Julie Magat

Macrophage skewing by Phd2 haplodeficiency prevents ischaemia by inducing arteriogenesis

PHD2 serves as an oxygen sensor that rescues blood supply by regulating vessel formation and shape in case of oxygen shortage. However, it is unknown whether PHD2 can influence arteriogenesis. Here we studied the role of PHD2 in collateral artery growth by using hindlimb ischaemia as a model, a process that compensates for the lack of blood flow in case of major arterial occlusion. We show that Phd2 (also known as Egln1) haplodeficient (Phd2+/−) mice displayed preformed collateral arteries that preserved limb perfusion and prevented tissue necrosis in ischaemia. Improved arteriogenesis in Phd2+/− mice was due to an expansion of tissue-resident, M2-like macrophages and their increased release of arteriogenic factors, leading to enhanced smooth muscle cell (SMC) recruitment and growth. Both chronic and acute deletion of one Phd2 allele in macrophages was sufficient to skew their polarization towards a pro-arteriogenic phenotype. Mechanistically, collateral vessel preconditioning relied on the activation of canonical NF-κB pathway in Phd2+/− macrophages. These results unravel how PHD2 regulates arteriogenesis and artery homeostasis by controlling a specific differentiation state in macrophages and suggest new treatment options for ischaemic disorders.

Hypoxia imaging with the nitroimidazole 18F-FAZA PET tracer: A comparison with Oxylite, EPR Oximetry and 19F-MRI Relaxometry

BACKGROUND AND PURPOSE (18)F-FAZA is a nitroimidazole PET tracer that can provide images of tumor hypoxia. However, it cannot provide absolute pO(2) values. To qualify (18)F-FAZA PET, we compared PET images to pO(2) measured by OxyLite, EPR oximetry and (19)F-MRI. MATERIALS AND METHODS Male WAG/Rij rats grafted with rhabdomyosarcoma were used. Tumor oxygenation was modified by gas breathing (air or carbogen). The same day of PET acquisition, the pO(2) was measured in the same tumor either by OxyLite probes (measurement at 10 different sites), EPR oximetry using low frequency EPR or (19)F-relaxometry using 15C5 on an 11.7T MR system. RESULTS There was a good correlation between the results obtained by PET and EPR (R = 0.93). In the case of OxyLite, although a weaker correlation was observed (R = 0.55), the trend for two values to agree was still related to the inverse function theoretically predicted. For the comparison of (18)F-FAZA PET and (19)F-MRI, no change in T(1) was observed. CONCLUSIONS A clear correlation between (18)F-FAZA PET image intensities and tumor oxygenation was demonstrated, suggesting that (18)F-FAZA PET is a promising imaging technique to guide cancer therapy.

Arsenic Trioxide Treatment Decreases the Oxygen Consumption Rate of Tumor Cells and Radiosensitizes Solid Tumors

Arsenic trioxide (As(2)O(3)) is an effective therapeutic against acute promyelocytic leukemia and certain solid tumors. Because As(2)O(3) inhibits mitochondrial respiration in leukemia cells, we hypothesized that As(2)O(3) might enhance the radiosensitivity of solid tumors by increasing tumor oxygenation [partial pressure of oxygen (pO(2))] via a decrease in oxygen consumption. Two murine models of radioresistant hypoxic cancer were used to study the effects of As(2)O(3). We measured pO(2) and the oxygen consumption rate in vivo by electron paramagnetic resonance oximetry and (19)fluorine-MRI relaxometry. Tumor perfusion was assessed by Patent blue staining. In both models, As(2)O(3) inhibited mitochondrial respiration, leading to a rapid increase in pO(2). The decrease in oxygen consumption could be explained by an observed decrease in glutathione in As(2)O(3)-treated cells, as this could increase intracellular reactive oxygen species that can disrupt mitochondrial membrane potential. When tumors were irradiated during periods of As(2)O(3)-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 As(2)O(3) acutely increases oxygen consumption and radiosensitizes tumors, providing a new rationale for clinical investigations of As(2)O(3) in irradiation protocols to treat solid tumors.

Noninvasive mapping of spontaneous fluctuations in tumor oxygenation using 19F MRI

PURPOSE Acute hypoxia (transient cycles of hypoxia-reoxygenation) is known to occur in solid tumors and may be a poorly appreciated therapeutic problem as it can be associated with resistance to radiation therapy, impaired delivery of chemotherapeutic agents, or metastasis development. The objective of the present study was to use MRF19 relaxometry maps to analyze the spontaneous fluctuations of partial pressure of oxygen (pO2) over time in experimental tumors. METHODS ThepO2 maps were generated after direct intratumoral administration of a fluorine compound (hexafluorobenzene) whose relaxation rate (1/T1) is proportional to the % O2. The authors used a SNAP inversion-recovery sequence at 4.7 T to acquire parametric images of the T1 relaxation time with a high spatial and temporal resolution. Homemade routines were developed to perform regions of interest analysis, as well as pixel by pixel analysis of pO2 over time. RESULTS The authors were able to quantify and probe the heterogeneity of spontaneous fluctuations in tumorpO2: (i) Spontaneous fluctuations in pO2 occurred regardless of the basal oxygenation state (i.e., both in oxygenated and in hypoxic regions) and (ii) spontaneous fluctuations occurred at a rate of 1 cycle/12-47 min. For validation, the analysis was performed in dead mice for which acute changes did not occur. The authors thereby demonstrated that F19 MRI technique is sensitive to acute change in pO2 in tumors. CONCLUSIONS This is the first approach that allows quantitative minimally invasive measurement of the spontaneous fluctuations of tumor oxygenation using a look-locker approach (e.g., SNAP IR). This approach could be an important tool to characterize the phenomenon of tumor acute hypoxia, to understand its physiopathology, and to improve therapies.

Hexafluorobenzene in comparison with perfluoro-15-crown-5-ether for repeated monitoring of oxygenation using19F MRI in a mouse model

Hexafluorobenzene (HFB) and perfluoro‐15‐crown‐5‐ether (15C5) were compared as fluorine reporter probes of tissue oxygenation using 19F 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 pO2 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, pO2 measurements assessed 2‐h postinjection showed consistent values with both probes, evidencing cross‐validation of the 19F MRI oximetry technique for acute measurements. However, the measurement at 36‐h was hampered for HFB, which showed significant lower values of muscle pO2, whereas 15C5 was able to reliably assess muscle pO2 at 36‐h postinjection. Magn Reson Med, 2013.

Mapping of oxygen by imaging lipids relaxation enhancement: A potential sensitive endogenous MRI contrast to map variations in tissue oxygenation

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.

Captopril and S-nitrosocaptopril as potent radiosensitizers: Comparative study and underlying mechanisms

In an effort to improve the issue of radiotherapy treatments, we tested whether S-nitrosocaptopril, a molecule combining a NO donor and an angiotensin converting enzyme inhibitor (ACE inhibitor), could temporarily improve the hemodynamic status of experimental tumors. We monitored the effect of S-nitrosocaptopril in TLT tumors using non rinvasive magnetic resonance techniques. We identified a time window during which tumor oxygenation was improved, as a result of a combined effect on tumor blood flow and oxygen consumption. Consequently, the administration of S-nitrosocaptopril contributed to the increase in efficacy of radiation therapy, an effect that was not observed with captopril alone.

Iron oxide particles covered with hexapeptides targeted at phosphatidylserine as MR biomarkers of tumor cell death

The aim of the study was to evaluate the ability of a new MR contrast agent to detect cell death as a biomarker of the efficacy of anti-cancer treatment. The phosphatidylserine-targeted hexapeptide (E3) was coupled to pegylated ultrasmall iron oxide nanoparticles (USPIO) that can be detected by magnetic resonance imaging (MRI) and by electron paramagnetic resonance (EPR). USPIO binding to staurosporine-treated TLT (transplantable liver tumor) cells, evaluated by X-Band EPR, indicated twice as much binding of USPIO grafted with the E3 peptide, compared with USPIO grafted with a scrambled peptide or ungrafted USPIO. In vivo experiments were carried out using TLT cells implanted intramuscularly into NMRI mice, and tumor cell death was induced by irradiation. After intravenous injection of the different types of USPIO, the accumulation of contrast agent was evaluated ex vivo by X-band EPR, in vivo by L-band EPR and by T(2)-weighted MRI. In irradiated tumors there was greater accumulation of the targeted USPIO particles compared with control particles or compared with the targeted particles in untreated tissues. In conclusion, phosphatidylserine-targeting of USPIO particles can detect dying tissues. This molecular targeted system should be evaluated further as a potential biomarker of tumor response to treatment.

In vivo mapping of tumor oxygen consumption using (19)F MRI relaxometry.

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 19F MRI relaxometry, to the mapping of the oxygen consumption in tumors. The protocol includes the continuous measurement of tumor pO2 during the following respiratory challenge: (i) basal values during air breathing; (ii) increasing pO2 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 pO2 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 19F 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, 19F 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

Electron paramagnetic resonance as a sensitive tool to assess the iron oxide content in cells for MRI cell labeling studies.

MRI cell tracking is a promising technique to track various cell types (stem cells, tumor cells, etc.) in living animals. Usually, cells are incubated with iron oxides (T(2) contrast agent) in order to take up the particles before being injected in vivo. Iron oxide quantification is important in such studies for validating the labeling protocols and assessing the dilution of the particles with cell proliferation. We here propose to implement electron paramagnetic resonance (EPR) as a very sensitive method to quantify iron oxide concentration in cells. Iron oxide particles exhibit a unique EPR spectrum, which directly reflects the number of particles in a sample. In order to compare EPR with existing methods (Perlss Prussian blue reaction, ICP-MS and fluorimetry), we labeled tumor cells (melanoma and renal adenocarcinoma cell lines) and fibroblasts with fluorescent iron oxide particles, and determined the limits of detection of the different techniques. We show that EPR is a very sensitive technique and is specific for iron oxide quantification as measurements are not affected by endogenous iron. As a consequence, EPR is well adapted to perform ex vivo analysis of tissues after cell tracking experiments in order to confirm MRI results.