In vivo magnetic resonance imaging of single cells in mouse brain with optical validation.

In the current work we demonstrate, for the first time, that single cells can be detected in mouse brain in vivo using magnetic resonance imaging (MRI). Cells were labeled with superparamagnetic iron oxide nanoparticles and injected into the circulation of mice. Individual cells trapped within the microcirculation of the brain could be visualized with high-resolution MRI using optimized MR hardware and the fast imaging employing steady state acquisition (FIESTA) pulse sequence on a 1.5 T clinical MRI scanner. Single cells appear as discrete signal voids on MR images. Direct optical validation was provided by coregistering signal voids on MRI with single cells visualized using high-resolution confocal microscopy. This work demonstrates the sensitivity of MRI for detecting single cells in small animals for a wide range of application from stem cell to cancer cell tracking.     

Imaging single mammalian cells with a 1.5 T clinical MRI scanner.

In the present work, we demonstrate that the steady-state free precession (SSFP) imaging pulse sequence FIESTA (fast imaging employing steady state acquisition) used in conjunction with a custom-built insertable gradient coil and customized RF coils can be used to detect individual SPIO-labeled cells using a commonly available 1.5 T clinical MRI scanner. This work provides the first evidence that single-cell tracking will be possible using clinical MRI scanners, opening up new possibilities for cell tracking and monitoring of cellular therapeutics in vivo in humans.     

Imaging of single human carcinoma cells in vitro using a clinical whole-body magnetic resonance scanner at 3.0 T.

The purpose of the present study was to examine whether single human carcinoma cells labeled with iron oxide nanoparticles could be detected by magnetic resonance (MR) imaging on a clinical 3-T scanner using a surface coil only. WiDr human colon carcinoma cells were loaded with two kinds of iron oxide nanoparticles differing by coating and size: aminosilan-coated (MagForce) and carboxy-dextran-coated particles (Resovist). The latter were preferred by the colon carcinoma cell line used here and taken up much faster (12 h) than the smaller carboxydextran-coated Resovist (48 h). Labeled single carcinoma cells, distributed in an agarose gel in a monodisperse layer as controlled by light microscopy, became detectable as punctuate signal extinctions when using a small circularly polarized surface coil in conjunction with a T(2)*-weighted GE sequence at 3 T. The threshold for the detectability of labeled colon carcinoma cells ranged at a load of 4-5 mug iron/10(6) cells. Obviating the need for special hardware additions, this study opens a new lane for single-cell tracking on clinical 3-T MR scanners amenable to patient studies.     

Iron-oxide labeling of hematogenous macrophages in a model of experimental autoimmune encephalomyelitis and the contribution to signal loss in fast imaging employing steady state acquisition (FIESTA) images

PURPOSE: To determine the contribution of blood-derived macrophages to the signal loss observed in MR images of inflammatory lesions in experimental autoimmune encephalomyelitis (EAE). MATERIALS AND METHODS: A relapsing-remitting form of EAE was induced in transgenic mice that express enhanced green fluorescent protein (EGFP) specifically in hematopoietic cells of the myelomonocytic lineage. Animals were injected with Feridex, a superparamagnetic iron oxide (SPIO) nanoparticle, 24 hours prior to in vivo MRI. MRI was performed using a 1.5T whole-body scanner; a high-performance, custom-built gradient coil insert; and a 3D steady-state free precession (SSFP) imaging pulse sequence. Comparisons were made between MR images and corresponding anti-GFP and Perl's Prussian blue (PPB)-stained brain sections. RESULTS: MR images revealed the presence of discrete regions of signal loss throughout the brains of EAE animals that were administered Feridex. Histological staining showed that regions of signal loss on MR images corresponded anatomically with regions of PPB- and GFP-positive cells. CONCLUSION: This experiment provides the first direct evidence that macrophages of hematogenous origin are labeled with SPIO after intravenous administration of Feridex, and contribute to the regions of signal loss detected in MR images of EAE brain.     

Cell tracking using iron oxide fails to distinguish dead from living transplanted cells in the infarcted heart

Recently, debate has arisen about the usefulness of cell tracking using iron oxide–labeled cells. Two important issues in determining the usefulness of cell tracking with MRI are generally overlooked; first, the effect of graft rejection in immunocompetent models, and second, the necessity for careful histological confirmation of the fate of the labeled cells in the presence of iron oxide. Therefore, both iron oxide–labeled living as well as dead epicardium‐derived cells (EPDCs) were investigated in ischemic myocardium of immunodeficient non‐obese diabetic (NOD)/acid: non‐obese diabetic severe combined immunodeficient (NOD/scid) mice with 9.4T MRI until 6 weeks after surgery, at which time immunohistochemical analysis was performed. In both groups, voids on MRI scans were observed that did not change in number, size, or localization over time. Based on MRI, no distinction could be made between living and dead injected cells. Prussian blue staining confirmed that the hypointense spots on MRI corresponded to iron‐loaded cells. However, in the dead‐EPDC recipients, all iron‐positive cells appeared to be macrophages, while the living‐EPDC recipients also contained engrafted iron‐loaded EPDCs. Iron labeling is inadequate for determining the fate of transplanted cells in the immunodeficient host, since dead cells produce an MRI signal indistinguishable from incorporated living cells. Magn Reson Med 63:817–821, 2010. © 2010 Wiley‐Liss, Inc.     

Gene expression profiling reveals early cellular responses to intracellular magnetic labeling with superparamagnetic iron oxide nanoparticles

With MRI (stem) cell tracking having entered the clinic, studies on the cellular genomic response toward labeling are warranted. Gene expression profiling was applied to C17.2 neural stem cells following superparamagnetic iron oxide/PLL (poly‐L ‐lysine) labeling over the course of 1 week. Relative to unlabeled cells, less than 1% of genes (49 total) exhibited greater than 2‐fold difference in expression in response to superparamagnetic iron oxide/PLL labeling. In particular, transferrin receptor 1 (Tfrc) and heme oxygenase 1 (Hmox1) expression was downregulated early, whereas genes involved in lysosomal function (Sulf1) and detoxification (Clu, Cp, Gstm2, Mgst1) were upregulated at later time points. Relative to cells treated with PLL only, cells labeled with superparamagnetic iron oxide/PLL complexes exhibited differential expression of 1399 genes. Though these differentially expressed genes exhibited altered expression over time, the overall extent was limited. Gene ontology analysis of differentially expressed genes showed that genes encoding zinc‐binding proteins are enriched after superparamagnetic iron oxide/PLL labeling relative to PLL only treatment, whereas members of the apoptosis/programmed cell death pathway did not display increased expression. Overexpression of the differentially expressed genes Rnf138 and Abcc4 were confirmed by quantitative real‐time polymerase chain reaction. These results demonstrate that, although early reactions responsible for iron homeostasis are induced, overall neural stem cell gene expression remains largely unaltered following superparamagnetic iron oxide/PLL labeling. Magn Reson Med 63:1031–1043, 2010. © 2010 Wiley‐Liss, Inc.     

Magnetic nanoparticle labeling of mesenchymal stem cells without transfection agent: cellular behavior and capability of detection with clinical 1.5 T magnetic resonance at the single cell level.

The purpose of this work was to evaluate the efficacy of labeling human mesenchymal stem cells (hMSCs) by ionic superparamagnetic iron oxide (SPIO) without a transfection agent and verifying its capability to be detected with clinical 1.5 T magnetic resonance (MR) at the single-cell level. Human hMSCs were incubated for 24 h with an ionic SPIO, Ferucarbotran. The labeling efficiency of hMSCs was determined by iron content measurement spectrophotometrically, and the influence of labeling on cell behavior was ascertained by examination of cell viability using the trypan blue exclusion method, cell proliferation analysis using MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay, mitochondrial membrane potential (MMP) change, differentiation capacity, and reactive oxygen species (ROS) production measured by dichlorofluorescein diacetate (DCFDA) fluorescent probe. Labeled hMSCs were scanned under 1.5 T MRI with three-dimensional (3D) and two-dimensional (2D) T(2)-weighted gradient echo (GRE) pulse sequences. Human hMSC labeling without transfection agent was efficient. The iron content in hMSCs was 23.4 pg Fe/cell. No significant change was found in viability, proliferation, MMP change, ROS production, or differentiation capacity. About 45.2% of the hMSCs could be detected using 1.5 T MRI at the single cell level with 3D GRE and four repetitions.     

Monitoring T-lymphocyte trafficking in tumors undergoing immune rejection.

Activated T cells, isolated from animals that had rejected a tumor (E.G7-OVA) expressing chicken ovalbumin, were labeled with citrated superparamagnetic iron oxide nanoparticles at an intracellular iron concentration of up to 0.5 pg/cell. Injection of these labeled T cells into animals bearing E.G7-OVA tumors undergoing immune rejection resulted in tumor infiltration of these cells, which was detectable as a heterogeneous decrease in intensity in T(2)-weighted MR images. T-cell infiltration was confirmed by immunohistochemical staining of tumor sections obtained postmortem and was shown to colocalize with iron that had been stained using Prussian blue. Tumor rejection was correlated with the uptake of labeled T cells, since the infiltration of labeled T cells was only observed in those tumors that went on to regress. This technique should assist in the elucidation of those factors that are important in mediating tumor immune rejection.     

In vivo MR imaging of bone marrow cells trafficking to atherosclerotic plaques

PURPOSE: To develop a magnetic resonance imaging (MRI)-based method to monitor in vivo trafficking of bone marrow (BM) cells to atherosclerotic lesions. MATERIALS AND METHODS: BM cells from LacZ-transgenic mice were labeled with a superparamagnetic iron oxide (Feridex) and then transplanted into ApoE(-/-) recipient mice that were fed an atherogenic diet. Twenty-four ApoE(-/-) mice were divided into three study groups: 1) group I with Feridex-labeled BM transplantation (BMT) cells (N = 9), 2) group II with unlabeled BMT cells (N = 10), and 3) group III with no BMT cells (N = 5). Migrated Feridex/LacZ-BM cells to atherosclerotic aortic walls were monitored in vivo using a 4.7T MR scanner and correlated with histopathological findings. RESULTS: In group I with Feridex-BMT cells, histology examination displayed plaques in five of nine animals. In four of these five animals, in vivo MRI showed large MR signal voids of the aorta walls (due to the "blooming" effect of migrated Feridex-BM cells in plaques), which were correlated with Feridex- and/or LacZ-positive cells detected in the atherosclerotic lesions. No signal voids could be visualized in the two control animal groups (groups II and III). CONCLUSION: This study demonstrates the potential use of in vivo MRI to monitor the trafficking of magnetically labeled BM cells to atherosclerotic lesions.     

High temporal resolution breathheld 3D FIESTA CINE imaging: validation of ventricular function in patients with chronic myocardial infarction

PURPOSE: To develop a gated single-breathhold, high temporal resolution three-dimensional (3D) CINE imaging technique and to evaluate its accuracy in volumetric and functional quantification in patients with chronic myocardial infarction. MATERIALS AND METHODS: A 3D CINE steady-state free precession (SSFP) pulse sequence was developed incorporating variable temporal sampling of the low and high spatial frequency k-space data to reduce breathhold time and parallel imaging to increase temporal resolution. Reconstruction with retrospective interpolation enabled complete R-R interval coverage. Feasibility was assessed in eight patients with chronic myocardial infarction and ventricular functional values were compared to those of a 2D CINE acquisition. RESULTS: There was no significant difference between the 3D CINE and 2D CINE for end-diastolic volume (168 +/- 73 vs. 177 +/- 59 mL, respectively; P < 0.27), end-systolic volume (81 +/- 62 vs. 79 +/- 53 mL; P < 0.81), and ejection fraction (EF) measurements (55 +/- 14% vs. 58 +/- 14%; P < 0.14). The mean difference in EF was less than 2.5%. A wall motion assessment indicated a good agreement, with a weighted kappa value of 0.62. CONCLUSION: High temporal resolution 3D CINE SSFP imaging of the whole heart can be obtained in a single breathhold and yield ventricular function measurements similar to 2D CINE methods.     

The mechanism of ring enhancement in hepatocellular carcinoma on superparamagnetic iron oxide-enhanced T1-weighted images: an investigation into peritumoral Kupffer cells

PURPOSE: To investigate the mechanism of ring enhancement on ferumoxides-enhanced T1-weighted (T1W) gradient echo images in malignant focal hepatic lesions. MATERIALS AND METHODS: Eighteen patients with hepatocellular carcinoma (HCC) underwent breath-hold T1-, T2-, and T2*-weighted magnetic resonance (MR) images at 1.5-Tesla after ferumoxides administration. The existence of ring enhancement on T1W, and the maximum size of the area showing decreased phagocytic activity on T2W and T2*W, and that of the area showing ring enhancement on T1W were evaluated. The Kupffer cell (KC) density of HCC itself and peritumoral liver parenchyma was assessed with KC stain sections. RESULTS: Ring enhancement was noted in 13 of 18 HCC (72%). Peritumoral KC density was increased in the ring enhancement (+) group as compared with the ring enhancement (-) group. In the ring enhancement (+) group, the tumor size measured on T2W was smaller than that measured on either T1W or T2*W, suggesting a sustained T1 relaxation effect and a decreased T2* relaxation effect in the peritumoral regions. CONCLUSION: Ring enhancement on superparamagnetic iron oxide (SPIO)-enhanced T1W may correlate with increased KC density and decreased SPIO clustering in KC in peritumoral regions.     

体外磁共振成像对神经干细胞标记后弛豫活性的实验研究

目的:研究超顺磁性氧化铁颗粒(SPIO)标记细胞后在细胞成像领域的应用价值。方法:SPIO与多聚赖氨酸(PLL)联合标记神经干细胞,使用4.7T磁共振对标记细胞进行T1WI、T2WI及T2*WI扫描,并测量标记细胞及未标记细胞的弛豫率R2和R2*。结果:①与未标记细胞相比,标记细胞于T1WI时信号强度平均上升24.06%,T2WI时信号强度平均下降50.66%,T2*WI时信号强度平均下降53.70%。②未标记细胞和标记细胞的T2分别为516 ms和77 ms,弛豫率R2分别为1.94/s及12.98/s;T2*分别为109 ms和22.9 ms,其弛豫率R2*分别为9.17/s及43.67/s。标记细胞的R2及R2*分别约增强了5倍及4倍。结论:SPIO能够有效的标记神经干细胞,明显提高标记细胞的R2及R2*,T2WI和T2*WI序列对显示标记细胞与未标记细胞间的信号差异较敏感。     

Depicting adoptive immunotherapy for prostate cancer in an animal model with magnetic resonance imaging

Genetically modified natural killer (NK) cells that recognize tumor‐associated surface antigens have recently shown promise as a novel approach for cancer immunotherapy. To determine NK cell therapy response early, a real‐time, noninvasive method to quantify NK cell homing to the tumor is desirable. The purpose of this study was to evaluate if MR imaging could provide a noninvasive, in vivo diagnosis of NK cell accumulation in epithelial cell adhesion molecule (EpCAM)‐positive prostate cancers in a rat xenograft model. Genetically engineered NK‐92‐scFv(MOC31)‐ζ cells, which express a chimeric antigen receptor specific to the tumor‐associated EpCAM antigen, and nontargeted NK‐92 cells were labeled with superparamagnetic particles of iron‐oxides (SPIO) ferumoxides. Twelve athymic rats with implanted EpCAM positive DU145 prostate cancers received intravenous injections of 1.5 × 10⁷ SPIO labeled NK‐92 and NK‐92‐scFv(MOC31)‐ζ cells. EpCAM‐positive prostate cancers demonstrated a progressive and a significant decline in contrast‐to‐noise‐ratio data at 1 and 24 h after injection of SPIO‐labeled NK‐92‐scFv(MOC31)‐ζ cells. Conversely, tumor contrast‐to‐noise‐ratio data did not change significantly after injection of SPIO‐labeled parental NK‐92 cells. Histopathology confirmed an accumulation of the genetically engineered NK‐92‐scFv(MOC31)‐ζ cells in prostate cancers. Thus, the presence or absence of a tumor accumulation of therapeutic NK cells can be monitored with cellular MR imaging. EpCAM‐directed, SPIO labeled NK‐92‐scFv(MOC31)‐ζ cells accumulate in EpCAM‐positive prostate cancers. Magn Reson Med, 2011. © 2010 Wiley‐Liss, Inc.     

Relaxometry model of strong dipolar perturbers for balanced-SSFP: application to quantification of SPIO loaded cells.

Magnetic resonance microscopy using magnetically labeled cells is an emerging discipline offering the potential for non-destructive studies targeting numerous cellular events in medical research. The present work develops a technique to quantify superparamagnetic iron-oxide (SPIO) loaded cells using fully balanced steady state free precession (b-SSFP) imaging. An analytic model based on phase cancellation was derived for a single particle and extended to predict mono-exponential decay versus echo time in the presence of multiple randomly distributed particles. Numerical models verified phase incoherence as the dominant contrast mechanism and evaluated the model using a full range of tissue decay rates, repetition times, and flip angles. Numerical simulations indicated a relaxation rate enhancement (DeltaR(2b)=0.412 gamma . LMD) proportional to LMD, the local magnetic dose (the additional sample magnetization due to the SPIO particles), a quantity related to the concentration of contrast agent. A phantom model of SPIO loaded cells showed excellent agreement with simulations, demonstrated comparable sensitivity to gradient echo DeltaR(*) (2) enhancements, and 14 times the sensitivity of spin echo DeltaR(2) measurements. We believe this model can be used to facilitate the generation of quantitative maps of targeted cell populations.     

Instant MR labeling of stem cells using magnetoelectroporation.

For cellular MR imaging, conventional approaches to intracellular magnetic labeling of nonphagocytic cells rely on the use of secondary compounds such as transfection agents and prolonged incubation of cells. Magnetoelectroporation (MEP) was investigated as an alternative method to achieve instant (<1 s) endosomal labeling with the FDA-approved formulation Feridex, without the need for adjunct agents or initiating cell cultures. While MEP was harmful at higher voltages or pulse durations, the procedure could be properly calibrated using a pulse of 130 V and 17 ms. Labeling was demonstrated for stem cells from mice, rats, and humans; the uptake of iron was in the picogram range and comparable to values obtained using transfection agents. MEP-labeled stem cells exhibited an unaltered viability, proliferation, and mitochondrial metabolic rate. Labeled mesenchymal stem cells (MSCs) and neural stem cells (NSCs) differentiated into adipogenic, osteogenic, and neural lineages in an identical fashion as unlabeled cells, while containing Feridex particles as demonstrated by double immunofluorescent staining. MEP-labeled NSCs proliferated normally following intrastriatal transplantation and could be readily detected by MR imaging in vivo. As MEP circumvents the use of secondary agents, obviating the need for clinical approval, MEP labeling may be ideally suitable for bedside implementation.     

Comparison of ferucarbotran‐enhanced fluid‐attenuated inversion‐recovery echo‐planar,T2‐weighted turbo spin‐echo,T2*‐weighted gradient‐echo,and diffusion‐weighted echo‐planar imaging for detection of malignant liver lesions

Purpose:

To compare the diagnostic accuracy of superparamagnetic iron oxide (SPIO)‐enhanced fluid‐attenuated inversion‐recovery echo‐planar imaging (FLAIR EPI) for malignant liver tumors with that of T2‐weighted turbo spin‐echo (TSE), T2*‐weighted gradient‐echo (GRE), and diffusion‐weighted echo‐planar imaging (DW EPI).

Materials and Methods:

SPIO‐enhanced magnetic resonance imaging (MRI) that included FLAIR EPI, T2‐weighted TSE, T2*‐weighted GRE, and DW EPI sequences was performed using a 3 T system in 54 consecutive patients who underwent surgical exploration with intraoperative ultrasonography. A total of 88 malignant liver tumors were evaluated. Images were reviewed independently by two blinded observers who used a 5‐point confidence scale to identify lesions. Results were correlated with results of histopathologic findings and surgical exploration with intraoperative ultrasonography. The accuracy of each MRI sequence was measured with jackknife alternative free‐response receiver operating characteristic analysis. The sensitivity of each observer with each MRI sequence was compared with McNemar's test.

Results:

Accuracy values were significantly higher with FLAIR EPI sequence (0.93) than with T2*‐weighted GRE (0.80) or DW EPI sequences (0.80) (P < 0.05). Sensitivity was significantly higher with the FLAIR EPI sequence than with any of the other sequences.

Conclusion:

SPIO‐enhanced FLAIR EPI sequence was more accurate in the diagnosis of malignant liver tumors than T2*‐weighted GRE and DW EPI sequences. SPIO‐enhanced FLAIR EPI sequence is helpful for the detection of malignant liver tumors. J. Magn. Reson. Imaging 2010;31:607–616. ©2010 Wiley‐Liss, Inc.