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.

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.

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.

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.

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.

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.

Three-dimensional alginate hydrogels for radiobiological and metabolic studies of cancer cells.

The purpose of this study is to demonstrate calcium alginate hydrogels as a system for in vitro radiobiological and metabolic studies of cancer cells. Previous studies have established calcium alginate as a versatile three-dimensional (3D) culturing system capable of generating areas of oxygen heterogeneity and modeling metabolic changes in vitro. Here, through dosimetry, clonogenic and viability assays, and pimonidazole staining, we demonstrate that alginate can model radiobiological responses that monolayer cultures do not simulate. Notably, alginate hydrogels with radii greater than 500 µm demonstrate hypoxic cores, while smaller hydrogels do not. The size of this hypoxic region correlates with hydrogel size and improved cell survival following radiation therapy. Hydrogels can also be utilized in hyperpolarized magnetic resonance spectroscopy and extracellular flux analysis. Alginate therefore offers a reproducible, consistent, and low-cost means for 3D culture of cancer cells for radiobiological studies that simulates important in vivo parameters such as regional hypoxia and enables long-term culturing and in vitro metabolic studies.

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.

Metabolic and Physiologic Imaging Biomarkers of the Tumor Microenvironment Predict Treatment Outcome with Radiation or a Hypoxia-Activated Prodrug in Mice.

Pancreatic ductal adenocarcinoma (PDAC) is characterized by hypoxic niches that lead to treatment resistance. Therefore, studies of tumor oxygenation and metabolic profiling should contribute to improved treatment strategies. Here, we define two imaging biomarkers that predict differences in tumor response to therapy: (i) partial oxygen pressure (pO2), measured by EPR imaging; and (ii) [1-13C] pyruvate metabolism rate, measured by hyperpolarized 13C MRI. Three human PDAC xenografts with varying treatment sensitivity (Hs766t, MiaPaCa2, and Su.86.86) were grown in mice. The median pO2 of the mature Hs766t, MiaPaCa2, and Su.86.86 tumors was 9.1 ± 1.7, 11.1 ± 2.2, and 17.6 ± 2.6 mm Hg, and the rate of pyruvate-to-lactate conversion was 2.72 ± 0.48, 2.28 ± 0.26, and 1.98 ± 0.51 per minute, respectively (n = 6, each). These results are in agreement with steady-state data of matabolites quantified by mass spectroscopy and histologic analysis, indicating glycolytic and hypoxia profile in Hs766t, MiaPaca2, and Su.86.86 tumors. Fractionated radiotherapy (5 Gy × 5) resulted in a tumor growth delay of 16.7 ± 1.6 and 18.0 ± 2.7 days in MiaPaca2 and Su.86.86 tumors, respectively, compared with 6.3 ± 2.7 days in hypoxic Hs766t tumors. Treatment with gemcitabine, a first-line chemotherapeutic agent, or the hypoxia-activated prodrug TH-302 was more effective against Hs766t tumors (20.0 ± 3.5 and 25.0 ± 7.7 days increase in survival time, respectively) than MiaPaCa2 (2.7 ± 0.4 and 6.7 ± 0.7 days) and Su.86.86 (4.7 ± 0.6 and 0.7 ± 0.6 days) tumors. Collectively, these results demonstrate the ability of molecular imaging biomarkers to predict the response of PDAC to treatment with radiotherapy and TH-302.Significance: pO2 imaging data and clinically available metabolic imaging data provide useful insight into predicting the treatment efficacy of chemotherapy, radiation, and a hypoxia-activated prodrug as monotherapies and combination therapies in PDAC tumor xenograft models. Cancer Res; 78(14); 3783-92. ©2018 AACR.

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.

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.

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.

Quantitative imaging of pO2 in orthotopic murine gliomas: hypoxia correlates with resistance to radiation.

Hypoxia is considered one of the microenvironmental factors associated with the malignant nature of glioblastoma. Thus, evaluating intratumoural distribution of hypoxia would be useful for therapeutic planning as well as assessment of its effectiveness during the therapy. Electron paramagnetic resonance imaging (EPRI) is an imaging technique which can generate quantitative maps of oxygen in vivo using the exogenous paramagnetic compound, triarylmethyl and monitoring its line broadening caused by oxygen. In this study, the feasibility of EPRI for assessment of oxygen distribution in the glioblastoma using orthotopic U87 and U251 xenograft model is examined. Heterogeneous distribution of pO2 between 0 and 50 mmHg was observed throughout the tumours except for the normal brain tissue. U251 glioblastoma was more likely to exhibit hypoxia than U87 for comparable tumour size (median pO2; 29.7 and 18.2 mmHg, p = .028, in U87 and U251, respectively). The area with pO2 under 10 mmHg on the EPR oximetry (HF10) showed a good correlation with pimonidazole staining among tumours with evaluated size. In subcutaneous xenograft model, irradiation was relatively less effective for U251 compared with U87. In conclusion, EPRI is a feasible method to evaluate oxygen distribution in the brain tumour.

Accelerated electron paramagnetic resonance imaging using partial Fourier compressed sensing reconstruction.

PURPOSE: Electron paramagnetic resonance (EPR) imaging has evolved as a promising tool to provide non-invasive assessment of tissue oxygenation levels. Due to the extremely short T2 relaxation time of electrons, single point imaging (SPI) is used in EPRI, limiting achievable spatial and temporal resolution. This presents a problem when attempting to measure changes in hypoxic state. In order to capture oxygen variation in hypoxic tissues and localize cycling hypoxia regions, an accelerated EPRI imaging method with minimal loss of information is needed. METHODS: We present an image acceleration technique, partial Fourier compressed sensing (PFCS), that combines compressed sensing (CS) and partial Fourier reconstruction. PFCS augments the original CS equation using conjugate symmetry information for missing measurements. To further improve image quality in order to reconstruct low-resolution EPRI images, a projection onto convex sets (POCS)-based phase map and a spherical-sampling mask are used in the reconstruction process. The PFCS technique was used in phantoms and in vivo SCC7 tumor mice to evaluate image quality and accuracy in estimating O2 concentration. RESULTS: In both phantom and in vivo experiments, PFCS demonstrated the ability to reconstruct images more accurately with at least a 4-fold acceleration compared to traditional CS. Meanwhile, PFCS is able to better preserve the distinct spatial pattern in a phantom with a spatial resolution of 0.6mm. On phantoms containing Oxo63 solution with different oxygen concentrations, PFCS reconstructed linewidth maps that were discriminative of different O2 concentrations. Moreover, PFCS reconstruction of partially sampled data provided a better discrimination of hypoxic and oxygenated regions in a leg tumor compared to traditional CS reconstructed images. CONCLUSIONS: EPR images with an acceleration factor of four are feasible using PFCS with reasonable assessment of tissue oxygenation. The technique can greatly enhance EPR applications and improve our understanding cycling hypoxia. Moreover this technique can be easily extended to various MRI applications.

13C-MR Spectroscopic Imaging with Hyperpolarized [1-13C]pyruvate Detects Early Response to Radiotherapy in SCC Tumors and HT-29 Tumors.

PURPOSE: X-ray irradiation of tumors causes diverse effects on the tumor microenvironment, including metabolism. Recent developments of hyperpolarized (13)C-MRI enabled detecting metabolic changes in tumors using a tracer [1-(13)C]pyruvate, which participates in important bioenergetic processes that are altered in cancers. Here, we investigated the effects of X-ray irradiation on pyruvate metabolism in squamous cell carcinoma (SCCVII) and colon cancer (HT-29) using hyperpolarized (13)C-MRI. EXPERIMENTAL DESIGN: SCCVII and HT-29 tumors were grown by injecting tumor cells into the hind legs of mice. [1-(13)C]pyruvate was hyperpolarized and injected intravenously into tumor-bearing mice, and (13)C-MR signals were acquired using a 4.7 T scanner. RESULTS: [1-(13)C]pyruvate and [1-(13)C]lactate were detected in the tumor-bearing legs immediately after hyperpolarized [1-(13)C]pyruvate administration. The [1-(13)C]lactate to [1-(13)C]pyruvate ratio (Lac/Pyr) increased as the tumors grew in nonirradiated SCCVII tumors. The increase in Lac/Pyr was suppressed modestly with a single 10 Gy of irradiation, but it significantly decreased by further irradiation (10 Gy × 3). Similar results were obtained in HT-29; Lac/Pyr significantly dropped with fractionated 30 Gy irradiation. Independent ex vivo measurements revealed that the lactate dehydrogenase (LDH) activity and protein level were significantly smaller in the irradiated SCCVII tumors compared with the nonirradiated tumors, indicating that a decrease in LDH activity was one of the main factors responsible for the decrease of Lac/Pyr observed on (13)C-MRI. CONCLUSIONS: Robust changes of Lac/Pyr observed in the HT-29 after the radiation suggested that lactate conversion from pyruvate monitored with hyperpolarized (13)C-MRI could be useful for the evaluation of early response to radiotherapy. See related commentary by Lai et al., p. 4996.

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.

Pyruvate induces transient tumor hypoxia by enhancing mitochondrial oxygen consumption and potentiates the anti-tumor effect of a hypoxia-activated prodrug TH-302.

BACKGROUND: TH-302 is a hypoxia-activated prodrug (HAP) of bromo isophosphoramide mustard that is selectively activated within hypoxic regions in solid tumors. Our recent study showed that intravenously administered bolus pyruvate can transiently induce hypoxia in tumors. We investigated the mechanism underlying the induction of transient hypoxia and the combination use of pyruvate to potentiate the anti-tumor effect of TH-302. METHODOLOGY/RESULTS: The hypoxia-dependent cytotoxicity of TH-302 was evaluated by a viability assay in murine SCCVII and human HT29 cells. Modulation in cellular oxygen consumption and in vivo tumor oxygenation by the pyruvate treatment was monitored by extracellular flux analysis and electron paramagnetic resonance (EPR) oxygen imaging, respectively. The enhancement of the anti-tumor effect of TH-302 by pyruvate treatment was evaluated by monitoring the growth suppression of the tumor xenografts inoculated subcutaneously in mice. TH-302 preferentially inhibited the growth of both SCCVII and HT29 cells under hypoxic conditions (0.1% O2), with minimal effect under aerobic conditions (21% O2). Basal oxygen consumption rates increased after the pyruvate treatment in SCCVII cells in a concentration-dependent manner, suggesting that pyruvate enhances the mitochondrial respiration to consume excess cellular oxygen. In vivo EPR oxygen imaging showed that the intravenous administration of pyruvate globally induced the transient hypoxia 30 min after the injection in SCCVII and HT29 tumors at the size of 500-1500 mm(3). Pretreatment of SCCVII tumor bearing mice with pyruvate 30 min prior to TH-302 administration, initiated with small tumors (∼ 550 mm(3)), significantly delayed tumor growth. CONCLUSIONS/SIGNIFICANCE: Our in vitro and in vivo studies showed that pyruvate induces transient hypoxia by enhancing mitochondrial oxygen consumption in tumor cells. TH-302 therapy can be potentiated by pyruvate pretreatment if started at the appropriate tumor size and oxygen concentration.

In vivo imaging of tumor physiological, metabolic, and redox changes in response to the anti-angiogenic agent sunitinib: longitudinal assessment to identify transient vascular renormalization.

AIMS: The tumor microenvironment is characterized by a highly reducing redox status, a low pH, and hypoxia. Anti-angiogenic therapies for solid tumors frequently function in two steps: the transient normalization of structurally and functionally aberrant tumor blood vessels with increased blood perfusion, followed by the pruning of tumor blood vessels and the resultant cessation of nutrients and oxygen delivery required for tumor growth. Conventional anatomic or vascular imaging is impractical or insufficient to distinguish between the two steps of tumor response to anti-angiogenic therapies. Here, we investigated whether the noninvasive imaging of the tumor redox state and energy metabolism could be used to characterize anti-angiogenic drug-induced transient vascular normalization. RESULTS: Daily treatment of squamous cell carcinoma (SCCVII) tumor-bearing mice with the multi-tyrosine kinase inhibitor sunitinib resulted in a rapid decrease in tumor microvessel density and the suppression of tumor growth. Tumor pO2 imaging by electron paramagnetic resonance imaging showed a transient increase in tumor oxygenation after 2-4 days of sunitinib treatment, implying improved tumor perfusion. During this window of vascular normalization, magnetic resonance imaging of the redox status using an exogenously administered nitroxide probe and hyperpolarized (13)C MRI of the metabolic flux of pyruvate/lactate couple revealed an oxidative shift in tumor redox status. INNOVATION: Redox-sensitive metabolic couples can serve as noninvasive surrogate markers to identify the vascular normalization window in tumors with imaging techniques. CONCLUSION: A multimodal imaging approach to characterize physiological, metabolic, and redox changes in tumors is useful to distinguish between the different stages of anti-angiogenic treatment.

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.