufficient amounts of naturally occuring paramagnetic compounds and the short lifetime of most free radicals is another source of difficulty. Another main difference between NMR and EPR lies in the short relaxation times of paramagnetic species (1000 times shorter than NMR relaxation times) with two consequences : EPR spectra are mostly obtained through continuous wave experiments, and EPR imaging requires magnetic gradient strengths more than one order of magnitude than those used in MRI (1). The development of spectrometers operating at low frequency as well as the development of stable paramagnetic substances that can be inserted inside tissues were necessary to develop in vivo applications of EPR. Principles of EPR oximetry Bimolecular collisions between oxygen and free radicals alter the resonance characteristics of the radical and consequently the EPR spectrum (2,3). These effects can be distinguished in T1and T2-sensitive experiments. The change in T2 modifies the EPR linewidth that can be directly calibrated as a function of the pO2. Two classes of paramagnetic materials are useful for oximetry purposes : soluble paramagnetic materials such as nitroxides or triarylmethyl radicals, and particulate materials. The spatial distribution of oxygen can be determined using the soluble materials. However, they have the limitation to be metabolically converted to diamagnetic species or cleared by excretion (4). Solid probes are usually metabolically inert, and are characterized by a higher sensitivity for oximetry. Variations of less than 1 mmHg can be detected. Within the last few years several new paramagnetic particulate materials have been found to exhibit a pO2-dependent EPR linewidth: lithium phthalocyanine crystals, lithium naphthalocyanine crystals, particles of natural coals such as fusinite or gloxy, analytical charcoals, synthetic carbohydrate chars, and carbon blacks. Once introduced inside the tissue, the microparticles enable repeated measurement of pO2 from the same site for weeks after implantation. Oxygen can be monitored from multiple sites inside a tissue using convenient additional gradients. Principles of DNP oximetry The DNP technique is a double resonance technique that couples the advantages of MRI with the sensitivity of EPR, by making use of the Overhauser effect. By saturating the EPR transition of a paramagnetic agent, the NMR signal intensities of the coupled water protons are enhanced by a significant factor by means of the Overhauser effect. The Overhauser enhancement depends on the EPR linewidth of the paramagnetic agent, which in turns depends on oxygen concentration. This effect is more pronounced using soluble paramagnetic materials possessing narrow-line such as trityl-based contrast agent (5). Applications to tumor oximetry EPR and DNP oximetry methods were applied by different groups to monitor the pO2 in tumors after pharmacological interventions (6,7), after irradiation (8,9), or to study the influence of physiological stimuli that contribute to modulate the tumor pO2 (10,11). Illustrative results will be presented with an emphasis on the relationships between the very subtle changes of pO2 in tumors that were detected and the modulation in the reponsiveness of tumors to treatments. PerspectivesIn vivo EPR oximetry has already produced very useful results that have contributed significantly to solve important biological problems. It should be of major interestto transpose this technique into the clinic. For that purpose, there is a critical need for paramagnetic sensors that are fully biocompatible. The strategies to improve thebiocompatibility of the exisiting sensors (12) as well the technical developments to achieve the clinical usefulness will be discussed. References1.Gruker D., Prog. Nucl. Magn. Reson. Spectrosc. 2000, 36, 241-270.2. Hyde JS and Subczynski in Spin Labeling : Theory and Applications, L. Berliner, ed., Plenum Press 1989, 3993. Swartz HM and Clarkson RB, Phys. Med. Biol. 1998, 43, 1957-1975.4. Gallez B et al. Magn. Reson. Med. 1996, 35, 97-1065. Golman K et al, JMRI 2000, 12, 929-938.6. Gallez B et al, Magn. Reson. Med. 1999, 42, 627-630.7. Krishna MC et al, PNAS 2002, 99, 2216-2221.8. Goda F et al, Cancer Res. 1996, 56, 3344-3349.9. Sonveaux P et al, FASEB J 2002 in press10. Jordan B et al, Cancer Res 2002, 62, 3555-3561.11. Jordan B et al, Int J Cancer 2002, in press12. Gallez B and Mader K, Free Rad Biol Med 2000, 29, 1078-1084. 722Proc. Intl. Soc. Mag. Reson. Med. 11 (2003)