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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- tumor. 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.

INTRODUCTION: Radiolabeled antibodies directed against endoglin (CD105) are promising tools for imaging and antiangiogenic cancer therapy. To validate iodinated antibodies as reliable tracers, we investigated the influence of the radiolabeling method (direct or indirect) on their in vivo stability. METHODS: Anti-CD105 mAbs were radioiodinated directly using chloramine-T ((125)I-anti-CD105-mAbs) or indirectly using D-KRYRR peptide as a linker ((125)I-KRYRR-anti-CD105-mAbs). The biodistribution was studied in B16 tumor-bearing mice via SPECT/CT imaging. RESULTS: Radioiodinated mAbs were stable in vitro. In vivo, thyroid showed the most important increase of uptake after 24h for (125)I-anti-CD105-mAbs (91.9±4.0%ID/ml) versus(125)I-KRYRR-anti-CD105-mAbs (4.4±0.6%ID/ml). Tumor uptake of (125)I-anti-CD105-mAbs (0.9±0.3%ID/ml) was significantly lower than that of (125)I-KRYRR-anti-CD105-mAbs (4.7±0.2%ID/ml). CONCLUSIONS: An accurate characterization of the in vivo stability of radioiodinated mAbs and the choice of an appropriate method for the radioiodination are required, especially for novel targets. The indirect radioiodination of internalizing anti-CD105 mAbs leads to more stable tracer by decreasing in vivo deiodination and improves the tumor retention of radioiodinated mAbs. ADVANCES IN KNOWLEDGE AND IMPLICATIONS FOR PATIENT CARE: To date, the only antiangiogenic antibody approved for clinical indications is bevacizumab. There is a need to develop more antibodies that have targets highly expressed on tumor endothelium. CD105 represents a promising marker of angiogenesis, but its therapeutic relevance in cancer needs to be further investigated. In this context, this study suggests the potential use of indirectly iodinated anti-CD105 mAbs for tumor imaging and for therapeutic purposes.

OBJECTIVES: To test the null hypotheses that photoactive resin composites containing a Type I photoinitiator would exhibit reduced DC or increased monomer elution at substantially short curing times compared with materials based on a Type 2 ketone/amine system. METHODS: Two experimental resin composites were prepared, using either Lucirin-TPO or camphorquinone/DMAEMA. Specimens were light-cured using appropriate spectral emission that coincided with the absorption properties of each initiator using different irradiation protocols (0.5, 1, 3, 9s at 500, 1000 and 2000mW/cm(2) for Lucirin-TPO based composites and 20 or 40s at 1000mW/cm(2) for Lucirin-TPO and camphorquinone-based composites). Degree of conversion (DC) was measured by Raman spectroscopy, propagating radical concentrations were collected by means of electron paramagnetic resonance (EPR) and monomer leaching was characterized using high-performance liquid chromatography (HPLC). RESULTS: The null hypotheses were rejected, except for a single irradiation protocol (0.5s @ 500mW/cm(2)). Lucirin-TPO-based composites could cure 20 times faster and release at least 4 times less monomers in comparison to camphorquinone-based composites. At 1000mW/cm(2), and 1s irradiation time for curing times of 1s, Lucirin-TPO based composites displayed 10% higher DC. The difference in polymerization efficiency of Lucirin-TPO compared with camphorquinone-based resin composites were explained using EPR; the former showing a significantly greater yield of radicals which varied logarithmically with radiant exposure. SIGNIFICANCE: Lucirin-TPO is substantially more efficient at absorbing and converting photon energy when using a curing-light with an appropriate spectral emission and otherwise a limitation noted in several previous publications. At concentrations of 0.0134mol/L, Lucirin-TPO-based composites require a minimum light intensity of 1000mW/cm(2) and an exposure time of 1s to provide significantly improved DC and minimal elu

Quinone-containing molecules have been developed against cancer mainly for their redox cycling ability leading to reactive oxygen species (ROS) formation. We have previously shown that donor-acceptor phenylaminonaphthoquinones are biologically active against a panel of cancer cells. In this report, we explored the mechanisms involved in cancer cell growth inhibition caused by two phenylaminonaphthoquinones, namely Q7 and Q9, with or without ascorbate (ASC). The results show that Q7 and Q9 are both redox cyclers able to form ROS, which strongly inhibit the proliferation of T24 cells. Q9 was a better redox cycler than Q7 because of marked stabilization of the semiquinone radical species arising from its reduction by ascorbate. Indeed, ASC dramatically enhances the inhibitory effect of Q9 on cell proliferation. Q9 plus ASC impairs the cell cycle, causing a decrease in the number of cells in the G2/M phase without involving other cell cycle regulating key proteins. Moreover, Q9 plus ASC influences the MAPK signaling pathways, provoking the appearance of a senescent cancer cell phenotype and ultimately leading to necrotic-like cell death. Because cellular senescence limits the replicative capacity of cells, our results suggest that induction of senescence may be exploited as a basis for new approaches to cancer therapy. © 2013 Elsevier Inc.

OBJECTIVES: To demonstrate that determination of the depth of cure of resin-based composites needs to take into account the depth at which the transition between glassy and rubbery states of the resin matrix occurs. METHODS: A commercially available nano-hybrid composite (Grandio) in a thick layer was light cured from one side for 10 or 40s. Samples were analyzed by Vickers indentation, Raman spectroscopy, atomic force microscopy, electron paramagnetic imaging and differential scanning calorimetry to measure the evolution of the following properties with depth: microhardness, degree of conversion, elastic modulus of the resin matrix, trapped free radical concentration and glass transition temperature. These measurements were compared to the composite thickness remaining after scraping off the uncured, soft composite. RESULTS: There was a progressive decrease in the degree of conversion and microhardness with depth as both properties still exhibited 80% of their upper surface values at 4 and 3.8mm, respectively, for 10s samples, and 5.6 and 4.8mm, respectively, for 40s samples. In contrast, there was a rapid decrease in elastic modulus at around 2.4mm for the 10s samples and 3.0mm for the 40s samples. A similar decrease was observed for concentrations of propagating radicals at 2mm, but not for concentrations of allylic radicals, which decreased progressively. Whereas the upper composite layers presented a glass transition temperature - for 10s, 55°C (±4) at 1mm, 56.3°C (±2.3) at 2mm; for 40s, 62.3°C (±0.6) at 1mm, 62°C (±1) at 2mm, 62°C (±1.7) at 3mm - the deeper layers did not display any glass transition. The thickness remaining after scraping off the soft composite was 7.01 (±0.07mm) for 10s samples and 9.48 (±0.22mm) for 40s samples. SIGNIFICANCE: Appropriate methods show that the organic matrix of resin-based composite shifts from a glassy to a gel state at a certain depth. Hence, we propose a new definition for the "depth of cure" as the depth at which the res

Purpose: Nitric oxide (NO), synthesized by the inducible nitric oxide synthase (iNOS), is known to inhibit metabolic oxygen consumption because of interference with mitochondrial respiratory activity. This study examined whether activation of iNOS (a) directly in tumor cells or (b) in bystander macrophages may improve radioresponse through sparing of oxygen. Methods and Materials: EMT-6 tumor cells and RAW 264.7 macrophages were exposed to bacterial lipopolysaccharide plus interferon-gamma, and examined for iNOS expression by reverse transcription polymerase chain reaction, Western blotting and enzymatic activity. Tumor cells alone, or combined with macrophages were subjected to metabolic hypoxia and analyzed for radiosensitivity by clonogenic assay, and for oxygen consumption by electron paramagnetic resonance and a Clark-type electrode. Results: Both tumor cells and macrophages displayed a coherent picture of iNOS induction at transcriptional/translational levels and NO/nitrite production, whereas macrophages showed also co-induction of the inducible heme oxygenase-1, which is associated with carbon monoxide (CO) and bilirubin production. Activation of iNOS in tumor cells resulted in a profound oxygen sparing and a 2.3-fold radiosensitization. Bystander NO-producing, but not CO-producing, macrophages were able to block oxygen consumption by 1.9-fold and to radiosensitize tumor cells by 2.2-fold. Both effects could he neutralized by aminoguanidine, a metabolic iNOS inhibitor. An improved radioresponse was clearly observed at macrophages to tumor cells ratios ranging between 1:16 to 1:1. Conclusions: Our study is the first, as far as we are aware, to provide evidence that iNOS may induce radiosensitization through oxygen sparing, and illuminates NO-producing macrophages as a novel determinant of tumor cell radioresponse within the hypoxic tumor microenvironment. (C) 2010 Elsevier Inc.

OBJECTIVE: To develop electron paramagnetic resonance (EPR) oximetry as a tool to characterize the oxygen environment in human ovarian xenografts in the early postgrafting period. DESIGN: Prospective experimental study. SETTING: Gynecology research unit in a university hospital. PATIENT(S): Biopsies were obtained from 6 women aged 22-35 years. INTERVENTION(S): Frozen-thawed human ovarian tissue fragments were grafted to an intraperitoneal site in nude mice. Before grafting, lithium phthalocyanine, an oxygen reporter, was implanted inside the fragments. MAIN OUTCOME MEASURE(S): To monitor partial pressure of oxygen (pO(2)) by EPR on postgrafting days 3, 5, 7, 10, 14, 17, and 21 and validate the technique by histologic assessment. RESULT(S): A period of hypoxia was identified before day 5, followed by gradual but significant oxygenation over the next 5 days, suggesting an active process of graft revascularization. Reoxygenation kinetics in human ovarian xenotransplants were quantified. CONCLUSION(S): Our data validated the EPR oximetry technique as a tool to monitor pO(2) in ovarian grafting. The critical early period of hypoxia was identified, and the first steps of reoxygenation were characterized. In the future, our model may be used to evaluate new freezing and grafting protocols with the aim of reducing potential cryoinjury and initial ischemia-reperfusion damage.

The aim of the present study was to evaluate the usefulness of electron paramagnetic resonance (EPR) spectroscopy and imaging in assessing the phagocytic activity of the liver after administration of India ink. We conducted experiments on livers from control rodents and from rodents in which the Kupffer cell population had been depleted by pretreatment with gadolinium chloride. The EPR signal intensity recorded in liver homogenates was about two times lower in GdCl(3) treated rats than in control rats. EPR imaging carried out on precision-cut liver slices indicated a good correlation between the depletion of Kupffer cells and the EPR signal intensity.

PURPOSE: To determine whether radiation-induced increases in nitric oxide (NO) production can influence tumor blood flow and improve delivery of Akt-targeting therapeutic DNA lipocomplexes to the tumor. METHODS AND MATERIALS: The contribution of NO to the endothelial response to radiation was identified using NO synthase (NOS) inhibitors and endothelial NOS (eNOS)-deficient mice. Reporter-encoding plasmids complexed with cationic lipids were used to document the tumor vascular specificity and the efficacy of in vivo lipofection after irradiation. A dominant-negative Akt gene construct was used to evaluate the facilitating effects of radiotherapy on the therapeutic transgene delivery. RESULTS: The abundance of eNOS protein was increased in both irradiated tumor microvessels and endothelial cells, leading to a stimulation of NO release and an associated increase in tumor blood flow. Transgene expression was subsequently improved in the irradiated vs. nonirradiated tumor vasculature. This effect was not apparent in eNOS-deficient mice and could not be reproduced in irradiated cultured endothelial cells. Finally, we combined low-dose radiotherapy with a dominant-negative Akt gene construct and documented synergistic antitumor effects. CONCLUSIONS: This study offers a new rationale to combine radiotherapy with gene therapy, by directly exploiting the stimulatory effects of radiation on NO production by tumor endothelial cells. The preferential expression of the transgene in the tumor microvasculature underscores the potential of such an adjuvant strategy to limit the angiogenic response of irradiated tumors.

PURPOSE: To characterize changes in the tumor microenvironment early after irradiation and determine the factors responsible for early reoxygenation. METHODS AND MATERIALS: Fibrosarcoma type II (FSaII) and hepatocarcinoma transplantable liver tumor tumor oxygenation were determined using electron paramagnetic resonance oximetry and a fiberoptic device. Perfusion was assessed by laser Doppler, dynamic contrast-enhanced MRI, and dye penetration. Oxygen consumption was determined by electron paramagnetic resonance. The interstitial fluid pressure was evaluated by the wick-in-needle technique. RESULTS: An increase in oxygen partial pressure was observed 3-4 h after irradiation. This increase resulted from a decrease in global oxygen consumption and an increase in oxygen delivery. The increase in oxygen delivery was due to radiation-induced acute inflammation (that was partially inhibited by the antiinflammatory agent diclofenac) and to a decrease in interstitial fluid pressure. The endothelial nitric oxide synthase pathway, identified as a contributing factor at 24 h after irradiation, did not play a role in the early stage after irradiation. We also observed that splitting a treatment of 18 Gy into two fractions separated by 4 h (time of maximal reoxygenation) had a greater effect on tumor regrowth delay than when applied as a single dose. CONCLUSION: Although the cell cycle redistribution effect is important for treatment protocols using multiple daily radiation fractions, the results of this work emphasize that the oxygen effect must be also considered to optimize the treatment strategy.

PURPOSE: Oxygen deficiency in tumors reduces the efficacy of nonsurgical treatment modalities. We tested the hypothesis that electrical stimulation of the sciatic nerve could modify the oxygenation status and the blood flow of tumors implanted in the thigh of mice. MATERIALS AND METHODS: The sciatic nerve was electrically stimulated at 5 Hz. Local transplantable liver tumor (TLT) and fibrosarcoma (FSaII) tumor oxygen pressure (pO(2)) and perfusion measurements were carried out using electron paramagnetic resonance (EPR) oximetry and the OxyLite/OxyFlo technique. The radiosensitizing effect of the protocol was assessed by irradiating FSaII tumors with X-rays. RESULTS: Tumor pO(2) increased from approximately 3 mm Hg to approximately 8 mm Hg, and relative tumor blood flow was increased by 241% and 162% for TLT and FSaII tumor models, respectively. The effect on the tumor oxygenation was inhibited by a nitric oxide synthase (NOS) inhibitor, and an increase in the tumor nitric oxide (NO) content was observed using EPR spin-trapping. The tumor oxygen consumption rate was decreased after the stimulation protocol. In addition, the electrical stimulation of the host tissue increased regrowth delays by a factor of 1.65. CONCLUSIONS: This increase in tumor oxygenation is due to the temporary increase in tumor blood flow, but particularly to a decrease in the tumor oxygen consumption rate (inhibition of respiration) that is mediated by a local production of NO during the protocol. Those tumor hemodynamic changes resulted in a radiosensitizing effect.

PURPOSE: In an effort to improve radiotherapy treatments, methods aimed at increasing the quantity of oxygen delivered to tumors were investigated. The aim of this study was to evaluate the effect of one nitric oxide (NO) donor (isosorbide dinitrate) on pO(2) and blood flow in a murine tumor model. The effect was compared to carbogen, used as a reference treatment. METHODS AND MATERIALS: Thirty-six liver tumors implanted in mouse thighs were imaged using magnetic resonance imaging (MRI) at 4.7 Tesla with dynamic Gd-DTPA and blood oxygen level-dependent (BOLD) contrast-enhanced imaging after administration of isosorbide dinitrate or carbogen. The effect on the pO(2) was also tested by EPR oximetry (1.1 GHz) on 52 mice. RESULTS: A significant increase in MRI intensity was observed for both treatments in comparison with the control group. EPR oximetry showed a dose-dependant increase in tumor pO(2) for isosorbide dinitrate (by 5.9 mmHg at 0.2 mg/kg) and a substantially greater change for carbogen breathing (by 23 mmHg). CONCLUSION: Both tumor blood flow and pO(2) were increased by isosorbide dinitrate and carbogen. Carbogen is more efficient than isosorbide dinitrate in increasing the BOLD image intensity, as well as the tumor pO(2), but as efficient as isosorbide dinitrate in the Gd-DTPA contrast-enhanced imaging. We conclude that the effects of carbogen on improving tumor pO(2) involve both improved blood flow and improved hemoglobin oxygenation, whereas the effects of isosorbide dinitrate are predominantly mediated by improved blood flow alone.

Manganese-dipyridoxal diphosphate (Mn-DPDP) is a liver-selective contrast agent selectively taken up by the hepatocytes. Because of the analogy of structure with pyridoxine (vitamin B6), it was previously suggested that this compound can be selectively taken up by the facilitated transport of vitamers B6. To understand the uptake mechanism, an in vivo binding study was performed based on a competition between 54Mn-DPDP and pyridoxine on the one hand, and Mn-DPDP and [3H]pyridoxine on the other. We found that the [3H]pyridoxine levels in the liver were not significantly different 5 min after intravenous administration of several doses of Mn-DPDP (5 nmol/kg to 50 mumol/kg): 5.0 +/- 0.3% of the injected dose/g tissue. The content of 54Mn (administered as 54Mn-DPDP) in the liver was not affected by a saturation dose of pyridoxine (1 mmol/kg) and was found to be constant (+/- 10% of the injected dose/g tissue) for 60 min. These experiments showed that the uptake of Mn-DPDP is not mediated by the transporter of pyridoxine.