Electron Paramagnetic Resonance Imaging of Melanin in Honey Bee.

Honey bees play a crucial role in the nature by pollinating wild flowers. Over the past years, there has been an increasing concern regarding the honey bee colony decline. Pesticides or environmental effects targeting the biochemistry of insect chitin and cuticle coating may be in part responsible for honey bee pathologies. We here propose the use of electron paramagnetic resonance imaging (EPRI) as a tool to image the melanin-chitin complexes as part of the exoskeleton of the honey bee. EPRI at 9.65 GHz was applied on intact freeze-dried bees. The imaging data were collected on the melanin peak. High-resolution images revealed that this compound is extensively distributed in the periphery of the animal, data consistent with the localization in the cuticle of the bee. While EPR of melanin has been so far explored in the context of melanoma characterization, it may offer new opportunities in research on honey bees and other insects.

Clinical and Statistical Considerations when Assessing Oxygen Levels in Tumors: Illustrative Results from Clinical EPR Oximetry Studies.

The success of treatment for malignancies, especially those undergoing radiation therapy or chemotherapy, has long been recognized to depend on the degree of hypoxia in the tumor. In addition to the prognostic value of knowing the tumor's initial level of hypoxia, assessing the tumor oxygenation during standard therapy or oxygen-related treatments (such as breathing oxygen-enriched gas mixtures or taking drugs that can increase oxygen supply to tissues) can provide valuable data to improve the efficacy of treatments. A series of early clinical studies of tumors in humans are ongoing at Dartmouth and Emory using electron paramagnetic resonance (EPR) oximetry to assess tumor oxygenation, initially and over time during either natural disease progression or treatment. This approach has the potential for reaching the long-sought goal of enhancing the effectiveness of cancer therapy. In order to effectively reach this goal, we consider the validity of the practical and statistical assumptions when interpreting the measurements made in vivo for patients undergoing treatment for cancer.

'Oxygen Level in a Tissue' - What Do Available Measurements Really Report?

The aim of the paper is to discuss what currently is feasible clinically to measure the level of oxygen and how that measurement can be clinically useful. Because oxygen in tissues is quite heterogeneous and all methods of measurement can only provide an average across heterogeneities at some spatial and temporal resolution, the values that are obtained may have limitations on their clinical utility. However, even if such limitations are significant, if one utilizes repeated measurements and focuses on changes in the measured levels, rather than 'absolute levels', it may be possible to obtain very useful clinical information. While these considerations are especially pertinent in cancer, they also pertain to most other types of pathology.

Acidosis-induced metabolic reprogramming in tumor cells enhances the anti-proliferative activity of the PDK inhibitor dichloroacetate.

Altered metabolic pathways in cancer such as exacerbated glycolytic flux and increased glutamine metabolism are promising targets for anti-cancer therapies. While commonly observed in glycolytic tumors, extracellular acidosis has never been considered as a potential modulator of anti-metabolic drug activity such as dichloroacetate (DCA). Using cancer cells from various origins selected for their ability to proliferate under acidic conditions, we found that DCA exerts greater inhibitory effects on the growth of these acid-adapted cells than in parental cells. Moreover, daily DCA administration to mice led to a significant decrease in tumor growth from acid-adapted cells but not from parental cells. 13C-tracer studies revealed that DCA induced a double metabolic shift, diminishing glycolysis and increasing intracellular glutamine in acid-adapted cells. As a consequence, DCA reduced the pentose phosphate pathway activity more extensively and increased apoptosis in acid-adapted cells. Finally, the combination of DCA with a glutaminase inhibitor significantly enhanced the cytotoxic effects of DCA. Overall, the interplay between acidosis and DCA exposure leads to metabolic reprogramming that considerably alters cellular fitness.

Injectable nanomedicine hydrogel for local chemotherapy of glioblastoma after surgical resection.

Glioblastoma (GBM) treatment includes, when possible, surgical resection of the tumor followed by radiotherapy and oral chemotherapy with temozolomide, however recurrences quickly develop around the resection cavity borders leading to patient death. We hypothesize that the local delivery of Lauroyl-gemcitabine lipid nanocapsule based hydrogel (GemC12-LNC) in the tumor resection cavity of GBM is a promising strategy as it would allow to bypass the blood brain barrier, thus reaching high local concentrations of the drug. The cytotoxicity and internalization pathways of GemC12-LNC were studied on different GBM cell lines (U251, T98-G, 9L-LacZ, U-87 MG). The GemC12-LNC hydrogel was well tolerated when injected in mouse brain. In an orthotopic xenograft model, after intratumoral administration, GemC12-LNC significantly increased mice survival compared to the controls. Moreover, its ability to delay tumor recurrences was demonstrated after perisurgical administration in the GBM resection cavity. In conclusion, we demonstrate that GemC12-LNC hydrogel could be considered as a promising tool for the post-resection management of GBM, prior to the standard of care chemo-radiation.

Improvement of the Off-Resonance Saturation, an MRI sequence for positive contrast with SPM particles: Theoretical and experimental study.

The SuperParaMagnetic particles (SPM particles) are used as contrast agents in MRI and produce negative contrast with conventional T2 or T2(∗)-weighted sequences. Unfortunately, the SPM particle detection on images acquired with such sequences is sometimes difficult because negative contrast can be created by artifacts such as air bubbles or calcification. To overcome this problem, new sequences as Off-Resonance Saturation (ORS) were developed to produce positive contrast with SPM particles. This work explores a new way to optimize the contrast generated by the ORS sequence by increasing the number of saturation pulses applied before the imaging sequence. This modified sequence is studied with numerical simulations and experiments on agarose gel phantoms. A theoretical model able to predict the contrast for different values of the sequence parameters is also developed. The results show that the contrast increases with the saturation pulses number with an optimal value of three saturation pulses in order to avoid artifacts and limit the Specific Absorption Rate (SAR) effect. The dependence of the contrast on the SPM particle concentration and sequence parameters is comparable to what was observed for the ORS sequence.

Monte Carlo Calculation of Radioimmunotherapy with (90)Y-, (177)Lu-, (131)I-, (124)I-, and (188)Re-Nanoobjects: Choice of the Best Radionuclide for Solid Tumour Treatment by Using TCP and NTCP Concepts.

Radioimmunotherapy has shown that the use of monoclonal antibodies combined with a radioisotope like (131)I or (90)Y still remains ineffective for solid and radioresistant tumour treatment. Previous simulations have revealed that an increase in the number of (90)Y labelled to each antibody or nanoobject could be a solution to improve treatment output. It now seems important to assess the treatment output and toxicity when radionuclides such as (90)Y, (177)Lu, (131)I, (124)I, and (188)Re are used. Tumour control probability (TCP) and normal tissue complication probability (NTCP) curves versus the number of radionuclides per nanoobject were computed with MCNPX to evaluate treatment efficacy for solid tumours and to predict the incidence of surrounding side effects. Analyses were carried out for two solid tumour sizes of 0.5 and 1.0 cm radius and for nanoobject (i.e., a radiolabelled antibody) distributed uniformly or nonuniformly throughout a solid tumour (e.g., Non-small-cell-lung cancer (NSCLC)). (90)Y and (188)Re are the best candidates for solid tumour treatment when only one radionuclide is coupled to one carrier. Furthermore, regardless of the radionuclide properties, high values of TCP can be reached without toxicity if the number of radionuclides per nanoobject increases.

Bottom-up study of the MRI positive contrast created by the Off-Resonance Saturation sequence.

Superparamagnetic iron oxide nanoparticles (SPM particles) are used in MRI to highlight regions such as tumors through negative contrast. Unfortunately, sources as air bubbles or tissues interfaces also lead to negative contrast, which complicates the image interpretation. New MRI sequences creating positive contrast in the particle surrounding, such as the Off-Resonance Saturation sequence (ORS), have thus been developed. However, a theoretical study of the ORS sequence is still lacking, which hampers the optimization of this sequence. For this reason, this work provides a self-consistent analytical expression able to predict the dependence of the contrast on the sequence parameters and the SPM particles properties. This expression was validated by numerical simulations and experiments on agarose gel phantoms on a 11.7 T scanner system. It provides a fundamental understanding of the mechanisms leading to positive contrast, which could allow the improvement of the sequence for future in vivo applications. The influence of the SPM particle relaxivities, the SPM particle concentration, the echo time and the saturation pulse parameters on the contrast were investigated. The best contrast was achieved with SPM particles possessing the smallest transverse relaxivity, an optimal particle concentration and for low echo times.

Theoretical and experimental study of ON-Resonance Saturation, an MRI sequence for positive contrast with superparamagnetic nanoparticles.

Superparamagnetic iron oxide nanoparticles (SPM particles) are widely used in MRI as negative contrast agents. Their detection is sometimes difficult because negative contrast can be caused by different artifacts. To overcome this problem, MRI protocols achieving positive contrast specific to SPM particles were developed such as the ON-Resonance Saturation (ONRS) sequence. The aim of the present work is to achieve a bottom-up study of the ONRS sequence by an understanding of the physical mechanisms leading to positive contrast. A complete theoretical modeling, a novel numerical simulation approach and experiments on agarose gel phantoms on a 11.7 T MRI system were carried out for this purpose. The influence of the particle properties and concentration - as well as the effect of the sequence parameters on the contrast - were investigated. It was observed that theory and experiments were in strong agreement. The tools developed in this work allowed to predict the parameters leading to the maximum contrast. For example, particles presenting a low transverse relaxivity can provide an interesting positive contrast after optimization of their concentration in the sample.

Dynamic contrast-enhanced MRI in mouse tumors at 11.7 T: comparison of three contrast agents with different molecular weights to assess the early effects of combretastatin A4.

Dynamic contrast-enhanced (DCE)-MRI is useful to assess the early effects of drugs acting on tumor vasculature, namely anti-angiogenic and vascular disrupting agents. Ultra-high-field MRI allows higher-resolution scanning for DCE-MRI while maintaining an adequate signal-to-noise ratio. However, increases in susceptibility effects, combined with decreases in longitudinal relaxivity of gadolinium-based contrast agents (GdCAs), make DCE-MRI more challenging at high field. The aim of this work was to explore the feasibility of using DCE-MRI at 11.7 T to assess the tumor hemodynamics of mice. Three GdCAs possessing different molecular weights (gadoterate: 560 Da, 0.29 mmol Gd/kg; p846: 3.5 kDa, 0.10 mmol Gd/kg; and p792: 6.47 kDa, 0.15 mmol Gd/kg) were compared to see the influence of the molecular weight in the highlight of the biologic effects induced by combretastatin A4 (CA4). Mice bearing transplantable liver tumor (TLT) hepatocarcinoma were divided into two groups (n = 5-6 per group and per GdCA): a treated group receiving 100 mg/kg CA4, and a control group receiving vehicle. The mice were imaged at 11.7 T with a T1 -weighted FLASH sequence 2 h after the treatment. Individual arterial input functions (AIFs) were computed using phase imaging. These AIFs were used in the Extended Tofts Model to determine K(trans) and vp values. A separate immunohistochemistry study was performed to assess the vascular perfusion and the vascular density. Phase imaging was used successfully to measure the AIF for the three GdCAs. In control groups, an inverse relationship between the molecular weight of the GdCA and K(trans) and vp values was observed. K(trans) was significantly decreased in the treated group compared with the control group for each GdCA. DCE-MRI at 11.7 T is feasible to assess tumor hemodynamics in mice. With K(trans) , the three GdCAs were able to track the early vascular effects induced by CA4 treatment.

Influence of paramagnetic melanin on the MRI contrast in melanoma: a combined high-field (11.7 T) MRI and EPR study.

Melanoma is the most dangerous form of skin cancer and its incidence is rising each year. Because the current methods of diagnosis based on the visual aspect of the tumor show limitations, several new techniques are emerging to help in this diagnosis, amongst which are magnetic resonance imaging (MRI) and electron paramagnetic resonance (EPR). The origin of the typical contrast pattern observable in melanoma in T1 - and T2 -weighted images remains to be elucidated and is a source of controversy. In addition, melanin could create sufficient magnetic inhomogeneities to allow its visualization on T2 *-weighted images using high-field MRI. In order to elucidate the possible role of melanin in the MRI contrast of melanoma, the present study was designed to correlate the paramagnetic content in melanin pigment to the contrast on T1 -, T2 - and T2 *-weighted images. MR images were obtained in vivo at 11.7 T using four types of experimental tumors with different pigmentations (B16, HBL, LND1 melanomas and KHT sarcomas). The paramagnetic content in melanin pigment was measured by EPR. No significant correlation was observed between the content in melanin and the relaxation times T1 , T2 and T2 *, emphasizing that the presence of pigment alone has negligible effect on the MRI contrast.

Dynamic contrast-enhanced MRI in mice at high field: estimation of the arterial input function can be achieved by phase imaging.

PURPOSE: Quantitative dynamic contrast-enhanced MRI requires an accurate arterial input function (AIF). At high field, increased susceptibility effects and decreased longitudinal relaxivity of contrast agents lead to predominant T2* effects in blood vessels, producing a dip in signal during passage of the contrast agent bolus. This study determined phase-derived AIFs in mice at 11.7 T. METHODS: AIFs were measured in aorta/vena cava for five FBV/N mice and in iliac arteries/veins for five NMRI mice with a fast low angle shot sequence, simultaneously with tumor imaging (temporal resolution: 1.19 s). Gadoterate was injected into the tail vein as a bolus (0.286 mmol Gd/kg). An in vitro study was also performed to calculate the relationship between ΔΦ and gadolinium concentration. RESULTS: The phantom system confirmed the linear relationship between measured ΔΦ and gadolinium concentration. In vivo, a dip in arterial magnitude signal made it impossible to quantify the AIF. With phase imaging, a clear quantifiable bolus peak was obtained; peak measured concentration in plasma was 4.9 ± 0.9 mM for FBV/N mice and 8.0 ± 0.6 mM for NMRI mice, close to the expected concentration of 6.8 mM. CONCLUSION: Phase imaging seems to be an appropriate means to measure the AIF of mice at high field.

Tumour hypoxia determines the potential of combining mTOR and autophagy inhibitors to treat mammary tumours.

BACKGROUND: Hypoxia can activate autophagy, a self-digest adaptive process that maintains cell turnover. Mammalian target of rapamycin (mTOR) inhibitors are used to treat cancer but also stimulate autophagy. METHODS: Human mammary cancer cells and derived xenografts were used to examine whether hypoxia could exacerbate autophagy-mediated resistance to the mTOR inhibitor rapamycin. RESULTS: Rapamycin exerted potent antitumour effects in MCF-7 and MDA-MB-231 mammary tumours through a marked inhibition of angiogenesis, but the autophagy inhibitor chloroquine (CQ) failed to further sensitise tumours to mTOR inhibition. Rapamycin treatment actually led to tumour reoxygenation, thereby preventing the development of autophagy. Chloroquine alone, however, blocked the growth of MCF-7 tumours and in vitro blunted the hypoxia-induced component of autophagy in these cells. Finally, when initiating CQ treatment in large, hypoxic tumours, a robust antitumour effect could be observed, which also further increased the antiproliferative effects of rapamycin. CONCLUSION: The mTOR inhibitor rapamycin significantly contributes to tumour growth inhibition and normalisation of the tumour vasculature through potent antiangiogenic effects. The resulting reduction in hypoxia accounts for a lack of sensitisation by the autophagy inhibitor CQ, except if the tumours are already at an advanced stage, and thus largely hypoxic at the initiation of the combination of rapamycin and CQ treatment.

Dual anticancer drug/superparamagnetic iron oxide-loaded PLGA-based nanoparticles for cancer therapy and magnetic resonance imaging.

We developed dual paclitaxel (PTX)/superparamagnetic iron oxide (SPIO)-loaded PLGA-based nanoparticles for a theranostic purpose. Nanoparticles presented a spherical morphology and a size of 240 nm. The PTX and iron loading were 1.84 ± 0.4 and 10.4 ± 1.93 mg/100 mg respectively. Relaxometry studies and phantom MRI demonstrated their efficacy as T2 contrast agent. Significant cellular uptake by CT26 cells of nanoparticles was shown by Prussian blue staining and fluorescent microscopy. While SPIO did not show any toxicity in CT-26 cells, PTX-loaded nanoparticles had a cytotoxic activity. PTX-loaded nanoparticle (5 mg/kg) with or without co-encapulated SPIO induced in vivo a regrowth delay of CT26 tumors. Together these multifunctional nanoparticles may be considered as future nanomedicine for simultaneous molecular imaging, drug delivery and real-time monitoring of therapeutic response.

Antiproliferative effects of phenylaminonaphthoquinones are increased by ascorbate and associated with the appearance of a senescent phenotype in human bladder cancer cells.

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.

Endotoxin-induced basal respiration alterations of renal HK-2 cells: a sign of pathologic metabolism down-regulation.

To study the mechanism of oxygen regulation in inflammation-induced acute kidney injury, we investigate the effects of a bacterial endotoxin (lipopolysaccharide, LPS) on the basal respiration of proximal tubular epithelial cells (HK-2) both by high-resolution respirometry and electron spin resonance spectroscopy. These two complementary methods have shown that HK-2 cells exhibit a decreased oxygen consumption rate when treated with LPS. Surprisingly, this cellular respiration alteration persists even after the stress factor was removed. We suggested that this irreversible decrease in renal oxygen consumption after LPS challenge is related to a pathologic metabolic down-regulation such as a lack of oxygen utilization by cells.

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 (Perls's 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.

Multimodal assessment of early tumor response to chemotherapy: comparison between diffusion-weighted MRI, 1H-MR spectroscopy of choline and USPIO particles targeted at cell death.

The aim of this study was to determine the value of different magnetic resonance (MR) protocols to assess early tumor response to chemotherapy. We used a murine tumor model (TLT) presenting different degrees of response to three different cytotoxic agents. As shown in survival curves, cyclophosphamide (CP) was the most efficient drug followed by 5-fluorouracil (5-FU), whereas the etoposide treatment had little impact on TLT tumors. Three different MR protocols were used at 9.4 Tesla 24 h post-treatment: diffusion-weighted (DW)-MRI, choline measurement by (1) H MRS, and contrast-enhanced MRI using ultrasmall iron oxide nanoparticles (USPIO) targeted at phosphatidylserine. Accumulation of contrast agent in apoptotic tumors was monitored by T(2) -weighted images and quantified by EPR spectroscopy. Necrosis and apoptosis were assessed by histology. Large variations were observed in the measurement of choline peak areas and could not be directly correlated to tumor response. Although the targeted USPIO particles were able to significantly differentiate between the efficiency of each cytotoxic agent and best correlated with survival endpoint, they present the main disadvantage of non-specific tumor accumulation, which could be problematic when transferring the method to the clinic. DW-MRI presents a better compromise by combining longitudinal studies with a high dynamic range; however, DW-MRI was unable to show any significant effect for 5-FU. This study illustrates the need for multimodal imaging in assessing tumor response to treatment to compensate for individual limitations.

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 MR 19F relaxometry maps to analyze the spontaneous fluctuations of partial pressure of oxygen (pO2) over time in experimental tumors. METHODS: The pO2 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 tumor pO2: (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 19F 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.

Experimental determination of the radial dose distribution in high gradient regions around 192Ir wires: comparison of electron paramagnetic resonance imaging, films, and Monte Carlo simulations.

PURPOSE: The experimental determination of doses at proximal distances from radioactive sources is difficult because of the steepness of the dose gradient. The goal of this study was to determine the relative radial dose distribution for a low dose rate 192Ir wire source using electron paramagnetic resonance imaging (EPRI) and to compare the results to those obtained using Gafchromic EBT film dosimetry and Monte Carlo (MC) simulations. METHODS: Lithium formate and ammonium formate were chosen as the EPR dosimetric materials and were used to form cylindrical phantoms. The dose distribution of the stable radiation-induced free radicals in the lithium formate and ammonium formate phantoms was assessed by EPRI. EBT films were also inserted inside in ammonium formate phantoms for comparison. MC simulation was performed using the MCNP4C2 software code. RESULTS: The radical signal in irradiated ammonium formate is contained in a single narrow EPR line, with an EPR peak-to-peak linewidth narrower than that of lithium formate (approximately 0.64 and 1.4 mT, respectively). The spatial resolution of EPR images was enhanced by a factor of 2.3 using ammonium formate compared to lithium formate because its linewidth is about 0.75 mT narrower than that of lithium formate. The EPRI results were consistent to within 1% with those of Gafchromic EBT films and MC simulations at distances from 1.0 to 2.9 mm. The radial dose values obtained by EPRI were about 4% lower at distances from 2.9 to 4.0 mm than those determined by MC simulation and EBT film dosimetry. CONCLUSIONS: Ammonium formate is a suitable material under certain conditions for use in brachytherapy dosimetry using EPRI. In this study, the authors demonstrated that the EPRI technique allows the estimation of the relative radial dose distribution at short distances for a 192Ir wire source.