In vivo monitoring of pH, redox status, and glutathione using L-band EPR for assessment of therapeutic effectiveness in solid tumors

Tissue oxygenation, acidosis, redox, and glutathione (GSH) are among the most crucial parameters related to metabolism and physiology of tumors (1–5). Areas of hypoxia and acidosis are common features of solid neoplasia that have been well documented. Cells that exist in such adverse microenvironmental conditions can appreciably alter the tumor response to cytotoxic anticancer treatments. Indeed, it has been established that the presence of oxygen-deficient or hypoxic cells can not only lead to therapeutic resistance in preclinical tumor models but also have a detrimental effect on the ability to control human malignancies treated with curative intent. The acidic extracellular pH in tumors has a number of important consequences, playing a role in tumor initiation, progression, and therapy (2). Recently, pHe has been identified as a significant prognostic factor not only in experimental transplantable tumor models but also in spontaneous tumors (6). Tumor cells are known to generate significant alterations in the redox status. This status is an important determinant in the response of the tumor to certain chemotherapeutic agents, radiation, and bioreductive hypoxic cell cytotoxins (4). Several studies have shown that GSH, a major contributor to intracellular redox status (7), is elevated in tumors (8,9). Intracellular GSH has been shown to be one of the major factors modulating tumor response to a variety of commonly used antineoplastic agents, including resistance toward cisplatin drugs (8). Therefore, development of approaches allowing for noninvasive assessment of these parameters in vivo provides an important tool for optimization of anticancer therapies and screening of anticancer drugs. In the last decades, significant progress has been made regarding in vivo EPR techniques. L-band EPR instruments, designed to increase the depth of microwave penetration and decrease nonresonant losses, have become commercially available. Moreover, even lower-field (down to 250 MHz) homemade radiofrequency (RF) EPR spectrometers as well as instrumentation for spatial and spectral-spatial EPR imaging of radicals have been constructed at several EPR centers (10–12). The advances in pulsed EPR techniques operating at 300 MHz frequency allow for the first time in vivo imaging of nitroxides with narrow EPR lines (13). Alternatively, we recently proposed a functional proton–electron double-resonance imaging (PEDRI) approach allowing for functional mapping of the aqueous samples with high-quality MRI spatial resolution and short acquisition time using specially designed probes, e.g., pH mapping using pH-sensitive nitroxides (14,15). However, EPR-based techniques are far from attaining their maximum potential because of the lack of stable in vivo exogenous spin probes. This situation started to change for the better in recent years after development of a number of potentially useful paramagnetic probes for in vivo functional studies (16,17). Here, we report an approach for in vivo tumor tissue multifunctional monitoring using L-band EPR-based techniques and specially designed paramagnetic probes. The approach allows for in vivo assessment of physiologically relevant tissue parameters, namely extracellular tissue acidity, redox, and intracellular GSH. In this study, we apply multifunctional EPR measurements in the model of breast cancer to assess changes of the tumor microenvironment induced by treatment with granulocyte macrophage colony-stimulating factor (GM-CSF) (18). In contrast to many anticancer agents, GM-CSF acts via recruiting more macrophages, also resulting in a fewer blood vessels and lower oxygen concentrations in tumors (18), which may affect tissue pH, redox, and GSH.