Noninvasive Identification of Viable Cell Populations in Docetaxel-Treated Breast Tumors Using Ferritin-Based Magnetic Resonance Imaging
et al. (2013) Noninvasive Identification of Viable Cell Populations in Docetaxel-Treated Breast Tumors Using
Ferritin-Based Magnetic Resonance Imaging. PLoS ONE 8(1): e52931. doi:10.1371/journal.pone.0052931
Noninvasive Identification of Viable Cell Populations in Docetaxel-Treated Breast Tumors Using Ferritin-Based Magnetic Resonance Imaging
YoonSeok Choi 0
Hoe Suk Kim 0
Kyoung-Won Cho 0
Kyung-Min Lee 0
Yoon Jung Yi 0
Sung-Jong Eun 0
Hyun Jin Kim 0
Jisu Woo 0
Seung Hong Choi 0
Taeg-Keun Whangbo 0
ChulSoo Choi 0
Dong-Young Noh 0
Woo Kyung Moon 0
Ilya Ulasov, University of Chicago, United States of America
0 1 Department of Radiology, Seoul National University Hospital , Seoul , Korea , 2 Department of Biomedical Science, College of Medicine, Seoul National University , Seoul , Korea , 3 The Institute of Radiation Medicine, Medical Research Center, Seoul National University , Seoul , Korea , 4 Department of Surgery, Seoul National University Hospital , Seoul , Korea , 5 Institute of LeeGilYeo Cancer and Diabetes Center, Gachon University of Medical Science , Incheon , Korea , 6 Department of Computer Science, Gachon University , Seongnam , Korea
Background: Cancer stem cells (CSCs) are highly tumorigenic and are responsible for tumor progression and chemoresistance. Noninvasive imaging methods for the visualization of CSC populations within tumors in vivo will have a considerable impact on the development of new CSC-targeting therapeutics. Methodology/Principal Findings: In this study, human breast cancer stem cells (BCSCs) transduced with dual reporter genes (human ferritin heavy chain [FTH] and enhanced green fluorescence protein [EGFP]) were transplanted into NOD/ SCID mice to allow noninvasive tracking of BCSC-derived populations. No changes in the properties of the BCSCs were observed due to ferritin overexpression. Magnetic resonance imaging (MRI) revealed significantly different signal intensities (R2* values) between BCSCs and FTH-BCSCs in vitro and in vivo. In addition, distinct populations of pixels with high R2* values were detected in docetaxel-treated FTH-BCSC tumors compared with control tumors, even before the tumor sizes changed. Histological analysis revealed that areas showing high R2* values in docetaxel-treated FTH-BCSC tumors by MRI contained EGFP+/FTH+ viable cell populations with high percentages of CD44+/CD242 cells. Conclusions/Significance: These findings suggest that ferritin-based MRI, which provides high spatial resolution and tissue contrast, can be used as a reliable method to identify viable cell populations derived from BCSCs after chemotherapy and may serve as a new tool to monitor the efficacy of CSC-targeting therapies in vivo.
Funding: This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MEST) (N0. 2012-01010846) and
the Second Stage of Brain Korea 21 program in 2012. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of
Competing Interests: The authors have declared that no competing interests exist.
Since the first identification of breast cancer stem cells (BCSCs)
from human tumor samples using CD44+/CD242 markers by
Al-Hajj et al., the role of BCSCs in tumor progression and
therapeutic resistance has been actively investigated to develop
better anti-cancer treatment strategies [1,2]. In breast cancer
patients, administration of chemotherapy or radiation therapy
increases the fraction of CD44+/CD242 tumor cells and
augments mammosphere formation in vitro and tumorigenicity in
xenotransplantation models [3,4]. The presence of BCSC markers
or gene expression signatures correlates with poor prognosis in
clinical tumor samples [5,6]. The therapeutic resistance of BCSCs
is associated with alterations in self-renewal and cell fate signaling
path ways, including Notch, Wnt, Hedgehog, and HER-2 .
New therapeutic regimens using single agents or a combination of
various drugs that target BCSCs are now under preclinical or
clinical trials. Monitoring the efficacy of cancer stem cell (CSC)
therapeutics in vivo, however, is challenging because the
conventional method of measuring tumor size is inadequate as an
endpoint . In vivo identification of BCSCs using cellular imaging
techniques will be extremely useful for this purpose because the
efficacy of treatment depends more on the fraction of viable cancer
cells in the tumor [8,9].
In vivo imaging methods, including intravital microscopy,
fluorescent imaging, luciferase imaging, positron emission
tomography (PET), and magnetic resonance imaging (MRI),
have been used to track cancer cells and monitor treatment
response . However, there have only been a few reports
of in vivo imaging of CSCs in different types of tumors [16,17].
Snyder et al. analyzed CSCs using quantum dot-conjugated
antibodies against CD44v6 and CD24 in tumors and suggested
the possibility of applying this approach to BCSC imaging .
Vlashi et al. demonstrated reduced 26S proteasome activity in
CSCs originating from glioma cells and monitored these CSCs
in vivo using a fluorescent protein (ZsGreen) fused to ornithine
decarboxylase, which is a target of the 26S proteasome .
Liu et al. longitudinally monitored CSCs derived from breast
cancer patients in an orthotopic xenograft mouse model using
ubiquitin promoter-driven luciferase and showed the role of
BCSCs in metastasis with imaging techniques . Recently,
Yoshii et al. showed that in a mouse colon carcinoma model,
Cu-64-ATSM, a PET imaging agent, localizes preferentially in
tumor regions with a high density of CD133+ cells with CSC
characteristics . However, in vivo imaging of BCSCs using
MRI or PET has not been reported to date. MRI can provide
tomographic or volumetric imaging of internal organs at high
anatomical resolutions and soft tissue contrast without using
ionizing radiation, which is not possible with other imaging
modalities. Clinically, MRI is routinely used to identify and
localize tumors before surgery and to monitor the response to
treatment in breast cancer .
There are two approaches to track and image cells of interest
in vivo with MRI. The first method uses a contrast agent as
a labeling or targeting agent. To date, superparamagnetic iron
oxide (SPIO) nanoparticles, due to their high relaxivity, have
been the most widely used contrast agents for tracking and
imaging diverse cells [19,20]. With surface modification of SPIO
nanoparticles, cells of interest can be targeted by an antibody,
peptide, or nucleotide conjugation . The presence of SPIO
nanoparticles in the magnetic field leads to low signal intensities
in T2 or T2* sensitive images. However, this method does not
enable the long-term imaging of the cells of interest because the
contrast agents become diluted as the cells divide, and the SPIO
nanoparticle signals can accumulate in sites within tumors,
where the cells are not viable . The use of the MRI reporter
gene ferritin can overcome these limitations . The
overexpression of ferritin enables cells to uptake more iron, and
this reporter produces low signal intensities in MRI. As MRI
reporters are stably expressed, even during cell division, they
can be used for studying dynamic processes, e.g., the migration
and invasion of cells of interest over an extended period of time
and can also be useful for providing temporal and spatial
information for anti-cancer treatment effects on a specific cell
population. The number of cancer cells or level of tumor
burden in deep tissues can be quantified by calculating R2*
( = 1/T2*) values from T2* mapping of MRI images [27,28]. In
addition, the introduction of optical reporter genes, such as
EGFP or luciferase, and ferritin together allows for the analysis
of cancer cells isolated from tumors in molecular biology and
In the present study, BCSCs isolated from human breast cancer
specimens were transduced with MRI (human ferritin heavy
chain, FTH) and fluorescence (enhanced green fluorescence
protein, EGFP) dual reporter genes and transplanted into
nonobese diabetic/severe combined immunodeficient (NOD/
SCID) mice to noninvasively track BCSC-derived populations
during tumor growth and monitor tumor responses after
chemotherapy. An MRI evaluation of ferritin-overexpressing
BCSCs (FTH-BCSCs) was performed, and the tumor response
to chemotherapy was determined by quantification of the R2*
values for entire tumors. In addition, viable cells were identified
and localized by volumetric MRI, and BCSC characteristics were
investigated by histological analysis.
All of the procedures were performed following approval by the
Institutional Review Board (IRB) at Seoul National University
Hospital. IRB approval number is H-0502-142-007. The
individual in this manuscript has given written informed consent (as
outlined in PLOS consent form) with the Declaration of Helsinki
to publish these case details.
In animal study, 6-week-old NOD/SCID female mice were
used. The animal study was reviewed and approved by the
Institutional Animal Care and Use Committee (IACUC;
No.110105) of the Seoul National University Hospital, and the
procedures for all animal experiments were performed according
to IACUC guidelines.
Isolation of Primary BCSCs and Establishment of
The human breast cancer tissues were obtained in operating
room within 30 minutes after excision from routine surgical
procedures for breast cancer patients. After excision from the
surgical procedures, tissues (ER-, PR-, HER2-) were minced into
1 mm3 sized pieces and digested with collagenases at 37uC for four
hour. After rinsing with the proper amount of medium used for
mammosphere culture, CD44+/CD242 cells were obtained with
fluorescence-activated cell sorter (FACS-Aria, BD Biosciences) and
maintained using an anchorage-independent culture method .
The BCSCs were incubated with DMEM mixed 3:1 with Hams
F12 medium (Invitrogen) supplemented with basic fibroblast
growth factor (10 ng/ml; Millipore), epidermal growth factor
(20 ng/ml; Invitrogen), leukemia inhibitory factor (10 ng/ml;
Millipore), B27 supplement (Invitrogen) and antibiotic-antimycotic
solution (Invitrogen) for mammosphere culture.
To establish the FTH-BCSCs, a lentivirus expressing
myctagged FTH (myc-FTH) and EGFP driven by the cytomegalovirus
(CMV) and phosphoglycerate kinase (PGK) promoters,
respectively, was introduced into cells by incubating the cells with 106
107 transduction units/ml for 610 hours in the presence of 8 mg/
ml polybrene. After three days of transduction, cells expressing
EGFP were sorted using a FACS-Aria, expanded and used in all
Analysis of Surface Markers on FTH-BCSCs
To analyze cell surface markers, 56105 BCSCs and
FTHBCSCs were dissociated from mammospheres and washed two
times with phosphate buffered saline (PBS) containing 1% BSA.
The cells were then incubated with anti-huCD44-phytoerythrin
(PE), anti-huCD24-PE, anti-huCD90-APC, anti-huCD49f-PE, or
anti-huCD34-PE antibodies (BD Biosciences) for one hour at
37uC. Cell-associated fluorescence was measured using a
FACSCalibur flow cytometer (BD Biosciences). The data were analyzed
using CellQuest software (BD Biosciences).
Western Blot Analysis
Cells were lysed in RIPA buffer with 1 mM
phenylmethylsulfonyl fluoride and a protease inhibitor cocktail (Sigma-Aldrich
Chemical Co.). The protein lysates were resolved by
SDSpolyacrylamide gel electrophoresis (SDS-PAGE) for four hours
at room temperature and were transferred to nitrocellulose
membranes for two hours at 4uC. After blocking, the membranes
were incubated with anti-FTH (Santa Cruz Biotechnology) and
anti-EGFP (Santa Cruz Biotechnology) antibodies overnight at
4uC followed by incubation with HRP-conjugated antibodies
(Santa Cruz Biotechnology) at room temperature for 30 minutes.
Blots were visualized using enhanced chemiluminescence reagents
Immunocytochemistry of Mammospheres
Mammospheres were fixed in 4% paraformaldehyde in PBS.
For the detection of myc-FTH expression, fixed mammospheres
were incubated with anti-c-myc antibody (Santa Cruz
Biotechnology) overnight at 4uC followed by incubation with the Alexa
Fluor 594-conjugated anti-mouse IgG antibody (Invitrogen) at
room temperature for one hour. Hoechst 33342 (Invitrogen) was
used to visualize cell nuclei. Images were scanned and analyzed
with a confocal laser microscope (LSM 5 META, Carl Zeiss).
Measurement of Iron Loading
BCSCs or FTH-BCSCs (26104) were placed in 12-well Petri
dishes, and 0 to 50 mM ferric ammonium citrate (FAC;
SigmaAldrich Chemical Co.) was added to the culture medium for 4
days. Harvested cells were counted and subsequently lysed with 6
N HCl to extract total iron. The amount of total iron was
determined using a total iron reagent kit (Pointe Scientific), and
the average iron amount in a cell was calculated by dividing the
total mean iron by the cell number.
To compare the mammosphere-forming abilities of the BCSCs
and FTH-BCSCs, dissociated cells from BCSC and FTH-BCSC
mammospheres were seeded in 96-well plates at a density of 100
cells/well and incubated for four days. The average number of
mammospheres was calculated by counting the number of
mammospheres in a well.
Cell Growth and Viability with Iron Supplementation
To evaluate the growth and viability of the cells under different
iron supply conditions, the trypan blue exclusion assay and flow
cytometric analysis with 7-amino-actinomycin D (7-AAD) (BD
Pharmingen) were performed. For the cell growth assays, 36104
BCSCs or FTH-BCSCs were initially seeded in 12-well plates
supplemented with FAC (up to 50 mM), and the average number
of cells was calculated on days 2, 4, 6 and 8. For the cell viability
assays, BCSCs and FTH-BCSCs were grown in medium
supplemented with increasing amounts of FAC for five days.
Next, both cell types were collected, incubated with 7-AAD for 5
10 minutes at 37uC and analyzed by flow cytometry.
Tumor Formation and Tumor Volume Measurements
To compare the tumor formation abilities of BCSCs and
FTHBCSCs, 2610216106 viable BCSCs and FTH-BCSCs were
implanted into the mammary fat pads of NOD/SCID mice
(BCSC tumors [n = 21]; FTH-BCSC tumors [n = 21]). Tumor
formation was monitored up to eight weeks after implantation,
depending on the number of injected cells.
To determine the volumes of the BCSC and FTH-BCSC
xenograft tumors, a modified ellipsoidal formula for volume
(volume = 1/2[length 6width2]) was used, in which length was the
measurement of the greatest longitudinal diameter and width was
the greatest transverse diameter.
At three weeks post-injection, the fluorescence imaging of
BCSC and FTH-BCSC tumors from a living mouse was assessed
using a Maestro imaging system (CRi Inc.) with excitation and
emission set at 445490 nm and 515 nm, respectively. The filter
was adjusted while the camera captured images, and the signals
from the tumors were merged with a GFP-filtered image.
Ex vivo fluorescence imaging of BCSC and FTH-BCSC tumors
excised from mice was performed by GFP fluorescence analysis
(excitation: 470 nm, emission: 535 nm) using a Kodak Image
Station 4000MM (Carestream Molecular Imaging).
Docetaxel Treatment and Cytotoxicity Assays
To investigate the in vitro cytotoxicity of docetaxel on BCSCs
and FTH-BCSCs, JC-1 staining and 3-2,5-diphenyltetrazolium
bromide (MTT) assays were performed following treatment with
docetaxel (110 nM, Sigma-Aldrich Chemical Co.) for 24 hours.
To evaluate the changes in the mitochondrial membrane potential
of both cell populations treated with docetaxel, the mitochondrial
vital dye JC-1 (10 mg/ml, Invitrogen) was used. JC-1 aggregates
with intense red fluorescence are known to accumulate in the
intact mitochondria of healthy cells. When the mitochondrial
membrane potential collapses in non-viable cells, JC-1 monomers
fluoresce green. The percentage of cells with intact mitochondrial
membranes was calculated by dividing the number of red
fluorescence-positive cells by the total cell number.
In vivo Docetaxel Treatment
The mice with BCSC (n = 18) and FTH-BCSC (n = 18) tumors
were divided into docetaxel-treated (n = 19) and untreated control
groups (n = 17). Docetaxel was treated when the tumor volumes
had 70 mm3, usually 16 to 18 days after tumor cell implantation.
In the docetaxel-treated groups, docetaxel (15 mg/kg) was injected
into the tail vein of mice three times at intervals of 72 hours to
evaluate the therapeutic effect of docetaxel on BCSC and
FTHBCSC tumors. The BCSC and FTH-BCSC tumors in the
docetaxel-treated group are represented as BCSC Doc tumors
(n = 10) and FTH-BCSC Doc tumors (n = 9), respectively. In the
control group (BCSC tumors [n = 8]; FTH-BCSC tumors [n = 9]),
saline was intravenously injected three times at the same intervals
as those used in the docetaxel-treated groups.
All MRI studies were performed on a 9.4-T BrukerBiospec
scanner (BrukerBiospin). A transmit-only volume coil and a
fourchannel surface coil (BrukerBiospin) were used for excitation and
signal reception, respectively. For in vitro MRI, phantoms
containing 26106 BCSCs and FTH-BCSCs, which were treated with or
without 25 mM FAC for 4 days, were prepared. A multi-slice,
multi-echo gradient echo sequence was used for in vitro T2*
mapping. The parameters were as follows: matrix size = 2566256,
repetition time (TR) = 5000 ms, slice thickness = 1 mm (no gap),
flip angle = 90u, field of view (FOV) = 25625 mm2, TE = 3.1
43.1 ms with a step size of 10 ms (five-point T2* mapping), 13
slices and 4 signal averages.
For in vivo MRI of mice bearing xenograft tumors, 16106
BCSCs or FTH-BCSCs were injected into the mammary fat pads
of NOD/SCID mice. For whole-animal MRI, mice were
anesthetized with isofluorane (1% in 100% oxygen). To stabilize
the body temperature of the mice during MRI experiments, an
animal warming system (BrukerBiospin) was used. Pre-treatment
images were obtained three weeks after injection, and longitudinal
follow-up images were obtained at 5 and 14 days after docetaxel
treatment. In vivo data were acquired for fat saturation. The
parameters were as follows: matrix size = 2566256,
TR = 5000 ms, slice thickness = 1 mm (no gap), flip angle = 90u,
FOV = 37637 mm2, TE = 3.134.4 ms with a step size of 4.4 ms
(eight-point T2* mapping), 17 slices and 1 signal average.
MRI Data Analysis
All data post-processing was performed with Matlab
(Mathworks Inc.). For both phantom and in vivo data, regions of interest
(ROIs) were defined in each individual, imaging slices were
acquired at the shortest TE and T2* maps of the ROIs were later
estimated by pixel-by-pixel analyses across the multi-point MRI
images, assuming single exponential decay. After performing the
R2* analyses, R2* color maps were merged with the anatomical
images acquired at the shortest TE. For in vivo data, volumetric
tumor images were reconstructed from the entire set of 2D slices
for each animal.
To investigate the differences in the R2* distribution among the
animal groups, we obtained R2* histograms for each animal by
including all pixels in the ROIs across all slices. Comparisons of
the mean R2* values among the animal groups were performed
based on the R2* distributions. Additionally, to represent the
differences in the R2* distribution better, we defined the mean plus
3 standard deviation (mean +3 SDs) as a threshold (Protocol S1).
The distribution of skewedness was analyzed using the threshold
values of each group and subsequent pixel percentages. In
addition, the mean R2* values of the pixels over the threshold
values of each group of tumors were calculated for the comparison
of the R2* value distribution of each group of tumors.
Histochemical Analysis of Tumors
After MRI examination, the excised tissues were fixed with 10%
buffered formalin and embedded in paraffin blocks. Tissues were
sectioned into 4-mm-thick sections. Hematoxylin and eosin
staining (H&E) was performed to distinguish the viable and
nonviable cell populations within the tumors. Immunofluorescence
staining for the surface markers CD44 and CD24 and the
proliferative marker phospho-histone 3 (PH3) were performed.
After incubation of tissue sections with a blocking solution for one
hour at room temperature, primary antibodies against CD44
(Thermo Scientific), CD24 (Novus Biologicals), or PH3 (Novus
Biologicals) were incubated overnight at 4uC. Next,
fluorescenceconjugated antibodies, namely, anti-mouse IgG Alexa 488, 647 or
anti-rabbit IgG Alexa 594 (Invitrogen), were incubated for 45
minutes at room temperature. Hoechst 33342 (Invitrogen) was
used to visualize the cell nuclei. Immunofluorescence images of the
tissue sections were obtained under a confocal laser microscope
(Carl Zeiss) and a fluorescence microscope (Leica).
All data are presented as the mean 6 SD for at least three
independent experiments. The mean values of the data were
statistically evaluated using ANOVA followed by an unpaired
ttest. A Fishers exact test was used to analyze the differences in R2*
value distributions. For all tests, P-values less than 0.05 or 0.01
were considered to be statistically significant.
The Transduction of FTH and EGFP does not Alter BCSC
BCSCs from a breast cancer patient with a CD44+/CD242
phenotype were isolated and maintained using mammosphere
culture media (Figure 1A). To establish ferritin-overexpressing
BCSCs, genes for myc-FTH and EGFP were transduced into cells
with a lentiviral vector, and fluorescence-activated cell sorting
(FACS) was used to collect the cells that expressed both myc-FTH
and EGFP. The levels of BCSC markers (CD44+/CD242) did
not change in the FTH-BCSCs (Figure 1B). Immunofluorescence
staining and western blot analysis revealed the expression of
mycFTH and EGFP in FTH-BCSCs (Figure 1C and D). Iron-loading
abilities were investigated with the addition of up to 50 mM of
FAC to both the FTH-BCSCs and BCSCs. The cellular iron levels
of both groups increased in a dose-dependent manner, but the
FTH-BCSCs had significantly higher iron levels compared with
the BCSCs (Figure 1E; P,0.05 in 25 and 50 mM of FAC).
Next, we sought to determine whether the FTH-BCSCs
retained their BCSC properties compared to control BCSCs.
The mammosphere-forming abilities and surface marker
expression of the BCSCs and FTH-BCSCs were compared. Four days
after single-cell dissociation, BCSCs and FTH-BCSCs generated
mammospheres, and no substantial differences were observed in
the average numbers of mammospheres (Figure S1A). The human
mammary stem cell marker CD49f was highly expressed in both
BCSCs and FTH-BCSCs . High levels of CD90
(mesenchymal lineage marker) and low levels of CD34 (hematopoietic stem
cell marker) were observed, and the levels of all of these surface
markers did not differ between the FTH-BCSCs and BCSCs
(Figure S1B and C).
Next, we performed a tumor forming ability assay with serially
diluted numbers of BCSCs and FTH-BCSCs to investigate
whether ferritin overexpression affects tumorigenesis in vivo (Figure
S2A). Various numbers of BCSCs or FTH-BCSCs were
xenografted into the mammary fat pads of mice, and the incidence
of the BCSC and FTH-BCSC tumors following engraftment with
26102 cells was approximately 25% with transplantation of more
than 16103 BCSCs or FTH-BCSCs, resulting in 100% tumor
formation (Table 1). The sizes of the BCSC and FTH-BCSC
tumors were similar. Ex vivo fluorescence imaging showed the
stable expression of EGFP in the FTH-BCSC tumors (Figure S2B).
These results demonstrated that the FTH-BCSCs retained the
characteristics of BCSCs, despite the overexpression of FTH and
We also investigated the effects of iron overload on the growth
and viability of the BCSCs and FTH-BCSCs. The growth rates of
the BCSCs and FTH-BCSCs were not affected by treatment with
FAC (Figure S3A). Cytotoxicity was not observed in either group
following treatment with 25 mM FAC (Figure S3B), which is
regarded as the physiological iron concentration in mouse serum
(250350 mg/dl) . Taken together, these findings demonstrate
that the FTH-BCSCs retained the biological properties of the
parent BCSCs and possessed an enhanced ability for iron storage,
suggesting the feasibility of FTH-BCSC MRI in vitro and in vivo.
FTH-BCSCs Exhibit a Significant Increase in R2* Values in
MRI Compared to BCSCs
To analyze the R2* values of the BCSCs and FTH-BCSCs, cells
were incubated with or without 25 mM FAC, and MRI images of
cell phantoms were obtained (Figure 2A). The mean R2* values of
the FTH-BCSCs (97.8560.51 s21) treated with 25 mM FAC were
significantly higher than those of the BCSCs (90.7260.21 s21).
However, the mean R2* values of the FTH-BCSCs and BCSCs in
the absence of FAC were not different (Figure 2B).
Next, we investigated the effect of ferritin overexpression in
a xenograft tumor model. Lower signal intensities were observed
in the MRI images of FTH-BCSC tumors compared with BCSC
tumors due to ferritin overexpression (Figure 2C, left). A
colorcoded map of BCSC and FTH-BCSC tumors revealed variable
R2* values (Figure 2C, right). Although both the BCSC and
FTHBCSC tumors exhibited differences in the R2* values within the
tumors, the FTH-BCSC tumors exhibited higher mean R2* values
compared with the BCSC tumors, and the FTH-BCSC tumors
exhibited shifted R2* distributions toward higher R2* values
Figure 1. Establishment of breast cancer stem cells (FTH-BCSCs) that overexpress the myc-tagged heavy chain subunit of ferritin
(myc-FTH) and EGFP. (A) Flow cytometric analysis of the surface markers, CD44 and CD24 in BCSCs isolated from human breast cancer specimens.
(B) Flow cytometric analysis of the surface markers, CD44 and CD24 in FTH-BCSCs expressing myc-FTH and EGFP. (C) Immunocytochemistry of BCSCs
and FTH-BCSCs (bar, 50 mm). (D) Western blot analysis for myc-FTH and EGFP in BCSCs and FTH-BCSCs. (E) Measurement of cellular iron in BCSCs and
FTH-BCSCs treated with increasing concentrations of ferric ammonium citrate (FAC). The results were obtained from 4 independent experiments and
are presented as means 6 SD. *P,0.05.
(Figure 2D; mean R2* values of BCSC tumors vs. FTH-BCSC
tumors; 87.262.7 s21 vs. 105.463.8 s21; P,0.01).
MRI Reveals Distinct Populations of Pixels with High R2*
Values within Docetaxel-treated FTH-BCSC Tumors
Next, we evaluated the potential use of FTH-based MRI for
monitoring the efficacy of an anti-cancer drug in xenografted
tumors. We found that the growth of docetaxel-treated BCSC Doc
and FTH-BCSC Doc tumors was attenuated compared to BCSC
and FTH-BCSC tumors. Additionally, overexpression of FTH did
not affect tumor growth (Figure S4).
We subsequently performed slice-by-slice MRI analysis of
tumors from each group and observed different distributions of
tumor R2* values in volumetric images with longitudinal follow-up
scans (Figure 3A and Figure S5). Before docetaxel treatment, the
R2* value distribution between BCSC and BCSC Doc tumors and
that between FTH-BCSC and FTH-BCSC Doc tumors were
similar (Figure 3B and Table 2). At day 5 of docetaxel treatment,
the mean R2* values of the BCSC Doc and FTH-BCSC Doc
tumors were significantly decreased compared with the BCSC and
FTH-BCSC tumors (P,0.05; Figure 3C and Table 2). At day 14
of docetaxel treatment, the BCSC Doc and FTH-BCSC Doc
tumors exhibited lower mean R2* values compared with the
BCSC and FTH-BCSC tumors (P,0.01; Figure 3D and Table 2).
Notably, histogram analysis revealed that only the FTH-BCSC
Doc tumors at day 14 of docetaxel treatment had distinct
populations of pixels with high R2* values and a distribution of
Figure 2. MRI images of phantom cells and xenograft tumors. (A) In vitro MRI images (left) and color-coded maps (right) of agarose phantoms
of BCSCs and FTH-BCSCs treated with or without 25 mM FAC. (B) R2* values measured from MRI images of BCSC and FTH-BCSC phantoms. *P,0.05.
(C) In vivo MRI images (left) and color-coded maps (right) of BCSC and FTH-BCSC xenograft tumors in the mammary fat pads of NOD/SCID mice. (D)
The distributions of the R2* values obtained for the BCSC and FTH-BCSC tumors at 3 weeks post-transplantation.
R2* values that was skewed compared with the other groups
(Figure 3D). In the FTH-BCSC Doc tumors, pixels that
represented a distinct population and were measured over the
threshold value (mean +3SDs; Table S1) occupied approximately
50% of the total pixels, while the percentages of all the other
groups were less than 30% (P,0.05, Figure 3E).
Histological Analysis Reveals that the Cell Populations
with High R2* Values are Localized in Viable Areas of the
Next, we performed histological analysis to investigate whether
ferritin-based MRI images reflects the tissue state after docetaxel
treatment. MRI showed that the R2* value pixels were similarly
distributed between the periphery and center of both BCSC and
FTH-BCSC tumors and H&E staining revealed that most of the
cells in the periphery and center of the BCSC and FTH-BCSC
tumors were viable (Figure 4A and C). In contrast, MRI showed
that the R2* value pixels were differently distributed between the
periphery and center of BCSC Doc and FTH-BCSC Doc tumors
and H&E staining revealed that the center of BCSC Doc tumors
with low R2* values had both viable and nonviable cell
populations (Figure 4B, right), whereas the center of the
FTHBCSC Doc tumors with mixed high and low R2* values matched
those of the viable and nonviable cells within the tumors,
respectively (Figure 4D, right). In addition, the periphery of
BCSC Doc and FTH-BCSC Doc tumors with high R2* values had
viable cells whereas viable cells were also found in the periphery of
the BCSC Doc tumors with low R2* values (Figure 4B, left). The
presence of viable cells in the FTH-BCSC and FTH-BCSC Doc
tumors was confirmed with EGFP fluorescence (Figure S6).
Additionally, we investigated the co-expression of myc-FTH and
EGFP in the FTH-BCSC and FTH-BCSC Doc tumors and
confirmed that the EGFP-positive cells had the myc-FTH
expression (Figure 4E).
To clarify whether ferritin overexpression alters the therapeutic
response to docetaxel, cell viability was evaluated by
mitochondrial membrane potential analysis and MTT assay after treatment
with docetaxel. Docetaxel induced a depolarization of the
mitochondrial membrane potentials and reduced the intact
mitochondria in both BCSCs and FTH-BCSCs (Figure S7A). A
decrease in cell viability in the FTH-BCSCs and BCSCs were
observed in a docetaxel dose-dependent manner using MTT assay
Mean R2* value 6SD (sec21)
(Figure S7B). There was no significant difference in the change of
mitochondrial membrane potential and cell viability between
FTH-BCSCs and BCSCs after treatment with docetaxel. Thus, we
conclude that the ferritin overexpression did not affect the BCSCs
response to docetaxel treatment.
The Viable Cell Populations within the Tumors Exhibit the
BCSC Phenotype and High Levels of Proliferative Markers
To investigate the effects of docetaxel treatment on FTH-BCSC
tumors, double staining for CD44 and CD24 was performed on
FTH-BCSC and FTH-BCSC Doc tumors. Because the cells
located in the periphery and center of the tumor responded
differently to docetaxel, CD44 and CD24 expression was analyzed
in both regions (Figure 5A). The periphery of the FTH-BCSC Doc
tumors contained significantly higher proportions of CD44+/
CD242 BCSCs compared to the periphery and center of the
FTH-BCSC tumors (FTH-BCSC periphery and center:
58.4662.07% and 44.3564.82%; FTH-BCSC Doc periphery
and center: 71.3863.75% and 56.1365.92%, P,0.05) (Figure 5B).
CD44+/CD24+ and CD442/CD242 cells were detected in the
periphery and center of FTH-BCSC and FTH-BCSC Doc
tumors. However, the proportion of these cells was not
significantly different between FTH-BCSC and FTH-BCSC DOC
We investigated the cell proliferation marker PH3 to determine
whether docetaxel alters tumor cell proliferation. The percentage
of cells expressing PH3 in the EGFP-positive cell population was
analyzed in the periphery and center of FTH-BCSC and
FTHBCSC Doc tumors. Significantly higher percentage of
PH3positive cells were observed in the periphery and center of the
FTH-BCSC Doc tumors (FTH-BCSC periphery and center:
0.9760.04% and 0.8360.19%, FTH-BCSC Doc periphery and
center: 11.4860.90% and 9.3261.38%, P,0.01) (Figure 5C and
The results of our study suggest that ferritin-based MRI can be
used as a noninvasive method to identify viable cell populations in
tumors after chemotherapy. In the present study, BCSCs
transduced with FTH and EGFP dual reporter genes were transplanted
into NOD/SCID mice to noninvasively track BCSC-derived
populations during tumor growth and monitor tumor responses
after chemotherapy. MRI showed distinct populations of pixels
with high R2* values in the docetaxel-treated FTH-BCSC tumors,
which correspond to EGFP+ viable cell populations with a high
percentage of CD44+/CD242 cells, as observed by histology. We
confirmed that lentiviral transduction of the reporter genes did not
alter the characteristics of BSCSs, as was revealed by the cell
surface marker analysis, proliferation assays, and in vivo tumor
growth. To the best of our knowledge, this study is the first to track
and image CSCs isolated from human tumor specimens and to
show viable cell populations of tumors after chemotherapy in
living mice with MRI reporter genes. We believe that our
experimental model system can be used to identify the most
effective treatments for tumors derived from BCSCs and to
develop new therapeutic strategies to target both BCSCs and
nonBCSCs to achieve durable remission [8,9,18,32].
In general, accumulation of iron in the cancer cells may have
adverse effects on the host since iron is pivotal nutrient for
proliferation and growth in normal cells as well as cancer cells.
Some studies reported high iron concentration promoted the
cancer cell proliferation by overexpressing transferrin receptor that
could elevate the level of reactive soluble iron in cells [33,34].
NOTE: Values in bold are statistically significant (*, P,0.05 and **, P,0.01).
Figure 3. Volumetric MRI images and distribution of R2* values in control (BCSC and FTH-BCSC) and docetaxel-treated (BCSC Doc
and FTH-BCSC Doc) xenograft tumors. (A) Follow-up volumetric MRI images of BCSC, BCSC Doc, FTH-BCSC and FTH-BCSC Doc tumors. (B-D)
Distribution of R2* values in BCSC, FTH-BCSC, BCSC Doc and FTH-BCSC Doc tumors at day 0, 5 and 14. The orange box indicates the pixels over the
threshold (mean +3 SDs) in the FTH-BCSC Doc tumors. E, analysis of the percentage of pixels over the threshold (mean +3SDs) in each tumor group.
However, the study reported by Cohen et al.  and our study
demonstrated iron accumulation by ferritin overexpression did not
alter breast cancer cell proliferation and viability in vitro .
These results suggest that ferritin overexpression can detoxify the
reactive free iron not to affect the proliferation and growth of
cancer cell, even the increase in net intracellular iron amount by
inducing iron uptake.
A notable finding in this MRI study was the distinctive R2*
value distribution found in FTH-BCSC tumors after
chemotherapy. After 14 days of docetaxel treatment, a population of pixels
with high R2* values appeared in the FTH-BCSC tumors while
the mean R2* values of tumors were significantly decreased. In
contrast, untreated BCSC, FTH-BCSC and docetaxel-treated
BCSC tumors did not show this distinctive R2* value distribution.
All R2* values for the control tumors showed a normal
distribution. Pixels that represented a distinct population and measured
over the threshold value (mean +3SDs) of the docetaxel-treated
FTH-BCSC tumors occupied approximately 50% of the total
pixels, while the percentages for control tumors were under 30%.
These findings support the result that cell populations with
different R2* value distributions were distinguished by FTH
overexpression. Finally, tissue analysis confirmed that the viable
cells in the FTH-BCSC tumors after chemotherapy were only
located in regions with high R2* values, while the viable cells in the
BCSC tumors were in both regions with low and high R2* values.
Together, these findings imply that MRI analysis using a ferritin
reporter can identify and quantify the existence of cells that survive
after chemotherapy even before the tumor changes in size.
To monitor the BCSC tumor response to chemotherapy, our
bimodal imaging approach based on FTH-EGFP dual reporter
the periphery and the center of each tumor. The white dotted lines on the MRI images indicate the boundary where the R2* values were differed in
the BCSC Doc and FTH-BCSC DOC tumors. The black dotted lines on H&E staining indicate the demarcation between viable and nonviable cells area
in the central portions of the BCSC Doc and FTH-BCSC Doc tumors (x100; bar in H&E, 100 mm, bar in MRI, 500 mm). MRI images before magnification
and cropping are in the Supplement (Figure S5). (E) Immunostaining of EGFP and myc-FTH in FTH-BCSC and FTH-BCSC Doc tumors. The white dotted
line indicates the demarcation between the areas of viable and nonviable cells in the central portion of FTH-BCSC Doc tumors (x400; bar, 50 mm).
genes has advantages over optical imaging or MRI alone because
areas of viable cells in tumors can be localized with 3D volumetric
analysis of MRI data while the use of EGFP as a reporter gene
enables the in vitro identification and molecular analysis of these
Figure 5. Immunohistochemical analysis of the peripheral and central portions of control (FTH-BCSC) and docetaxel-treated
(FTHBCSC Doc) xenograft tumors. (A) Double staining for CD44 and CD24 expression performed in FTH-BCSC and FTH-BCSC Doc tumors (x400; bar,
100 mm). (B) The percentage of CD44+/CD242, CD44+/CD24+, CD442/CD24+, CD442/CD242 and EGFP-positive cells in the periphery and center of
the FTH-BCSC and FTH-BCSC Doc tumors. *P,0.05. (C) Cell proliferation marker (phospho-histone 3, PH3) expression in the FTH-BCSC and FTH-BCSC
Doc tumors (x200; bar, 100 mm). (D) The percentage of PH3-positive/EGFP-positive cells in the periphery and center of the FTH-BCSC and FTH-BCSC
Doc tumors. **P,0.01.
viable cells [25,27,35,36]. Tissue samples from tumors could be
obtained without sacrifice of animals by using an MRI-guided
biopsy system . Furthermore, quantitative analyses of the MRI
data enabled the evaluation of anti-tumor effects in different areas
within the tumor, which is difficult with other in vivo methods. In
this study, a higher percentage of cells with the CD44+/CD242
phenotype was found in the remaining viable cells in the
docetaxel-treated FTH-BCSC tumors than in the untreated
FTH-BCSC tumors. This result demonstrates that more cells
with the BCSC phenotype are present in docetaxel-treated tumors,
which is consistent with results of previous studies . In the future
clinical trials, the efficacy of CSC-targeting therapies is likely be
monitored in vivo with MRI and SPIO nanoparticle-labeling of
anti-CSC markers while our imaging approach with bimodal
reporter genes is more suitable for studying dynamic processes or
tumor cells to stroma interaction in preclinical animal tumor
In conclusion, our results show that ferritin-based MRI, which
offers high spatial resolution and tissue contrast, can effectively
identify and localize remaining viable cell populations derived
from BCSCs after chemotherapy and may represent a novel tool
to monitor the efficacy of CSC-targeting therapies in vivo. The
experimental model system used in this study could be easily
applied to other cancer types, such as prostate, colon, pancreas,
liver and brain tumors.
Figure S1 Biological properties of the human BCSCs
and FTH-BCSCs. (A) The abilities to form mammospheres did
not differ between BCSCs and FTH-BCSCs. (B) A BCSC marker
(CD49fhigh), mesenchymal lineage markers (CD90high), and
a hematopoietic stem cell marker (CD342) were analyzed by
flow cytometry in BCSCs. (C) CD marker expression levels of
FTH-BCSCs were similar to those of BCSCs (CD49fhigh,
CD90high and CD342).
Figure S2 Comparison of tumor-forming abilities and
fluorescence imaging of BCSCs and FTH-BCSC-derived
tumors. (A) Tumor-forming abilities were similar for BCSCs and
FTH-BCSCs, and in vivo live imaging confirmed that only the
FTH-BCSC tumors expressed EGFP fluorescence. The red dotted
circle indicates the BCSC tumor and the yellow dotted circle
indicates the FTH-BCSC tumor. (B) Ex vivo EGFP fluorescence of
image excised tumors derived from BCSCs and FTH-BCSCs. The
sizes of the BCSCs and FTH-BCSC tumors were similar.
Figure S3 Cell growth analysis and the 7-AAD assay
with iron supplementation. (A) There was no significant
difference in the growth rates of the BCSCs and FTH-BCSCs in
the presence of an iron supplement (FAC). (B) The 7-AAD assay
revealed that the viabilities of the BCSCs and FTH-BCSCs in the
presence of the iron supplement were not significantly different.
Figure S4 Growth rates of BCSC, FTH-BCSC, BCSC Doc
and FTH-BCSC Doc tumors. BCSCs and FTH-BCSCs
(16106) were engrafted into the mammary fat pads of NOD/
SCID mice. Mice (n = 5 per group) were treated with i.v. injections
of docetaxel (15 mg/kg) at three-day intervals beginning the day
after the pre-treatment MRI. Tumor growth was attenuated in
BCSC Doc and FTH-BCSC Doc tumors 5 days after docetaxel
treatment, and FTH overexpression did not affect the tumor
growth rate in either the docetaxel-untreated or treated groups
(BCSC vs. FTH-BCSC tumors, BCSC Doc vs. FTH-BCSC Doc
tumors). The bars in the graph represent SDs.
Figure S6 Immunohistochemistry analysis of FTH in
FTH-BCSC and FTH-BCSC Doc tumors. H&E staining
images and EGFP fluorescence images were analyzed in
FTHBCSC and FTH-BCSC Doc tumors. Viable portions of H&E
staining and EGFP expressing cells that constituted the
FTHBCSC tumors and the viable portion of the FTH-BCSC Doc
tumors were well matched. Magnification: H&E, 406; and
fluorescence, 406. Scale bars: 200 mm.
Figure S7 In vitro cytotoxicity test in BCSCs and
FTHBCSCs with docetaxel treatment. (A) The percentages of
JC1 aggregates and cell viabilities after the docetaxel treatments were
evaluated by the calculation of JC1 aggregates numbers on the
fluorescence microscope images. (B) MTT assay was performed to
evaluate the toxicity of docetaxel in BCSCs and FTH-BCSCs.
Determination of R2* Threshold Values.
Conceived and designed the experiments: YC HSK KWC WKM.
Performed the experiments: YC KML JW YJY. Analyzed the data: YC
KWC SJE TKW SHC HJK. Contributed reagents/materials/analysis
tools: CSC DYN. Wrote the paper: YC HSK WKM.
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