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Brain metastasis from breast cancer is an increasingly important clinical problem. Here we assessed the role of CD44hi/CD24lo cells and pathways that regulate them, in an experimental model of brain metastasis. Notch signaling (mediated by γ-secretase) has been shown to contribute to maintenance of the cancer stem cell (CSC) phenotype. Cells sorted for a reduced stem-like phenotype had a reduced ability to form brain metastases compared with unsorted or CD44hi/CD24lo cells (P < 0.05; Kruskal–Wallis). To assess the effect of γ-secretase inhibition, cells were cultured with DAPT and the CD44/CD24 phenotypes quantified. 231-BR cells with a CD44hi/CD24lo phenotype was reduced by about 15% in cells treated with DAPT compared with DMSO-treated or untreated cells (P = 0.001, ANOVA). In vivo, mice treated with DAPT developed significantly fewer micro- and macrometastases compared with vehicle treated or untreated mice (P = 0.011, Kruskal–Wallis). Notch1 knockdown reduced the expression of CD44hi/CD24lo phenotype by about 20%. In vitro, Notch1 shRNA resulted in a reduction in cellular growth at 24, 48, and 72 hours time points (P = 0.033, P = 0.002, and P = 0.009, ANOVA) and about 60% reduction in Matrigel invasion was observed (P < 0.001, ANOVA). Cells transfected with shNotch1 formed significantly fewer macrometastases and micrometastases compared with scrambled shRNA or untransfected cells (P < 0.001; Kruskal–Wallis). These data suggest that the CSC phenotype contributes to the development of brain metastases from breast cancer, and this may arise in part from increased Notch activity. Mol Cancer Res; 9(7); 834–44. ©2011 AACR.

Supplementary Table 1, Figures 1-4 from Notch1 Inhibition Alters the CD44hi/CD24lo Population and Reduces the Formation of Brain Metastases from Breast Cancer

The magnetization-prepared 2 rapid acquisition gradient echo (MP2RAGE) sequence provides quantitative T1 maps in addition to high-contrast morphological images. Advanced acceleration techniques such as compressed sensing (CS) allow its acquisition time to be compatible with clinical applications. To consider its routine use in future neuroimaging protocols, the repeatability of the segmented brain structures was evaluated and compared with the standard morphological sequence (magnetization-prepared rapid gradient echo [MPRAGE]). The repeatability of the T1 measurements was also assessed.Thirteen healthy volunteers were scanned either 3 or 4 times at several days of interval, on a 3 T clinical scanner, with the 2 sequences (CS-MP2RAGE and MPRAGE), set with the same spatial resolution (0.8-mm isotropic) and scan duration (6 minutes 21 seconds). The reconstruction time of the CS-MP2RAGE outputs (including the 2 echo images, the MP2RAGE image, and the T1 map) was 3 minutes 33 seconds, using an open-source in-house algorithm implemented in the Gadgetron framework.Both precision and variability of volume measurements obtained from CAT12 and VolBrain were assessed. The T1 accuracy and repeatability were measured on phantoms and on humans and were compared with literature.Volumes obtained from the CS-MP2RAGE and the MPRAGE images were compared using Student t tests (P0.05 was considered significant).The CS-MP2RAGE acquisition provided morphological images of the same quality and higher contrasts than the standard MPRAGE images. Similar intravolunteer variabilities were obtained with the CS-MP2RAGE and the MPRAGE segmentations. In addition, high-resolution T1 maps were obtained from the CS-MP2RAGE. T1 times of white and gray matters and several deep gray nuclei are consistent with the literature and show very low variability (1%).The CS-MP2RAGE can be used in future protocols to rapidly obtain morphological images and quantitative T1 maps in 3-dimensions while maintaining high repeatability in volumetry and relaxation times.

T1 and T2 relaxation times combined with 31 P spectroscopy have been proven efficient for muscular disease characterization as well as for pre- and post-muscle stimulation measurements. Even though 31 P spectroscopy can already be performed during muscle exercise, no method for T1 and T2 measurement enables this possibility. In this project, a complete setup and protocol for multi-parametrical MRI of the rat gastrocnemius before, during and after muscle stimulation at 4.7 and 7 T is presented. The setup is fully MRI compatible and is composed of a cradle, an electro-stimulator and an electronic card in order to synchronize MRI sequences with muscle stimulation. A 2D triggered radial-encoded Look-Locker sequence was developed, and enabled T1 measurements in less than 2 min on stimulated muscle. Also, a multi-slice multi-echo sequence was adapted and synchronized for T2 measurements as well as 31 P spectroscopy acquisitions in less than 4 min in both cases on stimulated muscle. Methods were validated on young rats using different stimulation paradigms. Then they were applied on older rats to compare quantification results, using the different stimulation paradigms, and allowed observation of metabolic changes related to aging with good reproducibility. The robustness of the whole setup shows wide application opportunities.

To assess feasibility of a three-dimensional ultrashort echo time (3D-UTE)-sequence to evaluate normal and pathological disco-vertebral complex (DVC), with assessment of its different portions in a rat model of degenerative disk disease (DDD) with histological correlation. To assess whether this sequence, in comparison with long echo time T2-weighted sequence, is able to monitor DDD with differentiation of early from chronic DVC changes in pathological mechanical conditions. Five rats were induced with DDD model by percutaneous disk trituration of the tail with an 18-G needle under US-guidance and imaged at 4.7 T. MRI protocol included fat-saturated-T2 (RARE) and 3D-UTE-sequences performed at baseline (day 0. n = 5 animals /10 DVC) and each week (W) from W1 to W10 postoperatively. Visual analysis and signal intensity measurements of SNR and CNR of all DVC portions were performed on RARE and UTE images. Following killing (baseline, n = 1/2 DVC; W2, n = 2/4 DVC; W10, n = 2/4 DVC), histological analysis was performed and compared with MRI. In normal DVC, unlike conventional RARE-sequences, 3D-UTE allowed complete identification of DVC zonal anatomy including on visual analysis and CNR measurements. In pathological conditions, SNR and CNR measurements of the annulus fibrosus and nucleus pulposus on 3D-UTE distinguished early discitis at W1 from chronic discopathy (P

PURPOSE To develop a 2D radial multislice MP2RAGE sequence for fast and reliable T1 mapping at 7 T in mice and for MR thermometry. METHODS The 2D-MP2RAGE sequence was performed with the following parameters: TI1 -TI2 -MP2RAGETR = 1000-3000-9000 ms. The multiple dead times within the sequence were used for interleaved multislice acquisition, enabling one to acquire six slices in 9 seconds. The excitation pulse shape, inversion selectivity, and interslice gap were optimized. In vitro comparison with the inversion-recovery sequence was performed. The T1 variations with temperature were measured on tubes with T1 ranging from 800 ms to 2000 ms. The sequence was used to acquire T1 maps continuously during 30 minutes on the brain and abdomen of healthy mice. RESULTS A three-lobe cardinal sine excitation pulse, combined with an inversion slice thickness and an interslice gap of respectively 150% and 50% of the imaging slice thickness, led to a SD and bias of the T1 measurements below 1% and 2%, respectively. A linear dependence of T1 with temperature was measured between 10°C and 60°C. In vivo, less than 1% variation was measured between successive T1 maps in the mouse brain. In the abdomen, no obvious in-plane motion artifacts were observed but respiratory motion in the slice dimension led to 6% T1 underestimation. CONCLUSION The multislice MP2RAGE sequence could be used for fast whole-body T1 mapping and MR thermometry. Its reconstruction method would enable on-the-fly reconstruction.

Renal cell carcinoma (RCC) still lacks prognostic and predictive biomarkers to monitor the disease and the response to therapy. The usual strategy in translational research is to start from human samples, to identify molecular markers and gene networks and then to functionally validate themin vitroand in animal models. We devised herein a completely opposite strategy from “mouse to man” by performing an aggressiveness screen and used functional genomics, imaging, clinical data and computational approaches in order to discover molecular pathways and players in renal cancer development and metastasis. Multiple cell lines for primary tumor growth, survival in the blood circulation and lung metastasis or metastatic spread from the primary tumor were generated and analyzed using a multi-layered approach which includes large-scale transcriptome, genome and methylome analyses. Transcriptome and methylome analyses demonstrated distinct clustering in three different groups. Remarkably, DNA sequencing did not show significant genomic variations in the different groups which indicates absence of clonal selection during thein vivoamplification process. Transcriptome analysis revealed distinct signatures of tumor aggressiveness which were validated in patient cohorts. Methylome analysis of full-length DNA allowed clustering of the same groups and revealed clinically relevant signatures. Furthermore, we identified SAA2 and CFB as soluble prognostic and predictive biomarkers of the therapeutic response. We also uncovered IL34 as another soluble prognostic biomarker and key regulator of renal cell carcinoma (RCC) progression. This was also functionally validatedin vivo,and a mathematical model of IL34-dependent primary tumor growth and metastasis development was provided. These results indicate that such multilayered analysis in a RCC animal model leads to meaningful results that are of translational significance.One Sentence SummaryAn aggressiveness screen with multilayer systems analysis to identify signatures and biomarkers for renal cell carcinoma aggressiveness.

Recent advances in the field of Tissue Engineering allowed to control the three-dimensional organization of engineered constructs. Cell pattern imaging and in vivo follow-up remain a major hurdle in in situ bioprinting onto deep tissues. Magnetic Resonance Imaging (MRI) associated with Micron-sized superParamagnetic Iron Oxide (MPIO) particles constitutes a non-invasive method for tracking cells in vivo. To date, no studies have utilized Cellular MRI as a tool to follow cell patterns obtained via bioprinting technologies. Laser-Assisted Bioprinting (LAB) has been increasingly recognized as a new and exciting addition to the bioprinting’s arsenal, due to its rapidity, precision and ability to print viable cells. This non-contact technology has been successfully used in recent in vivo applications. The aim of this study was to assess the methodology of tracking MPIO-labeled stem cells using MRI after organizing them by Laser-Assisted Bioprinting. Optimal MPIO concentrations for tracking bioprinted cells were determined. Accuracy of printed patterns was compared using MRI and confocal microscopy. Cell densities within the patterns and MRI signals were correlated. MRI enabled to detect cell patterns after in situ bioprinting onto a mouse calvarial defect. Results demonstrate that MRI combined with MPIO cell labeling is a valuable technique to track bioprinted cells in vitro and in animal models.

The T1 longitudinal recovery time is regarded as a biomarker of cancer treatment efficiency. In this scope, the Magnetization Prepared 2 RApid Gradient Echo (MP2RAGE) sequence relevantly complies with fast 3D T1 mapping. Nevertheless, with its Cartesian encoding scheme, it is very sensitive to respiratory motion. Consequently, a radial encoding scheme was implemented for the detection and T1 measurement of hepatic metastases in mice at 7T. A 3D radial encoding scheme was developed using a golden angle distribution for the k-space trajectories. As in that case, each projection contributes to the image contrast, the signal equations had to be modified. Phantoms containing increasing gadoteridol concentrations were used to determine the accuracy of the sequence in vitro. Healthy mice were repetitively scanned to assess the reproducibility of the T1 values. The growth of hepatic metastases was monitored. Undersampling robustness was also evaluated. The accuracy of the T1 values obtained with the radial MP2RAGE sequence was > 90% compared to the Inversion-Recovery sequence. The motion robustness of this new sequence also enabled repeatable T1 measurements on abdominal organs. Hepatic metastases of less than 1-mm diameter were easily detected and T1 heterogeneities within the metastasis and between the metastases within the same animal were measured. With a twofold acceleration factor using undersampling, high-quality 3D T1 abdominal maps were achieved in 9 min. The radial MP2RAGE sequence could be used for fast 3D T1 mapping, to detect and characterize metastases in regions subjected to respiratory motion. • The Cartesian encoding of the MP2RAGE sequence was modified to a radial encoding. The modified sequence enabled accurate T 1 measurements on phantoms and on abdominal organs of mice. • Hepatic metastases were easily detected due to high contrast. Heterogeneity in T 1 was measured within the metastases and between each metastasis within the same animal. • As implementation of this sequence does not require specific hardware, we expect that it could be readily available for clinical practice in humans.

To develop a 2D radial multislice MP2RAGE sequence for fast and reliable TThe 2D-MP2RAGE sequence was performed with the following parameters: TIA three-lobe cardinal sine excitation pulse, combined with an inversion slice thickness and an interslice gap of respectively 150% and 50% of the imaging slice thickness, led to a SD and bias of the TThe multislice MP2RAGE sequence could be used for fast whole-body T

Purpose. To confirm the interest of 3-dimensional ultrashort echo-time (3D-UTE) sequences to assess morphologic aspects in normal and pathological Achilles entheses in a rat model of spondyloarthropathy (SpA) with histological correlations, in comparison with conventional RARE T2 Fat-Sat sequences, and, furthermore, to evaluate the feasibility of a 3D multiecho UTE sequence performed before and after the intravenous injection of ultrasmall superparamagnetic iron oxide (USPIO) particles to assess macrophagic involvement in the Achilles enthesis in the same rat model of SpA. Materials and Methods. Fourteen rats underwent in vivo MRI of the ankle at 4.7 T, including a 3D RARE T2 Fat-Sat sequence and a 3D ultrashort echo-time (UTE) sequence for morphologic assessment at baseline and day 3 after induction of an SpA model, leading to Achilles enthesopathy in the left paw (right paw serving as a control). A 3D multiecho UTE sequence was also performed at day 3 before and then 24 (4 rats) and 48 (2 rats) hours after intravenous injection of USPIO. Visual analysis and signal intensity measurements of all images were performed at different locations of the Achilles enthesis and preinsertional area. Visual analysis and T2∗ measurements were performed before and after USPIO injection, on the 3D multiecho UTE sequence in the same locations. Normal and pathological values were compared by Wilcoxon signed-rank tests. MR findings were compared against histological data. Results. 3D-UTE sequences enabled morphologic identification of the anterior fibrocartilage and posterior collagenic areas of the Achilles enthesis. Visual analysis and signal intensity measurements distinguished SpA-affected entheses from healthy ones at day 3 (P=0.02). After administration of USPIO, no differences in signals were detected. Similarly, both visual analysis and signal T2∗ measurements in the enthesis were unable to distinguish the SpA-affected tendons from healthy ones (P=0.914). Neither the normal anatomy of the enthesis nor its pathological pattern could be distinguished using the standard RARE sequence. Histology confirmed the absence of USPIO in Achilles entheses, despite marked signs of inflammation. Conclusion. Unlike conventional RARE T2 Fat-Sat sequences, 3D-UTE sequences enable morphologic assessment of normal enthesis anatomy and early detection of abnormalities in pathological conditions. However, 3D multiecho UTE sequences combined with USPIO injections with T2∗ measurements were unable to detect macrophagic involvement in these pathological conditions.

Background Due to the very short T2 of its components, the normal anatomy of Achilles enthesis is impossible to define with "conventional" long echo time (TE) T2 sequences. However, this is a common site affected by rheumatologic disease. Early abnormalities related to inflammatory processes are impossible to detect in this location. Purpose To assess the feasibility of a 3D-UTE (ultrashort echo time) sequence to evaluate normal and pathological Achilles entheses, determining both anterior fibrocartilaginous and posterior collagenic portions at 4.7T, in a rat model of spondyloarthropathy (SpA) with histological correlation. To assess whether this sequence detects SpA enthesopathy prior to long TE T2 sequences, enabling disease monitoring. Study type Prospective case-control study. Animal model Twelve immunocompetent Wistar male rats imaged before (controls); the model was induced in eight rats (16 tendons) imaged at day 6, day 13, and day 21 with regular sacrifice for ex vivo imaging and histological correlation. Field strength 4.7T Bruker Biospec Systems. 3D balanced steady-state free precession (bSSFP) and 3D-UTE sequences, performed at baseline (day 0, n = 12 animals / 24 tendons), day 6 (n = 8/16), 13 (n = 4/8), and day 21 (n = 2/4). Assessment Visual analysis and signal intensity measurements (signal to noise ratio, SNR) of both bSSFP and UTE images were performed by two independent musculoskeletal radiologists at different locations of the Achilles enthesis and preinsertional area. Statistical tests Normal and pathological rat values were compared by Wilcoxon signed-rank tests, as well as interobserver differences. MRI findings were compared against histological data. Results The 3D-UTE sequence identified the anterior fibrocartilage and posterior collagenic areas of Achilles entheses in all cases. Visual analysis and signal intensity measurements distinguished SpA-affected entheses from healthy ones at days 6 and 13 (P = 0.002 and P = 0.006, respectively). Neither the normal anatomy of the enthesis nor its pathological pattern could be identified on T2 bSSFP sequences. Data conclusion Unlike bSSFP T2 sequences, 3D-UTE sequences enable visualization of normal enthesis anatomy and early detection of abnormalities in pathological conditions. Level of evidence 2 Technical Efficacy: Stage 2 J. Magn. Reson. Imaging 2019;50:127-135.

International audience; To develop a Compressed Sensing (CS)-MP2RAGE sequence to drastically shorten acquisition duration and then detect and measure the T1 of brain metastases in mice at 7 T.METHODS:The encoding trajectory of the standard Cartesian MP2RAGE sequence has been modified (1) to obtain a variable density Poisson disk under-sampling distribution along the ky -kz plane, and (2) to sample the central part of the k-space exactly at TI1 and TI2 inversion times. In a prospective study, the accuracy of the T1 measurements was evaluated on phantoms containing increasing concentrations of gadolinium. The CS acceleration factors were increased to evaluate their influence on the contrast and T1 measurements of brain metastases in vivo. Finally, the 3D T1 maps were acquired with at 4-fold increased spatial resolution. The volumes and T1 values of the metastases were measured while using CS to reduce scan time.RESULTS:The implementation of the CS-encoding trajectory did not affect the T1 measurements in vitro. Accelerating the acquisition by a factor of 2 did not alter the contrast or the T1 values of the brain metastases. 3D T1 maps could be obtained in < 1 min using a CS factor of 6. Increasing the spatial resolution enabled more accurately measurement of the metastasis volumes while maintaining an acquisition duration below 5 min.CONCLUSION:The CS-MP2RAGE sequence could be of great interest in oncology to either rapidly obtain mouse brain 3D T1 maps or to increase the spatial resolution with no penalty on the scan duration.

Purpose To develop and assess a 3D-cine self-gated method for cardiac imaging of murine models. Materials and Methods A 3D stack-of-stars (SOS) short echo time (STE) sequence with a navigator echo was performed at 7T on healthy mice (n = 4) and mice with acute myocardial infarction (MI) (n = 4) injected with ultrasmall superparamagnetic iron oxide (USPIO) nanoparticles. In all, 402 spokes were acquired per stack with the incremental or the golden angle method using an angle increment of (360/402)° or 222.48°, respectively. A cylindrical k-space was filled and repeated with a maximum number of repetitions (NR) of 10. 3D cine cardiac images at 156 μm resolution were reconstructed retrospectively and compared for the two methods in terms of contrast-to-noise ratio (CNR). The golden angle images were also reconstructed with NR = 10, 6, and 3, to assess cardiac functional parameters (ejection fraction, EF) on both animal models. Results The combination of 3D SOS-STE and USPIO injection allowed us to optimize the identification of cardiac peaks on navigator signal and generate high CNR between blood and myocardium (15.3 ± 1.0). The golden angle method resulted in a more homogeneous distribution of the spokes inside a stack (P < 0.05), enabling reducing the acquisition time to 15 minutes. EF was significantly different between healthy and MI mice (P < 0.05). Conclusion The method proposed here showed that 3D-cine images could be obtained without electrocardiogram or respiratory gating in mice. It allows precise measurement of cardiac functional parameters even on MI mice. J. Magn. Reson. Imaging 2016;44:355–365.

International audience; 4D-MRI is a promising tool for organ exploration, target delineation and treatment planning. Intra-scan motion artifacts may be greatly reduced by increasing the imaging frame rate. However, poor signal-to-noise ratios (SNR) are observed when increasing spatial and/or frame number per physiological cycle, in particular in the abdomen. In the current work, the proposed 4D-MRI method favored spatial resolution, frame number, isotropic voxels and large field-of-view (FOV) during MR-acquisition. The consequential SNR penalty in the reconstructed data is addressed retrospectively using an iterative back-projection (IBP) algorithm. Practically, after computing individual spatial 3D deformations present in the images using a deformable image registration (DIR) algorithm, each 3D image is individually enhanced by fusing several successive frames in its local temporal neighborood, these latter being likely to cover common independent informations. A tuning parameter allows one to freely readjust the balance between temporal resolution and precision of the 4D-MRI. The benefit of the method was quantitatively evaluated on the thorax of 6 mice under free breathing using a clinically acceptable duration. Improved 4D cardiac imaging was also shown in the heart of 1 mice. Obtained results are compared to theoretical expectations and discussed. The proposed implementation is easily parallelizable and optimized 4D-MRI could thereby be obtained with a clinically acceptable duration.

The TA 3D radial encoding scheme was developed using a golden angle distribution for the k-space trajectories. As in that case, each projection contributes to the image contrast, the signal equations had to be modified. Phantoms containing increasing gadoteridol concentrations were used to determine the accuracy of the sequence in vitro. Healthy mice were repetitively scanned to assess the reproducibility of the TThe accuracy of the TThe radial MP2RAGE sequence could be used for fast 3D T• The Cartesian encoding of the MP2RAGE sequence was modified to a radial encoding. The modified sequence enabled accurate T

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 © 2015 John Wiley & Sons, Ltd.

The development of new non-invasive diagnostic and therapeutic approaches is of paramount importance in order to improve the outcome of patients with glioblastoma (GBM). In this work we investigated a completely non-invasive pre-clinical protocol to effectively target and detect brain tumors through the orotracheal route, using ultra-small nanoparticles (USRPs) and MRI. A mouse model of GBM was developed. In vivo MRI acquisitions were performed before and after intravenous or orotracheal administration of the nanoparticles to identify and segment the tumor. The accumulation of the nanoparticles in neoplastic lesions was assessed ex vivo through fluorescence microscopy. Before the administration of contrast agents, MR images allowed the identification of the presence of abnormal brain tissue in 73% of animals. After orotracheal or intravenous administration of USRPs, in all the mice an excellent co-localization of the position of the tumor with MRI and histology was observed. The elimination time of the USRPs from the tumor after the orotracheal administration was approximately 70% longer compared with intravenous injection. MRI and USRPs were shown to be powerful imaging tools able to detect, quantify and longitudinally monitor the development of GBMs. The absence of ionizing radiation and high resolution of MRI, along with the complete non-invasiveness and good reproducibility of the proposed protocol, make this technique potentially translatable to humans. To our knowledge, this is the first time that the advantages of a needle-free orotracheal administration route have been demonstrated for the investigation of the pathomorphological changes due to GBMs.

Background 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. Methods Mice were injected intravenously with iron-labeled melanoma cells and imaged at 7 Tesla (T). Respiration peaks were identified using Free Induction Decay acquired immediately after the radiofrequency pulse. Respiration-corrupted k-space lines were deleted. Four images were acquired to reconstruct final images using the Sum-Of-Square technique. Image sharpness, metastasis contrast and iron-labeled cell detection with SG-bSSFP sequence (acquired with echo time [TE] = 3 ms or TE = 6 ms) were compared with standard methods (gradient echo (GRE) and RARE). Results After reconstruction, the 3D SG-bSSFP images were 75–80% sharper, free from banding (75% liver signal-to-noise ratio recovery) and respiratory motion (26–42% improvement in signal homogeneity) artifacts. Metastasis contrast was twice higher on SG-bSSFP with TE = 3 ms than on RARE images. Iron-labeled cells and metastases were simultaneously detected on SG-bSSFP images with TE = 6 ms, with similar void intensity and tumor contrast to GRE and RARE, respectively. Halving acquisition time preserved iron sensitivity and metastasis contrast, allowing for 3D abdomen imaging in 13 min (TE = 3 ms) or 26 min (TE = 6 ms). Conclusion Combining a self-gating technique with bSSFP sequences at 7T provides high-resolution 3D artifact-free abdominal images of small animals. J. Magn. Reson. Imaging 2015;41:1413–1421. © 2014 Wiley Periodicals, Inc.

Purpose To develop an undersampled anatomical, three-dimensional (3-D) time-resolved magnetic resonance angiography (MRA) method for small animals based on time-of-flight (TOF) effect and radial sampling. Methods Mouse carotid arteries and Circle of Willis images were acquired on a 7T scanner with an electrocardiogram (ECG)-triggered sequence. Preliminary experiments were used to generate an approximately uniform distribution of radial projections with a first golden angle and to produce anatomical TOF images. A second golden angle ratio between consecutive projections of cine acquisitions was added to make it possible to use a temporal filter during reconstruction of time-resolved angiography. A decreasing number of projections were tested, and their impact on signal-to-noise ratio (SNR) and spatial resolution was assessed. Results In anatomical MRA, the undersampled radial approach efficiently allows fast acquisition of mouse angiogram in 3D (22 sec). It was also only slightly sensitive to motion and flow artifacts. The time-resolved sequence can be performed with only 2,500 projections per cine and a temporal resolution under 4 ms in a relatively short acquisition time (less than 5 min). Conclusion This technique simultaneously provided high 3D isotropic spatial resolution and excellent temporal resolution with a good SNR level, allowing blood flow to be visualized in a restricted acquisition time. Magn Reson Med 73:984–994, 2015. © 2014 Wiley Periodicals, Inc.

Purpose To assess anatomic and functional magnetic resonance imaging (MRI) for monitoring of tumor volume and metabolism of orthotopic xenograft prostate cancer tumors. Materials and Methods Human-derived PC-3M cells were implanted into the prostate in 22 nude mice. Tumor volume and MRI appearance were monitored for up to 29 days. Histology was performed to detect metastases. Hyperpolarized [1-13C]pyruvate MRI was used to measure tumor metabolism on day 22. Results Tumors were visible by MRI 9 days after tumor cell implantation. Tumor volume increased to 720 ± 190 mm3 on day 29 of imaging. Metastasis was seen in the iliac lymph nodes at all timepoints, and in more distant lymph nodes at later timepoints, but was not detectable by MRI. Regions with low pyruvate uptake corresponded to regions with necrosis and had a higher lactate/pyruvate ratio (0.98 ± 0.4 vs. 1.6 ± 1.1). Conclusion MRI using the balanced steady-state free precession (bSSFP) sequence can be used to monitor tumor growth in orthotopic PC-3M tumors as early as 9 days post-injection. Hyperpolarized pyruvate MRI has potential to assess tumor metabolism and necrosis. J. Magn. Reson. Imaging 2014;40:848–856. © 2013 Wiley Periodicals, Inc.

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Purpose To develop a fast three-dimensional (3D) k-space encoding method based on spiral projection imaging (SPI) with an interleaved golden-angle approach and to validate this novel sequence on small animal models. Methods A disk-like trajectory, in which each disk contained spirals, was developed. The 3D encoding was performed by tilting the disks with a golden angle. The sharpness was first calculated at different T2* values. Then, the sharpness was measured on phantom using variable undersampling ratios. Finally, the sampling method was validated by whole brain time-of-flight angiography and ultrasmall superparamagnetic iron oxide (USPIO) enhanced free-breathing liver angiography on mouse. Results The in vitro results demonstrated the robustness of the method for short T2* and high undersampling ratios. In vivo experiments showed the ability to properly detect small vessels in the brain with an acquisition time shorter than 1 min. Free-breathing mice liver angiography showed the insensitivity of this protocol toward motions and flow artifacts, and enabled the visualization of liver motion during breathing. Conclusions The method implemented here allowed fast 3D k-space sampling with a high undersampling ratio. Combining the advantages of center-out spirals with the flexibility of the golden angle approach could have major implications for real-time imaging. Magn Reson Med 77:1831-1840, 2017. © 2016 International Society for Magnetic Resonance in Medicine.

International audience; The goal of this study was to develop a 3D diffusion weighted sequence for free breathing liver imaging in small animals at high magnetic field. Hepatic metastases were detected and the apparent diffusion coefficients (ADC) were measured. A 3D SE-EPI sequence was developed by (i) inserting a water-selective excitation radiofrequency pulse to suppress adipose tissue signal and (ii) bipolar diffusion gradients to decrease the sensitivity to respiration motion. Mice with hepatic metastases were imaged at 7T by applying b values from 200 to 1100 s/mm 2. 3D images with high spatial resolution (182 9 156 9 125 lm) were obtained in only 8 min 32 s. The modified DW-SE-EPI sequence allowed to obtain 3D abdominal images of healthy mice with fat SNR 2.5 times lower than without any fat suppression method and sharpness 2.8 times higher than on respiration-triggered images. Due to the high spatial resolution , the core and the periphery of disseminated hepatic metastases were differentiated at high b-values only, demonstrating the presence of edema and proliferating cells (with ADC of 2.65 9 10-3 and 1.55 9 10-3 mm 2 /s, respectively). Furthermore, these metastases were accurately distinguished from proliferating ones within the same animal at high b-values (mean ADC of 0.38 9 10-3 mm 2 /s). Metastases of less than 1.7 mm 3 diameter were detected. The new 3D SE-EPI sequence enabled to obtain diffusion information within liver metastases. In addition of intra-metastasis heterogeneity, differences in diffusion were measured between metastases within an animal. This sequence could be used to obtain diffusion information at high magnetic field.

Gamma delta T cells (GDTc) comprise a small subset of cytolytic T cells shown to kill malignant cells in vitro and in vivo. We have developed a novel protocol to expand GDTc from human blood whereby GDTc were initially expanded in the presence of alpha beta T cells (ABTc) that were then depleted prior to use. We achieved clinically relevant expansions of up to 18,485-fold total GDTc, with 18,849-fold expansion of the Vδ1 GDTc subset over 21 days. ABTc depletion yielded 88.1 ± 4.2 % GDTc purity, and GDTc continued to expand after separation. Immunophenotyping revealed that expanded GDTc were mostly CD27-CD45RA- and CD27-CD45RA+ effector memory cells. GDTc cytotoxicity against PC-3M prostate cancer, U87 glioblastoma and EM-2 leukemia cells was confirmed. Both expanded Vδ1 and Vδ2 GDTc were cytotoxic to PC-3M in a T cell antigen receptor- and CD18-dependent manner. We are the first to label GDTc with ultrasmall superparamagnetic iron oxide (USPIO) nanoparticles for cellular MRI. Using protamine sulfate and magnetofection, we achieved up to 40 % labeling with clinically approved Feraheme (Ferumoxytol), as determined by enumeration of Perls' Prussian blue-stained cytospins. Electron microscopy at 2,800× magnification verified the presence of internalized clusters of iron oxide; however, high iron uptake correlated negatively with cell viability. We found improved USPIO uptake later in culture. MRI of GDTc in agarose phantoms was performed at 3 Tesla. The signal-to-noise ratios for unlabeled and labeled cells were 56 and 21, respectively. Thus, Feraheme-labeled GDTc could be readily detected in vitro via MRI.

The goal of this study was to evaluate the ability of balanced steady state free precession (b-SSFP) magnetic resonance imaging sequence to distinguish between live and lysed iron-labelled cells. Human breast cancer cells were labelled with iron oxide nanoparticles. Cells were lysed using sonication. Imaging was performed at 3 T. The timing parameters for b-SSFP and the number of iron-labelled cells in samples were varied to optimise the b-SSFP signal difference between live and lysed iron-labelled cell samples. For in vivo experiments, cells were mixed with Matrigel and implanted into nude mice. Three mice implanted with live labelled cancer cells were irradiated to validate this method. Lysed iron-labelled cells have a significantly higher signal compared with live, intact iron-labelled cells in bSSFP images. The contrast between live and dead cells can be maximised by careful optimisation of timing parameters. A change in the b-SSFP signal was measured 6 days after irradiation, reflecting cell death in vivo. Histology confirmed the presence of dead cells in the implant. Our results show that the b-SSFP sequence can be optimised to allow for the discrimination of live iron-labelled cells and lysed iron-labelled cells in vitro and in vivo. • Balanced steady state free precession (b-SSFP) MRI can assess iron-labelled cells. • bSSFP can discriminate between live and lysed iron-labelled cells. • The compartmentalisation of iron in cells influences the bSSFP signal. • The bSSFP signal increases after cell death by irradiation in vivo.

Brain metastasis from breast cancer is an increasingly important clinical problem. Here we assessed the role of CD44hi/CD24lo cells and pathways that regulate them, in an experimental model of brain metastasis. Notch signaling (mediated by γ-secretase) has been shown to contribute to maintenance of the cancer stem cell (CSC) phenotype. Cells sorted for a reduced stem-like phenotype had a reduced ability to form brain metastases compared with unsorted or CD44hi/CD24lo cells (P < 0.05; Kruskal–Wallis). To assess the effect of γ-secretase inhibition, cells were cultured with DAPT and the CD44/CD24 phenotypes quantified. 231-BR cells with a CD44hi/CD24lo phenotype was reduced by about 15% in cells treated with DAPT compared with DMSO-treated or untreated cells (P = 0.001, ANOVA). In vivo, mice treated with DAPT developed significantly fewer micro- and macrometastases compared with vehicle treated or untreated mice (P = 0.011, Kruskal–Wallis). Notch1 knockdown reduced the expression of CD44hi/CD24lo phenotype by about 20%. In vitro, Notch1 shRNA resulted in a reduction in cellular growth at 24, 48, and 72 hours time points (P = 0.033, P = 0.002, and P = 0.009, ANOVA) and about 60% reduction in Matrigel invasion was observed (P < 0.001, ANOVA). Cells transfected with shNotch1 formed significantly fewer macrometastases and micrometastases compared with scrambled shRNA or untransfected cells (P < 0.001; Kruskal–Wallis). These data suggest that the CSC phenotype contributes to the development of brain metastases from breast cancer, and this may arise in part from increased Notch activity. Mol Cancer Res; 9(7); 834–44. ©2011 AACR.

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 T(2) *-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 © 2011 John Wiley & Sons, Ltd.

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 HSP(70) 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 r(2) was 224 mM(-1).s(-1). Such results could generate a very high contrast between loaded and unloaded cells on T(2)-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.

Purpose To examine the ability of TrueFISP imaging for evaluating tumor size in mouse brain at high field. Materials and Methods Brains of healthy and glioma-implanted nude mice were imaged at 4.7T and 9.4T. 3D imaging was performed with TrueFISP and T2w-RARE. Radiofrequency pulses with alternating phase and the sum-of-squares reconstruction method were used in TrueFISP imaging. The contrast-to-noise ratio (CNR) was evaluated in healthy and in tumoral brain regions. Results Banding artifact was correctly suppressed in TrueFISP images thanks to the method used. The signal-to-noise ratio (SNR) and CNR were comparable in TrueFISP and T2w-RARE images at 4.7T, but acquisition was around 4 times shorter with TrueFISP. Tumor was well detected at 4.7T with TrueFISP, but CNR between tumor and healthy tissue was low at 9.4T. Conclusion TrueFISP is efficient and fast to perform brain imaging at 4.7T. Multiple TrueFISP acquisitions with phase-cycled radiofrequency (RF) pulses and sum-of-squares reconstruction allow suppression of banding artifact. Tumoral regions were well detected and the method allows longitudinal studies of tumor growth. J. Magn. Reson. Imaging 2008;28:497–503. © 2008 Wiley-Liss, Inc.

To develop and assess a 3D-cine self-gated method for cardiac imaging of murine models.A 3D stack-of-stars (SOS) short echo time (STE) sequence with a navigator echo was performed at 7T on healthy mice (n = 4) and mice with acute myocardial infarction (MI) (n = 4) injected with ultrasmall superparamagnetic iron oxide (USPIO) nanoparticles. In all, 402 spokes were acquired per stack with the incremental or the golden angle method using an angle increment of (360/402)° or 222.48°, respectively. A cylindrical k-space was filled and repeated with a maximum number of repetitions (NR) of 10. 3D cine cardiac images at 156 μm resolution were reconstructed retrospectively and compared for the two methods in terms of contrast-to-noise ratio (CNR). The golden angle images were also reconstructed with NR = 10, 6, and 3, to assess cardiac functional parameters (ejection fraction, EF) on both animal models.The combination of 3D SOS-STE and USPIO injection allowed us to optimize the identification of cardiac peaks on navigator signal and generate high CNR between blood and myocardium (15.3 ± 1.0). The golden angle method resulted in a more homogeneous distribution of the spokes inside a stack (P0.05), enabling reducing the acquisition time to 15 minutes. EF was significantly different between healthy and MI mice (P0.05).The method proposed here showed that 3D-cine images could be obtained without electrocardiogram or respiratory gating in mice. It allows precise measurement of cardiac functional parameters even on MI mice. J. Magn. Reson. Imaging 2016;44:355-365.

Background Mapping the longitudinal relaxation time (T1) is a promising quantitative tool for the detection of myocardial pathologies either in mouse or human. T1 maps are usually obtained in 2D in order to reduce the total scan time. However, to diagnose myocardial infarction it might be useful to obtain the T1 maps in three dimensions (3D) which require a long acquisition time. In this project a new 3D T1 mapping method based on Look-Locker protocol using continuous radio frequency (RF) excitation and spiral gradient shape has been developed. This method allowed to obtain 3D maps with high spatial (200 x 200 x 300 μm) & temporal resolution (

BACKGROUND: In this study we used cellular magnetic resonance imaging (MRI) to detect mesenchymal stem cells (MSC) labeled with a Fluorine-19 (19F) agent. 19F-MRI offers unambiguous detection and in vivo quantification of labeled cells. METHODS: We investigated two common stem cell transplant mouse models: an immune competent, syngeneic transplant model and an immune compromised, xenograft transplant model. 19F labelled stem cells were implanted intramuscularly into the hindlimb of healthy mice. The transplant was then monitored for up to 17 days using 19F-MRI, after which the tissue was excised for fluorescence microscopy and immunohistochemisty. RESULTS: Immediately following transplantation, 19F-MRI quantification correlated very well with the expected cell number in both models. The 19F signal decreased over time in both models, with a more rapid decrease in the syngeneic model. By endpoint, only 2/7 syngeneic mice had any detectable 19F signal. In the xenograft model, all mice had detectable signal at endpoint. Fluorescence microscopy and immunohistochemistry were used to show that the 19F signal was related to the presence of bystander labeled macrophages, and not original MSC. CONCLUSIONS: Our results show that 19F-MRI is an excellent tool for verifying the delivery of therapeutic cells early after transplantation. However, in certain circumstances the transfer of cellular label to other bystander cells may confuse interpretation of the long-term fate of the transplanted cells.

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 µm(3) 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.

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.Mice were injected intravenously with iron-labeled melanoma cells and imaged at 7 Tesla (T). Respiration peaks were identified using Free Induction Decay acquired immediately after the radiofrequency pulse. Respiration-corrupted k-space lines were deleted. Four images were acquired to reconstruct final images using the Sum-Of-Square technique. Image sharpness, metastasis contrast and iron-labeled cell detection with SG-bSSFP sequence (acquired with echo time [TE] = 3 ms or TE = 6 ms) were compared with standard methods (gradient echo (GRE) and RARE).After reconstruction, the 3D SG-bSSFP images were 75-80% sharper, free from banding (75% liver signal-to-noise ratio recovery) and respiratory motion (26-42% improvement in signal homogeneity) artifacts. Metastasis contrast was twice higher on SG-bSSFP with TE = 3 ms than on RARE images. Iron-labeled cells and metastases were simultaneously detected on SG-bSSFP images with TE = 6 ms, with similar void intensity and tumor contrast to GRE and RARE, respectively. Halving acquisition time preserved iron sensitivity and metastasis contrast, allowing for 3D abdomen imaging in 13 min (TE = 3 ms) or 26 min (TE = 6 ms).Combining a self-gating technique with bSSFP sequences at 7T provides high-resolution 3D artifact-free abdominal images of small animals.

To develop an undersampled anatomical, three-dimensional (3-D) time-resolved magnetic resonance angiography (MRA) method for small animals based on time-of-flight (TOF) effect and radial sampling.Mouse carotid arteries and Circle of Willis images were acquired on a 7T scanner with an electrocardiogram (ECG)-triggered sequence. Preliminary experiments were used to generate an approximately uniform distribution of radial projections with a first golden angle and to produce anatomical TOF images. A second golden angle ratio between consecutive projections of cine acquisitions was added to make it possible to use a temporal filter during reconstruction of time-resolved angiography. A decreasing number of projections were tested, and their impact on signal-to-noise ratio (SNR) and spatial resolution was assessed.In anatomical MRA, the undersampled radial approach efficiently allows fast acquisition of mouse angiogram in 3D (22 sec). It was also only slightly sensitive to motion and flow artifacts. The time-resolved sequence can be performed with only 2,500 projections per cine and a temporal resolution under 4 ms in a relatively short acquisition time (less than 5 min).This technique simultaneously provided high 3D isotropic spatial resolution and excellent temporal resolution with a good SNR level, allowing blood flow to be visualized in a restricted acquisition time.

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.

To assess anatomic and functional magnetic resonance imaging (MRI) for monitoring of tumor volume and metabolism of orthotopic xenograft prostate cancer tumors.Human-derived PC-3M cells were implanted into the prostate in 22 nude mice. Tumor volume and MRI appearance were monitored for up to 29 days. Histology was performed to detect metastases. Hyperpolarized [1-(13) C]pyruvate MRI was used to measure tumor metabolism on day 22.Tumors were visible by MRI 9 days after tumor cell implantation. Tumor volume increased to 720 ± 190 mm(3) on day 29 of imaging. Metastasis was seen in the iliac lymph nodes at all timepoints, and in more distant lymph nodes at later timepoints, but was not detectable by MRI. Regions with low pyruvate uptake corresponded to regions with necrosis and had a higher lactate/pyruvate ratio (0.98 ± 0.4 vs. 1.6 ± 1.1).MRI using the balanced steady-state free precession (bSSFP) sequence can be used to monitor tumor growth in orthotopic PC-3M tumors as early as 9 days post-injection. Hyperpolarized pyruvate MRI has potential to assess tumor metabolism and necrosis.

INTRODUCTION: The overall goal of this study was to assess the utility of three-dimensional magnetic resonance imaging (MRI) for monitoring the temporal and spatial development of experimental brain metastasis in mice. MATERIALS AND METHODS: Brain metastatic human breast cancer cells (231-BR or 231-BR-HER2) were injected intracardially in nude mice for delivery to the brain. Mouse brains were imaged in vivo at different time points using a balanced steady-state-free precession (bSSFP) pulse sequence at 1.5 T. Brains were categorized into four regions: cortex, central brain, olfactory, and posterior. The number of metastases and their volumes were quantified for both cell lines. RESULTS: There was no difference in the mean number of metastases for either cell line. The volumes of metastases in mice injected with 231-BR-HER2 cells were significantly larger than those for mice injected with 231-BR cells. The growth rate for 231-BR-HER2 metastases was 67.5% compared with 54.4% for the 231-BR metastases. More than 50% of metastases were located in the cortex and 25% to 30% of metastases were identified in the central brain for each time point and for mice injected with either cell line. The volumes of metastases were significantly larger in mice with fewer metastases at end point. SIGNIFICANT CONCLUSIONS: MRI provided a comprehensive accounting of the number and size of experimental brain metastases in the whole mouse brain at multiple time points. This approach has provided new information about the temporal and spatial development of metastases in the brain not possible by other histopathologic or imaging methods.

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., Author Summary We used mathematical modelling to formalize the standard theory of metastatic initiation, under which secondary tumours, after establishment in a distant organ, grow independently from each other and from the primary tumour. When calibrated on the experimental data of primary tumour and total metastatic burden in the lungs in an animal model of renal cell carcinoma, the initial model predicted a size distribution of metastatic foci that did not fit with observations obtained experimentally using magnetic resonance imaging (which provided size and number of macro-metastases). The model predicted an increase in the number of lesions, but of smaller size when compared to the data. This led us to revise the standard theory and to propose two hypotheses in order to explain the observations: 1) small metastatic foci merge into larger ones and/or 2) circulating tumour cells may join already established tumours. We then derived a spatial model of tumour growth in order to explore the quantitative implications of tumours merging on global tumour growth and estimated the numbers of required metastatic foci to obtain the observed metastatic volumes.

Emeline Julie Ribot1,2, Line Pourtau1,3, Philippe Massot1,3, Pierre Voisin1,3, Eric Thiaudiere1,3, JeanMichel Franconi1,3 and Sylvain Miraux1,3 1Centre de Resonance Magnetique des Systemes Biologiques, Universite Bordeaux Segalen, CNRS 2Imaging Research Laboratories, Robarts Research Institute, The University of Western Ontario 3Laboratory of Excellence TRAIL, Translational Research and Advanced Imaging Laboratory, University of Bordeaux 1,3France 2Canada

OBJECTIVES: The current lack of efficacy for any chemo- or molecular therapeutic in the treatment of brain metastases is thought to be due, in part, to the heterogeneous permeability of the blood-brain-barrier (BBB). Little is known about how heterogeneous permeability develops, or how it varies among individual metastases. Understanding the BBB’s role in metastasis will be crucial to the development of new, more effective therapies. In this article, we developed the first magnetic resonance imaging-based strategy to detect and measure the volumes of BBB permeable and nonpermeable metastases and studied the development of altered BBB permeability in metastases in vivo, over time in a mouse model of breast cancer metastasis to the brain. MATERIALS AND METHODS: Animals bearing human experimental brain metastases of breast cancer (231-BR cells) were imaged, using 3-dimensional balanced steady-state free precession to visualize total metastases, and contrast-enhanced T1-weighted spin echo with gadopentetic acid (Gd-DTPA) to visualize which of these displayed contrast enhancement, as Gd-DTPA leakage is indicative of altered BBB permeability. RESULTS: Metastases detected 20 days after injection showed no Gd-DTPA enhancement. At day 25, 6.1% ± 6.3% (mean ± standard deviation) of metastases enhanced, and by day 30, 28.1% ± 14.2% enhanced (P < 0.05). Enhancing metastases (mid: 0.14 ± 0.18 mm(3), late: 0.24 ± 0.32 mm(3)) had larger volumes than nonenhancing (mid: 0.04 ± 0.04 mm(3), late: 0.09 ± 0.09 mm(3), P < 0.05); however, there was no significant difference between the growth rates of the 2. CONCLUSIONS: A significant number of brain metastases were uniformly nonpermeable, which highlights the need for developing treatment strategies that can overcome the permeability of the BBB. The model developed herein can provide the basis for in vivo evaluation of both BBB permeable and nonpermeable metastases response to therapy.

THE INCIDENCE OF BREAST CANCER patients with brain metastases is increasing (1,2). This is due in part to the pharmaceutical focus on development of therapeutic agents that successfully treat systemic, but not brain metastases. Still, the median survival time with aggressive treatment is extended by only 4–12 months. To study the mechanisms of breast cancer metastasis to the brain, mouse models have been developed using human breast cancer cell lines (3,4). Histology, immunohistochemistry, and microscopy are typically used to estimate the numbers and the cross-sectional area of metastases, and for analysis of cellular markers (5–7). The limitations of these methods are the need to sacrifice the animal, allowing only an endpoint analysis, and the small subset of tissue volume assessed. Microimaging technologies, including high-resolution magnetic resonance imaging (MRI), micro-computed tomography, and positron emission tomography have shown great utility for characterizing small-animal models of disease. MRI is particularly well suited for studies of brain metastasis because it is noninvasive, three-dimensional, has excellent soft-tissue contrast, and no limitation in depth of penetration at clinical magnetic field strengths. Cellular MRI combines the use of high-resolution MRI with cell labels to track cells of interest. The most commonly used labels are iron-based nanoparticles including superparamagnetic, ultrasmall, and micron-sized iron oxide particles (MPIO). Stem cells (8), macrophages (9), dendritic cells (10), cancer cells (11), and lymphocytes (12) have been tracked in vivo with MRI using this approach. The presence of the label causes a distortion in the magnetic field and leads to abnormal signal hypointensities in iron-sensitive images. Most studies have employed T2-weighted (T2w) spin echo, and T2*-weighted gradient echo sequences to detect iron-labeled cells. The balanced steady-state free precession (b-SSFP) imaging sequence has been used to detect single iron-labeled macrophages and cancer cells in vivo as signal voids in the mouse brain (11,13). The major advantage of the b-SSFP sequence is its high signal-to-noise ratio (SNR) efficiency, allowing for imaging at high spatial resolution in reasonable scan times (14). Another important feature of b-SSFP is its high sensitivity to magnetic field inhomogeneities (15,16). Miraux et al (17) also investigated the b-SSFP sequence on mouse brains at 4.7 and 9.4 T. They compared b-SSFP images of implanted gliomas with T2w SE images, the most commonly employed contrast for the visualization of brain tumors in mice. At 4.7 T the SNR and brain-to-tumor contrast-to-noise ratio (CNR) were comparable in b-SSFP and T2w images, but the scan time was ≈4 times shorter with b-SSFP. At 9.4 T, the CNR was attenuated and tumors were barely visible. Bernas et al (18) developed a b-SSFP protocol at 3 T to optimally visualize iron-loaded glioma in the mouse brain. This protocol consists of a set of two b-SSFP image acquisitions with complementary contrasts, allowing delineation of tumors, which appear as regions of signal hyperintensity due to longer T2 of tumor tissue compared to brain parenchyma, and providing high sensitivity to iron-labeled cells, which are detected as regions of signal void. The purpose of this study was to optimize b-SSFP at 1.5 T with the goal of generating image contrast for the simultaneous detection of single iron-labeled cancer cells and brain tumors in vivo in one scan. A single scan would reduce the acquisition time and allow for monitoring of both nonproliferative cancer cells and developing metastases. For this purpose, MPIO-labeled cancer cells were injected into the left ventricle of the heart in nude mice. After intracardiac injection, cells become trapped in the brain microvasculature. With time, some cancer cells proliferate into metastases that appear as regions of signal hyperintensity in b-SSFP images, primarily due to increased T2 relaxation, while other cells remain dormant (nonproliferative) and are detected as persistent signal voids (11).

11173; International audience; 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 T(2) *-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 © 2011 John Wiley & Sons, Ltd.

International audience; 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.