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Magnetic Particle Imaging (MPI) is a new imaging modality that sensitively and specifically detects superparamagnetic iron oxide nanoparticles (SPIONs). Many labs have been developing cellular magnetic resonance imaging (MRI) tools using both SPIONs and fluorine-19 (19F)-based contrast agents for numerous important applications, including tracking of immune and stem cells used for cellular therapies. SPION-based MRI cell tracking has very high sensitivity, but low specificity. SPIONs produce negative contrast in MRI, or signal voids. SPIO is not directly detected by MRI, but indirectly through its relaxation effects on protons, therefore, it is not possible to reliably quantify the local tissue concentration of SPION particles and cell number cannot be determined. 19F based cell tracking uses perfluorocarbons (PFC) to label cells. The number of 19F atoms can be directly measured from 19F MR images and related to cell number. 19F MRI has high specificity, but low sensitivity. MPI cell tracking displays great potential for overcoming the challenges of MRI-based cell tracking allowing for both high cellular sensitivity and high specificity and quantification of SPIO labeled cell number. In this paper we describe nanoparticle and MPI system factors that influence MPI sensitivity and resolution, quantification methods and give our perspective on testing and applying MPI for cell tracking.

Magnetic particle imaging (MPI) is an emerging molecular imaging technique that directly detects iron nanoparticles distributed in living subjects. Compared with imaging iron with magnetic resonance imaging (MRI), MPI signal can be measured to determine iron content in specific regions. In this paper, the detection of iron-labeled macrophages associated with cancer by MRI and MPI was compared. Imaging was performed on 4T1 tumor-bearing mice 16–21 days post-cancer cell implantation, 24 h after intravenous injection of Ferucarbotran, a superparamagnetic iron oxide (SPIO) or Ferumoxytol, an ultra-small SPIO. Images of living mice were acquired on a 3T clinical MRI (General Electric, n = 6) or MPI (Magnetic Insight, n = 10) system. After imaging, tumors and lungs were removed, imaged by MPI and examined by histology. MRI signal voids were observed within all tumors. In vivo, MPI signals were observed in the tumors of 4 of 5 mice after the administration of each contrast agent and in all excised tumors. Signal voids visualized by MRI were more apparent in tumors of mice injected with Ferumoxytol than those that received Ferucarbotran; this was consistent with iron content measured by MPI. Signal voids relating to macrophage uptake of iron were not detected in lungs by MRI, since air also appears hypointense. In vivo, MPI could not differentiate between iron in the lungs vs the high signal from iron in the liver. However, once the lungs were excised, MPI signal was detectable and quantifiable. Histologic examination confirmed iron within macrophages present in the tumors. MPI provides quantitative information on in vivo iron labeling of macrophages that is not attainable with MRI. The optimal iron nanoparticle for MPI in general is still under investigation; however, for MPI imaging of macrophages labeled in vivo by intravenous administration, Ferumoxytol nanoparticles were superior to Ferucarbotran.

The use of imaging to detect and monitor the movement and accumulation of cells in living subjects can provide significant insights that can improve our understanding of metastasis and guide therapeutic development. For cell tracking using Magnetic Resonance Imaging (MRI), cells are labeled with iron oxides and the effects of the iron on water provides contrast. However, due to low specificity and difficulties in quantification with MRI, other modalities and approaches need to be developed. Magnetic Particle Imaging (MPI) is an emerging imaging technique which directly detects magnetic iron, allowing for a specific, quantitative and sensitive readout. Here, we use MPI to image iron-labeled tumor cells longitudinally, from implantation and growth at a primary site to movement to distant anatomic sites. In vivo bioluminescent imaging (BLI) was used to localize tumor metastases and computed tomography (CT) allowed for correlation of these signals to anatomic locations. These three imaging modalities provide information on immune escape and metastasis of iron-labeled, and unlabeled, tumor cells, and the accumulation of cell-free iron contrast over time. We identified iron signals by MPI and tumor cells via BLI, and correlated these positive contrast images with CT scans to reveal the anatomic sites with cancer cells; histologic analysis confirmed the presence of iron-labeled tumor cells in the tissues, suggesting that the metastatic cells retained enough iron for MPI detection. The use of multi-modality cell tracking reveals the movement, accumulation and fates of labeled cells that will be helpful understanding cancer progression and guiding the development of targeted therapies.

PurposeMagnetic particle imaging (MPI) and fluorine-19 (19F) MRI produce images which allow for quantification of labeled cells. MPI is an emerging instrument for cell tracking, which is expected to have superior sensitivity compared to 19F MRI. Our objective is to assess the cellular sensitivity of MPI and 19F MRI for detection of mesenchymal stem cells (MSC) and breast cancer cells.MethodsCells were labeled with ferucarbotran or perfluoropolyether, for imaging on a preclinical MPI system or 3 Tesla clinical MRI, respectively. In vivo sensitivity with MPI and 19F MRI was evaluated by imaging MSC that were administered by different routes.ResultsUsing the same imaging time, as few as 4000 MSC (76 ng iron) and 8000 breast cancer cells (74 ng iron) were reliably detected with MPI, and 256,000 MSC (9.01 × 101619F atoms) were detected with 19F MRI, with SNR > 5. In vivo imaging revealed reduced sensitivity compared to ex vivo cell pellets of the same cell number.ConclusionMPI has the potential to be more sensitive than 19F MRI for cell tracking. We attribute reduced MPI and 19F MRI cell detection in vivo to the effect of cell dispersion among other factors, which are described.

Metastasis is the leading cause of mortality in breast cancer patients, with brain metastases becoming increasingly prevalent. Studying this disease is challenging due to the limited experimental models and methods available. Here, we used iron-based cellular MRI to track the fate of a mammary carcinoma cell line (MDA-MB-231-BR)in vivoto characterize the growth of brain metastases in the nude and severely immune-compromised NOD/SCID/ILIIrg−/− (NSG) mouse.Nude and NSG mice received injections of iron-labeled MDA-MB-231-BR cells. Images were acquired with a 3T MR system and assessed for signal voids and metastases. The percentage of signal voids and the number and volume of metastases were quantified.Ex vivoimaging of the liver, histology, and immunofluorescence labeling was performed.On day 0, iron-labeled cells were visualized as signal voids throughout the brain. The percentage of voids decreased significantly between day 0 and endpoint. At endpoint, there was no difference in the number of brain metastases or tumour burden in NSG mice compared to nudes. Tumour volumes in nude mice were significantly larger than in NSG mice. Body images indicated that the NSG mice had metastases in the liver, lungs, and lymph nodes.Characterization of the NSG and nude mouse is necessary to study breast cancer brain metastasisin vivo. Here, we show that the 231BR cell line grew differently in NSG mice compared to nude mice. This work demonstrates the role that imaging can play toward credentialing these models that cannot be done with otherin vitroor histopathologic methods alone.

Triple negative breast cancer (TNBC), when associated with poor outcome, is aggressive in nature with a high incidence of brain metastasis and the shortest median overall patient survival after brain metastasis development compared to all other breast cancer subtypes. As therapies that control primary cancer and extracranial metastatic sites improve, the incidence of brain metastases is increasing and the management of patients with breast cancer brain metastases continues to be a significant clinical challenge. Mouse models have been developed to permit in depth evaluation of breast cancer metastasis to the brain. In this study, we compare the efficiency and metastatic potential of two experimental mouse models of TNBC. Longitudinal MRI analysis and end point histology were used to quantify initial cell arrest as well as the number and volume of metastases that developed in mouse brain over time. We showed significant differences in MRI appearance, tumor progression and model efficiency between the syngeneic 4T1-BR5 model and the xenogeneic 231-BR model. Since TNBC does not respond to many standard breast cancer treatments and TNBC brain metastases lack effective targeted therapies, these preclinical TNBC models represent invaluable tools for the assessment of novel systemic therapeutic approaches. Further pursuits of therapeutics designed to bypass the blood tumor barrier and permit access to the brain parenchyma and metastatic cells within the brain will be paramount in the fight to control and treat lethal metastatic cancer.

The therapeutic potential of mesenchymal stem cells (MSCs) is limited, as many cells undergo apoptosis following administration. In addition, the attraction of immune cells (predominately macrophages) to the site of implantation can lead to MSC rejection. We implemented a trimodal imaging technique to monitor the fate of transplanted MSCs and infiltrating macrophages in vivo. MSCs were labeled with an iron oxide nanoparticle (ferumoxytol) and then implanted within the hind limb muscle of 10 C57BI/6 mice. Controls received unlabeled MSCs (n = 5). A perfluorocarbon agent was administered intravenously for uptake by phagocytic macrophages in situ, 1 and 12 days later, the ferumoxytol-labeled MSCs were detected by proton (1H) magnetic resonance imaging (MRI) and magnetic particle imaging (MPI). Perfluorocarbon-labeled macrophages were detected by fluorine-19 (19F) MRI. 1H/19F MRI was acquired on a clinical scanner (3 T) using a dual-tuned surface coil and balanced steady-state free precession (bSSFP) sequence. The measured volume of signal loss and MPI signal declined over 12 days, which is consistent with the death and clearance of iron-labeled MSCs. 19F signal persisted over 12 days, suggesting the continuous infiltration of perfluorocarbon-labeled macrophages. Because MPI and 19F MRI signals are directly quantitative, we calculated estimates of the number of MSCs and macrophages present over time. The presence of MSCs and macrophages was validated with histology following the last imaging session. This is the first study to combine the use of iron- and fluorine-based MRI with MPI cell tracking.

Purpose Incidence of brain metastasis attributed to breast cancer is increasing and prognosis is poor. It is thought that disseminated dormant cancer cells persist in metastatic organs and may evade treatments, thereby facilitating a mechanism for recurrence. Radiotherapy is used to treat brain metastases clinically, but assessment has been limited to macroscopic tumor volumes detectable by clinical imaging. Here, we use cellular MRI to understand the concurrent responses of metastases and nonproliferative or slowly cycling cancer cells to radiotherapy. Methods MRI cell tracking was used to investigate the impact of early cranial irradiation on the fate of individual iron-labeled cancer cells and outgrowth of breast cancer brain metastases in the human MDA-MB-231-BR-HER2 cell model. Results Early whole-brain radiotherapy significantly reduced the outgrowth of metastases from individual disseminated cancer cells in treated animals compared to controls. However, the numbers of nonproliferative iron-retaining cancer cells in the brain were not significantly different. Conclusions Radiotherapy, when given early in cancer progression, is effective in preventing the outgrowth of solitary cancer cells to brain metastases. Future studies of the nonproliferative cancer cells’ clonogenic potentials are warranted, given that their persistent presence suggests that they may have evaded treatment. Magn Reson Med 78:1506–1512, 2017. © 2016 International Society for Magnetic Resonance in Medicine

Purpose Cellular MRI) was used to detect implanted human mesenchymal stem cells (hMSCs) and the resulting macrophage infiltration that occurs in response to xenotransplantation. Methods Human mesenchymal stem cells were prelabeled with a fluorine-19 (19F) agent prior to implantation, allowing for their visualization and quantification over time. Following implantation of 1 × 106 19F-labeled hMSCs into the mouse hind limb, longitudinal imaging was performed to monitor the stem cell graft. Macrophages were labeled in situ by the intravenous administration of an ultrasmall superparamagentic iron oxide (USPIO), allowing for tracking of the inflammatory response. Results Quantification of 19F MRI on day 0 agreed with the implanted number of cells, and 19F signal decreased over time. By day 14, only 22% ± 11% of the original 19F signal remained. In a second group, USPIO were administered intravenously after implantation of 19F-labeled hMSCs. When imaged on day 2, a significant decrease in 19F signal was observed compared to the first group alongside a large signal void region in the corresponding proton images. Immunohistochemistry confirmed the presence of iron-labeled macrophages in the stem cell tract. Conclusion A dual-labeling technique was used to noninvasively track two distinct cell populations simultaneously. This information could be used to provide additional insight into the cause of graft failure. © 2016 International Society for Magnetic Resonance in Medicine.

Fluorine-19 (19F)-based contrast agents for magnetic resonance imaging stand to revolutionize imaging-based research and clinical trials in several fields of medical intervention. First, their use in characterizing in vivo cell behavior may help bring cellular therapy closer to clinical acceptance. Second, their use in lung imaging provides novel noninvasive interrogation of the ventilated airspaces without the need for complicated, hard-to-distribute hardware. This article reviews the current state of 19F-based cell tracking and lung imaging using magnetic resonance imaging and describes the link between the methods across these fields and how they may mutually benefit from solutions to mutual problems encountered when imaging 19F-containing compounds, as well as hardware and software advancements.

Cellular magnetic resonance imaging (MRI) enables visualization of cells in vivo. This is accomplished by labeling cells with superparamagnetic iron oxide nanoparticles. Here, we describe the steps for labeling human cancer cells with iron for tracking them after injection into nude mice. We also provide details for validation of cell labeling, ultrasound guided intra-cardiac injection, and MRI.

The mechanisms that influence metastatic growth rates are poorly understood. One mechanism of interest known as concomitant tumour resistance (CTR) can be defined as the inhibition of metastasis by existing tumour mass. Conversely, the presence of a primary tumour has also been shown to increase metastatic outgrowth, termed concomitant tumour enhancement (CTE). The majority of studies evaluating CTR/CTE in preclinical models have relied on endpoint histological evaluation of tumour burden. The goal of this research was to use conventional magnetic resonance imaging (MRI), cellular MRI, and bioluminescence imaging to study the impact of a primary tumour on the development of brain metastases in a syngeneic mouse model. Here, we report that the presence of a 4T1 primary tumour significantly enhances total brain tumour burden in Balb/C mice. Using in vivo BLI/MRI we could determine this was not related to differences in initial arrest or clearance of viable cells in the brain, which suggests that the presence of a primary tumour can increase the proliferative growth of brain metastases in this model. The continued application of our longitudinal cellular and molecular imaging tools will yield a better understanding of the mechanism(s) by which this physiological inhibition (CTR) and/or enhancement (CTE) occurs.

Background Brain metastasis is becoming increasingly prevalent in breast cancer due to improved extra-cranial disease control. With emerging availability of modern image-guided radiation platforms, mouse models of brain metastases and small animal magnetic resonance imaging (MRI), we examined brain metastases’ responses from radiotherapy in the pre-clinical setting. In this study, we employed half brain irradiation to reduce inter-subject variability in metastases dose-response evaluations. Methods Half brain irradiation was performed on a micro-CT/RT system in a human breast cancer (MDA-MB-231-BR) brain metastasis mouse model. Radiation induced DNA double stranded breaks in tumors and normal mouse brain tissue were quantified using γ-H2AX immunohistochemistry at 30 min (acute) and 11 days (longitudinal) after half-brain treatment for doses of 8, 16 and 24 Gy. In addition, tumor responses were assessed volumetrically with in-vivo longitudinal MRI and histologically for tumor cell density and nuclear size. Results In the acute setting, γ-H2AX staining in tumors saturated at higher doses while normal mouse brain tissue continued to increase linearly in the phosphorylation of H2AX. While γ-H2AX fluorescence intensities returned to the background level in the brain 11 days after treatment, the residual γ-H2AX phosphorylation in the radiated tumors remained elevated compared to un-irradiated contralateral tumors. With radiation, MRI-derived relative tumor growth was significantly reduced compared to the un-irradiated side. While there was no difference in MRI tumor volume growth between 16 and 24 Gy, there was a significant reduction in tumor cell density from histology with increasing dose. In the longitudinal study, nuclear size in the residual tumor cells increased significantly as the radiation dose was increased. Conclusions Radiation damages to the DNAs in the normal brain parenchyma are resolved over time, but remain unrepaired in the treated tumors. Furthermore, there is a radiation dose response in nuclear size of surviving tumor cells. Increase in nuclear size together with unrepaired DNA damage indicated that the surviving tumor cells post radiation had continued to progress in the cell cycle with DNA replication, but failed cytokinesis. Half brain irradiation provides efficient evaluation of dose-response for cancer cell lines, a pre-requisite to perform experiments to understand radio-resistance in brain metastases.

OBJECTIVES: Brain metastases due to breast cancer are increasing, and the prognosis is poor. Lack of effective therapy is attributed to heterogeneity of breast cancers and their resulting metastases, as well as impermeability of the blood–brain barrier (BBB), which hinders delivery of therapeutics to the brain. This work investigates three experimental models of HER2+ breast cancer brain metastasis to better understand the inherent heterogeneity of the disease. We use magnetic resonance imaging (MRI) to quantify brain metastatic growth and explore its relationship with BBB permeability. DESIGN: Brain metastases due to breast cancer cells (SUM190-BR3, JIMT-1-BR3, or MDA-MB-231-BR-HER2) were imaged at 3 T using balanced steady-state free precession and contrast-enhanced T1-weighted spin echo sequences. The histology and immunohistochemistry corresponding to MRI were also analyzed. RESULTS: There were differences in metastatic tumor appearance by MRI, histology, and immunohistochemistry (Ki67, CD31, CD105) across the three models. The mean volume of an MDA-MB-231-BR-HER2 tumor was significantly larger compared to other models (F2,12 = 5.845, P < .05); interestingly, this model also had a significantly higher proportion of Gd-impermeable tumors (F2,12 = 22.18, P < .0001). Ki67 staining indicated that Gd-impermeable tumors had significantly more proliferative nuclei compared to Gd-permeable tumors (t[24] = 2.389, P < .05) in the MDA-MB-231-BR-HER2 model. CD31 and CD105 staining suggested no difference in new vasculature patterns between permeable and impermeable tumors in any model. CONCLUSION: Significant heterogeneity is present in these models of brain metastases from HER2+ breast cancer. Understanding this heterogeneity, especially as it relates to BBB permeability, is important for improvement in brain metastasis detection and treatment delivery.

Whole-brain radiotherapy is the standard of care for patients with breast cancer with multiple brain metastases and, although this treatment has been essential in the management of existing brain tumors, there are many known negative consequences associated with the irradiation of normal brain tissue. In our study, we used in vivo magnetic resonance imaging analysis to investigate the influence of radiotherapy-induced damage of healthy brain on the arrest and growth of metastatic breast cancer cells in a mouse model of breast cancer brain metastasis. We observed that irradiated, but otherwise healthy, neural tissue had an increased propensity to support metastatic growth compared with never-irradiated controls. The elucidation of the impact of irradiation on normal neural tissue could have implications in clinical patient management, particularly in patients with residual systemic disease or with residual radio-resistant brain cancer.

In general, brain metastases occur in about 10% of metastatic cancer patients, but the incidence may be as high as 40–50% for some diagnoses such as lung cancer or HER2+ breast cancer. The brain is considered a “sanctuary site” for metastatic growth due to the presence of the blood–brain barrier, which excludes most systemic anticancer drugs from entering the brain to inhibit tumor growth. In the absence of effective drugs for brain metastases, treatment options are limited to surgery or radiation therapy. The prognosis for this disease is poor and survival is estimated in months, not years. Experimental models of brain metastases, along with novel preclinical imaging strategies, provide excellent tools for studying the biology of this disease over time and investigating response to new therapies. Improved understanding of brain metastatic progression is desperately needed to develop treatment strategies for the eradication of this disease.

ABSTRACT: Cellular MRI uses superparamagnetic iron oxide nanoparticles to label cells (in vitro or in vivo) for detection in magnetic resonance images. The infiltration of inflammatory macrophages can be visualized in brain diseases, such as multiple sclerosis, stroke and Alzheimer‘s disease, and correlates with disease severity and responses to treatments. Mesenchymal stromal cells, neural stem cells and immune cells used as cell therapies in CNS diseases can be tracked in vivo over time to determine their migration and dispersion. Tracking labeled cancer cells provides information about metastasis and proliferative status in preclinical tumor models. Ongoing technical improvements come from the development of new particles, the use of fluorine-based contrast agents and the refinement of high-field MRI for cell tracking.

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.

The bacterial gene MagA imparts magnetic properties to mammalian cells and provides a basis for cell tracking by magnetic resonance imaging (MRI). In a mouse model of tumor growth from transplanted cells, we used repetitive MRI to demonstrate the in vivo imaging potential of MagA expression relative to a modified ferritin overexpression system, lacking regulation through iron response elements (HF + LF).Subcutaneous tumor xenografts were monitored weekly from days 2 to 34 post-injection. Small animal MRI employed balanced steady-state free precession. Imaging was correlated with tumor histology using hematoxylin, Prussian Blue, Ki-67, and BS-1 lectin.Tumor heterogeneity with respect to tissue morphology and magnetic resonance (MR) contrast was apparent within a week of cell transplantation. In MagA- and HF + LF-expressing tumors, MR contrast enhancement was recorded up to day 20 post-injection and 0.073-cm(3) tumor volumes. MagA-expressing tumors showed increases in both quantity and quality of MR contrast as measured by fractional void volume and contrast-to-noise ratio, respectively. MR contrast in both MagA- and HF + LF-expressing tumors was maximal by day 13, doubling fractional void volume 1 week ahead of controls.MagA- and HF + LF-expressing tumor xenografts augment MR contrast after 1 week of growth. MagA expression increases MR contrast within days of cell transplantation and provides MR contrast comparable to HF + LF. MagA has utility for monitoring cell growth and differentiation, with potential for in vivo detection of reporter gene expression using MRI.

Cell tracking with magnetic resonance imaging (MRI) and iron nanoparticles is commonly used to monitor the fate of implanted cells in preclinical disease models. Few studies have employed these methods to study cancer cells because proliferative iron-labeled cancer cells will lose the label as they divide. In this study, we evaluate the potential for retention of the iron nanoparticle label, and resulting MRI signal, to serve as a marker for slowly dividing cancer cells. Green fluorescent protein-transfected MDA-MB-231 breast cancer cells were labeled with red fluorescent micron-sized superparamagnetic iron oxide (MPIO) nanoparticles. Cells were examined in vitro at multiple time points after labeling by staining for iron-labeled cells and by flow cytometric detection of the fluorescent MPIO. Severe combined immune deficiency (SCID) mice were implanted with 5 x 105 MPIO-labeled or unlabeled cells in the mammary fat pad and MRI was performed weekly until 28 days after injection. Microscopy was performed to validate MRI. In vitro assays revealed a very small percentage of cells that retained MPIO at 14 days after labeling. Regions of signal loss were observed in MRI of primary tumors that developed from iron-labeled cancer cells. Small focal regions of signal loss were detected in images of the axillary and brachial nodes in six of eight mice, at day 14 or later, with microscopy confirming the presence of iron-labeled cancer cells. Our data suggest an interesting role for cell tracking with iron particles since label retention leads to persistent signal void, allowing proliferative status to be determined.

Cellular MRI involves sensitive visualization of iron-labeled cells in vivo but cannot differentiate between dead and viable cells. Bioluminescence imaging (BLI) measures cellular viability, and thus we explored combining these tools to provide a more holistic view of metastatic cancer cell fate in mice. Human breast carcinoma cells stably expressing Firefly luciferase were loaded with iron particles, injected into the left ventricle, and BLI and MRI were performed on days 0, 8, 21 and 28. The number of brain MR signal voids (i.e., iron-loaded cells) on day 0 significantly correlated with BLI signal. Both BLI and MRI signals decreased from day 0 to day 8, indicating a loss of viable cells rather than a loss of iron label. Total brain MR tumour volume on day 28 also correlated with BLI signal. Overall, BLI complemented our sensitive cellular MRI technologies well, allowing us for the first time to screen animals for successful injections, and, in addition to MR measures of cell arrest and tumor burden, provided longitudinal measures of cancer cell viability in individual animals. We predict this novel multimodality molecular imaging framework will be useful for evaluating the efficacy of emerging anti-cancer drugs at different stages of the metastatic cascade.

Cellular magnetic resonance imaging (MRI) is an evolving field of imaging with strong translational and research potential. The ability to detect, track, and quantify cells in vivo and over time allows for studying cellular events related to disease processes and may be used as a biomarker for decisions about treatments and for monitoring responses to treatments. In this review, we discuss methods for labeling cells, various applications for cellular MRI, the existing limitations, strategies to address these shortcomings, and clinical cellular MRI.

Stem cells, cancer cells and immune cells were labeled by co-incubation with a new ultra-small iron oxide nanoparticle called Molday ION Rhodamine-B (MIRB). Iron staining, fluorescence imaging, transmission electron microscopy and flow cytometry were used to assess cell viability, function and labeling efficiency. This study has shown that MIRB can be used to label both adherent and nonadherent cell lines, with high viability and loading levels sufficient for their detection in vivo by MRI at 3 T.

An optimized non-invasive imaging modality capable of tracking and quantifying in vivo DC migration in patients would provide clinicians with valuable information regarding therapeutic DC-based vaccine outcomes. Superparamagnetic iron oxide (SPIO) nanoparticles were used to label bone marrow-derived DC. In vivo DC migration was tracked and quantified non-invasively using cellular magnetic resonance imaging (MRI) in a mouse model. Labelling DC with SPIO reflects the kinetics of DC migration in vivo but appears to reduce overall DC migration, in part due to nanoparticle size. Magnetic separation of SPIO-labelled (SPIO(+)) DC from unlabelled (SPIO(-)) DC prior to injection improves SPIO(+) DC migration to the lymph node. Corresponding MR image data better correlate with the presence of DC in vivo; an improved immunological response is also seen. Cellular MRI is a viable, non-invasive imaging tool that can routinely track DC migration in vivo. Consideration should be given to optimizing MRI contrast agent-labelling of clinical-grade DC in order to accurately correlate DC fate to immunological outcomes in patients.

This study seeks to assess the use of labeling with micron-sized iron oxide (MPIO) particles for the detection and quantification of the migration of dendritic cells (DCs) using cellular magnetic resonance imaging (MRI).DCs were labeled with red fluorescent MPIO particles for detection by cellular MRI and a green fluorescent membrane dye (PKH67) for histological detection. MPIO-labeled DCs or unlabeled control DCs were injected into mice footpads at two doses (0.1 × 10(6) and 1 × 10(6)). Images were acquired at 3 Tesla before DC injection and 2, 3, and 7 days post-DC injection.Labeling DCs with MPIO particles did not affect viability, but it did alter markers of DC activation and maturation. MRI and fluorescence microscopy allowed for the detection of MPIO-labeled DCs within the draining popliteal nodes after their injection into the footpad.This paper presents the first report of the successful use of fluorescent MPIO particles to label and track DC migration.

The objective of this study was to track the fate of iron-labeled, multipotent stromal cells (MSC) after their direct transplantation into mice with spinal cord injuries using magnetic resonance imaging (MRI). Mice with spinal cord injuries received a direct transplant of (1) live MSC labeled with micron-sized iron oxide particles (MPIO); (2) dead, MPIO-labeled MSC; (3) unlabeled MSC; or (4) free MPIO and were imaged at 3 T for 6 weeks after transplantation. Live, iron-labeled MSC appeared as a well-defined region of signal loss in the mouse spinal cord at the site of transplant. However, the MR appearance of dead, iron-labeled MSC and free iron particles was similar and persisted for the 6 weeks of the study. Iron-labeled stem cells can be detected and monitored in vivo after direct transplantation into the injured spinal cord of mice. However, the fate of the iron label is not clear. Our investigation indicates that caution should be taken when interpreting MR images after direct transplantation of iron-labeled cells.

The metastatic cell population, ranging from solitary cells to actively growing metastases, is heterogeneous and unlikely to respond uniformly to treatment. However, quantification of the entire experimental metastatic cell population in whole organs is complicated by requirements of an imaging modality with the large field of view and high spatial resolution necessary to detect both single cells and metastases in the same organ. Thus, it is difficult to assess differential responses of these distinct metastatic populations to therapy. Here, we develop a magnetic resonance imaging (MRI) technique capable of quantifying the full population of metastatic cells in a secondary organ. B16F1 mouse melanoma cells were labeled with micron-sized iron oxide particles (MPIO) and injected into mouse liver via the mesenteric vein. Livers were removed immediately or at day 9 or 11, following doxorubicin or vehicle control treatment, and imaged using a 3T clinical magnetic resonance scanner and custom-built gradient coil. Both metastases (>200 μm) and MPIO-labeled single cells were detected and quantified from MR images as areas of hyperintensity or hypointensity (signal voids), respectively. We found that 1mg/kg doxorubicin treatment inhibited metastasis growth (n = 11 per group; P = 0.02, t test) but did not decrease the solitary metastatic cell population in the same livers (P > 0.05). Thus, the technique presented here is capable of quickly quantifying the majority of the metastatic cell population, including both growing metastases and solitary cells, in whole liver by MRI and can identify differential responses of growing metastases and solitary cells to therapy. [Cancer Res 2009;69(21):8326–31]

Object An understanding of single glioma cell invasion has been limited by the static picture provided by histological studies. The ability to nondestructively assess cell invasion dynamically in a full 3D volume would improve the quality and quantity of information available from both in vivo and in vitro experiments. The purpose of this study was to observe glioma cell invasion in a 3D in vitro model using a microimaging protocol at 1.5 tesla and to assess the uptake of micron-sized particles of iron oxide (MPIO) and the consequent effects on cell function. Methods Rat C6 glioma cells were labeled with MPIO to a sufficient extent to allow single cell detection in vitro without significant effects on cell proliferation or plating efficiency. When placed on agar-coated plates, the cells formed stable multicellular tumor spheroids (MCTSs), which were embedded in collagen type I gel and serially visualized using magnetic resonance (MR) imaging and phase-contrast microscopy over 8 days. The MCTSs initially appeared as large susceptibility artifacts on MR images, but within 2 days, as cells moved away from the main MCTS, small discrete areas of signal loss, possibly due to single cells, could be observed and tracked. Conclusions Glioma cell invasion can be nondestructively observed using MR imaging. The sensitivity of MR imaging, along with its ability to represent full 3D volumes noninvasively over time, makes it ideal for longitudinal in vivo cell tracking studies.

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. J. Magn. Reson. Imaging 2007;26:144–151. © 2007 Wiley-Liss, Inc.

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.

Purpose To directly correlate spinal cord pathology of guinea pigs with experimental allergic encephalomyelitis (EAE) to the MRI data obtained at 1.5T. Materials and Methods Spinal cords from EAE animals were imaged in vivo with the following MRI sequences: T2-FSE, PD-FSE, fluid-attenuated inversion recovery (FLAIR)-FSE, T2-CSE, T1-CSE, T1-CSE + gadolinium-DTPA (Gd-DTPA), PD-CSE, and short-tau inversion recovery (STIR)-FSE. The spinal cords were removed and the lesions with specific pathological compositions were identified by histological analysis. Regions of interest (ROIs) were drawn on the corresponding MR images, and signal-to-noise ratios (SNRs) were measured for each MR sequence and compared with controls. Results The receiver operating characteristic (ROC) analysis of STIR-FSE and PD-CSE was able to differentiate tissue that contained cellular infiltrates with a high degree of accuracy. The SNRs of T2-FSE, STIR-FSE, T2-CSE, PD-CSE, and T1-CSE + Gd-DTPA were elevated in lesions that contained cellular infiltrates alone, whereas the SNRs of PD-CSE and T1-CSE + Gd-DTPA were reduced in demyelinated lesions that also contained inflammation. Conclusion The SNR difference between the two lesion groups suggests that the combination of STIR-FSE, PD-CSE, and T1-CSE + Gd-DTPA sequences may be useful for differentiating inflammatory lesions containing demyelination from lesions with inflammation alone. J. Magn. Reson. Imaging 2005;22:180–188. © 2005 Wiley-Liss, Inc.

Brain metastases are an important clinical problem. Few animal models exist for melanoma brain metastases; many of which are not clinically relevant. Longitudinal MRI was implemented to examine the development of tumors in a clinically relevant mouse model of melanoma brain metastases. Fifty thousand human metastatic melanoma (A2058) cells were injected intracardially into nude mice. Three Tesla MRI was performed using a custom-built gradient insert coil and a mouse solenoid head coil. Imaging was performed on consecutive days at four time points. Tumor burden and volumes of metastases were measured from balanced steady-state free precession image data. Metastases with a disrupted blood-tumor barrier were identified from T1-weighted spin echo images acquired after administration of gadopentetic acid (Gd-DTPA). Metastases permeable to Gd-DTPA showed signal enhancement. The number of enhancing metastases was determined by comparing balanced steady-state free precession images with T1-weighted spin echo images. After the final imaging session, ex-vivo permeability and histological analyses were carried out. Imaging showed that both enhancing and nonenhancing brain metastases coexist in the brain, and that most metastases switched from the nonenhancing to the enhancing phenotype. Small numbers of brain metastases were enhancing when first detected by MRI and remained enhancing, whereas other metastases remained nonenhancing to Gd-DTPA throughout the experiment. No clear relationship existed between the permeability of brain metastases and size, brain location and age. Longitudinal in-vivo MRI is key to studying the complex and dynamic processes of metastasis and changes in the blood-tumor barrier permeability, which may lead to a better understanding of the variable responses of brain metastases to treatments.

Breast cancer that has metastasized to the brain presents difficult clinical challenges. This diagnosis comes with high mortality rates, largely due to complexities in early detection and ineffective therapies associated with both dormancy and impermeability of the blood–brain barrier (BBB). Magnetic resonance imaging (MRI) is the current gold standard for diagnosis and assessment of brain tumors. It has been used clinically to investigate metastatic development as well as monitor response to therapy. Here, we describe preclinical imaging strategies that we have used to study the development of brain metastases due to breast cancer. Using this approach, we have identified three subsets of metastatic disease: permeable metastases, nonpermeable metastases, and solitary, dormant cancer cells, which likely have very different biology and responses to therapy. The ability to simultaneously monitor the spatial and temporal distribution of dormant cancer cells, metastatic growth, and associated tumor permeability can provide great insight into factors that contribute to malignant proliferation. Our preclinical findings suggest that standard clinical detection strategies may underestimate the true metastatic burden of breast cancer that has metastasized to the brain. A better understanding of true metastatic burden in brains will be important to assist in the development of more effective chemotherapeutics—particularly those targeted to cross the BBB—as well as detection of small nonpermeable metastases.

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.

Studies of the metastatic process and potential cancer therapies have been advanced by the use of imaging technology that enables the noninvasive assessment of tumor development over time. Several imaging modalities have been used to examine brain metastases in preclinical cancer models. Magnetic resonance imaging (MRI) is the clinical gold standard for anatomical evaluation of brain metastases. New advances in MRI and MR spectroscopy (MRS) have now enabled physiological characteristics of tumors to be investigated including tumor permeability, vascularity, cellularity and metabolism as well as cerebral blood flow and blood volume. MRI can also be used to detect single iron-labeled cancer cells after their initial arrest in mouse brain and subsequent tumor development. Nuclear imaging techniques including positron emission tomography (PET) and single photon emission computed tomography (SPECT) are popular tools for classifying tumors and monitoring their treatment. Brain tumors can be assessed for bio­chemical alterations such as glucose use, DNA synthesis, amino acid transport and oxygenation state. Optical imaging techniques based on the use of fluorescent or bioluminescent reporters have been found advantageous for monitoring metastatic tumor burden in experimental animals. Fluorescent entities have ­further been used in intravital microscopy to track and monitor the relationship between tumor cells and brain vasculature, including cancer cell arrest, early extravasation, perpetuation of a perivascular position and either angiogenesis or vessel co-option. Finally, imaging studies of brain metastases are often improved by using multiple imaging techniques concurrently, thereby exploiting the best features of separate modalities to acquire multilayered information and provide further insights into the evolution of metastases.

The clinical application of dendritic cells (DC) as adjuvants in immunotherapies such as the cell-based cancer vaccine continues to gain interest. The overall efficacy of this emerging immunotherapy, however, remains low. Studies suggest the stage of maturation and activation of ex vivo-prepared DC immediately prior to patient administration is critical to subsequent DC migration in vivo, which ultimately affects overall vaccine efficacy. While it is possible to generate mature and activated DC ex vivo using various stimulatory cocktails, in the case of cancer patients, the qualitative and quantitative assessment of which DC stimulatory cocktail works most effectively to enhance subsequent DC migration in vivo is difficult. Thus, a non-invasive imaging modality capable of monitoring the real-time migration of DC in long-term studies is required. In this paper, we address whether cellular magnetic resonance imaging (MRI) is sufficiently sensitive to quantitatively detect differences in the migratory abilities of two different DC preparations: untreated (resting) versus ex vivo matured in a mouse model. In order to distinguish our ex vivo-generated DC of interest from surrounding tissues in magnetic resonance (MR) images, DC were labeled in vitro with the superparamagnetic iron oxide (SPIO) nanoparticle FeREX®. Characterization of DC phenotype and function following addition of a cytokine maturation cocktail and the toll-like receptor ligand CpG, both in the presence and in the absence of SPIO, were also carried out. Conventional histological techniques were used to verify the quantitative data obtained from MR images. This study provides important information relevant to tracking the in vivo migration of ex vivo-prepared and stimulated DC.

BACKGROUND AIMS. The use of dendritic cells (DC) as an adjuvant in cell-based immunotherapeutic cancer vaccines is a growing field of interest. A reliable and non-invasive method to track the fate of autologous DC following their administration to patients is required in order to confirm that clinically sufficient numbers are reaching the lymph node (LN). We demonstrate that an immunocompromised mouse model can be used to conduct translational studies employing cellular magnetic resonance imaging (MRI). Such studies can provide clinically relevant information regarding the migration potential of clinical-grade DC used in cancer immunotherapies. METHODS. Human monocyte-derived dendritic cells (mo-DC) were generated from negatively selected monocytes obtained from either healthy donors or cancer patients. DC were labeled with superparamagnetic iron oxide (SPIO) nanoparticles in order to track them in vivo in a CB17scid mouse model using cellular MRI. SPIO did not have any adverse effects on DC phenotype or function, independent of donor type. Cellular MRI readily detected migration of SPIO-loaded DC in CB17scid mice. No differences in migration were observed between DC obtained from healthy donors and those obtained from donors undergoing autologous stem cell transplant for cancer therapy. CONCLUSIONS. Cellular MRI provided semi-quantitative image data that corresponded with data obtained by digital morphometry, validating cellular MRI's potential to assess DC migration in DC-based cancer immunotherapy clinical trials.

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).

The goal of this study was to investigate the normal MRI appearance of lymphoid organs in immuno-competent and immuno-deficient mice commonly used in research. Four mice from each of four different mouse strains (nude, NOG, C57BL/6, CB-17 SCID (SCID)) were imaged weekly for one month. Images were acquired with a 3D balanced steady state free precession (bSSFP) sequence. The volume of the lymph nodes and spleens were measured from MR images. In images of nude and SCID mice, lymph nodes sometimes contained a hyperintense region visible on MRI images. Volumes of the nodes were highly variable in nude mice. Nodes in SCID mice were smaller than in nude or C57Bl/6 mice (p

Molecular medicine is focusing its attention on developing immunotherapeutic strategies that engage the immune system to combat a number of human diseases, including cancer. As a result, great emphasis has been placed on enhancing existing imaging modalities and developing new imaging techniques in order to assess the in vivo consequences of a given immunotherapy. Recently, improvements in the resolution and sensitivity of existing in vivo imaging modalities, including computed tomography (CT), ultrasound (US), positron emission tomography (PET), single positron emission tomography (SPECT), optical imaging (OI), and magnetic resonance imaging (MRI), have evolved enormously. In this chapter, each modality, used either individually or together as multi-modal hybrid imaging techniques, will be evaluated in the context of how they contribute to assessing immunotherapies in vivo in preclinical and clinical settings.

Molecular imaging with magnetic resonance imaging (MRI) may benefit from the ferrimagnetic properties of magnetosomes, membrane-enclosed iron biominerals whose formation in magnetotactic bacteria is encoded by multiple genes. One such gene is MagA, a putative iron transporter. We have examined expression of MagA in mouse neuroblastoma N2A cells and characterized their response to iron loading and cellular imaging by MRI. MagA expression augmented both Prussian blue staining and the elemental iron content of N2A cells, without altering cell proliferation, in cultures grown in the presence of iron supplements. Despite evidence for iron incorporation in both MagA and a variant, MagAE137V, only MagA expression produced intracellular contrast detectable by MRI at 11 Tesla. We used this stable expression system to model a new sequence for cellular imaging with MRI, using the difference between gradient and spin echo images to distinguish cells from artifacts in the field of view. Our results show that MagA activity in mammalian cells responds to iron supplementation and functions as a contrast agent that can be deactivated by a single point mutation. We conclude that MagA is a candidate MRI reporter gene that can exploit more fully the superior resolution of MRI in noninvasive medical imaging.

The feasibility of performing high-resolution in vivo magnetic resonance imaging (MRI) to visualize the injured mouse spinal cord using a three-dimensional (3D)-FIESTA (fast imaging employing steady state acquisition) pulse sequence, in a clip compression injury model, is presented. Images were acquired using a 3-Tesla clinical whole-body MR system equipped with a high-performance gradient coil insert. High-resolution mouse cord images were used to detect and monitor the cord lesions for 6 weeks after spinal cord injury (SCI). The epicenter of the injury appeared as a region of mixed signal intensities on day 2 post-SCI. Regions of signal hypointensity appeared at the lesion site by 2 weeks post-SCI and became more apparent with time. In some mice, large cyst-like lesions were detected rostral to the lesion epicenter, as early as 2 weeks post-SCI, and increased in volume with time. In addition, MRI was used to detect and monitor iron-labeled mesenchymal stem cells (MSCs) after their transplantation into the injured cord. MSCs appeared as large, obvious regions of signal loss in the cord, which decreased in size over time.

There is a clinical need to be able to assess graft loss of transplanted pancreatic islets (PI) non-invasively with clear-cut quantification of islet survival. We tracked transplanted PI in diabetic mice during the early post-transplant period by magnetic resonance imaging (MRI) and quantified the islet loss using automatic segmentation technique. Magnetically labeled islet iso-, allo- and xenografts were injected into the right liver lobes. Animals underwent MRI scanning during 14 days after PI transplantation. MR images were processed using custom-made software, which automatically detects hypointense regions representing PI. It is based on morphological top-hat and bottom-hat transforms. Manually and automatically detected areas, corresponding to PI, differed by 4% in phantoms. Signal loss regions due to PI decreased comparably in all groups during the first week post transplant. Throughout the second week post-transplant, the signal loss area continued in a steep decline in case of allografts and xenografts, whereas the decline in case of isografts slowed down. Automatic segmentation allows for the more reproducible, objective assessment of transplanted PI. Quantification confirms the assumption that a significant number of islets are destroyed in the first week following transplantation irrespective of allografts, xenografts or isografts.

Object The purpose of this study was to compare measures of lesion volume obtained by means of 1.5-T MR imaging to those obtained by the Cavalieri method, 6 weeks after experimental spinal cord injury. Methods Nine male Wistar rats were subjected to spinal cord injury by clip compression (50 g) at the T-4 level. Six weeks postinjury, the rats were sacrificed, and spinal cords were analyzed ex vivo for lesion volume by means of 1.5-T MR imaging and subsequently, by the Cavalieri method. In the latter method, cords were cut longitudinally in 25-μm sections and stained with solochrome cyanin for myelin. The area of the lesion was determined for each serial section, and the distance-weighted sum of all area measures was then calculated to estimate the total lesion volume. Results Bland–Altman analysis showed that the 2 methods had an acceptable level of agreement for lesion volume estimation, but the Cavalieri method was prone to an overestimation bias. The MR imaging estimates of lesion volume were greater than the Cavalieri method estimates in 3 spinal cords, but the difference between measures was within 1 standard deviation of perfect agreement in these 3 lesions, and the mean difference between measures was 18.3%. In contrast, in those lesions in which the Cavalieri method yielded larger lesion volumes (5 lesions), the difference between measures was 2 standard deviations away from perfect agreement for 2 animals and the mean difference between measures was 72.4%. Conclusions The results illustrate that the overestimation bias of the Cavalieri method is due, in part, to artifacts produced during processing of the spinal cord tissue.

Introduction Pancreatic islets (PI) labeled with Feridex can be visualized using magnetic resonance imaging (MRI) after transplantation into the liver. However, there is still no accurate method of quantifying the signal loss caused by the iron contrast agent within transplanted tissue. The aim of this study was to test a new method for quantifying signal loss during the early posttransplantation period. Methods Isolated mouse PI (C57BL/6 and BALB/c) were labeled in CMRL-1066 culture media supplemented with Feridex. Two hundred twenty PI were injected directly into the right liver lobes of BALB/c diabetic (streptozotocine 220 mg/kg) recipients (isografts, n = 3; allografts, n = 3). Animals were scanned at 3 T, on a whole body scanner equipped with a high-performance gradient insert and using the 3D FIESTA sequence, on days 1, 7, and 14. Signal loss was quantified by comparison of liver tissue with and without labeled PI. Signal loss detected on the first scan was rated as 100% and subsequent measurements were recalculated as relative numbers. Results While the function of the isografts remained stable throughout the study, the allografts failed on days 5 and 10. A decrease in the amount of signal loss was observed in all animals and was comparable after the first week in both groups. However, there was a difference between groups after the second week (mean ± SD; isografts, 100% ⇒ 61.8 ± 6.74% ⇒ 47.18 ± 7.14%; allografts, 100% ⇒ 59.39 ± 8.54% ⇒ 38.16 ± 6.81%). Conclusion Disappearance of signal loss is comparable in all animals during the first week and seems to be independent of acute rejection.

Metastasis is responsible for most deaths due to malignant melanoma. The clinical significance of micrometastases in the lymph is a hotly debated topic, but an improved understanding of the lymphatic spread of cancer remains important for improving cancer survival. Cellular magnetic resonance imaging (MRI) is a newly emerging field of imaging research that is expected to have a large impact on cancer research. In this study, we demonstrate the cellular MRI technology required to reliably image the lymphatic system in mice and to detect iron-labeled metastatic melanoma cells within the mouse lymph nodes. Melanoma cells were implanted directly into the inguinal lymph nodes in mice, and micro-MRI was performed using a customized 1.5-T clinical MRI system. We show cell detection of as few as 100 iron-labeled cells within the lymph node, with injections of larger cell numbers producing increasingly obvious regions of signal void. In addition, we show that cellular MRI allows monitoring of the fate of these cells over time as they develop into intranodal tumors. This technology will allow noninvasive investigations of cellular events in cancer metastasis within an entire animal and will facilitate progress in understanding the mechanisms of metastasis within the lymphatic system.

We have developed a magnetic resonance imaging (MRI) technique for imaging Feridex (superparamagnetic iron oxide [SPIO])-labeled islets of Langerhans using a standard clinical 1.5-Tesla (T) scanner and employing steady-state acquisition imaging sequence (3DFIESTA). Both porcine and rat islets were labeled with SPIO by a transfection technique using a combination of poly-l-lysine and electroporation. Electron microscopy demonstrated presence of SPIO particles within the individual islet cells, including β-cells and particles trapped between cell membranes. Our labeling method produced a transfection rate of 860 pg to 3.4 ng iron per islet, dependent on the size of the islet. The labeling procedure did not disrupt either the function or viability of the islets. In vitro 3DFIESTA magnetic resonance images of single-labeled islets corresponded with their optical images. In vivo T2*-weighted scan using 1.5 T detected as few as 200 SPIO-labeled islets transplanted under rat kidney capsule, which correlated with immunohistochemistry of the transplant for insulin and iron. Ex vivo 3DFIESTA images of kidneys containing 200, 800 or 2,000 SPIO-labeled islet isografts showed good correlation between signal loss and increasing numbers of islets. These data provide evidence that islets can be labeled with SPIO and imaged using clinically available 1.5- T MRI.

MRI of superparamagnetic iron oxide (SPIO)-labeled cells has become a valuable tool for studying the in vivo trafficking of transplanted cells. Cellular detection with MRI is generally considered to be orders of magnitude less sensitive than other techniques, such as positron emission tomography (PET), single photon emission-computed tomography (SPECT), or optical fluorescence microscopy. However, an analytic description of the detection threshold for single SPIO-labeled cells and the parameters that govern detection has not been adequately provided. In the present work, the detection threshold for single SPIO-labeled cells and the effect of resolution and SNR were studied for a balanced steady-state free precession (SSFP) sequence (3D-FIESTA). Based on the results from both theoretical and experimental analyses, an expression that predicts the minimum detectable mass of SPIO (m(c)) required to detect a single cell against a uniform signal background was derived: m(c) = 5v/(K(fsl) x SNR), where v is the voxel volume, SNR is the image signal-to-noise ratio, and K(fsl) is an empirical constant measured to be 6.2 +/- 0.5 x 10(-5) microl/pgFe. Using this expression, it was shown that the sensitivity of MRI is not very different from that of PET, requiring femtomole quantities of SPIO iron for detection under typical micro-imaging conditions (100 microm isotropic resolution, SNR = 60). The results of this work will aid in the design of cellular imaging experiments by defining the lower limit of SPIO labeling required for single cell detection at any given resolution and SNR.