Evaluation of a deuterated triarylmethyl spin probe for in vivo R2 ∗-based EPR oximetric imaging with enhanced dynamic range.

PURPOSE: In this study, we compared two triarylmethyl (TAM) spin probes, Ox071 and Ox063 for their efficacy in measuring tissue oxygen levels under hypoxic and normoxic conditions by R2 *-based EPR oximetry. METHODS: The R2 * dependencies on the spin probe concentration and oxygen level were calibrated using deoxygenated 1, 2, 5, and 10 mM standard solutions and 2 mM solutions saturated at 0%, 2%, 5%, 10%, and 21% of oxygen. For the hypoxic model, in vivo imaging of a MIA PaCa-2 tumor implanted in the hind leg of a mouse was performed on successive days by R2 *-based EPR oximetry using either Ox071 or Ox063. For the normoxic model, renal imaging of healthy athymic mice was performed using both spin probes. The 3D images were reconstructed by single point imaging and multi-gradient technique was used to determine R2 * maps. RESULTS: The signal intensities of Ox071 were approximately three times greater than that of Ox063 in the entire partial pressure of oxygen (pO2 ) range investigated. The histograms of the tumor pO2 images were skewed for both spin probes, and Ox071 showed more frequency counts at pO2 > 32 mm Hg. In the normoxic kidney model, there was a clear delineation between the high pO2 cortex and the low pO2 medulla regions. The histogram of high-resolution kidney oximetry image using Ox071 was nearly symmetrical and frequency counts were seen up to 55 mm Hg, which were missed in Ox063 imaging. CONCLUSION: As an oximetric probe, Ox071 has clear advantages over Ox063 in terms of sensitivity and the pO2 dynamic range.

Continuous monitoring of postirradiation reoxygenation and cycling hypoxia using electron paramagnetic resonance imaging.

Reoxygenation has a significant impact on the tumor response to radiotherapy. With developments in radiotherapy technology, the relevance of the reoxygenation phenomenon in treatment efficacy has been a topic of interest. Evaluating the reoxygenation in the tumor microenvironment throughout the course of radiation therapy is important in developing effective treatment strategies. In the current study, we used electron paramagnetic resonance imaging (EPRI) to directly map and quantify the partial oxygen pressure (pO2 ) in tumor tissues. Human colorectal cancer cell lines, HT29 and HCT116, were used to induce tumor growth in female athymic nude mice. Tumors were irradiated with 3, 10, or 20 Gy using an x-ray irradiator. Prior to each EPRI scan, magnetic resonance imaging (MRI) was performed to obtain T2-weighted anatomical images for reference. The differences in the mean pO2 were determined through two-tailed Student's t-test and one-way analysis of variance. The median pO2 60 min after irradiation was found to be lower in HCT116 than in HT29 (9.1 ± 1.5 vs. 14.0 ± 1.0 mmHg, p = 0.045). There was a tendency for delayed and incomplete recovery of pO2 in the HT29 tumor when a higher dose of irradiation (10 and 20 Gy) was applied. Moreover, there was a dose-dependent increase in the hypoxic areas (pO2  < 10 mmHg) 2 and 24 h after irradiation in all groups. In addition, an area that showed pO2 fluctuation between hypoxia and normoxia (pO2  > 10 mmHg) was also identified surrounding the region with stable hypoxia, and it slightly enlarged after recovery from acute hypoxia. In conclusion, we demonstrated the reoxygenation phenomenon in an in vivo xenograft model study using EPRI. These findings may lead to new knowledge regarding the reoxygenation process and possibilities of a new radiation therapy concept, namely, reoxygenation-based radiation therapy.

EPR-based oximetric imaging: a combination of single point-based spatial encoding and T1 weighting.

PURPOSE: Spin-lattice relaxation rate (R1 )-based time-domain EPR oximetry is reported for in vivo applications using a paramagnetic probe, a trityl-based Oxo71. METHODS: The R1 dependence of the trityl probe Oxo71 on partial oxygen pressure (pO2 ) was assessed using single-point imaging mode of spatial encoding combined with rapid repetition, similar to T1 -weighted MRI, for which R1 was determined from 22 repetition times ranging from 2.1 to 40.0 µs at 300 MHz. The pO2 maps of a phantom with 3 tubes containing 2 mM Oxo71 solutions equilibrated at 0%, 2%, and 5% oxygen were determined by R1 and apparent spin-spin relaxation rate ( R2*) simultaneously. RESULTS: The pO2 maps derived from R1 and R2* agreed with the known pO2 levels in the tubes of Oxo71. However, the histograms of pO2 revealed that R1 offers better pO2 resolution than R2* in low pO2 regions. The SDs of pixels at 2% pO2 (15.2 mmHg) were about 5 times lower in R1 -based estimation than R2*-based estimation (mean ± SD: 13.9 ± 1.77 mmHg and 18.3 ± 8.70 mmHg, respectively). The in vivo pO2 map obtained from R1 -based assessment displayed a homogeneous profile in low pO2 regions in tumor xenografts, consistent with previous reports on R2*-based oximetric imaging. The scan time to obtain the R1 map can be significantly reduced using 3 repetition times ranging from 4.0 to 12.0 µs. CONCLUSION: Using the single-point imaging modality, R1 -based oximetry imaging with useful spatial and oxygen resolutions for small animals was demonstrated.

Wireless implantable coil with parametric amplification for in vivo electron paramagnetic resonance oximetric applications.

PURPOSE: To develop an implantable wireless coil with parametric amplification capabilities for time-domain electron paramagnetic resonance (EPR) spectroscopy operating at 300 MHz. METHODS: The wireless coil and lithium phthalocyanine (LiPc), a solid paramagnetic probe, were each embedded individually in a biocompatible polymer polydimethoxysiloxane (PDMS). EPR signals from the LiPc embedded in PDMS (LiPc/PDMS) were generated by a transmit-receive surface coil tuned to 300 MHz. Parametric amplification was configured with an external pumping coil tuned to 600 MHz and placed between the surface coil resonator and the wireless coil. RESULTS: Phantom studies showed significant enhancement in signal to noise using the pumping coil. However, no influence of the pumping coil on the oxygen-dependent EPR spectral linewidth of LiPc/PDMS was observed, suggesting the validity of parametric amplification of EPR signals for oximetry by implantation of the encapsulated wireless coil and LiPc/PDMS in deep regions of live objects. In vivo studies demonstrate the feasibility of this approach to longitudinally monitor tissue pO2 in vivo and also monitor acute changes in response to pharmacologic challenges. The encapsulated wireless coil and LiPc/PDMS engendered no host immune response when implanted for ∼3 weeks and were found to be well tolerated. CONCLUSIONS: This approach may find applications for monitoring tissue oxygenation to better understand the pathophysiology associated with wound healing, organ transplantation, and ischemic diseases.

Accelerated 4D quantitative single point EPR imaging using model-based reconstruction.

PURPOSE: Electron paramagnetic resonance imaging has surfaced as a promising noninvasive imaging modality that is capable of imaging tissue oxygenation. Due to extremely short spin-spin relaxation times, electron paramagnetic resonance imaging benefits from single-point imaging and inherently suffers from limited spatial and temporal resolution, preventing localization of small hypoxic tissues and differentiation of hypoxia dynamics, making accelerated imaging a crucial issue. METHODS: In this study, methods for accelerated single-point imaging were developed by combining a bilateral k-space extrapolation technique with model-based reconstruction that benefits from dense sampling in the parameter domain (measurement of the T2 (*) decay of a free induction delay). In bilateral kspace extrapolation, more k-space samples are obtained in a sparsely sampled region by bilaterally extrapolating data from temporally neighboring k-spaces. To improve the accuracy of T2 (*) estimation, a principal component analysis-based method was implemented. RESULTS: In a computer simulation and a phantom experiment, the proposed methods showed its capability for reliable T2 (*) estimation with high acceleration (8-fold, 15-fold, and 30-fold accelerations for 61×61×61, 95×95×95, and 127×127×127 matrix, respectively). CONCLUSION: By applying bilateral k-space extrapolation and model-based reconstruction, improved scan times with higher spatial resolution can be achieved in the current single-point electron paramagnetic resonance imaging modality.

Four-channel surface coil array for 300-MHz pulsed EPR imaging: proof-of-concept experiments.

Time-domain electron paramagnetic resonance imaging is currently a useful preclinical molecular imaging modality in experimental animals such as mice and is capable of quantitatively mapping hypoxia in tumor implants. The microseconds range relaxation times (T1 and T2) of paramagnetic tracers and the large bandwidths (tens of MHz) to be excited by electron paramagnetic resonance pulses for spatial encoding makes imaging of large objects a challenging task. The possibility of using multiple array coils to permit studies on large sized object is the purpose of the present work. Toward this end, the use of planar array coils in different configurations to image larger objects than cannot be fully covered by a single resonator element is explored. Multiple circular surface coils, which are arranged in a plane or at suitable angles mimicking a volume resonator, are used in imaging a phantom and a tumor-bearing mouse leg. The image was formed by combining the images collected from the individual coils with suitable scaling. The results support such a possibility. By multiplexing or interleaving the measurements from each element of such array resonators, one can scale up the size of the subject and at the same time reduce the radiofrequency power requirements and increase the sensitivity.

Single acquisition quantitative single-point electron paramagnetic resonance imaging.

PURPOSE: Electron paramagnetic resonance imaging has emerged as a promising noninvasive technology to dynamically image tissue oxygenation. Owing to its extremely short spin-spin relaxation times, electron paramagnetic resonance imaging benefits from a single-point imaging scheme where the entire free induction decay signal is captured using pure phase encoding. However, direct T2 (*)/pO2 quantification is inhibited owing to constant magnitude gradients which result in time-decreasing field of view. Therefore, conventional acquisition techniques require repeated imaging experiments with differing gradient amplitudes (typically 3), which results in long acquisition time. METHODS: In this study, gridding was evaluated as a method to reconstruct images with equal field of view to enable direct T2 (*)/pO2 quantification within a single imaging experiment. Additionally, an enhanced reconstruction technique that shares high spatial k-space regions throughout different phase-encoding time delays was investigated (k-space extrapolation). RESULTS: The combined application of gridding and k-space extrapolation enables pixelwise quantification of T2 (*) from a single acquisition with improved image quality across a wide range of phase-encoding time delays. The calculated T2 (*)/pO2 does not vary across this time range. CONCLUSIONS: By utilizing gridding and k-space extrapolation, accurate T2 (*)/pO2 quantification can be achieved within a single data set to allow enhanced temporal resolution (by a factor of 3).

Evaluation of partial k-space strategies to speed up time-domain EPR imaging.

Narrow-line spin probes derived from the trityl radical have led to the development of fast in vivo time-domain EPR imaging. Pure phase-encoding imaging modalities based on the single-point imaging scheme have demonstrated the feasibility of three-dimensional oximetric images with functional information in minutes. In this article, we explore techniques to improve the temporal resolution and circumvent the relatively short biological half-lives of trityl probes using partial k-space strategies. There are two main approaches: one involves the use of the Hermitian character of the k-space by which only part of the k-space is measured and the unmeasured part is generated using the Hermitian symmetry. This approach is limited in success by the accuracy of numerical estimate of the phase roll in the k-space that corrupts the Hermiticy. The other approach is to measure only a judicially chosen reduced region of k-space (a centrosymmetric ellipsoid region) that more or less accounts for >70% of the k-space energy. Both of these aspects were explored in Fourier transform-EPR imaging with a doubling of scan speed demonstrated by considering ellipsoid geometry of the k-space. Partial k-space strategies help improve the temporal resolution in studying fast dynamics of functional aspects in vivo with infused spin probes.

EPR oxygen imaging and hyperpolarized 13C MRI of pyruvate metabolism as noninvasive biomarkers of tumor treatment response to a glycolysis inhibitor 3-bromopyruvate.

The hypoxic nature of tumors results in treatment resistance and poor prognosis. To spare limited oxygen for more crucial pathways, hypoxic cancerous cells suppress mitochondrial oxidative phosphorylation and promote glycolysis for energy production. Thereby, inhibition of glycolysis has the potential to overcome treatment resistance of hypoxic tumors. Here, EPR imaging was used to evaluate oxygen dependent efficacy on hypoxia-sensitive drug. The small molecule 3-bromopyruvate blocks glycolysis pathway by inhibiting hypoxia inducible enzymes and enhanced cytotoxicity of 3-bromopyruvate under hypoxic conditions has been reported in vitro. However, the efficacy of 3-bromopyruvate was substantially attenuated in hypoxic tumor regions (pO2<10 mmHg) in vivo using squamous cell carcinoma (SCCVII)-bearing mouse model. Metabolic MRI studies using hyperpolarized 13C-labeled pyruvate showed that monocarboxylate transporter-1 is the major transporter for pyruvate and the analog 3-bromopyruvate in SCCVII tumor. The discrepant results between in vitro and in vivo data were attributed to biphasic oxygen dependent expression of monocarboxylate transporter-1 in vivo. Expression of monocarboxylate transporter-1 was enhanced in moderately hypoxic (8-15 mmHg) tumor regions but down regulated in severely hypoxic (<5 mmHg) tumor regions. These results emphasize the importance of noninvasive imaging biomarkers to confirm the action of hypoxia-activated drugs.

Evofosfamide and Gemcitabine Act Synergistically in Pancreatic Cancer Xenografts by Dual Action on Tumor Vasculature and Inhibition of Homologous Recombination DNA Repair.

Aims: Pancreatic ductal adenocarcinomas (PDACs) form hypovascular and hypoxic tumors, which are difficult to treat with current chemotherapy regimens. Gemcitabine (GEM) is often used as a first-line treatment for PDACs but has issues with chemoresistance and penetration in the interior of the tumor. Evofosfamide, a hypoxia-activated prodrug, has been shown to be effective in combination with GEM, although the mechanism of each drug on the other has not been established. We used mouse xenografts from two cell lines (MIA Paca-2 and SU.86.86) with different tumor microenvironmental characteristics to probe the action of each drug on the other. Results: GEM treatment enhanced survival times in mice with SU.86.86 leg xenografts (hazard ratio [HR] = 0.35, p = 0.03) but had no effect on MIA Paca-2 mice (HR = 0.91, 95% confidence interval = 0.37-2.25, p = 0.84). Conversely, evofosfamide did not improve survival times in SU.86.86 mice to a statistically significant degree (HR = 0.57, p = 0.22). Electron paramagnetic resonance imaging showed that oxygenation worsened in MIA Paca-2 tumors when treated with GEM, providing a direct mechanism for the activation of the hypoxia-activated prodrug evofosfamide by GEM. Sublethal amounts of either treatment enhanced the toxicity of other treatment in vitro in SU.86.86 but not in MIA Paca-2. By the biomarker γH2AX, combination treatment increased the number of double-stranded DNA lesions in vitro for SU.86.86 but not MIA Paca-2. Innovation and Conclusion: The synergy between GEM and evofosfamide appears to stem from the dual action of GEMs effect on tumor vasculature and inhibition by GEM of the homologous recombination DNA repair process. Antioxid. Redox Signal. 39, 432-444.

Transient decrease in tumor oxygenation after intravenous administration of pyruvate.

MRI using hyperpolarized (13) C-labeled pyruvate is a promising tool to biochemically profile tumors and monitor their response to therapy. This technique requires injection of pyruvate into tumor-bearing animals. Pyruvate is an endogenous entity but the influence of exogenously injected bolus doses of pyruvate on tumor microenvironment is not well understood. In this study, the effect of injecting a bolus of pyruvate on tumor oxygen status was investigated. EPR oxygen imaging revealed that the partial pressure of oxygen (pO(2)) in squamous cell carcinoma implanted in mice decreased significantly 30 min after [1-(13) C]pyruvate injection, but recovered to preinjection levels after 5 h. Dynamic contrast-enhanced-MRI studies showed that, at the dose of pyruvate used, no changes in tumor perfusion were noticed. Immunohistochemical analysis of hypoxic marker pimonidazole independently verified that the squamous cell carcinoma tumor transiently became more hypoxic by pyruvate injection. Efficacy of radiotherapy was suppressed when X-irradiation was delivered during the period of pyruvate-induced transient hypoxia. These results suggest importance of taking into account the transient decrease in tumor pO(2) after pyruvate injection in hyperpolarized (13) C MRI, because tumor oxygen status is an important factor in determining outcomes of therapies.

Simultaneous imaging of tumor oxygenation and microvascular permeability using Overhauser enhanced MRI.

Architectural and functional abnormalities of blood vessels are a common feature in tumors. A consequence of increased vascular permeability and concomitant aberrant blood flow is poor delivery of oxygen and drugs, which is associated with treatment resistance. In the present study, we describe a strategy to simultaneously visualize tissue oxygen concentration and microvascular permeability by using a hyperpolarized (1)H-MRI, known as Overhauser enhanced MRI (OMRI), and an oxygen-sensitive contrast agent OX63. Substantial MRI signal enhancement was induced by dynamic nuclear polarization (DNP). The DNP achieved up to a 7,000% increase in MRI signal at an OX63 concentration of 1.5 mM compared with that under thermal equilibrium state. The extent of hyperpolarization is influenced mainly by the local concentration of OX63 and inversely by the tissue oxygen level. By collecting dynamic OMRI images at different hyperpolarization levels, local oxygen concentration and microvascular permeability of OX63 can be simultaneously determined. Application of this modality to murine tumors revealed that tumor regions with high vascular permeability were spatio-temporally coincident with hypoxia. Quantitative analysis of image data from individual animals showed an inverse correlation between tumor vascular leakage and median oxygen concentration. Immunohistochemical analyses of tumor tissues obtained from the same animals after OMRI experiments demonstrated that lack of integrity in tumor blood vessels was associated with increased tumor microvascular permeability. This dual imaging technique may be useful for the longitudinal assessment of changes in tumor vascular function and oxygenation in response to chemotherapy, radiotherapy, or antiangiogenic treatment.

Hypoxia Imaging As a Guide for Hypoxia-Modulated and Hypoxia-Activated Therapy.

Significance: Oxygen imaging techniques, which can probe the spatiotemporal heterogeneity of tumor oxygenation, could be of significant clinical utility in radiation treatment planning and in evaluating the effectiveness of hypoxia-activated prodrugs. To fulfill these goals, oxygen imaging techniques should be noninvasive, quantitative, and capable of serial imaging, as well as having sufficient temporal resolution to detect the dynamics of tumor oxygenation to distinguish regions of chronic and acute hypoxia. Recent Advances: No current technique meets all these requirements, although all have strengths in certain areas. The current status of positron emission tomography (PET)-based hypoxia imaging, oxygen-enhanced magnetic resonance imaging (MRI), 19F MRI, and electron paramagnetic resonance (EPR) oximetry are reviewed along with their strengths and weaknesses for planning hypoxia-guided, intensity-modulated radiation therapy and detecting treatment response for hypoxia-targeted prodrugs. Critical Issues: Spatial and temporal resolution emerges as a major concern for these areas along with specificity and quantitative response. Although multiple oxygen imaging techniques have reached the investigative stage, clinical trials to test the therapeutic effectiveness of hypoxia imaging have been limited. Future Directions: Imaging elements of the redox environment besides oxygen by EPR and hyperpolarized MRI may have a significant impact on our understanding of the basic biology of the reactive oxygen species response and may extend treatment possibilities.

PEGPH20, a PEGylated human hyaluronidase, induces radiosensitization by reoxygenation in pancreatic cancer xenografts. A molecular imaging study.

PURPOSE: PEGylated human hyaluronidase (PEGPH20) enzymatically depletes hyaluronan, an important component of the extracellular matrix, increasing the delivery of therapeutic molecules. Combinations of chemotherapy and PEGPH20, however, have been unsuccessful in Phase III clinical trials. We hypothesize that by increasing tumor oxygenation by improving vascular patency and perfusion, PEGPH20 will also act as a radiosensitization agent. EXPERIMENTAL DESIGN: The effect of PEGPH20 on radiation treatment was analyzed with respect to tumor growth, survival time, p02, local blood volume, and the perfusion/permeability of blood vessels in a human pancreatic adenocarcinoma BxPC3 mouse model overexpressing hyaluronan synthase 3 (HAS3). RESULTS: Mice overexpressing HAS3 developed fast growing, radiation resistant tumors that became rapidly more hypoxic as time progressed. Treatment with PEGPH20 increased survival times when used in combination with radiation therapy, significantly more than either radiation therapy or PEGPH20 alone. In mice that overexpressed HAS3, EPR imaging showed an increase in local pO2 that could be linked to increases in perfusion/permeability and local blood volume immediately after PEGPH20 treatment. Hyperpolarized [1-13C] pyruvate suggested PEGPH20 caused a metabolic shift towards decreased glycolytic flux. These effects were confined to the mice overexpressing HAS3 - no effect of PEGPH20 on survival, radiation treatment, or pO2 was seen in wild type BxPC3 tumors. CONCLUSIONS: PEGPH20 may be useful for radiosensitization of pancreatic cancer but only in the subset of tumors with substantial hyaluronan accumulation. The response of the treatment may potentially be monitored by non-invasive imaging of the hemodynamic and metabolic changes in the tumor microenvironment.

Low-field paramagnetic resonance imaging of tumor oxygenation and glycolytic activity in mice.

A priori knowledge of spatial and temporal changes in partial pressure of oxygen (oxygenation; pO(2)) in solid tumors, a key prognostic factor in cancer treatment outcome, could greatly improve treatment planning in radiotherapy and chemotherapy. Pulsed electron paramagnetic resonance imaging (EPRI) provides quantitative 3D maps of tissue pO(2) in living objects. In this study, we implemented an EPRI set-up that could acquire pO(2) maps in almost real time for 2D and in minutes for 3D. We also designed a combined EPRI and MRI system that enabled generation of pO(2) maps with anatomic guidance. Using EPRI and an air/carbogen (95% O(2) plus 5% CO(2)) breathing cycle, we visualized perfusion-limited hypoxia in murine tumors. The relationship between tumor blood perfusion and pO(2) status was examined, and it was found that significant hypoxia existed even in regions that exhibited blood flow. In addition, high levels of lactate were identified even in normoxic tumor regions, suggesting the predominance of aerobic glycolysis in murine tumors. This report presents a rapid, noninvasive method to obtain quantitative maps of pO(2) in tumors, reported with anatomy, with precision. In addition, this method may also be useful for studying the relationship between pO(2) status and tumor-specific phenotypes such as aerobic glycolysis.

Intracellular hypoxia of tumor tissue estimated by noninvasive electron paramagnetic resonance oximetry technique using paramagnetic probes.

Electron paramagnetic resonance (EPR) oximetry at 700 MHz operating frequency employing a surface coil resonator is used to assess tissue partial pressure of oxygen (pO(2)) using paramagnetic media whose linewidth and decay constant are related to oxygen concentration. Differences in extracellular and intracellular pO(2) in squamous cell carcinoma (SCC) tumor tissue were tested using several types of water-soluble paramagnetic media, which localize extracellularly or permeate through the cell membrane. The nitroxide carboxy-PROXYL (CxP) can only be distributed in blood plasma and extracellular fluids whereas the nitroxides carbamoyl-PROXYL (CmP) and TEMPOL (TPL) can permeate cell membranes and localize intracellularly. EPR signal decay constant and the linewidth of the intravenously administered nitroxides in SCC tumor tissues implanted in mouse thigh and the contralateral normal muscle of healthy mice breathing gases with different pO(2) were compared. The pO(2) in the blood can depend on the oxygen content in the breathing gas while tissue pO(2) was not directly influenced by pO(2) in the breathing gas. The decay constants of CmP and TPL in tumor tissue were significantly larger than in the normal muscles, and lower linewidths of CmP and TPL in tumor tissue was observed. The SCC tumor showed intracellular hypoxia even though the extracellular pO(2) is similar to normal tissue in the peripheral region.

Molecular Imaging of the Tumor Microenvironment Reveals the Relationship between Tumor Oxygenation, Glucose Uptake, and Glycolysis in Pancreatic Ductal Adenocarcinoma.

Molecular imaging approaches for metabolic and physiologic imaging of tumors have become important for treatment planning and response monitoring. However, the relationship between the physiologic and metabolic aspects of tumors is not fully understood. Here, we developed new hyperpolarized MRI and electron paramagnetic resonance imaging procedures that allow more direct assessment of tumor glycolysis and oxygenation status quantitatively. We investigated the spatial relationship between hypoxia, glucose uptake, and glycolysis in three human pancreatic ductal adenocarcinoma tumor xenografts with differing physiologic and metabolic characteristics. At the bulk tumor level, there was a strong positive correlation between 18F-FDG-PET and lactate production, while pO2 was inversely related to lactate production and 18F-2-fluoro-2-deoxy-D-glucose (18F-FDG) uptake. However, metabolism was not uniform throughout the tumors, and the whole tumor results masked different localizations that became apparent while imaging. 18F-FDG uptake negatively correlated with pO2 in the center of the tumor and positively correlated with pO2 on the periphery. In contrast to pO2 and 18F-FDG uptake, lactate dehydrogenase activity was distributed relatively evenly throughout the tumor. The heterogeneity revealed by each measure suggests a multimodal molecular imaging approach can improve tumor characterization, potentially leading to better prognostics in cancer treatment. SIGNIFICANCE: Novel multimodal molecular imaging techniques reveal the potential of three interrelated imaging biomarkers to profile the tumor microenvironment and interrelationships of hypoxia, glucose uptake, and glycolysis.

Towards reduction of SAR in scaling up in vivo pulsed EPR imaging to larger objects.

An excessive RF power requirement is one of the main obstacles in the clinical translation of EPR imaging. The radio frequency (RF) pulses used in EPR imaging to excite electron spins must be very short to match their fast relaxation. With traditional pulse schemes and ninety degree flip angles, this can lead to either unsafe specific absorption rate (SAR) levels or unfeasibly long repetition times. In spectroscopy experiments, it has been shown that stochastic excitation and correlation detection can reduce the power while maintaining sensitivity but have yet to be applied to imaging experiments. Stochastic excitation is implemented using a pseudo-random phase modulation of the input stimulus. Using a crossed coil resonator assembly comprised of an outer saddle coil and an inner surface coil, it was possible to obtain a minimum isolation of ∼50 dB across a 12 MHz bandwidth. An incident peak RF power of 5 mW was used to excite the system. The low background signal obtained from this resonator allowed us to generate images with 32 dB (>1000:1) signal-to-noise ratio (SNR) while exciting with a traditional pulse sequence in a phantom containing the solid paramagnetic probe NMP-TCNQ (N-methyl pyridinium tetracyanoquinodimethane). Using two different stochastic excitation schemes, we were able to achieve a greater than 4-fold increase in SNR at the same peak power and number of averages, compared to single pulse excitation. This procedure allowed imaging at significantly lower RF power levels than used in conventional EPR imaging system configurations. Similar techniques may enable clinical applications for EPR imaging by facilitating the use of larger RF coils while maintaining a safe SAR level.

Pulsed Electron Paramagnetic Resonance Imaging: Applications in the Studies of Tumor Physiology.

SIGNIFICANCE: Electron paramagnetic resonance imaging (EPRI) is capable of generating images of tissue oxygenation using exogenous paramagnetic probes such as trityl radicals or nitroxyl radicals. The spatial distribution of the paramagnetic probe can be generated using magnetic field gradients as in magnetic resonance imaging and, from its spectral features, spatial maps of oxygen can be obtained from live objects. In this review, two methods of signal acquisition and image formation/reconstruction are described. The probes used and its application to study tumor physiology and monitor treatment response with chemotherapy drugs in mouse models of human cancer are summarized. Recent Advances: By implementing phase encoding/Fourier reconstruction in EPRI in time domain mode, the frequency contribution to the spatial resolution was avoided and images with improved spatial resolution were obtained. The EPRI-generated pO2 maps in tumor were useful to detect and evaluate the effects of various antitumor therapies on tumor physiology. Coregistration with other imaging modalities provided a better understanding of hypoxia-related alteration in physiology. CRITICAL ISSUES: The high radiofrequency (RF) power of EPR irradiation and toxicity profile of radical probes are the main obstacles for clinical application. The improvement of RF low power pulse sequences may allow for clinical translation. FUTURE DIRECTIONS: Pulsed EPR oximetry can be a powerful tool to research various diseases involving hypoxia such as cancer, ischemic heart diseases, stroke, and diabetes. With appropriate paramagnetic probes, it can also be applied for various other purposes such as detecting local acid-base balance or oxidative stress. Antioxid. Redox Signal. 28, 1378-1393.

Co-imaging of the tumor oxygenation and metabolism using electron paramagnetic resonance imaging and 13-C hyperpolarized magnetic resonance imaging before and after irradiation.

To examine the relationship between local oxygen partial pressure and energy metabolism in the tumor, electron paramagnetic resonance imaging (EPRI) and magnetic resonance imaging (MRI) with hyperpolarized [1-13C] pyruvate were performed. SCCVII and HT29 solid tumors implanted in the mouse leg were imaged by EPRI using OX063, a paramagnetic probe and 13C-MRI using hyperpolarized [1-13C] pyruvate. Local partial oxygen pressure and pyruvate metabolism in the two tumor implants were examined. The effect of a single dose of 5-Gy irradiation on the pO2 and metabolism was also investigated by sequential imaging of EPRI and 13C-MRI in HT29 tumors. A phantom study using tubes filled with different concentration of [1-13C] pyruvate, [1-13C] lactate, and OX063 at different levels of oxygen confirmed the validity of this sequential imaging of EPRI and hyperpolarized 13C-MRI. In vivo studies revealed SCCVII tumor had a significantly larger hypoxic fraction (pO2 < 8 mmHg) compared to HT29 tumor. The flux of pyruvate-to-lactate conversion was also higher in SCCVII than HT29. The lactate-to-pyruvate ratio in hypoxic regions (pO2 < 8 mmHg) 24 hours after 5-Gy irradiation was significantly higher than those without irradiation (0.76 vs. 0.36) in HT29 tumor. The in vitro study showed an increase in extracellular acidification rate after irradiation. In conclusion, co-imaging of pO2 and pyruvate-to-lactate conversion kinetics successfully showed the local metabolic changes especially in hypoxic area induced by radiation therapy.