Macrophages Homing to Metastatic Lymph Nodes Can Be Monitored with Ultrasensitive Ferromagnetic Iron-Oxide Nanocubes and a 1.5T Clinical MR Scanner
et al. (2012) Macrophages Homing to Metastatic Lymph Nodes Can Be Monitored with Ultrasensitive
Ferromagnetic Iron-Oxide Nanocubes and a 1.5T Clinical MR Scanner. PLoS ONE 7(1): e29575. doi:10.1371/journal.pone.0029575
Macrophages Homing to Metastatic Lymph Nodes Can Be Monitored with Ultrasensitive Ferromagnetic Iron- Oxide Nanocubes and a 1.5T Clinical MR Scanner
Hye Rim Cho 0
Seung Hong Choi 0
Nohyun Lee 0
Taeghwan Hyeon 0
Hyeonjin Kim 0
Woo Kyung 0
Wing-Kin Syn, Institute of Hepatology London, United Kingdom
0 1 Department of Radiology, Seoul National University College of Medicine , Seoul , Korea , 2 Department of Radiation Applied Life Science, Seoul National University College of Medicine , Seoul , Korea , 3 National Creative Research Initiative Center for Oxide Nanocrystalline Materials and School of Chemical and Biological Engineering, Seoul National University , Seoul , Korea
Background: Due to the ability of macrophages to specifically home to tumors, their potential use as a delivery vehicle for cancer therapeutics has been suggested. Tracking the delivery and engraftment of macrophages into human tumors with a 1.5T clinical MR scanner requires the development of sensitive contrast agents for cell labeling. Therefore, this study aimed to determine whether intravenously injected macrophages could target a primary tumor as well as metastatic LNs, and whether these cells could be detected in vivo by MRI. Methodology: Peritoneal macrophages were obtained from BALB/c nude mice. The viability, phagocytotic capacity and migratory activity of the macrophages were assessed. MR imaging was performed using a clinical 1.5 T MR scanner and we estimated the T2* of the labeled macrophages. Metastatic lymph nodes were produced in BALB/c nude mice. We administrated 26106 macrophages labeled with 50 mg Fe/mL FIONs intravenously into the mice. In the 3D T2* GRE MR images obtained one day after the injection of the labeled macrophages or FION solution, the percentages of pixels in the tumors or LNs below the minimum normalized SI (signal intensity) threshold were summated and reported as the black pixel count (%) for the FION hypointensity. Tumors in the main tumor model as well as the brachial, axillary and inguinal lymph nodes in the metastatic LN models were removed and stained. For all statistical analyses, single-group data were assessed using t test or the Mann-Whitney test. Repeated measurements analysis of variance (ANOVA) with Tukey-Kramer post hoc comparisons were performed for multiple comparisons. Conclusions: The FION-labeled macrophages, which could be non-invasively monitored using a 1.5 T clinical MR scanner, targeted both the main tumors and LN metastases. Overall, the results of this study suggest that the use of macrophages may have many future applications in the clinic for vectorizing therapeutic agents toward main tumors as well as LN metastases.
Funding: This work was supported by the Mid-Career Researcher Program through a NRF grant funded by the MEST (No. 20090080219), a grant from the
National R&D Program for Cancer Control, the Ministry of Health & Welfare, Republic of Korea (A01185), and a grant from the National R&D Program for Cancer
Control, Ministry of Health & Welfare, Republic of Korea (1120300). The funders had no role in study design, data collection and analysis, decision to publish, or
preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
. These authors contributed equally to this work.
Many primary malignancies spread through the lymphatic
system. Tumor cells passing through, or residing in, lymph nodes
(LNs) can serve as a reservoir of cells that can lead to lethal distant
metastases. The detection of metastases in the sentinel LNs and
other LNs within the regional bed provides clinically important
information for tumor staging, the choice of treatment, and the
prediction of patient outcomes [1,2]. Sensitive and specific
noninvasive imaging techniques to visualize LN metastasis in vivo
are critical for gaining information about tumor progression [3,4].
Of the various in vivo imaging techniques, magnetic resonance
imaging (MRI), with its high resolution, exquisite soft-tissue
contrast and ability to produce images of entire organs/organisms
without the use of ionizing radiation,  is the most common
method to evaluate a tumors size, location, and metastatic
burden. MRI is especially useful to study cancer dynamics in deep
tissues, which makes it readily translatable to clinical applications.
MRI has recently emerged as a powerful tool for in vivo cell
tracking [5,6] and the simultaneous development of new
intracellular contrast agents has allowed the in vivo detection of
very small cellular populations [7,8].
The first description of the presence of leukocytes within human
tumors, which was thought to reflect the onset of cancer at sites of
previous chronic inflammation, was presented by Virchow in 1863
. It has now been established that the majority of malignant
tumors contain numerous macrophages as a major component of
the host leukocytic infiltrate . These macrophages are referred
to as tumor-associated macrophages and although most are
derived from peripheral blood monocytes that are recruited into
the tumor mass from the circulation [11,12], there is also evidence
of local proliferation of macrophages within the tumor tissue
[13,14]. Chemokines (chemotactic cytokines) provide the
directional stimulus for the movement of leukocytes during
development, hemostasis, and inflammation, and are believed to be
important for the recruitment of monocytes into tumors. Some
studies have also shown the tumor-targeting potential of
macrophages in animal models [15,16].
This study aimed to determine whether intravenously
injected syngeneic macrophages could target a primary tumor
as well as metastatic LNs in a mouse model, and whether these
cells could be detected in vivo by MRI. We utilized macrophages
that had been labeled with ferromagnetic iron-oxide nanocubes
(FIONs) to facilitate detection by MRI and histology. After an
in vitro assessment of the efficiency and innocuity of the labeling
procedure, we used in vivo MRI in combination with histology
to demonstrate and monitor the capacity of metastatic cancer
cells in the LNs to attract intravenously administrated
Identification of macrophages within the intraperitoneal
cells by FACS
Over 95% of the intraperitoneal cells were F4/80 positive
Determination of intracellular FION uptake
After FION labeling using at a concentration of 50 mg Fe/mL
for 2 h, Prussian blue staining of the labeled macrophages revealed
an abundant uptake of the FIONs into the cytoplasm (Figure 2A).
TEM of the FION-labeled macrophages revealed FIONs in the
cytoplasmic organelles (Figure 2B).
The viability, phagocytosis, and migration of
In terms of cell viability, the MTT assays indicated no
statistically significant difference between the macrophages labeled
with FIONs at concentrations of 12.550 mg Fe/mL and the
unlabeled cells. However, the macrophages exposed to the 100 mg
Fe/mL FION concentration manifested significantly reduced
viability compared with the control cells (Figure 3A). When the
macrophages were treated with 50 mg Fe/mL FIONs for up to
24 h, we did not detect any differences in the cell viability
The macrophages treated with 50 mg Fe/mL FIONs exhibited
reduced phagocytotic efficiency as the incubation time increased
from 0 to 12 h. However, no significant difference was detected
between the macrophages treated with FIONs for 2 h and the
unlabeled cells (Figure 3C).
Additionally, no significant differences in the migratory ability
were evident between the unlabeled and FION-labeled
macrophages after 2, 6, or 12 h treatments (50 mg Fe/mL FION)
In vitro MR imaging
The T2* of the macrophages decreased from 10066.40 ms in
the absence of FIONs to 9.0966.20 ms in the presence of 50 Fe
mg/mL FIONs in the culture medium. This T2* was significantly
lower than the T2* (71.4368.84 ms) of the macrophages labeled
with Feridex (Figure 4A). Increasing the iron concentration to 100
Fe mg/mL FIONs did not further reduce the T2* (Figure 4B).
Based on our in vitro results, a concentration of 50 mg Fe/mL
FIONs and a 2-h incubation time was implemented for the
FIONlabeling of macrophages for the in vivo experiments. These
conditions did not affect the physiologic activity of the
In vivo MR imaging and histological analysis
The main tumor model. In T2* GRE MR images of the
mice with main tumors, the hypointensities from the
FIONlabeled macrophages were detected within the tumors (n = 6) on
the day after the intravenous administration and were found to be,
significantly higher than the hypointensities within the main
tumors of the mice (n = 6) that received only a FION solution
(P = 0.00268) (Figure 5A). The percentages of hypointense pixels
within the tumors of the mice treated with the FION-labeled
macrophages or only a FION solution were 12.09364.139 and
2.07461.461, respectively (Figure 5B). Moreover, a histological
examination of the main tumors using Prussian blue staining
revealed intracellular FIONs within the macrophages (Figure 5C),
which was more prominent from the tumors of the mice injected
with the FION-labeled macrophages than the FION solution
The metastatic lymph node model. A total of 12 brachial,
12 axillary, and 12 inguinal LNs were isolated from the six mice
used for the metastatic LN model, and these LNs were evaluated
by MRI and histological analysis. In all brachial and axillary LNs,
metastases were detected, but metastatic foci were not found in
any of the inguinal LNs. The mean sizes of the metastatic
melanomas in the brachial and axillary LNs were 8.3 mm (range:
5.210.3 mm) and 3.4 mm (range: 1.15.4 mm), respectively.
The T2* GRE MR images showed that the hypointensities of
the FION-labeled macrophages were detected within the
metastatic brachial and axillary LNs on the day after the intravenous
administration of the macrophages. In contrast, fewer
hypointensities were detected in the non-metastatic inguinal LNs revealed
Figure 2. The identification of the intracellular distribution of FIONs in macrophages. (A) Microscopic view (4006) of macrophages
treated with FIONs (50 mg Fe/mL). The ingested FIONs were visualized by Prussian blue staining (left: phase, right: Prussian blue). (B) The location of
the FIONs was further confirmed by electron microscopy (left: 150006, right: 300006), which revealed that the ingested FIONs were located in
cytoplasmic organelles (arrows).
than in the brachial and axillary LNs (Figure 6A). The percentages
of hypointense pixels within the metastatic brachial and axillary
LNs as well as, the non-metastatic inguinal LNs were
45.064611.932, 34.242611.456 and 8.41365.449, respectively
(P,0.05). In addition, we could clearly differentiate metastatic
LNs from normal LNs using a threshold of 20% hypointense
pixels; all LNs exhibiting over 20% of hypointense pixels
contained metastases (Figure 6B). Although the FION-labeled
cells were present in both the metastatic melanomas and normal
LN tissues, Prussian blue staining demonstrated an abundant
presence of FION-loaded cells within the metastatic LNs
(Figure 6C). The FION-labeled macrophages in the LNs also
were also positively stained with the F4/80 antibody (Figure 6C).
The mean numbers of F4/80 positive cells in the brachial, axillary
and inguinal LNs were 40.968.4, 12.865.3, and 1.662.4 cells/
0.01 mm2, respectively, and exhibited statistically significant
differences (P,0.001) (Figure 6D).
Herein, we have demonstrated the feasibility of using
FIONlabeled macrophages to target main tumors and melanoma LN
metastases in mice. Although previous investigations have used
macrophages or stem cells labeled with iron oxide particles to
target tumors [16,17], this study is the first to report the use of
macrophages labeled with iron-particles to target LN metastases.
Additionally, in this study, we have demonstrated that the homing
of iron-labeled macrophages can be detected and visualized in vivo
using a clinical 1.5T MR scanner.
Macrophages have enormous potential as vehicles for directed
therapeutic delivery. The mechanism responsible for the homing
of activated macrophages to tumors likely involves chemokine
ligands and receptors similar to the recruitment of other
leukocytes to areas of inflammation. The most important of
these chemokines produced by human tumors appear to be
monocyte chemotactic protein-1 (MCP-1), macrophage colony
stimulating factor (M-CSF or CSF-1), and vascular endothelial
growth factor (VEGF) . Hypoxia is also known to recruit of
macrophages to the tumor regions . Valable et al 
visualized macrophages labeled with micrometer-sized iron-oxide
particles using MRI and demonstrated that the labeled cells,
accumulated in the brain tumors in rats. However, to our
knowledge, the present study is the first to assess the applicability
of a systemic delivery of iron-particle-labeled macrophages to
metastatic tumors in the LNs.
As a non-invasive imaging technique that uses non-ionizing
radiation, MRI and the general use of iron particles may have
important future roles in human applications. In the case of
cellular MR imaging, the labeling method used in this study has
several advantages. First, the macrophages could be easily labeled
with the FIONs, within a two-hour time periods. Although
previous studies have reported that an overnight incubation was
required to label the macrophages with Feridex [20,21,22], the
intracellular iron concentration in the macrophages labeled with
FIONs for 2 h was higher than the concentration found after a
24 h Feridex labeling (Figure S2). In theory, the short labeling
time would improve the viability and functionality of the
transplanted macrophages. Second, the FIONs did not affect the
viability or functions, including phagocytosis and migration, of the
labeled macrophages. Third, we could visualize the injected
FION-labeled macrophages using a clinical 1.5 T MR scanner due
to the large magnetization and resulting high relaxivity of the
FIONs [23,24]. Furthermore, the feasibility of using FION
particles for tracking a variety of transplanted cells in vivo after
labeling is known . Until now, however, the use of direct
intravenous FION injections has been limited in vivo because of the
tendency of FIONs to aggregate. For clinical applications, the
ability to track the homing of macrophages to primary tumors and
LN metastases using a simple non-invasive clinical scanner would
be greatly beneficial.
Currently, key challenges for anti-tumoral therapy include
maintaining an elevated concentration of therapeutic agents at the
tumor site and preventing the spread of these agents into the
surrounding healthy tissue [25,26]. Cell-based targeting strategies
for the delivery of therapeutic agents have great potential for
overcoming these challenges. Using genetic engineering, cellular
therapy may facilitate the sustained production of a desired
molecule. For example, genetically modified mesenchymal or
neural stem cells overexpressing interleukin-2  or
interleukin12  may be used to inhibit tumor growth. One of the bodys
responses to the presence of a malignant neoplasm is the
recruitment of peripheral blood monocytes to the tumor by a
chemoattractive gradient into the tumor. Once the monocytes
cross the endothelial basement membrane, they differentiate into
macrophages . Macrophages are important components of the
innate immune response against tumors and are attracted by
locally secreted chemokines as described above [29,30]. Thus, we
believe the approach described here could be utilized for the
delivery of anti-tumor agents towards primary tumors as well as
Figure 4. In vitro T2* measurement of macrophages labeled with iron oxide. (A) MR phantom of the labeled macrophages was constructed
in 1% agar/PBS. T2* MR images showed that the SI significantly decreased when the macrophages were labeled with FIONs. However, the SI of
Feridex labeled macrophages was poorly differentiated. (B) The T2* of the macrophages was found to decrease from 10066.40 ms in the absence of
FIONs. The T2* of the FION-labeled macrophages was decreased when the incubation concentration reached 50 mg Fe/mL (9.0966.02 ms), however,
the T2* of the macrophages labeled with 100 (mg Fe/mL) Feridex (62.5067.69 ms) was higher than that of the macrophages labeled 12.5 mg Fe/mL
FIONs (33.3366.02 ms).
Other studies have suggested that some of the MRI signal may
be the result of either the release of free iron, or the uptake of iron
by endogenous macrophages after the death of labeled cells
[31,32]. In our experiments, we did not detect MRI signal with the
use of free FIONs, suggesting that the iron is cleared under such
circumstances. Thus, the MRI signal is generated exclusively by
viable, labeled cells.
In the present study, murine B16F10 melanoma cells were
chosen because of their high potential to metastasize to regional
LNs [33,34]. Although the murine models employed in this study
have implicated macrophages as promising agents for cancer
targeting, some uncertainties still remain. Further study of the
homing ability of macrophages to different tumor entities and their
hematogenous or lymphatic metastases would be of great benefit
for clinical applications.
Bone marrow-derived macrophages, resident or induced
peritoneal macrophages, or macrophages from the spleen, liver,
and lung can be isolated from mice. The phenotypic and
functional data obtained from studies using macrophages isolated
from these various sites have indicated that all macrophages are
not equal with respect to cytokine production, migratory capacity,
and the ability to ingest and kill pathogens . In the present
study, we used peritoneal macrophages activated by treatment
with thioglycollate broth for the following reasons. First, peritoneal
lavage is a common method for obtaining relatively large numbers
of fully differentiated macrophages from mice, and approximately
107 macrophages can be recovered per mouse after treatment with
thioglycollate broth . Second, the main purpose of the present
study was to investigate whether the macrophages labeled with
FION can be used to target tumors in vivo, so we did not include
experiments to determine which type of macrophage is
appropriate for targeting tumors. Thus, we believe that further study using
macrophages isolated from various sites is warranted for future
In this study, metastatic LN mouse model was obtained by
injecting melanoma cells near the brachial LNs, which induced
metastasis in brachial and axillary LNs. This method is limited by
the difficulty of simulating the metastatic spread condition because
of the proximity of the injection site to the LNs. However, we
believe that this method has strengths in regard to the study of
sentinel LNs and the high yield of metastatic LNs.
In conclusion, this study has demonstrated that macrophages
can be simply and efficiently labeled with FIONs. The labeled
macrophages, which could be monitored non-invasively using a
1.5 T clinical MR scanner, targeted both main tumors and LN
metastases. Overall, the results of this study suggest that the use of
macrophages may have many future applications in the clinic for
vectorizing therapeutic agents towards main tumors as well as LN
Materials and Methods
These experiments were approved by the animal care
committee at Seoul National University Hospital.
The isolation of peritoneal macrophages
Activated peritoneal macrophages were obtained from
six-weekold male BALB/c nude mice. For the murine MR imaging
experiments, activated peritoneal macrophages were obtained
from syngeneic mice 5 days after the intraperitoneal injection of
2 mL of a 3% aged Brewer thioglycollate solution (Sigma) using
peritoneal washings. The peritoneal washings were centrifuged at
1500 g for 10 min and then resuspended in 1 mL of red blood cell
lysis buffer (Sigma) for 7 min. Cells were then centrifuged again
and resuspended in RPMI-1640 medium (WelGENE) containing
10% fetal bovine serum (WelGENE) and 1% a
penicillinstreptomycin mixture (Gibco). The cell suspensions were then
plated in tissue culture flasks and allowed to adhere to it .
Identification of peritoneal macrophages by FACS
To identify the macrophages within the intraperitoneal cells, we
used a FACSCalibur flow cytometer (BD Biosciences) equipped
with a 530-nm filter (bandwidth615 nm) and a 585-nm filter
(bandwidth621 nm) and Cell-Quest software (BD Bioscience),
and the analysis was performed using an F4/80 antibody (Abcam).
After removing the red blood cells as described above, 16104 cells
were fixed by 4% paraformaldehyde for 30 min at room
temperature. The cells were washed with PBS and stained with
primary F4/80 antibody for 1 h. Control cells were not stained
with antibody. After 1 h, the cells were washed several times and
analyzed by fluorescence after staining with Alexa Flour
488conjugated secondary antibody.
Contrast agents for macrophage labeling
For labeling macrophages, FIONs were synthesized and used
according to the previously reported methods [23,24]. Two
milliliters of the synthesized FIONs in chloroform were mixed with
1 mL of chloroform containing 10 mg of PEG-phospholipid and
1 mg of 1,
2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethyleneglycol)-2000] (NH2-PEG-phospholipid, Avanti
Polar Lipids, Inc.) and dispersed in carbonate buffer (pH 9.0). The size
of the PEG-phospholipid encapsulated FIONs was 57.869.9 nm.
The magnetization of the FIONs at 300 K was measured to be
132.1 emu per gram of Fe. The r2 relaxivity of the FIONs dispersed
in a 1% agarose solution was 324 mM21 s21 at 1.5 T, which was
higher than had been reported for FeridexH (Advanced Magnetics,
Cambridge, Mass) and MPIOs (Bangs LaboratoryH, Fisher, IN,
USA) dispersed in a 1% agarose solution (133 and 169 mM21 s21
at 1.5 T, respectively) [23,24].
The labeling of macrophages and the measurement of
The macrophages were magnetically labeled with FIONs or
Feridex, and the intracellular concentration of iron was measured
(Figures S1, S2, and Files S1, S2).
Transmission electron microscopy (TEM)
The labeled macrophages were washed with PBS and then fixed
with 2.5% glutaraldehyde in 0.1 M PBS (pH 7.4) for 2 h.
Subsequently, the macrophages were treated with 2% osmium
tetroxide in 0.1 mM cacodylate buffer for 2 h. The macrophages
were then dehydrated using a graded ethanol series (from 50 to 100%
ethanol) in propylene oxide (EM Sciences, Fort Washington, PA).
The samples were then embedded in pure Epon resin (EM Sciences)
for 3 days at 60uC. Next, ultrathin sections were prepared using glass
knives and a Diatome diamond knife (Reichert-Jung, Vienna,
Austria) on an RMC MTXL ultramicrotome (Tucson, AZ). The
prepared sections were then stained with lead citrate and uranyl
acetate (both from EM Sciences) and visualized by TEM (JEM-1400).
Cell viability assay
To determine cell viabilities, macrophages were initially seeded
in 96-well tissue culture plates at a density of 16105 cells per well.
The macrophages were washed three times using PBS after
exposure to FIONs at concentrations of 12.5, 25, 50, or100 mg Fe/
mL for 2 h, or FIONs at a concentration of 50 mg Fe/mL for 2, 6
or 24 h. The viabilities of the macrophages were assessed using a
standard 3-,5-diphenyltetrazolium bromide (MTT) assay. The
optical densities were read at 540 nm.
The bacterial phagocytotic capacity of the macrophages was
assayed using a commercially available kit (Vybrant phagocytosis
assay kit; Molecular Probes). Macrophages were incubated with or
without FIONs dissolved in RPMI-1640 at a 50 mg Fe/mL
concentration for varying time (2, 6, or 12 h) at a cell density of
16105 cells per well in 96-well plates. The media was then removed,
fluorescein-labeled Escherichia coli were added, and the procedure
was performed according the manufacturers instructions.
Macrophage migration assays were performed in 96-well
chambers (CytoSelect CBA-105; Cell Biolabs) according to the
manufacturers instructions. Briefly, 16105 macrophages that had
been treated with 50 mg Fe/mL FIONs for 2, 6 or 12 h were
suspended in the upper chamber, and the media from a 48 h
culture of B16F10 cells was placed in the lower compartment of
the chamber. After 24 h, the migrated cells were detected with
CyQuant GR Dye (Molecular Probes).
In vitro MR imaging of phantom
MR phantom of the labeled macrophages for stable and
homogeneous MRI measurement was constructed in 1% agar/
PBS. Briefly, peritoneal macrophages were incubated with FIONs
or Feridex at concentrations of 12.5, 25, 50, or 100 mg Fe/mL for
2 h in standard tissue culture incubators with 5% CO2. After the
incubation, 16103 cells were washed, harvested, transferred to
0.2 mL thin wall strip tubes (Axygen) that had been, coated with
50 mL molten 1% agar/PBS on the bottom and centrifuged
(1000 rpm, 5 min). The supernatants were removed, and 50 mL of
molten 1% agar/PBS was added and given 30 min at room
temperature to settle. All agar/PBS solutions were sterilized
through 0.45 mm pore size filter paper (Whatman). MR imaging
was performed using a clinical 1.5 T MR scanner (Signa Excite,
GE healthcare) with a wrist coil. For the estimation of T2*, we
used a gradient echo (GE) pulse sequence with the following
imaging parameters: TR/TE = 800/4.9, 13.6, 22.3, or 57 ms; flip
angle = 20u, FOV = 50650 mm, matrix = 2566256, slice
thickness = 2.1 mm, and the number of excitation = 2. Regions of
interest (ROIs) were defined in the representative slices acquired at
the shortest TE, and this data was used to generate the T2* maps
of the ROIs using a pixel-by-pixel analysis across the four-point
MR images in MATLABTM (MathWorks Inc., Natick, USA)
assuming single exponential decay (i.e., SI = SI06e2TE/T2*, where
SI represents signal intensity and SI0 represents proton density).
B16F10 cells were obtained from the American Type Culture
Collection (ATCC, Rockville, MD) and maintained in DMEM
with 10% fetal bovine serum (FBS) at 37uC.
The main tumor model. B16F10 melanoma cells were
prepared in 100 mL serum- free DMEM and then subcutaneously
transplanted into the shoulders of 6-week old BALB/c nude mice
(n = 12; 26106 cells/100 mL medium/each mouse). In vivo MR
imaging of the tumors was performed 2 weeks after the cell
The metastatic lymph node model. Metastatic lymph
nodes were produced in 6-week old BALB/c nude mice (n = 6)
according to the following step: A. a superficial skin incision,
approximately 1 cm in length, was made in the bilateral upper
brachial area of anesthetized mice. B. the skin was inverted to
expose the brachial lymph nodes. C. a 5 mL volume of 26106
B16F10 cells was then slowly injected in the fatty area adjacent to
the bilateral brachial area using a Hamilton syringe and an
investigational microneedle (Hamilton Syringe), and D. the dermal
incision was closed with a 5-0 Prolene purse string (Harrell
Medical). In vivo MR imaging of the metastatic lymph nodes was
performed 1 week after the cell implantation.
In vivo MR imaging
In vivo MR imaging was performed using a clinical 1.5 T MR
scanner (Signa Excite, GE Healthcare) with a wrist coil. The
imaging protocol consisted of a sagittal and coronal 3D T2* GRE
sequence with the following imaging parameters: TR/TE = 58/
12, flip angle = 10u, FOV = 80680 mm, matrix = 2566192, slice
thickness = 0.7 mm, and the number of excitations = 6. Mice were
anesthetized by the intraperitoneal injection of a solution
containing zolazepam (5 mg/kg, ZoletilH, Virbac) and xylazine
(10 mg/kg, RompunH, Bayer-Schering Pharma). We obtained
MR images before and one day after the intravenous
administration of the labeled macrophages or a FION solution.
Main tumor model. Mice were intravenously injected with
26106 macrophages that had been labeled with 50 mg Fe/mL
FIONs for 2 h (n = 6) or 26106 macrophages that had been
labeled with a FION solution of identical Fe amount (9.1 mg
FIONs) (n = 6).
Metastatic lymph node model. We administered 26106
macrophages that had been labeled with 50 mg Fe/mL FIONs for
2 h intravenously into the mice (n = 6).
In vivo MR imaging analysis
The MR data were digitally transferred from a PACS
workstation to a personal computer and processed with ImageJ
(available at http://rsb.info.nih.gov/ij/ ) and software developed in
house using Microsoft Visual C++. One author performed all of
the image processing, region-of-interest (ROI) drawing, and data
ROIs that contained the entire tumor or lymph node (brachial,
axillary and inguinal LNs were included) were drawn in each
section of the T2* GRE images. Using the software developed
inhouse, the data acquired from each slice were summated to derive
the pixel-by-pixel SI values for the entire tumor. Previous studies
[37,38] have shown that muscle tissue remains unchanged by the
contrast agent; therefore, the pixel-by-pixel SI values were then
normalized to the muscle to cancel the SI fluctuations related to
variations in the technical parameters between the MRI sequences
obtained both pre- and post-injection of the labeled macrophages
or the FION solution. The SI of muscle was measured within a
single ROI measuring 35 mm2 placed in the shoulder muscle.
Finally, SI histograms for the tumors and LNs were plotted using a
bin size of 1% with normalized SI (nSI) value (i.e., normalized SI
value (%) = [SI of tumors or lymph nodes]/[SI of muscles]6100)
on the x-axis, and the percentage of the total lesion volume was
expressed on the y axis by dividing the frequency of each bin by
the total percentages of pixels analyzed.
A baseline pixel histogram using the nSI of a tumor or LN was
created from MR images obtained before the injection of the
labeled macrophages or FION solution to establish the minimum
nSI in the absence of any FIONs. On the MR images obtained
one day after the injection of the labeled macrophages or FION
solution, the percentages of pixels in the tumors or LNs below the
minimum nSI threshold was summated and reported as the black
pixel count (%) for the FION hypointensity.
Tumors from the main tumor models (n = 12) as well as
brachial, axillary and inguinal lymph nodes from the metastatic
LN models (n = 6) were removed and fixed in 10% buffered
formalin. Paraffin-embedded tumors and lymph nodes were
sectioned into 4-mm thick sections. Staining methods included
hematoxylin and eosin (H&E) to visualize the tumor and lymph
node morphology, and Prussian blue and immunohistochemical
staining. To detect the presence of FIONs in the tissues, prepared
paraffin sections were dewaxed, hydrated, and treated with 0.01%
protease XXIV (Sigma) in PBS for 20 min at 37uC. Sections were
then incubated with a 1:1 (vol/vol) mixture of 1% potassium
ferrous cyanide (kaliumhexacyanoferrat [II]) and 5% HCl for 1 h.
Slides were then rinsed in distilled water and counterstained with
Nuclear Fast Red for 10 min. The presence of iron oxides was
qualitatively assessed with a microscope.
For the detection of mature macrophages in LNs,
immunohistochemical staining was performed using the following steps were
performed using the F4/80 antibody (Abcam). First, endogenous
peroxidase and protein were blocked with a solution of 0.3%
H2O2 and goat serum (Dako) to prevent nonspecific antibody
binding. After 30 min of blocking, the tissues were incubated with
the primary F4/80 antibody for 1 h, and after several brief washes,
a HRP-conjugated goat anti-rat secondary antibody (Santacruz)
was applied for 2 h. After additional washings, the macrophages
were evaluated by staining with the peroxidase DAB substrate
(Dako). The F4/80-positive cells in each LN were counted in a
square unit of surface with an area of 0.01 mm2. The mean
numbers of F4/80 positive cells/surface unit were calculated from
50 measurements in each LN from all of the mice with metastatic
For all statistical analyses, a two-tailed P value of less than 0.05
was considered to be statistically significant. Statistical analyses
were performed using commercially available software (MedCalc,
version 22.214.171.124, MedCalc Software, Mariakerke, Belgium).
Single-group data were assessed using Students t test or the
Mann-Whitney test. Repeated measurements analysis of variance
(ANOVA) with TukeyKramer post hoc comparisons were
performed for multiple comparisons.
Figure S1 The Prussian blue staining of macrophages
after incubation with various concentrations of iron
oxide for different times. With increases in the concentration
of iron oxide (from 12.5 to 100 mg Fe/mL) and exposure time
(from 2 to 12 h), the macrophages phagocytosed more particles.
When compared with Feridex-labeling, the FION-labeled
macrophages showed a higher intracellular uptake of iron after a 2
Figure S2 The intracellular iron content (pg Fe/cell)
after incubation with increasing iron oxide
concentrations (mg Fe/mL). The iron concentration in the macrophages
correlated to the FION incubation concentration. Up to 4.539 (pg)
of FIONs could be ingested by one macrophage when the FION
incubation concentration reached 50 (mg Fe/mL) for 2 h. The
amount of iron was 2.214 (pg) when the FIONs concentration was
25 (mg Fe/mL) for 2 h (N). However, when the macrophages were
incubated with Feridex in concentration 100 (mg Fe/mL) for 24 h,
the amount of iron ingested was 1.284 (pg/cell) (# with the dotted
line), which was significantly lower (* P,0.001) than the iron
content of the macrophages treated with different concentration of
FIONs (from 12.550 mg Fe/mL) for 2 h.
The authors will share the software developed in-house for pixel analysis in
the present study ().
Conceived and designed the experiments: HRC SHC WKM. Performed
the experiments: HRC SHC. Analyzed the data: HRC SHC HK.
Contributed reagents/materials/analysis tools: NL TH. Wrote the paper:
HRC SHC WKM.
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