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Imaging macrophage distribution and density in mammary tumors and lung metastases using fluorine‐19 MRI cell tracking

The presence of tumor-associated macrophages (TAMs) correlates with breast cancer progression and metastatic spread. Metastasis-associated macrophages (MAMs) are also recruited to distant sites, where they support metastatic growth. In this study, we demonstrate that in vivo fluorine-19 (19 F)-based MRI cell tracking can evaluate the density and distribution of macrophages within murine breast cancer tumors and associated metastases.

Visualization of macrophage recruitment in head and neck carcinoma model using fluorine‐19 magnetic resonance imaging

PURPOSE: To evaluate the role of infiltrating macrophages in murine models of single and double mutation head and neck tumors using a novel fluorine-19 (19 F) MRI technology.

METHODS: Tumor cell lines single-hit/SCC4 or double-hit/Cal27, with mutations of TP53 and TP53 & FHIT, respectively, were injected bilaterally into the flanks of (n = 10) female mice. With tumors established, perfluorocarbon nanoemulsion was injected intravenously, which labels in situ predominantly monocytes and macrophages. Longitudinal spin density-weighted 19 F MRI data enabled quantification of the macrophage burden in tumor and surrounding tissue.

Quantifying tumor associated macrophages in breast cancer: a comparison of iron and fluorine-based MRI cell tracking

Tumor associated macrophages (TAMs) are associated with tumor growth and metastasis. MRI can detect TAMs labeled with iron oxide (USPIO) or perfluorocarbon (PFC) agents. This study compared these two cell tracking approaches for imaging TAMs in vivo. 4T1 tumors were imaged with MRI at 4 days or 3 weeks post cell implantation after intravenous (i.v.) administration of either USPIO or PFC. Signal loss was detected within tumors at both time points post USPIO. Images acquired at 4 days demonstrated signal loss encompassing the entire tumor and around the periphery at 3 weeks. Number of black voxels suggested higher numbers of TAMs in the tumor at the later time point. After PFC administration, Fluorine-19 (19F) signal was detected in a similar spatial distribution as signal loss post USPIO.

In Vivo 19F MR Imaging Cell Tracking of Inflammatory Macrophages and Site-specific Development of Colitis-associated Dysplasia

Fluorine 19 MR imaging can be used to track bowel inflammation, and its magnitude is associated with colitis-associated dysplasia development.

To investigate whether the magnitude of in vivo fluorine 19 (19F) magnetic resonance (MR) imaging signal is associated with subsequent development of colitis-associated dysplasia after in situ fluorination of inflammatory macrophages in a mouse model of inflammatory bowel disease (IBD).

A novel probe for the non-invasive detection of tumor-associated inflammation

A novel dual-mode contrast agent was formulated through the addition of an optical near infrared (NIR) probe to a perfluorocarbon (PFC)-based 19F magnetic resonance imaging (MRI) agent, which labels inflammatory cells in situ. A single PFC-NIR imaging agent enables both a qualitative, rapid optical monitoring of an inflammatory state and a quantitative, detailed and tissue-depth independent magnetic resonance imaging (MRI). The feasibility of in vivo optical imaging of the inflammatory response was demonstrated in a subcutaneous murine breast carcinoma model. Ex vivo optical imaging was used to quantify the PFC-NIR signal in the tumor and organs, and results correlated well with quantitative 19F NMR analyses of intact tissues. 19F MRI was employed to construct a three-dimensional image of the cellular microenvironment at the tumor site. Flow cytometry of isolated tumor cells was used to identify the cellular localization of the PFC-NIR probe within the tumor microenvironment. Contrast is achieved through the labeling of host cells involved in the immune response, but not tumor cells. The major cellular reservoir of the imaging agent were tumor-infiltrating CD11b+ F4/80low Gr-1low cells, a cell subset sharing immunophenotypic features with myeloid-derived suppressor cells (MDSCs). These cells are recruited to sites of inflammation and are implicated in immune evasion and tumor progression. This PFC-NIR contrast agent coupled to non-invasive, quantitative imaging techniques could serve as a valuable tool for evaluating novel anticancer agents.

Assaying macrophage activity in a murine model of inflammatory bowel disease using fluorine-19 MRI

Macrophages have an important role in the pathogenesis of most chronic inflammatory diseases. A means of non-invasively quantifying macrophage migration would contribute significantly towards our understanding of chronic inflammatory processes and aid the evaluation of novel therapeutic strategies. We describe the use of a perfluorocarbon tracer reagent and in vivo (19)F magnetic resonance imaging (MRI) to quantify macrophage burden longitudinally. We apply these methods to evaluate the severity and three-dimensional distribution of macrophages in a murine model of inflammatory bowel disease (IBD).

Selective in-vivo visualization of immune-cell infiltration in a mouse model of autoimmune myocarditis by fluorine-19 cardiac magnetic resonance

The goal of this study was to characterize the performance of fluorine-19 ((19)F) cardiac magnetic resonance (CMR) for the specific detection of inflammatory cells in a mouse model of myocarditis. Intravenously administered perfluorocarbons are taken up by infiltrating inflammatory cells and can be detected by (19)F-CMR. (19)F-labeled cells should, therefore, generate an exclusive signal at the inflamed regions within the myocardium.

Visualizing arthritic inflammation and therapeutic response by fluorine-19 magnetic resonance imaging (19F MRI)

Non-invasive imaging of inflammation to measure the progression of autoimmune diseases, such as rheumatoid arthritis (RA), and to monitor responses to therapy is critically needed. V-Sense, a perfluorocarbon (PFC) contrast agent that preferentially labels inflammatory cells, which are then recruited out of systemic circulation to sites of inflammation, enables detection by 19F MRI. With no 19F background in the host, detection is highly-specific and can act as a proxy biomarker of the degree of inflammation present.

Rapid quantification of inflammation in tissue samples using perfluorocarbon emulsion and fluorine-19 nuclear magnetic resonance

Quantification of inflammation in tissue samples can be a time-intensive bottleneck in therapeutic discovery and preclinical endeavors. We describe a versatile and rapid approach to quantitatively assay macrophage burden in intact tissue samples. Perfluorocarbon (PFC) emulsion is injected intravenously, and the emulsion droplets are effectively taken up by monocytes and macrophages. These ‘in situ’ labeled cells participate in inflammatory events in vivo resulting in PFC accumulation at inflammatory loci.

19F detection of acute allograft rejection with in vivo perfluorocarbon labeling of immune cells

Current diagnosis of organ rejection following transplantation relies on tissue biopsy, which is not ideal due to sampling limitations and risks associated with the invasive procedure. We have previously shown that cellular magnetic resonance imaging (MRI) of iron-oxide labeled immune-cell infiltration can provide a noninvasive measure of rejection status by detecting areas of hypointensity on T 2*-weighted images. In this study, we tested the feasibility of using a fluorine-based cellular tracer agent to detect macrophage accumulation in rodent models of acute allograft rejection by fluorine-19 ((19) F) MRI and magnetic resonance spectroscopy.

T‐cells and macrophages peak weeks after experimental stroke: Spatial and temporal characteristics

The activities of the central and peripheral immune systems impact neurological outcome after ischemic stroke. However, studies investigating the temporal profile of leukocyte infiltration, especially T-cell recruitment, are sparse. Our aim was to investigate leukocyte infiltration at different time points after experimental stroke in mice. Permanent middle cerebral artery occlusion was performed on 11 weeks old C57BL/6J mice, allowed to survive for 1, 3, 8, 14 or 28 days. In addition to infarct size measurements, detailed immunohistochemical analyses of T-cell and macrophage influx were performed. A recently introduced F-19 MR probe (V-sense), designed to track macrophages, was furthermore tested. Fourteen and 28 days after permanent middle cerebral artery occlusion a significant increase in CD3+ T-cells was found within the ipsilateral hemisphere compared to controls, especially within the infarct core and the corpus callosum. The number of CD68+ cells within the infarct core was significantly increased at days 8, 14 and 28. This temporal pattern was also seen in MRI. After experimental stroke within the infarcted cortex we found a delayed (day 14) infiltration of T-cells and macrophages. Furthermore, our data show that T-cells are present in higher numbers in the corpus callosum compared to the rest of the brain (except from the infarct core where they were highest).

Quantifying tumor associated macrophages in breast cancer: a comparison of iron and fluorine-based MRI cell tracking

Tumor associated macrophages (TAMs) are associated with tumor growth and metastasis. MRI can detect TAMs labeled with iron oxide (USPIO) or perfluorocarbon (PFC) agents. This study compared these two cell tracking approaches for imaging TAMs in vivo. 4T1 tumors were imaged with MRI at 4 days or 3 weeks post cell implantation after intravenous (i.v.) administration of either USPIO or PFC. Signal loss was detected within tumors at both time points post USPIO. Images acquired at 4 days demonstrated signal loss encompassing the entire tumor and around the periphery at 3 weeks. Number of black voxels suggested higher numbers of TAMs in the tumor at the later time point. After PFC administration, Fluorine-19 (19F) signal was detected in a similar spatial distribution as signal loss post USPIO. 19F signal quantification revealed that the number of 19F spins was not significantly different at the two time points, suggesting a similar number of TAMs were present in tumors but accumulated in different regions. 19F signal was higher centrally in tumors at 4 days and heterogenous around the periphery at 3 weeks. This study revealed that 19F-based cell tracking methods better represent TAM density and provides additional information not achievable with iron-based methods.

Spinal cord injury suppresses cutaneous inflammation: implications for peripheral wound healing

People who suffer a traumatic spinal cord injury (SCI) are at increased risk for developing dermatological complications. These conditions increase cost of care, incidence of rehospitalization, and the risk for developing other infections. The consequences of dermatological complications after SCI are likely exacerbated further by post-injury deficits in neural-immune signaling. Indeed, a functional immune system is essential for optimal host defense and tissue repair. Here, we tested the hypothesis that SCI at high spinal levels, which causes systemic immune suppression, would suppress cutaneous inflammation below the level of injury.

Imaging neuroinflammation in vivo in a neuropathic pain rat model with near-infrared fluorescence and 19F magnetic resonance

Chronic neuropathic pain following surgery represents a serious worldwide health problem leading to life-long treatment and the possibility of significant disability. In this study, neuropathic pain was modeled using the chronic constriction injury (CCI). The CCI rats exhibit mechanical hypersensitivity (typical neuropathic pain symptom) to mechanical stimulation of the affected paw 11 days post surgery, at a time when sham surgery animals do not exhibit hypersensitivity. Following a similar time course, TRPV1 gene expression appears to rise with the hypersensitivity to mechanical stimulation.

Fluorine-19 magnetic resonance angiography of the mouse

To implement and characterize a fluorine-19 ((19)F) magnetic resonance imaging (MRI) technique and to test the hypothesis that the (19)F MRI signal in steady state after intravenous injection of a perfluoro-15-crown-5 ether (PCE) emulsion may be exploited for angiography in a pre-clinical in vivo animal study.

Bioluminescence and 19F magnetic resonance imaging visualize the efficacyof lysostaphin alone and in combination with oxacillin against Staphylococcus aureus in murine thigh and catheter-associated infection models

Staphylococci are the leading cause of hospital-acquired infections worldwide. Increasingly, they resist antibiotic treatment owing to the development of multiple antibiotic resistance mechanisms in most strains. Therefore, the activity and efficacy of recombinant lysostaphin as a drug against this pathogen have been evaluated. Lysostaphin exerts high levels of activity against antibiotic-resistant strains of Staphylococcus aureus, including methicillin-resistant S. aureus (MRSA).

19F magnetic resonance imaging of perfluorocarbons for the evaluation of response to antibiotic therapy in a staphylococcus aureus infection model

The emergence of antibiotic resistant bacteria in recent decades has highlighted the importance of developing new drugs to treat infections. However, in addition to the design of new drugs, the development of accurate preclinical testing methods is essential. In vivo imaging technologies such as bioluminescence imaging (BLI) or magnetic resonance imaging (MRI) are promising approaches. In a previous study, we showed the effectiveness of (19)F MRI using perfluorocarbon (PFC) emulsions for detecting the site of Staphylococcus aureus infection. In the present follow-up study, we investigated the use of this method for in vivo visualization of the effects of antibiotic therapy.

Visualization of inflammation using 19F-magnetic resonance imaging and perfluorocarbons

Inflammation plays a central pathophysiological role in a large number of diseases. While conventional magnetic resonance imaging (MRI) can depict gross tissue alterations due to proton changes, specific visualization of inflammation is an unmet task in clinical medicine. (19) F/(1) H MRI is a novel technology that allows tracking of stem and immune cells in experimental disease models after labelling with perfluorocarbon (PFC) emulsions. (19) F markers such as PFC compounds provide a unique signal in vivo due to the negligible (19) F background signal of the body.

Imaging of intratumoral inflammation during oncolytic virotherapy of tumors by 19F-magnetic resonance imaging (MRI)

Oncolytic virotherapy of tumors is an up-coming, promising therapeutic modality of cancer therapy. Unfortunately, non-invasive techniques to evaluate the inflammatory host response to treatment are rare. Here, we evaluate (19)F magnetic resonance imaging (MRI) which enables the non-invasive visualization of inflammatory processes in pathological conditions by the use of perfluorocarbon nanoemulsions (PFC) for monitoring of oncolytic virotherapy.

In vivo imaging of stepwise vessel occlusion in cerebral photothrombosis of mice by 19F MRI

(19)F magnetic resonance imaging (MRI) was recently introduced as a promising technique for in vivo cell tracking. In the present study we compared (19)F MRI with iron-enhanced MRI in mice with photothrombosis (PT) at 7 Tesla. PT represents a model of focal cerebral ischemia exhibiting acute vessel occlusion and delayed neuroinflammation.

Visualization of abscess formation in a murine thigh infection model of Staphylococcus aureus by 19F-magnetic resonance imaging (MRI)

During the last years, (19)F-MRI and perfluorocarbon nanoemulsion (PFC) emerged as a powerful contrast agent based MRI methodology to track cells and to visualize inflammation. We applied this new modality to visualize deep tissue abscesses during acute and chronic phase of inflammation caused by Staphylococcus aureus infection.

In vivo imaging of inflammation in the peripheral nervous system by (19)F MRI

Visualization of neuroinflammation is still a major task in neuroscience and neurology since inflammatory processes play a central pathophysiological role in many disorders of the nervous system but are not yet covered by conventional imaging techniques. Recently, (19)F magnetic resonance imaging (MRI) was introduced as a new cellular imaging technology. In the present study, we established (19)F high field MRI for cell tracking in the peripheral nervous system (PNS) of rats using dedicated MR coils. To mimic focal neuroinflammation, lysolecithin was locally injected into the left sciatic nerve inducing demyelination followed by severe infiltration of monocytes/macrophages from the circulation.

Monitoring of Intracerebellarly-Administered Natural Killer cells with Fluorine-19 MRI

Purpose
Medulloblastoma (MB) is the most common malignant brain tumor in children. Recent studies have shown the ability of natural killer (NK) cells to lyse MB cell lines in vitro, but in vivo successes remain elusive and the efficacy and fate of NK cells in vivo remain unknown.

Methods
To address these questions, we injected MB cells into the cerebellum of immunodeficient mice and examined tumor growth at various days after tumor establishment via bioluminescence imaging. NK cells were labeled with a fluorine-19 (19F) MRI probe and subsequently injected either intratumorally or contralaterally to the tumor in the cerebellum and effect on tumor growth was monitored.

Perfluorocarbon Labeling of Human Glial‐Restricted Progenitors for 19F Magnetic Resonance Imaging

One of the fundamental limitations in assessing potential efficacy in Central Nervous System (CNS) transplantation of stem cells is the capacity for monitoring cell survival and migration noninvasively and longitudinally. Human glial‐restricted progenitor (hGRP) cells (Q‐Cells) have been investigated for their utility in providing neuroprotection following transplantation into models of amyotrophic lateral sclerosis (ALS) and have been granted a Food and Drug Administration (FDA) Investigational New Drug (IND) for intraspinal transplantation in ALS patients. Furthermore, clinical development of these cells for therapeutic use will rely on the ability to track the cells using noninvasive imaging methodologies as well as the verification that the transplanted GRPs have disease‐relevant activity. As a first step in development, we investigated the use of a perfluorocarbon (PFC) dual‐modal (19F magnetic resonance imaging [MRI] and fluorescence) tracer agent to label Q‐Cells in culture and following spinal cord transplantation.

Fluorine-19 MRI for detection and quantification of immune cell therapy for cancer

Over the past two decades, immune cell therapy has emerged as a potent treatment for multiple cancers, first through groundbreaking leukemia therapy, and more recently, by tackling solid tumors. Developing successful therapeutic strategies using live cells could benefit from the ability to rapidly determine their in vivo biodistribution and persistence. Assaying cell biodistribution is unconventional compared to traditional small molecule drug pharmacokinetic readouts used in the pharmaceutical pipeline, yet this information is critical towards understanding putative therapeutic outcomes and modes of action. Towards this goal, efforts are underway to visualize and quantify immune cell therapy in vivo using advanced magnetic resonance imaging (MRI) techniques.

19 F-perfluorocarbon-labeled human peripheral blood mononuclear cells can be detected in vivo using clinical MRI parameters in a therapeutic cell setting

A 19Fluorine (19F) perfluorocarbon cell labeling agent, when employed with an appropriate cellular MRI protocol, allows for in vivo cell tracking. 19F cellular MRI can be used to non-invasively assess the location and persistence of cell-based cancer vaccines and other cell-based therapies. This study was designed to determine the feasibility of labeling and tracking peripheral blood mononuclear cells (PBMC), a heterogeneous cell population. Under GMP-compliant conditions human PBMC were labeled with a 19F-based MRI cell-labeling agent in a manner safe for autologous re-injection. Greater than 99% of PBMC labeled with the 19F cell-labeling agent without affecting functionality or affecting viability.

Improvement of 19F MR image uniformity in a mouse model of cellular therapy using inductive coupling

OBJECTIVE: Improve 19F magnetic resonance imaging uniformity of perfluorocarbon (PFC)-labeled cells by using a secondary inductive resonator tuned to 287 MHz to enhance the induced radio frequency (RF) magnetic field (B1) at 7.05 T.

Fluorine-19 nuclear magnetic resonance of chimeric antigen receptor T cell biodistribution in murine cancer model

Discovery of effective cell therapies against cancer can be accelerated by the adaptation of tools to rapidly quantitate cell biodistribution and survival after delivery. Here, we describe the use of nuclear magnetic resonance (NMR) ‘cytometry’ to quantify the biodistribution of immunotherapeutic T cells in intact tissue samples. In this study, chimeric antigen receptor (CAR) T cells expressing EGFRvIII targeting transgene were labeled with a perfluorocarbon (PFC) emulsion ex vivo and infused into immunocompromised mice bearing subcutaneous human U87 glioblastomas expressing EGFRvIII and luciferase.

Integrating a 19F MRI Tracer Agent into the Clinical Scale Manufacturing of a T-Cell Immunotherapy

Leukocyte immunotherapies have made great progress in the treatment of cancer. Recent reports on the treatment of B-cell malignancies using Chimeric Antigen Receptor and affinity enhanced T-Cell Receptor therapies have demonstrated encouraging clinical results. As investigators begin to explore the treatment of solid tumors with these cells, the hurdle of evaluating T-cell homing to and persistence at the site of disease remain. Significant challenges regarding the GMP manufacture and administration of a therapeutic dose of millions to billions of transduced T-cells remain. Here we report on the application of a clinically authorized 19F MRI tracer agent to human T-cells, employing state-of-the-art methods and equipment in the manufacture of a cellular therapy. Using a general T-cell expansion protocol and clinical scale industrial bioreactors, we show 19F labeling without detriment to the product +/− cryopreservation. While the incorporation of the 19F tracer is not trivial, it is just one of the many steps that can aid in progression of a therapeutic to and though the clinic. Combining the MRI tracking capabilities, safety profiles, and clinical sensitivity of this method, this application demonstrates the ability of 19F MRI to be used in industrial scale applications to visualize the spatial fate of cellular therapeutics.

19F-MRI for monitoring human NK cells in vivo

The availability of clinical-grade cytokines and artificial antigen-presenting cells has accelerated interest in using natural killer (NK) cells as adoptive cellular therapy (ACT) for cancer. One of the technological shortcomings of translating therapies from animal models to clinical application is the inability to effectively and non-invasively track these cells after infusion in patients.

Clinical cell therapy imaging using a perfluorocarbon tracer and fluorine‐19 MRI

Cellular therapeutics are emerging as a treatment option for a host of serious human diseases. To accelerate clinical translation, noninvasive imaging of cell grafts in clinical trials can potentially be used to assess the initial delivery and behavior of cells.

Tracking the Fate of Stem Cell Implants with Fluorine-19 MRI

In this study we used cellular magnetic resonance imaging (MRI) to detect mesenchymal stem cells (MSC) labeled with a Fluorine-19 (19F) agent. 19F-MRI offers unambiguous detection and in vivo quantification of labeled cells.

Tracking immune cells in vivo using magnetic resonance imaging

The increasing complexity of in vivo imaging technologies, coupled with the development of cell therapies, has fuelled a revolution in immune cell tracking in vivo. Powerful magnetic resonance imaging (MRI) methods are now being developed that use iron oxide- and ¹⁹F-based probes. These MRI technologies can be used for image-guided immune cell delivery and for the visualization of immune cell homing and engraftment, inflammation, cell physiology and gene expression.

Functional assessment of human dendritic cells labeled for in vivo (19)F magnetic resonance imaging cell tracking

Dendritic cells (DC) are increasingly being used as cellular vaccines to treat cancer and infectious diseases. While there have been some promising results in early clinical trials using DC-based vaccines, the inability to visualize non-invasively the location, migration and fate of cells once adoptively transferred into patients is often cited as a limiting factor in the advancement of these therapies. A novel perflouropolyether (PFPE) tracer agent was used to label human DC ex vivo for the purpose of tracking the cells in vivo by (19)F magnetic resonance imaging (MRI). We provide an assessment of this technology and examine its impact on the health and function of the DC.

In vitro labelling and detection of mesenchymal stromal cells: a comparison between magnetic resonance imaging of iron-labelled cells and magnetic resonance spectroscopy of fluorine-labelled cells

Among the various stem cell populations used for cell therapy, adult mesenchymal stromal cells (MSCs) have emerged as a major new cell technology. These cells must be tracked after transplantation to monitor their migration within the body and quantify their accumulation at the target site. This study assessed whether rat bone marrow MSCs can be labelled with superparamagnetic iron oxide (SPIO) nanoparticles and perfluorocarbon (PFC) nanoemulsion formulations without altering cell viability and compared magnetic resonance imaging (MRI) and magnetic resonance spectroscopy (MRS) results from iron-labelled and fluorine-labelled MSCs, respectively.

In-Vivo Detection and Tracking of T Cells in Various Organs in a Melanoma Tumor Model by 19 F-Fluorine MRS/MRI.

19F-MRI and 19F-MRS can identify specific cell types after in-vitro or in-vivo 19F-labeling. Knowledge on the potential to track in-vitro 19F-labeled immune cells in tumor models by 19F-MRI/MRS is scarce.

Application of dual 19F and iron cellular MRI agents to track the infiltration of immune cells to the site of a rejected stem cell transplant.

Cellular MRI) was used to detect implanted human mesenchymal stem cells (hMSCs) and the resulting macrophage infiltration that occurs in response to xenotransplantation. Human mesenchymal stem cells were prelabeled with a fluorine‐19 (19F) agent prior to implantation, allowing for their visualization and quantification over time.

In Vivo 19 F-Magnetic Resonance Imaging of Adoptively Transferred NK Cells.

In order to assess the biodistribution, homing, and persistence of adoptively transferred natural killer (NK) cell immunotherapies, there is a need for imaging methodology suitable for use in preclinical studies with relevance to clinical translation.

Cellular Imaging with MRI

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.

Fluorine-19 Labeling of Stromal Vascular Fraction Cells for Clinical Imaging Applications

Stromal vascular fraction (SVF) cells are used clinically for various therapeutic targets. The location and persistence of engrafted SVF cells are important parameters for determining treatment failure versus success. We used the GID SVF-1 platform and a clinical protocol to harvest and label SVF cells with the fluorinated ((19)F) agent CS-1000 as part of a first-in-human phase I trial (clinicaltrials.gov identifier NCT02035085) to track SVF cells with magnetic resonance imaging during treatment of radiation-induced fibrosis in breast cancer patients. Flow cytometry revealed that SVF cells consisted of 25.0% ± 15.8% CD45+, 24.6% ± 12.5% CD34+, and 7.5% ± 3.3% CD31+ cells, with 2.1 ± 0.7 × 10⁵ cells per cubic centimeter of adipose tissue obtained. Fluorescent CS-1000 (CS-ATM DM Green) labeled 87.0% ± 13.5% of CD34+ progenitor cells compared with 47.8% ± 18.5% of hematopoietic CD45+ cells, with an average of 2.8 ± 2.0 × 10¹² ¹⁹F atoms per cell, determined using nuclear magnetic resonance spectroscopy. The vast majority (92.7% ± 5.0%) of CD31+ cells were also labeled, although most coexpressed CD34. Only 16% ± 22.3% of CD45-/CD31-/CD34- (triple-negative) cells were labeled with CS-ATM DM Green. After induction of cell death by either apoptosis or necrosis, >95% of ¹⁹F was released from the cells, indicating that fluorine retention can be used as a surrogate marker for cell survival. Labeled-SVF cells engrafted in a silicone breast phantom could be visualized with a clinical 3-Tesla magnetic resonance imaging scanner at a sensitivity of approximately 2 × 10⁶ cells at a depth of 5 mm. The current protocol can be used to image transplanted SVF cells at clinically relevant cell concentrations in patients.

Combining perfluorocarbon and superparamagnetic iron-oxide cell labeling for improved and expanded applications of cellular MRI

The ability to detect the migration of cells in living organisms is fundamental in understanding biological processes and important for the development of novel cell-based therapies to treat disease. MRI can be used to detect the migration of cells labeled with superparamagnetic iron-oxide (SPIO) or perfluorocarbon (PFC) agents. In this study, we explored combining these two cell-labeling approaches to overcome current limitations and enable new applications for cellular MRI.

A multi-modality platform to image stem cell graft survival in the naïve and stroke damaged mouse brain

Neural stem cell implantations have been extensively investigated for treatment of brain diseases such as stroke. In order to follow the localization and functional status of cells after implantation noninvasive imaging is essential. Therefore, we developed a comprehensive multi-modality platform for in vivo imaging of graft localization, density, and survival using 19F magnetic resonance imaging in combination with bioluminescence imaging.

In vivo intracellular oxygen dynamics in murine brain glioma and immunotherapeutic response of cytotoxic T cells observed by fluorine-19 magnetic resonance Imaging

Noninvasive biomarkers of anti-tumoral efficacy are of great importance to the development of therapeutic agents. Tumor oxygenation has been shown to be an important indicator of therapeutic response. We report the use of intracellular labeling of tumor cells with perfluorocarbon (PFC) molecules, combined with quantitative ¹⁹F spin-lattice relaxation rate (R₁) measurements, to assay tumor cell oxygen dynamics in situ. In a murine central nervous system (CNS) GL261 glioma model, we visualized the impact of Pmel-1 cytotoxic T cell immunotherapy, delivered intravenously, on intracellular tumor oxygen levels.

Direct labeling of 19F-perfluorocarbon onto multilayered cell sheet for MRI-based non-invasive cell tracking

Autologous stem cell transplantation for eye diseases is immunologically preferable to avoid allograft rejection. However, the fate of the grafted cells has never been studied. Here, we propose to use 19F-perfluorocarbon magnetic resonance imaging tracer agent, to label cell sheet in vitro. This labeling enables non-invasive visualization of possible migration of grafted cells.

Combining perfluorocarbon and superparamagnetic iron-oxide cell labeling for improved and expanded applications of cellular MRI

The ability to detect the migration of cells in living organisms is fundamental in understanding biological processes and important for the development of novel cell-based therapies to treat disease. MRI can be used to detect the migration of cells labeled with superparamagnetic iron-oxide (SPIO) or perfluorocarbon (PFC) agents. In this study, we explored combining these two cell-labeling approaches to overcome current limitations and enable new applications for cellular MRI.

In vivo MR detection of fluorine-labeled human MSC using the bSSFP sequence

Mesenchymal stem cells (MSC) are used to restore deteriorated cell environments. There is a need to specifically track these cells following transplantation in order to evaluate different methods of implantation, to follow their migration within the body, and to quantify their accumulation at the target. Cellular magnetic resonance imaging (MRI) using fluorine-based nanoemulsions is a great means to detect these transplanted cells in vivo because of the high specificity for fluorine detection and the capability for precise quantification.

In vivo MRI cell tracking using perfluorocarbon probes and fluorine-19 detection

This article presents a brief review of preclinical in vivo cell-tracking methods and applications using perfluorocarbon (PFC) probes and fluorine-19 ((19) F) MRI detection. Detection of the (19) F signal offers high cell specificity and quantification ability in spin density-weighted MR images. We discuss the compositions of matter, methods and applications of PFC-based cell tracking using ex vivo and in situ PFC labeling in preclinical studies of inflammation and cellular therapeutics. We also address the potential applicability of (19) F cell tracking to clinical trials.

A multi-modality platform to image stem cell graft survival in the naïve and stroke-damaged mouse brain

Neural stem cell implantations have been extensively investigated for treatment of brain diseases such as stroke. In order to follow the localization and functional status of cells after implantation noninvasive imaging is essential. Therefore, we developed a comprehensive multi-modality platform for in vivo imaging of graft localization, density, and survival using 19F magnetic resonance imaging in combination with bioluminescence imaging.

Combining perfluorocarbon and superparamagnetic iron oxide cell labeling for improved and expanded applications of cellular MRI

The ability to detect the migration of cells in living organisms is fundamental in understanding biological processes and important for the development of novel cell-based therapies to treat disease. MRI can be used to detect the migration of cells labeled with superparamagnetic iron-oxide (SPIO) or perfluorocarbon (PFC) agents. In this study, we explored combining these two cell-labeling approaches to overcome current limitations and enable new applications for cellular MRI.

Labeling cells for in vivo tracking using 19F MRI

Noninvasive in vivo cell tracking is crucial to fully understand the function of mobile and/or transplanted cells, particularly immune cells and cellular therapeutics. 19F MRI for cell tracking has several advantages; chief among them are its noninvasive nature which allows longitudinal data acquisition, use of a stable, non-radioactive isotope permitting long-term tracking, the absence of confounding endogenous signal, and the ability to quantify cell numbers from image data.

Visualizing brain inflammation with a shingled-leg radio-frequency head probe for 19F/1H MRI

Magnetic resonance imaging (MRI) provides the opportunity of tracking cells in vivo. Major challenges in dissecting cells from the recipient tissue and signal sensitivity constraints albeit exist. In this study, we aimed to tackle these limitations in order to study inflammation in autoimmune encephalomyelitis. We constructed a very small dual-tunable radio frequency (RF) birdcage probe tailored for 19F (fluorine) and 1H (proton) MR mouse neuroimaging. The novel design eliminated the need for extra electrical components on the probe structure and afforded a uniform -field as well as good SNR.

In vivo intracellular oxygen dynamics in murine brain glioma and immunotherapeutic response of cytotoxic T cells observed by fluorine-19 magnetic resonance imaging

Noninvasive biomarkers of anti-tumoral efficacy are of great importance to the development of therapeutic agents. Tumor oxygenation has been shown to be an important indicator of therapeutic response. We report the use of intracellular labeling of tumor cells with perfluorocarbon (PFC) molecules, combined with quantitative ¹⁹F spin-lattice relaxation rate (R₁) measurements, to assay tumor cell oxygen dynamics in situ. In a murine central nervous system (CNS) GL261 glioma model, we visualized the impact of Pmel-1 cytotoxic T cell immunotherapy, delivered intravenously, on intracellular tumor oxygen levels. GL261 glioma cells were labeled ex vivo with PFC and inoculated into the mouse striatum. The R₁ of ¹⁹F labeled cells was measured using localized single-voxel magnetic resonance spectroscopy, and the absolute intracellular partial pressure of oxygen (pO₂) was ascertained. Three days after tumor implantation, mice were treated with 2×10⁷ cytotoxic T cells intravenously. At day five, a transient spike in pO₂ was observed indicating an influx of T cells into the CNS and putative tumor cell apoptosis. Immunohistochemistry and quantitative flow cytometry analysis confirmed that the pO₂ was causally related to the T cells infiltration. Surprisingly, the pO₂ spike was detected even though few (∼4×10⁴) T cells actually ingress into the CNS and with minimal tumor shrinkage. These results indicate the high sensitivity of this approach and its utility as a non-invasive surrogate biomarker of anti-cancer immunotherapeutic response in preclinical models.

(19)F MRI tracer preserves in vitro and in vivo properties of hematopoietic stem cells

Hematopoietic stem cells (HSCs) have numerous therapeutic applications including immune reconstitution, enzyme replacement, regenerative medicine, and immunomodulation. The trafficking and persistence of these cells after administration is a fundamental question for future therapeutic applications of HSCs. Here, we describe the safe and efficacious labeling of human CD34(+) HSCs with a novel, self-delivering perfluorocarbon ¹⁹F magnetic resonance imaging (MRI) tracer, which has recently been authorized for use in a clinical trial to track therapeutic cells.

Non-invasive imaging of transplanted human neural stem cells and ECM scaffold remodeling in the stroke-damaged rat brain by (19)F- and diffusion-MRI

Transplantation of human neural stem cells (hNSCs) is emerging as a viable treatment for stroke related brain injury. However, intraparenchymal grafts do not regenerate lost tissue, but rather integrate into the host parenchyma without significantly affecting the lesion cavity. Providing a structural support for the delivered cells appears important for cell based therapeutic approaches. The non-invasive monitoring of therapeutic methods would provide valuable information regarding therapeutic strategies but remains a challenge.

In vivo tracking of human neural stem cells with 19F magnetic resonance imaging

Magnetic resonance imaging (MRI) is a promising tool for monitoring stem cell-based therapy. Conventionally, cells loaded with ironoxide nanoparticles appear hypointense on MR images. However, the contrast generated by ironoxide labeled cells is neither specific due to ambiguous background nor quantitative. A strategy to overcome these drawbacks is 19F MRI of cells labeled with perfluorocarbons.

A novel (19)F agent for detection and quantification of human dendritic cells using magnetic resonance imaging

Monitoring of cell therapeutics in vivo is of major importance to estimate its efficacy. Here, we present a novel intracellular label for (19)F magnetic resonance imaging (MRI)-based cell tracking, which allows for noninvasive, longitudinal cell tracking without the use of radioisotopes. A key advantage of (19)F MRI is that it allows for absolute quantification of cell numbers directly from the MRI data. The (19)F label was tested in primary human monocyte-derived dendritic cells.

Inflammation driven by overexpression of the hypoglycosylated abnormal mucin 1 (MUC1) links inflammatory bowel disease and pancreatitis

Pancreatitis occurs as an extraintestinal complication of inflammatory bowel disease (IBD), but the cause is poorly understood. Mucin 1 (MUC1) is overexpressed in an abnormal, hypoglycosylated form on the colonic epithelium in human IBD where it contributes to inflammation. MUC1 is also expressed on pancreatic ductal epithelia. We tested the possibility that in IBD, MUC1 expression on pancreatic ducts is also abnormal leading to inflammation and pancreatitis.

In vivo observation of intracellular oximetry in perfluorocarbon-labeled glioma cells and chemotherapeutic response in the CNS using fluorine-19 MRI

Preclinical development of therapeutic agents against cancer could greatly benefit from noninvasive markers of tumor killing. Potentially, the intracellular partial pressure of oxygen (pO(2) ) can be used as an early marker of antitumor efficacy. Here, the feasibility of measuring intracellular pO(2) of central nervous system glioma cells in vivo using (19) F magnetic resonance techniques is examined. Rat 9L glioma cells were labeled with perfluoro-15-crown-5-ether ex vivo and then implanted into the rat striatum. (19) F MRI was used to visualize tumor location in vivo.

In vivo cytometry of antigen-specific t cells using 19F MRI

Noninvasive methods to image the trafficking of phenotypically defined immune cells are paramount as we attempt to understand adaptive immunity. A (19)F MRI-based methodology for tracking and quantifying cells of a defined phenotype is presented. These methods were applied to a murine inflammation model using antigen-specific T cells. The T cells that were intracellularly labeled ex vivo with a perfluoropolyether (PFPE) nanoemulsion and cells were transferred to a host receiving a localized inoculation of antigen. Longitudinal (19)F MRI over 21 days revealed a dynamic accumulation and clearance of T cells in the lymph node (LN) draining the antigen.

Fluorine-19 MRI for visualization and quantification of cell migration in a diabetes model

This article describes an in vivo imaging method for visualizing and quantifying a specific cell population. Cells are labeled ex vivo with a perfluoropolyether nanoparticle tracer agent and then detected in vivo using (19)F MRI following cell transfer. (19)F MRI selectively visualizes only the labeled cells with no background, and a conventional (1)H image taken in the same imaging session provides anatomical context. Using the nonobese diabetic mouse, an established model of type 1 diabetes, (19)F MRI data were acquired showing the early homing behavior of diabetogenic T cells to the pancreas.

In vivo imaging platform for tracking immunotherapeutic cells

Cellular therapeutics show great promise for the treatment of disease, but few noninvasive techniques exist for monitoring the cells after administration. Here we present a magnetic resonance imaging (MRI) technology that uses perfluoropolyether (PFPE) agents to track cells in vivo. Fluorine MRI selectively images only the labeled cells, and a ‘conventional’ (1)H image places the cells in their anatomical context. We labeled phenotypically defined dendritic cells (DCs) with PFPE ex vivo and observed efficient intracellular uptake of the PFPE with little effect on DC function.

Celsense, Inc. Products for Cellular Imaging

Celsense, Inc. offers novel imaging agents used to non-invasively visualize and measure biological processes at the cellular level in humans and other living organisms. The core innovations of the Company involve imaging agents that can be used to observe cell trafficking using fluorine magnetic resonance imaging (19F MRI) techniques. Applications include the discovery and development of therapeutic agents (small molecules, recombinant proteins, cells, or genes), monitoring therapeutic delivery in clinical trials, and monitoring delivery for clinically approved therapeutics.

Cell Sense: Enabling in vivo Cell Tracking

A challenge in the development and translation of new cellular therapies is effective tracking of cells post-transfer in both animal and human subjects. Cell Sense reagents are engineered to safely and efficiently label cells ex vivo, rendering them detectable with fluorine-19 magnetic
resonance imaging (19F MRI), a safe, highly specific and quantitative imaging modality. Cell Sense reagents provide positive contrast independent of the anatomical image and without confounding
background. A variety of therapeutic cell types have been labeled using Cell Sense, enabling applications in regenerative medicine, immunotherapy, and diagnosis of inflammation using ex vivo labeled immune cells.

V-Sense: Imaging Inflammation

The inflammatory process plays a role in the onset and progression of a variety of acute and chronic diseases. The quantification of inflammation using rapid ex vivo measurements or non-invasive in vivo imaging can facilitate research and aid development of novel therapeutics. V-Sense 1000H (V-Sense) is a perfluorocarbon emulsion (manufactured by Celsense, Inc.) which is phagocytosed by immune cells in situ, allowing detection of a general aspect of inflammation: immune cell infiltration to a distal location. A specific and quantitative label, V-Sense has the capacity to simplify and streamline experimental workflow and enable in vivo imaging, contributing vivid insight into the inflammatory process.

Improved compressed sensing reconstruction for 19 F magnetic resonance imaging

Objective — In magnetic resonance imaging (MRI), compressed sensing (CS) enables the reconstruction of undersampled sparse data sets. Thus, partial acquisition of the underlying k-space data is sufficient, which significantly reduces measurement time. While 19F MRI data sets are spatially sparse, they often suffer from low SNR. This can lead to artifacts in CS reconstructions that reduce the image quality. We present a method to improve the image quality of undersampled, reconstructed CS data sets.

A chemical shift encoding (CSE) approach for spectral selection in fluorine‐19 MRI

PURPOSE: To develop a chemical shift encoding (CSE) approach for fluorine-19 MRI of perfluorocarbons in the presence of multiple known fluorinated chemical species.

THEORY AND METHODS: A multi-echo CSE technique is applied for spectral separation of the perfluorocarbon perfluoro-15-crown-5-ether (PFCE) and isoflurane (ISO) based on their chemical shifts at 4.7 T. Cramér-Rao lower bound analysis is used to identify echo combinations with optimal signal-to-noise performance. Signal contributions are fit with a multispectral fluorine signal model using a non-linear least squares estimation reconstruction directly from k-space data. This CSE approach is tested in fluorine-19 phantoms and in a mouse with a 2D and 3D spoiled gradient-echo acquisition using multiple echo times determined from Cramér-Rao lower bound analysis.

Chemical shift encoding (CSE) for sensitive fluorine‐19 MRI of perfluorocarbons with complex spectra

PURPOSE: To implement a fluorine-19 (19 F) chemical shift encoding (CSE) approach for the sensitive imaging of molecules with multi-resonance spectra to remove their chemical shift displacement (CSD) artifacts, and to characterize its sensitivity versus established pulse sequences.

Hydrophilic fluorinated molecules for spectral 19F MRI

Fluorine-19 (19F) Magnetic Resonance Imaging (MRI) is an emerging modality for molecular imaging and cell tracking. The hydrophobicity of current exogenous probes, perfluorocarbons (PFCs) and perfluoropolyethers (PFPEs), limits the formulation options available for in vivo applications. Hydrophilic probes permit more formulation flexibility. Further, the broad Nuclear Magnetic Resonance (NMR) chemical shift range of organofluorine species enables multiple probes with unique 19F MR signatures for simultaneous interrogation of distinct molecular targets in vivo. We report herein a flexible approach to stable liposomal formulations of hydrophilic fluorinated molecules (each bearing numerous magnetically equivalent 19F atoms), with 19F encapsulation of up to 22.7 mg/mL and a per particle load of 3.6 × 106 19F atoms. Using a combination of such probes, we demonstrate, with no chemical shift artifacts, the simultaneous imaging of multiple targets within a given target volume by spectral 19F MRI.

Novel Nanoimaging Strategies for Noninvasive Graft Monitoring in Vascularized Composite Allotransplantation

This article focuses on the unmet needs and key challenges in vascularized composite allotransplantation (VCA) and the opportunities for current nanotechnology advances in therapeutics and diagnostics for personalized treatment and optimization of clinical outcomes. The transformative role of nanoimaging as a noninvasive tool in the longitudinal surveillance of acute and chronic rejection after VCA is critically reviewed. Nanoimaging can inform management decisions and guide continuous treatment adjustments over time in patients to improve safety and efficacy in VCA. Nanoimaging signatures can be unbiased and quantitative measures of treatment effectiveness as well as medication adherence, both of which are critical prerequisites for overall graft survival and patient quality of life.

Characterization of perfluorocarbon relaxation times and their influence on the optimization of fluorine‐19 MRI at 3 tesla

PURPOSE: To characterize and optimize 19 F MRI for different perfluorocarbons (PFCs) at 3T and quantify the loss of acquisition efficiency as a function of different temperature and cellular conditions.

METHODS: The T1 and T2 relaxation times of the commonly used PFCs perfluoropolyether (PFPE), perfluoro-15-crown-5-ether (PFCE), and perfluorooctyl bromide (PFOB) were measured in phantoms and in several different conditions (cell types, presence of fixation agent, and temperatures). These relaxation times were used to optimize pulse sequences through numerical simulations. The acquisition efficiency in each cellular condition was then determined as the ratio of the signal after optimization with the reference relaxation times and after optimization with its proper relaxation times. Finally, PFC detection limits were determined.

Comparison of different compressed sensing algorithms for low SNR 19 F MRI applications-Imaging of transplanted pancreatic islets and cells labeled with perfluorocarbons

Transplantation of pancreatic islets is a possible treatment option for patients suffering from Type I diabetes. In vivo imaging of transplanted islets is important for assessment of the transplantation site and islet distribution. Thanks to its high specificity, the absence of intrinsic background signal in tissue and its potential for quantification, 19 F MRI is a promising technique for monitoring the fate of transplanted islets in vivo. In order to overcome the inherent low sensitivity of 19 F MRI, leading to long acquisition times with low signal-to-noise ratio (SNR), compressed sensing (CS) techniques are a valuable option. We have validated and compared different CS algorithms for acceleration of 19 F MRI acquisition in a low SNR regime using pancreatic islets labeled with perfluorocarbons both in vitro and in vivo. Using offline simulation on both in vitro and in vivo low SNR fully sampled 19 F MRI datasets of labeled islets, we have shown that CS is effective in reducing the image acquisition time by a factor of three to four without seriously affecting SNR, regardless of the particular algorithms used in this study, with the exception of CoSaMP. Using CS, signals can be detected that might have been missed by conventional 19 F MRI. Among different algorithms (SPARSEMRI, OMMP, IRWL1, Two-level and CoSAMP), the two-level l1 method has shown the best performance if computational time is taken into account. We have demonstrated in this study that different existing CS algorithms can be used effectively for low SNR 19 F MRI. An up to fourfold gain in SNR/scan time could be used either to reduce the scan time, which is beneficial for clinical and translational applications, or to increase the number of averages, to potentially detect otherwise undetected signal when compared with conventional 19 F MRI acquisitions. Potential applications in the field of cell therapy have been demonstrated.

Characterization of perfluorocarbon relaxation times and their influence on the optimization of fluorine‐19 MRI at 3 tesla

To characterize and optimize 19F MRI for different perfluorocarbons (PFCs) at 3T and quantify the loss of acquisition efficiency as a function of different temperature and cellular conditions.

In vivo MRI cell tracking using perfluorocarbon probes and fluorine-19 detection

This article presents a brief review of preclinical in vivo cell-tracking methods and applications using perfluorocarbon (PFC) probes and fluorine-19 ((19) F) MRI detection. Detection of the (19) F signal offers high cell specificity and quantification ability in spin density-weighted MR images. We discuss the compositions of matter, methods and applications of PFC-based cell tracking using ex vivo and in situ PFC labeling in preclinical studies of inflammation and cellular therapeutics. We also address the potential applicability of (19) F cell tracking to clinical trials.

Accelerated Fluorine-19 MRI Cell Tracking Using Compressed Sensing

Cell tracking using perfluorocarbon labels and fluorine-19 (19F) MRI is a noninvasive approach to visualize and quantify cell populations in vivo. In this study, we investigated three-dimensional compressed sensing methods to accelerate 19F MRI data acquisition for cell tracking and evaluate the impact of acceleration on 19F signal quantification. We show that a greater than 8-fold reduction in imaging time was feasible without pronounced image degradation and with minimal impact on the image signal-to-noise ratio and 19F quantification accuracy. In 19F phantom studies, we show that apparent feature topology is maintained with compressed sensing reconstruction, and false positive signals do not appear in areas devoid of fluorine.

Fluorine (19F) MRS and MRI in biomedicine

Shortly after the introduction of (1)H MRI, fluorinated molecules were tested as MR-detectable tracers or contrast agents. Many fluorinated compounds, which are nontoxic and chemically inert, are now being used in a broad range of biomedical applications, including anesthetics, chemotherapeutic agents, and molecules with high oxygen solubility for respiration and blood substitution. These compounds can be monitored by fluorine ((19)F) MRI and/or MRS, providing a noninvasive means to interrogate associated functions in biological systems. As a result of the lack of endogenous fluorine in living organisms, (19)F MRI of ‘hotspots’ of targeted fluorinated contrast agents has recently opened up new research avenues in molecular and cellular imaging.

Inflammation driven by overexpression of the hypoglycosylated abnormal mucin 1 (MUC1) links inflammatory bowel disease and pancreatitis

OBJECTIVE: Pancreatitis occurs as an extraintestinal complication of inflammatory bowel disease (IBD), but the cause is poorly understood. Mucin 1 (MUC1) is overexpressed in an abnormal, hypoglycosylated form on the colonic epithelium in human IBD where it contributes to inflammation. MUC1 is also expressed on pancreatic ductal epithelia. We tested the possibility that in IBD, MUC1 expression on pancreatic ducts is also abnormal leading to inflammation and pancreatitis.

(19)F MRI for quantitative in vivo cell tracking

Cellular therapy, including stem cell transplants and dendritic cell vaccines, is typically monitored for dosage optimization, accurate delivery, and localization using noninvasive imaging, of which magnetic resonance imaging (MRI) is a key modality. (19)F MRI retains the advantages of MRI as an imaging modality, and also allows direct detection of labeled cells for unambiguous identification and quantification, unlike typical metal-based contrast agents. Recent developments in (19)F MRI-based in vivo cell quantification, the existing clinical use of (19)F compounds and current explosive interest in cellular therapeutics have brought (19)F imaging technology closer to clinical application.

In vivo observation of intracellular oximetry in perfluorocarbon-labeled glioma cells and chemotherapeutic response in the CNS using fluorine-19 MRI

Preclinical development of therapeutic agents against cancer could greatly benefit from noninvasive markers of tumor killing. Potentially, the intracellular partial pressure of oxygen (pO(2) ) can be used as an early marker of antitumor efficacy. Here, the feasibility of measuring intracellular pO(2) of central nervous system glioma cells in vivo using (19) F magnetic resonance techniques is examined. Rat 9L glioma cells were labeled with perfluoro-15-crown-5-ether ex vivo and then implanted into the rat striatum. (19) F MRI was used to visualize tumor location in vivo. The mean (19) F T(1) of the implanted cells was measured using localized, single-voxel spectroscopy. The intracellular pO(2) in tumor cells was determined from an in vitro calibration curve. The basal pO(2) of 9L cells (day 3) was determined to be 45.3 ± 5 mmHg (n = 6). Rats were then treated with a 1 × LD10 dose of bischloroethylnitrosourea intravenously and changes in intracellular pO(2) were monitored. The pO(2) increased significantly (P = 0.042, paired T-test) to 141.8 ± 3 mmHg within 18 h after bischloroethylnitrosourea treatment (day 4) and remained elevated (165 ± 24 mmHg) for at least 72 h (day 6). Intracellular localization of the perfluoro-15-crown-5-ether emulsion in 9L cells before and after bischloroethylnitrosourea treatment was confirmed by histological examination and fluorescence microscopy. Overall, noninvasive (19) F magnetic resonance techniques may provide a valuable preclinical tool for monitoring therapeutic response against central nervous system or other deep-seated tumors.

In vivo cytometry of antigen-specific t cells using 19F MRI

Noninvasive methods to image the trafficking of phenotypically defined immune cells are paramount as we attempt to understand adaptive immunity. A (19)F MRI-based methodology for tracking and quantifying cells of a defined phenotype is presented. These methods were applied to a murine inflammation model using antigen-specific T cells. The T cells that were intracellularly labeled ex vivo with a perfluoropolyether (PFPE) nanoemulsion and cells were transferred to a host receiving a localized inoculation of antigen. Longitudinal (19)F MRI over 21 days revealed a dynamic accumulation and clearance of T cells in the lymph node (LN) draining the antigen. The apparent T-cell numbers were calculated in the LN from the time-lapse (19)F MRI data. The effect of in vivo T-cell division on the (19)F MRI cell quantification accuracy was investigated using fluorescence assays. Overall, in vivo cytometry using PFPE labeling and (19)F MRI is broadly applicable to studies of whole-body cell biodistribution.

Fluorine-containing nanoemulsions for MRI cell tracking

In this article we review the chemistry and nanoemulsion formulation of perfluorocarbons used for in vivo(19)F MRI cell tracking. In this application, cells of interest are labeled in culture using a perfluorocarbon nanoemulsion. Labeled cells are introduced into a subject and tracked using (19)F MRI or NMR spectroscopy. In the same imaging session, a high-resolution, conventional ((1)H) image can be used to place the (19)F-labeled cells into anatomical context. Perfluorocarbon-based (19)F cell tracking is a useful technology because of the high specificity for labeled cells, ability to quantify cell accumulations, and biocompatibility. This technology can be widely applied to studies of inflammation, cellular regenerative medicine, and immunotherapy.

Self-delivering nanoemulsions for dual fluorine-19 MRI and fluorescence detection

We report the design, synthesis, and biological testing of highly stable, nontoxic perfluoropolyether (PFPE) nanoemulsions for dual 19F MRI-fluorescence detection. A linear PFPE polymer was covalently conjugated to common fluorescent dyes (FITC, Alexa647 and BODIPy-TR), mixed with pluronic F68 and linear polyethyleneimine (PEI), and emulsified by microfluidization. Prepared nanoemulsions (<200 nm) were readily taken up by both phagocytic and non-phagocytic cells in vitro after a short (approximately 3 h) co-incubation. Following cell administration in vivo, 19F MRI selectively visualizes cell migration. Exemplary in vivo MRI images are presented of T cells labeled with a dual-mode nanoemulsion in a BALB/c mouse. Fluorescence detection enables fluorescent microscopy and FACS analysis of labeled cells, as demonstrated in several immune cell types including Jurkat cells, primary T cells and dendritic cells.

Fluorine-19 MRI for visualization and quantification of cell migration in a diabetes model

This article describes an in vivo imaging method for visualizing and quantifying a specific cell population. Cells are labeled ex vivo with a perfluoropolyether nanoparticle tracer agent and then detected in vivo using (19)F MRI following cell transfer. (19)F MRI selectively visualizes only the labeled cells with no background, and a conventional (1)H image taken in the same imaging session provides anatomical context. Using the nonobese diabetic mouse, an established model of type 1 diabetes, (19)F MRI data were acquired showing the early homing behavior of diabetogenic T cells to the pancreas. A computational algorithm provided T cell counts in the pancreas. Approximately 2% of the transferred cells homed to the pancreas after 48 hr. The technique allows for both unambiguous detection of labeled cells and quantification directly from the in vivo images. The in vivo quantification and cell trafficking patterns were verified using (19)F spectroscopy and fluorescence microscopy in excised pancreata. The labeling procedure did not affect T-cell migration in vivo. This imaging platform is applicable to many cell types and disease models and can potentially be used for monitoring the trafficking of cellular therapeutics.

In vivo imaging platform for tracking immunotherapeutic cells

Cellular therapeutics show great promise for the treatment of disease, but few noninvasive techniques exist for monitoring the cells after administration. Here we present a magnetic resonance imaging (MRI) technology that uses perfluoropolyether (PFPE) agents to track cells in vivo. Fluorine MRI selectively images only the labeled cells, and a ‘conventional’ (1)H image places the cells in their anatomical context. We labeled phenotypically defined dendritic cells (DCs) with PFPE ex vivo and observed efficient intracellular uptake of the PFPE with little effect on DC function. We injected labeled DCs into tissue or intravenously in mice and then tracked the cells in vivo using (19)F MRI. Although we focused on DCs, which are being developed as immunotherapeutics for cancer and autoimmune diseases, this technology should be useful for monitoring a wide range of cell types in vivo.

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