In vivo tracking of adenoviral-transduced iron oxide-labeled bone marrow-derived dendritic cells using magnetic particle imaging.

BACKGROUND: Despite widespread study of dendritic cell (DC)-based cancer immunotherapies, the in vivo postinjection fate of DC remains largely unknown. Due in part to a lack of quantifiable imaging modalities, this is troubling as the amount of DC migration to secondary lymphoid organs correlates with therapeutic efficacy. Magnetic particle imaging (MPI) has emerged as a suitable modality to quantify in vivo migration of superparamagnetic iron oxide (SPIO)-labeled DC. Herein, we describe a popliteal lymph node (pLN)-focused MPI scan to quantify DC in vivo migration accurately and consistently. METHODS: Adenovirus (Ad)-transduced SPIO+ (Ad SPIO+) and SPIO+ C57BL/6 bone marrow-derived DC were generated and assessed for viability and phenotype, then fluorescently labeled and injected into mouse hind footpads (n = 6). Two days later, in vivo DC migration was quantified using whole animal, pLN-focused, and ex vivo pLN MPI scans. RESULTS: No significant differences in viability, phenotype and in vivo pLN migration were noted for Ad SPIO+ and SPIO+ DC. Day 2 pLN-focused MPI quantified DC migration in all instances while whole animal MPI only quantified pLN migration in 75% of cases. Ex vivo MPI and fluorescence microscopy confirmed that pLN MPI signal was due to originally injected Ad SPIO+ and SPIO+ DC. CONCLUSION: We overcame a reported limitation of MPI by using a pLN-focused MPI scan to quantify pLN-migrated Ad SPIO+ and SPIO+ DC in 100% of cases and detected as few as 1000 DC (4.4 ng Fe) in vivo. MPI is a suitable preclinical imaging modality to assess DC-based cancer immunotherapeutic efficacy. RELEVANCE STATEMENT: Tracking the in vivo fate of DC using noninvasive quantifiable magnetic particle imaging can potentially serve as a surrogate marker of therapeutic effectiveness. KEY POINTS: • Adenoviral-transduced and iron oxide-labeled dendritic cells are in vivo migration competent. • Magnetic particle imaging is a suitable modality to quantify in vivo dendritic cell migration. • Magnetic particle imaging focused field of view overcomes dynamic range limitation.

Inter-user Comparison for Quantification of Superparamagnetic Iron Oxides with Magnetic Particle Imaging Across Two Institutions Highlights a Need for Standardized Approaches.

PURPOSE: Magnetic particle imaging (MPI) is being explored in biological contexts that require accurate and reproducible quantification of superparamagnetic iron oxide nanoparticles (SPIONs). While many groups have focused on improving imager and SPION design to improve resolution and sensitivity, a few have focused on improving quantification and reproducibility of MPI. The aim of this study was to compare MPI quantification results by two different systems and the accuracy of SPION quantification performed by multiple users at two institutions. PROCEDURES: Six users (3 from each institute) imaged a known amount of Vivotrax + (10 µg Fe), diluted in a small (10 µL) or large (500 µL) volume. These samples were imaged with or without calibration standards in the field of view, to create a total of 72 images (6 users × triplicate samples × 2 sample volumes × 2 calibration methods). These images were analyzed by the respective user with two region of interest (ROI) selection methods. Image intensities, Vivotrax + quantification, and ROI selection were compared across users, within and across institutions. RESULTS: MPI imagers at two different institutes produce significantly different signal intensities, that differ by over 3 times for the same concentration of Vivotrax + . Overall quantification yielded measurements that were within [Formula: see text] 20% from ground truth; however, SPION quantification values obtained at each laboratory were significantly different. Results suggest that the use of different imagers had a stronger influence on SPION quantification compared to differences arising from user error. Lastly, calibration conducted from samples in the imaging field of view gave the same quantification results as separately imaged samples. CONCLUSIONS: This study highlights that there are many factors that contribute to the accuracy and reproducibility of MPI quantification, including variation between MPI imagers and users, despite pre-defined experimental setup, image acquisition parameters, and ROI selection analysis.

Inter-user comparison for quantification of superparamagnetic iron oxides with magnetic particle imaging across two institutions highlights a need for standardized approaches.

Purpose: Magnetic particle imaging (MPI) is being explored in biological contexts that require accurate and reproducible quantification of superparamagnetic iron oxide nanoparticles (SPIONs). While many groups have focused on improving imager and SPION design to improve resolution and sensitivity, few have focused on improving quantification and reproducibility of MPI. The aim of this study was to compare MPI quantification results by two different systems and the accuracy of SPION quantification performed by multiple users at two institutions. Procedures: Six users (3 from each institute) imaged a known amount of Vivotrax+ (10 µg Fe), diluted in a small (10 µL) or large (500 µL) volume. These samples were imaged with or without calibration standards in the field of view, to create a total of 72 images (6 users x triplicate samples x 2 sample volumes x 2 calibration methods). These images were analyzed by the respective user with two region of interest (ROI) selection methods. Image intensities, Vivotrax+ quantification, and ROI selection was compared across users, within and across institutions. Results: MPI imagers at two different institutes produce significantly different signal intensities, that differ by over 3 times for the same concentration of Vivotrax+. Overall quantification yielded measurements that were within ± 20% from ground truth, however SPION quantification values obtained at each laboratory were significantly different. Results suggest that the use of different imagers had a stronger influence on SPION quantification compared to differences arising from user error. Lastly, calibration conducted from samples in the imaging field of view gave the same quantification results as separately imaged samples. Conclusions: This study highlights that there are many factors that contribute to the accuracy and reproducibility of MPI quantification, including variation between MPI imagers and users, despite pre-defined experimental set up, image acquisition parameters, and ROI selection analysis.

Dual Magnetic Particle Imaging and Akaluc Bioluminescence Imaging for Tracking Cancer Cell Metastasis.

Magnetic particle imaging (MPI) provides hotspot tracking and direct quantification of superparamagnetic iron oxide nanoparticle (SPIO)-labelled cells. Bioluminescence imaging (BLI) with the luciferase reporter gene Akaluc can provide complementary information on cell viability. Thus, we explored combining these technologies to provide a more holistic view of cancer cell fate in mice. Akaluc-expressing 4T1Br5 cells were labelled with the SPIO Synomag-D and injected into the mammary fat pads (MFP) of four nude mice. BLI was performed on days 0, 6 and 13, and MPI was performed on days 1, 8 and 14. Ex vivo histology and fluorescence microscopy of MFP and a potential metastatic site was conducted. The BLI signal in the MFP increased significantly from day 0 to day 13 (p < 0.05), mirroring tumor growth. The MPI signal significantly decreased from day 1 to day 14 (p < 0.05) due to SPIO dilution in proliferating cells. Both modalities detected secondary metastases; however, they were visualized in different anatomical regions. Akaluc BLI complemented MPI cell tracking, allowing for longitudinal measures of cell viability and sensitive detection of distant metastases at different locations. We predict this multimodal imaging approach will help to evaluate novel therapeutics and give a better understanding of metastatic mechanisms.

Complementary early-phase magnetic particle imaging and late-phase positron emission tomography reporter imaging of mesenchymal stem cells in vivo.

Stem cell-based therapies have demonstrated significant potential in clinical applications for many debilitating diseases. The ability to non-invasively and dynamically track the location and viability of stem cells post administration could provide important information on individual patient response and/or side effects. Multi-modal cell tracking provides complementary information that can offset the limitations of a single imaging modality to yield a more comprehensive picture of cell fate. In this study, mesenchymal stem cells (MSCs) were engineered to express human sodium iodide symporter (NIS), a clinically relevant positron emission tomography (PET) reporter gene, as well as labeled with superparamagnetic iron oxide nanoparticles (SPIOs) to allow for detection with magnetic particle imaging (MPI). MSCs were additionally engineered with a preclinical bioluminescence imaging (BLI) reporter gene for comparison of BLI cell viability data to both MPI and PET data over time. MSCs were implanted into the hind limbs of immunocompromised mice and imaging with MPI, BLI and PET was performed over a 30-day period. MPI showed sensitive detection that steadily declined over the 30-day period, while BLI showed initial decreases followed by later rapid increases in signal. The PET signal of MSCs was significantly higher than the background at later timepoints. Early-phase imaging (day 0-9 post MSC injections) showed correlation between MPI and BLI data (R2 = 0.671), while PET and BLI showed strong correlation for late-phase (day 10-30 post MSC injections) imaging timepoints (R2 = 0.9817). We report the first use of combined MPI and PET for cell tracking and show the complementary benefits of MPI for sensitive detection of MSCs early after implantation and PET for longer-term measurements of cell viability.

Tracking the fates of iron-labeled tumor cells in vivo using magnetic particle imaging.

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 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 localized 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.

Magnetic Particle Imaging Is a Sensitive In Vivo Imaging Modality for the Detection of Dendritic Cell Migration.

PURPOSE: The purpose of this study was to evaluate magnetic particle imaging (MPI) as a method for the in vivo tracking of dendritic cells (DC). DC are used in cancer immunotherapy and must migrate from the site of implantation to lymph nodes to be effective. The magnitude of the ensuing T cell response is proportional to the number of lymph node-migrated DC. With current protocols, less than 10% of DC are expected to reach target nodes. Therefore, imaging techniques for studying DC migration must be sensitive and quantitative. Here, we describe the first study using MPI to detect and track DC injected into the footpads of C57BL/6 mice migrating to the popliteal lymph nodes (pLNs). PROCEDURES: DC were labelled with Synomag-D™ and injected into each hind footpad of C57BL/6 mice (n = 6). In vivo MPI was conducted immediately and repeated 48 h later. The MPI signal was measured from images and related to the signal from a known number of cells to calculate iron content. DC numbers were estimated by dividing iron content in the image by the iron per cell measured from a separate cell sample. The presence of SPIO-labeled DC in nodes was validated by ex vivo MPI, histology, and fluorescence microscopy. RESULTS: Day 2 imaging showed a decrease in MPI signal in the footpads and an increase in signal at the pLNs, indicating DC migration. MPI signal was detected in the left pLN in four of the six mice and two of the six mice showed MPI signal in the right pLN. Ex vivo imaging detected signal in 11/12 nodes. We report a sensitivity of approximately 4000 cells (0.015 µg Fe) in vivo and 2000 cells (0.007 µg Fe) ex vivo. CONCLUSIONS: Here, we describe the first study to use MPI to detect and track DC in a migration model with immunotherapeutic applications. We also bring attention to the issue of resolving unequal signals within close proximity, a challenge for any pre-clinical study using a highly concentrated tracer bolus that shadows nearby lower signals.

Glial activation positron emission tomography imaging in radiation treatment of breast cancer brain metastases.

Brain metastases affect more breast cancer patients than ever before due to increased overall patient survival with improved molecularly targeted treatments. Approximately 25-34% of breast cancer patients develop brain metastases in their lifetime. Due to the blood-brain barrier (BBB), the standard treatment for breast cancer brain metastases (BCBM) is surgery, stereotactic radiosurgery (SRS) and/or whole brain radiation therapy (WBRT). At the cost of cognitive side effects, WBRT has proven efficacy in treating brain metastases when used with local therapies such as SRS and surgery. This review investigated the potential use of glial activation positron emission tomography (PET) imaging for radiation treatment of BCBM. In order to put these studies into context, we provided background on current radiation treatment approaches for BCBM, our current understanding of the brain microenvironment, its interaction with the peripheral immune system, and alterations in the brain microenvironment by BCBM and radiation. We summarized preclinical literature on the interactions between glial activation and cognition and clinical studies using translocator protein (TSPO) PET to image glial activation in the context of neurological diseases. TSPO-PET is not employed clinically in assessing and guiding cancer therapies. However, it has gained traction in preclinical studies where glial activation was investigated from primary brain cancer, metastases and radiation treatments. Novel glial activation PET imaging and its applications in preclinical studies using breast cancer models and glial immunohistochemistry are highlighted. Lastly, we discuss the potential clinical application of glial activation imaging to improve the therapeutic ratio of radiation treatments for BCBM.

Preclinical Models of Brain Metastases in Breast Cancer.

Breast cancer remains a leading cause of mortality among women worldwide. Brain metastases confer extremely poor prognosis due to a lack of understanding of their specific biology, unique physiologic and anatomic features of the brain, and limited treatment strategies. A major roadblock in advancing the treatment of breast cancer brain metastases (BCBM) is the scarcity of representative experimental preclinical models. Current models are predominantly based on the use of animal xenograft models with immortalized breast cancer cell lines that poorly capture the disease's heterogeneity. Recent years have witnessed the development of patient-derived in vitro and in vivo breast cancer culturing systems that more closely recapitulate the biology from individual patients. These advances led to the development of modern patient-tissue-based experimental models for BCBM. The success of preclinical models is also based on the imaging technologies used to detect metastases. Advances in animal brain imaging, including cellular MRI and multimodality imaging, allow sensitive and specific detection of brain metastases and monitoring treatment responses. These imaging technologies, together with novel translational breast cancer models based on patient-derived cancer tissues, represent a unique opportunity to advance our understanding of brain metastases biology and develop novel treatment approaches. This review discusses the state-of-the-art knowledge in preclinical models of this disease.

MAIT cells accumulate in ovarian cancer-elicited ascites where they retain their capacity to respond to MR1 ligands and cytokine cues.

The low mutational burden of epithelial ovarian cancer (EOC) is an impediment to immunotherapies that rely on conventional MHC-restricted, neoantigen-reactive T lymphocytes. Mucosa-associated invariant T (MAIT) cells are MR1-restricted T cells with remarkable immunomodulatory properties. We sought to characterize intratumoral and ascitic MAIT cells in EOC. Single-cell RNA sequencing of six primary human tumor specimens demonstrated that MAIT cells were present at low frequencies within several tumors. When detectable, these cells highly expressed CD69 and VSIR, but otherwise exhibited a transcriptomic signature inconsistent with overt cellular activation and/or exhaustion. Unlike mainstream CD8+ T cells, CD8+ MAIT cells harbored high transcript levels of TNF, PRF1, GZMM and GNLY, suggesting their arming and cytotoxic potentials. In a congenic, MAIT cell-sufficient mouse model of EOC, MAIT and invariant natural killer T cells amassed in the peritoneal cavity where they showed robust IL-17A and IFN-γ production capacities, respectively. However, they gradually lost these functions with tumor progression. In a cohort of 23 EOC patients, MAIT cells were readily detectable in all ascitic fluids examined. In a sub-cohort in which we interrogated ascitic MAIT cells for functional impairments, several exhaustion markers, most notably VISTA, were present on the surface. However, ascitic MAIT cells were capable of producing IFN-γ, TNF-α and granzyme B, but neither IL-17A nor IL-10, in response to an MR1 ligand, bacterial lysates containing MR1 ligands, or a combination of IL-12 and IL-18. In conclusion, ascitic MAIT cells in EOC possess inducible effector functions that may be modified in future immunotherapeutic strategies.

Magnetic microspheres can be used for magnetic particle imaging of cancer cells arrested in the mouse brain.

PURPOSE: Magnetic particle imaging (MPI) is a new imaging modality that sensitively and specifically detects superparamagnetic iron oxide nanoparticles (SPIOs). MRI cell tracking with SPIOs has very high sensitivity, but low specificity and quantification is difficult. MPI could overcome these limitations. There are no reports of micron-sized iron oxide particles (MPIO) for cell tracking by MPI. Therefore, the goal was to evaluate if MPIO can be used for in vivo detection and quantification of cancer cells distributed in the mouse brain by MPI. METHODS: In the first experiment mice were injected with either 2.5 × 105 or 5.0 × 105 MPIO-labeled cancer cells and MPI was performed ex vivo. In a second experiment, mice received either 2.5 × 105 or 5.0 × 104 MPIO-labeled cells and MPI was performed in vivo. In a third experiment, mice were injected with 5.0 × 104 cells, labeled with either MPIO or ferucarbotran, and MPI was performed in vivo. RESULTS: MPIO-labeled cells were visible in all MPI images of the mouse brain. The MPI signal and iron content measurements were greater for brains of mice that were injected with higher numbers of MPIO-labeled cells. Ferucarbotran-labeled cells were not detected in the brain by MPI. CONCLUSION: This is the first example of the use of MPIO for cell tracking with MPI. With an intracardiac cell injection, ~15% of cells will arrest in the brain vasculature. For our lowest cell injection of 5.0 × 104 cells, this was ~10 000 cells, distributed throughout the brain.

Visualizing CAR-T cell Immunotherapy Using 3 Tesla Fluorine-19 MRI.

PURPOSE: Chimeric antigen receptor (CAR) T cell cancer immunotherapies have shown remarkable results in patients with hematological malignancies and represent the first approved genetically modified cellular therapies. However, not all blood cancer patients respond favorably, serious side effects have been reported, and the treatment of solid tumors has been a challenge. An imaging tool for visualizing the variety of CAR-T cell products in use and being explored could provide important patient-specific data on CAR-T cell location to inform on potential success or failure of treatment as well as off-target toxicities. Fluorine-19 (19F) magnetic resonance imaging (MRI) allows for the noninvasive detection of 19F perfluorocarbon (PFC) labeled cells. Our objective was to visualize PFC-labeled (PFC +) CAR-T cells in a mouse model of leukemia using clinical field strength (3 Tesla) 19F MRI and compare the cytotoxicity of PFC + versus unlabeled CAR-T cells. PROCEDURES: NSG mice (n = 17) received subcutaneous injections of CD19 + human B cell leukemia cells (NALM6) expressing firefly luciferase in their left hind flank (1 × 106). Twenty-one days later, each mouse received an intratumoral injection of 10 × 106 PFC + CD19-targeted CAR-T cells (n = 6), unlabeled CD19-targeted CAR-T cells (n = 3), PFC + untransduced T cells (n = 5), or an equivalent volume of saline (n = 3). 19F MRI was performed on mice treated with PFC + CAR-T cells days 1, 3, and 7 post-treatment. Bioluminescence imaging (BLI) was performed on all mice days - 1, 5, 10, and 14 post-treatment to monitor tumor response. RESULTS: PFC + CAR-T cells were successfully detected in tumors using 19F MRI on days 1, 3, and 7 post-injection. In vivo BLI data revealed that mice treated with PFC + or PFC - CAR-T cells had significantly lower tumor burden by day 14 compared to untreated mice and mice treated with PFC + untransduced T cells (p < 0.05). Importantly, mice treated with PFC + CAR-T cells showed equivalent cytotoxicity compared to mice receiving PFC - CAR-T cells. CONCLUSIONS: Our studies demonstrate that clinical field strength 19F MRI can be used to visualize PFC + CAR-T cells for up to 7 days post-intratumoral injection. Importantly, PFC labeling did not significantly affect in vivo CAR-T cell cytotoxicity. These imaging tools may have broad applications for tracking emerging CAR-T cell therapies in preclinical models and may eventually be useful for the detection of CAR-T cells in patients where localized injection of CAR-T cells is being pursued.

The sensitivity of magnetic particle imaging and fluorine-19 magnetic resonance imaging for cell tracking.

Magnetic 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. Cells were labeled with ferucarbotran or perfluoropolyether, for imaging on a preclinical MPI system or 3 Tesla clinical MRI, respectively. Using 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 × 1016 19F atoms) were detected with 19F MRI, with SNR > 5. MPI has the potential to be more sensitive than 19F MRI for cell tracking. In vivo sensitivity with MPI and 19F MRI was evaluated by imaging MSC that were administered by different routes. In vivo imaging revealed reduced sensitivity compared to ex vivo cell pellets of the same cell number. We attribute reduced MPI and 19F MRI cell detection in vivo to the effect of cell dispersion among other factors, which are described.

A method for the efficient iron-labeling of patient-derived xenograft cells and cellular imaging validation.

There is momentum towards implementing patient-derived xenograft models (PDX) in cancer research to reflect the histopathology, tumor behavior, and metastatic properties observed in the original tumor. To study PDX cells preclinically, we used both bioluminescence imaging (BLI) to evaluate cell viability and magnetic particle imaging (MPI), an emerging imaging technology to allow for detection and quantification of iron nanoparticles. The goal of this study was to develop the first successful iron labeling method of breast cancer cells derived from patient brain metsastases and validate this method with imaging during tumor development. The overall workflow of this labeling method is as follows: adherent and non-adherent luciferase expressing human breast cancer PDX cells (F2-7) are dissociated and concurrently labeled after incubation with micron-sized iron oxide particles (MPIO; 25 µg Fe/ml), with labeling validated by cellular imaging with MPI and BLI. In this study, NOD/SCID/ILIIrg-/- (n = 5) mice Received injections of 1 × 106 iron-labeled F2-7 cells into the fourth mammary fat pad (MFP). BLI was performed longitudinally to day 49 and MPI was performed up to day 28. In vivo BLI revealed that signal increased over time with tumor development. MPI revealed decreasing signal in the tumors over time. Here, we demonstrate the first application of MPI to monitor the growth of a PDX MFP tumor and the first successful labeling of PDX cells with iron oxide particles. Imaging of PDX cells provides a powerful system to better develop personalized therapies targeting breast cancer brain metastasis.

Chronic stress physically spares but functionally impairs innate-like invariant T cells.

The deleterious effects of psychological stress on mainstream T lymphocytes are well documented. However, how stress impacts innate-like T cells is unclear. We report that long-term stress surprisingly abrogates both T helper 1 (TH1)- and TH2-type responses orchestrated by invariant natural killer T (iNKT) cells. This is not due to iNKT cell death because these cells are unusually refractory to stress-inflicted apoptosis. Activated iNKT cells in stressed mice exhibit a "split" inflammatory signature and trigger sudden serum interleukin-10 (IL-10), IL-23, and IL-27 spikes. iNKT cell dysregulation is mediated by cell-autonomous glucocorticoid receptor signaling and corrected upon habituation to predictable stressors. Importantly, under stress, iNKT cells fail to potentiate cytotoxicity against lymphoma or to reduce the burden of metastatic melanoma. Finally, stress physically spares mouse mucosa-associated invariant T (MAIT) cells but hinders their TH1-/TH2-type responses. The above findings are corroborated in human peripheral blood and hepatic iNKT/MAIT cell cultures. Our work uncovers a mechanism of stress-induced immunosuppression.

Visualizing tumour self-homing with magnetic particle imaging.

Due to their innate tumour homing capabilities, in recent years, circulating tumour cells (CTCs) have been engineered to express therapeutic genes for targeted treatment of primary and metastatic lesions. Additionally, previous studies have incorporated optical or PET imaging reporter genes to enable noninvasive monitoring of therapeutic CTCs in preclinical tumour models. An alternative method for tracking cells is to pre-label them with imaging probes prior to transplantation into the body. This is typically more sensitive to low numbers of cells since large amounts of probe can be concentrated in each cell. The objective of this work was to evaluate magnetic particle imaging (MPI) for the detection of iron-labeled experimental CTCs. CTCs were labeled with micro-sized iron oxide (MPIO) particles, administered via intra-cardiac injection in tumour bearing mice and were detected in the tumour region of the mammary fat pad. Iron content and tumour volumes were calculated. Ex vivo MPI of the tumours and immunohistochemistry were used to validate the imaging data. Here, we demonstrate for the first time the ability of MPI to sensitively detect systemically administered iron-labeled CTCs and to visualize tumour self-homing in a murine model of human breast cancer.

A Perspective on Cell Tracking with Magnetic Particle Imaging.

Many labs have been developing cellular magnetic resonance imaging (MRI), using both superparamagnetic iron oxide nanoparticles (SPIONs) and fluorine-19 (19F)-based cell labels, to track immune and stem cells used for cellular therapies. Although SPION-based MRI cell tracking has very high sensitivity for cell detection, SPIONs are indirectly detected owing to relaxation effects on protons, producing negative magnetic resonance contrast with low signal specificity. 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 has high specificity for perfluorocarbon-labeled cells, and 19F signal is directly related to cell number. However, 19F MRI has low sensitivity. Magnetic particle imaging (MPI) is a new imaging modality that directly detects SPIONs. SPION-based cell tracking using MPI displays great potential for overcoming the challenges of MRI-based cell tracking, allowing for both high cellular sensitivity and specificity, and quantification of SPION-labeled cell number. Here 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.

Engineering Circulating Tumor Cells as Novel Cancer Theranostics.

New ways to target and treat metastatic disease are urgently needed. Tumor "self-homing" describes the recruitment of circulating tumor cells (CTCs) back to a previously excised primary tumor location, contributing to tumor recurrence, as well as their migration to established metastatic lesions. Recently, self-homing CTCs have been exploited as delivery vehicles for anti-cancer therapeutics in preclinical primary tumor models. However, the ability of CTCs to self-home and treat metastatic disease is largely unknown. Methods: Here, we used bioluminescence imaging (BLI) to explore whether systemically administered CTCs home to metastatic lesions and if CTCs armed with both a reporter gene and a cytotoxic prodrug gene therapy can be used to visualize and treat metastatic disease. Results: BLI performed over time revealed a remarkable ability of CTCs to home to and treat tumors throughout the body. Excitingly, metastatic tumor burden in mice that received therapeutic CTCs was lower compared to mice receiving control CTCs. Conclusion: This study demonstrates the noteworthy ability of experimental CTCs to home to disseminated breast cancer lesions. Moreover, by incorporating a prodrug gene therapy system into our self-homing CTCs, we show exciting progress towards effective and targeted delivery of gene-based therapeutics to treat both primary and metastatic lesions.

Comparing the fate of brain metastatic breast cancer cells in different immune compromised mice with cellular magnetic resonance imaging.

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 vivo to 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 vivo imaging of the liver, histology, and immunofluorescence labeling was performed. Brain metastases grew more rapidly in NSG mice. At day 21 post cell injection, the average number of brain tumors in NSG mice was approximately four times greater than in nude mice. The persistence of iron-labeled cells, visualized as signal voids by MRI, was also examined. The percentage of voids decreased significantly over time for both nude and NSG mice. Body images revealed that the NSG mice also had metastases in the liver, lungs, and lymph nodes while tumors were only detected in the brains of nude mice. This work demonstrates the advantages of using the highly immune-compromised NSG mouse to study breast cancer metastasis, treatments aimed at inhibiting metastasis and outgrowth of breast cancer metastases in multiple organs, and the role that imaging can play toward credentialing these models that cannot be done with other in vitro or histopathologic methods alone.

Magnetic Particle Imaging of Macrophages Associated with Cancer: Filling the Voids Left by Iron-Based Magnetic Resonance Imaging.

PURPOSE: 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. PROCEDURES: 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. RESULTS: 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. CONCLUSIONS: 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.