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In this paper, we describe an assay to analyze simultaneously the oxygen consumption rate (OCR) and superoxide production in a biological system. The analytical set-up uses electron paramagnetic resonance (EPR) spectroscopy with two different isotopically-labelled sensors: 15N-PDT (4-oxo-2,2,6,6-tetramethylpiperidine-d16-15N-1-oxyl) as oxygen-sensing probe and 14N-CMH (1-hydroxy-3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine, a cyclic hydroxylamine, as sensor of reactive oxygen species (ROS). The superoxide contribution to CMH oxidation is assessed using SOD or PEGSOD as controls. Because the EPR spectra are not superimposable, the variation of EPR linewidth of 15N-PDT (linked to OCR) and the formation of the nitroxide from 14N-CMH (linked to superoxide production) can be recorded simultaneously over time on a single preparation. The EPR toolbox was qualified in biological systems of increasing complexity. First, we used an enzymatic assay based on the hypoxanthine (HX)/xanthine oxidase (XO) which is a well described model of oxygen consumption and superoxide production. Second, we used a cellular model of superoxide production using macrophages exposed to phorbol 12-myristate 13-acetate (PMA) which stimulates the NADPH oxidase (NOX) to consume oxygen and produce superoxide. Finally, we exposed isolated mitochondria to established inhibitors of the electron transport chain (rotenone and metformin) in order to assess their impact on OCR and superoxide production. This EPR toolbox has the potential to screen the effect of intoxicants or drugs targeting the mitochondrial function., Graphical abstract Image 1, Highlights • OCR and superoxide production are crucial to assess mitochondrial (dys)function. • The EPR toolbox analyzes simultaneously the OCR and superoxide production. • The EPR toolbox was validated in enzymatic system, cells and isolated mitochondria. • The EPR toolbox has the potential to screen compounds altering mitochondrial function.

Hypoxia is a crucial factor in cancer therapy, determining prognosis and the effectiveness of treatment. Although efforts are being made to develop methods for assessing tumor hypoxia, no markers of hypoxia are currently used in routine clinical practice. Recently, we showed that the combined endogenous MR biomarkers, R1 and R2*, which are sensitive to [dissolved O2] and [dHb], respectively, were able to detect changes in tumor oxygenation induced by a hyperoxic breathing challenge. In this study, we further validated the ability of the combined MR biomarkers to assess the change in tumor oxygenation induced by an allosteric effector of hemoglobin, myo‐inositol trispyrophosphate (ITPP), on rat tumor models. ITPP induced an increase in tumor pO2, as observed using L‐band electron paramagnetic resonance oximetry, as well as an increase in both R1 and R2* MR parameters. The increase in R1 indicated an increase in [O2], whereas the increase in R2* resulted from an increase in O2 release from blood, inducing an increase in [dHb]. The impact of ITPP was then evaluated on factors that can influence tumor oxygenation, including tumor perfusion, saturation rate of hemoglobin, blood pH and oxygen consumption rate (OCR). ITPP decreased blood [HbO2] and significantly increased blood acidity, which is also a factor that right‐shifts the oxygen dissociation curve. No change in tumor perfusion was observed after ITPP treatment. Interestingly, ITPP decreased OCR in both tumor cell lines. In conclusion, ITPP increased tumor pO2 via a combined mechanism involving a decrease in OCR and an allosteric effect on hemoglobin that was further enhanced by a decrease in blood pH. MR biomarkers could assess the change in tumor oxygenation induced by ITPP. At the intra‐tumoral level, a majority of tumor voxels were responsive to ITPP treatment in both of the models studied., The combined endogenous MR biomarkers, R1 and R2*, arepromising tools for assessing tumor hypoxia. In this study, R1 and R2* successfully tracked the increase in tumor oxygenation induced by an allostericeffector of hemoglobin, ITPP. ITPP causes a combined decrease in the bindingaffinity of Hb‐O2, in blood pH and in the oxygen consumption rate oftumor cells, which translates into a significant increase in R1 and R2*.

In an effort to develop noninvasive in vivo methods for mapping tumor oxygenation, magnetic resonance (MR)-derived parameters are being considered, including global R1, water R1, lipids R1, and R2*. R1 is sensitive to dissolved molecular oxygen, whereas R2* is sensitive to blood oxygenation, detecting changes in dHb. This work compares global R1, water R1, lipids R1, and R2* with pO2 assessed by electron paramagnetic resonance (EPR) oximetry, as potential markers of the outcome of radiation therapy (RT).R1, R2*, and EPR were performed on rhabdomyosarcoma and 9L-glioma tumor models, under air and carbogen breathing conditions (95% O2, 5% CO2). Because the models demonstrated different radiosensitivity properties toward carbogen, a growth delay (GD) assay was performed on the rhabdomyosarcoma model and a tumor control dose 50% (TCD50) was performed on the 9L-glioma model.Magnetic resonance imaging oxygen-sensitive parameters detected the positive changes in oxygenation induced by carbogen within tumors. No consistent correlation was seen throughout the study between MR parameters and pO2. Global and lipids R1 were found to be correlated to pO2 in the rhabdomyosarcoma model, whereas R2* was found to be inversely correlated to pO2 in the 9L-glioma model (P=.05 and .03). Carbogen increased the TCD50 of 9L-glioma but did not increase the GD of rhabdomyosarcoma. Only R2* was predictive (P.05) for the curability of 9L-glioma at 40 Gy, a dose that showed a difference in response to RT between carbogen and air-breathing groups. (18)F-FAZA positron emission tomography imaging has been shown to be a predictive marker under the same conditions.This work illustrates the sensitivity of oxygen-sensitive R1 and R2* parameters to changes in tumor oxygenation. However, R1 parameters showed limitations in terms of predicting the outcome of RT in the tumor models studied, whereas R2* was found to be correlated with the outcome in the responsive model.

Tumour hypoxia is a well‐established factor of resistance in radiation therapy (RT). Myo‐inositol trispyrophosphate (ITPP) is an allosteric effector that reduces the oxygen‐binding affinity of haemoglobin and facilitates the release of oxygen by red blood cells. We investigated herein the oxygenation effect of ITPP in six tumour models and its radiosensitizing effect in two of these models. The evolution of tumour pO2 upon ITPP administration was monitored on six models using 1.2 GHz Electron Paramagnetic Resonance (EPR) oximetry. The effect of ITPP on tumour perfusion was assessed by Hoechst staining and the oxygen consumption rate (OCR) in vitro was measured using 9.5 GHz EPR. The therapeutic effect of ITPP with and without RT was evaluated on rhabdomyosarcoma and 9L‐glioma rat models. ITPP enhanced tumour oxygenation in six models. The administration of 2 g/kg ITPP once daily for 2 days led to a tumour reoxygenation for at least 4 days. ITPP reduced the OCR in six cell lines but had no effect on tumour perfusion when tested on 9L‐gliomas. ITPP plus RT did not improve the outcome in rhabdomyosarcomas. In 9L‐gliomas, some of tumours receiving the combined treatment were cured while other tumours did not benefit from the treatment. ITPP increased oxygenation in six tumour models. A decrease in OCR could contribute to the decrease in tumour hypoxia. The association of RT with ITPP was beneficial for a few 9L‐gliomas but was absent in the rhabdomyosarcomas.

Availability of an innocuous and repeatable technique for monitoring tumor oxygenation throughout therapeutic course should be a key factor for adaptative therapeutic strategies. We previously qualified lipids R1 as a marker of oxygen level on experimental tumor models. The objectives of the present study were to assess the applicability of measuring lipids R1 in primary central nervous system malignancies in a clinical setting as well as to compare lipids R1 with global (water+lipids) R1 and R2* which are also sensitive to the oxygen environment. 25 patients with brain neuroepithelial tumors were examined on a clinical 3T MR system. Values obtained within regions of interest contouring contrast-enhanced tumor (C+), unenhanced tumor (C-), peritumoral edema, and normal appearing white matter (NAWM) were compared to those obtained for the normal brain parenchyma of 17 healthy volunteers. Global R1 and lipids R1 values were significantly lower in tumors than in NAWM of patients or healthy brain of normal volunteers. In contrast, R2* values were not significantly different in tumors compared to NAWM or healthy brains. None of them showed significant difference between C+ and C- tumors. Global R1 values within NAWM were significantly different from that of both tumor and peritumoral edema, but lacked sensitivity to differentiate between tumor and peritumoral edema. In turn, lipids R1 measurements enabled discrimination between tumor areas and peritumoral edema. In conclusion, global R1 and lipids R1 deserve further attention as potential markers of tumor hypoxia in primary brain tumors.

Lactate exchange between glycolytic and oxidative cancer cells is proposed to optimize tumor growth. Blocking lactate uptake through monocarboxylate transporter 1 (MCT1) represents an attractive therapeutic strategy but may stimulate glucose consumption by oxidative cancer cells. We report here that inhibition of mitochondrial pyruvate carrier (MPC) activity fulfils the tasks of blocking lactate use while preventing glucose oxidative metabolism. Using in vitro 13C-glucose and in vivo hyperpolarized 13C-pyruvate, we identify 7ACC2 as a potent inhibitor of mitochondrial pyruvate transport which consecutively blocks extracellular lactate uptake by promoting intracellular pyruvate accumulation. Also, while in spheroids MCT1 inhibition leads to cytostatic effects, MPC activity inhibition induces cytotoxic effects together with glycolysis stimulation and uncompensated inhibition of mitochondrial respiration. Hypoxia reduction obtained with 7ACC2 is further shown to sensitize tumor xenografts to radiotherapy. This study positions MPC as a control point for lactate metabolism and expands on the anticancer potential of MPC inhibition., Tumor cells can fuel their metabolism with lactate. Here the authors show that inhibition of mitochondrial pyruvate carrier (MPC) blocks extracellular lactate uptake by promoting intracellular pyruvate accumulation and inhibits oxidative metabolism, ultimately resulting in cytotoxicity and radiosensitization.

PURPOSE: To benchmark MOBILE (Mapping of Oxygen By Imaging Lipid relaxation Enhancement), a recent noninvasive MR method of mapping changes in tumor hypoxia, electron paramagnetic resonance (EPR) oximetry, and dynamic contrast-enhanced MRI (DCE-MRI) as biomarkers of changes in tumor hemodynamics induced by the antivascular agent combretastatin A4 (CA4). METHODS: NT2 and MDA-MB-231 mammary tumors were implanted subcutaneously in FVB/N and nude NMRI mice. Mice received 100 mg/kg of CA4 intraperitoneally 3 hr before imaging. The MOBILE sequence (assessing R1 of lipids) and the DCE sequence (assessing Ktrans hemodynamic parameter), were assessed on different cohorts. pO2 changes were confirmed on matching tumors using EPR oximetry consecutive to the MOBILE sequence. Changes in tumor vasculature were assessed using immunohistology consecutive to DCE-MRI studies. RESULTS: Administration of CA4 induced a significant decrease in lipids R1 (P = 0.0273) on pooled tumor models and a reduction in tumor pO2 measured by EPR oximetry. DCE-MRI also exhibited a significant drop of Ktrans (P

Tumor hypoxia has long been considered as a detrimental factor for the response to irradiation. In order to improve the sensitivity of tumors cells to radiation therapy, tumor hypoxia may theoretically be alleviated by increasing the oxygen delivery or by decreasing the oxygen consumption by tumor cells. Mathematical modelling suggested that decreasing the oxygen consumption should be more efficient than increasing oxygen delivery in order to alleviate tumor hypoxia. In this paper, we review several promising strategies targeting the mitochondrial respiration for which alleviation of tumor hypoxia and increase in sensitivity to irradiation have been demonstrated. Because the translation of these approaches into the clinical arena requires the use of pharmacodynamics biomarkers able to identify shift in oxygen consumption and tumor oxygenation, we also discuss the relative merits of imaging biomarkers (Positron Emission Tomography and Magnetic Resonance) that may be used for therapeutic guidance. This article is part of a Special Issue entitled Mitochondria in Cancer, edited by Giuseppe Gasparre, Rodrigue Rossignol and Pierre Sonveaux.

Purpose: Because of its paramagnetic properties, oxygen may act as an endogenous magnetic resonance imaging contrast agent by changing proton relaxation rates. Changes in tissue oxygen concentrations have been shown to produce changes in relaxation rate R1 of water. The aim of the study was to improve the sensitivity of oxygen enhanced R1 imaging by exploiting the higher solubility of oxygen in lipids (as compared with water) to sensitively monitor changes in tissue oxygen levels by selectively measuring the R1 of lipids. Methods: The method, with the acronym ‘‘MOBILE’’ (mapping of oxygen by imaging lipids relaxation enhancement), was applied in different mouse models of hypoxic processes on a 11.7 T magnetic resonance imaging system. MOBILE was compared with R*2, R1 of water, and with pO2 measurements (using electron paramagnetic resonance oximetry). MOBILE was also applied in the brain of healthy human volunteers exposed to an oxygen breathing challenge on a 3 T magnetic resonance imaging system. Results: MOBILE was shown to be able to monitor changes in oxygenation in tumor, peripheral, liver, and brain tissues. The clinical translation was demonstrated in human volunteers. Conclusion: MOBILE arises as a promising noninvasive and sensitive tool for diagnosis and therapeutic guidance in disorders involving hypoxia. Magn Reson Med 70:732–744, 2013. V C 2012 Wiley Periodicals, Inc.

Hexafluorobenzene (HFB) and perfluoro-15-crown-5-ether (15C5) were compared as fluorine reporter probes of tissue oxygenation using (19)F MRI for dynamic assessment of muscle oxygenation, with special focus on muscle tissue toxicity of the probes, and consecutive alteration of animal behavior. The latter were also compared in terms of sensitivity to changes in oxygenation as well as of signal-to-noise ratio for accurate pO(2) measurements. For that purpose, mouse muscles were imaged at 11.7 T, at 2- and 36-h after intramuscular injection of HFB or 15C5. Histological analysis of the muscle tissue revealed a lack of toxicity for 15C5 from 2 up to 36-h postinjection, whereas HFB induced tissue necrosis, blood clots and thrombosis as soon as 24-h postinjection. This muscle toxicity led to a limitation in mice mobility 24-h after injection of HFB as evidenced by behavioral testing (open-field, grip strength, and catwalk tests), which was not the case after 15C5 intramuscular injection. Finally, pO(2) measurements assessed 2-h postinjection showed consistent values with both probes, evidencing cross-validation of the (19)F MRI oximetry technique for acute measurements. However, the measurement at 36-h was hampered for HFB, which showed significant lower values of muscle pO(2), whereas 15C5 was able to reliably assess muscle pO(2) at 36-h postinjection.

The study of magnetic resonance (MR) markers over the past decade has provided evidence that the tumor microenvironnement and hemodynamics play a major role in determining tumor response to therapy. The aim of the present work is to predict and monitor the efficacy of co-treatments to radio- and chemotherapy by noninvasive MR imaging. Ten different co-treatments were involved in this retrospective analysis of our previously published data, including NO-mediated co-treatments (insulin and isosorbide dinitrate), anti-inflammatory drugs (hydrocortisone, NS-398), anti-angiogenic agents (thalidomide, SU5416 and ZD6474), a vasoactive agent (xanthinol nicotinate), botulinum toxin and carbogen breathing. Dynamic contrast enhanced (DCE) MRI, intrinsic susceptibility-weighted (BOLD) MRI and electronic paramagnetic resonance (EPR) oximetry all reflect tumor microenvironment hemodynamic variables that are known to influence tumor response. Eight MR-derived parameters (markers) were tested for their ability to predict therapeutic outcome (factor of increase in regrowth delay) in experimental tumor models (TLT and FSaII) after radiation therapy and/or chemotherapy with cyclophosphamide, namely tumor pO(2) and O(2) consumption rate (using EPR oximetry); tumor blood flow and permeability, i.e. V (p), K (trans), K (ep) and percentage of perfused vessels (using DCE-MRI); and BOLD signal intensity and R (2)* (using functional MRI). This multi-modal comparison of co-treatment efficacy points out the limitations of each MR marker and identifies in vivo pO(2) as a relevant endpoint for radiation therapy. DCE parameters (V (p) and K (ep)) were identified as a relevant endpoints for cyclophosphamide chemotherapy in our tumor models. This study helps qualify relevant imaging endpoints in the preclinical setting of cancer therapy. Copyright (c) 2010 John Wiley & Sons, Ltd.

Recently, we have developed a new electron paramagnetic resonance (EPR) protocol in order to estimate tissue oxygen consumption in vivo. Because it is crucial to probe the heterogeneity of response in tumors, the aim of this study was to apply our protocol, together with (19)F MRI relaxometry, to the mapping of the oxygen consumption in tumors. The protocol includes the continuous measurement of tumor pO(2) during the following respiratory challenge: (i) basal values during air breathing; (ii) increasing pO(2) values during carbogen breathing until saturation of tissue with oxygen; (iii) switching back to air breathing. We have demonstrated previously using EPR oximetry that the kinetics of return to the basal value after oxygen saturation are mainly governed by tissue oxygen consumption. This challenge was applied in hyperthyroid mice (generated by chronic treatment with L-thyroxine) and control mice, as hyperthyroidism is known to dramatically affect the oxygen consumption rate of tumor cells. Our recently developed snapshot inversion recovery MRI fluorocarbon oximetry technique allowed the pO(2) return kinetics to be measured with a high temporal resolution. The kinetic constants (i.e. oxygen consumption rates) were higher for tumors from hyperthyroid mice than from control mice, data that are consistent with our previous EPR study. The corresponding histograms of the (19)F MRI data showed that the kinetic constants displayed a shift to the right for the hyperthyroid group, indicating a higher oxygen consumption in these tumors. The color maps showed a large heterogeneity in terms of oxygen consumption rate within a tumor. In conclusion, (19)F MRI relaxometry allows the noninvasive mapping of the oxygen consumption in tumors. The ability to assess the heterogeneity of tumor response is critical in order to identify potential tumor regions that might be resistant to treatment and therefore produce a poor response to therapy. Copyright © 2010 John Wiley & Sons, Ltd.

Islet encapsulation requires several properties including (1) biocompatibility, (2) immunoprotection, and (3) oxygen diffusion for islet survival and diabetes correction. New chemical alginates were tested in vivo and compared with traditional high-mannuronate and -guluronate alginates. New alginates with coupled peptide sequence (sterile lyophilized high mannuronate [SLM]-RGD3% and sterile lyophilized high guluronate [SLG]-RGD3%), to improve encapsulated cell adherence in the matrix, and alginates with a very low viscosity (VLDM7% and VLDG7%), to reduce implant size by loading a higher number of islets per volume of polymer, were implanted subcutaneously in 70 Wistar rats for comparison with alginates of high viscosity and high content of mannuronic (SLM3%) or guluronic acids (SLG3%). Permeability of alginates to 36-, 75-, and 150-kDa lectins coupled to fluorescein isothiocynate was quantified before implantation and at 2, 4, and 12 weeks after implantation. Biocompatibility (fibrosis, graft stability, immunologic infiltration by CD3/CD68 cells, and neovascularization) was assessed at each explantation time. Permeability to small molecules was found for all alginates. Impermeability to 150-kDa molecules, such as IgG, was observed only for SLM3% before implantation and was maintained up to 12 weeks after implantation. SLM3% and SLG3% demonstrated better graft stability with lower CD3/CD68 recruitment and fibrosis than the other alginates. SLM3% induced a significantly higher angiogenesis and maintained oxygen pressure at approximately 40 mm Hg for up to 4 weeks after implantation as measured by in vivo electronic paramagnetic resonance oximetry. SLM-encapsulated pig islets implanted subcutaneously in rats demonstrated no inflammatory/immunologic reactions and islets functioned for up to 60 days without immunosuppression. A traditional alginate made of high mannuronic content (SLM3%) is an adapted material to immunoprotect islets in subcutaneous tissue. No improvement was found with lower viscosity and use of GRGDSP-peptide sequence.

The oxygen consumption rate of tumor cells affects tumor oxygenation and response to therapies. Highly sensitive methods for determining cellular oxygen consumption are, therefore, needed to identify treatments that can modulate this parameter. We compared the performances of three different methods for measuring cellular oxygen consumption: electron paramagnetic resonance (EPR) oximetry, the Clark electrode, and the MitoXpress fluorescent assay. To compare the assays, we used K562 cells in the presence of rotenone and hydrocortisone, compounds that are known to inhibit the mitochondrial electron transport chain to different extents. The EPR method was the only one that could identify both rotenone and hydrocortisone as inhibitors of tumor cell oxygen consumption. The Clark electrode and the fluorescence assay demonstrated a significant decrease in cellular oxygen consumption after administration of the most potent inhibitor (rotenone) but failed to show any significant effect of hydrocortisone. EPR oximetry is, therefore, the most sensitive method for identifying inhibitors of oxygen consumption on cell assays, whereas the Clark electrode offers the unique opportunity to add external compounds during experiments and still shows great sensitivity in studying enzyme and chemical reactions that consume oxygen (non-cell assays). Finally, the MitoXpress fluorescent assay has the advantage of a high-sample throughput and low bulk requirements but at the cost of a lower sensitivity.

Hypoxia and blood flow heterogeneities are characteristics of solid tumours and are major obstacles for therapy. Exploiting the biology of nitric oxide (NO), a small radical with multiple functions, is particularly attractive to circumvent these sources of resistance and to sensitise tumour to cytotoxic treatments such as radiotherapy and chemotherapy. Indeed, while NO mediates angiogenic effects, NO may also promote tumour perfusion, drug delivery and oxygenation. Different strategies to deliver NO to tumours and pertaining to the FECS-EJC award laureate's work are reviewed, with a focus on their therapeutic potential. The development of techniques to monitor how and to which extent NO delivery influences the phenotype of a given tumour in a given patient is also discussed.

The aim of this study was to develop an MRI fluorocarbon oximetry technique using snapshot inversion recovery and compare it with fluorescence quenching fiber-optic probe oximetry (OxyLite) performed simultaneously in experimental mouse tumors. The oxygen reporter probe hexafluorobenzene (HFB) was injected directly into the tumors, along with the insertion of the OxyLite probe. Tumor oxygenation (pO(2)) was modified using carbogen or lethal doses of the anesthetic gas. MRI pO(2) maps were generated in 1.5 min with an in-plane spatial resolution of 1.88 mm. MRI and OxyLite showed consistent baseline and postmortem pO(2) values. Increases in tumor pO(2) during carbogen breathing showed similar kinetics for the two methods. The pO(2) values observed using the OxyLite corresponded with relatively hypoxic values observed by MRI. The apparent discrepancy between mean values might be due to the difference in sampling volumes of the techniques and the observation of multiple locations using (19)F MRI versus a single location using the large optical fiber. Overall, the present method provides a rapid way to map the tumor oxygenation and is particularly suitable to monitor acute changes of pO(2) in tumors. Magn Reson Med, 2009. (c) 2008 Wiley-Liss, Inc.

The study of alterations of tumor metabolism should allow the identification of new targets for innovative anticancer strategies. Metabolic alterations are generally established in vitro, and conclusions are often extrapolated to the in vivo situation without further tumor metabolic phenotyping. To highlight the key role of microenvironment on tumor metabolism, we studied the response of glycolytic and oxidative tumor models to metabolic modulations in vitro and in vivo. MDA-MB-231 and SiHa tumor models, characterized in vitro as glycolytic and oxidative, respectively, were studied. Theoretically, when passing from a hypoxic state to an oxygenated state, a Warburg phenotype should conserve a glycolytic metabolism, whereas an oxidative phenotype should switch from glycolytic to oxidative metabolism (Pasteur effect). This challenge was applied in vitro and in vivo to evaluate the impact of different oxic conditions on glucose metabolism. 18F-fluorodeoxyglucose uptake, lactate production, tumor oxygenation, and metabolic fluxes were monitored in vivo using positron emission tomography, microdialysis, electron paramagnetic resonance imaging, and 13C-hyperpolarizated magnetic resonance spectroscopy, respectively. In vitro, MDA-MB-231 cells were glycolytic under both hypoxic and oxygenated conditions, whereas SiHa cells underwent a metabolic shift after reoxygenation. On the contrary, in vivo, the increase in tumor oxygenation (induced by carbogen challenge) led to a similar metabolic shift in glucose metabolism in both tumor models. The major discordance in metabolic patterns observed in vitro and in vivo highlights that any extrapolation of in vitro metabolic profiling to the in vivo situation should be taken cautiously and that metabolic phenotyping using molecular imaging is mandatory in vivo.

Purpose: We hypothesized that glucocorticoids may enhance tumor radiosensitivity by increasing tumor oxygenation (pO2) through inhibition of mitochondrial respiration.Experimental Design: The effect of three glucocorticoids (hydrocortisone, dexamethasone, and prednisolone) on pO2 was studied in murine TLT liver tumors and FSaII fibrosarcomas. At the time of maximum pO2 (tmax, 30 min after administration), perfusion, oxygen consumption, and radiation sensitivity were studied. Local pO2 measurements were done using electron paramagnetic resonance. The oxygen consumption rate of tumor cells after in vivo glucocorticoid administration was measured using high-frequency electron paramagnetic resonance. Tumor perfusion and permeability measurements were assessed by dynamic contrast-enhanced magnetic resonance imaging.Results: All glucocorticoids tested caused a rapid increase in pO2. At tmax, tumor perfusion decreased, indicating that the increase in pO2 was not caused by an increase in oxygen supply. Also at tmax, global oxygen consumption decreased. When irradiation (25 Gy) was applied at tmax, the tumor radiosensitivity was enhanced (regrowth delay increased by a factor of 1.7).Conclusion: These results show the potential usefulness of the administration of glucocorticoids before irradiation.

Emerging preclinical studies support the concept of a transient “normalization” of tumor vasculature during the early stage of antiangiogenic treatment, with possible beneficial effects on associated radiotherapy or chemotherapy. One key issue in this area of research is to determine whether this feature is common to all antiangiogenic drugs and whether the phenomenon occurs in all types of tumors. In the present study, we characterized the evolution of the tumor oxygenation (in transplantable liver tumor and FSAII tumor models) after administration of SU5416, an antagonist of the vascular endothelial growth factor receptor. SU5416 induced an early increase in tumor oxygenation [measured by electronic paramagnetic resonance (EPR)], which did not correlate with remodeling of the tumor vasculature (assessed by CD31 labeling using immunohistochemistry) or with tumor perfusion (measured by dynamic contrast enhanced-magnetic resonance imaging). Inhibition of mitochondrial respiration (measured by EPR) was responsible for this early reoxygenation. Consistent with these unique findings in the tumor microenvironment, we found that SU5416 potentiated tumor response to radiotherapy but not to chemotherapy. In addition to the fact that the characterization of the tumor oxygenation is essential to enable correct application of combined therapies, our results show that the long-term inhibition of oxygen consumption is a potential novel target in this class of compounds. (Cancer Res 2006; 66(19): 9698-704)

We hypothesized that nonsteroidal antiinflammatory drugs (NSAIDs) might enhance tumor radiosensitivity by increasing tumor oxygenation (pO2), via either a decrease in the recruitment of macrophages or from inhibition of mitochondrial respiration. The effect of four NSAIDs (diclofenac, indomethacin, piroxicam, and NS-398) on pO2 was studied in murine TLT liver tumors and FSaII fibrosarcomas. At the time of maximum pO2 (tmax, 30 minutes after administration), perfusion, oxygen consumption, and radiation sensitivity were studied. Local pO2 measurements were done using electron paramagnetic resonance. Tumor perfusion and permeability measurements were assessed by dynamic contrast-enhanced magnetic resonance imaging. The oxygen consumption rate of tumor cells after in vivo NSAID administration was measured using high-frequency electron paramagnetic resonance. Tumor-infiltrating macrophage localization was done with immunohistochemistry using CD11b antibody. All the NSAIDs tested caused a rapid increase in pO2. At tmax, tumor perfusion decreased, indicating that the increase in pO2 was not caused by an increase in oxygen supply. Also at tmax, global oxygen consumption decreased but the amount of tumor-infiltrating macrophages remained unchanged. Our study strongly indicates that the oxygen effect caused by NSAIDs is primarily mediated by an effect on mitochondrial respiration. When irradiation (18 Gy) was applied at tmax, the tumor radiosensitivity was enhanced (regrowth delay increased by a factor of 1.7). These results show the potential utility of an acute administration of NSAIDs for radiosensitizing tumors, and shed new light on the mechanisms of NSAID radiosensitization. These results also provide a new rationale for the treatment schedule when combining NSAIDs and radiotherapy.

The potential physiological and therapeutic applications of functional MRI (fMRI) in skeletal muscle will depend on our ability to identify factors that may contribute to fluctuations in the BOLD signal. Until now, interpretations of signal changes in fMRI studies of muscle have mostly relied on the increase in muscle T2 associated with osmotically driven fluid shifts. However, recent studies have documented increases in BOLD signal intensity (SI) after single contractions, coinciding with increases in muscle hemoglobin saturation. In this study, the factors that contribute to variations in the intensity of the BOLD signal in exercising muscle are further addressed. For this purpose, BOLD imaging was performed during and after a moderate electrical stimulation was applied to the sciatic nerve in mice. In addition, oxygen pressure (pO2), blood flow, and skeletal muscle T2 (fast and slow components: T2 and T'2, respectively) were monitored. A comparison between mice lacking eNOS (eNOS-/- mice) and their wild-type (WT) littermates was performed. In WT mice, the BOLD SI, as well as muscle oxygenation and T'2, were significantly increased for a prolonged time in response to this moderate exercise protocol. Blood flow immediately dropped after the electrical stimulation was stopped. In eNOS-/- mice, the high BOLD SI did not persist after the exercise protocol ended. This finding correlates well with the evolution of muscle oxygenation, which progressively decreases after stimulation in eNOS-/- mice. However, T'2 remained high for a prolonged time after stimulation. We therefore concluded that the maintenance of BOLD SI in moderately exercising skeletal muscle depends mainly on changes in pO2, rather than on blood flow or T2 effects.

We investigated the impact of oxygenation status (measured by EPR oximetry) on the uptake of (18)F-FDG (measured by PET) in two different tumor models during a carbogen breathing challenge. We observed a significant drop in (18)F-FDG uptake under carbogen breathing that suggests a rapid metabolic adaptation to the oxygen environment.

The clinical applicability of brain oxygenation mapping using the MOBILE (Mapping of Oxygen By Imaging Lipids relaxation Enhancement) magnetic resonance (MR) technique was assessed in the clinical setting of normal brain and of acute cerebral ischemia as a founding proof-of-concept translational study. Changes in the oxygenation level within healthy brain tissue can be detected by analyzing the spin-lattice proton relaxation (‘Global T 1 ’ combining water and lipid protons) because of the paramagnetic properties of molecular oxygen. It was hypothesized that selective measurement of the relaxation of the lipid protons (‘Lipids T 1 ’) would result in enhanced sensitivity of pO2 mapping because of higher solubility of oxygen in lipids than in water, and this was demonstrated in pre-clinical models using the MOBILE technique. In the present study, 12 healthy volunteers and eight patients with acute (48–72 hours) brain infarction were examined with the same clinical 3T MR system. Both Lipids R1 (R1 = 1/T1) and Global R1 were significantly different in the infarcted area and the contralateral unaffected brain tissue, with a higher statistical significance for Lipids R1 (median difference: 0.408 s-1; p

Monitoring of oxygenation in tumours is an important issue in predicting the success of anti-cancer treatments such as radiotherapy. Gradient echo (GE) imaging sequences can be used for monitoring changes in tumour blood flow and oxygenation. However, the application of this method in head and neck tumours is hampered by significant artefacts and losses of the MR signal near air-tissue interfaces. We investigated the usefulness of a gradient-echo slice excitation profile (GESEPI) sequence that should keep the oxygen contrast while recovering the signal loss caused by susceptibility artefacts. A tumour model was implanted in the neck and in the leg of mice. MR imaging was performed at 4.7 T. GE and GESEPI sequences were used for monitoring the blood oxygen level dependent (BOLD) contrast after carbogen breathing. The pO2 was also monitored in tumours using an OxyLite probe (Oxford Optronics). Using the tumours implanted in the leg, we found that the variations of signal intensity after carbogen breathing were similarin both sequences. In the tumour implanted in the neck, it was possible, using GESEPI sequences, to recover the signal loss caused by susceptibility artefacts and to monitor the effect of carbogen-induced changes in the tumour.

Hypoxia is a common feature of solid tumors, which translates into increased angiogenesis, malignant phenotype cell selection, change in gene expression and greater resistance to radiotherapy and chemotherapy. Therefore, there is a need for markers of hypoxia to stratify patients, in order to personalize treatment to improve therapeutic outcome. However, no modality has yet been validated for the screening of hypoxia in routine clinical practice. Magnetic resonance imaging (MRI) R1 and R2 * relaxation parameters are sensitive to tissue oxygenation: R1 is sensitive to dissolved oxygen and R2 * is sensitive to intravascular deoxyhemoglobin content. Two rat tumor models with distinct levels of hypoxia, 9L-glioma and rhabdomyosarcoma, were imaged for R1 and R2 * under air and carbogen (95% O2 and 5% CO2 ) breathing conditions. It was observed that the basal tumor oxygenation level had an impact on the amplitude of response to carbogen in the vascular compartment (R2 *), but not in the tissue compartment (R1 ). In addition, the change in tissue oxygenation estimated by ΔR1 correlated with the change in vascular oxygenation estimated by ΔR2 *, which is consistent with an increase in oxygen supply generating an elevated tumor pO2 . At the intra-tumoral level, we identified four types of voxel to which a hypoxic feature was attributed (mild hypoxia, severe hypoxia, normoxia and vascular steal), depending on the carbogen-induced change in R1 and R2 * values for each voxel. The results showed that 9L-gliomas present more normoxic fractions, whereas rhabdomyosarcomas present more hypoxic fractions, which is in accordance with a previous study using 18 F-fluoroazomycin arabinoside (18 F-FAZA) and electron paramagnetic resonance (EPR) oximetry. The response of the combined endogenous MRI contrasts to carbogen challenge could be a useful tool to predict different tumor hypoxic fractions.

Although oxygen consumption is a key factor in metabolic phenotyping, its assessment in tumors remains critical, as current technologies generally display poor specificity. The objectives of this study were to explore the feasibility of direct (17) O nuclear magnetic resonance (NMR) spectroscopy to assess oxygen metabolism in tumors and its modulations. To investigate the impact of hypometabolism induction in the murine fibrosarcoma FSAII tumor model, we monitored the oxygen consumption of normothermic (37°C) and hypothermic (32°C) tumor-bearing mice. Hypothermic animals showed an increase in tumor pO2 (measured by electron paramagnetic resonance oximetry) contrary to normothermic animals. This was related to a decrease in oxygen consumption rate (assessed using (17) O magnetic resonance spectroscopy (MRS) after the inhalation of (17) O2 -enriched gas). This study highlights the ability of direct (17) O MRS to measure oxygen metabolism in tumors and modulations of tumor oxygen consumption rate.

Purpose: Although hypoxia has been long recognized as a crucial factor impairing tumor response in many therapeutic schemes, atraumatic and reliable methods of individually quantifying tumor oxygenation are still lacking in day-to-day clinical practice. The aim of this work was to investigate the potentially quantitative properties of our recently described noninvasive magnetic resonance (MR) technique “MOBILE” (mapping of oxygen by imaging lipids relaxation enhancement) and to qualify this endogenous contrast as a tumor hypoxia marker. Experimental Design: The “MOBILE” technique, which assesses the longitudinal MR relaxation rate, R1, of lipid protons, was benchmarked with the parent technique which assesses the global (or water) R1, in response to a hyperoxic challenge (carbogen breathing) and to a hypoxic challenge (combretastatin A4) in MDA-MB-231 xenografts and in NT2 mammary tumors. Electron paramagnetic resonance (EPR) oximetry was used to quantitatively assess the tumor pO2 in matching tumors longitudinally. Results and Conclusion: Our study evidenced that (i) positive and negative changes in tumor oxygenation can be detected using MOBILE; (ii) a change in the R1 of lipids is positively correlated with a change in the tumor pO2 (P = 0.0217, r = 0.5097); (iii) measured lipid R1 values are positively correlated with absolute pO2 values in both tumor models (P = 0.0275, r = 0.3726); and (iv) changes in the R1 of lipids are more sensitive than changes in the global R1. As this technique presents unique translational properties, it seems promising for the individual longitudinal monitoring of tumor oxygenation in a clinical setting. Clin Cancer Res; 20(21); 5403–11. ©2014 AACR.

EPR oximetry using an implantable paramagnetic probe was used to quantify the partial pressure of oxygen (pO(2)) in tissues in a transplantable mouse tumor model (TLT) after administration of 34 different vasodilators belonging to one of the following classes: angiotensin-converting enzyme inhibitors, calcium antagonists, alpha antagonists, potassium channel openers, beta-blockers, NO donors, and peripheral vasoactive agents. Twenty-four compounds were efficient in significantly increasing the local pO(2) in a majority of tumors. The increase of local pO(2) using pharmacological treatments was lower than that achieved by using oxygen or carbogen breathing. This technique offers an unprecedented tool for rapidly and accurately measuring treatment-induced modifications of pO(2) in tumors. Magn Reson Med 42:627-630, 1999.

It is known that some charcoals possess paramagnetic centers with an electron paramagnetic resonance (EPR) linewidth which can be broadened by oxygen. In order to identify potential candidates as sensors for in vivo EPR oximetry, we carried out a systematic study among commercially available charcoals. A total of 34 charcoals were tested. The steps used for the screening were: (1) to check the presence of paramagnetic centers in the material; (2) to measure the EPR linewidth in nitrogen and in air on the dry material and on a aqueous suspension of particles; (3) to calibrate the oxygen sensitive materials (EPR linewidth vs. pO2); (4) to test the sensitivity and stability of the response to changes of pO2 in a simple model of hypoxia induced in mice. Seventeen charcoals contained paramagnetic centers detectable by low-frequency EPR (1.1 GHz). The EPR spectrum consist of one single line which is typical of carbon-centered radicals (g-factor approximately 2). Eight charcoals presented sufficient interesting EPR properties (linewidth in nitrogen0.1 mT, linewidth in air for an aqueous suspension of particles0.15 mT) to be further characterized in vivo. Only three charcoals presented a stable, reproducible, and sensitive response to pO2 for more than 2 months. These three coals should be considered as good candidates to be used as oxygen sensor using in vivo EPR spectroscopy.

Hypoxia-driven intervention (oxygen manipulation or dose escalation) could overcome radiation resistance linked to tumor hypoxia. Here, we evaluated the value of hypoxia imaging using (18)F-FAZA PET to predict the outcome and guide hypoxia-driven interventions.Two hypoxic rat tumor models were used: rhabdomyosarcoma and 9L-glioma. For the irradiated groups, the animals were divided into two subgroups: breathing either room air or carbogen. (18)F-FAZA PET images were obtained just before the irradiation to monitor the hypoxic level of each tumor. Absolute pO2 were also measured using EPR oximetry. Dose escalation was used in Rhabdomyosarcomas.For 9L-gliomas, a significant correlation between (18)F-FAZA T/B ratio and tumor growth delay was found; additionally, carbogen breathing dramatically improved the tumor response to irradiation. On the contrary, Rhabdomyosarcomas were less responsive to hyperoxic challenge. For that model, an increase in growth delay was observed using dose escalation, but not when combining irradiation with carbogen.(18)F-FAZA uptake may be prognostic of outcome following radiotherapy and could assess the response of tumor to carbogen breathing. (18)F-FAZA PET may help to guide the hypoxia-driven intervention with irradiation: carbogen breathing in responsive tumors or dose escalation in tumors non-responsive to carbogen.

Most solid tumors are characterized by unstable perfusion patterns, creating regions of hypoxia that are detrimental to radiotherapy treatment response. Because postsurgical radiotherapy, alone or in combination with other interventions, is a first-line treatment for many malignancies, strategies aimed at homogeneously increasing tumor pO 2 have been the focus of intense research over the past decades. Among other approaches of demonstrable clinical and preclinical utility, this review focuses on those directly targeting oxygen consumption to redirect oxygen from a metabolic fate to the stabilization of radiation-induced DNA damage, more particularly drugs targeting glucose and lactate metabolism, nitric oxide donors or inducers, and mitogen-activated protein kinase pathway inhibitors. Their utility as adjuvant treatments with radiotherapy has been proven preclinically, which should foster further their clinical development and evaluation.

It is well recognized that tumor hypoxia is a critical determinant for response to therapy. The effect of an EGFR inhibitor/gefitinib (Iressa®) on tumor oxygenation was monitored daily using in vivo EPR (electron paramagnetic resonance) oximetry on TLT and FSaII tumor models. An increase in pO2 was shown at a dose of 45 mg/kg i.p. (n = 4/group/tumor model). This allowed the identification of a window of reoxygenation in both tumor models (with a maximum between 15 and 20 mmHg after 2 days of treatment). The increase in tumor oxygenation was shown to be the result of a decrease in oxygen consumption. This is the first report on the effect of gefitinib on oxygen consumption by tumor cells and subsequent increase in tumor oxygenation in vivo.

The aim of the study was to sensitively monitor changes in tumor oxygen using the MOBILE (mapping of oxygen by imaging lipids relaxation enhancement) technique. This method was applied in mammary tumor mouse models on an 11.7T Bruker MRI system. MOBILE was compared with functional imaging R2*, R1 of water and with pO2 measurements (using EPR oximetry and O2-dependent fluorescence quenching measurements). MOBILE was shown to be capable to monitor changes in oxygenation in tumor tissues.

It is well recognized that tumor hypoxia is a critical determinant for response to therapy. The effect of an EGFR inhibitor/gefitinib (Iressa®) on tumor oxygenation was monitored daily using in vivo EPR (electron paramagnetic resonance) oximetry on TLT and FSaII tumor models. An increase in pO2 was shown at a dose of 45 mg/kg i.p. (n = 4/group/tumor model). This allowed the identification of a window of reoxygenation in both tumor models (with a maximum between 15 and 20 mmHg after 2 days of treatment). The increase in tumor oxygenation was shown to be the result of a decrease in oxygen consumption. This is the first report on the effect of gefitinib on oxygen consumption by tumor cells and subsequent increase in tumor oxygenation in vivo.

The aim of the study was to sensitively monitor changes in tumor oxygen using the MOBILE (mapping of oxygen by imaging lipids relaxation enhancement) technique. This method was applied in mammary tumor mouse models on an 11.7T Bruker MRI system. MOBILE was compared with functional imaging R2*, R1 of water and with pO2 measurements (using EPR oximetry and O2-dependent fluorescence quenching measurements). MOBILE was shown to be capable to monitor changes in oxygenation in tumor tissues.

Because of its paramagnetic properties, oxygen may act as an endogenous magnetic resonance imaging contrast agent by changing proton relaxation rates. Changes in tissue oxygen concentrations have been shown to produce changes in relaxation rate R1 of water. The aim of the study was to improve the sensitivity of oxygen enhanced R1 imaging by exploiting the higher solubility of oxygen in lipids (as compared with water) to sensitively monitor changes in tissue oxygen levels by selectively measuring the R1 of lipids.The method, with the acronym "MOBILE" (mapping of oxygen by imaging lipids relaxation enhancement), was applied in different mouse models of hypoxic processes on a 11.7 T magnetic resonance imaging system. MOBILE was compared with R*2, R1 of water, and with pO2 measurements (using electron paramagnetic resonance oximetry). MOBILE was also applied in the brain of healthy human volunteers exposed to an oxygen breathing challenge on a 3 T magnetic resonance imaging system.MOBILE was shown to be able to monitor changes in oxygenation in tumor, peripheral, liver, and brain tissues. The clinical translation was demonstrated in human volunteers.MOBILE arises as a promising noninvasive and sensitive tool for diagnosis and therapeutic guidance in disorders involving hypoxia.

The ultimate goal of radiotherapy is to induce irreversible damages in genetically unstable, fast-growing cancer cells while minimizing the cytotoxic effects on host tissues. The satus of the tumor vasculature is particular because it is located within the tumor but mostly arises from host cells. The aim of this study was to characterize the effects of low-dose irradiation on the function of endothelial cells lining tumor vessels. Using isolated arterioles mounted on a pressure myograph, we first documented that the nitric oxide (NO)-mediated vasorelaxation that was defective in tumor vessels was completely restored following local tumor irradiation. Immunoblot analyses revealed that this was attributable to an increase in the abundance of the endothelial NO synthase while the expression of its physiological inhibitor, caveolin-1, was reduced. We further showed that the potentiation of the NO-dependent pathway induced a marked increase in tumor blood flow and oxygenation that determined the higher sensitivity of the tumor to further irradiation. Finally, we documented that the NO-mediated effects of irradiation on the tumor vasculature increased the delivery and expression of a reporter gene into the tumor. Thus, low-dose irradiation of endothelial cells within a tumor is a key determinant of the effectiveness of radiotherapy and may offer a new strategy to increase gene and/or drug delivery to the tumor.

Arsenic trioxide (As2O3) is an effective therapeutic against acute promyelocytic leukemia and certain solid tumors. Because As2O3 inhibits mitochondrial respiration in leukemia cells, we hypothesized that As2O3 might enhance the radiosensitivity of solid tumors by increasing tumor oxygenation [partial pressure of oxygen (pO2)] via a decrease in oxygen consumption. Two murine models of radioresistant hypoxic cancer were used to study the effects of As2O3. We measured pO2 and the oxygen consumption rate in vivo by electron paramagnetic resonance oximetry and 19fluorine-MRI relaxometry. Tumor perfusion was assessed by Patent blue staining. In both models, As2O3 inhibited mitochondrial respiration, leading to a rapid increase in pO2. The decrease in oxygen consumption could be explained by an observed decrease in glutathione in As2O3-treated cells, as this could increase intracellular reactive oxygen species that can disrupt mitochondrial membrane potential. When tumors were irradiated during periods of As2O3-induced augmented oxygenation, radiosensitivity increased by 2.2-fold compared with control mice. Notably, this effect was abolished when temporarily clamped tumors were irradiated. Together, our findings show that As2O3 acutely increases oxygen consumption and radiosensitizes tumors, providing a new rationale for clinical investigations of As2O3 in irradiation protocols to treat solid tumors. Cancer Res; 72(2); 482–90. ©2011 AACR.

Recently, we have developed a new electron paramagnetic resonance (EPR) protocol in order to estimate tissue oxygen consumption in vivo. Because it is crucial to probe the heterogeneity of response in tumors, the aim of this study was to apply our protocol, together with (19)F MRI relaxometry, to the mapping of the oxygen consumption in tumors. The protocol includes the continuous measurement of tumor po(2) during the following respiratory challenge: (i) basal values during air breathing; (ii) increasing po(2) values during carbogen breathing until saturation of tissue with oxygen; (iii) switching back to air breathing. We have demonstrated previously using EPR oximetry that the kinetics of return to the basal value after oxygen saturation are mainly governed by tissue oxygen consumption. This challenge was applied in hyperthyroid mice (generated by chronic treatment with L-thyroxine) and control mice, as hyperthyroidism is known to dramatically affect the oxygen consumption rate of tumor cells. Our recently developed snapshot inversion recovery MRI fluorocarbon oximetry technique allowed the po(2) return kinetics to be measured with a high temporal resolution. The kinetic constants (i.e. oxygen consumption rates) were higher for tumors from hyperthyroid mice than from control mice, data that are consistent with our previous EPR study. The corresponding histograms of the (19)F MRI data showed that the kinetic constants displayed a shift to the right for the hyperthyroid group, indicating a higher oxygen consumption in these tumors. The color maps showed a large heterogeneity in terms of oxygen consumption rate within a tumor. In conclusion, (19)F MRI relaxometry allows the noninvasive mapping of the oxygen consumption in tumors. The ability to assess the heterogeneity of tumor response is critical in order to identify potential tumor regions that might be resistant to treatment and therefore produce a poor response to therapy.

The oxygen consumption rate in tumors affects tumor oxygenation and response to therapies. A new EPR method was developed to measure tissue oxygen consumption non-invasively. The protocol was based on the measurement of pO(2) during a carbogen challenge. The following sequence was used: (1) basal value during air breathing; (2) saturation of tissue with oxygen by carbogen breathing; (3) switch back to air breathing. The assumption was that the kinetics of the return to the basal value after oxygen saturation would be governed mainly by tissue oxygen consumption. This challenge was applied in hyperthyroid mice (generated by chronic treatment with l-thyroxine) and control mice because hyperthyroidism is known to dramatically affect the oxygen consumption rate of tumor and muscle cells. Muscle and tumor cells from the hyperthyroid mice consumed oxygen faster than muscle and tumor cells from the control mice, which is consistent with the results of in vitro studies. Tumor perfusion was not affected by the treatment with l-thyroxine. This method gives an index that may reasonably be ascribed to the local oxygen consumption and has the unique advantage of being adaptable to in vivo studies.

SummaryA key function of blood vessels, to supply oxygen, is impaired in tumors because of abnormalities in their endothelial lining. PHD proteins serve as oxygen sensors and may regulate oxygen delivery. We therefore studied the role of endothelial PHD2 in vessel shaping by implanting tumors in PHD2+/− mice. Haplodeficiency of PHD2 did not affect tumor vessel density or lumen size, but normalized the endothelial lining and vessel maturation. This resulted in improved tumor perfusion and oxygenation and inhibited tumor cell invasion, intravasation, and metastasis. Haplodeficiency of PHD2 redirected the specification of endothelial tip cells to a more quiescent cell type, lacking filopodia and arrayed in a phalanx formation. This transition relied on HIF-driven upregulation of (soluble) VEGFR-1 and VE-cadherin. Thus, decreased activity of an oxygen sensor in hypoxic conditions prompts endothelial cells to readjust their shape and phenotype to restore oxygen supply. Inhibition of PHD2 may offer alternative therapeutic opportunities for anticancer therapy.

The aim of this study was to develop an MRI fluorocarbon oximetry technique using snapshot inversion recovery and compare it with fluorescence quenching fiber-optic probe oximetry (OxyLite) performed simultaneously in experimental mouse tumors. The oxygen reporter probe hexafluorobenzene (HFB) was injected directly into the tumors, along with the insertion of the OxyLite probe. Tumor oxygenation (pO(2)) was modified using carbogen or lethal doses of the anesthetic gas. MRI pO(2) maps were generated in 1.5 min with an in-plane spatial resolution of 1.88 mm. MRI and OxyLite showed consistent baseline and postmortem pO(2) values. Increases in tumor pO(2) during carbogen breathing showed similar kinetics for the two methods. The pO(2) values observed using the OxyLite corresponded with relatively hypoxic values observed by MRI. The apparent discrepancy between mean values might be due to the difference in sampling volumes of the techniques and the observation of multiple locations using (19)F MRI versus a single location using the large optical fiber. Overall, the present method provides a rapid way to map the tumor oxygenation and is particularly suitable to monitor acute changes of pO(2) in tumors.

Tumors contain oxygenated and hypoxic regions, so the tumor cell population is heterogeneous. Hypoxic tumor cells primarily use glucose for glycolytic energy production and release lactic acid, creating a lactate gradient that mirrors the oxygen gradient in the tumor. By contrast, oxygenated tumor cells have been thought to primarily use glucose for oxidative energy production. Although lactate is generally considered a waste product, we now show that it is a prominent substrate that fuels the oxidative metabolism of oxygenated tumor cells. There is therefore a symbiosis in which glycolytic and oxidative tumor cells mutually regulate their access to energy metabolites. We identified monocarboxylate transporter 1 (MCT1) as the prominent path for lactate uptake by a human cervix squamous carcinoma cell line that preferentially utilized lactate for oxidative metabolism. Inhibiting MCT1 with alpha-cyano-4-hydroxycinnamate (CHC) or siRNA in these cells induced a switch from lactate-fueled respiration to glycolysis. A similar switch from lactate-fueled respiration to glycolysis by oxygenated tumor cells in both a mouse model of lung carcinoma and xenotransplanted human colorectal adenocarcinoma cells was observed after administration of CHC. This retarded tumor growth, as the hypoxic/glycolytic tumor cells died from glucose starvation, and rendered the remaining cells sensitive to irradiation. As MCT1 was found to be expressed by an array of primary human tumors, we suggest that MCT1 inhibition has clinical antitumor potential.

Insulin and NS-398 have been reported to inhibit oxygen consumption in experimental tumor models, thereby increasing oxygenation and radiosensitization. The aim of this work was to use MRI to study changes in murine FSaII tumor hemodynamics after administration of those oxygen consumption inhibitors. A multiple-echo gradient-echo (GRE) MRI sequence (4.7 T) was used to map changes in three factors: the GRE signal (at TE=20 ms), the parameter S0 (theoretical signal at TE=0 ms), and the relaxation rate R*2. Perfusion maps were obtained by dynamic contrast-enhanced (DCE) MRI. Insulin caused a significant decrease in the tumor blood oxygen level-dependent (BOLD) signal over time. factor This was likely the result of decreased blood flow, since both S0 and the percentage of perfused tumor decreased as well. Tumor R*2 did not change significantly in response to the treatments, which is surprising considering that other non-MRI techniques (electron paramagnetic resonance (EPR) oximetry and fiber-optic probes) have shown that tumor oxygenation increases after treatment. This suggests that metabolic changes associated with vasoactive challenges may have an unpredictable influence on blood saturation and R*2. In conclusion, this study further emphasizes the fact that changes in BOLD signal and R*2 in tumors do not depend uniquely on changes in oxygenation status.

BACKGROUND AND PURPOSE: We have previously reported that insulin significantly enhances tumor oxygenation (pO(2)) and increases radiation-induced tumor regrowth delay in experimental models. Considering the large radiosensitizing effect, clinical trials might be envisioned. The aim of the present pre-clinical study was to obtain a more complete set of safety and efficacy data which would further justify the commencement of such clinical trials. MATERIAL AND METHODS: Toxicity on normal (early and late-responding) tissues was measured by the intestinal crypt regeneration assay and the late leg contracture assay. Efficacy in terms of enhancement of pO(2) (measured by in vivo EPR oximetry) and increase in radiation-induced tumor regrowth delay was evaluated with a dose-response study on mice bearing FSaII fibrosarcoma. RESULTS: The effect on regrowth delay was directly correlated with the effect on the tumor pO(2), with a maximal effect using 400 mU kg(-1) insulin. Importantly, there was no increase in the radiation toxicity for normal tissues. Finally, we found that the hypoglycaemia induced by insulin can be corrected by simultaneous glucose infusion without modification of efficacy. CONCLUSION: Insulin here demonstrated a therapeutic gain and a lack of toxicity to normal tissues. The results of this study fully justify further larger preclinical assays such as the use of fractionated irradiation and a tumor control dose assay, before determining the utility of insulin as a radiosensitizer for human patients in the clinic.

This review paper attempts to provide an overview of the principles and techniques that are often termed electron paramagnetic resonance (EPR) oximetry. The paper discusses the potential of such methods and illustrates they have been successfully applied to measure oxygen tension, an essential parameter of the tumor microenvironment. To help the reader understand the motivation for carrying out these measurements, the importance of tumor hypoxia is first discussed: the basic issues of why a tumor is hypoxic, why these hypoxic microenvironments promote processes driving malignant progression and why hypoxia dramatically influences the response of tumors to cytotoxic treatments will be explained. The different methods that have been used to estimate the oxygenation in tumors will be reviewed. To introduce the basics of EPR oximetry, the specificity of in vivo EPR will be discussed by comparing this technique with NMR and MRI. The different types of paramagnetic oxygen sensors will be presented, as well as the methods for recording the information (EPR spectroscopy, EPR imaging, dynamic nuclear polarization). Several applications of EPR for characterizing tumor oxygenation will be illustrated, with a special emphasis on pharmacological interventions that modulate the tumor microenvironment. Finally, the challenges for transposing the method into the clinic will also be discussed.

Different nitric oxide (NO)-mediated treatments (e.g., isosorbide dinitrate, insulin and electrical stimulation of the host tissue) have been investigated for their effects on tumor oxygenation and radiation sensitivity. We further address the issue of the role played by modulation of the NO-pathway in tumor radiosensitivity. For this purpose, the local concentration of NO was monitored after treatment in FSaII tumors and a comparison between the sensitivity of LLC tumors implanted both on eNOS(-/-) and wild-type (WT) mice was carried out. First, we demonstrate the central role played by eNOS in the radiosensitizing effect after application of insulin treatment and electrical stimulation: a significant increase in tumor NO content is induced by these treatments and the increase in tumor oxygenation, as well as the radiosensitizing effect are abolished in eNOS knock-out mice, in contrast to WT mice. Second, by comparing the level of oxygen and NO achieved in tumors after NO-mediated treatments and carbogen, we provide evidence that these NO-mediated treatments are not simply acting by a single oxygen effect. These treatments induced significant regrowth delays compared to carbogen, despite a smaller increase in tumor oxygenation. For the NO-mediated treatments, there was a direct correlation between the NO content and the radiosensitizing effect. These data strongly suggest that NO is a complementary factor additive to oxygen in determining the sensitivity to irradiation and we therefore propose that NO acts as an intrinsic radiosensitizer in vivo.

Oxygen deficiency in tumors reduces the efficacy of nonsurgical treatment modalities such as conventional radiotherapy and chemotherapy. Since tumor perfusion is directly affected by the vascular resistance to flow of vessels feeding the tumor, vasodilator drugs might be a way to increase tumor blood flow and oxygenation. The effects of nitric oxide (NO) donor administration on tumor oxygenation, perfusion and radiation sensitivity were studied in the FSaII tumor model. Local tumor oxygenation was measured using electron paramagnetic resonance oximetry and a fiberoptic probe, OxyLite. We concomitantly measured the modulation of tumor blood flow by laser Doppler flowmetry. We determined FSaII tumor regrowth delay after isosorbide dinitrate administration and irradiation compared to carbogen breathing before irradiation and with X-rays alone. Administration of the NO donor improved the FSaII tumor pO(2) concomitant with an increase in tumor blood flow. We also demonstrated an increase in FSaII tumor radiation sensitivity after isosorbide dinitrate administration, which was similar to the effect of carbogen breathing in the same tumor model. Administration of isosorbide dinitrate could be considered in terms of improvement in tumor blood flow and a possible concomitant increase in accessibility of chemosensitizing agents to the tumor, particularly in terms of modification of the tumor response to irradiation.

The effects of insulin on tumor oxygenation, perfusion, oxygen consumption,and radiation sensitivity were studied on two different mouse tumor models (TLT, a liver tumor, and FSAII, a fibrosarcoma). Anesthetized mice were infused with insulin i.v. at a rate of 16 milliUnits/kg/min for 25 min. Local tumor oxygenation measurements were carried out using two independent techniques: electron paramagnetic resonance oximetry and a fiber-optic device (OxyLite). Two complementary techniques were also used to assess the blood flow inside the tumor: a laser Doppler system (OxyFlo) and contrast-enhanced magnetic resonance imaging. The oxygen consumption rate of tumor cells after in vivo insulin infusion was measured using high frequency electron paramagnetic resonance oximetry. To know if insulin was able to enhance radiation-induced tumor regrowth delay, tumor-bearing mice were treated with 16 Gy of 250 kV radiation dose after insulin infusion. We provide evidence that insulin increases the local pressure of oxygen of tumors (from 0-3 mm Hg to 8-11 mm Hg) as well as the tumor response to irradiation (increasing regrowth delay by a factor of 2.11). We found that the insulin-induced increase of tumor pressure of oxygen: (a) is not caused by an increase in the tumor blood flow, which is even decreased after insulin infusion; (b) is because of a decrease in the tumor cell oxygen consumption (in vivo insulin consumed oxygen three times slower than control cells); and (c) is inhibited by a nitric oxide (NO) synthase inhibitor, Nomega-nitro-L-arginine methyl ester, when injected i.p. at 15 micromol/kg(-1), 1 h before insulin infusion. We demonstrate by immunoblotting that the NO pathway involves a phosphorylation of endothelial NO synthase and showed a concomitant increase in the cyclic GMP tumor level. These findings provide unique insights into biological processes in tumors, new possible management for treating cancer patients, and raise major questions about the role of insulin secretion (fasting status and diabetes) in the clinical response of tumors to radiation therapy.