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Rapid expansion of tumor cells that outpace existing vasculature essential for nutrient and oxygen support as well as waste removal, correlates with profound changes in the microenvironment including angiogenesis, vasodilation, glucose metabolism, and cell cycle perturbations. Since hypoxic cells are up to three times more radioresistant than normoxic cells, identification of hypoxic populations to predict radiotherapeutic outcome is important. The consequences of hypoxia and activated proteins contribute to radioresistant tumors and radiotherapeutic failure. Stereotactic MCa4 tumor tissue biopsies from mouse tumors that were guided by electron paramagnetic resonance (EPR) O 2 imaging were examined for hypoxia-induced proteins. The oxygen broadening of narrow EPR spectral lines or, equivalently, the increase in relaxation rates of electron magnetization, report pO 2 with 1-2 torr resolution in image voxels less than 1 mm 3 . The pO 2 reporter molecule OX063d64 (trityl) was used to acquire the data described here. Trityl appears to be selectively retained in tumors with a half-life of ~30 min. We used an inversion recovery electron spin echo (IRESE) to measure the T1 rate of the trityl inside the tumor bearing leg. We estimate our uncertainty in pO 2 measurement to be 1-3 torr per voxel. Three hypoxic cell biomarkers, hypoxic-induced factor 1-alpha (HIF-1α), vascular endothelial growth factor (VEGF), and carbonic anhydrase IX (CA9), were examined using the ELISA assay. Quantification of these proteins based on results from the ELISA immunoassay kits indicate a strong correlation between EPR pO 2 -identified hypoxic fractions (<10 torr) and HIF-1α, VEGF, and CA9. We clearly demonstrate that hypoxic regions in tumors generate substantial amounts of HIF- 1α, VEGF, and CA9 protein.

Modern standards for radiation treatment do not take into account tumor oxygenation for radiation treatment planning. Strong correlation between tumor oxygenation and radiation treatment success suggests that oxygen-guided radiation therapy (OGRT) may be a promising enhancement of cancer radiation treatment. We have developed an OGRT protocol for rodents. Electron paramagnetic resonance (EPR) imaging is used for recording oxygen maps with high spatial resolution and excellent accuracy better than 1 torr. Radiation is delivered with an animal intensity modulated radiation therapy (IMRT) XRAD225Cx micro-CT/ therapy system. The radiation plan is delivered in two steps. First, a uniform 15% tumor control dose (TCD 15 ) is delivered to the whole tumor. In the second step, an additional booster dose amounting to the difference between TCD 98 and TCD 15 is delivered to radio-resistant, hypoxic tumor regions. Delivery of the booster dose is performed using a multiport conformal beam protocol. For radiation beam shaping we used individual radiation blocks 3D-printed from tungsten infused ABS polymer. Calculation of beam geometry and the production of blocks is performed next to the EPR imager, immediately after oxygen imaging. Preliminary results demonstrate the sub-millimeter precision of the radiation delivery and high dose accuracy. The efficacy of the radiation treatment is currently being tested on syngeneic FSa fibrosarcoma tumors grown in the legs of C3H mice.

The triarylmethyl radical OX063d24 is currently used for pulsed electron paramagnetic resonance oximetry at 250 MHz. Both 1/T 1 and 1/T 2 increase with increasing oxygen concentration. The dependence of 1/T 1 on probe concentration is smaller than for 1/T 2 . To inform the selection of the optimum frequency for in vivo oximetry 1/T 1 , 1/T 2 and signal-to-noise were measured as a function of frequency between 400 and 1000 MHz on a variable-frequency spectrometer with an adjustable-frequency cross-loop resonator. 1/T 1 and 1/T 2 decrease with increasing frequency and signal-to-noise increases with increasing frequency, which are all favourable for imaging at higher frequencies. However, depth of penetration of the radio frequency (RF) into an animal decreases with increasing frequency. Assuming that the RF loss in the animal to be studied determines the resonator Q, our results indicate that the optimum frequency for in vivo imaging will be determined by the desired depth of penetration in the tissue.

The outcome of cancer radiation treatment is strongly correlated with tumor oxygenation. The aim of this study is to use oxygen tension distributions in tumors obtained using Electron Paramagnetic Resonance (EPR) imaging to devise better tumor radiation treatment. The proposed radiation plan is delivered in two steps. In the first step, a uniform 50% tumor control dose (TCD50) is delivered to the whole tumor. For the second step an additional dose boost is delivered to radioresistant, hypoxic tumor regions. FSa fibrosarcomas grown in the gastrocnemius of the legs of C3H mice were used. Oxygen tension images were obtained using a 250 MHz pulse imager and injectable partially deuterated trityl OX63 (OX71) spin probe. Radiation was delivered with a novel animal intensity modulated radiation therapy (IMRT) XRAD225Cx microCT/radiation therapy delivery system. In a simplified scheme for boost dose delivery, the boost area is approximated by a sphere, whose radius and position are determined using an EPR O2 image. The sphere that irradiates the largest fraction of hypoxic voxels in the tumor was chosen using an algorithm based on Receiver Operator Characteristic (ROC) analysis. We used the fraction of irradiated hypoxic volume as the true positive determinant and the fraction of irradiated normoxic volume as the false positive determinant in the terms of that analysis. The most efficient treatment is the one that demonstrates the shortest distance from the ROC curve to the upper left corner of the ROC plot. The boost dose corresponds to the difference between TCD90 and TCD50 values. For the control experiment an identical radiation dose to the normoxic tumor area is delivered.

Electron paramagnetic resonance imaging (EPRI) has been used to noninvasively provide 3D images of absolute oxygen concentration (pO2) in small animals. These oxygen images are well resolved both spatially (~1 mm) and in pO2 (1-3 mmHg). EPRI preclinical images of pO2 have demonstrated extremely promising results for various applications investigating oxygen related physiologic and biologic processes as well as the dependence of various disease states on pO2, such as the role of hypoxia in cancer. Recent developments have been made that help to progress EPRI towards the eventual goal of human application. For example, a bimodal crossed-wire surface coil has been developed. Very preliminary tests demonstrated a 20 dB isolation between transmit and receive for this coil, with an anticipated additional 20 dB achievable. This could potentially be used to image local pO2 in human subjects with superficial tumors with EPRI. Local excitation and detection will reduce the specific absorption rate limitations on images and eliminate any possible power deposition concerns. Additionally, a large 9 mT EPRI magnet has been constructed which can fit and provide static main and gradient fields for imaging local anatomy in an entire human. One potential obstacle that must be overcome in order to use EPRI to image humans is the approved use of the requisite EPRI spin probe imaging agent (trityl). While nontoxic, EPRI trityl spin probes have been injected intravenously when imaging small animals, and require relatively high total body injection doses that would not be suitable for human imaging applications. Work has been done demonstrating the alternative use of intratumoral (IT) injections, which can reduce the amount of trityl required for imaging by a factor of 2000- relative to a whole body intravenous injection. The development of a large magnet that can accommodate human subjects, the design of a surface coil for imaging of superficial pO2, and the reduction of required spin probe using IT injections all are crucial steps towards the eventual use of EPRI to image pO2 in human subjects. In the future this can help investigate the oxygenation status of superficial tumors (e.g., breast tumors). The ability to image pO2 in humans has many other potential applications to diseases such as peripheral vascular disease, heart disease, and stroke.

Distributions of oxygen concentration (pO₂) are a critical determinant of normal tissue health as well as tumor aggressiveness and response to therapy. A number of studies show the value of normal tissue and tumor tissue oxygenation images and some of these will be discussed here. A strong correlation between tumor hypoxic fraction as measured with electron paramagnetic resonance oxygen imaging and radiation treatment success or failure has been found in two separate cancer types. Oxygen images of the torso of wild type mice show initial reduction of lung, liver, visceral, and muscle pO₂ with cyclic halving of fraction of inspired oxygen (FiO₂), but variation is blunted over an hour. Spontaneous breast cancers in Mouse Mammary Tumor Viral (MMTV) promoted-polyoma middle T antigen (PyMT) mice with BNIP3, a major factor in promotion of mitochondrial autophagy, knocked out will be compared with wild type animals. Preliminary studies for the BNIP3 knock out animals show extremely low pO₂. The wide variety of studies, in which oxygen images can play an integral role, serve to demonstrate the importance of oxygen images.

Chronic hypoxia strongly affects the malignant state and resistance to therapy for tumors. Transient hypoxia has been hypothesized, but not proven to be more deleterious. Electron paramagnetic resonance imaging (EPRI) provides non-invasive, quantitative imaging of static pO₂ in vivo. Dynamic EPRI produces pO₂ movies, enabling non-invasive assessment of in vivo pO₂ changes, such as transient hypoxia. Recent developments have been made to enable Dynamic EPRI. Maximally spaced projection sequencing has been implemented to allow for more accurate and versatile acquisition of EPRI data when studying dynamic systems. Principal component analysis filtering has been employed to enhance SNR. Dynamic EPRI studies will provide temporally resolved oxygen movies necessary to perform in vivo studies of physiologically relevant pO₂ changes in tumors. These oxygen movies will allow for the localization/quantification of transient hypoxia and will therefore help to disentangle the relationship between chronic and transient hypoxia, in order to better understand their roles in therapeutic optimization and outcome.

The partial pressure of oxygen (pO₂) in tissues plays an important role in the pathophysiology of many diseases and influences outcome of cancer therapy, ischemic heart and cerebrovascular disease treatments and wound healing. Over the years a suite of EPR techniques for reliable oxygen measurements has been developed. This is a mini-review of pulse EPR in vivo oxygen imaging methods that utilize soluble spin probes. Recent developments in pulse EPR imaging technology have brought an order of magnitude increase in image acquisition speed, enhancement of sensitivity and considerable improvement in the precision and accuracy of oxygen measurements.