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

Abstract

Significance: 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 pO₂, 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.

Critical issues: Here we overview the current status of EPR approaches used in combination with functional paramagnetic probes that provide quantitative information on chemical TME and redox (pO₂, pH, redox status, Pi, and GSH). In particular, an application of a recently developed dual-function pH and redox nitroxide probe and multifunctional trityl probe provides unsurpassed opportunity for in vivo concurrent measurements of several TME parameters in preclinical studies. The measurements of several parameters using a single probe allow for their correlation analyses independent of probe distribution and time of measurements.

Future directions: The recent progress in clinical EPR instrumentation and development of biocompatible paramagnetic probes for in vivo multifunctional TME profiling eventually will make possible translation of these EPR techniques into clinical settings to improve prediction power of early diagnostics for the malignant transition and for future rational design of TME-targeted anticancer therapeutics. Antioxid. Redox Signal. 28, 1365-1377.

Keywords: electron paramagnetic resonance; paramagnetic probes; tumor acidosis; tumor hypoxia; tumor microenvironment; tumor redox.

FIG. 1.

Chemical structures of the EPR oximetric probes discussed in the text. Particulate LiNc-BuO, soluble nitroxide probes, mHCTPO, PDT, ¹⁵N-DCP, and ¹⁴N-DCP, and soluble trityl radicals, OX063 and OX071. EPR, electron paramagnetic resonance.

FIG. 2.

Chemical structures of the soluble paramagnetic probes for multifunctional TME profiling discussed in the text. pH and redox probes, NR1 and its deuterated analog, NR2; disulfide biradical probe for GSH detection, RSSR; and multifunctional pH, pO₂, and Pi probe of extracellular microenvironment, HOPE. GSH, glutathione; TME, tumor microenvironment.

FIG. 3.

Three-dimensional oxygen map of mouse fibrosarcoma tumor bearing leg. Tumor outline, from a registered MRI image, is shown in red. Reproduced from Ref. (29) with permission. MRI, magnetic resonance imaging. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars

FIG. 4.

pH-Dependence of the observed hyperfine splitting constant, a_N, of the NR1 probe. L-band EPR spectra were acquired at 37°C. The solid line is the fit of experimental data with standard titration curve yielding pK_a = 6.6, a_N(NR1-H⁺) = 14.24 G, and a_N(NR1) =15.27 G. Insert: Exemplified EPR spectrum of 1 mM NR1 solution. The measured hyperfine splitting constant, 14.63 G. Adapted from Ref. (64) with permission from John Wiley & Sons, Inc.

FIG. 5.

Illustration of the nitroxide/hydroxylamine redox couple. In living tissue, one-electron reduction of the nitroxides prevails over hydroxylamine oxidation and the equilibrium is strongly shifted toward the hydroxylamine (63).

FIG. 6.

In vivo pH and redox assessment of TME in mouse model of breast cancer using dual-function NR1 probe. (a) EPR insert: L-band EPR spectrum of the NR1 probe measured after i.t. injection (10 μL, 10 mM) in mammary tumor tissue of anesthetized female FVB/N mice. The hyperfine splitting, a_N, was found to be equal to 14.72 G, which corresponds to the value of pH_e = 6.52 assuming tumor tissue temperature 34°C and pK_a = 6.65. NR1 reduction rate is assessed by the following: the decay of central-field spectral component. The exemplified kinetics measured in mammary tumor (■) and mammary glands (○) demonstrates a higher reducing capacity of the tumor tissue versus normal mammary gland. The analysis of the initial part of the kinetics yields the rates of the EPR signal reduction, k_red, in extracellular media of the tissues being equal to 2.5 × 10⁻³ s⁻¹ in tumor versus 0.5 × 10⁻³ s⁻¹ in normal gland. (b) Extracellular tissue pH (filled bars) and the reduction rate (empty bars) values of NR1 nitroxide in normal mammary glands and mammary tumors of female FVB/N mice measured by in vivo EPR. Error bars denote SE. Adapted from Ref. (10) with permission.

FIG. 7.

Mapping of extracellular tumor pH. (a) Coregistered EPR pH map and MRI of right hind leg of C3H mouse bearing squamous cell carcinoma. A 4D (1 × spectral, 3 × spatial) 750 MHz continuous waves EPRI of low-field and high-field EPR peaks were performed using the following parameters: scan time, 0.3 s; modulation amplitude, 1.5 G; Gmax, 16 G/cm; FOV, 5.09 cm; projections, 576; and total acquisition time, ≈6 min. EPR pH map was superimposed with six Varian 7T MRI image. (b) pH distributions from 3D volume measurement of right hind leg from control and tumor-bearing mice. Adapted from Goodwin et al. (38) with permission. EPRI, EPR imaging. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars

FIG. 8.

Redox mapping of tumor. Two-dimensional spatial mapping of pseudofirst-order rate constants (left panels) and frequency plot (right panels) of the nitroxide reduction rate constants in the TME of untreated and BSO-treated mice are shown. Reproduced from Ref. (70) with permission. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars

FIG. 9.

EPR assessment of intracellular glutathione. (a) The X-band EPR spectra of 100 μM RSSR measured at various time points after incubation with 2.5 mM GSH in 0.1 M Na-phosphate buffer, pH 7.2, and 1 mM DTPA at 34°C. The kinetics analysis provides the observed rate constant value of the reaction between GSH and RSSR, k_obs(pH 7.2, 34°C) = (2.8 ± 0.2) M⁻¹ s⁻¹. (b) Kinetics of the monoradical spectral peak intensity change measured by L-band EPR in mammary tumor (●) and normal mammary gland (○) of FVB/N mice immediately after i.t. injection of RSSR probe. The solid lines are the fits of the initial part of the kinetics by the monoexponent supposing k_obs (pH 7.2, 34°C) = 2.8 M⁻¹s⁻¹ and yielding [GSH] = 10.7 and 3.3 mM for the tumor and normal mammary gland, correspondingly. (c) Intracellular [GSH] measured using R₂SSR₂ probe in vivo in normal mammary glands and mammary tumors of female FVB/N mice. Adapted from Ref. (10) with permission from John Wiley & Sons, Inc.

FIG. 10.

Multifunctional assessment of chemical microenvironment using HOPE probe. (a) The scheme of pH-dependent equilibrium between two ionization states of the probe. (b) L-band EPR spectrum of HOPE. (c) The EPR linewidth of the HOPE is a pO₂ marker (accuracy, ≈1 mmHg; pO₂ range, 1–100 mmHg). (d) The fraction of HOPE^3- form is a pH marker in the range from 6 to 8.0 (accuracy, ±0.05). (e) Dependence of proton exchange rate (expressed in mG) of the HOPE with inorganic phosphate on Pi concentration extracted by spectra simulation (accuracy, ±0.1 mM, range, 0.1–20 mM) (8, 20). Reproduced from ref. (9) with permission from Nature Publishing Group. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars

FIG. 11.

(a) Setup for in vivo L-band EPR measurements of the tissue microenvironment parameters in living mice. Photograph shows the anesthetized mouse between the magnets of the EPR spectrometer with the insert on the right showing placement and positioning of the loop resonator on top of the measured tissue. The values of pO₂ (b), pH_e (c), and [Pi] (d) measured in normal mammary glands of FVB/N wild-type mice (MG, n = 23) and in the TME of breast cancer in MMTV-PyMT transgenic mice (T, n = 18). Error bars are SD.*p = 0.065, **p = 0.006, ***p = 6 × 10⁻⁶. (e–h) Correlation between interstitial pO₂, pH_e, and Pi values measured in MG and in the TME. To extend the range of oxygen variations, anoxic conditions in interstitial space were established by i.t. injection of oxygen-consuming enzymatic system of glucose/glucose oxidase (red symbols). Blue lines represent linear fit for the total data sets. (e) A positive correlation between pO₂ and pH_e in normal tissue (r = 0.5, p = 0.014 for black symbols; r = 0.64, p = 1.8 × 10⁻⁴ for total data set) versus (f) no significant correlation between pO₂ and pH_e in TME (r = 0.01, p = 0.97 for black symbols; r = 0.23, p = 0.3 for total data set) was found. (g) A negative correlation between pO₂ and Pi both in normal tissue (r = −0.51, p = 0.013 for black symbols; r = −0.7, p = 2.3 × 10⁻⁵ for total data set) and (h) in TME (b, bottom: r = −0.4, p = 0.079 for black symbols; r = −0.62, p = 0.001 for total data set) was found. Adapted from Ref. (9) with permission. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars

FIG. 12.

Intratumor GM-CSF injection slows tumor growth and normalizes TME parameters. (a) PyMT+ FVB female mice with palpable tumors were treated with PBS or 100 ng GM-CSF by intratumor injection thrice per week. Points, mean tumor size from 8 (GM-CSF) and 10 (PBS) mice; bars, SE. Adapted from Ref. (34) with permission. (b–d) Normalization of TME of GM-CSF-treated tumors as measured by in vivo EPR (measurements started 1 week after treatment initiation). The values of pH (b) and reducing capacity of extracellular TME (c) were measured using NR1 probe, and intracellular GSH values were measured using RSSR probe (d). Error bars denote SE. Adapted from Ref. (10) with permission. GM-CSF, granulocyte macrophage colony-stimulating factor; PBS, phosphate-buffered saline. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars