Emeline J. Ribot

3D TrueFISP imaging of mouse brain at 4.7T and 9.4T

To examine the ability of TrueFISP imaging for evaluating tumor size in mouse brain at high field.

In vivo MR tracking of therapeutic microglia to a human glioma model

A knowledge of the spatial localization of cell vehicles used in gene therapy against glioma is necessary before launching therapy. For this purpose, MRI cell tracking is performed by labeling the cell vehicles with contrast agents. In this context, the goal of this study was to follow noninvasively the chemoattraction of therapeutic microglial cells to a human glioma model before triggering therapy. Silica nanoparticles grafted with gadolinium were used to label microglia. These vehicles, expressing constitutively the thymidine kinase suicide gene fused to the green fluorescent protein gene, were injected intravenously into human glioma‐bearing nude mice. MRI was performed at 4.7 T to track noninvasively microglial accumulation in the tumor. This was followed by microscopy on brain slices to assess the presence in the glioma of the contrast agents, microglia and fusion gene through the detection of silica nanoparticles grafted with tetramethyl rhodamine iso‐thiocyanate, 3,3′‐dioctadecyloxacarbocyanine perchlorate and green fluorescent protein fluorescence, respectively. Finally, gancyclovir was administered systemically to mice. Human microglia were detectable in living mice, with strong negative contrast on T2*‐weighted MR images, at the periphery of the glioma only 24 h after systemic injection. The location of the dark dots was identical in MR microscopy images of the extracted brains at 9.4 T. Fluorescence microscopy confirmed the presence of the contrast agents, exogenous microglia and suicide gene in the intracranial tumor. In addition, gancyclovir treatment allowed an increase in mice survival time. This study validates the MR tracking of microglia to a glioma after systemic injection and their use in a therapeutic strategy against glioma. Copyright

Nanoparticle phagocytosis and cellular stress: involvement in cellular imaging and in gene therapy against glioma.

In gene therapy against glioma, targeting tumoral tissue is not an easy task. We used the tumor infiltrating property of microglia in this study. These cells are well adapted to this therapy since they can phagocyte nanoparticles and allow their visualization by MRI. Indeed, while many studies have used transfected microglia containing a suicide gene and other internalized nanoparticles to visualize microglia, none have combined both approaches during gene therapy. Microglia cells were transfected with the TK‐GFP gene under the control of the HSP70 promoter. First, the possible cellular stress induced by nanoparticle internalization was checked to avoid a non‐specific activation of the suicide gene. Then, MR images were obtained on tubes containing microglia loaded with superparamagnetic nanoparticles (VUSPIO) to characterize their MR properties, as well as their potential to track cells in vivo. VUSPIO were efficiently internalized by microglia, were found non‐toxic and their internalization did not induce any cellular stress. VUSPIO relaxivity r2 was 224 mM−1.s−1. Such results could generate a very high contrast between loaded and unloaded cells on T2‐weighted images. The intracellular presence of VUSPIO does not prevent suicide gene activity, since TK is expressed in vitro and functional in vivo. It allows MRI detection of gene modified macrophages during cell therapy strategies. Copyright

Study of the MR relaxation of microglia cells labeled with Gd-DTPA-bearing nanoparticles

Therapies involving cells as vehicles need to visualize in situ the trafficking of the cells concerned. This cellular imaging can be driven by cell contrast agent-based nanoparticle internalization and non-invasive MRI (magnetic resonance imaging) detection. Here, microglial cells, that would transport a suicide gene to a glioma, were incubated for different times, with various concentrations of silica nanoparticles on which numerous Gd-DTPA were grafted. The goal of this study was to investigate the repartition of cell-associated particles. MRI was used to quantitatively follow the particle uptake process. Fluorescence microscopy images showed that, although most of the nanoparticles were internalized, some remained adsorbed on the extracellular membrane surface. The cells were then submitted to various treatments: glycine to release bound nanoparticles and/or ultrasound to destroy the cell membranes. The R(1) relaxation rates were measured at 4.7 T. R(1) was maximal for 4 h of incubation, decreased after 8 h and remained stable for the 24 following hours. The magnetic resonance signal of ultrasonicated and glycine-treated cells made it possible to quantify the loss of bound nanoparticles after 8 h. Nevertheless, this release did not prevent cell detection since the internalized nanoparticles are enough concentrated to visualize the labeled cells even after 4 days of cell growth. These results highlight the compartmentalization of nanoparticles in microglia and the evolution of the MR signal of the labeled cells. This study could be of importance to interpret in vivo the MR signal changes that could occur after administration of such nanoparticle-labeled cells in therapeutic strategies.

Application of MRI phase-difference mapping to assessment of vascular concentrations of BMS agent in mice.

Direct quantitation of contrast agent concentration can be performed using dynamic susceptibility contrast MRI. This method is based on phase imaging and administration of paramagnetic agents such as gadolinium-chelates. This technique has only been applied on humans or primates. However, numerous research models have been developed on small animals like mice. For this reason, the aim of this work was the application of this MRI technique, allowing the direct quantitation of the contrast agent concentrations in vivo, in the mouse vascular system at high field. For this purpose, Dy-DOTA has been preferred to Gd-DOTA due to a lower T(2)* effect. Dy-DOTA shifts in Larmor frequency were measured by phase difference mapping, using fast gradient-echo imaging at short echo times. Such an acquisition sequence allowed the limitation of susceptibility artifacts at high magnetic fields and phase wrapping. As demonstrated in a phantom oriented parallel to the static magnetic field, it is possible to measure contrast agent concentrations between 0 and 10 mm with an uncertainty of about 100 microm. Finally, the method was applied on living mice at 4.7 T. After the bolus injection, the evolution of contrast agent concentrations was assessed in brain blood vessels parallel to B(0). Long-term disappearance of contrast agent was monitored at high spatial resolution every 15 s. Alternatively, lower resolved images at 0.72 s time-resolution allowed preliminary assessment of arterial input functions. The feasibility of quantitative bolus-tracking in small rodents opens the way for comprehensive descriptions of flow and over time-dependent biological processes, especially in pathological murine models.

Cellular Density Effect on RGD Ligand Internalization in Glioblastoma for MRI Application

Cellular density is a parameter measured for glioma grade and invasiveness diagnosis. The characterization of the cellular density can be performed, non invasively, by magnetic resonance imaging (MRI), since, this technique displays a good resolution. Nevertheless MRI sensitivity is critical. Development of smart contrast agents appears useful to increase MRI signal to noise ratio (SNR). Tumor invasiveness is correlated with high expression of integrins that can be targeted by RGD motif. In this study, MRI contrast agents or fluorescent probes linked to RGD-peptides were used, in a glioma model, to assess the relation between RGD uptake/signal improvement/cell density and consequently tumor invasiveness. Experiments were performed in vitro with U87-MG glioma cells. Flow cytometry and microscopy experiments with RGD and iRGD-alexa488 demonstrated that cell internalization was dependent on cell density. The internalization involved a clathrin-dependent endocytosis. Cytoskeleton and particularly the microtubules were concerned. Actin filaments played a minor role. The internalization was also dependent on the glycolysis and the oxidative phosphorylations. The cellular density modulated the importance of the endocytosis pathways and of the metabolism but not the cytoskeleton contribution. The internalization of the RGD-peptide associated to gadolinium chelate increased the SNR of U87 cells. Moreover, following the cell density augmentation, the SNR increased with a low amplitude but a trend was clearly determined. In conclusion, RGD-peptide internalization appeared, in vitro, as a marker of cellular density. In perspective, the combination of these peptides with contrast agents associated to more sensitive MRI techniques could improve the MRI signal allowing the characterization of cellular density for tumor diagnosis.

Computational Modelling of Metastasis Development in Renal Cell Carcinoma.

The biology of the metastatic colonization process remains a poorly understood phenomenon. To improve our knowledge of its dynamics, we conducted a modelling study based on multi-modal data from an orthotopic murine experimental system of metastatic renal cell carcinoma. The standard theory of metastatic colonization usually assumes that secondary tumours, once established at a distant site, grow independently from each other and from the primary tumour. Using a mathematical model that translates this assumption into equations, we challenged this theory against our data that included: 1) dynamics of primary tumour cells in the kidney and metastatic cells in the lungs, retrieved by green fluorescent protein tracking, and 2) magnetic resonance images (MRI) informing on the number and size of macroscopic lesions. Critically, when calibrated on the growth of the primary tumour and total metastatic burden, the predicted theoretical size distributions were not in agreement with the MRI observations. Moreover, tumour expansion only based on proliferation was not able to explain the volume increase of the metastatic lesions. These findings strongly suggested rejection of the standard theory, demonstrating that the time development of the size distribution of metastases could not be explained by independent growth of metastatic foci. This led us to investigate the effect of spatial interactions between merging metastatic tumours on the dynamics of the global metastatic burden. We derived a mathematical model of spatial tumour growth, confronted it with experimental data of single metastatic tumour growth, and used it to provide insights on the dynamics of multiple tumours growing in close vicinity. Together, our results have implications for theories of the metastatic process and suggest that global dynamics of metastasis development is dependent on spatial interactions between metastatic lesions.

Fast and robust 3D T1 mapping using spiral encoding and steady RF excitation at 7 T: application to cardiac manganese enhanced MRI (MEMRI) in mice.

Mapping longitudinal relaxation times in 3D is a promising quantitative and non‐invasive imaging tool to assess cardiac remodeling. Few methods are proposed in the literature allowing us to perform 3D T1 mapping. These methods often require long scan times and use a low number of 3D images to calculate T1. In this project, a fast 3D T1 mapping method using a stack‐of‐spirals sampling scheme and regular RF pulse excitation at 7 T is presented. This sequence, combined with a newly developed fitting procedure, allowed us to quantify T1 of the whole mouse heart with a high spatial resolution of 208 × 208 × 315 µm3 in 10–12 min acquisition time. The sensitivity of this method for measuring T1 variations was demonstrated on mouse hearts after several injections of manganese chloride (doses from 25 to 150 µmol kg−1). T1 values were measured in vivo in both pre‐ and post‐contrast experiments. This protocol was also validated on ischemic mice to demonstrate its efficiency to visualize tissue damage induced by a myocardial infarction. This study showed that combining spiral gradient shape and steady RF excitation enabled fast and robust 3D T1 mapping of the entire heart with a high spatial resolution. Copyright

Water Selective Imaging and bSSFP Banding Artifact Correction in Humans and Small Animals at 3T and 7T, Respectively

Introduction The purpose of this paper is to develop an easy method to generate both fat signal and banding artifact free 3D balanced Steady State Free Precession (bSSFP) images at high magnetic field. Methods In order to suppress fat signal and bSSFP banding artifacts, two or four images were acquired with the excitation frequency of the water-selective binomial radiofrequency pulse set On Resonance or shifted by a maximum of 3/4TR. Mice and human volunteers were imaged at 7T and 3T, respectively to perform whole-body and musculoskeletal imaging. “Sum-Of-Square” reconstruction was performed and combined or not with parallel imaging. Results The frequency selectivity of 1-2-3-2-1 or 1-3-3-1 binomial pulses was preserved after (3/4TR) frequency shifting. Consequently, whole body small animal 3D imaging was performed at 7T and enabled visualization of small structures within adipose tissue like lymph nodes. In parallel, this method allowed 3D musculoskeletal imaging in humans with high spatial resolution at 3T. The combination with parallel imaging allowed the acquisition of knee images with ~500μm resolution images in less than 2min. In addition, ankles, full head coverage and legs of volunteers were imaged, demonstrating the possible application of the method also for large FOV. Conclusion In conclusion, this robust method can be applied in small animals and humans at high magnetic fields. The high SNR and tissue contrast obtained in short acquisition times allows to prescribe bSSFP sequence for several preclinical and clinical applications.

Self‐gated bSSFP sequences to detect iron‐labeled cancer cells and/or metastases in vivo in mouse liver at 7 Tesla

To develop and evaluate three‐dimensional (3D) self‐gated balanced steady state free precession (bSSFP) imaging at high magnetic fields to track iron‐labeled cells and metastases in murine abdomens.