Background We investigated the role of A(2B)-adenosine receptor in regulating immunosuppressive metabolic stress in the tumor microenvironment. Novel A(2B)-adenosine receptor antagonist PBF-1129 was tested for antitumor activity in mice and evaluated for safety and immunologic efficacy in a phase I clinical trial of patients with non-small cell lung cancer. Methods The antitumor efficacy of A(2B)-adenosine receptor antagonists and their impact on the metabolic and immune tumor microenvironment were evaluated in lung, melanoma, colon, breast, and epidermal growth factor receptor-inducible transgenic cancer models. Employing electron paramagnetic resonance, we assessed changes in tumor microenvironment metabolic parameters, including pO(2), pH, and inorganic phosphate, during tumor growth and evaluated the immunologic effects of PBF-1129, including its pharmacokinetics, safety, and toxicity, in patients with non-small cell lung cancer. Results Levels of metabolic stress correlated with tumor growth, metastasis, and immunosuppression. Tumor interstitial inorganic phosphate emerged as a correlative and cumulative measure of tumor microenvironment stress and immunosuppression. A(2B)-adenosine receptor inhibition alleviated metabolic stress, downregulated expression of adenosine-generating ectonucleotidases, increased expression of adenosine deaminase, decreased tumor growth and metastasis, increased interferon gamma production, and enhanced the efficacy of antitumor therapies following combination regimens in animal models (anti-programmed cell death 1 protein vs anti-programmed cell death 1 protein plus PBF-1129 treatment hazard ratio = 11.74 [95% confidence interval=3.35 to 41.13], n=10, P<.001, 2-sided F test). In patients with non-small cell lung cancer, PBF-1129 was well tolerated, with no dose-limiting toxicities; demonstrated pharmacologic efficacy; modulated the adenosine generation system; and improved antitumor immunity. Conclusions Data identify A(2B)-adenosine receptor as a valuable therapeutic target to modify metabolic and immune tumor microenvironment to reduce immunosuppression, enhance the efficacy of immunotherapies, and support clinical application of PBF-1129 in combination therapies.

Linearity of the magnetic field sweep is important for high resolution continuous wave EPR imaging. Driving the field with triangular wave function is the most efficient way to scan EPR projections. However, the magnetic field sweep profile can be significantly distorted during fast millisecond projection scan. In this work, we introduce a method to generate highly linear and properly symmetrical triangular sweeps of the magnetic field using calibrated harmonics of the triangular wave function. First, the frequency response function of the EPR magnet and its power circuitry was obtained. For this, the field sweeping coil was driven with sinusoidal signals of different frequencies and the actual magnetic field inside the magnet was recorded. To cover wide range of frequencies, the measurements were carried out independently using gaussmeter, Hall-effect linear sensor integrated circuit, and an inductance coil. For each frequency, the system gain and the phase delay were determined. These data were used to adjust the amplitudes and the phases of individual harmonics of the triangular wave function. After the calibration, the maximum deviation of the magnetic field from the linear function was 0.05% of sweep width for 4 ms scan. The maximum discrepancy between the forward and the reverse scan was less than 0.04%. Sweep overhead time for changing the scan direction was 5%. The proposed approach allows generation of high fidelity triangular magnetic field sweeps with accuracy better than 0.1% for the range of the magnetic field sweep widths up to 48 G and scan duration from 10 s down to 1 ms. (c) 2021 Elsevier Inc. All rights reserved.

Nitroxide probes have been commonly used for biomedical EPR imaging applications; however, image quality has been limited by the number of image projections, as well as the probe linewidth and hyperfine structure. In the current study, we evaluate the use of fast millisecond scan EPR projection acquisition along with a novel reconstruction algorithm optimized for 3D spatial EPR image reconstruction from a high number of noisy projections. This reconstruction method utilizes the raw image projection data and zero gradient spectrum to account for EPR line shape and hyperfine structure of any given paramagnetic probe without the need for deconvolution that is poorly suited for high noise data. Using fast scan EPR imaging with this reconstruction method, we image non-deuterated, deuterated and 15N substituted nitroxide probes in experimental phantoms of complex geometries. We evaluate the image resolution that can be obtained and the imaging time required. With 16,384 projections acquired over 1 min, and a field gradient of 8 G/cm, with a 2503 voxel 3D matrix, spatial resolutions of up to 100 µm are theoretically possible for a cubical volume of 25 × 25 × 25 mm3. In experiments with a variety of phantoms with mM nitroxide radical probes, resolutions of 600–250 µm were obtained with 1–10 min acquisitions, respectively. The presently obtainable signal sensitivity and noise levels of these acquisitions limited the obtainable resolution. With longer time acquisitions or further improvements in sensitivity and noise reduction, image resolutions approaching 100 µm should be possible.

Purpose To develop a novel resonator for high‐quality fast scan electron paramagnetic resonance (EPR) and EPR/NMR co‐imaging of the head and brain of mice at 1.25 GHz. Methods Resonator dimensions were scaled to fit the mouse head with maximum filling factor. A single‐loop 6‐gap resonator of 20 mm diameter and 20 mm length was constructed. High resonator stability was achieved utilizing a fixed position double coupling loop. Symmetrical mutually inverted connections rendered it insensitive to field modulation and fast scan. Coupling adjustment was provided by a parallel‐connected variable capacitor located at the feeding line at λ/4 distance. To minimize radiation loss, the shield around the resonator was supplemented with a planar conductive disc that focuses return magnetic flux. Results Coupling of the resonator loaded with the mouse head was efficient and easy. This resonator enabled high‐quality in vivo 3D EPR imaging of the mouse head following intravenous infusion of nitroxide probes. With this resonator and rapid scan EPR system, 4 ms scans were acquired in forward and reverse directions so that images with 2‐scan 3,136 projections were acquired in 25 s. Head images were achieved with resolutions of 0.4 mm, enabling visualization of probe localization and uptake across the blood–brain barrier. Conclusions This resonator design provides good sensitivity, high stability, and B 1 field homogeneity for in vivo fast scan EPR of the mouse head and brain, enabling faster measurements and higher resolution imaging of probe uptake, localization, and metabolism than previously possible.

Significance We identified a novel function of cytoglobin (Cygb) as an efficient superoxide dismutase (SOD) with a high–bimolecular dismutation rate on the order of 10 8 M −1 ⋅ s −1 . It is the first Fe-centered SOD identified in mammalian cells and the only member of the globin family shown to have potent SOD activity. This SOD function of Cygb may serve to explain the prior reports of its antioxidative activity and the toxicities and disease seen with its genetic deletion, including the increased incidence of tumors. Combined with its potent NO dioxygenase activity, Cygb would be highly effective in decreasing both O 2 •− and NO levels, serving to decrease the formation of peroxynitrite and secondary oxidative injury.

A novel method for reconstructing 3D spatial EPR images from large numbers of noisy projections was developed that minimizes mean square error between the experimental projections and those from the reconstructed image. The method utilizes raw projection data and zero gradient spectrum to account for EPR line shape and hyperfine structure of the paramagnetic probe without the need for deconvolution techniques that are poorly suited for processing of high noise projections. A numerical phantom was reconstructed for method validation. Reconstruction time for the matrix of 128(3) voxels and 16,384 noiseless projections was 4.6 min for a single iteration. The algorithm converged quickly, reaching R-2 similar to 0.99975 after the very first iteration. An experimental phantom sample with nitroxyl radical was measured. With 16,384 projections and a field gradient of 8 G/cm, resolutions of 0.4 mm were achieved for a cubical area of 25 x 25 x 25 mm(3). Reconstruction was sufficiently fast and memory efficient making it suitable for applications with large 3D matrices and fully determined system of equations. The developed algorithm can be used with any gradient distribution and does not require adjustable filter parameters that makes for simple application. A thorough analysis of the strengths and limitations of this method for 3D spatial EPR imaging is provided. (C) 2020 Elsevier Inc. All rights reserved.

Objectives: To develop and test a new system for whole body exposure of small animals to support investigation of the biological effects of aerosol generated by electronic cigarette (e-cig) products under diverse inhalation conditions with improved control and monitoring of the e-cig vape exposure and nicotine delivered to the animal's systemic circulation. Methods: A computer-controlled design, with built-in sensors for real time monitoring of O2, CO2, relative humidity, and temperature within the exposure chambers and port for measuring total particulate matter (TPM) was developed, constructed and tested. This design accommodates a variety of commercial vaping devices, offers software flexibility to adjust exposure protocols to mimic different users' puffing patterns, enables variable nicotine delivery to the animal's systemic circulation; minimizes travel time and alterations of aerosol quality or quantity by delivering aerosol directly to the exposure chamber, offers local or remote operation of up to six distinct exposure chambers from a single control unit, and can simultaneously test different exposure conditions or products in diverse animal groups, which reduces inter-run variability, saves time, and increases productivity. Results: The time course pattern of TPM concentration during different phases of the exposure cycle was measured. With increased puffing duration or number of exposure cycles, higher TPM exposure and plasma cotinine levels were observed with plasma cotinine levels in the range reported in light or heavy smokers. Conclusion: Overall, this novel, versatile, and durable exposure system facilitates high-throughput evaluation of the relative safety and potential toxicity of a variety of e-cig devices and liquids.

PurposeIn continuous wave EPR imaging, the acquisition of high-quality images was previously limited by the requisite long acquisition times of each image projection that was typically greater than 1 second. To accelerate the process of image acquisition facilitating greater numbers of projections and higher image resolution, instrumentation was developed to greatly accelerate the magnetic field scan that is used to obtain each EPR image projection. MethodsA low-inductance solenoidal coil for field scanning was used along with a spherical solenoid air core magnet, and scans were driven by triangular symmetric waves, allowing forward and reverse spectrum acquisition as rapid as 3.8 ms. The uniform distribution of projections was used to optimize the contribution of projections for 3D image reconstruction. ResultsUsing this fast-scan EPR system, high-quality EPR images of phantoms and perfused rat hearts were performed using trityl or nanoparticulate LiNcBuO (lithium octa-n-butoxy-substituted naphthalocyanine) probes with fast-scan EPR imaging at L-band, achieving spatial resolutions of up to 250 micrometers in 1 minute. ConclusionFast-scan EPR imaging can greatly facilitate the efficient and precise mapping of the spatial distribution of free radical and other paramagnetic probes in living systems.

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.

EPR oximetry with the use of trityl radicals can enable sensitive O2 measurement in biological cells and tissues. However, in vitro cellular and in vivo biological applications are limited by rapid trityl probe degradation or biological clearance and the need to enhance probe O2 sensitivity. We synthesized novel perfluorocarbon (PFC) emulsions, ∼200nm droplet size, containing esterified perchlorinated triphenyl methyl (PTM) radicals dispersed in physiological aqueous buffers. These formulations exhibit excellent EPR signal stability, over 20-fold greater than free PTM probes, with high oxygen sensitivity ∼17mG/mmHg enabling pO2 measurement in aqueous solutions or cell suspensions with sensitivity >0.5mmHg. Thus, PFC-PTM probes hold great promise to enable combined O2 delivery and sensing as needed to restore or enhance tissue oxygenation in disease.

Electron paramagnetic resonance (EPR) allows quantitative imaging of tissue redox status, which provides important information about ischemic syndromes, cancer and other pathologies. For continuous wave EPR imaging, however, poor signal-to-noise ratio and low acquisition efficiency limit its ability to image dynamic processes in vivo including tissue redox, where conditions can change rapidly. Here, we present a data acquisition and processing framework that couples fast acquisition with compressive sensing-inspired image recovery to enable EPR-based redox imaging with high spatial and temporal resolutions. The fast acquisition (FA) allows collecting more, albeit noisier, projections in a given scan time. The composite regularization based processing method, called spatio-temporal adaptive recovery (STAR), not only exploits sparsity in multiple representations of the spatio-temporal image but also adaptively adjusts the regularization strength for each representation based on its inherent level of the sparsity. As a result, STAR adjusts to the disparity in the level of sparsity across multiple representations, without introducing any tuning parameter. Our simulation and phantom imaging studies indicate that a combination of fast acquisition and STAR (FASTAR) enables high-fidelity recovery of volumetric image series, with each volumetric image employing less than 10 s of scan. In addition to image fidelity, the time constants derived from FASTAR also match closely to the ground truth even when a small number of projections are used for recovery. This development will enhance the capability of EPR to study fast dynamic processes that cannot be investigated using existing EPR imaging techniques.

Variable radio frequency proton-electron double-resonance imaging (VRF PEDRI) enables extracting a functional map from a limited number of images acquired at pre-selected EPR frequencies using specifically designed paramagnetic probes with high-quality spatial resolution and short acquisition times. In this work we explored the potential of VRF PEDRI for pH mapping of aqueous samples using recently synthesized pH-sensitive phosphonated trityl radical, pTR. The ratio of Overhauser enhancements measured at each pixel at two different excitation frequencies corresponding to the resonances of protonated and deprotonated forms of pTR probe allows for a pH map extraction. Long relaxation times of pTR allow for pH mapping at EPR irradiation power as low as 1.25 W during 130 s acquisition time with spatial resolution of about 1 mm. This is particularly important for in vivo applications enabling one to avoid sample overheating by reducing RF power deposition.

PurposeTo improve image quality and reduce data requirements for spatial electron paramagnetic resonance imaging (EPRI) by developing a novel reconstruction approach using compressed sensing (CS).MethodsEPRI is posed as an optimization problem, which is solved using regularized least‐squares with sparsity promoting penalty terms, consisting of the l1 norms of the image itself and the total variation of the image. Pseudo‐random sampling was employed to facilitate recovery of the sparse signal. The reconstruction was compared with the traditional filtered back‐projection reconstruction for simulations, phantoms, isolated rat hearts, and mouse gastrointestinal (GI) tracts labeled with paramagnetic probes.ResultsA combination of pseudo‐random sampling and CS was able to generate high‐fidelity EPR images at high acceleration rates. For three‐dimensional (3D) phantom imaging, CS‐based EPRI showed little visual degradation at nine‐fold acceleration. In rat heart datasets, CS‐based EPRI produced high quality images with eight‐fold acceleration. A high resolution mouse GI tract reconstruction demonstrated a visual improvement in spatial resolution and a doubling in signal‐to‐noise ratio (SNR).ConclusionA novel 3D EPRI reconstruction using compressed sensing was developed and offers superior SNR and reduced artifacts from highly undersampled data. Magn Reson Med 72:893–901, 2014. © 2013 Wiley Periodicals, Inc.

PurposeTo improve the quality and speed of electron paramagnetic resonance imaging (EPRI) acquisition by combining a uniform sampling distribution with spinning gradient acquisition.Theory and MethodsA uniform sampling distribution was derived for spinning gradient EPRI acquisition (uniform spinning sampling, USS) and compared to the existing (equilinear spinning sampling, ESS) acquisition strategy. Novel corrections were introduced to reduce artifacts in experimental data.ResultsSimulations demonstrated that USS puts an equal number of projections near each axis whereas ESS puts excessive projections at one axis, wasting acquisition time. Artifact corrections added to the magnetic gradient waveforms reduced noise and correlation between projections. USS images had higher SNR (85.9 ± 0.8 vs. 56.2 ± 0.8) and lower mean‐squared error than ESS images. The quality of the USS images did not vary with the magnetic gradient orientation, in contrast to ESS images. The quality of rat heart images was improved using USS compared to that with ESS or traditional fast‐scan acquisitions.ConclusionA novel EPRI acquisition which combines spinning gradient acquisition with a uniform sampling distribution was developed. This USS spinning gradient acquisition offers superior SNR and reduced artifacts compared to prior methods enabling potential improvements in speed and quality of EPR imaging in biological applications. Magn Reson Med 71:893–900, 2014. © 2013 Wiley Periodicals, Inc.

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.

In vivo or ex vivo electron paramagnetic resonance imaging (EPRI) is a powerful technique for determining the spatial distribution of free radicals and other paramagnetic species in living organs and tissues. However, applications of EPRI have been limited by long projection acquisition times and the consequent fact that rapid gated EPRI was not possible. Hence in vivo EPRI typically provided only time‐averaged information. In order to achieve direct gated EPRI, a fast EPR acquisition scheme was developed to decrease EPR projection acquisition time down to 10–20 ms, along with corresponding software and instrumentation to achieve fast gated EPRI of the isolated beating heart with submillimeter spatial resolution in as little as 2–3 min. Reconstructed images display temporal and spatial variations of the free‐radical distribution, anatomical structure, and contractile function within the rat heart during the cardiac cycle. Magn Reson Med, 2013. © 2012 Wiley Periodicals, Inc.

Approach for in vivo real-time assessment of tumor tissue extracellular pH (pH(e)), redox, and intracellular glutathione based on L-band EPR spectroscopy using dual function pH and redox nitroxide probe and disulfide nitroxide biradical, is described. These parameters were monitored in PyMT mice bearing breast cancer tumors during treatment with granulocyte macrophage colony-stimulating factor. It was observed that tumor pH(e) is about 0.4 pH units lower than that in normal mammary gland tissue. Treatment with granulocyte macrophage colony-stimulating factor decreased the value of pH(e) by 0.3 units compared with PBS control treatment. Tumor tissue reducing capacity and intracellular glutathione were elevated compared with normal mammary gland tissue. Granulocyte macrophage colony-stimulating factor treatment resulted in a decrease of the tumor tissue reducing capacity and intracellular glutathione content. In addition to spectroscopic studies, pH(e) mapping was performed using recently proposed variable frequency proton-electron double-resonance imaging. The pH mapping superimposed with MRI image supports probe localization in mammary gland/tumor tissue, shows high heterogeneity of tumor tissue pH(e) and a difference of about 0.4 pH units between average pH(e) values in tumor and normal mammary gland. In summary, the developed multifunctional approach allows for in vivo, noninvasive pH(e), extracellular redox, and intracellular glutathione content monitoring during investigation of various therapeutic strategies for solid tumors.

In vivo mapping of alterations in redox status is important for understanding organ specific pathology and disease. While electron paramagnetic resonance imaging (EPRI) enables spatial mapping of free radicals, it does not provide anatomic visualization of the body. Proton MRI is well suited to provide anatomical visualization. We applied EPR/NMR co-imaging instrumentation to map and monitor the redox state of living mice under normal or oxidative stress conditions induced by secondhand cigarette smoke (SHS) exposure. A hybrid co-imaging instrument, EPRI (1.2 GHz)/proton MRI (16.18 MHz), suitable for whole-body co-imaging of mice was utilized with common magnet and gradients along with dual EPR/NMR resonators that enable co-imaging without sample movement. The metabolism of the nitroxide probe, 3-carbamoyl-proxyl (3-CP), was used to map the redox state of control and SHS-exposed mice. Co-imaging allowed precise 3D mapping of radical distribution and reduction in major organs such as the heart, lungs, liver, bladder and kidneys. Reductive metabolism was markedly decreased in SHS-exposed mice and EPR/NMR co-imaging allowed quantitative assessment of this throughout the body. Thus, in vivo EPR/NMR co-imaging enables in vivo organ specific mapping of free radical metabolism and redox stress and the alterations that occur in the pathogenesis of disease.

Proton–electron double resonance imaging (PEDRI) has been utilized for indirect determination of oxygen concentrations in aqueous samples and living systems. Due to the complexity of the problem, there are seven oxygen related parameters that need to be measured to determine the distribution of oxygen. We present an improved approach in which image intensities from only two PEDRI acquisitions with different EPR irradiation powers are required to determine the distribution of a paramagnetic probe and oxygen in an analyzed sample. This is achieved using three reference samples with known concentrations of a paramagnetic probe and oxygen placed inside the resonator together with the measurement sample. An EPR-off image, which has low signal intensity at low magnetic field (0.02T) is not required for the calculations, significantly reducing the total time of the experiments and the noise while enhancing the accuracy of these oxygen measurements. The Finland trityl radical was used as the paramagnetic probe and oxygen concentrations could be accurately measured and imaged over the physiological range from 0 to 240μM.

Electron Paramagnetic Resonance Imaging (EPRI) can provide insight into in vivo anatomic and functional imaging of free radicals and paramagnetic molecules and their role in disease in small animal models. However, there is a need to expedite the data acquisition and post-processing to enable new EPRI applications. While previous work has used a fixed-rate master clock to pace all A/D and D/A conversion activities of the data acquisition electronics, Adaptive Heterogeneous Clocking (AHC) significantly reduces communication between the host computer and gradient hardware by using different clocks to pace the A/D and D/A functions of our acquisition cards. Projections containing up to 4096 points can be acquired in as little as 10 – 20 ms using AHC. Nearly real-time acquisition can be performed for complex computer-generated gradient waveforms, which will enable a variety of new sampling patterns.