An efficient noninvasive method for in vivo imaging of tumor oxygenation by using a low-field magnetic resonance scanner and a paramagnetic contrast agent is described. The methodology is based on Overhauser enhanced magnetic resonance imaging (OMRI), a functional imaging technique. OMRI experiments were performed on tumor-bearing mice (squamous cell carcinoma) by i.v. administration of the contrast agent Oxo63 (a highly derivatized triarylmethyl radical) at nontoxic doses in the range of 2-7 mmol/kg either as a bolus or as a continuous infusion. Spatially resolved pO 2 (oxygen concentration) images from OMRI experiments of tumor-bearing mice exhibited heterogeneous oxygenation profiles and revealed regions of hypoxia in tumors (<10 mmHg; 1 mmHg ‫؍‬ 133 Pa). Oxygenation of tumors was enhanced on carbogen (95% O 2͞5% CO2) inhalation. The pO2 measurements from OMRI were found to be in agreement with those obtained by independent polarographic measurements using a pO 2 Eppendorf electrode. This work illustrates that anatomically coregistered pO 2 maps of tumors can be readily obtained by combining the good anatomical resolution of water proton-based MRI, and the superior pO 2 sensitivity of EPR. OMRI affords the opportunity to perform noninvasive and repeated pO 2 measurements of the same animal with useful spatial (Ϸ1 mm) and temporal (2 min) resolution, making this method a powerful imaging modality for small animal research to understand tumor physiology and potentially for human applications.A bnormal values of pO 2 (the partial pressure of O 2 ) are linked to many pathophysiological conditions (e.g., ischemic diseases, reperfusion injury, and oxygen toxicity). Approximately one-third of human tumors evaluated for oxygen status have shown significant oxygen deficiency, and oxygen deficiency increases the tumor's resistance toward cancer treatment modalities, including radiation and chemotherapy (1, 2). Additionally, hypoxic microenvironments in tumors are known to promote processes driving malignant progression, such as angiogenesis, elimination of p53 tumor suppressor activity, genetic instability, and metastasis (3-5). Understanding of tumor hypoxia could lead to the discovery of diagnostic and prognostic markers for malignant progression, discovery of novel therapeutic targets, and the development of new constructs for gene therapy applications in human cancer. Hence, a noninvasive technique that could accurately and repetitively measure tissue oxygenation would find broad application in clinical and basic research. Unfortunately, the currently used electrochemical method (6) for in vivo oxygen measurement is an invasive technique applicable only to accessible tumors. Further, the technique is hampered by measurements of only a small part of the total tumor, which cannot be re-evaluated. Several magnetic resonance techniques (7, 8) have been developed for in vivo oximetry, including spin label oximetry (9), MRI (10), and electron paramagnetic resonance imaging (EPRI) (11,12). The blood oxygen level-dependent...

Tumors exhibit fluctuations in blood flow that influence oxygen concentrations and therapeutic resistance. To assist therapeutic planning and improve prognosis, noninvasive dynamic imaging of spatial and temporal variations in oxygen partial pressure (pO 2 ) would be useful. Here, we illustrate the use of pulsed electron paramagnetic resonance imaging (EPRI) as a novel imaging method to directly monitor fluctuations in oxygen concentrations in mouse models. A common resonator platform for both EPRI and magnetic resonance imaging (MRI) provided pO 2 maps with anatomic guidance and microvessel density. Oxygen images acquired every 3 minutes for a total of 30 minutes in two different tumor types revealed that fluctuation patterns in pO 2 are dependent on tumor size and tumor type. The magnitude of fluctuations in pO 2 in SCCVII tumors ranged between 2-to 18-fold, whereas the fluctuations in HT29 xenografts were of lower magnitude. Alternating breathing cycles with air or carbogen (95% O 2 plus 5% CO 2 ) distinguished higher and lower sensitivity regions, which responded to carbogen, corresponding to cycling hypoxia and chronic hypoxia, respectively. Immunohistochemical analysis suggests that the fluctuation in pO 2 correlated with pericyte density rather than vascular density in the tumor. This EPRI technique, combined with MRI, may offer a powerful clinical tool to noninvasively detect variable oxygenation in tumors.

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.

The time-domain (TD) mode of electron paramagnetic resonance (EPR) data collection offers a means of estimating the concentration of a paramagnetic probe and the oxygen-dependent linewidth (LW) to generate pO 2 maps with minimal errors. A methodology for noninvasive pO 2 imaging based on the application of TD-EPR using oxygen-induced LW broadening of a triarylmethyl (TAM)-based radical is presented. The decay of pixel intensities in an image is used to estimate T* 2 , which is inversely proportional to pO 2 . Factors affecting T* 2 in each pixel are critically analyzed to extract the contribution of dissolved oxygen to EPR line-broadening. Suitable experimental and image-processing parameters were obtained to produce pO 2 maps with minimal artifacts. Image artifacts were also minimized with the use of a novel data collection strategy using multiple gradients. Results from a phantom and in vivo imaging of tumor-bearing mice validated this novel method of noninvasive oximetry. The current imaging protocols achieve a spatial resolution of ϳ1.0 mm and a temporal resolution of ϳ9 s for 2D pO 2 mapping, with a reliable oxygen resolution of ϳ1 mmHg (0.12% oxygen in gas phase). This work demonstrates that in vivo oximetry can be performed with good sensitivity, accuracy, and high spatial and temporal resolution.

Structural and functional abnormalities in tumor blood vessels impact the delivery of oxygen and nutrients to solid tumors, resulting chronic and cycling hypoxia. While chronically hypoxic regions exhibit treatment resistance, more recently it has been shown that cycling hypoxic regions acquire pro-survival pathways. Angiogenesis inhibitors have been shown to transiently normalize the tumor vasculatures and enhance tumor response to treatments. However, the effect of anti-angiogenic therapy on cycling tumor hypoxia remains unknown. Using electron paramagnetic resonance imaging (EPRI) and magnetic resonance imaging (MRI) in tumor bearing mice, we have examined the vascular re-normalization process by longitudinally mapping tumor partial pressure of oxygen (pO2) and microvessel density during treatments with a multi-tyrosine kinase inhibitor sunitinib. Transient improvement in tumor oxygenation was visualized by EPRI 2–4 days following anti-angiogenic treatments, accompanied by a 45% decrease in microvessel density. Radiation treatment during this time period of improved oxygenation by anti-angiogenic therapy resulted in a synergistic delay in tumor growth. Additionally, dynamic oxygen imaging obtained every 3 minutes was conducted to distinguish tumor regions with chronic and cycling hypoxia. Sunitinib treatment suppressed the extent of temporal fluctuations in tumor pO2 during the vascular normalization window, resulting in the decrease of cycling tumor hypoxia. Overall, the findings suggest that longitudinal and noninvasive monitoring of tumor pO2 makes it possible to identify a window of vascular renormalization to maximize the effects of combination therapy with anti-angiogenic drugs.

This study describes the use of the single-point imaging (SPI) modality, also known as constant-time imaging (CTI), in radiofrequency (RF) Fourier transform (FT) electron paramagnetic resonance (EPR). The SPI technique, commonly used for highresolution solid-state nuclear magnetic resonance (NMR) imaging, has been successfully applied to 2D and 3D RF-FT-EPR imaging of phantoms containing narrow-line EPR spin probes. The SPI scheme is essentially a phase-encoding technique that operates by acquiring a single data point in the free induction decay (FID) after a fixed delay (phase-encoding time), following the pulsed RF excitation, in the presence of static magnetic field gradients. Since the phase-encoding time remains constant for a given image data set, the spectral information is automatically deconvolved, providing well-resolved pure spatial images. Therefore, images obtained using SPI are artifactfree and the resolution is not significantly limited by the line width, compared to the images obtained using the conventional filtered back-projection (FBP) scheme, suggesting that the SPI modality may have advantages for EPR imaging of large objects. In this work the advantages and limitations of SPI as compared to FBP are investigated by imaging suitable phantom objects. Although SPI takes longer to perform than the FBP method, optimization of the data collection scheme may increase the temporal resolution, rendering this technique suitable for in vivo studies. Spectral information can also be extracted from a series of SPI images that are generated as a function of the delay from the excitation pulse. Magn Reson Med 48:370 -379,

Imaging of free radicals by electron paramagnetic resonance (EPR) spectroscopy using time domain acquisition as in nuclear magnetic resonance (NMR) has not been attempted because of the short spin-spin relaxation times, typically under 1 microsecond, of most biologically relevant paramagnetic species. Recent advances in radiofrequency (RF) electronics have enabled the generation of pulses of the order of 10-50 ns. Such short pulses provide adequate spectral coverage for EPR studies at 300 MHz resonant frequency. Acquisition of free induction decays (FID) of paramagnetic species possessing inhomogenously broadened narrow lines after pulsed excitation is feasible with an appropriate digitizer/averager. This report describes the use of time-domain RF EPR spectrometry and imaging for in vivo applications. FID responses were collected from a water-soluble, narrow line width spin probe within phantom samples in solution and also when infused intravenously in an anesthetized mouse. Using static magnetic field gradients and back-projection methods of image reconstruction, two-dimensional images of the spin-probe distribution were obtained in phantom samples as well as in a mouse. The resolution in the images was better than 0.7 mm and devoid of motional artifacts in the in vivo study. Results from this study suggest a potential use for pulsed RF EPR imaging (EPRI) for three-dimensional spatial and spectral-spatial imaging applications. In particular, pulsed EPRI may find use in vivo studies to minimize motional artifacts from cardiac and lung motion that cause significant problems in frequency-domain spectral acquisition, such as in continuous wave (cw) EPR techniques.

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.angiogenesis ͉ dynamic nuclear polarization ͉ hyperpolarized MRI ͉ tumor hypoxia ͉ DCE-MRI