Search

Purpose: Spatial heterogeneity in tumor hypoxia is one of the most important factors regulating tumor growth, development, aggressiveness, metastasis, and affecting treatment outcome. Most solid tumors are known to have hypoxia or low oxygen levels (pO2 ≤10 torr). Electron paramagnetic resonance oxygen imaging (EPROI) is an emerging oxygen mapping technology. EPROI utilizes the linear relationship between the relaxation rates of the injectable OX071 trityl spin probe and the partial oxygen pressure (pO2). However, most of the EPROI studies have been limited to mouse models of solid tumors because of the instrument-size limitations. The purpose of this work was to develop a human-sized 9-mT (250 MHz resonance frequency, 60 cm bore size) pulse EPROI instrument and evaluate its performance with rabbit VX-2 tumor oxygen imaging. Methods: A New Zealand white rabbit with a 3.2-cm VX-2 tumor in the calf muscle was imaged using the human-sized EPROI instrument and a 2.25-in. ID volume coil. The animal received a ~8-min intravenous injection of OX071 (5.2 mL total volume at 72 mM concentration) and, after 75 min, an intratumoral injection (120 μL total at 5 mM OX071 concentration) and underwent EPROI. At the end of the experiments, MRI was performed using a preclinical 9.4-T MRI system to outline the tumor boundaries. Results: For the first time, a human-sized pulse EPROI instrument with a 60-cm bore size/250-MHz frequency was built and evaluated using rabbit tumor oxygen imaging. For the first time, the systemic IV injection of the oxygen-sensitive trityl OX071 spin probe was used for an animal of this size. The resulting EPROI image from the IV injection showed complete tumor coverage. The image obtained after intratumoral injection showed localized coverage in the upper lobe of the tumor, demonstrating the need for improved intratumoral injection protocol. Conclusions: This study demonstrates the performance of the world's first human-sized pulse EPROI instrument. It also demonstrates that the EPROI of larger animals can be performed using the systemic injection of a manageable amount of the spin probe. This brings EPROI one step closer to clinical applications in cancer therapies. Oxygen imaging is a platform technology, and the instrument and techniques developed here will also be useful for other clinical applications. [ABSTRACT FROM AUTHOR]

Purpose: This study aimed to develop a biocompatible oximetric electron paramagnetic resonance (EPR) spin probe with reduced self-relaxation, and sensitivity to oxygen for a higher signal-to-noise ratio and longer relaxation times at high oxygen concentration, compared to the reference spin probe OX071. Procedures: SOX71 was synthesized by succinylation of the twelve alcohol groups of OX071 spin probe and characterized by EPR at X-Band (9.5 GHz) and at low field (720 MHz). The biocompatibility of SOX71 was tested in vitro and in vivo in mice. A pharmacokinetic study was performed to determine the best time frame for EPR imaging. Finally, a proof-of-concept EPR oxygen imaging was performed on a mouse model of a fibrosarcoma tumor. Results: SOX71 was synthesized in one step from OX071. SOX71 exhibits a narrow line EPR spectrum with a peak-to-peak linewidth of 66 mG, similar to OX071. SOX71 does not bind to albumin nor show cell toxicity for the concentrations tested up to 5 mM. No toxicity was observed after systemic delivery via intraperitoneal injection in mice at twice the dose required for EPR imaging. After the injection, the probe is readily absorbed into the bloodstream, with a peak blood concentration half an hour, post-injection. Then, the probe is quickly cleared by the kidney with a half-life of ~ 45 min. SOX71 shows long relaxation times under anoxic condition (T1e = 9.5 µs and T2e = 5.1 µs; [SOX71] = 1 mM in PBS at 37 °C, pO2 = 0 mmHg, 720 MHz). Both the relaxation rates R1e and R2e show a decreased sensitivity to pO2, leading to twice longer relaxation times under room air conditions (pO2 = 159 mmHg) compared to OX071. This is ideal for oxygen imaging in samples with a wide range of pO2. Both the relaxation rates R1e and R2e show a decreased sensitivity to self-relaxation compared to OX071, with a negligible effect of the probe concentration on R1e. SOX71 was successfully applied to image oxygen in a tumor. Conclusion: SOX71, a succinylated derivative of OX071 was synthesized, characterized, and applied for in vivo EPR tumor oxygen imaging. SOX71 is highly biocompatible, and shows decreased sensitivity to oxygen and self-relaxation. This first report suggests that SOX71 is superior to OX071 for absolute oxygen mapping under a broad range of pO2 values. [ABSTRACT FROM AUTHOR]

Purpose: Progress toward developing a novel radiocontrast agent for determining pO2 in tumors in a clinical setting is described. The imaging agent is designed for use with electron paramagnetic resonance imaging (EPRI), in which the collision of a paramagnetic probe molecule with molecular oxygen causes a spectroscopic change which can be calibrated to give the real oxygen concentration in the tumor tissue. Procedures: The imaging agent is based on a nanoscaffold of aluminum hydroxide (boehmite) with sizes from 100 to 200 nm, paramagnetic probe molecule, and encapsulation with a gas permeable, thin (10–20 nm) polymer layer to separate the imaging agent and body environment while still allowing O2 to interact with the paramagnetic probe. A specially designed deuterated Finland trityl (dFT) is covalently attached on the surface of the nanoparticle through 1,3-dipolar addition of the alkyne on the dFT with an azide on the surface of the nanoscaffold. This click-chemistry reaction affords 100% efficiency of the trityl attachment as followed by the complete disappearance of the azide peak in the infrared spectrum. The fully encapsulated, dFT-functionalized nanoparticle is referred to as RADI-Sense. Results: Side-by-side in vivo imaging comparisons made in a mouse model made between RADI-Sense and free paramagnetic probe (OX-071) showed oxygen sensitivity is retained and RADI-Sense can create 3D pO2 maps of solid tumors Conclusions: A novel encapsulated nanoparticle EPR imaging agent has been described which could be used in the future to bring EPR imaging for guidance of radiotherapy into clinical reality. [ABSTRACT FROM AUTHOR]

Purpose: Tumor hypoxia contributes to aggressive phenotypes and diminished therapeutic responses to radiation therapy (RT) with hypoxic tissue being 3-fold less radiosensitive than normoxic tissue. A major challenge in implementing hypoxic radiosensitizers is the lack of a high-resolution imaging modality that directly quantifies tissue-oxygen. The electron paramagnetic resonance oxygen-imager (EPROI) was used to quantify tumor oxygenation in two murine tumor models: E0771 syngeneic transplant breast cancers and primary p53/MCA soft tissue sarcomas, with the latter autochthonous model better recapitulating the tumor microenvironment in human malignancies. We hypothesized that tumor hypoxia differs between these models. We also aimed to quantify the absolute change in tumor hypoxia induced by the mitochondrial inhibitor papaverine (PPV) and its effect on RT response. Procedures: Tumor oxygenation was characterized in E0771 and primary p53/MCA sarcomas via EPROI, with the former model also being quantified indirectly via diffuse reflectance spectroscopy (DRS). After confirming PPV's effect on hypoxic fraction (via EPROI), we compared the effect of 0 versus 2 mg/kg PPV prior to 20 Gy on tumor growth delay and survival. Results: Hypoxic sarcomas were more radioresistant than normoxic sarcomas (p=0.0057, 2-way ANOVA), and high baseline hypoxic fraction was a significant (p=0.0063, Cox Regression Model) hazard in survivability regardless of treatment. Pre-treatment with PPV before RT did not radiosensitize tumors in the sarcoma or E0771 model. In the sarcoma model, EPROI successfully identified baseline hypoxic tumors. DRS quantification of total hemoglobin, saturated hemoglobin, changes in mitochondrial potential and glucose uptake showed no significant difference in E0771 tumors pre- and post-PPV. Conclusion: EPROI provides 3D high-resolution pO2 quantification; EPR is better suited than DRS to characterize tumor hypoxia. PPV did not radiosensitize E0771 tumors nor p53/MCA sarcomas, which may be related to the complex pattern of vasculature in each tumor. Additionally, understanding model-dependent tumor hypoxia will provide a much-needed foundation for future therapeutic studies with hypoxic radiosensitizers. [ABSTRACT FROM AUTHOR]

Type 1 diabetes (T1D) is an autoimmune disease that leads to the loss of insulin-producing beta cells. Bioartificial pancreas (BAP) or beta cell replacement strategies have shown promise in curing T1D and providing long-term insulin independence. Hypoxia (low oxygen concentration) that may occur in the BAP devices due to cell oxygen consumption at the early stages after implantation damages the cells, in addition to imposing limitations to device dimensions when translating promising results from rodents to humans. Finding ways to provide cells with sufficient oxygenation remains the major challenge in realizing BAP devices' full potential. Therefore, in vitro oxygen imaging assessment of BAP devices is crucial for predicting the devices' in vivo efficiency. Electron paramagnetic resonance oxygen imaging (EPROI, also known as electron MRI or eMRI) is a unique imaging technique that delivers absolute partial pressure of oxygen (pO2) maps and has been used for cancer hypoxia research for decades. However, its applicability for assessing BAP devices has not been explored. EPROI utilizes low magnetic fields in the mT range, static gradients, and the linear relationship between the spin–lattice relaxation rate (R1) of oxygen-sensitive spin probes such as trityl OX071 and pO2 to generate oxygen maps in tissues. With the support of the Juvenile Diabetes Research Foundation (JDRF), an academic-industry partnership consortium, the "Oxygen Measurement Core" was established at O2M to perform oxygen imaging assessment of BAP devices originated from core members' laboratories. This article aims to establish the protocols and demonstrate a few examples of in vitro oxygen imaging of BAP devices using EPROI. All pO2 measurements were performed using a recently introduced 720 MHz/25 mT preclinical oxygen imager instrument, JIVA-25™. We began by performing pO2 calibration of the biomaterials used in BAPs at 25 mT magnetic field since no such data exist. We compared the EPROI pO2 measurement with a single-point probe for a few selected materials. We also performed trityl OX071 toxicity studies with fibroblasts, as well as insulin-producing cells (beta TC6, MIN6, and human islet cells). Finally, we performed proof-of-concept in vitro pO2 imaging of five BAP devices that varied in size, shape, and biomaterials. We demonstrated that EPROI is compatible with commonly used biomaterials and that trityl OX071 is nontoxic to cells. A comparison of the EPROI with a fluorescent-based point oxygen probe in selected biomaterials showed higher accuracy of EPROI. The imaging of typically heterogenous BAP devices demonstrated the utility of obtaining oxygen maps over single-point measurements. In summary, we present EPROI as a quality control tool for developing efficient cell transplantation devices and artificial tissue grafts. Although the focus of this work is encapsulation systems for diabetes, the techniques developed in this project are easily transferable to other biomaterials, tissue grafts, and cell therapy devices used in the field of tissue engineering and regenerative medicine (TERM). In summary, EPROI is a unique noninvasive tool to experimentally study oxygen distribution in cell transplantation devices and artificial tissues, which can revolutionize the treatment of degenerative diseases like T1D. [ABSTRACT FROM AUTHOR]

Purpose: To identify the optimal threshold in 18F-fluoromisonidazole (FMISO) PET images to accurately locate tumor hypoxia by using electron paramagnetic resonance imaging (pO2 EPRI) as ground truth for hypoxia, defined by pO2 ≤ 10 mmHg. Methods: Tumor hypoxia images in mouse models of SCCVII squamous cell carcinoma (n = 16) were acquired in a hybrid PET/EPRI imaging system 2 h post-injection of FMISO. T2-weighted MRI was used to delineate tumor and muscle tissue. Dynamic contrast enhanced (DCE) MRI parametric images of Ktrans and ve were generated to model tumor vascular properties. Images from PET/EPR/MRI were co-registered and resampled to isotropic 0.5 mm voxel resolution for analysis. PET images were converted to standardized uptake value (SUV) and tumor-to-muscle ratio (TMR) units. FMISO uptake thresholds were evaluated using receiver operating characteristic (ROC) curve analysis to find the optimal FMISO threshold and unit with maximum overall hypoxia similarity (OHS) with pO2 EPRI, where OHS = 1 shows perfect overlap and OHS = 0 shows no overlap. The means of dice similarity coefficient, normalized Hausdorff distance, and accuracy were used to define the OHS. Monotonic relationships between EPRI/PET/DCE-MRI were evaluated with the Spearman correlation coefficient (ρ ) to quantify association of vasculature on hypoxia imaged with both FMISO PET and pO2 EPRI. Results: FMISO PET thresholds to define hypoxia with maximum OHS (both OHS = 0.728 ± 0.2) were SUV ≥ 1.4 × SUVmean and SUV ≥ 0.6 × SUVmax. Weak-to-moderate correlations (| ρ |< 0.70) were observed between PET/EPRI hypoxia images with vascular permeability (Ktrans) or fractional extracellular-extravascular space (ve) from DCE-MRI. Conclusion: This is the first in vivo comparison of FMISO uptake with pO2 EPRI to identify the optimal FMISO threshold to define tumor hypoxia, which may successfully direct hypoxic tumor boosts in patients, thereby enhancing tumor control. [ABSTRACT FROM AUTHOR]

Inadequate oxygenation is a major challenge in cell encapsulation, a therapy which holds potential to treat many diseases including type I diabetes. In such systems, cellular oxygen (O2) delivery is limited to slow passive diffusion from transplantation sites through the poorly O2-soluble encapsulating matrix, usually a hydrogel. This constrains the maximum permitted distance between the encapsulated cells and host site to within a few hundred micrometers to ensure cellular function. Inspired by the natural gas-phase tracheal O2 delivery system of insects, we present herein the design of a biomimetic scaffold featuring internal continuous air channels endowed with 10,000-fold higher O2 diffusivity than hydrogels. We incorporate the scaffold into a bulk hydrogel containing cells, which facilitates rapid O2 transport through the whole system to cells several millimeters away from the device-host boundary. A computational model, validated by in vitro analysis, predicts that cells and islets maintain high viability even in a thick (6.6 mm) device. Finally, the therapeutic potential of the device is demonstrated through the correction of diabetes in immunocompetent mice using rat islets for over 6 months. Cell encapsulation into biomaterials for transplantation is currently limited by inadequate oxygenation. Here the authors present a biomimetic scaffold featuring internal continuous air channels endowed with 10,000-fold higher O2 diffusivity than hydrogels and demonstrate correction of diabetes in immunocompetent mice using rat islets for over 6 months. [ABSTRACT FROM AUTHOR]

Electron paramagnetic resonance oxygen imaging, EPROI, is established as a method for quantitative oxygen imaging. For tissue identification, in vivo oxygen images need to be paired with higher tissue contrast images such as MRI, CT, or ultrasound. All these images are acquired using different instruments and a method to represent them in a common coordinate frame, image registration, is required. Registered EPROI can be used for directing cancer treatment or validation of other oxygen-related imaging methods such as PET or histological staining. We present an image registration and analysis system comprised of two components: an animal bed with registration guides and an ArbuzGUI MATLAB toolbox developed in our laboratory. The toolbox includes components for image registration, segmentation, and oxygen analysis. [ABSTRACT FROM AUTHOR]

A pulse 720 MHz EPR imager with a permanent magnet, arbitrary waveform generator based excitation, and detection system operating in the direct sampling and signal down-conversion modes is presented. This flexible instrument allowed testing various acquisition modes and demonstrating its signal to noise ratio advantage over the previously used 250 MHz instrument. [ABSTRACT FROM AUTHOR]

Yakov Sergeevich Lebedev was a pioneer in high-frequency EPR, taking advantage of the separation of g-factor anisotropy effects from nuclear hyperfine splitting and the higher-frequency molecular motion sensitivity from higher-frequency measurements (Appl Magn Reson 7: 339–362, 1994). This article celebrates a second EPR subfield in which Prof. Lebedev pioneered, EPR imaging (Chem Phys Lett 99: 301–304, 1983). We celebrate the clinical enhancements that are suggested in this low-frequency work and imaging application to animal physiology at lower-than-standard EPR frequencies. [ABSTRACT FROM AUTHOR]

This paper presents a survey of electron paramagnetic resonance (EPR) image registration. Image registration is the process of overlaying images (two or more) of the same scene taken at different times, from different viewpoints and/or different techniques. EPR-imaging (EPRI) techniques belong to the functional-imaging modalities and therefore suffer from a lack of anatomical reference which is mandatory in preclinical imaging. For this reason, it is necessary to merging EPR images with other modalities which allow for obtaining anatomy images. Methodological analysis and review of the literature were done, providing a summary for developing a good foundation for research study in this field which is crucial in understanding the existing levels of knowledge. Out of these considerations, the aim of this paper is to enhance the scientific community's understanding of the current status of research in EPR preclinical image registration and also communicate to them the contribution of this research in the field of image processing. [ABSTRACT FROM AUTHOR]

Resonators for preclinical electron paramagnetic resonance imaging have been designed primarily for rodents and rabbits and have internal diameters between 16 and 51 mm. Lumped-circuit resonators include loop-gap, Alderman-Grant, and saddle coil topologies and surface coils. Bimodal resonators are useful for isolating the detected signal from incident power and reducing dead time in pulse experiments. Resonators for continuous wave, rapid scan, and pulse experiments are described. Experience at the University of Chicago and University of Denver in design of resonators for in vivo imaging is summarized. [ABSTRACT FROM AUTHOR]

Radiation treatment success and high tumor oxygenation and success have been known to be highly correlated. This suggests that radiation therapy guided by images of tumor regions with low oxygenation, oxygen-guided radiation therapy (OGRT) may be a promising enhancement of cancer radiation treatment. Before applying the technique to human subjects, OGRT needs to be tested in animals, most easily in rodents. Electron paramagnetic resonance imaging provides quantitative maps of tissue and tumor oxygen in rodents with 1 mm spatial resolution and 1 torr pO resolution at low oxygen levels. The difficulty of using mouse models is their small size and that of their tumors. To overcome this we used XRAD225Cx micro-CT/ therapy system and 3D printed conformal blocks. Radiation is delivered first to a uniform 15% tumor control dose for the whole tumor and then a boost dose to either hypoxic tumor regions or equal volumes of well oxygenated tumor. Delivery of the booster dose used a multiple beam angles to deliver radiation beams whose shape conforms to that of all hypoxic regions or fully avoids those regions. To treat/avoid all hypoxic regions we used individual radiation blocks 3D-printed from acrylonitrile butadiene styrene polymer infused with tungsten particles fabricated immediately after imaging to determine regions with pO less than 10 torr. Preliminary results demonstrate the efficacy of the radiation treatment with hypoxic boosts with syngeneic FSa fibrosarcoma tumors in the legs of C3H mice. [ABSTRACT FROM AUTHOR]

A magnetometer designed for permanent magnet manufacturing and operated around 25 mT with 10 ppm absolute accuracy is described. The magnetometer uses pulse electron paramagnetic resonance methodology. The use of a pulsed broadband acquisition allowed reliable measurements in the presence of the magnetic field gradient and in relatively inhomogeneous magnetic fields of un-shimmed magnets. [ABSTRACT FROM AUTHOR]

Purpose: Tumor microenvironments show remarkable tumor pO(2) heterogeneity, as seen in prior EPR pO(2) images (EPROI). pO(2) correlation with hypoxia response proteins is frustrated by large rapid pO(2) changes with position.Procedures: To overcome this limitation, biopsies stereotactically located in the EPROI were used to explore the relationship between vascular endothelial growth factor A (VEGF) concentrations in living mouse tumors and the local EPROI pO(2).Results: Quantitative ELISA VEGF concentrations correlated (p = 0.0068 to 0.019) with mean pO(2), median pO(2), and the fraction of voxels in the biopsy volume with pO(2) less than 3, 6, and 10 Torr.Conclusions: This validates EPROI hypoxic fractions at the molecular level and provides a new paradigm for the assessment of the relationship, in vivo, between hypoxia and hypoxia response proteins. When translated to human subjects, this will enhance understanding of human tumor pathophysiology and cancer response to therapy. [ABSTRACT FROM AUTHOR]

Photosystem I (PS I) contains two symmetric branches of electron transfer cofactors. In both the A- and B-branches, the phylloquinone in the A1 site is π-stacked with a tryptophan residue and is H-bonded to the backbone nitrogen of a leucine residue. In this work, we use optical and electron paramagnetic resonance (EPR) spectroscopies to investigate cyanobacterial PS I complexes, where these tryptophan residues are changed to phenylalanine. The time-resolved optical data show that backward electron transfer from the terminal electron acceptors to P is affected in the A- and B-branch mutants, both at ambient and cryogenic temperatures. These results suggest that the quinones in both branches take part in electron transport at all temperatures. The electron-nuclear double resonance (ENDOR) spectra of the spin-correlated radical pair PA and the photoaccumulated radical anion A, recorded at cryogenic temperature, allowed the identification of characteristic resonances belonging to protons of the methyl group, some of the ring protons and the proton hydrogen-bonded to phylloquinone in the wild type and both mutants. Significant changes in PS I isolated from the A-branch mutant are detected, while PS I isolated from the B-branch mutant shows the spectral characteristics of wild-type PS I. A possible short-lived B-branch radical pair cannot be detected by EPR due to the available time resolution; therefore, only the A-branch quinone is observed under conditions typically employed for EPR and ENDOR spectroscopies. [ABSTRACT FROM AUTHOR]

A detailed analysis of the EPR signatures at X-band and Q-band of an enzyme (SoxB) involved in sulfur oxidation from Paracoccus pantotrophus is presented. EPR spectra are attributed to an exchange-coupled dimanganese Mn2(II,II) complex. An antiferromagnetic exchange interaction of J=−7.0 (±1) cm−1 ( H=−2 JS 1 S 2 ) is evidenced by a careful examination of the temperature dependence of the EPR spectra. The spin Hamiltonian parameters for a total spin of S T =1, 2 and 3 are obtained and an inter-manganese distance of 3.4 (±0.1) Å is estimated. The comparison with exchange coupling and inter-manganese distance data of other dimanganese proteins and model compounds leads to a tentative assignment of the Mn bridging ligands to bis(μ-hydroxo) (μ-carboxylato). [ABSTRACT FROM AUTHOR]

Pulse EPR, 55Mn-ENDOR and ELDOR-detected NMR experiments were performed on the S2-state of the oxygen-evolving complex from spinach Photosystem II. The novel technique of random acquisition in ENDOR was used to suppress heating artefacts. Our data unambiguously shows that four Mn ions have significant hyperfine coupling constants. Numerical simulation of the 55Mn-ENDOR spectrum allowed the determination of the principal values of the hyperfine interaction tensors for all four Mn ions of the oxygen-evolving complex. The results of our 55Mn-ENDOR experiments are in good agreement with previously published data [Peloquin JM et al. (2000) J Am Chem Soc 122: 10926–10942]. For the first time ELDOR-detected NMR was applied to the S2-state and revealed a broad peak that can be simulated numerically with the same parameters that were used for the simulation of the 55Mn-ENDOR spectrum. This provides strong independent support for the assigned hyperfine parameters. [ABSTRACT FROM AUTHOR]

Cellular therapies for type-1 diabetes can leverage cell encapsulation to dispense with immunosuppression. However, encapsulated islet cells do not survive long, particularly when implanted in poorly vascularized subcutaneous sites. Here we show that the induction of neovascularization via temporary controlled inflammation through the implantation of a nylon catheter can be used to create a subcutaneous cavity that supports the transplantation and optimal function of a geometrically matching islet-encapsulation device consisting of a twisted nylon surgical thread coated with an islet-seeded alginate hydrogel. The neovascularized cavity led to the sustained reversal of diabetes, as we show in immunocompetent syngeneic, allogeneic and xenogeneic mouse models of diabetes, owing to increased oxygenation, physiological glucose responsiveness and islet survival, as indicated by a computational model of mass transport. The cavity also allowed for the in situ replacement of impaired devices, with prompt return to normoglycemia. Controlled inflammation-induced neovascularization is a scalable approach, as we show with a minipig model, and may facilitate the clinical translation of immunosuppression-free subcutaneous islet transplantation., (© 2023. The Author(s), under exclusive licence to Springer Nature Limited.)