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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.

For over a century, it has been known that tumor hypoxia, regions of a tumor with low levels of oxygenation, are important contributors to tumor resistance to radiation therapy and failure of radiation treatment of cancer. Recently, using novel pulse electron paramagnetic resonance (EPR) oxygen imaging, near absolute images of the partial pressure of oxygen (pO2) in tumors of living animals have been obtained. We discuss here the means by which EPR signals can be obtained in living tissues and tumors. We review development of EPR methods to image the pO2 in tumors and the potential for the pO2 image acquisition in human subjects., (© 2015 Elsevier Inc. All rights reserved.)

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.

Clinical trials to ameliorate hypoxia as a strategy to relieve the radiation resistance it causes have prompted a need to assay the precise extent and location of hypoxia in tumors. Electron paramagnetic resonance oxygen imaging (EPR O2 imaging) provides a noninvasive means to address this need. To obtain a preclinical proof-of-principle that EPR O2 images could predict radiation control, we treated mouse tumors at or near doses required to achieve 50% control (TCD50). Mice with FSa fibrosarcoma or MCa4 carcinoma were subjected to EPR O2 imaging and immediately radiated to a TCD50 or TCD50 ± 10 Gy. Statistical analysis was permitted by collection of approximately 1,300 tumor pO2 image voxels, including the fraction of tumor voxels with pO2 less than 10 mm Hg (HF10). Tumors were followed for 90 days (FSa) or 120 days (MCa4) to determine local control or failure. HF10 obtained from EPR images showed statistically significant differences between tumors that were controlled by the TCD50 and those that were not controlled for both FSa and MCa4. Kaplan-Meier analysis of both types of tumors showed that approximately 90% of mildly hypoxic tumors were controlled (HF10%< 10%), and only 37% (FSA) and 23% (MCa4) tumors controlled if hypoxic. EPR pO2 image voxel distributions in these approximately 0.5 mL tumors provide a prediction of radiation curability independent of radiation dose. These data confirm the significance of EPR pO2 hypoxic fractions. The 90% control of low HF10 tumors argue that 0.5 mL subvolumes of tumors may be more sensitive to radiation and may need less radiation for high tumor control rates. Cancer Res; 73(17); 5328-35. ©2013 AACR.

Hypoxic tumors are resistant to radiotherapy, motivating the development of tools to image local oxygen concentrations. It is generally believed that stable or chronic hypoxia is the source of resistance, but more recent work suggests a role for transient hypoxia. Conventional EPR imaging (EPRI) is capable of imaging tissue pO2in vivo, with high pO2 resolution and 1mm spatial resolution but low imaging speed (10min temporal resolution for T1-based pO2 mapping), which makes it difficult to investigate the oxygen changes, e.g., transient hypoxia. Here we describe a new imaging method which accelerates dynamic EPR oxygen imaging, allowing 3D imaging at 2 frames per minute, fast enough to image transient hypoxia at the "speed limit" of observed pO2 change. The method centers on a low-rank tensor model that decouples the tradeoff between imaging speed, spatial coverage/resolution, and number of inversion times (pO2 accuracy). We present a specialized sparse sampling strategy and image reconstruction algorithm for use with this model. The quality and utility of the method is demonstrated in simulations and in vivo experiments in tumor bearing mice.

One of the major drawbacks associated with autologous fat grafting is unpredictable graft retention. Various efforts to improve the survivability of these cells have been explored, but these methods are time‐consuming, complex, and demand significant technical skill. In our study, we examine the use of cryopreserved amniotic membrane as a source of exogenous growth factors to improve adipocyte survivability under normal and hypoxic conditions. Human primary preadipocytes were cultured in a gelatin‐ferulic acid (Gtn‐FA) hydrogel with variable oxygen concentration and treated with amniotic membrane‐derived condition medium (CM) for 7 days. This hydrogel provides a hypoxic environment and also creates a 3D cell culture to better mimic recipient site conditions. The O2 concentration in the hydrogel was measured by electron paramagnetic resonance oxygen imaging (EPROI). The conjugation of FA was confirmed by FTIR and NMR spectroscopy. The cell viability and adipocyte differentiation were analyzed by alamarBlue™ assay, Oil Red O staining, and RT‐qPCR. The expression of genes: Pref‐1, C/EBP β, C/EBP α, PPAR‐ƴ, SLC2A4, and VEGF‐A were quantified. The cell viability results show that the 50% CM showed significantly higher cell pre‐adipocyte cell viability. In addition, compared to normal conditions, hypoxia/CM provided higher PPAR‐ƴ (p <.05), SLC2A4, and VEGF‐A (p <.05) (early and terminal differentiating markers) mRNA expression. This finding demonstrates the efficacy of amniotic CM supplementation as a novel way to promote adipocyte survival and retention via the expression of key gene markers for differentiation and angiogenesis. [ABSTRACT FROM AUTHOR]

Purpose: Application of in vivo electron paramagnetic resonance (EPR) oxygen imaging (EPROI) to tumors larger than those of mice requires development of both instrumental and medical aspects of imaging. Methods: 250 MHz EPR oxygen imaging was performed using a loop-gap resonator with a volume exceeding 100 cm3. The paramagnetic spin probe was injected directly into the femoral artery feeding the rabbit leg/tumor. Results: The authors present continuous wave and electron spin echo EPR oxygen images of a large size (4 cm) VX-2 tumor located on the leg of a New Zealand white rabbit. Conclusions: This study demonstrates the feasibility of continuous wave and electron spin echo oxygen imaging modalities for investigation of volumes of tumor and normal tissue relevant to large animals. The injection of the spin probe directly into the artery feeding a rabbit leg will allow one to reduce, by over one order of magnitude, the amount of spin probe used as compared to whole animal IV injection. [ABSTRACT FROM AUTHOR]