Nihms 541476
Nihms 541476
Nihms 541476
Author Manuscript
Nat Rev Immunol. Author manuscript; available in PMC 2014 January 09.
Published in final edited form as:
NIH-PA Author Manuscript
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
NIH-PA Author Manuscript
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.
NIH-PA Author Manuscript
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
NIH-PA Author Manuscript
(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
NIH-PA Author Manuscript
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
Nat Rev Immunol. Author manuscript; available in PMC 2014 January 09.
Ahrens and Bulte Page 3
Nat Rev Immunol. Author manuscript; available in PMC 2014 January 09.
Ahrens and Bulte Page 4
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.
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
NIH-PA Author Manuscript
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.
Nat Rev Immunol. Author manuscript; available in PMC 2014 January 09.
Ahrens and Bulte Page 5
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.
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
Nat Rev Immunol. Author manuscript; available in PMC 2014 January 09.
Ahrens and Bulte Page 6
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
NIH-PA Author Manuscript
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.
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.
Nat Rev Immunol. Author manuscript; available in PMC 2014 January 09.
Ahrens and Bulte Page 7
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
NIH-PA Author Manuscript
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.
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.
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
Nat Rev Immunol. Author manuscript; available in PMC 2014 January 09.
Ahrens and Bulte Page 8
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
NIH-PA Author Manuscript
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.
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.
and biomedical research, but there are still substantial efforts underway to devise next-
generation cell imaging technologies.
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
Nat Rev Immunol. Author manuscript; available in PMC 2014 January 09.
Ahrens and Bulte Page 9
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
NIH-PA Author Manuscript
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.
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.
Nat Rev Immunol. Author manuscript; available in PMC 2014 January 09.
Ahrens and Bulte Page 10
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.
NIH-PA Author Manuscript
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.
References
1. Bousso P, Moreau HD. Functional immunoimaging: the revolution continues. Nature Rev Immunol.
2012; 12:858–864. [PubMed: 23175230]
2. Thakur ML, Lavender JP, Arnot RN, Silvester DJ, Segal AW. Indium-111-labeled autologous
leukocytes in man. J Nuclear Med. 1977; 18:1014–1021.
3. Tjuvajev JG, et al. Imaging the expression of transfected genes in vivo. Cancer Res. 1995; 55:6126–
6132. [PubMed: 8521403]
4. Koehne G, et al. Serial in vivo imaging of the targeted migration of human HSV-TK-transduced
antigen-specific lymphocytes. Nature Biotech. 2003; 21:405–413.
5. Dubey P, et al. Quantitative imaging of the T cell antitumor response by positron-emission
tomography. Proc Natl Acad Sci USA. 2003; 100:1232–1237. [PubMed: 12547911]
6. Yaghoubi SS, et al. Noninvasive detection of therapeutic cytolytic T cells with [18F]-FHBG PET in
a patient with glioma. Nature Clin Pract Oncol. 2009; 6:53–58. [PubMed: 19015650]
7. Aoki I, et al. Cell labeling for magnetic resonance imaging with the T-1 agent manganese chloride.
NMR Biomed. 2006; 19:50–59. [PubMed: 16411253]
NIH-PA Author Manuscript
12819345]
16. Arbab AS, et al. Labeling of cells with ferumoxides-protamine sulfate complexes does not inhibit
function or differentiation capacity of hematopoietic or mesenchymal stem cells. NMR Biomed.
2005; 18:553–559. [PubMed: 16229060]
17. Thu MS, et al. Self-assembling nanocomplexes by combining ferumoxytol, heparin and protamine
for cell tracking by magnetic resonance imaging. Nature Med. 2012; 18:463–467. [PubMed:
22366951]
18. Ahrens ET, Feili-Hariri M, Xu H, Genove G, Morel PA. Receptor-mediated endocytosis of iron-
oxide particles provides efficient labeling of dendritic cells for in vivo MR imaging. Magn Reson
Med. 2003; 49:1006–1013. [PubMed: 12768577]
19. Walczak P, et al. Magnetoelectroporation: improved labeling of neural stem cells and leukocytes
for cellular magnetic resonance imaging using a single FDA-approved agent. Nanomedicine.
2006; 2:89–94. [PubMed: 17292120]
Nat Rev Immunol. Author manuscript; available in PMC 2014 January 09.
Ahrens and Bulte Page 12
20. Kircher MF, et al. In vivo high resolution three-dimensional imaging of antigen-specific cytotoxic
T-lymphocyte trafficking to tumors. Cancer Res. 2003; 63:6838–6846. [PubMed: 14583481]
21. Daldrup-Link HE, et al. In vivo tracking of genetically engineered, anti-HER2/neu directed natural
NIH-PA Author Manuscript
killer cells to HER2/neu positive mammary tumors with magnetic resonance imaging. Eur Radiol.
2005; 15:4–13. [PubMed: 15616814]
22. Moore A, et al. MRI of insulitis in autoimmune diabetes. Magn Reson Med. 2002; 47:751–758.
[PubMed: 11948737]
23. Anderson SA, et al. Magnetic resonance imaging of labeled T-cells in a mouse model of multiple
sclerosis. Ann Neurol. 2004; 55:654–659. [PubMed: 15122705]
24. Baumjohann D, et al. In vivo magnetic resonance imaging of dendritic cell migration into the
draining lymph nodes of mice. Eur J Immunol. 2006; 36:2544–2555. [PubMed: 16909432]
25. Rohani R, et al. In vivo cellular MRI of dendritic cell migration using micrometer-sized iron oxide
(MPIO) particles. Mol Imaging Biol. 2011; 13:679–694. [PubMed: 20803172]
26. Long CM, van Laarhoven HW, Bulte JW, Levitsky HI. Magnetovaccination as a novel method to
assess and quantify dendritic cell tumor antigen capture and delivery to lymph nodes. Cancer Res.
2009; 69:3180–3187. [PubMed: 19276358]
27. Shapiro EM, Medford-Davis LN, Fahmy TM, Dunbar CE, Koretsky AP. Antibody-mediated cell
labeling of peripheral T cells with micron-sized iron oxide particles (MPIOs) allows single cell
detection by MRI. Contrast Media Mol Imaging. 2007; 2:147–153. [PubMed: 17541955]
28. Heyn C, et al. In vivo magnetic resonance imaging of single cells in mouse brain with optical
validation. Magn Reson Med. 2006; 55:23–29. [PubMed: 16342157]
NIH-PA Author Manuscript
29. Chan TW, Eley C, Liberti P, So A, Kressel HY. Magnetic resonance imaging of abscesses using
lipid-coated iron oxide particles. Investigative Radiol. 1992; 27:443–449.
30. Dousset V, et al. MR imaging of relapsing multiple sclerosis patients using ultra-small-particle iron
oxide and compared with gadolinium. AJNR Am J Neuroradiol. 2006; 27:1000–1005. [PubMed:
16687532]
31. Vellinga MM, et al. Pluriformity of inflammation in multiple sclerosis shown by ultra-small iron
oxide particle enhancement. Brain. 2008; 131:800–807. [PubMed: 18245785]
32. Sosnovik DE, Nahrendorf M. Cells and iron oxide nanoparticles on the move: magnetic resonance
imaging of monocyte homing and myocardial inflammation in patients with ST-elevation
myocardial infarction. Circ Cardiovasc Imaging. 2012; 5:551–554. [PubMed: 22991284]
33. Gaglia JL, et al. Noninvasive imaging of pancreatic islet inflammation in type 1A diabetes patients.
J Clin Invest. 2011; 121:442–445. [PubMed: 21123946]
34. Luciani A, et al. Adipose tissue macrophages: MR tracking to monitor obesity-associated
inflammation. Radiology. 2012; 263:786–793. [PubMed: 22523321]
35. Kanno S, et al. Macrophage accumulation associated with rat cardiac allograft rejection detected
by magnetic resonance imaging with ultrasmall superparamagnetic iron oxide particles.
Circulation. 2001; 104:934–938. [PubMed: 11514382]
36. Harisinghani MG, et al. Noninvasive detection of clinically occult lymph-node metastases in
NIH-PA Author Manuscript
Nat Rev Immunol. Author manuscript; available in PMC 2014 January 09.
Ahrens and Bulte Page 13
42. Riess JG. Oxygen carriers (“blood substitutes”) — raison d’etre, chemistry, and some physiology.
Chem Rev. 2001; 101:2797–2919. [PubMed: 11749396]
43. Janjic JM, Ahrens ET. Fluorine-containing nanoemulsions for MRI cell tracking. Wiley Interdiscip
NIH-PA Author Manuscript
[PubMed: 20084048]
52. Ahrens ET, Young WB, Xu H, Pusateri LK. Rapid quantification of inflammation in tissue
samples using perfluorocarbon emulsion and fluorine-19 nuclear magnetic resonance.
Biotechniques. 2011; 50:229–234. [PubMed: 21548906]
53. Kadayakkara DK, Ranganathan S, Young WB, Ahrens ET. Assaying macrophage activity in a
murine model of inflammatory bowel disease using fluorine-19 MRI. Lab Invest. 2012; 92:636–
645. [PubMed: 22330343]
54. Hertlein T, et al. Visualization of abscess formation in a murine thigh infection model of
Staphylococcus aureus by F-19-magnetic resonance imaging (MRI). PLoS ONE. 2011; 6:e18246.
[PubMed: 21455319]
55. Hitchens TK, et al. 19F MRI detection of acute allograft rejection with in vivo perfluorocarbon
labeling of immune cells. Magn Reson Med. 2011; 65:1144–1153. [PubMed: 21305593]
56. Flogel U, et al. Noninvasive detection of graft rejection by in vivo 19F MRI in the early stage. Am
J Transplant. 2011; 11:235–244. [PubMed: 21214858]
57. Noth U, et al. Perfluoro-15-crown-5-ether labelled macrophages in adoptive transfer experimental
allergic encephalomyelitis. Artif Cells Blood Substit Immobil Biotechnol. 1997; 25:243–254.
[PubMed: 9167839]
58. Weise G, Basse-Luesebrink TC, Wessig C, Jakob PM, Stoll G. In vivo imaging of inflammation in
NIH-PA Author Manuscript
the peripheral nervous system by 19F MRI. Exp Neurol. 2011; 229:494–501. [PubMed: 21459088]
59. Ebner B, et al. Early assessment of pulmonary inflammation by 19F MRI in vivo. Circ Cardiovasc
Imag. 2010; 3:U202–U109.
60. Flogel U, et al. In vivo monitoring of inflammation after cardiac and cerebral ischemia by fluorine
magnetic resonance imaging. Circulation. 2008; 118:140–148. [PubMed: 18574049]
61. O’Hanlon CE, Amede KG, O’Hear MR, Janjic JM. NIR-labeled perfluoropolyether nanoemulsions
for drug delivery and imaging. J Fluor Chem. 2012; 137:27–33. [PubMed: 22675234]
62. Morel PA, et al. Gene expression analysis of dendritic cells that prevent diabetes in NOD mice:
analysis of chemokines and costimulatory molecules. J Leukoc Biol. 2011; 90:539–550. [PubMed:
21628331]
63. Tannous BA, et al. Metabolic biotinylation of cell surface receptors for in vivo imaging. Nature
Methods. 2006; 3:391–396. [PubMed: 16628210]
64. Weissleder R, et al. In vivo magnetic resonance imaging of transgene expression. Nature Med.
2000; 6:351–355. [PubMed: 10700241]
Nat Rev Immunol. Author manuscript; available in PMC 2014 January 09.
Ahrens and Bulte Page 14
65. Bar-Shir A, et al. Transforming thymidine into a magnetic resonance imaging probe for monitoring
gene expression. J Am Chem Soc. 2013; 135:1617–1624. [PubMed: 23289583]
66. Ahrens, ET. Contrast agents for magnetic resonance imaging and methods related thereto. US
NIH-PA Author Manuscript
the CNS using fluorine-19 MRI. Magn Reson Med. 2010; 64:1252–1259. [PubMed: 20860007]
75. Sotak CH, et al. A new perfluorocarbon for use in fluorine-19 magnetic resonance imaging and
spectroscopy. Magn Reson Med. 1993; 29:188–195. [PubMed: 8429782]
76. Zhong J, Sakaki M, Okada H, Ahrens ET. In vivo intracellular oxygen dynamics in murine brain
glioma and immunotherapeutic response of cytotoxic T cells observed by fluorine-19 magnetic
resonance imaging. PLoS One. 2013; 8:e59479. [PubMed: 23667419]
77. Partlow KC, et al. F-19 magnetic resonance imaging for stem/progenitor cell tracking with
multiple unique perfluorocarbon nanobeacons. FASEB J. 2007; 21:1647–1654. [PubMed:
17284484]
78. Gilad AA, et al. Feasibility of concurrent dual contrast enhancement using CEST contrast agents
and superparamagnetic iron oxide particles. Magn Reson Med. 2009; 61:970–974. [PubMed:
19189296]
79. Liu G, et al. In vivo multicolor molecular MR imaging using diamagnetic chemical exchange
saturation transfer liposomes. Magn Reson Med. 2012; 67:1106–1113. [PubMed: 22392814]
80. Bulte JW. In vivo MRI cell tracking: clinical studies. AJR Am J Roentgenol. 2009; 193:314–325.
[PubMed: 19620426]
81. Richards JMJ, et al. Clinical cell tracking of mononuclear cells using magnetic resonance imaging
and superparamagnetic particles of iron oxide. Br J Surg. 2011; 98:4–4. [PubMed: 20812233]
NIH-PA Author Manuscript
82. Keupp J, et al. Simultaneous dual-nuclei imaging for motion corrected detection and quantification
of 19F imaging agents. Magn Reson Med. 2011; 66:1116–1122. [PubMed: 21394779]
83. Aarntzen EH, et al. In vivo tracking techniques for cellular regeneration, replacement, and
redirection. J Nucl Med. 2012; 53:1825–1828. [PubMed: 23143090]
84. Bulte JW, et al. MPI cell tracking: what can we learn from MRI? Proc Soc Photo-Opt Instrum Eng.
2011; 7965:79650z.
Nat Rev Immunol. Author manuscript; available in PMC 2014 January 09.
Ahrens and Bulte Page 15
NIH-PA Author Manuscript
NIH-PA Author Manuscript
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
NIH-PA Author Manuscript
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.
Nat Rev Immunol. Author manuscript; available in PMC 2014 January 09.
Ahrens and Bulte Page 16
NIH-PA Author Manuscript
NIH-PA Author Manuscript
and detected.
Nat Rev Immunol. Author manuscript; available in PMC 2014 January 09.
Ahrens and Bulte Page 17
NIH-PA Author Manuscript
NIH-PA Author Manuscript
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
NIH-PA Author Manuscript
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.
Nat Rev Immunol. Author manuscript; available in PMC 2014 January 09.
NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript
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?
BLI + + ++ +++ 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.
Nat Rev Immunol. Author manuscript; available in PMC 2014 January 09.
Page 18
NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript
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?
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)
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.
Nat Rev Immunol. Author manuscript; available in PMC 2014 January 09.
Page 19