In Vivo EPR Assessment of pH, pO2, Redox Status, and Concentrations of Phosphate and Glutathione in the Tumor Microenvironment

This protocol demonstrates the capability of low-field electron paramagnetic resonance (EPR)-based techniques in combination with functional paramagnetic ...

West Virginia University

West Virginia University School of Medicine

Electron paramagnetic resonance (EPR) imaging in the spectral-spatial domain with application of soluble paramagnetic probes provides an opportunity for spatially resolved functional measurements of living objects. The purpose of this study was to develop EPR methods for visualization of oxygenation and acidosis of ischemic myocardium.

Functional electron paramagnetic resonance imaging of ischemic rat heart: Monitoring of tissue oxygenation and pH.

A one step synthesis of fluorescent 8-aryl-(7-deazaguanines) has been accomplished. Probes exhibit blue to green high quantum yield fluorescence in a variety of organic and aqueous solutions, high extinction coefficients, and large Stokes shifts often above 100 nm. The probes are highly cell permeable, and exhibit stable bright fluorescence once intracellular; therefore are suited to the design of biosensors.

New class of 8-aryl-7-deazaguanine cell permeable fluorescent probes.

Stable triarylmethyl radicals are ideal spin labels used for biomedical electron paramagnetic resonance applications. Previously reported structures exhibit polar charged functions for water solubilization preventing them from crossing the cell membrane. We report the synthesis of a triarylmethyl radical conjugated to poly-arginine peptide allowing intracellular delivery of the paramagnetic label.

Poly-arginine conjugated triarylmethyl radical as intracellular spin label.

Tissue oxygenation, extracellular acidity, and tissue reducing capacity are among crucial parameters of tumor microenvironment (TME) of significant importance for tumor pathophysiology. In this paper, we demonstrate the complementary application of particulate lithium octa-n-butoxy-naphthalocyanine and soluble nitroxide paramagnetic probes for monitoring of these TME parameters using electron paramagnetic resonance (EPR) technique. Two different types of therapeutic interventions were studied: hypothermia and systemic administration of metabolically active drug. In summary, the results demonstrate the utility of EPR technique for non-invasive concurrent longitudinal monitoring of physiologically relevant chemical parameters of TME in mouse xenograft tumor models, including that under therapeutic intervention.

Concurrent Longitudinal EPR Monitoring of Tissue Oxygenation, Acidosis, and Reducing Capacity in Mouse Xenograft Tumor Models.

Superoxide radical is involved in numerous physiological and pathophysiological processes. Tetrathiatriarylmethyl (TAM) radicals are known to react with superoxide allowing measurement of superoxide production in biological media. We report the synthesis of a Nitro conjugated TAM radical showing a rate constant of 7 × 10 M swhich is two order of magnitude higher than other TAMs, allowing high sensitivity measurement of superoxide.

Nitro-Triarylmethyl Radical as Dual Oxygen and Superoxide Probe.

The aim of this study was to demonstrate the feasibility of in vivo three-dimensional (3D) relaxation time Tmapping of a dicarboxy-PROXYL radical using continuous-wave electron paramagnetic resonance (CW-EPR) imaging.

Feasibility of in vivo three-dimensional Tmapping using dicarboxy-PROXYL and CW-EPR-based single-point imaging.

Noninvasive in vivo assessment of chemical tumor microenvironment (TME) parameters such as oxygen (pO), extracellular acidosis (pH), and concentration of interstitial inorganic phosphate (Pi) may provide unique insights into biological processes in solid tumors. In this work, we employ a recently developed multifunctional trityl paramagnetic probe and electron paramagnetic resonance (EPR) technique for in vivo concurrent assessment of these TME parameters in various mouse models of cancer. While the data support the existence of hypoxic and acidic regions in TME, the most dramatic differences, about 2-fold higher concentrations in tumors vs. normal tissues, were observed for interstitial Pi - the only parameter that also allowed for discrimination between non-metastatic and highly metastatic tumors. Correlation analysis between [Pi], pO, pHand tumor volumes reveal an association of high [Pi] with changes in tumor metabolism and supports different mechanisms of protons and Pi accumulation in TME. Our data identifies interstitial inorganic phosphate as a new TME marker for tumor progression. Pi association with tumor metabolism, buffer-mediated proton transport, and a requirement of high phosphorus content for the rapid growth in the "growth rate hypothesis" may underline its potential role in tumorigenesis and tumor progression.

Interstitial Inorganic Phosphate as a Tumor Microenvironment Marker for Tumor Progression.

This Feature overviews the basic principles of using stable organic radicals involved in reversible exchange processes as functional paramagnetic probes. We demonstrate that these probes in combination with electron paramagnetic resonance (EPR)-based spectroscopy and imaging techniques provide analytical tools for quantitative mapping of critical parameters of local chemical microenvironment. The Feature is written to be understandable to people who are laymen to the EPR field in anticipation of future progress and broad application of these tools in biological systems, especially in vivo, over the next years.

Exchange Phenomena in the Electron Paramagnetic Resonance Spectra of the Nitroxyl and Trityl Radicals: Multifunctional Spectroscopy and Imaging of Local Chemical Microenvironment.

Superoxide radical represents one of the most biologically relevant reactive oxygen species involved in numerous physiological and pathophysiological processes. Superoxide measurement through the decay of an electron paramagnetic resonance (EPR) signal of a triarylmethyl (TAM) radical possesses the advantage of a high selectivity and relatively high rate constant of TAM reaction with the superoxide. Hereby we report a straightforward synthesis and characterization of a TAM-TAM biradical showing a high reactivity with superoxide (second-order rate constant, (6.7 ± 0.2) × 10Ms) enabling the measurement of superoxide radical by following the increase of a sharp EPR signal associated with the formation of a TAM-quinone-methide monoradical product.

Triarylmethyl-based biradical as a superoxide probe.

Proton-electron double-resonance imaging (PEDRI) employs electron paramagnetic resonance irradiation with low-field magnetic resonance imaging so that the electron spin polarization is transferred to nearby protons, resulting in higher signals. PEDRI provides information about free radical distribution and, indirectly, about the local microenvironment such as partial pressure of oxygen (pO), tissue permeability, redox status, and acid-base balance. Recent Advances: Local acid-base balance can be imaged by exploiting the different resonance frequency of radical probes between R and RHforms. Redox status can also be imaged by using the loss of radical-related signal after reduction. These methods require optimized radical probes and pulse sequences.

In Vivo Application of Proton-Electron Double-Resonance Imaging.

A key role of the tumor microenvironment (TME) in cancer progression, treatment resistance, and as a target for therapeutic intervention is increasingly appreciated. Among important physiological components of the TME are tissue hypoxia, acidosis, high reducing capacity, elevated concentrations of intracellular glutathione (GSH), and interstitial inorganic phosphate (Pi). Noninvasive in vivo pO, pH, GSH, Pi, and redox assessment provide unique insights into biological processes in the TME, and may serve as a tool for preclinical screening of anticancer drugs and optimizing TME-targeted therapeutic strategies. Recent Advances: A reasonable radiofrequency penetration depth in living tissues and progress in development of functional paramagnetic probes make low-field electron paramagnetic resonance (EPR)-based spectroscopy and imaging the most appropriate approaches for noninvasive assessment of the TME parameters.

In Vivo Molecular Electron Paramagnetic Resonance-Based Spectroscopy and Imaging of Tumor Microenvironment and Redox Using Functional Paramagnetic Probes.

Electron paramagnetic resonance (EPR)-based spectroscopic and imaging techniques allow for the study of free radicals-molecules with one or more unpaired electrons. Biological EPR applications include detection of endogenous biologically relevant free radicals as well as use of specially designed exogenous radicals to probe local microenvironments. This Forum focuses on recent advances in the field of in vivo EPR applications discussed at the International Conference on Electron Paramagnetic Resonance Spectroscopy and Imaging of Biological Systems (EPR-2017). Although direct EPR detection of endogenous free radicals such as reactive oxygen species (ROS) in vivo remains unlikely in most cases, alternative approaches based on applications of advanced spin traps and probes for detection of paramagnetic products of ROS reactions often allow for specific assessment of free radical production in living subjects. In recent decades, significant progress has been achieved in the development and in vivo application of specially designed paramagnetic probes as "molecular spies" to assess and map physiologically relevant functional information such as tissue oxygenation, redox status, pH, and concentrations of interstitial inorganic phosphate and intracellular glutathione. Recent progress in clinical EPR instrumentation and development of biocompatible paramagnetic probes for in vivo multifunctional tissue profiling will eventually make translation of the EPR techniques into clinical settings possible. Antioxid. Redox Signal. 00, 000-000.

Valery V. Khramtsov

In Vivo Multifunctional Magnetic Resonance center,
Robert C. Byrd Health Sciences Center,
In Vivo Multifunctional Magnetic Resonance center, Robert C. Byrd Health Sciences Center

West Virginia University

In Vivo EPR Assessment of pH, pO₂, Redox Status, and Concentrations of Phosphate and Glutathione in the Tumor Microenvironment

Valery V. Khramtsov

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West Virginia University

In Vivo EPR Assessment of pH, pO2, Redox Status, and Concentrations of Phosphate and Glutathione in the Tumor Microenvironment

In Vivo Multifunctional Magnetic Resonance center,
Robert C. Byrd Health Sciences Center,
In Vivo Multifunctional Magnetic Resonance center, Robert C. Byrd Health Sciences Center

In Vivo EPR Assessment of pH, pO₂, Redox Status, and Concentrations of Phosphate and Glutathione in the Tumor Microenvironment