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Purpose: Preclinical radiation replicating clinical intensity modulated radiation therapy (IMRT) techniques can provide data translatable to clinical practice. For this work, treatment plans were created for oxygen-guided dose-painting in small animals using inverse-planned IMRT. Spatially varying beam intensities were achieved using 3-dimensional (3D)-printed compensators.Methods and Materials: Optimized beam fluence from arbitrary gantry angles was determined using a verified model of the XRAD225Cx treatment beam. Compensators were 3D-printed with varied thickness to provide desired attenuation using copper/polylactic-acid. Spatial resolution capabilities were investigated using printed test-patterns. Following American Association of Physicists in Medicine TG119, a 5-beam IMRT plan was created for a miniaturized (∼1/8th scale) C-shape target. Electron paramagnetic resonance imaging of murine tumor oxygenation guided simultaneous integrated boost (SIB) plans conformally treating tumor to a base dose (Rx1) with boost (Rx2) based on tumor oxygenation. The 3D-printed compensator intensity modulation accuracy and precision was evaluated by individually delivering each field to a phantom containing radiochromic film and subsequent per-field gamma analysis. The methodology was validated end-to-end with composite delivery (incorporating 3D-printed tungsten/polylactic-acid beam trimmers to reduce out-of-field leakage) of the oxygen-guided SIB plan to a phantom containing film and subsequent gamma analysis.Results: Resolution test-patterns demonstrate practical printer resolution of ∼0.7 mm, corresponding to 1.0 mm bixels at the isocenter. The miniaturized C-shape plan provides planning target volume coverage (V95% = 95%) with organ sparing (organs at risk Dmax < 50%). The SIB plan to hypoxic tumor demonstrates the utility of this approach (hypoxic tumor V95%,Rx2 = 91.6%, normoxic tumor V95%,Rx1 = 95.7%, normal tissue V100%,Rx1 = 7.1%). The more challenging SIB plan to boost the normoxic tumor rim achieved normoxic tumor V95%,Rx2 = 90.9%, hypoxic tumor V95%,Rx1 = 62.7%, and normal tissue V100%,Rx2 = 5.3%. Average per-field gamma passing rates using 3%/1.0 mm, 3%/0.7 mm, and 3%/0.5 mm criteria were 98.8% ± 2.8%, 96.6% ± 4.1%, and 90.6% ± 5.9%, respectively. Composite delivery of the hypoxia boost plan and gamma analysis (3%/1 mm) gave passing results of 95.3% and 98.1% for the 2 measured orthogonal dose planes.Conclusions: This simple and cost-effective approach using 3D-printed compensators for small-animal IMRT provides a methodology enabling preclinical studies that can be readily translated into the clinic. The presented oxygen-guided dose-painting demonstrates that this methodology will facilitate studies driving much needed biologic personalization of radiation therapy for improvements in patient outcomes. [ABSTRACT FROM AUTHOR]

The Finland trityl paramagnet is characterized by magnetic susceptibility and a new form of quantitative electron paramagnetic resonance (EPR) that utilizes a superconducting quantum interference device (SQUID) as a detection method. This radical is of interest due to its use as a dynamic nuclear polarization agent as well as a potential magnetic refrigerant and quantum computing bit. The SQUID-EPR data show that the EPR linewidth of a concentrated trityl powder decreases dramatically from 4.4 to 1.4 mT as the temperature is increased from 1.8 to 10 K. The quantitative nature of SQUID-EPR is used to thermodynamically quantify the EPR energy transfer times and saturated fractions. At 95 GHz and 1.8 K, only 40% of the spins are in resonance at the onset of saturation. Conventional dc magnetic susceptibility over 1.8–150 K indicates an S=1/2 Curie–Weiss relationship with little long range interaction. Magnetization versus applied field at 1.8 and 4 K fits a Brillouin function with >80% electronic polarization at a normalized field of gμBμ0H/kT≈3. These results provide information required for theoretical modeling and engineering of the trityl radical for a wide range of applications. [ABSTRACT FROM AUTHOR]

Purpose: It has been known for over 100 years that tumor hypoxia, a near-universal characteristic of solid tumors, decreases the curative effectiveness of radiation therapy. However, to date, there are no reports that demonstrate an improvement in radiation effectiveness in a mammalian tumor on the basis of tumor hypoxia localization and local hypoxia treatment.Methods and Materials: For radiation targeting of hypoxic subregions in mouse fibrosarcoma, we used oxygen images obtained using pulse electron paramagnetic resonance pO2 imaging combined with 3D-printed radiation blocks. This achieved conformal radiation delivery to all hypoxic areas in FSa fibrosarcomas in mice.Results: We demonstrate that treatment delivering a radiation boost to hypoxic volumes has a significant (P = .04) doubling of tumor control relative to boosts to well-oxygenated volumes. Additional dose to well-oxygenated tumor regions minimally increases tumor control beyond the 15% control dose to the entire tumor. If we can identify portions of the tumor that are more resistant to radiation, it might be possible to reduce the dose to more sensitive tumor volumes without significant compromise in tumor control.Conclusions: This work demonstrates in a single, intact mammalian tumor type that tumor hypoxia is a local tumor phenomenon whose treatment can be enhanced by local radiation. Despite enormous clinical effort to overcome hypoxic radiation resistance, to our knowledge this is the first such demonstration, even in preclinical models, of targeting additional radiation to hypoxic tumor to improve the therapeutic ratio. [ABSTRACT FROM AUTHOR]

In rapid-scan EPR the magnetic field or frequency is repeatedly scanned through the spectrum at rates that are much faster than in conventional continuous wave EPR. The signal is directly-detected with a mixer at the source frequency. Rapid-scan EPR is particularly advantageous when the scan rate through resonance is fast relative to electron spin relaxation rates. In such scans, there may be oscillations on the trailing edge of the spectrum. These oscillations can be removed by mathematical deconvolution to recover the slow-scan absorption spectrum. In cases of inhomogeneous broadening, the oscillations may interfere destructively to the extent that they are not visible. The deconvolution can be used even when it is not required, so spectra can be obtained in which some portions of the spectrum are in the rapid-scan regime and some are not. The technology developed for rapid-scan EPR can be applied generally so long as spectra are obtained in the linear response region. The detection of the full spectrum in each scan, the ability to use higher microwave power without saturation, and the noise filtering inherent in coherent averaging results in substantial improvement in signal-to-noise relative to conventional continuous wave spectroscopy, which is particularly advantageous for low-frequency EPR imaging. This overview describes the principles of rapid-scan EPR and the hardware used to generate the spectra. Examples are provided of its application to imaging of nitroxide radicals, diradicals, and spin-trapped radicals at a Larmor frequency of ca. 250 MHz. [ABSTRACT FROM AUTHOR]

Thiol redox status is an important physiologic parameter that affects the success or failure of cancer treatment. Rapid scan electron paramagnetic resonance (RS EPR) is a novel technique that has shown higher signal-to-noise ratio than conventional continuous-wave EPR in in vitro studies. Here we used RS EPR to acquire rapid three-dimensional images of the thiol redox status of tumors in living mice. This work presents, for the first time, in vivo RS EPR images of the kinetics of the reaction of 2 H, 15 N-substituted disulfide-linked dinitroxide (PxSSPx) spin probe with intracellular glutathione. The cleavage rate is proportional to the intracellular glutathione concentration. Feasibility was demonstrated in a FSa fibrosarcoma tumor model in C3H mice. Similar to other in vivo and cell model studies, decreasing intracellular glutathione concentration by treating mice with l -buthionine sulfoximine (BSO) markedly altered the kinetic images. [ABSTRACT FROM AUTHOR]

In pulsed magnetic resonance, the excitation power is many orders of magnitude larger than that induced by the spin system in the receiving coil or resonator. The receiver must be protected during and immediately after the excitation pulse to allow for the energy stored in the resonator to dissipate to a safe level. The time during which the signal is not detected, the instrumental dead-time, can be shortened by using magnetically decoupled excitation and receive coils. Such coils are oriented, with respect to each other, in a way that minimizes the total magnetic flux produced by one coil in the other. We suggest that magnetically decoupled coils can be isolated to a larger degree by tuning them to separate frequencies. Spins are excited at one frequency, and the echo signal is detected at another. Sinusoidal magnetic field modulation that rapidly changes the Larmor frequency of the spins between the excitation and detection events is used to ensure the resonance conditions for both coils. In this study, the relaxation times of trityl-CD3 were measured in a field-modulated pulsed EPR experiment and compared to results obtained using a standard spin echo method. The excitation and receive coils were tuned to 245 and 256.7 MHz, respectively. Using an available rapid-scan, cross-loop EPR resonator, we demonstrated an isolation improvement of approximately 20–30 dB due to frequency decoupling. Theoretical analysis, numerical simulations, and proof-of-concept experiments demonstrated that substantial excitation-detection decoupling can be achieved. A pulsed L-band system, including a small volume bi-modal resonator equipped with modulation coils, was constructed to demonstrate fivefold dead-time reduction in comparison with the standard EPR experiment. This was achieved by detuning of the excitation and receive coils by 26 MHz and using sinusoidal modulation at 480 kHz. [ABSTRACT FROM AUTHOR]

Hypoxic tumors are resistant to radiotherapy, motivating the development of tools to image local oxygen concentrations. It is generally believed that stable or chronic hypoxia is the source of resistance, but more recent work suggests a role for transient hypoxia. Conventional EPR imaging (EPRI) is capable of imaging tissue pO 2 in vivo , with high pO 2 resolution and 1 mm spatial resolution but low imaging speed (10 min temporal resolution for T 1 -based pO 2 mapping), which makes it difficult to investigate the oxygen changes, e.g., transient hypoxia. Here we describe a new imaging method which accelerates dynamic EPR oxygen imaging, allowing 3D imaging at 2 frames per minute, fast enough to image transient hypoxia at the “speed limit” of observed pO 2 change. The method centers on a low-rank tensor model that decouples the tradeoff between imaging speed, spatial coverage/resolution, and number of inversion times (pO 2 accuracy). We present a specialized sparse sampling strategy and image reconstruction algorithm for use with this model. The quality and utility of the method is demonstrated in simulations and in vivo experiments in tumor bearing mice. [ABSTRACT FROM AUTHOR]

Site-directed spin labeling (SDSL) in combination with electron paramagnetic resonance (EPR) spectroscopy has become an important tool for measuring distances in proteins on the order of a few nm. For this purpose pairs of spin labels, most commonly nitroxides, are site-selectively introduced into the protein. Recent efforts to develop new spin labels are focused on tailoring the intrinsic properties of the label to either extend the upper limit of measurable distances at physiological temperature, or to provide a unique spectral lineshape so that selective pairwise distances can be measured in a protein or complex containing multiple spin label species. Triarylmethyl (TAM) radicals are the foundation for a new class of spin labels that promise to provide both capabilities. Here we report a new methanethiosulfonate derivative of a TAM radical that reacts rapidly and selectively with an engineered cysteine residue to generate a TAM containing side chain (TAM1) in high yield. With a TAM1 residue and Cu 2+ bound to an engineered Cu 2+ binding site, enhanced T 1 relaxation of TAM should enable measurement of interspin distances up to 50 Å at physiological temperature. To achieve favorable TAM1-labeled protein concentrations without aggregation, proteins are tethered to a solid support either site-selectively using an unnatural amino acid or via native lysine residues. The methodology is general and readily extendable to complex systems, including membrane proteins. [ABSTRACT FROM AUTHOR]

ABSTRACT The importance of tissue oxygenation has led to a great interest in methods for imaging pO2 in vivo. Electron paramagnetic resonance imaging (EPRI) provides noninvasive, near absolute 1 mm-resolved 3D images of pO2 in the tissues and tumors of living animals. Current EPRI image reconstruction methods tend to be time consuming and preclude real-time visualization of information. Methods are presented to significantly accelerate the reconstruction process in order to enable real-time reconstruction of EPRI pO2 images. These methods are image reconstruction using graphics processing unit (GPU)-based 3D filtered back-projection and lookup table parameter fitting. The combination of these methods leads to acceleration factors of over 650 compared to current methods and allows for real-time reconstruction of EPRI images of pO2 in vivo. © 2015 Wiley Periodicals, Inc. Concepts Magn Reson Part B (Magn Reson Engineering) 45B: 46-57, 2015 [ABSTRACT FROM AUTHOR]

Pulse electron paramagnetic resonance imaging (Pulse EPRI) is a robust method for noninvasively measuring local oxygen concentrations in vivo . For 3D tomographic EPRI, the most commonly used reconstruction algorithm is filtered back projection (FBP), in which the parabolic filtration process strongly influences image quality. In this work, we designed and compared 7 parabolic filtration methods to reconstruct both simulated and real phantoms. To evaluate these methods, we designed 3 error criteria and 1 spatial resolution criterion. It was determined that the 2 point derivative filtration method and the two-ramp-filter method have unavoidable negative effects resulting in diminished spatial resolution and increased artifacts respectively. For the noiseless phantom the rectangular-window parabolic filtration method and sinc -window parabolic filtration method were found to be optimal, providing high spatial resolution and small errors. In the presence of noise, the 3 point derivative method and Hamming-window parabolic filtration method resulted in the best compromise between low image noise and high spatial resolution. The 3 point derivative method is faster than Hamming-window parabolic filtration method, so we conclude that the 3 point derivative method is optimal for 3D FBP. [ABSTRACT FROM AUTHOR]

Clinical trials to ameliorate hypoxia as a strategy to relieve the radiation resistance it causes have prompted a need to assay the precise extent and location of hypoxia in tumors. Electron paramagnetic resonance oxygen imaging (EPR O2 imaging) provides a noninvasive means to address this need. To obtain a preclinical proof-of-principle that EPR O2 images could predict radiation control, we treatedmouse tumors at or near doses required to achieve 50% control (TCD50). Mice with FSa fibrosarcoma or MCa4 carcinoma were subjected to EPR O2 imaging and immediately radiated to a TCD50 or TCD50 10Gy. Statistical analysiswas permitted by collection of approximately 1,300 tumor pO2 image voxels, including the fraction of tumor voxels with pO2 less than 10mmHg (HF10). Tumors were followed for 90 days (FSa) or 120 days (MCa4) to determine local control or failure. HF10 obtained fromEPR images showed statistically significant differences between tumors thatwere controlled by the TCD50 and those that were not controlled for both FSa andMCa4. Kaplan-Meier analysis of both types of tumors showed that approximately 90%ofmildly hypoxic tumorswere controlled (HF10% 10%), and only 37%(FSA) and 23% (MCa4) tumors controlled if hypoxic. EPR pO2 image voxel distributions in these approximately 0.5 mL tumors provide a prediction of radiation curability independent of radiation dose. These data confirm the significance of EPR pO2 hypoxic fractions. The 90% control of low HF10 tumors argue that 0.5 mL subvolumes of tumors may be more sensitive to radiation and may need less radiation for high tumor control rates. [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]

Abstract: The Frank polyphase sequence has been applied to pulsed EPR of triarylmethyl radicals at 256MHz (9.1mT magnetic field), using 256 phase pulses. In EPR, as in NMR, use of a Frank sequence of phase steps permits pulsed FID signal acquisition with very low power microwave/RF pulses (ca. 1.5mW in the application reported here) relative to standard pulsed EPR. A 0.2mM aqueous solution of a triarylmethyl radical was studied using a 16mm diameter cross-loop resonator to isolate the EPR signal detection system from the incident pulses. [Copyright &y& Elsevier]

A rapid method to determine the systemic incorporation of amifostine has been sought in order to determine the effectiveness of different administration routes without the delay inherent in awaiting therapeutic results. Consistent changes in animal measurements of nitroxide signal decay were monitored using in vivo EPR at frequencies low enough to ensure uniform sensitivity to organs deep in 20-g C3H mice. Conditions included both co-administration of the amifostine with the carbamoyl-proxyl spin probe (CP) via i.p. injection (n=6) and oral administration (n=8) of the amifostine. These decreased the first order rate of decay of the CP EPR signal after a dose of 13.5 Gy radiation, by 23% and 18%, respectively. These changes were significantly different from the rate of decay of the CP EPR signal without amifostine, but were statistically indistinguishable from each other. These data demonstrate: (1) condition-dependent exponential decay of CP EPR signal allowing its use to determine systemic availability of a drug, and (2) that oral administration and i.p. injection of amifostine are both effective in affecting the CP EPR signal decay rate in a mouse model. This is a strong indicator of similar bioavailability in mice from both routes of administration. [Copyright &y& Elsevier]

Molecular oxygen and its thermodynamic transformation drive nearly all life processes. Quantitative measurement and imaging of oxygen in living systems is of fundamental importance for the study of life processes and their aberrations-disease- many of which are affected by hypoxia, or low levels of oxygen. Cancer is among the disease processes profoundly affected by hypoxia. Electron paramagnetic resonance has been shown to provide remarkably accurate images of normal and cancerous tissue. In this review, we emphasize the reactivity of molecular oxygen particularly highlighting the metabolic processes of living systems to store free energy in the reactants. The history of hypoxic resistance of living systems to cytotoxic therapy, particularly radiation therapy is also reviewed. The measurement and imaging of molecular oxygen with pulse spin lattice relaxation (SLR) electron paramagnetic resonance (EPR) is reviewed briefly. This emphasizes the advantages of the spin lattice relaxation based measurement paradigm to reduce the sensitivity of the measurement to the presence of the oxygen sensing probe itself. The involvement of a novel small mammal external beam radiation delivery system is described. This enables an experimental paradigm based on control by radiation of the last resistant clonogen. This is much more specific for tumor cure than growth delay assays which primarily reflects control of tumor cells most sensitive to therapy., (© 2024. The Author(s), under exclusive licence to World Molecular Imaging Society.)

Significance: Fundamental to the application of tissue redox status to human health is the quantification and localization of tissue redox abnormalities and oxidative stress and their correlation with the severity and local extent of disease to inform therapy. The centrality of the low-molecular-weight thiol, glutathione, in physiological redox balance has long been appreciated, but direct measurement of tissue thiol status in vivo has not been possible hitherto. Recent advances in instrumentation and molecular probes suggest the feasibility of real-time redox assessment in humans. Recent Advances: Recent studies have demonstrated the feasibility of using low-frequency electron paramagnetic resonance (EPR) techniques for quantitative imaging of redox status in mammalian tissues in vivo . Rapid-scan (RS) EPR spectroscopy and imaging, new disulfide-dinitroxide spin probes, and novel analytic techniques have led to significant advances in direct, quantitative imaging of thiol redox status. Critical Issues: While novel RS EPR imaging coupled with first-generation molecular probes has demonstrated the feasibility of imaging thiol redox status in vivo , further technical advancements are desirable and ongoing. These include developing spin probes that are tailored for specific tissues with response kinetics tuned to the physiological environment. Equally critical are RS instrumentation with higher signal-to-noise ratio and minimal signal distortion, as well as optimized imaging protocols for image acquisition with sparsity adapted to image information content. Future Directions: Quantitative images of tissue glutathione promise to enable acquisition of a general image of mammalian and potentially human tissue health.

Background: Clinical attempts to find benefit from specifically targeting and boosting resistant hypoxic tumor subvolumes have been promising but inconclusive. While a first preclinical murine tumor type showed significant improved control with hypoxic tumor boosts, a more thorough investigation of efficacy from boosting hypoxic subvolumes defined by electron paramagnetic resonance oxygen imaging (EPROI) is necessary. The present study confirms improved hypoxic tumor control results in three different tumor types using a clonogenic assay and explores potential confounding experimental conditions., Materials and Methods: Three murine tumor models were used for multi-modal imaging and radiotherapy: MCa-4 mammary adenocarcinomas, SCC7 squamous cell carcinomas, and FSa fibrosarcomas. Registered T2-weighted MRI tumor boundaries, hypoxia defined by EPROI as pO 2 ≤ 10 mmHg, and X-RAD 225Cx CT boost boundaries were obtained for all animals. 13 Gy boosts were directed to hypoxic or equal-integral-volume oxygenated tumor regions and monitored for regrowth. Kaplan-Meier survival analysis was used to assess local tumor control probability (LTCP). The Cox proportional hazards model was used to assess the hazard ratio of tumor progression of Hypoxic Boost vs. Oxygenated Boost for each tumor type controlling for experimental confounding variables such as EPROI radiofrequency, tumor volume, hypoxic fraction, and delay between imaging and radiation treatment., Results: An overall significant increase in LTCP from Hypoxia Boost vs. Oxygenated Boost treatments was observed in the full group of three tumor types ( p  < 0.0001). The effects of tumor volume and hypoxic fraction on LTCP were dependent on tumor type. The delay between imaging and boost treatments did not have a significant effect on LTCP for all tumor types., Conclusion: This study confirms that EPROI locates resistant tumor hypoxic regions for radiation boost, increasing clonogenic LTCP, with potential enhanced therapeutic index in three tumor types. Preclinical absolute EPROI may provide correction for clinical hypoxia images using additional clinical physiologic MRI., Competing Interests: HH holds US patent 8,664,955 (US-8664955-B1) and HH and BE hold US patent 9,392,957 (US-9392957-B1) for aspects of the pO2 imaging technology. MK, HH, and BE are members of a start-up company O2M Technologies, LLC. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Gertsenshteyn, Epel, Giurcanu, Barth, Lukens, Hall, Martinez, Grana, Maggio, Miller, Sundramoorthy, Krzykawska-Serda, Pearson, Aydogan, Weichselbaum, Tormyshev, Kotecha and Halpern.)