In Vivo Electron Paramagnetic Resonance Molecular Profiling of Tumor Microenvironment upon Tumor Progression to Malignancy in an Animal Model of Breast Cancer

Abstract

Purpose: Hypoxia and acidosis are recognized tumor microenvironment (TME) biomarkers of cancer progression. Alterations in cancer redox status and metabolism are also associated with elevated levels of intracellular glutathione (GSH) and interstitial inorganic phosphate (Pi). This study aims to evaluate the capability of these biomarkers to discriminate between stages and inform on a switch to malignancy.

Procedures: These studies were performed using MMTV-PyMT( +) female transgenic mice that spontaneously develop breast cancer and emulate human tumor staging. In vivo assessment of oxygen concentration (pO₂), extracellular acidity (pH_e), Pi, and GSH was performed using L-band electron paramagnetic resonance spectroscopy and multifunctional trityl and GSH-sensitive nitroxide probes.

Results: Profiling of the TME showed significant deviation of measured biomarkers upon tumor progression from pre-malignancy (pre-S4) to the malignant stage (S4). For the combined marker, HOP: (pH_e × pO₂)/Pi, a value > 186 indicated that the tumors were pre-malignant in 85% of the mammary glands analyzed, and when < 186, they were malignant 42% of the time. For GSH, a value < 3 mM indicated that the tumors were pre-malignant 74% of the time, and when > 3 mM, they were malignant 80% of the time. The only marker that markedly deviated as early as stage 1 (S1) from its value in pre-S1 was elevated Pi, followed by a decrease of pH_e and pO₂ and increase in GSH at later stages.

Conclusion: Molecular TME profiling informs on alteration of tumor redox and metabolism during tumor staging. Early elevation of interstitial Pi at S1 may reflect tumor metabolic alterations that demand elevated phosphorus supply in accordance with the high rate growth hypothesis. These metabolic changes are supported by the following decrease of pH_e due to a high tumor reliance on glycolysis and increase of intracellular GSH, a major intracellular redox buffer. The appreciable decrease in TME pO₂ was observed only at malignant S4, apparently as a consequence of tumor mass growth and corresponding decrease in perfusion efficacy and increase in oxygen consumption as the tumor cells proliferate.

Keywords: Electron paramagnetic resonance; Interstitial inorganic phosphate; Intracellular glutathione; Molecular cancer biomarkers; Nitroxides; Spin probes; Trityl radicals; Tumor hypoxia and acidosis; Tumor microenvironment.

Fig. 1.

(a). The scheme of pH-dependent equilibrium between two ionization states of the HOPE probe, HOPE^3- and HOPE^4-. (b). L-band EPR spectrum of HOPE representing superposition of two forms of the radical with the different phosphorus hyperfine splittings, a_P=3.63 G for HOPE^3- and a_P= 3.37 G for HOPE^4-. Insert shows the high-field component. (c). The EPR linewidth of the HOPE is a pO₂ marker (accuracy, ≈ 1 mmHg for pO₂ range, 1–100 mmHg). (d). The fraction of HOPE^3- ionization form (p_a) from total probe concentration, is a pH marker with accuracy, ± 0.05, for the pH range from 6 to 8.0. (e). A dependence on the proton exchange rate (expressed in mG) of the HOPE with inorganic phosphate on Pi concentration extracted by spectra simulation (accuracy, ± 0.1 mM, range, 0.1–8 mM) [41, 42]. Note interstitial extracellular localization of the HOPE probe: it does not penetrate into the cells due to bulky charged structures and the HOPE signal from the blood is not detected by EPR due to signal broadening by pTAM complexation with plasma albumin [43]. Reproduced from ref. [15] with permission of Nature Publishing Group.

Fig. 2.

(a). The structure of the imidazolidine disulfide biradical RSSR and scheme of thiol-disulfide exchange reaction of the RSSR probe with GSH that results in the formation of two monoradicals. (b). L-band EPR spectra measured in vivo in mammary tumor of female FVB/N mice 10 s and 150 s after intratissue injection of the RSSR probe. (c). Kinetics of the monoradical peak intensity increase measured in tumor (●) and normal mammary gland (o). The solid lines are the fits of the initial part of the kinetics by the monoexponent supposing k_GSH (pH 7.2, 34°C)=2.8 M⁻¹ s⁻¹ [10] and yielding [GSH] =10.7 mM and 3.3 mM for the tumor and normal mammary gland, correspondingly. Adapted from [10] with permission from John Wiley & Sons, Inc.

Fig. 3.. Tumor parameters change with stage.

(a) [Pi]: One-Way ANOVA: Overall P=0.0262 for differences across all stages, and Tukey’s Post-Hoc test: *P=0.0129 for pS1 vs. S3 and **P=0.0147 for pS1 vs. S4. n=5 (pS1), n=23 (S1), n=25 (S2), n=21 (S3), n=23 (S4). (b) pH: One-Way ANOVA: Overall P<0.0001 for differences across all stages, and Tukey’s Post-Hoc test: *P=0.0004 for pS1 vs S4, **P=0.0104 for S2 vs S4, and ***P=0.0167 for S3 vs S4. n=5 (pS1), n=23 (S1), n=25 (S2), n=21 (S3), n=23 (S4). (c) Oxygen (pO₂): One-way ANOVA: Overall P=0.0269 for differences across all stages. One statistical outlier in S4 (134 mmHg) was removed by Grubb’s Test. n=5 (pS1), n=23 (S1), n=25 (S2), n=21 (S3), n=22 (S4). Error bars represent Mean ± SEM.

Fig. 4.. Correlations of pO₂ and pH_e as tumors stage.

Tumor pO₂ and pH_e correlate in stages 1, 2, and 3 in the MMTV-PyMT breast cancer mice: Pearson r correlation: S1: R² =0.2466 and P=0.0159, n=23; S2: R² =0.2122 and Pearson r P=0.0208, n=25; S3: R² =0.3988 and Pearson r P=0.0021, n=21, while they lose correlation in S4 (malignancy): R² =0.0255 and Pearson r P=0.4780 (NS), n=22.

Fig. 5.. Intracellular GSH change with stage.

One-Way ANOVA: Overall P=0.0191 for differences across all stages and Tukey’s Post-Hoc test: *P=0.023 for S2 vs S4. n=16 (S1), n=13 (S2), n=13 (S3), n=15 (S4). Error bars represent Mean ± SEM.

Fig. 6.. Discrimination between premalignant stages (S1-S3) and malignancy, S4.

(a) Combination of three parameters measured by the HOPE probe, HOP=(pH_e×pO₂)/[Pi] where pH_e is in units of pH, pO₂ in mmHg, and [Pi] in mM. HOP-Statistic: Mann Whitney Test for non-parametric data, *P=0.0251 for difference between pre-malignant stages and malignant stage. X: mean values for pre-malignant=334, malignant=210. White line: median values for pre-malignant=226, malignant=181. Red dotted line at 186 HOP value indicates that when >186, the samples were 85% pre-malignant staged mice, and when <186, the samples were 42% malignant staged mice. n=69 for pre-malignant, n=23 for malignant. (b) [GSH] in mM: Mann Whitney Test for non-parametric data, *P=0.0003 for difference between pre-malignant stages and malignant stage. White line: median values for pre-malignant=0.6 mM, malignant=7.1 mM. X: mean values for pre-malignant=2.6 mM, malignant=6.7 mM. Red dotted line at 3 mM indicates that when <3 mM, the samples were 74% pre-malignant staged mice, and when >3.0 mM, the samples were 80% malignant staged mice. Outliers were excluded from graph. n=42 for pre-malignant, n=15 for malignant.