Dose-Specific Intratumoral GM-CSF Modulates Breast Tumor Oxygenation and Antitumor Immunity.

GM-CSF has been employed as an adjuvant to cancer immunotherapy with mixed results based on dosage. We previously showed that GM-CSF regulated tumor angiogenesis by stimulating soluble vascular endothelial growth factor (VEGF) receptor-1 from monocytes/macrophages in a dose-dependent manner that neutralized free VEGF, and intratumoral injections of high-dose GM-CSF ablated blood vessels and worsened hypoxia in orthotopic polyoma middle T Ag (PyMT) triple-negative breast cancer (TNBC). In this study, we assessed both immunoregulatory and oxygen-regulatory components of low-dose versus high-dose GM-CSF to compare effects on tumor oxygen, vasculature, and antitumor immunity. We performed intratumoral injections of low-dose GM-CSF or saline controls for 3 wk in FVB/N PyMT TNBC. Low-dose GM-CSF uniquely reduced tumor hypoxia and normalized tumor vasculature by increasing NG2+ pericyte coverage on CD31+ endothelial cells. Priming of "cold," anti-PD1-resistant PyMT tumors with low-dose GM-CSF (hypoxia reduced) sensitized tumors to anti-PD1, whereas high-dose GM-CSF (hypoxia exacerbated) did not. Low-dose GM-CSF reduced hypoxic and inflammatory tumor-associated macrophage (TAM) transcriptional profiles; however, no phenotypic modulation of TAMs or tumor-infiltrating lymphocytes were observed by flow cytometry. In contrast, high-dose GM-CSF priming increased infiltration of TAMs lacking the MHC class IIhi phenotype or immunostimulatory marker expression, indicating an immunosuppressive phenotype under hypoxia. However, in anti-PD1 (programmed cell death 1)-susceptible BALB/c 4T1 tumors (considered hot versus PyMT), high-dose GM-CSF increased MHC class IIhi TAMs and immunostimulatory molecules, suggesting disparate effects of high-dose GM-CSF across PyMT versus 4T1 TNBC models. Our data demonstrate a (to our knowledge) novel role for low-dose GM-CSF in reducing tumor hypoxia for synergy with anti-PD1 and highlight why dosage and setting of GM-CSF in cancer immunotherapy regimens require careful consideration.

Biocompatible Monophosphonated Trityl Spin Probe, HOPE71, for In Vivo Measurement of pO2, pH, and [Pi] by Electron Paramagnetic Resonance Spectroscopy.

Hypoxia, acidosis, and elevated inorganic phosphate concentration are characteristics of the tumor microenvironment in solid tumors. There are a number of methods for measuring each parameter individually in vivo, but the only method to date for noninvasive measurement of all three variables simultaneously in vivo is electron paramagnetic spectroscopy paired with a monophosphonated trityl radical, pTAM/HOPE. While HOPE has been successfully used for in vivo studies upon intratissue injection, it cannot be delivered intravenously due to systemic toxicity and albumin binding, which causes significant signal loss. Therefore, we present HOPE71, a monophosphonated trityl radical derived from the very biocompatible trityl probe, Ox071. Here, we describe a straightforward synthesis of HOPE71 starting with Ox071 and report its EPR sensitivities to pO2, pH, and [Pi] with X-band and L-band EPR spectroscopy. We also confirm that HOPE71 lacks albumin binding, shows low cytotoxicity, and has systemic tolerance. Finally, we demonstrate its ability to profile the tumor microenvironment in vivo in a mouse model of breast cancer.

Electron Paramagnetic Resonance Implemented with Multiple Harmonic Detections Successfully Maps Extracellular pH In Vivo.

Extracellular acidification indicates a metabolic shift in cancer cells and is, along with tissue hypoxia, a hallmark of tumor malignancy. Thus, non-invasive mapping of extracellular pH (pHe) is essential for researchers to understand the tumor microenvironment and to monitor tumor response to metabolism-targeting drugs. While electron paramagnetic resonance (EPR) has been successfully used to map pHe in mouse xenograft models, this method is not sensitive enough to map pHe with a moderate amount of exogenous pH-sensitive probes. Here, we show that a modified EPR system achieves twofold higher sensitivity by using the multiple harmonic detection (MHD) method and improves the robustness of pHe mapping in mouse xenograft models. Our results demonstrate that treatment of a mouse xenograft model of human-derived pancreatic ductal adenocarcinoma cells with the carbonic anhydrase IX (CAIX) inhibitor U-104 delays tumor growth with a concurrent tendency toward further extracellular acidification. We anticipate that EPR-based pHe mapping can be expanded to monitor the response of other metabolism-targeting drugs. Furthermore, pHe monitoring can also be used for the development of improved metabolism-targeting cancer treatments.

EPR Viscometric Measurements Using a 13C-Labeled Triarylmethyl Radical in Protein-based Biotherapeutics and Human Synovial Fluids.

The viscosity measurements are of clinical significance for evaluation of the potential pathological conditions of biological lubricants such as synovial fluids of joints, and for formulation and characterization of peptide- and protein-based biotherapeutics. Due to inherent potential therapeutic activity, protein drugs have proven to be one of the most efficient therapeutic agents in treatment of several life-threatening disorders, such as diabetes and autoimmune diseases. However, home-use applications for treating chronic inflammatory diseases, such as diabetes and rheumatoid arthritis, necessitate the development of high-concentration insulin and monoclonal antibodies formulations for patient self-administration. High protein concentrations can affect viscosity of the corresponding drug solutions complicating their manufacture and administration. The measurements of the viscosity of new insulin analogs and monoclonal antibodies solutions under development is of practical importance to avoid unwanted highly viscous, and therefore, painful for injection drug formulations. Recently, we have demonstrated capability of the electron paramagnetic resonance (EPR) viscometry using viscosity-sensitive 13C-labeled trityl spin probe (13C1-dFT) to report the viscosity of human blood, and interstitial fluids measured in various organs in mice ex-vivo and in anesthetized mice, in vivo. In the present work, we demonstrate utility of the EPR viscometry using 13C1-dFT to measure microviscosity of commercial insulin samples, antibodies solution, and human synovial fluids using small microliter volume samples (5-50 µL). This viscometry analysis approach provides useful tool to control formulations and administration of new biopharmaceuticals, and for evaluation of the state of synovial fluids of importance for clinical applications.

Biological Applications of Electron Paramagnetic Resonance Viscometry Using a 13C-Labeled Trityl Spin Probe.

Alterations in viscosity of biological fluids and tissues play an important role in health and diseases. It has been demonstrated that the electron paramagnetic resonance (EPR) spectrum of a 13C-labeled trityl spin probe (13C-dFT) is highly sensitive to the local viscosity of its microenvironment. In the present study, we demonstrate that X-band (9.5 GHz) EPR viscometry using 13C-dFT provides a simple tool to accurately measure the microviscosity of human blood in microliter volumes obtained from healthy volunteers. An application of low-field L-band (1.2 GHz) EPR with a penetration depth of 1-2 cm allowed for microviscosity measurements using 13C-dFT in the living tissues from isolated organs and in vivo in anesthetized mice. In summary, this study demonstrates that EPR viscometry using a 13C-dFT probe can be used to noninvasively and rapidly measure the microviscosity of blood and interstitial fluids in living tissues and potentially to evaluate this biophysical marker of microenvironment under various physiological and pathological conditions in preclinical and clinical settings.

Microsecond Exchange Processes Studied by Two-Dimensional ESR at 95 GHz.

Exchange processes which include conformational change, protonation/deprotonation, and binding equilibria are routinely studied by 2D exchange NMR techniques, where information about the exchange of nuclei between environments with different NMR shifts is obtained from the development of cross-peaks. Whereas 2D NMR enables the real time study of millisecond and slower exchange processes, 2D ESR in the form of 2D-ELDOR (two-dimensional electron-electron double resonance) has the potential for such studies over the nanosecond to microsecond real time scales. Cross-peak development due to chemical exchange has been seen previously for semiquinones in ESR, but this is not possible for most common ESR probes, such as nitroxides, studied at typical ESR frequencies because, unlike NMR, the exchanging states yield ESR signals that are not resolved from each other within their respective line widths. But at 95 GHz, it becomes possible to resolve them in many cases because of the increased g-factor resolution. The 95 GHz instrumental developments occurring at ACERT now enable such studies. We demonstrate these new capabilities in two studies: (A) the protonation/deprotonation process for a pH-sensitive imidazoline spin label in aqueous solution where the exchange rate and the population ratio of the exchanging states are controlled by the concentration and pH of the buffer solution, respectively, and (B) a nitroxide radical partitioning between polar (aqueous) and nonpolar (phospholipid) environments in multilamellar lipid vesicles, where the cross-peak development arises from the exchange of the nitroxide between the two phases. This work represents the first example of the observation and analysis of cross-peaks arising from chemical exchange processes involving nitroxide spin labels.

Synthesis, Characterization, and Application of a Highly Hydrophilic Triarylmethyl Radical for Biomedical EPR.

Stable tetrathiatriarylmethyl radicals have significantly contributed to the recent progress in biomedical electron paramagnetic resonance (EPR) due to their unmatched stability in biological media and long relaxation times. However, the lipophilic core of the most commonly used structure (Finland trityl) is responsible for its interaction with plasma biomacromolecules, such as albumin, and self-aggregation at high concentrations and/or low pH. While Finland trityl is generally considered inert toward many reactive radical species, we report that sulfite anion radical efficiently substitutes the three carboxyl moieties of Finland trityl with a high rate constant of 3.53 × 108 M-1 s-1, leading to a trisulfonated Finland trityl radical. This newly synthesized highly hydrophilic trityl radical shows an ultranarrow linewidth (ΔBpp = 24 mG), a lower affinity for albumin than Finland trityl, and a high aqueous solubility even at acidic pH. Therefore, this new tetrathiatriarylmethyl radical can be considered as a superior spin probe in comparison to the widely used Finland trityl. One of its potential applications was demonstrated by in vivo mapping oxygen in a mouse model of breast cancer. Moreover, we showed that one of the three sulfo groups can be easily substituted with S-, N-, and P-nucleophiles, opening access to various monofunctionalized sulfonated trityl radicals.

In vitro simultaneous mapping of the partial pressure of oxygen, pH and inorganic phosphate using electron paramagnetic resonance.

The partial pressure of oxygen (pO2) and the extracellular pH in the tumour microenvironment are essential parameters for understanding the physiological state of a solid tumour. Also, phosphate-containing metabolites are involved in energy metabolism, and interstitial inorganic phosphate (Pi) is an informative marker for tumour growth. This article describes the simultaneous mapping of pO2, pH and Pi using 750 MHz continuous-wave (CW) electron paramagnetic resonance (EPR) and a multifunctional probe, monophosphonated trityl radical p1TAM-D. The concept was demonstrated by acquiring three-dimensional (3D) maps of pO2, pH and Pi for multiple solution samples. This was made possible by combining a multifunctional radical probe, fast CW-EPR spectral acquisition, four-dimensional (4D) spectral-spatial image reconstruction, and spectral fitting. The experimental results of mapping pO2, pH and Pi suggest that the concept of simultaneous mapping using EPR is potentially applicable for the multifunctional measurements of a mouse tumour model.

Dextran-conjugated tetrathiatriarylmethyl radicals as biocompatible spin probes for EPR spectroscopy and imaging.

Tetrathiatriarylmethyl (TAM) radicals represent soluble paramagnetic probes for biomedical electron paramagnetic resonance (EPR)-based spectroscopy and imaging. There is an increasing demand in the development of multifunctional, biocompatible and targeted trityl probes hampered by the difficulties in derivatization of the TAM structure. We proposed a new straightforward synthetic strategy using click chemistry for the covalent conjugation of the TAM radical with a water-soluble biocompatible carrier exemplified here by dextran. A set of dextran-grafted probes varied in the degrees of Finland trityl radical loading and dextran modification by polyethelene glycol has been synthesized. The EPR spectrum of the optimized macromolecular probe exhibits a single narrow line with high sensitivity to oxygen and has advantages over the unbound Finland trityl of being insensitive to interactions with albumin. In vivo EPR imaging of tissue oxygenation performed in breast tumor-bearing mouse using dextran-grafted probe demonstrates its utility for preclinical oximetric applications.

In Vivo Extracellular pH Mapping of Tumors Using Electron Paramagnetic Resonance.

An electron paramagnetic resonance (EPR)-based method for noninvasive three-dimensional extracellular pH mapping was developed using a pH-sensitive nitroxyl radical as an exogenous paramagnetic probe. Fast projection scanning with a constant magnetic field sweep enabled the acquisition of four-dimensional (3D spatial +1D spectral) EPR images within 7.5 min. Three-dimensional maps of pH were reconstructed by processing the pH-dependent spectral information on the images. To demonstrate the proposed method of pH mapping, the progress of extracellular acidosis in tumor-bearing mouse legs was studied. Furthermore, extracellular pH mapping was used to visualize the spatial distribution of acidification in different tumor xenograft mouse models of human-derived pancreatic ductal adenocarcinoma cells. The proposed EPR-based pH mapping method enabled quantitative visualization of regional changes in extracellular pH associated with altered tumor metabolism.

Imaging of Enzyme Activity by Electron Paramagnetic Resonance: Concept and Experiment Using a Paramagnetic Substrate of Alkaline Phosphatase.

Enzyme activities are well established biomarkers of many pathologies. Imaging enzyme activity directly in vivo may help to gain insight into the pathogenesis of various diseases but remains extremely challenging. In this communication, we report the use of EPR imaging (EPRI) in combination with a specially designed paramagnetic enzymatic substrate to map alkaline phosphatase activity with a high selectivity, thereby demonstrating the potential of EPRI to map enzyme activity.

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

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.

Feasibility of in vivo three-dimensional T 2* mapping using dicarboxy-PROXYL and CW-EPR-based single-point imaging.

OBJECTIVES: The aim of this study was to demonstrate the feasibility of in vivo three-dimensional (3D) relaxation time T 2* mapping of a dicarboxy-PROXYL radical using continuous-wave electron paramagnetic resonance (CW-EPR) imaging. MATERIALS AND METHODS: Isotopically substituted dicarboxy-PROXYL radicals, 3,4-dicarboxy-2,2,5,5-tetra(2H3)methylpyrrolidin-(3,4-2H2)-(1-15N)-1-oxyl (2H,15N-DCP) and 3,4-dicarboxy-2,2,5,5-tetra(2H3)methylpyrrolidin-(3,4-2H2)-1-oxyl (2H-DCP), were used in the study. A clonogenic cell survival assay was performed with the 2H-DCP radical using squamous cell carcinoma (SCC VII) cells. The time course of EPR signal intensities of intravenously injected 2H,15N-DCP and 2H-DCP radicals were determined in tumor-bearing hind legs of mice (C3H/HeJ, male, n = 5). CW-EPR-based single-point imaging (SPI) was performed for 3D T 2* mapping. RESULTS: 2H-DCP radical did not exhibit cytotoxicity at concentrations below 10 mM. The in vivo half-life of 2H,15N-DCP in tumor tissues was 24.7 ± 2.9 min (mean ± standard deviation [SD], n = 5). The in vivo time course of the EPR signal intensity of the 2H,15N-DCP radical showed a plateau of 10.2 ± 1.2 min (mean ± SD) where the EPR signal intensity remained at more than 90% of the maximum intensity. During the plateau, in vivo 3D T 2* maps with 2H,15N-DCP were obtained from tumor-bearing hind legs, with a total acquisition time of 7.5 min. CONCLUSION: EPR signals of 2H,15N-DCP persisted long enough after bolus intravenous injection to conduct in vivo 3D T 2* mapping with CW-EPR-based SPI.

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

PURPOSE: 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. METHODS: EPR oxygen measurements were performed using isotopically substituted (2) H,(15) N-dicarboxyproxyl. The radical has an EPR line width of 320 mG and oxygen-induced line broadening of 0.53 mG/mm Hg, providing oxygen sensitivity down to 5 µM. pH measurements were performed using previously developed pH-sensitive imidazoline nitroxide. The radical has an EPR spectrum with pH-dependable hyperfine splitting, pK = 6.6, providing pH sensitivity of approximately 0.05 U in the physiological range. RESULTS: EPR imaging of isolated and perfused rat hearts was performed in the two-dimensional + spectral domain. The spatial resolution of the measurements was about 1.4 mm. Marked tissue hypoxia was observed in the ischemic area of the heart after occlusion of the left anterior descending coronary artery. Tissue oxygenation was partly restored upon reperfusion. EPR mapping of myocardial pH indicated acidosis of the ischemic area down to pH 6.7-6.8. CONCLUSION: This study demonstrates the capability of low-field EPR and the nitroxide spin probes for mapping of myocardial oxygenation and pH. The developed approaches might be used for noninvasive investigation of microenvironment on living objects. Magn Reson Med 76:350-358, 2016. © 2015 Wiley Periodicals, Inc.

Poly-arginine conjugated triarylmethyl radical as intracellular spin label.

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.

Detection of nitric oxide production in cell cultures by luciferin-luciferase chemiluminescence.

A chemiluminescent method is proposed for quantitation of NO generation in cell cultures. The method is based on activation of soluble guanylyl cyclase by NO. The product of the guanylyl cyclase reaction, pyrophosphate, is converted to ATP by ATP sulfurylase and ATP is detected in a luciferin-luciferase system. The method has been applied to the measurement of NO generated by activated murine macrophages (RAW 264.7) and bovine aortic endothelial cells. For macrophages activated by lipopolysaccharide and γ-interferon, the rate of NO production is about 100 amol/(cell·min). The rate was confirmed by the measurements of nitrite, the product of NO oxidation. For endothelial cells, the basal rate of NO generation is 5 amol/(cell·min); the rate approximately doubles upon activation by bradykinin, Ca(2+) ionophore A23187 or mechanical stress. For both types of cells the measured rate of NO generation is strongly affected by inhibitors of NO synthase. The sensitivity of the method is about 50 pM/min, allowing the registration of NO generated by 10(2)-10(4) cells. The enzyme-linked chemiluminescent method is two orders of magnitude more sensitive than fluorescent detection using 4-amino-5-methylamino-2',7'-difluorofluorescein (DAF-FM).

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

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.

Electron spin relaxation times and rapid scan EPR imaging of pH-sensitive amino-substituted trityl radicals.

Carboxy-substituted trityl (triarylmethyl) radicals are valuable in vivo probes because of their stability, narrow lines, and sensitivity of their spectroscopic properties to oxygen. Amino-substituted trityl radicals have the potential to monitor pH in vivo, and the suitability for this application depends on spectral properties. Electron spin relaxation times T1 and T2 were measured at X-band for the protonated and deprotonated forms of two amino-substituted triarylmethyl radicals. Comparison with relaxation times for carboxy-substituted triarylmethyl radicals shows that T1 exhibits little dependence on protonation or the nature of the substituent, which makes it useful for measuring O2 concentration, independent of pH. Insensitivity of T1 to changes in substituents is consistent with the assignment of the dominant contribution to spin lattice relaxation as a local mode that involves primarily atoms in the carbon and sulfur core. Values of T2 vary substantially with pH and the nature of the aryl group substituent, reflecting a range of dynamic processes. The narrow spectral widths for the amino-substituted triarylmethyl radicals facilitate spectral-spatial rapid scan electron paramagnetic resonance imaging, which was demonstrated with a phantom. The dependence of hyperfine splittings patterns on pH is revealed in spectral slices through the image.

In vivo proton-electron double-resonance imaging of extracellular tumor pH using an advanced nitroxide probe.

A variable radio frequency proton-electron double-resonance imaging (VRF PEDRI) approach for pH mapping of aqueous samples has been recently developed (Efimova et al. J. Magn. Reson. 2011, 209, 227-232). A pH map is extracted from two PEDRI acquisitions performed at electron paramagnetic resonance (EPR) frequencies of protonated and unprotonated forms of a pH-sensitive probe. To translate VRF PEDRI to an in vivo setting, an advanced pH probe was synthesized. Probe deuteration resulted in a narrow spectral line of 1.2 G compared to a nondeuterated analogue line width of 2.1 G allowing for an increase of Overhauser enhancements and reduction in rf power deposition. Binding of the probe to the cell-impermeable tripeptide, glutathione (GSH), allows for targeting to extracellular tissue space for monitoring extracellular tumor acidosis, a prognostic factor in tumor pathophysiology. The probe demonstrated pH sensitivity in the 5.8-7.8 range, optimum for measurement of acidic extracellular tumor pH (pH(e)). In vivo VRF PEDRI was performed on Met-1 tumor-bearing mice. Compared to normal mammary glands with a neutral mean pH(e) (7.1 ± 0.1), we observed broader pH distribution with acidic mean pH(e) (6.8 ± 0.1) in tumor tissue. In summary, VRF PEDRI in combination with a newly developed pH probe provides an analytical approach for spatially resolved noninvasive pHe monitoring, in vivo.