Mangala Srinivas

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 19F MRI following cell transfer. 19F MRI selectively visualizes only the labeled cells with no background, and a conventional 1H image taken in the same imaging session provides anatomical context. Using the nonobese diabetic mouse, an established model of type 1 diabetes, 19F 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 19F 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. Magn Reson Med 58:725–734, 2007.

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. We review the application of (19)F MRI to cell tracking, discussing intracellular (19)F labels, cell labeling and in vivo quantification, as well as the potential clinical uses of (19)F MRI.

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. The intracellular fluorescence signal is directly proportional to the 19F NMR signal and can be used to calibrate cell loading in vitro.

Imaging of cellular therapies.

Cellular therapy promises to revolutionize medicine, by restoring tissue and organ function, and combating key disorders including cancer. As with all major developments, new tools must be introduced to allow optimization. For cell therapy, the key tool is in vivo imaging for real time assessment of parameters such as cell localization, numbers and viability. Such data is critical to modulate and tailor the therapy for each patient. In this review, we discuss recent work in the field of imaging cell therapies in the clinic, including preclinical work where clinical examples are not yet available. Clinical trials in which transferred cells were imaged using magnetic resonance imaging (MRI), nuclear scintigraphy, single photon emission computed tomography (SPECT), and positron emission tomography (PET) are evaluated from an imaging perspective. Preclinical cell tracking studies that focus on fluorescence and bioluminescence imaging are excluded, as these modalities are generally not applicable to clinical cell tracking. In this review, we assess the advantages and drawbacks of the various imaging techniques available, focusing on immune cells, particularly dendritic cells. Both strategies of prelabeling cells before transplant and the use of an injectable label to target cells in situ are covered. Finally, we discuss future developments, including the emergence of multimodal imaging technology for cell tracking from the preclinical to the clinical realm.

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 19F 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 19F 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 19F MRI data. The effect of in vivo T‐cell division on the 19F MRI cell quantification accuracy was investigated using fluorescence assays. Overall, in vivo cytometry using PFPE labeling and 19F MRI is broadly applicable to studies of whole‐body cell biodistribution. Magn Reson Med, 2009.

Customizable, multi-functional fluorocarbon nanoparticles for quantitative in vivo imaging using 19F MRI and optical imaging.

Monitoring cell trafficking in vivo noninvasively is critical to improving cellular therapeutics, drug delivery, and understanding disease progression. In vivo imaging, of which magnetic resonance imaging (MRI) is a key modality, is commonly used for such monitoring. (19)F MRI allows extremely specific detection and quantification of cell numbers directly from in vivo image data, longitudinally and without ionizing radiation. We used fluorocarbons previously used in blood substitutes and imaging agents for ultrasound and computed tomography to synthesize monodisperse nanoparticles that are stable at 37 degrees C and can be frozen for storage. These large (19)F labeling compounds are insoluble in aqueous environments and often emulsified, typically forming emulsions unsuitable for long-term storage. Instead, we used a non-toxic polymer already in clinical use, poly(D,L-lactide-co-glycolide), to encapsulate a range of (19)F compounds. These nanoparticles can be customized in terms of content (imaging agent, fluorescent dye, drug), size (200-2000 nm), coating (targeting agent, antibody) and surface charge (-40 to 30 mV). We added a fluorescent dye and antibody to demonstrate the versatility of this modular imaging agent. These nanoparticles are adaptable to multimodal imaging, although here we focused on MRI and fluorescence imaging. Here, we imaged primary human dendritic cells, as used in clinical vaccines.

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. (19)F 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. However, generation of sufficient signal i.e. (19)F cell loading is a key challenge, particularly with non-phagocytic cells such as lymphocytes and stem cells. A range of (19)F cell labels have been developed, including emulsions, particles, polymers, and agents for clinical use. Various animal and primary human cells, such as dendritic cells, lymphocytes and phagocytes have been successfully labeled and studied in models of autoimmune disease, inflammation and transplant rejection. Primary human cells, particularly dendritic cells as used in vaccine therapy have been tested for imminent clinical application. Here, we summarize current cell loading strategies and sensitivity of in vivo cell imaging with (19)F MRI, and discuss the processing of image data for accurate quantification of cell numbers. This novel technology is uniquely applicable to the longitudinal and quantitative tracking of cells in vivo.

A novel 19F 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 19F magnetic resonance imaging (MRI)‐based cell tracking, which allows for noninvasive, longitudinal cell tracking without the use of radioisotopes. A key advantage of 19F MRI is that it allows for absolute quantification of cell numbers directly from the MRI data. The 19F label was tested in primary human monocyte‐derived dendritic cells. These cells took up label effectively, resulting in a labeling of 1.7 ± 0.1 × 1013 19F atoms per cell, with a viability of 80 ± 6%, without the need for electroporation or transfection agents. This results in a minimum detection sensitivity of about 2,000 cells/voxel at 7 T, comparable with gadolinium‐labeled cells. Comparison of the detection sensitivity of cells labeled with 19F, iron oxide and gadolinium over typical tissue background showed that unambiguous detection of the 19F‐labeled cells was simpler than with the contrast agents. The effect of the 19F agent on cell function was minimal in the context of cell‐based vaccines. From these data, we calculate that detection of 30,000 cells in vivo at 3 T with a reasonable signal to noise ratio for 19F images would require less than 30 min with a conventional fast spin echo sequence, given a coil similar to the one used in this study. This is well within acceptable limits for clinical studies, and thus, we conclude that 19F MRI for quantitative cell tracking in a clinical setting has great potential.

Obstacles on the way to the clinical visualisation of beta cells: looking for the Aeneas of molecular imaging to navigate between Scylla and Charybdis.

For more than a decade, researchers have been trying to develop non-invasive imaging techniques for the in vivo measurement of viable pancreatic beta cells. However, in spite of intense research efforts, only one tracer for positron emission tomography (PET) imaging is currently under clinical evaluation. To many diabetologists it may remain unclear why the imaging world struggles to develop an effective method for non-invasive beta cell imaging (BCI), which could be useful for both research and clinical purposes. Here, we provide a concise overview of the obstacles and challenges encountered on the way to such BCI, in both native and transplanted islets. We discuss the major difficulties posed by the anatomical and cell biological features of pancreatic islets, as well as the chemical and physical limits of the main imaging modalities, with special focus on PET, SPECT and MRI. We conclude by indicating new avenues for future research in the field, based on several remarkable recent results.

Early identification of antigen-specific immune responses in vivo by [18F]-labeled 3′-fluoro-3′-deoxy-thymidine ([18F]FLT) PET imaging

Current biomarkers are unable to adequately predict vaccine-induced immune protection in humans with infectious disease or cancer. However, timely and adequate assessment of antigen-specific immune responses is critical for successful vaccine development. Therefore, we have developed a method for the direct assessment of immune responses in vivo in a clinical setting. Melanoma patients with lymph node (LN) metastases received dendritic cell (DC) vaccine therapy, injected intranodally, followed by [18F]-labeled 3′-fluoro-3′-deoxy-thymidine ([18F]FLT) PET at varying time points after vaccination. Control LNs received saline or DCs without antigen. De novo immune responses were readily visualized in treated LNs early after the prime vaccination, and these signals persisted for up to 3 wk. This selective [18F]FLT uptake was markedly absent in control LNs, although tracer uptake in treated LNs increased profoundly with as little as 4.5 × 105 DCs. Immunohistochemical staining confirmed injected DC dispersion to T-cell areas and resultant activation of CD4+ and CD8+ T cells. The level of LN tracer uptake significantly correlates to the level of circulating antigen-specific IgG antibodies and antigen-specific proliferation of T cells in peripheral blood. Furthermore, this correlation was not observed with [18F]-labeled fluoro-2-deoxy-2-d-glucose. Therefore, [18F]FLT PET offers a sensitive tool to study the kinetics, localization, and involvement of lymphocyte subsets in response to vaccination. This technique allows for early discrimination of responding from nonresponding patients in anti-cancer vaccination and aid physicians in individualized decisionmaking.