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Nat Rev Immunol. Author manuscript; available in PMC 2014 January 09.
Published in final edited form as:
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Nat Rev Immunol. 2013 October ; 13(10): . doi:10.1038/nri3531.

Tracking immune cells in vivo using magnetic resonance


imaging
Eric T. Ahrens and
Department of Radiology, University of California at San Diego, San Diego, California 92093–
0695, USA
Jeff W. M. Bulte
Russell H. Morgan Department of Radiology and Radiological Science, Division of MR Research,
Department of Oncology, Department of Biomedical Engineering, Department of Chemical &
Biomolecular Engineering, and Cellular Imaging Section and Vascular Biology Program, Institute
for Cell Engineering, The Johns Hopkins University School of Medicine, 217 Traylor Building, 720
Rutland Avenue, Baltimore, Maryland 21205, USA
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Eric T. Ahrens: eta@ucsd.edu; Jeff W. M. Bulte: jwmbulte@mri.jhu.edu

Abstract
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. MRI-based cell tracking is now also being applied to evaluate therapeutics that
modulate endogenous immune cell recruitment and to monitor emerging cellular
immunotherapies. These recent uses show that MRI has the potential to be developed in many
applications to follow the fate of immune cells in vivo.

Non-invasive cell tracking is an emerging approach for imaging cells in their native
environment. The phenotype of an immune cell is partly defined by the patterns and
expression levels of cell surface molecules and secreted factors. These molecules are
commonly assayed in vitro using techniques such as flow cytometry and
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immunohistochemistry. However, determining which cell surface molecules and secreted


factors are present, and at what levels, under various culture conditions or disease states, is
only one piece of the puzzle. A more challenging question to answer relates to the biological
roles of these molecules in vivo. Non-invasive imaging of the trafficking patterns of
phenotypically defined populations of immune cells can have an important role in answering
this question. Moreover, immune cells are increasingly being used as next-generation
therapeutics to treat chronic conditions such as autoimmune disease and cancer. A common
requirement for the development of nearly all cell therapies is a non-invasive means to
visualize the biodistribution of cells following injection. Imaging of cell trafficking can
provide crucial information regarding the persistence and the motility of transferred cells, as
well as the optimal routes of delivery and the therapeutic doses for individuals. On the
regulatory side, emerging therapies using immune cells can be slow to gain regulatory
approval partly because clinical researchers are challenged to verify where the cells traffic to

© 2013 Macmillan Publishers Limited. All rights reserved


Competing interests statement
The authors declare competing financial interests: see Web version for details.
Ahrens and Bulte Page 2

immediately after inoculation and where they migrate to over time. Cell tracking in vivo can
potentially provide this information and might help to overcome regulatory barriers.
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Inflammation is a hallmark of many of the major diseases on which biomedical research


commonly focuses, such as autoimmune diseases, neurological disorders, transplant
rejection and cancer. Measuring the effectiveness of treatment in terms of the inflammatory
burden can be challenging. Conventional methods often include invasive biopsies and
histology or imaging methods that are nonspecific for inflammation or that lack quantitative
measures. There is a need for improved inflammation-specific imaging diagnostics, as well
as surrogate biomarkers of inflammation, that could enable researchers to determine the
efficacy of an anti-inflammatory therapy safely, quickly, quantitatively and in a longitudinal
manner. There is also a need for pharmacological safety profiling to detect off-target
inflammatory side effects in preclinical and clinical drug trials. Vital imaging can help to
steer the decision-making process at the preclinical and clinical trial stages; it can facilitate
smaller, less costly trials by enabling the enrolment of fewer patients. Imaging can
potentially yield a rich data set from each patient in terms of inflammation severity and its
time course in a three-dimensional anatomical context.

Given the clear need for in vivo cell tracking, much progress has been made in this area in
recent years. Imaging methods using radionuclides have traditionally been used for the non-
invasive imaging of leukocytes. However, technologies using magnetic resonance imaging
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(MRI) (BOX 1) are now emerging, and the field is experiencing a rapid expansion in the
development of new imaging probes and genetically encoded reporters that enable the
visualization of specific cell populations and molecular events in vivo in both animals and
humans. These new capabilities have been made possible by next-generation, non-toxic cell
labelling probes and by MRI methods. MRI has the advantage that it does not use ionizing
radiation and can safely image deep tissues at high resolution.

Box 1
Magnetic resonance imaging
The signal used for magnetic resonance imaging (MRI) is derived from endogenous
mobile water protons (1H) or fluorinated molecules (such as 19F) that are present or
introduced in the subject. When the subject is placed in a large static magnetic field, the
magnetic moment associated with 1H or 19F tends to align along the direction of the
magnetic field. The 1H or 19F nuclei are perturbed from this equilibrium by pulsed radio-
frequency radiation. Following the removal of the radio-frequency radiation, the nuclei
recover to equilibrium and induce a transient voltage in a receiver antenna; this transient
voltage constitutes the nuclear magnetic resonance (NMR) signal. The physical
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properties of a specific tissue, such as the density of nuclei, the nuclear spin–lattice
relaxation time (T1) and the spin–spin relaxation time (T2), often determine the amount
of signal that is available. The alignment of the nuclei along the magnetic field direction
is not instantaneous, but occurs gradually over a period that is parameterized by the time
constant T1. T2 is the characteristic time constant for which nuclei remain in ‘phase’
with each other, and its value is reflected in the duration of the transient NMR signal.
MRI-based cell tracking involves detecting cells that exhibit a differential signal. The
MRI signal can be controlled in four ways, as discussed below.
Positive contrast agents containing paramagnetic metals
Paramagnetic contrast agents primarily affect T1. Most often, T1 contrast agents contain
Gd3+ that is chelated to a low-molecular-mass molecule to limit toxicity. The
surrounding water protons rapidly exchange with the complex, which results in a

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reduction of T1 and an increase in signal intensity (positive contrast) of Gd3+-labelled


cells on T1-weighted magnetic resonance images.
Negative contrast agents containing superparamagnetic iron oxides
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Superparamagnetic iron oxide (SPIO) contrast agents primarily affect T2 by virtue of


their iron oxide crystals, which have a strong magnetic moment. These agents commonly
consist of small crystalline particles of ferrous and ferric oxides (FeO–Fe2O3) that are
coated with dextran. These particulates strongly perturb the magnetic field that they are
in proximity to. Surrounding water molecules subsequently experience a highly
inhomogeneous magnetic field, which results in a local signal loss (negative contrast) of
SPIO-labelled cells on T2-weighted magnetic resonance images.
Molecular probes that induce chemical exchange saturation transfer
Certain protons that are loosely bound at specific chemical sites, such as amide protons,
have a slightly different resonance frequency than water protons, from which the MRI
signal is always collected. When these labile non-water protons are irradiated with a
saturation pulse at their specific off-resonance frequency, the protons lose the ability to
create an MRI signal. The saturated labile non-water protons exchange position with the
water protons, which leads to a loss of MRI signal of the labelled cells on chemical
exchange saturation transfer (CEST) images. The CEST irradiation can be turned on and
off (see the figure).
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Molecular probes containing 19F


The physical principles behind detection and image formation are the same for both 1H
and 19F MRI. Unlike metal ion-based magnetic resonance contrast agents, which are
detected through their indirect effects on the surrounding water protons, the 19F probe
functions as a tracer agent in that 19F MRI directly detects the 19F nuclei that are
associated with the labelled cells, with no background. The 19F signal is directly
proportional to the number of fluorine atoms and the number of labelled cells that are
present (see the figure). A composite 19F and 1H image is generally constructed, which
shows the regions containing labelled cells within their anatomical context.
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In this Innovation article, we describe emerging methods and applications of MRI-based


tracking of immune cells. These methods use exogenous cell labels that are comprised of

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iron oxide nano-particles or perfluorocarbon (PFC) nano-emulsions (FIG. 1), or genetically-


encoded MRI reporters (FIG. 2). The field of MRI-based cell tracking is rapidly evolving,
and in the future we believe it will be possible to visualize not only the location and the
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number of cells in vivo but also more sophisticated biological processes, such as immune
cell viability and activation status. These advances indicate a future in which MRI platforms
will be used to elucidate a wide range of human cellular immunological processes in vivo.

Available in vivo cell tracking techniques


Much of our current knowledge of immune cell trafficking has come from static end points
that have been obtained using conventional light microscopy and flow cytometry. In recent
years, a wide range of imaging modalities have become available that can image immune
cells in their native environment; however, each of these techniques has inherent advantages
and limitations (TABLE 1). About a decade ago, the field saw a revolution with the
introduction of intravital, two-photon microscopy, which enables precise determination of
the movements of immune cells within lymph nodes and tumours1. However, because of
limited tissue opacity, it is not possible to non-invasively look into tissues at a depth beyond
~1 mm using two-photon microscopy. Deeper tissue penetration and whole-body non-
invasive tomography of small animals can be achieved by capturing the photons emitted by
fluorescent or bioluminescent probes. Such bioluminescence imaging has become one of the
most widely used techniques for non-invasive tracking of immune cells and inflammation in
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small animals, but it cannot be used in larger animals or clinically because of the physical
limits of light penetration in deep tissues.

Radionuclide labelling of cells is the oldest technique for tracking immune cells in larger
animals and humans2; for example, 111In-oxine labelling of autologous white blood cells is
used as a diagnostic agent for the imaging of occult inflammation and infection in humans
and is currently the only cell tracking technique approved by the United States Food and
Drug Administration Nuclear imaging is particularly useful for whole-body distribution
studies, as there is no background signal.

Reporter genes became available in the field of nuclear medicine in the early 1990s
following the development of the thymidine kinase enzyme that is derived from herpes
simplex virus (HSV)3. After administration of positron-emitting substrates, such as 18FIAU
(1-(2′-deoxy-2′-[18F]-β-D-arabinofuranosyl)-5-iodouracil) or 18FHBG (9-[4-[18F]fluoro-3-
(hydroxymethyl)butyl]guanine), HSV thymidine kinase phosphorylates the radioactive
probe and is responsible for its prolonged retention in transfected cells. In a. few studies, this
approach has been used to monitor T cell trafficking4,5, including the use of positron
emission tomography (PET) to visualize cytotoxic T cell homing to the tumour in a patient
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with glioma6.

However, there are cytotoxicity and patient safety concerns that limit the use of
radionuclide-based methods for cell tracking. Moreover, radionuclide-based techniques are
unable to provide anatomical imaging by themselves, and these scans must be combined, for
example, with computed tomography (CT) or MRI, which adds to the methodological
complexity and the cost. In addition, for the diagnostic imaging of inflammation using 111In-
oxine labelling of white blood cells, there is a considerable cost associated with the invasive
leukophoresis procedure and ex vivo labelling of the patient’s cells that is necessary before
reinfusion. For applications in which the transferred cells, having been labelled ex vivo, are
expected to persist in the body for extended periods of time (for example, in T cell and stem
cell therapies), the effect of radio-cytotoxicity on cell viability is a concern. In addition, the
finite half-life of the radioisotopes that are used precludes extended longitudinal studies.

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These limitations of radionuclide-based techniques have helped to stimulate the


development of alternative imaging methods, particularly MRI-based methods (TABLE 2).
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Metal ion-based MRI contrast agents


Positive and negative contrast agents
Intravascular MRI contrast agents that incorporate metal ions (such as Gd3+) are commonly
used as positive agents to enhance the signal of lesions in clinical scans. The paramagnetic
metal ions in these agents accelerate the relaxation of nearby water protons (1H), and this
signal enhancement can be detected by 1H MRI (BOX 1) as a localized increase of image
contrast. MRI contrast agents can also be used to label immune cells for cell tracking studies
(TABLE 2). After cellular internalization by simple co-incubation, electroporation or
conjugation to cell-labelling moieties, paramagnetic metal ions such as Mn2+ and Gd3+ can
provide cells with positive contrast7. Positive contrast is generally preferred for image
interpretation because the signal of the underlying anatomical structure is enhanced; the
alternative, negative contrast (see below), decreases the signal, which makes the tissue
invisible at times. However, labelling with paramagnetic metal ions only provides modest
sensitivity in terms of the number of cells that can be detected (TABLE 2). Particulate
formulations of metal ions, such as superparamagnetic iron oxide (SPIO) nanoparticles,
create a very strong localized magnetic field disturbance in the MRI scanner because of the
synergistic magnetic alignment of the individual iron ions. This localized magnetic
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perturbation affects nearby water protons, which causes a very strong negative contrast, in
which the areas containing SPIO particles become dark on the images8,9. Immune cells can
be labelled with SPIO nanoparticles using two main approaches (FIG. 1): sorted or mixed
cell populations can be labelled ex vivo by incubation with SPIO nanoparticles in culture
media; or non-selected phagocytic cell populations (for example, macrophages) can be
labelled in situ following intravenous injection of SPIO nanoparticles. In many cases, SPIO
particles have been conjugated to fluorochromes, which creates dual-mode agents that
enable the validation of in vivo MRI cell tracking by post-mortem light microscopy10,11.

Labelling immune cells ex vivo


Inspired by clinical studies using radionuclide-based tracking of tumour-infiltrating
lymphocytes, one of the first experimental applications of ex vivo SPIO labelling of cells
was to label lymphocytes (FIG. 3a), first by labelling the cell membrane12, followed by
labelling intra-cellular endosomes13,14. Membrane labelling can be achieved by targeting
antibodies or peptides to specific cell surface epitopes, whereas intracellular labelling
requires the use of cell-penetrating peptides or transfection agents if cells have a low level of
phagocytosis or macropinocytosis activity. Intracellular labelling of lymphocytes has proven
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to be challenging, owing to the fairly small cytoplasmic volume of non-activated cells and
the low phagocytic activity. The most widely used method for SPIO labelling of immune
cells is to complex the negatively charged SPIO particles with various cationic transfection
agents, such as poly-L-lysine or protamine sulphate, followed by co-incubation with cells in
culture15–17. Receptor-mediated endocytosis using specific SPIO–antibody conjugates, such
as conjugates that are specific for CD11c, has also been reported for the ex vivo labelling of
both immature and mature dendritic cells (DCs)18. Electroporation of the imaging agent into
immune cells is also feasible19.

MRI-based immune cell tracking using SPIO particles has been applied to many types of
preclinical study, ranging from the tumour homing of cytotoxic T cells20 and natural killer
cells21 to the organ-specific homing of autoimmune T cells22,23 (FIG. 3b) and to studies
investigating the migratory patterns of DCs used in cancer vaccines24–26. Iron oxide
particles with a large, micrometre-sized diameter have been used to extracellularly label

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lymphocytes with greater sensitivity than SPIO nanoparticles27. As these iron oxide particles
have a tenfold larger radius than SPIO nanoparticles, the labelled cells become much more
magnetic, which enables the detection of single cells. However, the clinical translation
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potential of these fairly large particles remains uncertain. When dedicated gradient insert
coils are used to enhance the performance of the MRI scanner, SPIO-labelled macrophages
can also be imaged at the single-cell level28.

Labelling macrophages in vivo


Labelling of cells in vivo following systemic injection of SPIO particles (FIG. 1) is generally
achieved by nanoparticles being taken up by the phagocytic cells of the reticuloendothelial
system (RES), including circulating blood monocytes and tissue macrophages (and in much
smaller numbers, neutrophils and DCs), which are frequently found at inflammatory sites.
This approach has been broadly applied to image inflammatory events in both preclinical
and clinical settings29–35. In a clinical context, this approach has been most widely used to
image lymph nodes for tumour staging36 and to detect atherosclerotic plaques37. Immune
cell labelling in situ can also be achieved by the direct injection of SPIO agents into a tissue.
In a mouse study, SPIO labelling of DCs was accomplished following vaccination with
irradiated labelled tumour cells from which antigens and SPIO were co-captured by the
DCs. Labelled DCs could be visualized homing to the sentinel lymph node several days after
this so-called ‘magnetovaccination’26 (FIG. 3c).
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Concerns and limitations


It is important that any cell labelling protocol that is used for imaging does not substantially
alter the immunological properties of the cells, and that it does not cause marked
cytotoxicity or changes in the phenotype and the function of the cells. Changes in the
phenotype of immune cells after labelling could reduce the efficacy of immunotherapeutic
strategies. Overall, very few studies have noted an adverse effect of SPIO labelling on cell
function or phenotype. In one study38, following SPIO labelling in culture, macrophages
were found to have altered cytokine production, which was shifted towards an anti-
inflammatory, less responsive phenotype. In other studies, the cytokine profile, surface
expression of phenotypic markers, migratory capacity and ability to present antigens did not
differ in SPIO-labelled DCs compared with in unlabelled DCs26,39,40. SPIO particles are
biodegradable and, depending on the cell type, are broken down quickly; for example, when
SPIO particles are taken up by Küpffer cells in the liver, the iron is metabolized and can be
found in haemoglobin as soon as one week after administration41. Thus, SPIO-based cell
labelling is best suited to short-term imaging studies; for example, MRI-guided injection, in
which the infusion of cells and their initial engraftment can be imaged in real-time10, is
viewed as a key early clinical use of SPIO-based cell tracking.
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There are other limitations to SPIO-based cell tracking that are also common to essentially
all nanoparticle-based and PFC nanoemulsion-based (see below) imaging reagents: in
mitotic cells, cell division and subsequent dilution of the intracellular label can potentially
limit long-term cell visualization; cell death can lead to dispersion of the reagent and loss of
MRI detectability; the imaging reagent could potentially be transferred to resident
phagocytes (such as macrophages); and if a large number of these labelled phagocytes
remain in a region of interest, false positive contrast could result.

PFC cell labels and 19F MRI detection


In an effort to design next-generation cell tracking probes, there has recently been
considerable interest in the use of PFC nano-emulsions. PFC emulsions can be used to track
cells using ex vivo and in situ cell labelling approaches that are analogous to those used for

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SPIO nanoparticles (FIG. 1). PFC-based cell tracking enables high specificity cell detection
and the quantification of cells. PFC-labelled cells are detected using 19F MRI (BOX 1),
whereas metal ion-based contrast agents (such as SPIO particles) are detected using 1H
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MRI. The PFC functions as a tracer agent rather than as a contrast agent, as 19F MRI
directly detects the 19F nuclei that are associated with the labelled cells. Importantly,
because of the extremely low concentration of naturally occurring 19F in the body, 19F MRI
has no background signal from the host’s tissues and therefore only labelled cells are
observed. Thus, false positive cell detection is unlikely, which overcomes one of the major
limitations of metal ion-based cell labelling approaches. Moreover, quantification of the 19F
MRI signal is directly related to the apparent number of cells in the regions of interest or,
alternatively, to the inflammation severity.

PFCs are among the most biologically inert organic molecules that have ever been
produced42. They are water insoluble and immiscible in cell membranes and, for cellular
use, they must be formulated into colloidal suspensions, such as nanoemulsions, ideally with
a small droplet diameter (<200 nm)43. There are no known enzymes that metabolize PFCs in
vivo and they are not degraded at typical lysosomal pH values42; thus, cell labelling with
PFCs can potentially be long lasting.

Labelling immune cells ex vivo


The idea of using PFCs as tracer agents for 19F MRI followed shortly after the discovery
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of 1H MRI44, but their use in cell tracking is a relatively recent development45,46. Different
nanoemulsion formulations have been designed for ex vivo (FIG. 3d) and in situ cell
labelling43. For ex vivo labelling, an important innovation has been the formulation of
nanoemulsions that intracellularly label cells in culture without the use of transfection
methods47 — through the inclusion of charged moieties on the nanoemulsion droplet surface
— thereby improving overall cell viability and the ease of use of PFCs. Numerous in vitro
studies have investigated the effects of PFC labelling on cellular phenotype and function in
primary immune cells, for example, using mouse DCs45 and T cells48. The most detailed
study so far involved PFC-labelled primary human DCs49; cells were assayed for viability,
maturation phenotype, cytokine production, T cell stimulatory capacity and chemotaxis, and
no differences in these parameters were observed between labelled and unlabelled cells in
vitro.

Tracking immune cells in vivo


Tracking cells in vivo using PFCs was first used to visualize DC migration in mice45.
Several T cell studies have used PFC-based cell tracking to examine early pancreas
inflammation in non-obese diabetic (NOD) mice50, which are an acute inflammation
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model48, and the biodistribution of mucin 1 (MUC1)-specific T cells in inflammatory bowel


disease (IBD)51. A surprising finding of the IBD study was the localization of MUC1-
specific T cells in the pancreatic duct, which indicates that pancreatitis, which is often
diagnosed in patients with IBD, might be a true extra-intestinal manifestation of the disease.
In addition, primary human DCs that are relevant to immunotherapeutic clinical trials have
been labelled using PFCs and longitudinally tracked in vivo into draining lymph nodes in
NOD severe combined immunodeficient (SCID) mice49.

Imaging of macrophage recruitment to inflammatory sites in vivo could help to elucidate the
host inflammatory response, could provide considerable diagnostic value for a wide range of
diseases and could be used as a surrogate marker and to monitor therapeutic interventions.
By using PFC nanoemulsions that have been formulated for intravenous injection and that
have a long blood half-life, nanoemulsion droplets enter into the RES and are intrinsically
taken up in situ by monocytes and macrophages and, to a lesser extent, by neutrophils and

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DCs. As these in situ-labelled cells participate in inflammatory events in the body, the result
is 19F accumulation at inflammatory sites. Importantly, the in vivo 19F MRI signal can be
quantified in inflammatory sites, and this signal is linearly proportional to the macrophage
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burden52,53. 19F labelling in situ has been widely used to visualize inflammation in a large
number of preclinical models of human disease, such as IBD53 (FIG. 3e), bacterial
infection54, organ transplant rejection55,56, experimental allergic encephalomyelitis52,57,
peripheral nerve inflammation58, pulmonary inflammation59 and cardiac and cerebral
ischaemia60.

Extending the use of PFC emulsions


Similar to dual-mode SPIO agents, PFC nanoemulsions that can be detected by both 19F
MRI and fluorescence have also been devised45,47; these reagents contain a bright
fluorescent dye that is directly conjugated to the PFC molecule prior to nanoemulsion
formulation, which thereby ensures that the originating 19F MRI and fluorescent signals are
coincident in labelled cells. The fluorescent moiety enables the identification of the fate and
the phenotype of labelled cells using flow cytometry, fluorescence microscopy or in vivo
optical imaging; for example, dual-mode fluorescent PFC emulsions have been used to label
primary murine CD4+ T cells that were then adoptively transferred into wild-type mice and
imaged in multiple lymph nodes using MRI, followed by analysis using optical methods47.
Near-infrared dyes have also been incorporated into PFC emulsions, which enables in vivo
imaging at moderate tissue depths61. Moreover, fluorescent PFC emulsions have
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considerable potential as an optical-only cell labelling probe because of their brightness, low
toxicity and long retention time in cells.

Conventional 19F nuclear magnetic resonance (NMR) spectroscopy can also be used to
assay the biodistribution of PFC-labelled immune cells with high sensitivity in fixed, intact
tissue panels. 19F NMR can measure the total cell count (or cell density) of labelled cells in
tissue samples; for example, NMR cytometry has been used in T cell biodistribution studies
in rodent IBD and diabetes models50,51,62. As NMR is non-destructive, the same tissues can
then be processed for histology to further refine the tissue analysis. 19F-capable NMR
spectrometers are ubiquitous in research laboratories and are commonly used for molecular
structure determination, therefore the implementation of NMR cytometry is fairly
straightforward. NMR instrumentation can also be used to rapidly and quantitatively assay
inflammation in intact, excised tissue samples, which yields an inflammatory index52 that is
proportional to the macrophage burden.

Future directions in immune cell tracking


Current MRI-based cell tracking technologies are poised to have a broad impact on basic
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and biomedical research, but there are still substantial efforts underway to devise next-
generation cell imaging technologies.

MRI reporter genes


One important aim of these efforts is the development of robust MRI reporter genes. An
ideal reporter gene would not be diluted by cell mitosis and would be cleared rapidly after
cell death and could potentially elucidate cell viability, activation status and/or
differentiation status. In recent years, researchers have been exploring schemes using
exogenous (that is, injected) substrates, endogenous substrates or those that do not require a
substrate (FIG. 2) in their quest to define optimal nucleic acid-based MRI reporters.

The first exogenous substrate approach involves genetically encoded, cell surface ligands
that bind to MRI-detectable probes; for example, suitable ligands could include biotin63 or

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the transferrin receptor64, for use with a probe that consists of a SPIO nano-particle bound to
streptavidin or transferrin, respectively. Alternatively, transfection of cells with the HSV
thymidine kinase reporter gene used for PET imaging can be used to accumulate a
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thymidine analogue probe in these cells that is detectable by chemical exchange saturation
transfer (CEST) MRI65 (BOX 1). One of the challenges of using ligand–probe-based
reporter schemes is the efficient delivery of the probe to the tissues of interest.

The second class of MRI reporters use endogenous substrates (FIG. 2); several distinct
technologies have been developed in this area. In one approach, transgenic or vector
technologies are used to induce cells to express genes that encode engineered iron-binding
proteins, for example, those in the ferritin family66–69. Following transgene expression in
situ, the ferritin outer protein shell sequesters physiologically available iron, and the
endogenous formation of an iron oxide crystal in the ferritin core renders the complex
paramagnetic, which produces MRI contrast. Reporters that do not require a substrate have
more recently been developed that involve the expression of amide-rich proteins that can be
detected by CEST MRI70. So far, MRI reporter genes have not been used for immune cell
tracking and their use has been mostly limited to tumour cells65,70 and to stem cells69.

Sensing the extracellular and intracellular environment


Another key area of innovation in the use of MRI to track cells has been the development of
probes that can sense enzymatic activity and/or changes in the microenvironment, for
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example, changes in the local pH. Using arginine-filled liposomes as pH-sensitive contrast
agents, changes in tissue pH that are associated with an immune response can potentially be
detected by CEST MRI71,72. In addition, there are in vivo MRI probe technologies to detect
enzymes that are overexpressed following cellular activation, such as protein kinase A
(PKA). Following PKA-mediated phosphorylation of its peptide substrates, which can
function as CEST contrast agents, a 50% change in the CEST MRI signal was observed73.

MRI measurements of intracellular oximetry have also been used to assay cell viability and
metabolism74. Certain PFC molecules used for in vivo cytometry readily dissolve
paramagnetic oxygen, which alters the MRI property (that is, the relaxation time) of the 19F
signal in a manner that linearly increases with the absolute partial pressure of oxygen75. In a
rodent brain glioma model, intracellular oximetry technology has recently been used to
monitor the apoptosis dynamics of tumour cells as a result of their interaction with CD8+ T
cells that are specific for glioma-associated antigens76.

Tracking cell–cell interactions


MRI also has the potential to visualize multiple cell populations in vivo to follow the spatial
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dynamics of cell–cell interactions, such as antigen presentation to B cells or T cells, and


macrophage-mediated recruitment of lymphocytes; for example, ex vivo cell-labelling agents
containing PFC molecules with different 19F NMR chemical shifts (that is, molecular
signatures) can be used to label different immune cell subsets. Following injection of these
labelled subsets, in vivo multicolour magnetic resonance images can be constructed, which
show the distribution of each cell species according to its unique 19F spectral signature77 (a
process known as multichannel in vivo cytometry). Alternatively, discrete immune cell
populations could be labelled with two different modes of contrast, such as a positive
contrast agent with a negative contrast agent (TABLE 2), a metal-based contrast agent with
a CEST agent78, or multiple CEST agents of different off-resonance frequencies, which
enables the generation of multiple colours79.

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Prospects and challenges for translation


The use of MRI-based cell tracking in clinical trials is still in its infancy. Only a few small
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clinical pilot studies using SPIO-based cell tracking have been reported so far80. In the first
clinical trial to use MRI-based cell tracking, an immunotherapeutic DC vaccine was labelled
ex vivo with SPIO nanoparticles, and ultrasound imaging was used to guide the delivery of
these cells into the inguinal lymph nodes in patients with melanoma40. Using MRI it was
shown that the target lymph node was missed in four out of eight patients; this low success
rate was not detected when 111In-oxine-labelled DCs were tracked using low-resolution
radioscintigraphy40. In another study, peripheral blood mono-nuclear cells were labelled ex
vivo with SPIO particles, injected systemically and visualized at sites of cutaneous
inflammation81. As a setback for further clinical use, the SPIO agents that have been used
for cell labelling — ferumoxides injectable solution (Feridex; Berlex laboratories in the
United States (also known as Endorem in Europe)) and ferucarbotran (Resovist; Bayer
Schering Parma AG) — are no longer being manufactured because of economic
considerations. There are other experimental SPIO agents available but they would need
large investments in order to be developed as a clinical MRI contrast agent.

Instead, there has recently been a technological push to use PFC nanoemulsions for clinical
MRI-based cell tracking49. PFC labelling is clinically attractive because of the very low
toxicity of the probes, the exceptionally high cell specificity in images and the cell
NIH-PA Author Manuscript

quantification capacity in vivo. The sensitivity of PFC labelling to detect small cell numbers
is potentially less than for SPIO agents (TABLE 2), but this disadvantage may be offset by
the fact that there is no background signal in 19F images, cell detection is unambiguous (in
other words, there are no false positives) and images can be readily quantitated to yield
apparent cell numbers in regions of interest. The technical barriers that are associated with
the implementation and the quantification of 19F MRI on a clinical scanner are
surmountable; prior studies have shown the feasibility of efficient data collection schemes
and the quantification of PFC signal intensity at clinical field strengths82.

Our view is that MRI-based cell tracking will routinely be used in the future for clinical
trials to monitor therapeutic cell delivery and inflammation. We expect that MRI-based cell
tracking will provide unexpected results regarding how cells behave in vivo following
delivery and that this information will be crucial to obtain clinical success. Moreover, we
foresee real-time SPIO-based MRI-guided immune cell delivery as a routine application of
cell tracking, in conjunction with MRI-compatible injection catheters83. Finally, the
development of cell tracking software for automated analysis of MRI scans may further
facilitate image interpretation and intercomparison studies.
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Conclusion
Overall, MRI-based cell tracking has great potential, not only in basic immunological
research but also to guide the development of experimental immune cell-based therapies.
SPIO-based cell tracking has already been used in the clinic and PFC cell labels, which are
detected using 19F MRI, are currently being incorporated into clinical trials in the United
States; these methods are likely to gain momentum in the future. In the meantime, emerging
MRI-based cell tracking approaches could enable the visualization of cell survival, the
activation status of immune cells and the identification of cell–cell interactions in vivo in
ways that were previously impossible.

Nat Rev Immunol. Author manuscript; available in PMC 2014 January 09.
Ahrens and Bulte Page 11

Acknowledgments
The authors are supported by US National Institutes of Health grants R01-CA134633, P41-EB0019772, R01-
NIH-PA Author Manuscript

NS045062, R01-EB007825, R01-DA026299 and U54-CA151838, the Maryland Stem Cell Research Foundation
and the California Institute for Regenerative Medicine.

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Figure 1. Schematic showing ex vivo and in situ labelling of cells with magnetic resonance
nanoparticle contrast agents
Cell labelling can be achieved ex vivo in pre-selected and cultured cells by adding the
labelling reagent — superparamagnetic iron oxide (SPIO) nanoparticles or perfluorocarbon
(PFC) emulsion — directly to the media followed by co-incubation. Often the labelling
reagent is complexed with a cationic transfection agent before it is added to the cell culture
to label non-phagocytic cells. Self-delivering formulation s of PFC emulsions have also been
devised that do not require a transfection agent. Alternatively, electroporation can be used to
label cells in culture. After collection and washing, labelled cells are then administered to
the subject. In addition, the labelling agent can be intravenously injected; in this in situ
labelling approach, the agent is intrinsically taken up by phagocytic cells of the
reticuloendothelial (RES) system, particularly by monocytes and macrophages, which then
accumulate at sites of inflammation. For both ex vivo and in situ labelling approaches an 1H
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magnetic resonance imaging (MRI) scan is then carried out to visualize the anatomy. The
resulting images have a decreased signal in regions containing SPIO-labelled cells (known
as negative contrast). In the case of PFC probes, a 19F MRI scan is also acquired in the same
imaging session. A composite, pseudo-coloured 19F and 1H image is then constructed, in
which labelled cells appear in the 19F colour channel (known as hot spot contrast). By
quantifying the 19F MRI signal in individual organs using using nuclear magnetic resonance
(NMR) spectroscopy an inflammation index can be calculated, which is a direct correlation
between the 19F signal and the number of labelled macrophages.

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Figure 2. The development of MRI reporter genes


Certain nucleic acid-based reporters encode cell surface receptors that bind to specific
ligands that are conjugated to superparamagnetic iron oxide (SPIO) to render them magnetic
resonance imaging (MRI)-detectable. They require delivery of the ligand–SPIO complex as
an exogenous substrate. An example of this approach is an engineered transferrin receptor
that binds transferrin-ligated SPIO. Another exogenous substrate-based approach involves a
reporter gene encoding the thymidine kinase enzyme derived from herpes simplex virus
(HSV). This enzyme phosphorylates thymidine analogues, which causes them to remain
trapped in transduced cells. When thymidine analogues are used that are rich in specific
(imino) protons, chemical exchange saturation transfer (CEST) MRI can be used to track the
transduced cells. Other approaches use endogenous substrates, whereby the encoded reporter
binds iron (Fe2+) that is naturally present in the body as the contrasting metal ion. Finally, a
CEST reporter (such as lysine-rich protein (LRP)) can be used that does not require a
substrate, as the protein itself contains multiple labile amide protons that can be saturated
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and detected.

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Figure 3. Tracking immune cells with MRI using SPIO nanoparticles and PFC emulsions
a | Intracellular labelling of mononuclear cells with magnetoliposomes is shown13. The
electron micrograph shows superparamagnetic iron oxide (SPIO) particles in secondary
lysosomes (small arrows) and in primary lysosomes that are fusing with endosomes (large
arrow). The scale bar represents 200 nm. b | Imaging of a non-obese diabetic (NOD) severe
combined immunodeficient (SCID) mouse pancreas ex vivo is shown following the adoptive
transfer of SPIO-labelled T cells22. Image hypointensities represent infiltrating T cells that
are observed at 24 hours post transfer. c | Imaging of in vivo antigen capture and trafficking
of dendritic cells (DCs) is shown26. Sentinel DCs were labelled in situ by intradermal
injection of unlabelled (dashed arrow) or SPIO-labelled (solid arrow) irradiated cancer cells,
which function as a vaccine. Following phagocytosis of both SPIO particles and tumour
antigens in a process known as magnetovaccination, the hypointense DCs migrate into the
medulla of the draining popliteal lymph node, as observed on day 8. d | An electron
micrograph of a perfluorocarbon (PFC)-labelled DC is shown. Numerous bright spots (PFC
droplets) are observed inside the cell. Particles appear as smooth spheroids45. Arrowheads
indicate vesicles. The scale bar represents 200 nm. e | Inflammatory bowel disease (IBD) in
an interleukin-10 (Il10)−/− mouse model was visualized using in situ PFC labelling and 19F
magnetic resonance imaging (MRI) in vivo53. The 19F image (pseudo-colour) is shown on
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the far left, the composite 1H and 19F image is shown in the middle and a three-dimensional
rendering of the in vivo 19F MRI data from the abdomen is shown on the far right. A
reference tube that contains PFC emulsion (R) was placed alongside the torso of the mouse.
The images through the abdomen show PFC accumulation (indicating inflammation) in the
ascending colon (ac) and descending colon (dc), where (a) is the anus. Part a is reproduced,
with permission, from REF. 13 © (1993) John Wiley and Sons. Part b is reproduced, with
permission, from REF. 22 © (2002) John Wiley and Sons. Part c is reproduced, with
permission, from REF. 26 © (2009) American Association for Cancer Research. Part d is
reproduced, with permission, from REF. 45 © (2005) Macmillan Publishers Ltd. All rights
reserved. Part e is reproduced, with permission, from REF. 52 © (2012) Macmillan
Publishers Ltd. All rights reserved.

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Table 1
Comparison of non-invasive imaging modalities for in vivo cell tracking

Imaging technique Resolution* Tissue penetration depth* Sensitivity of cell Possibility of longitudinal Used generally in the Used in clinical cell
detection* studies* clinic? tracking?

MRI +++ +++ +++ ++ Yes Yes


Ahrens and Bulte

SPECT + +++ ++ + Yes Yes

PET + +++ ++ +++ Yes Yes


CT or X-ray +++ +++ + + Yes No

Ultrasound imaging ++ ++ ++ + Yes No

BLI + + ++ +++ No No

Fluorescence imaging or NIR + + ++ ++ No No

2PLSM +++ + +++ ++ No No

MPI84 + +++ ++ ++ No No

2PLSM, two-photon laser scanning microscopy; BLI, bioluminescence imaging; CT, computed tomography; MPI, magnetic particle imaging; MRI, magnetic resonance imaging; NIR, near-infrared
imaging; PET, positron emission tomography; SPECT, single-photon emission computed tomography.
*
Strengths and weaknesses are given using a relative scale in which + = poor, ++ = moderate and +++ = excellent.

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Table 2
Overview of available in vivo MRI-based cell tracking techniques

Probe MRI technique Type of contrast Minimum number of Quantification of cell Clinical trial approval?
detectable cells number?

Gd3+ or Mn2+ labelling T1-weighted 1H MRI* Positive ~103 No No


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SPIO labelling T2-weighted 1H MRI‡ Negative 1 No Yes (in the Netherlands, China, Switzerland,
Czech Republic, Israel, Poland and United
Kingdom)
PFC labelling 19F MRI Hot spot (also known as tracer) 103–105 Yes Yes (in USA)

Metal-binding reporter genes T2-weighted 1H MRI‡ Negative 104 No No

CEST agents and reporter genes 1H CEST MRI Differential (can be colour 104 No No
encoded)

CEST, chemical exchange saturation transfer; MRI, magnetic resonance imaging; PFC, perfluorocarbon; SPIO, superparamagnetic iron oxide.
*
T1 is the nuclear spin–lattice relaxation time.

T2 is the spin–spin relaxation time.

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