Corrigendum: Absolute oxygen-guided radiation therapy improves tumor control in three preclinical tumor models.

[This corrects the article DOI: 10.3389/fmed.2023.1269689.].

Absolute oxygen-guided radiation therapy improves tumor control in three preclinical tumor models.

Background: Clinical attempts to find benefit from specifically targeting and boosting resistant hypoxic tumor subvolumes have been promising but inconclusive. While a first preclinical murine tumor type showed significant improved control with hypoxic tumor boosts, a more thorough investigation of efficacy from boosting hypoxic subvolumes defined by electron paramagnetic resonance oxygen imaging (EPROI) is necessary. The present study confirms improved hypoxic tumor control results in three different tumor types using a clonogenic assay and explores potential confounding experimental conditions. Materials and methods: Three murine tumor models were used for multi-modal imaging and radiotherapy: MCa-4 mammary adenocarcinomas, SCC7 squamous cell carcinomas, and FSa fibrosarcomas. Registered T2-weighted MRI tumor boundaries, hypoxia defined by EPROI as pO2 ≤ 10 mmHg, and X-RAD 225Cx CT boost boundaries were obtained for all animals. 13 Gy boosts were directed to hypoxic or equal-integral-volume oxygenated tumor regions and monitored for regrowth. Kaplan-Meier survival analysis was used to assess local tumor control probability (LTCP). The Cox proportional hazards model was used to assess the hazard ratio of tumor progression of Hypoxic Boost vs. Oxygenated Boost for each tumor type controlling for experimental confounding variables such as EPROI radiofrequency, tumor volume, hypoxic fraction, and delay between imaging and radiation treatment. Results: An overall significant increase in LTCP from Hypoxia Boost vs. Oxygenated Boost treatments was observed in the full group of three tumor types (p < 0.0001). The effects of tumor volume and hypoxic fraction on LTCP were dependent on tumor type. The delay between imaging and boost treatments did not have a significant effect on LTCP for all tumor types. Conclusion: This study confirms that EPROI locates resistant tumor hypoxic regions for radiation boost, increasing clonogenic LTCP, with potential enhanced therapeutic index in three tumor types. Preclinical absolute EPROI may provide correction for clinical hypoxia images using additional clinical physiologic MRI.

Toward a Nanoencapsulated EPR Imaging Agent for Clinical Use.

PURPOSE: Progress toward developing a novel radiocontrast agent for determining pO2 in tumors in a clinical setting is described. The imaging agent is designed for use with electron paramagnetic resonance imaging (EPRI), in which the collision of a paramagnetic probe molecule with molecular oxygen causes a spectroscopic change which can be calibrated to give the real oxygen concentration in the tumor tissue. PROCEDURES: The imaging agent is based on a nanoscaffold of aluminum hydroxide (boehmite) with sizes from 100 to 200 nm, paramagnetic probe molecule, and encapsulation with a gas permeable, thin (10-20 nm) polymer layer to separate the imaging agent and body environment while still allowing O2 to interact with the paramagnetic probe. A specially designed deuterated Finland trityl (dFT) is covalently attached on the surface of the nanoparticle through 1,3-dipolar addition of the alkyne on the dFT with an azide on the surface of the nanoscaffold. This click-chemistry reaction affords 100% efficiency of the trityl attachment as followed by the complete disappearance of the azide peak in the infrared spectrum. The fully encapsulated, dFT-functionalized nanoparticle is referred to as RADI-Sense. RESULTS: Side-by-side in vivo imaging comparisons made in a mouse model made between RADI-Sense and free paramagnetic probe (OX-071) showed oxygen sensitivity is retained and RADI-Sense can create 3D pO2 maps of solid tumors CONCLUSIONS: A novel encapsulated nanoparticle EPR imaging agent has been described which could be used in the future to bring EPR imaging for guidance of radiotherapy into clinical reality.

The optimal 18F-fluoromisonidazole PET threshold to define tumor hypoxia in preclinical squamous cell carcinomas using pO2 electron paramagnetic resonance imaging as reference truth.

PURPOSE: To identify the optimal threshold in 18F-fluoromisonidazole (FMISO) PET images to accurately locate tumor hypoxia by using electron paramagnetic resonance imaging (pO2 EPRI) as ground truth for hypoxia, defined by pO2 [Formula: see text] 10 mmHg. METHODS: Tumor hypoxia images in mouse models of SCCVII squamous cell carcinoma (n = 16) were acquired in a hybrid PET/EPRI imaging system 2 h post-injection of FMISO. T2-weighted MRI was used to delineate tumor and muscle tissue. Dynamic contrast enhanced (DCE) MRI parametric images of Ktrans and ve were generated to model tumor vascular properties. Images from PET/EPR/MRI were co-registered and resampled to isotropic 0.5 mm voxel resolution for analysis. PET images were converted to standardized uptake value (SUV) and tumor-to-muscle ratio (TMR) units. FMISO uptake thresholds were evaluated using receiver operating characteristic (ROC) curve analysis to find the optimal FMISO threshold and unit with maximum overall hypoxia similarity (OHS) with pO2 EPRI, where OHS = 1 shows perfect overlap and OHS = 0 shows no overlap. The means of dice similarity coefficient, normalized Hausdorff distance, and accuracy were used to define the OHS. Monotonic relationships between EPRI/PET/DCE-MRI were evaluated with the Spearman correlation coefficient ([Formula: see text]) to quantify association of vasculature on hypoxia imaged with both FMISO PET and pO2 EPRI. RESULTS: FMISO PET thresholds to define hypoxia with maximum OHS (both OHS = 0.728 [Formula: see text] 0.2) were SUV [Formula: see text] 1.4 [Formula: see text] SUVmean and SUV [Formula: see text] 0.6 [Formula: see text] SUVmax. Weak-to-moderate correlations (|[Formula: see text]|< 0.70) were observed between PET/EPRI hypoxia images with vascular permeability (Ktrans) or fractional extracellular-extravascular space (ve) from DCE-MRI. CONCLUSION: This is the first in vivo comparison of FMISO uptake with pO2 EPRI to identify the optimal FMISO threshold to define tumor hypoxia, which may successfully direct hypoxic tumor boosts in patients, thereby enhancing tumor control.

Improving Tumor Hypoxia Location in 18F-Misonidazole PET with Dynamic Contrast-enhanced MRI Using Quantitative Electron Paramagnetic Resonance Partial Oxygen Pressure Images.

Purpose: To enhance the spatial accuracy of fluorine 18 (18F) misonidazole (MISO) PET imaging of hypoxia by using dynamic contrast-enhanced (DCE) MR images as a basis for modifying PET images and by using electron paramagnetic resonance (EPR) partial oxygen pressure (pO2) as the reference standard. Materials and Methods: Mice (n = 10) with leg-borne MCa4 mammary carcinomas underwent EPR imaging, T2-weighted and DCE MRI, and 18F-MISO PET/CT. Images were registered to the same space for analysis. The thresholds of hypoxia for PET and EPR images were tumor-to-muscle ratios greater than or equal to 2.2 mm Hg and less than or equal to 14 mm Hg, respectively. The Dice similarity coefficient (DSC) and Hausdorff distance (d H ) were used to quantify the three-dimensional overlap of hypoxia between pO2 EPR and 18F-MISO PET images. A training subset (n = 6) was used to calculate optimal DCE MRI weighting coefficients to relate EPR to the PET signal; the group average weights were then applied to all tumors (from six training mice and four test mice). The DSC and d H were calculated before and after DCE MRI-corrected PET images were obtained to quantify the improvement in overlap with EPR pO2 images for measuring tumor hypoxia. Results: The means and standard deviations of the DSC and d H between hypoxic regions in original PET and EPR images were 0.35 mm ± 0.23 and 5.70 mm ± 1.7, respectively, for images of all 10 mice. After implementing a preliminary DCE MRI correction to PET data, the DSC increased to 0.86 mm ± 0.18 and the d H decreased to 2.29 mm ± 0.70, showing significant improvement (P < .001) for images of all 10 mice. Specifically, for images of the four independent test mice, the DSC improved with correction from 0.19 ± 0.28 to 0.80 ± 0.29 (P = .02), and the d H improved from 6.40 mm ± 2.5 to 1.95 mm ± 0.63 (P = .01). Conclusion: Using EPR information as a reference standard, DCE MRI information can be used to correct 18F-MISO PET information to more accurately reflect areas of hypoxia.Keywords: Animal Studies, Molecular Imaging, Molecular Imaging-Cancer, PET/CT, MR-Dynamic Contrast Enhanced, MR-Imaging, PET/MR, Breast, Oncology, Tumor Mircoenvironment, Electron Paramagnetic ResonanceSupplemental material is available for this article.© RSNA, 2021.

Highly sensitive electron paramagnetic resonance nanoradicals for quantitative intracellular tumor oxymetric images.

Purpose: Tumor oxygenation is a critical parameter influencing the efficacy of cancer therapy. Low levels of oxygen in solid tumor have been recognized as an indicator of malignant progression and metastasis, as well as poor response to chemo- and radiation therapy. Being able to measure oxygenation for an individual's tumor would provide doctors with a valuable way of identifying optimal treatments for patients. Methods: Electron paramagnetic resonance imaging (EPRI) in combination with an oxygen-measuring paramagnetic probe was performed to measure tumor oxygenation in vivo. Triarylmethyl (trityl) radical exhibits high specificity, sensitivity, and resolution for quantitative measurement of O2 concentration. However, its in vivo applications in previous studies have been limited by the required high dosage, its short half-life, and poor intracellular permeability. To address these limitations, we developed high-capacity nanoformulated radicals that employed fluorescein isothiocyanate-labeled mesoporous silica nanoparticles (FMSNs) as trityl radical carriers. The high surface area nanostructure and easy surface modification of physiochemical properties of FMSNs enable efficient targeted delivery of highly concentrated, nonself-quenched trityl radicals, protected from environmental degradation and dilution. Results: We successfully designed and synthesized a tumor-targeted nanoplatform as a carrier for trityl. In addition, the nanoformulated trityl does not affect oxygen-sensing capacity by a self-relaxation or broadening effect. The FMSN-trityl exhibited high sensitivity/response to oxygen in the partial oxygen pressure range from 0 to 155 mmHg. Furthermore, MSN-trityl displayed outstanding intracellular oxygen mapping in both in vitro and in vivo animal studies. Conclusion: The highly sensitive nanoformulated trityl spin probe can profile intracellular oxygen distributions of tumor in a real-time and quantitative manner using in vivo EPRI.

Oxygen-Guided Radiation Therapy.

PURPOSE: It has been known for over 100 years that tumor hypoxia, a near-universal characteristic of solid tumors, decreases the curative effectiveness of radiation therapy. However, to date, there are no reports that demonstrate an improvement in radiation effectiveness in a mammalian tumor on the basis of tumor hypoxia localization and local hypoxia treatment. METHODS AND MATERIALS: For radiation targeting of hypoxic subregions in mouse fibrosarcoma, we used oxygen images obtained using pulse electron paramagnetic resonance pO2 imaging combined with 3D-printed radiation blocks. This achieved conformal radiation delivery to all hypoxic areas in FSa fibrosarcomas in mice. RESULTS: We demonstrate that treatment delivering a radiation boost to hypoxic volumes has a significant (P = .04) doubling of tumor control relative to boosts to well-oxygenated volumes. Additional dose to well-oxygenated tumor regions minimally increases tumor control beyond the 15% control dose to the entire tumor. If we can identify portions of the tumor that are more resistant to radiation, it might be possible to reduce the dose to more sensitive tumor volumes without significant compromise in tumor control. CONCLUSIONS: This work demonstrates in a single, intact mammalian tumor type that tumor hypoxia is a local tumor phenomenon whose treatment can be enhanced by local radiation. Despite enormous clinical effort to overcome hypoxic radiation resistance, to our knowledge this is the first such demonstration, even in preclinical models, of targeting additional radiation to hypoxic tumor to improve the therapeutic ratio.

Spin Lattice Relaxation EPR pO2 Images May Direct the Location of Radiation Tumor Boosts to Enhance Tumor Cure.

Radiation treatment success and high tumor oxygenation and success have been known to be highly correlated. This suggests that radiation therapy guided by images of tumor regions with low oxygenation, oxygen-guided radiation therapy (OGRT) may be a promising enhancement of cancer radiation treatment. Before applying the technique to human subjects, OGRT needs to be tested in animals, most easily in rodents. Electron paramagnetic resonance imaging provides quantitative maps of tissue and tumor oxygen in rodents with 1 mm spatial resolution and 1 torr pO2 resolution at low oxygen levels. The difficulty of using mouse models is their small size and that of their tumors. To overcome this we used XRAD225Cx micro-CT/ therapy system and 3D printed conformal blocks. Radiation is delivered first to a uniform 15% tumor control dose for the whole tumor and then a boost dose to either hypoxic tumor regions or equal volumes of well oxygenated tumor. Delivery of the booster dose used a multiple beam angles to deliver radiation beams whose shape conforms to that of all hypoxic regions or fully avoids those regions. To treat/avoid all hypoxic regions we used individual radiation blocks 3D-printed from acrylonitrile butadiene styrene polymer infused with tungsten particles fabricated immediately after imaging to determine regions with pO2 less than 10 torr. Preliminary results demonstrate the efficacy of the radiation treatment with hypoxic boosts with syngeneic FSa fibrosarcoma tumors in the legs of C3H mice.

Correlation Between Hypoxia Proteins and EPR-Detected Hypoxia in Tumors.

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) O2 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 pO2 with 1-2 torr resolution in image voxels less than 1 mm3. The pO2 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 pO2 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 pO2-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.

EPR Imaging Spin Probe Trityl Radical OX063: A Method for Its Isolation from Animal Effluent, Redox Chemistry of Its Quinone Methide Oxidation Product, and in Vivo Application in a Mouse.

We report herein a method for the recovery, purification, and application of OX063, a costly, commercially available nontoxic spin probe widely used for electron paramagnetic resonance (EPR) imaging, as well as its corresponding quinone methide (QM) form. This precious probe can be successfully recovered after use in animal model experiments (25-47% recovery from crude lyophilizate with 98.5% purity), even from samples that are >2 years old. Significantly, the recovered trityl can be reused in further animal model EPR imaging experiments. The work also describes support for the observed formation of an air-sensitive radical derived from the QM under reducing conditions.

In vivo electron paramagnetic resonance imaging of differential tumor targeting using cis-3,4-di(acetoxymethoxycarbonyl)-2,2,5,5-tetramethyl-1-pyrrolidinyloxyl.

PURPOSE: Electron paramagnetic resonance spectroscopy promises quantitative images of important physiologic markers of animal tumors and normal tissues, such as pO(2), pH, and thiol redox status. These parameters of tissue function are conveniently reported by tailored nitroxides. For defining tumor physiology, it is vital that nitroxides are selectively localized in tumors relative to normal tissue. Furthermore, these paramagnetic species should be specifically taken up by cells of the tumor, thereby reporting on both the site of tumor formation and the physiological status of the tissue. This study investigates the tumor localization of the novel nitroxide, cis-3,4-di(acetoxymethoxycarbonyl)-2,2,5,5-tetramethyl-1-pyrrolidin-yloxyl 3 relative to the corresponding di-acid 4. METHODS: We obtained images of nitroxide 3 infused intravenously into C3H mice bearing 0.5-cm(3) FSa fibrosarcoma on the leg, and compared these with images of similar tumors infused with nitroxide 4. RESULTS: The ratio of spectral intensity from within the tumor-bearing region to that of normal tissue was higher in the mice injected with 3 relative to 4. CONCLUSION: This establishes the possibility of tumor imaging with a nitroxide with intracellular distribution and provides the basis for EPR images of animal models to investigate the relationship between crucial aspects of tumor microenvironment and malignancy and its response to therapy.

EPR oxygen images predict tumor control by a 50% tumor control radiation dose.

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.

Radiation oxygen biology with pulse electron paramagnetic resonance imaging in animal tumors.

The reduced oxygen in tumors (hypoxia) generates radiation resistance and limits tumor control probability (TCP) at radiation doses without significant normal tissue complication. Modern radiation therapy delivery with intensity-modulated radiation therapy (IMRT) enables complex, high-dose gradient patterns, which avoid sensitive human tissues and organs. EPR oxygen images may allow selection of more resistant parts of a tumor to which to deliver more radiation dose to enhance TCP. EPR O2 images are obtained using injected narrow-line, low relaxation rate trityl spin probes that enable pulse radiofrequency EPR O2 images of tumors in the legs of mice, rats, and rabbits, the latter exceeding 4 cm in size. Low relaxation rates of trityls have enabled novel T1-, rather than T2-, based oximetry, which provides near absolute pO2 imaging. Tomographic image formation and filtered back projection reconstruction are used to generate these images with fixed, linear stepped gradients. Images obtained both with T2 and T1 oximetric images have demonstrated the complex in vivo mechanism explaining the unexpected efficacy of TNFerade, a radiation-inducible adenoviral construct to locally produce TNF-induced vascular as well as radiation damage [1, 2]. The unexpected efficacy of large-dose radiation fractions is seen to be due to an interaction between host microvasculature and tumor cells producing a prompt (15 min) postradiation hypoxia, paralyzing tumor cell repair, and sensitizing tumors. Finally, cure of tumors treated to a single 50 % control dose shows a significant dependence on EPR O2 image hypoxic fractions, best shown with the fraction of voxels less than 10 Torr (HF10). We show that these O2 images provide a quantitative basis for measuring tumor and normal tissue response to abnormally low O2 levels. Measurements of vascular endothelial growth factor (VEGF) production in a specific syngeneic mouse fibrosarcoma, FSa versus fraction of tissue voxels with pO2 less than 10 Torr, produced a slope of 0.14 pg VEGF protein/mg total protein/% HF10. We argue that this quantification may be diagnostic of tumor versus normal tissue, and it may be etiologic in the development of malignancy.

Comparison of 250 MHz electron spin echo and continuous wave oxygen EPR imaging methods for in vivo applications.

PURPOSE: The authors compare two electron paramagnetic resonance imaging modalities at 250 MHz to determine advantages and disadvantages of those modalities for in vivo oxygen imaging. METHODS: Electron spin echo (ESE) and continuous wave (CW) methodologies were used to obtain three-dimensional images of a narrow linewidth, water soluble, nontoxic oxygen-sensitive trityl molecule OX063 in vitro and in vivo. The authors also examined sequential images obtained from the same animal injected intravenously with trityl spin probe to determine temporal stability of methodologies. RESULTS: A study of phantoms with different oxygen concentrations revealed a threefold advantage of the ESE methodology in terms of reduced imaging time and more precise oxygen resolution for samples with less than 70 torr oxygen partial pressure. Above 100 torr, CW performed better. The images produced by both methodologies showed pO2 distributions with similar mean values. However, ESE images demonstrated superior performance in low pO2 regions while missing voxels in high pO2 regions. CONCLUSIONS: ESE and CW have different areas of applicability. ESE is superior for hypoxia studies in tumors.

Where it's at really matters: in situ in vivo vascular endothelial growth factor spatially correlates with electron paramagnetic resonance pO2 images in tumors of living mice.

PURPOSE: Tumor microenvironments show remarkable tumor pO(2) heterogeneity, as seen in prior EPR pO(2) images (EPROI). pO(2) correlation with hypoxia response proteins is frustrated by large rapid pO(2) changes with position. PROCEDURES: To overcome this limitation, biopsies stereotactically located in the EPROI were used to explore the relationship between vascular endothelial growth factor A (VEGF) concentrations in living mouse tumors and the local EPROI pO(2). RESULTS: Quantitative ELISA VEGF concentrations correlated (p = 0.0068 to 0.019) with mean pO(2), median pO(2), and the fraction of voxels in the biopsy volume with pO(2) less than 3, 6, and 10 Torr. CONCLUSIONS: This validates EPROI hypoxic fractions at the molecular level and provides a new paradigm for the assessment of the relationship, in vivo, between hypoxia and hypoxia response proteins. When translated to human subjects, this will enhance understanding of human tumor pathophysiology and cancer response to therapy.

Electron paramagnetic resonance oxygen imaging of a rabbit tumor using localized spin probe delivery.

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 i.v. injection.

Anti-HER2 immunoliposomes for selective delivery of electron paramagnetic resonance imaging probes to HER2-overexpressing breast tumor cells.

Electron paramagnetic resonance (EPR) imaging is an emerging modality that can detect and localize paramagnetic molecular probes (so-called spin probes) in vivo. We previously demonstrated that nitroxide spin probes can be encapsulated in liposomes at concentrations exceeding 100 mM, at which nitroxides exhibit a concentration-dependent quenching of their EPR signal that is analogous to the self-quenching of fluorescent molecules. Therefore, intact liposomes encapsulating high concentrations of nitroxides exhibit greatly attenuated EPR spectral signals, and endocytosis of such liposomes represents a cell-activated contrast-generating mechanism. After endocytosis, the encapsulated nitroxide is liberated and becomes greatly diluted in the intracellular milieu. This dequenches the nitroxides to generate a robust intracellular EPR signal. It is therefore possible to deliver a high concentration of nitroxides to cells while minimizing background signal from unendocytosed liposomes. We report here that intracellular EPR signal can be selectively generated in a specific cell type by exploiting its expression of Human Epidermal Growth Factor Receptor 2 (HER2). When targeted by anti-HER2 immunoliposomes encapsulating quenched nitroxides, Hc7 cells, which are novel HER2-overexpressing cells derived from the MCF7 breast tumor cell line, endocytose the liposomes copiously, in contrast to the parent MCF7 cells or control CV1 cells, which do not express HER2. HER2-dependent liposomal delivery enables Hc7 cells to accumulate 750 µM nitroxide intracellularly. Through the use of phantom models, we verify that this concentration of nitroxides is more than sufficient for EPR imaging, thus laying the foundation for using EPR imaging to visualize HER2-overexpressing Hc7 tumors in animals.

Cellular uptake of electron paramagnetic resonance imaging probes through endocytosis of liposomes.

Electron paramagnetic resonance imaging (EPRI) allows detection and localization of paramagnetic spin probes in vivo and in real time. We have shown that nitroxide spin probes entrapped in the intracellular milieu can be imaged by EPRI. Therefore, with the development of a tumor-targetable vehicle that can efficiently deliver nitroxides into cells, it should be possible to use nitroxide spin probes to label and image cells in a tumor. In this study, we assess the potential of liposomes as a delivery vehicle for imaging probes. We demonstrate that liposomes can stably encapsulate nitroxides at very high concentrations (>100 mM), at which nitroxides exhibit concentration-dependent quenching of their EPR signal-a process analogous to the quenching of fluorescent molecules. The encapsulating liposomes thus appear spectroscopically "dark". When the liposomes are endocytosed and degraded by cells, the encapsulated nitroxides are liberated and diluted into the much larger intracellular volume. The consequent relief of quenching generates a robust intracellular nitroxide signal that can be imaged. We show that through endocytosis of nitroxide-loaded liposomes, CV1 cells can achieve intracellular nitroxide concentrations of approximately 1 mM. By using tissue phantom models, we verify that this concentration is more than sufficient for in vivo EPR imaging.

Electron paramagnetic resonance oxygen image hypoxic fraction plus radiation dose strongly correlates with tumor cure in FSa fibrosarcomas.

PURPOSE: Tumor hypoxia has long been known to produce resistance to radiation. In this study, electron paramagnetic resonance (EPR) oxygen imaging was investigated for its power to predict the success of tumor control according to tumor oxygenation level and radiation dose. METHODS AND MATERIALS: A total of 34 EPR oxygen images were obtained from the legs of C3H mice bearing 0.5-cm(3) FSa fibrosarcomas under both normal (air breathing) and clamped tumor conditions. Under the same conditions as those during which the images were obtained, the tumors were irradiated to a variety of doses near the FSa dose at which 50% of tumors were cured. Tumor tissue was distinguished from normal tissue using co-registration of the EPR oxygen images with spin-echo magnetic resonance imaging of the tumor and/or stereotactic localization. The tumor voxel statistics in the EPR oxygen image included the mean and median partial pressure of oxygen and the fraction of tumor voxels below the specified partial pressure of oxygen values of 3, 6, and 10 mm Hg. Bivariate logistic regression analysis using the radiation dose and each of the EPR oxygen image statistics to determine which best separated treatment failure from success. RESULTS: The measurements of the dose at which 50% of tumors were cured were similar to those found in published data for this syngeneic tumor. Bivariate analysis of 34 tumors demonstrated that tumor cure correlated with dose (p = 0.004) and with a <10 mm Hg hypoxic fraction (p = 0.023). CONCLUSION: Our results have shown that, together, radiation dose and EPR image hypoxic fraction separate the population of FSa fibrosarcomas that are cured from those that fail, thus predicting curability.

Very-low-frequency electron paramagnetic resonance (EPR) imaging of nitroxide-loaded cells.

Recent advances in electron paramagnetic resonance (EPR) imaging have made it possible to image, in real time in vivo, cells that have been labeled with nitroxide spin probes. We previously reported that cells can be loaded to high (millimolar) intracellular concentrations with (2,2,5,5-tetramethylpyrrolidin-1-oxyl-3-ylmethyl)amine-N,N-diacetic acid by incubation with the corresponding acetoxymethyl (AM) ester. Furthermore, the intracellular lifetime (t(1/e)) of this nitroxide is 114 min-sufficiently long to permit in vivo imaging studies. In the present study, at a gradient of approximately 50 mT/m, we acquire and compare EPR images of a three-tube phantom, filled with either a 200-microM solution of the nitroxide, or a suspension of cells preincubated with the nitroxide AM ester. In both cases, 3-mm resolution images can be acquired with excellent signal-to-noise ratios (SNRs). These findings indicate that cells well-loaded with nitroxide are readily imageable by EPR imaging, and that in vivo tracking studies utilizing such cells should be feasible.