Journal of Controlled Release 264 (2017) 127–135

Contents lists available at ScienceDirect

Journal of Controlled Release

journal homepage: www.elsevier.com/locate/jconrel

Effective treatment of drug resistant recurrent breast tumors harboring MARK

cancer stem-like cells by staurosporine/epirubicin co-loaded polymeric
micelles
Juanjuan Zhanga, Hiroaki Kinohb, Louise Hespela, Xueying Liub, Sabina Quaderb, John Martina,
Tsukasa Chidaa, Horacio Cabrala,⁎⁎, Kazunori Kataokab,c,⁎
a
Department of Bioengineering, Graduate School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan
b
Innovation Center of NanoMedicine, Kawasaki Institute of Industrial Promotion, 3-25-14, Tonomachi, Kawasaki-ku, Kawasaki 210-0821, Japan
c
Policy Alternatives Research Institute, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan

A R T I C L E I N F O A B S T R A C T

Keywords: Breast cancer recurrence and resistance are associated with cancer stem-like cell (CSC) sub-populations. As
Breast cancer conventional therapies fail to treat CSCs, institution of novel therapeutic strategies capable of eradicating both
Polymeric micelles cancer cells and CSCs is central for achieving effective treatments with long-term survival. Here, we studied the
Drug-resistance ability of polymeric micelles cooperatively loading the cytotoxic drug epirubicin (Epi) and the CSC inhibitor
Cancer stem-like cells
staurosporine (STS) to treat breast tumors, particularly when tumors relapsed after chemotherapy. The STS/Epi-
Epirubicin
Staurosporine
loaded micelles (STS/Epi/m) demonstrated potent therapeutic efficacy against both naïve orthotopic 4T1-luc
breast tumors and their recurrent Epi-resistant counterparts, significantly prolonging survival. This efficacy
enhancement of STS/Epi/m was correlated with the ability of the micelles to suppress the CSC-associated sub-
populations of breast cancer, i.e. the aldehyde dehydrogenase-positive (ALDH+) population and the CD44+/
CD24− fraction, in Epi-resistant cells and tumors. These results demonstrated STS/Epi/m as a promising strategy
for effective management of breast cancer.

1. Introduction avoid recurrence and increase patient survival.

Breast cancer is the predominant cancer in women with 25% of new
cases and 15% of deaths from all cancers [1]. Breast tumors are treated
with various therapeutic strategies, including surgery, radiotherapy,
immunotherapy and chemotherapy [2]. Particularly, treatment with
epirubicin (Epi) offers considerable advantages for managing breast
cancer, including its spectrum of activity, dosage, toxicity profile and
the possibility to be combined with taxanes and/or trastuzumab for
increasing efficacy [3,4]. However, despite showing good therapeutic
responses, breast cancer patients present high relapse rate [5–7]. Such
breast cancer recurrence has been associated with the presence of a
subpopulation of cancer cells, so called cancer stem-like cells (CSCs),
showing self-renewal ability, high proliferation rate and the capacity to
produce heterogeneous cancer cells [8,9]. CSCs are also resistant to
conventional therapies due to their enhanced detoxification mechan-
isms, including increased membrane transport, DNA repair and ROS
scavenging systems [10,11]. Therefore, novel therapeutic approaches
capable of eradicating both breast cancer cells and CSCs are needed to
Nanomedicines have potential for enhancing therapeutic outcomes
by concurrently treating both cancer cells and CSCs [12-15]. Among
clinically translationable nanomedicines, polymeric micelles have
shown unique properties for delivering drugs to solid tumors, including
their stable and high drug loading, controlled drug release, relative
small size, and high tumor accumulation [16,17]. Moreover, due to the
core-shell compartmentalized structure of polymeric micelles, it is
possible to cooperatively load various therapeutic agents aimed to exert
synergistic efficacies. Thus, we have developed micellar nanomedicines
capable of attaining coordinated effects of CSCs inhibitors and cytotoxic
drugs within tumor cells to achieve synergistic activity against cancer
cells and CSCs [18]. These nanomedicines profit from the synergistic
anticancer effects of Epi and staurosporine (STS), a broadly multi-ki-
nase inhibitor with high potency against CSCs [5], by co-incorporating
STS in the core of our pH-sensitive Epi-loaded polymeric micelles (Epi/
m) through manipulation of the interaction between STS and the
polymer-conjugated Epi molecules [18]. The Epi/m used as platform
have demonstrated high accumulation in tumor tissues, selective

⁎
Correspondence to: K. Kataoka, Innovation Center of NanoMedicine, Kawasaki Institute of Industrial Promotion, 3-25-14, Tonomachi, Kawasaki-ku, Kawasaki 210-0821, Japan.
⁎⁎
Corresponding author.
E-mail addresses: horacio@bmw.t.u-tokyo.ac.jp (H. Cabral), kataoka@bmw.t.u-tokyo.ac.jp (K. Kataoka).

http://dx.doi.org/10.1016/j.jconrel.2017.08.025
Received 17 July 2017; Received in revised form 21 August 2017; Accepted 22 August 2017
Available online 24 August 2017
0168-3659/ © 2017 Published by Elsevier B.V.
J. Zhang et al.
Fig. 1. Scheme of staurosporine/epirubicin-loaded micelles (STS/Epi/m). The micelles are assembled after mixing STS and Epi-conjugated block copolymers in DMF, which is then
evaporated and replaced with HEPES buffer. The resulting self-assembled polymeric micelles are 50 nm in diameter.
intratumoral drug activation and potent antitumor effects [19,20],
leading to their progress into Phase I/II clinical trials [17,21]. By co-
operatively delivering both Epi and STS inside cancer cells, the STS/
Epi-loaded micelles (STS/Epi/m; Fig. 1) facilitate the elimination of
cancer cells and CSCs through endosomal pH-triggered drug release and
reversal of drug resistance mechanisms [18]. These STS/Epi/m de-
monstrated enhanced antitumor activity in a model of recalcitrant
mesothelioma, treating both cancer cells and CSCs, which resulted in
tumor eradication [18]. In this way, STS/Epi/m appear as an attractive
strategy for dealing with drug resistant tumors after relapse.
Herein, we studied the capability of STS/Epi/m for treating breast
tumors, particularly after developing resistance to free Epi in vivo. The
loading of STS in the micelles was tuned to maximize the STS/Epi in-
corporation ratio, while preserving the original diameter of Epi/m. By
evaluating the ability of STS/Epi/m to treat both naïve and Epi-pre-
treated orthotopic breast tumors, we demonstrated the effective sup-
pression of tumor growth of STS/Epi/m through enhanced efficacy
against the CSC fraction.
2. Materials and methods
2.1. Materials
STS was purchased from Funakoshi Co. (Tokyo, Japan). Epi and
poly(ethylene glycol)-b-poly(aspartate-hydrazide-epirubicin) copo-
lymer were obtained from Nanocarrier Co. (Tsukuba, Japan). N, N-
Dimethylformamide (DMF), methanol (MeOH), dimethyl sulfoxide
(DMSO), penicillin-streptomycin, Dulbecco's phosphate buffered saline
(D-PBS), hydrochloric acid (HCl) and other common use chemicals
were purchased from Wako Pure Chemicals Industries, Ltd. (Tokyo,
Japan). Amicon stirred cells, ultrafilter tubes and dialysis membrane
(molecular weight cut-off size (MWCO): 30,000), Syringe Filters
(Polyethersulfone (PES), Sterile, 0.22 μm) were purchased from
Millipore Co. (Massachusetts, USA). Cell culture medium and 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was
purchased from Sigma-Aldrich (Sigma-St. Louis, USA). Fetal bovine
serum (FBS) was bought from Invitrogen (Carlsbad, CA). Anti-CD44
antibody (ab112178), Anti-CD24 antibody (ab202963), Donkey Anti-
Rabbit IgG H & L (Alexa Fluor 488) (ab150073) and Goat Anti-Rat IgG
H & L (Alexa Fluor 594) (ab150160) were purchased from Abcam
(Cambridge, UK). Blocking One Buffer was purchased from Nakalai
Tesque Co., Ltd. (Tokyo, Japan). ALDEFLUOR Kit and ALDEFLUOR
DEAB reagent were purchased from STEMCELL Technologies Inc.
(Vancouver, Canada).
2.2. Cell lines
Murine breast adenocarnicoma 4T1 cells expressing luciferase (4T1-
128
Journal of Controlled Release 264 (2017) 127–135
luc) were purchased from Japanese Collection of Research Bioresources
Cell Bank (Osaka, Japan). The cells were cultured in Dulbecco's
Modified Eagle Medium (DMEM) plus 10% FBS and 1% streptomycin/
penicillin, and maintained at 37 °C and 5% CO2.
2.3. Animals
Balb-c mice (female; 6 week-old) were purchased from Charles
River Co. (Tokyo, Japan). All the experiments were conducted under
the ethical guidelines of The University of Tokyo.
2.4. Preparation of STS/Epi/m
pH-sensitive STS/Epi/m having different loading of STS were pre-
pared by mixing PEG-b-poly(aspartate-hydrazide-epirubicin) copo-
lymer (Mw of PEG = 12,000 Da; poly(aspartate) units = 40; Epi
units = 8) at 1 mg/ml on an Epi-basis with STS at different drug weight
ratios of STS/Epi (0.0625:1, 0.2:1, 0.3:1, 0.5:1 and 1.25:1) in DMF, and
stirred for 30 min at room temperature in dark. Then, DMF was eva-
porated using a rotatory evaporator to form a thin film on the surface of
the flask, followed by addition of 10 mL of HEPES buffer (10 mM,
pH 7.4) into the flask containing the dried sample. The mixture was
sonicated for 30 min. The resulting micelles were then purified by ul-
trafiltration (MWCO = 30,000). Finally, the micelles were filtered by
using a PES filter (0.22 μm). The size of the micelles was measured by
dynamic light scattering (DLS) by using a Zetasizer Nano ZS (Malvern,
UK).
2.5. Drug loading of STS/Epi/m
pH-sensitive STS/Epi/m were disrupted by incubation in 1 mol/L
HCl for 1 h at 37 °C to release both STS and Epi. The concentration of
the drugs inside micelles was determined by HPLC (Column: Tosoh with
TSK gel 80-TM; injection volume: 10 μL; mobile phase; 40% 1 mM
formic acid (pH = 3) and 60% HPLC grade methanol; flow rate:
0.8 mL/min; temperature: 40 °C). STS and Epi were detected by UV
absorption at 290 nm and 254 nm, respectively. The concentration of
STS was interpolated from a standard curve, and the concentration of
Epi was calculated according to area of Epi standard solution de-
termined each time.
2.6. In vitro cytotoxicity study against 4T1-luc cells
The cytotoxic effects of the drugs, including free Epi, Epi/m, free
STS plus free Epi, free STS plus Epi/m and STS/Epi/m, on 4T1-luc cells
was evaluated, as follows: 4T1-luc cells were seeded in 96-well plates in
DMEM medium with 10% fetal bovine serum at 37 °C. Twenty-four
hours later, the drugs were added and the cells were incubated for
J. Zhang et al.
Table 1
Size of STS/Epi/m prepared with different STS/Epi feeding ratio. The concentration of
Epi-conjugated block copolymer was fixed at 1 mg/ml on an Epi-basis.
STS/Epi feeding ratio STS/Epi loading ratio in the Diameter (nm)b
in weight micelles in weighta
0.06 0.06 48
0.2 0.2 53
0.3 0.3 77
0.5 0.4 78
1.25 1 119
a
Determined by HPLC.
b
Determined by DLS.
another 48 h. The cell viability was evaluated by using 3-(4,5-di-
methylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and the
detection of formazan at 570 nm.
2.7. Assessment of Epi concentration in plasma and tumor tissues
To study the concentration of the micelles in plasma, Balb-c mice
(n = 3) were intravenously injected with Epi/m and STS/Epi/m (Epi:
10 mg/kg), and the blood was collected at 1, 4, 8 and 24 h. Then, the
blood samples were centrifuged to obtain the plasma. To determine the
drug concentration in tumors, mice bearing orthotopic 4T1-luc tumors
with 6 mm diameter were randomly divided into 4 groups (n = 3), and
intravenously injected with free Epi, free STS, Epi/m and STS/Epi/m
(Epi: 10 mg/kg), after 24 h, tumor tissues were collected and homo-
genized with PBS (pH 7.4). The plasma samples and tumor tissue
homogenates were treated with 1 N HCl to cleave the pH-sensitive
bonds and release the Epi molecules conjugated to the block copoly-
mers. Then, Epi was extracted from the samples, and the concentration
was measured by HPLC as described in our previous report [19].
2.8. Antitumor activity evaluation against orthotopic 4T1-luc tumors
Syngeneic orthotopic breast tumors were prepared by inoculating
4T1-luc cells (1 × 106 cells suspended in 100 μl PBS) into the mam-
mary fat pad of Balb-c mice (female, 18–20 g; 6 week old). Tumors were
allowed to grow until their diameter reached 6 mm. Then, mice were
randomly divided into 5 groups (n = 8). Mice were injected in-
travenously 3 times with free Epi, Epi/m, free STS plus free Epi, and
STS/Epi/m every 4 days, i.e. on days 0, 4 and 8. The dose of the drugs
was fixed at 6 mg/kg based on Epi and 1.2 mg/kg based on STS. The
tumor volume was measured every 2 days and calculated based on the
following equation: V = (L × W2)/2, where L is the longest dimension
and W is the shortest dimension. The body weight was measured to
evaluate the systemic toxicities.
2.9. Antitumor activity evaluation against Epi-pretreated orthotopic 4T1-luc
tumors
Mice bearing orthotopic 4T1-luc tumors with 6 mm diameter were
treated with free Epi (6 mg/kg) by intravenous injections 3 times on
days 0, 4 and 8. After the tumor growth relapsed 8 days after the last
injection of Epi, the mice were randomly divided into 5 groups (n = 7)
and were treated intravenously 3 times with free Epi, Epi/m, free STS
plus free Epi, and STS/Epi/m every 4 days, i.e. on days 16, 20 and 24.
Table 2
Fifty-percent inhibitory concentration of free Epi, free Epi plus free STS, Epi/m and STS/Epi/m against 4T1-luc cells based of Epi and STS concentrations. STS concentration was fixed at
the ratio of 5:1 of Epi: STS. Data are expressed as the mean ± S.D. (n = 4).
Epi
IC50 (ng/ml) Based on Epi 60 ± 7
Based on STS − −
Journal of Controlled Release 264 (2017) 127–135
The drug dosage was fixed at 6 mg/kg based on Epi and 1.2 mg/kg
based on STS. Tumor volume and body weight were measured every
2 days to evaluate the antitumor activities and systematic toxicities of
PDI various formulations.
2.10. In vitro cytotoxicity against cells from naïve tumors and Epi-treated
0.1
0.1
tumors
0.2
0.2 Cells of naïve tumors or Epi-treated tumors were collected from the
0.2 mice of above described models, i.e. with and without Epi pretreatment.
The tumor tissues were dissociated after 1 h incubation with 0.05%
collagenase at 37 °C and by pipetting the tumor debris several times.
Thereafter, filtered cell suspensions were prepared by using cell strai-
ners (40 μm) and washed three times by repeated centrifugation in PBS.
Cells were transferred into a flask with DMEM medium plus 10% FBS
and 1% streptomycin/penicillin. The cells were incubated at 37 °C for
1 week, and their proliferation rate was confirmed to be comparable to
4T1-luc cells. Then, the cells from naïve tumors and Epi-treated tumors
were seeded into 96-well plates (5000 cells/well). Twenty-four hours
later, 100 μL medium containing free Epi, Epi/m, free STS plus free Epi,
and STS/Epi/m were added. After 48 h incubation, the cytotoxicity was
measured as described previously.
2.11. Immunofluorescence staining of tumor tissues
Tissue samples from the orthotopic 4T1-luc tumor model were
collected and fixed in paraformaldehyde for 1 h. Then, the samples
were transferred to 30% sucrose solution and incubated overnight at
4 °C. To make frozen blocks of tumor tissues, the samples were dried,
put into molds filled with Tissue Tek O.C.T. compound, and freezed.
The frozen tissue blocks were stored at − 80 °C until cryosectioning. For
immunofluorescence studies, tumor tissue sections were marked with
anti-CD44 and anti-CD24 antibodies, and subsequently stained with
secondary antibodies conjugating Alexa594 or Alexa488, respectively.
Immunofluorescent images were obtained by confocal laser scanning
microscopy with a Zeiss LSM780 Meta confocal microscope (Carl Zeiss,
Germany).
2.12. Flow cytometry analysis of CD44+/CD24− subpopulation
To identify the CD44 +/CD24 − cancer cell population, flow cyto-
metry analysis was performed. Cells collected from naïve 4T1-luc tu-
mors and Epi-pretreated 4T1-luc tumors, as well as in vitro cultured
4T1-luc cells were used. Single cells at a concentration of 1 × 106 cells/
ml were prepared, and incubated with FITC-conjugated anti-CD44 and
PE-conjugated anti-CD24 antibodies, and mouse IgG isotype (all from
BD Bioscience, San Diego, CA) for 30 min on ice. After staining, the cells
were washed by centrifugation at 500g for 5 min, and resuspended in
flow cytometry analysis buffer. The cells were analyzed on a BD FACS
ARIA. Sample analysis was performed using BD FACS Diva software.
The experiment was done in triplicate and the statistical significance
was evaluated by Student's t-test.
2.13. Aldehyde dehydrogenase (ALDH) activity assay
Cells of Epi-treated 4T1-luc tumor were collected as described
above. The cells were seeded in six-well plates and incubated for 48 h
Epi/m Epi + STS Epi/m + STS STS/Epi/m
2400 ± 300 10 ± 1 10 ± 1 2 ± 0.1
2 ± 0.2 2 ± 0.2 0.4 ± 0.02
129
J. Zhang et al.
A 70 B intravenous injection. B. Tumor accumulation of free Epi,
STS/Epi/m
60 Epi/m
% dose/ml of plasma
%dose/gram tissue
50
40
30
20
10
0 0
0 4 8 12 16 20 24
Time (h)
with free Epi, free Epi plus free STS, EPI/m, and STS/Epi/m at the 90%
inhibitory concentration of each drug. The cells were then collected and
the subpopulation with high aldehyde dehydrogenase (ALDH) activity
was detected by using the Aldefluor kit (StemCell Technologies,
Durham, NC, USA). Accordingly, cells were suspended in Aldefluor
assay buffer containing BODIPY-aminoacetaldehyde and an aliquot of
each sample was treated with 50 mM diethylaminobenzaldehyde
(DEAB) as a negative control. The samples were then incubated for
30 min at 37 °C. The sorting of the ALDH+ cell subpopulations was
done by using a MoFlo Astrios FACS instrument (Beckman Coulter,
Brea, CA, USA) after setting the sorting gates with the negative controls.
3. Results and discussion
3.1. Preparation of STS/Epi-loaded micelles
The interaction between STS and Epi allows co-loading both drugs
in the core structure of Epi/m [18]. In our previous report, the STS/Epi/
m were prepared from PEG-b-poly(aspartate) copolymers, on which Epi
molecules have been conjugated through hydrazone links for controlled
release of both Epi and STS at endosomal pH (Fig. 1) [18], by fixing the
mixing ratio of STS to Epi at 1:5 in weight. Here, we varied the feeding
ratio of STS to determine the maximum drug loading without affecting
the size distribution compared to the original Epi/m. Thus, the con-
centration of Epi-containing block copolymers was fixed at 1 mg/ml on
an Epi-basis and STS at different concentrations was mixed in DMF to
make STS/Epi weight ratios of 0.0625:1, 0.2:1, 0.3:1, 0.5:1 and 1.25:1
in weight. After evaporating DMF, 10 mM HEPES buffer (pH 7.4) was
added, and the resulting solutions were sonicated and ultrafiltered for
obtaining the STS/Epi/m. DLS results showed that the size of the mi-
celles increased with the loading of STS ranging from 45-nm for STS/
Epi = 0.06 to 119-nm for STS/Epi = 1 (Table 1). Moreover, the PDI of
the micelles increased at STS/Epi feeding ratio higher than 0.3
(Table 1). Note that the diameter of Epi/m is 50-nm, thereby, the
maximum loading of STS that can be achieved without affecting the
original size of Epi/m [20] is at an STS/Epi ratio of 0.2. Importantly,
the size of nanomedicines has been indicated to affect the accumulation
and penetration in tumor tissues [22–24]. In this regard, polymeric
micelles with sub-50-nm diameter have shown deep penetration in
poorly permeable tumors, such as pancreatic and stomach cancers,
which allowed them to reach distant cancer cells and exert improved
antitumor effects [22,24]. Thus, in the following biological studies, we
used the STS/Epi mixing ratio of 0.2 for preparing 50-nm STS/Epi/m
with narrow PDI (0.1).
3.2. In vitro cytotoxicity against 4T1-luc cells
We first evaluated the cytotoxicity of the drugs in 4T1-luc cells to
Journal of Controlled Release 264 (2017) 127–135
Fig. 2. A. Plasma clearance of Epi/m and STS/Epi/m after
8 Epi/m and STS/Epi/m 24 h after intravenous injection. Data
shown as average ± S.D. (n = 3). **p < 0.01 by Student's
**
t-test.
6
**
4
2
Epi Epi/m STS/Epi/m
confirm their activity. Thus, the cells were exposed to free Epi, free Epi
plus free STS, Epi/m plus free STS, and STS/Epi/m for 48 h. The 50%
inhibitory concentration (IC50) of free Epi was found to be approxi-
mately 40-fold lower than that Epi/m (Table 2). This difference in the
cytotoxicity between free Epi and Epi/m can be associated with the
different cell uptake routes, i.e. diffusion through the cellular mem-
brane for free Epi and endocytosis for the micelles, as well as with the
gradual drug release from the micelles at endosomal conditions to at-
tain active Epi molecules. The combination of free Epi plus STS was
more cytotoxic than the Epi formulations alone, showing IC50 values of
around 0.01 μg/ml on an Epi-basis (Table 2). These results are in
agreement with previous reports showing the high potency of STS
against cancer cells [25], as well as our observations indicating the
synergistic efficacy of Epi with STS [18]. Importantly, STS/Epi/m
presented the highest activity against 4T1-luc cells, with an IC50 value
5-fold lower than free Epi plus STS or Epi/m plus free STS (Table 2). In
addition, the higher efficacy of STS/Epi/m over free Epi plus STS or
Epi/m plus free STS could be related to the protection of STS within the
core of the micelles at the extracellular space, and the co-delivery of
both STS and Epi for cooperative actions once inside the cancer cells
[18].
3.2.1. Plasma clearance and tumor accumulation
The blood circulation of the micelles was evaluated to determine if
the loading of STS in the core of the micelles affected their clearance
from the bloodstream. Thus, Epi/m and STS/Epi/m were intravenously
injected in mice at 10 mg/kg on an Epi basis, and the blood was sam-
pled at defined time points. The Epi concentrations in plasma showed
that both Epi/m and STS/Epi/m were long circulating (Fig. 2A), with
STS/Epi/m showing slightly higher plasma levels than Epi/m at 8 h
after administration. These results indicated that the co-loading of STS
in the core of Epi/m did not hamper the prolonged half-life of Epi/m.
The Epi concentration in 4T1-luc orthotopic tumors after in-
travenous administration of free Epi, Epi/m and STS/Epi/m was also
studied to ascertain the enhancement of the drug delivery to the tu-
mors. Accordingly, tumor-bearing mice were injected with the drugs,
and 24 h later the mice were sacrificed, the tumors were collected and
the drug concentrations in the tumors were determined by HPLC. The
results showed that both Epi/m and STS/Epi/m attained significantly
higher levels of Epi in the tumors than that reached after free Epi in-
jection (Fig. 2B). Moreover, while Epi/m delivered approximately 3%
of the injected dose per gram of tumor tissue, which corresponded with
the reported accumulation of Epi/m (NC-6300) in MDA-MB-231 tumors
[19], the STS/Epi/m showed higher accumulation than Epi/m, i.e.
around 5% of injected dose per gram of tumor tissue (Fig. 2B), which
could further promote the enhanced antitumor effects of STS/Epi/m.
The higher accumulation of STS/Epi/m than Epi/m in the tumors might
be associated with the higher availability of STS/Epi/m in the
130
J. Zhang et al. Journal of Controlled Release 264 (2017) 127–135

grow until their diameter reached 6 mm in diameter. Then, the mice

A were injected intravenously 3 times with free Epi, Epi/m, free STS plus
free Epi, and STS/Epi/m every 4 days, i.e. on days 0, 4 and 8. Thus,
STS/Epi/m were found to effectively inhibit the growth of the tumors,
whereas mice treated with free Epi, Epi/m, or the combination of free
Epi and free STS showed significantly lower suppression of the tumor
growth (Fig. 3A). Moreover, despite the repeated administration of
***
STS/Epi/m, mice did not show any body weight loss during the ex-
periment (Fig. 3B), indicating the safe profile of the micelles. On the
*** other hand, 4 mice treated with the combination of free Epi and free
STS died soon after the first injection (Fig. 3C), probably because of the
strong toxicity of the combination. Survival curves indicated that STS/
*** Epi/m significantly extended survival, with all mice living for > 2
months (Fig. 3C). This superior activity of STS/Epi/m could be related
to the effective eradication of both cancer cells and CSCs through co-
operative drug interactions [18]. Therefore, to confirm this efficacy of
STS/Epi/m against the CSC fraction, we next evaluated the effect of the
treatments on the CSC population within tumors.

B 3.4. Effect of drugs on the CD44+/CD24− population in orthotopic breast

tumors

The CD44+/CD24− phenotype has been used as a reliable pheno-

type of CSCs from breast cancer [26,27]. Thus, herein, we evaluated the
effect of the drugs on the CD44+/CD24− population by immuno-
fluorescence microscopy. Thus, mice bearing 4T1-luc tumors were in-
travenously injected with HEPES, Epi, Epi/m, STS plus Epi, and STS/
Epi/m at the same dosage used in the antitumor activity experiment
(Fig. 3), that is, 6 mg/kg for Epi and 1.2 mg/kg for STS. Forty eight-
hours after injection, the tumors were collected, cryo-sectioned and
stained for CD44 and CD24 for immunofluorescence analysis. The his-
tology results showed that the 4T1-luc tumors present discrete areas of
CD44+/CD24− cells (Fig. 4A; HEPES), which represent approximately
10% of the total tumor area, indicating the occurrence of CSC in these
tumors. After treatment with Epi or Epi/m, the levels CD44+/CD24− in
the tumors slightly increased (Fig. 4), which could be associated with
the enrichment of the CSC fraction after chemotherapy, as reported in
C the clinic [28]. The tumors treated with free STS plus Epi showed
100 CD44+/CD24− levels comparable to those of HEPES treated tumors
(Fig. 4). Conversely, STS/Epi/m significantly reduced the CD44+/
Percentage survival

HEPES CD24− fraction in 4T1-luc tumors (Fig. 4B), substantiating their en-
EPI
hanced activity against CSCs and their superior antitumor effect against
naïve tumors. Moreover, the tumors treated with STS/Epi/m showed
EPI/m
50 homogeneous eradication of the CD44+ subpopulation throughout the
EPI+STS
tissue section (Fig. 4A, red), which suggests the ability of the micelles to
STS/EPI/m reach CSCs distributed in the tumor mass. The enhanced activity of
STS/Epi/m on breast CSCs could be associated with the inhibitory ef-
fects of STS against protein kinase C α (PKCα) [29]. PKCα is associated
with the aggressive triple-negative breast cancers, and its inhibition has
0
0 20 40 60 80 been shown to suppress breast CSCs [30]. Moreover, the STS-induced
Time (day) apoptosis in breast cancer cells has been associated with the inhibition
of the JAK2/STAT3 pathway [31], which is necessary for the growth of
Fig. 3. Antitumor activity against naïve orthotopic 4T1-luc tumors. A. Tumor growth of CD44+/CD24− CSCs in human breast tumors [32]. Thus, these results
tumors treated with HEPES, Epi/m, STS/Epi/m, Epi, STS plus Epi. The concentration of
support our strategy of cooperatively delivering STS and Epi within a
Epi was fixed at 6 mg/kg and the dose of STS was 1.2 mg/kg. Arrowheads: injection
points. Data are expressed as the mean ± S.D. (n = 8). ***p < 0.001 by Student's t-test.
single micelle platform for allowing effective inhibition of both cancer
B. Relative Body weight changes during the antitumor experiment. Data are expressed as cells and the CSC fraction.
the mean ± S.D. (n = 8). C. Survival of mice during the experiment. From the histology results, it appears that the tumors treated with
Epi showed a higher fraction of CD44+/CD24− cells. Thus, to further
bloodstream (Fig. 2A). confirm such expansion of the CSC subpopulation after Epi treatment, in
vitro cultured 4T1-luc cells, cells from naïve tumors and cells from Epi
treated tumors were marked with FITC-conjugated anti-CD44 and PE-
3.3. In vivo antitumor activity against orthotopic breast tumors conjugated anti-CD24, and the extent of the CD44+/CD24− fraction
was determined by flow cytometry analysis. The results showed that the
The in vivo antitumor activity of the drugs was firstly evaluated CD44+/CD24− fraction is present in the in vitro cultured 4T1-luc cells,
against naïve 4T1-luc orthotopic tumors without any drug pretreatment being approximately 5% of the total cells. The cells from the naïve 4T1-
to determine the efficacy of the treatments. Tumors were allowed to luc tumors showed around 10% of CD44+/CD24− cells, which

131
J. Zhang et al.
A Epi-pretreated
4T1-luc cells Naïve 4T1-luc tumor cells
CD24-PE
5.2%
CD44-FITC
Fig. 5. Flow cytometry analysis of the CD44+/CD24− subpopulation in in vitro cultured 4T1-luc cells, cells from naïve 4T1-luc tumors and Epi-pretreated tumors. A. Representative flow
cytometer scatterplots. B. CD44+/CD24− fractions in the different groups. Data shown as average ± S.D. (n = 3). **p < 0.01 by Student's t-test.
correlated with our histological observations, while the several cycles
of Epi significantly increased the CSC subpopulation in the tumors, with
approximately 16% CD44+/CD24− cells (Fig. 5, p < 0.01 by Student's
t-test; n = 3). Such increase in the CD44+/CD24− fraction after Epi
treatment corresponds with the expansion of this subpopulation in
breast tumors in the clinic after treatment with anthracyclines [33].
3.5. In vivo antitumor activity against Epirubicin-pretreated orthotopic
breast tumors
After confirming the activity of STS/Epi/m against naïve breast
tumors and their ability to suppress the CD44+/CD24− phenotype, we
evaluated the efficacy of the drugs in Epi-pretreated tumors to assess
their ability for suppressing tumor relapse after conventional therapy.
To prepare the animal model, 4T1-luc cells were inoculated in the
Journal of Controlled Release 264 (2017) 127–135
Fig. 4. Immunofluorescent histological study of the CD44+
and CD24+ cells in the orthotopic 4T1-luc tumors after
drug treatment. A. Microdistribution of CD44+ cells (red)
and CD24+ (green) cells in tumor tissues 48 h after treat-
ment with HEPES, Epi, Epi/m, STS plus Epi, and STS/Epi/
m. The concentration of Epi was fixed at 6 mg/kg and the
dose of STS was 1.2 mg/kg. Scale bars are 2 mm. B.
Quantification of the CD44+/CD24− fraction per μm2 in
tumor tissue sections. Data are expressed as the
mean ± S.D. from 4 pictures obtained from 2 different
tumor sections. **p < 0.01 by Student's t-test. (For inter-
pretation of the references to colour in this figure legend,
the reader is referred to the web version of this article.)
20
B
4T1-luc tumor cells
**
CD44+/CD24- population
15
10
9.77% 15.9%
5
0
4T1-luc Naïve Epi-retreated
cells tumor cells tumor cells
mammary fat pad of Balb-c mice, and the tumors were allowed to grow
until their diameter reached 6 mm in diameter. Then, the mice were
injected intravenously 3 times with free Epi (6 mg/kg) every second
day. Sixteen-days later, when the tumor growth relapsed, mice were
treated intravenously 4 times with free Epi, Epi/m, free STS plus free
Epi, and STS/Epi/m every 4 days, and then with another intravenous
injection on day 20. Thus, while free Epi showed some efficacy against
naïve tumors, its effect was negligible against the recurrent tumors
(Fig. 6A). Moreover, the combination of free Epi and free STS could not
delay the growth of the tumors. Conversely, Epi/m achieved higher
efficacy than the free drugs, probably due to their ability to deliver
higher amounts of drugs to tumors via the enhanced permeability and
retention (EPR) effect [19]. Although the extent of EPR effect in hu-
mans is being debated, the ability of nanomedicines to target human
tumors through this route has been demonstrated for various
132
J. Zhang et al. Journal of Controlled Release 264 (2017) 127–135

A HEPES buffer
A 100
EPI
***

Concentration of EPI (µg/ml)

2000
EPI/m
STS/EPI/m 10
Tumor size (mm3)

STS+EPI **
1000
1

0.1
0
0 10 20 30 40
Time (day) 0.01
B
100 HEPES
EPI 0.001
Percent survival

EPI/m

S
I/m

/m
EP

ST
EPI+STS

I
EP

EP
I+
STS/EPI/m

S/
EP
50

ST
B 100
***

Concentration of EPI (µg/ml)

0
0 20 40 60 10
Time (day)

Fig. 6. Antitumor activity against orthotopic 4T1-luc tumors pretreated with Epirubicin.
A. Antitumor activity against Epi-pretreated 4T1-luc orthotopic tumors after intravenous
1
injection with HEPES buffer, Epi/m, STS/Epi/m, Epi, STS plus Epi. The concentration of
Epi was fixed at 6 mg/kg and the dose of STS was 1.2 mg/kg. Arrowheads: injection
points. Data are expressed as the mean ± S.D. (n = 8). **p < 0.001 by Student's t-test. 0.1
The tumor size was followed until 50% of animals were alive. B. Survival of mice during
the experiment.
0.01
nanomedicine platforms in a wide range of human tumors [34–36].
Moreover, a recent report has also demonstrated the incidence of EPR
effect in breast cancer metastases of human patients [36]. While the 0.001
I

S
I/m

I/m
enhanced activity of Epi/m could only extend the survival slightly
EP

ST
EP

EP
(Fig. 6B), STS/Epi/m effectively reduced the growth rate of the tumors
I+

S/
EP

(Fig. 6A), significantly increasing the survival of mice compared to the

other groups (Fig. 6B), with 50% of mice still alive after 2 months. Even
though there is still room for improvement, e.g. optimization of dose,
the enhanced activity of STS/Epi/m against relapsed tumors validates
their utility. Indeed, lengthening the time to relapse and increasing the
overall survival have been considered as main goals for anti-CSCs
therapies [37], and our results indicate the high potential of STS/Epi/m
for achieving these objectives.
3.6. In vitro cytotoxicity against cancer cells from naïve and Epirubicin-
pretreated tumors
To further study the efficacy enhancement of STS/Epi/m against the
tumors, we evaluated the cytotoxicity of the drugs against the cells
collected from naïve and Epi-pretreated 4T1-luc tumors. The cells were
exposed to free Epi, free Epi plus free STS, Epi/m, and STS/Epi/m for
48 h. Thus, the IC50 values of the drugs against the cells from naïve
tumors were found to be comparable to the IC50 values against 4T1-luc
cells (Table 2 and Fig. 7A), with STS/Epi/m showing the highest cy-
totoxicity. Conversely, the IC50 value of free Epi against the cells from
Epi-pretreated 4T1-luc tumors increased > 10 times, indicating the
development of Epi-resistance (Fig. 7B). Adding free STS to free Epi
improved the efficacy against the cells from pretreated tumors, though
the IC50 value was still higher than that against the naïve cells
(Fig. 7B). The IC50 of Epi/m against the cells from Epi-pretreated 4T1-
luc tumors was also higher than that against the cells from naïve tu-
mors, though the increase was relatively lower than for free Epi (Fig. 7),
ST
Fig. 7. Fifty-percent inhibitory concentrations of free Epi, Epi/m, free Epi plus free STS,
and STS/Epi/m against cells from naïve 4T1-luc tumors (A) and cells from Epi-pretreated
tumors (B). Data presented as the mean ± S.E. (n = 4). ***p < 0.001 and **p < 0.01
by Student's t-test.
suggesting that the encapsulation of Epi in the micelles may facilitate
overcoming certain resistance mechanisms. However, STS/Epi/m was
the only treatment to effectively maintain the activity, and their IC50
values in cells from naïve and pretreated tumors were comparable
(Fig. 7). The ability of STS/Epi/m to overcome Epi resistance could be
associated with the strong inhibitory effect of STS against the ABC
transporters [38], which mediate the resistance to anthracyclines, in-
cluding epirubicin, through efflux mechanisms [39]. We have recently
demonstrated the strong inhibition of ABC transporters by STS/Epi/m
in breast cancer resistant cells, i.e. Epi-resistant human breast adeno-
carcinoma MCF-7 cells [18], thereby, preventing Epi efflux and cir-
cumventing Epi resistance. As the CSC fraction of 4T1 cells, including
the CD44+/CD24− and ALDH+ subpopulations [40,41], present the
ABC transporters, the STS/Epi/m could inhibit the Epi efflux also in
these cells to overcome Epi resistance. Thus, the superior activity of
STS/Epi/m against Epi-resistant cells, and their ability to safely and
cooperatively deliver both STS and Epi to breast tumor tissues, resulted
in the therapeutic improvement against Epi-pretreated tumors.

133
J. Zhang et al.
Fig. 8. Effect of drug treatment on the ALDH levels in cells from Epi-treated tumors. A. Flow cytometry study of Aldefluor stained cells incubated with HEPES (control) or 90% inhibitory
concentrations of free Epi, free Epi plus free STS, Epi/m, and STS/Epi/m for 48 h. The Aldefluor-positive cell population was determined by setting the gate sorting with a negative control
(+ DEAB; lower panel). B. Quantification of the ALDH+ positive population. Each bar represents mean ± S.E. (n = 4). **p < 0.01 compared with control cells by Student's t-test.
3.7. Effect of drugs on ALDH+ population in Epi-resistant cancer cells
The enhancement in the cytotoxic effects of STS/Epi/m was also
assessed by evaluating the remaining CSC fraction in the cells collected
from Epi-pretreated tumors after drug exposure. Here, we focused on
the ALDH-positive subpopulation, as ALDH-positive breast cancer cells
display properties of CSC [42]. The cells of Epi-pretreated 4T1-luc tu-
mors were collected and incubated with free Epi, free Epi plus free STS,
Epi/m, and STS/Epi/m at the 90% inhibitory concentration of each
treatment for 48 h. Thus, except for the cells treated with STS/Epi/m,
the levels of ALDH-positive subpopulation were increased after the
various in vitro treatments (Fig. 8). These results further indicate that
STS/Epi/m effectively reduced the CSC population in the Epi-pretreated
cells, which could lead to improve therapeutic outcomes even after
tumor relapse following Epi treatment.
4. Conclusion
STS/Epi/m effectively treated breast tumors bearing recalcitrant
CSC subpopulation, demonstrating potent therapeutic effects by eradi-
cating primary orthotopic breast tumors and inhibiting the growth of
Epi-resistant breast tumors. The STS/Epi/m eliminated the CSC fraction
in these tumors through the cooperatively delivery of STS and Epi,
leading to the extended survival of mice. Considering the high in-
cidence of breast tumor recurrence after chemotherapy, our STS/Epi/m
appears as an attractive strategy capable of achieving prolonged sur-
vival.
Acknowledgements
This work was partially supported by Grants-in-Aid for Scientific
Research B (JP16H03179; H.C.) and Young Scientists B (JP25750172;
H.C.) from the Japan Society for the Promotion of Science (JSPS), the
Project of Cancer Research And Therapeutic Evolution (P-CREATE)
(Project No. 16cm0106202h0001; H.C. and K.K.) from Japan Agency
for Medical Research and Development (AMED) and “the Center of
Innovation, Science and Technology based Radical Innovation and
Entrepreneurship Program (COI STREAM)” from the Ministry of
Education, Culture, Sports, Science and Technology (MEXT) of Japan
(K.K.).
References
[1] L.A. Torre, F. Bray, R.L. Siegel, J. Ferlay, J. Lortet-Tieulent, A. Jemal, Global cancer
statistics, 2012, CA Cancer J. Clin. 65 (2015) 87–108.
134
Journal of Controlled Release 264 (2017) 127–135
[2] R. Siegel, J. Ma, Z. Zou, A. Jemal, Cancer statistics, 2014, CA Cancer J. Clin. 64
(2014) 9–29.
[3] S. Glück, Adjuvant chemotherapy for early breast cancer: optimal use of epirubicin,
Oncologist 10 (2005) 780–791.
[4] French Epirubicin Study Group, Epirubicin-based chemotherapy in metastatic
breast cancer patients: role of dose-intensity and duration of treatment, J. Clin.
Oncol. Off. J. Am. Soc. Clin. Oncol. 18 (2000) 3115–3124.
[5] Early Breast Cancer Trialists' Collaborative Group (EBCTCG), Effects of che-
motherapy and hormonal therapy for early breast cancer on recurrence and 15-year
survival: an overview of the randomised trials, Lancet 365 (2005) 1687–1717.
[6] J.A. Crozier, A. Swaika, A. Moreno-Aspitia, Adjuvant chemotherapy in breast
cancer: to use or not to use, the anthracyclines, World J. Clin. Oncol. 5 (2014)
529–538.
[7] M. Untch, M. Rezai, S. Loibl, P.A. Fasching, J. Huober, H. Tesch, I. Bauerfeind,
J. Hilfrich, H. Eidtmann, B. Gerber, C. Hanusch, T. Kuhn, A. du Bois, J.U. Blohmer,
C. Thomssen, S. Dan Costa, C. Jackisch, M. Kaufmann, K. Mehta, G. von Minckwitz,
Neoadjuvant treatment with trastuzumab in HER2-positive breast cancer: results
from the GeparQuattro study, J. Clin. Oncol. Off. J. Am. Soc. Clin. Oncol. 28 (2010)
2024–2031.
[8] M. Al-Hajj, M.S. Wicha, A. Benito-Hernandez, S.J. Morrison, M.F. Clarke,
Prospective identification of tumorigenic breast cancer cells, Proc. Natl. Acad. Sci.
100 (2003) 3983–3988.
[9] F. Yu, H. Yao, P. Zhu, X. Zhang, Q. Pan, C. Gong, Y. Huang, X. Hu, F. Su,
J. Lieberman, E. Song, let-7 regulates self renewal and tumorigenicity of breast
cancer cells, Cell 131 (2007) 1109–1123.
[10] L.A. Doyle, W. Yang, L.V. Abruzzo, T. Krogmann, Y. Gao, A.K. Rishi, D.D. Ross, A
multidrug resistance transporter from human MCF-7 breast cancer cells, Proc. Natl.
Acad. Sci. 95 (1998) 15665–15670.
[11] M. Diehn, R.W. Cho, N.A. Lobo, T. Kalisky, M.J. Dorie, A.N. Kulp, D. Qian, J.S. Lam,
L.E. Ailles, M. Wong, B. Joshua, M.J. Kaplan, I. Wapnir, F.M. Dirbas, G. Somlo,
C. Garberoglio, B. Paz, J. Shen, S.K. Lau, S.R. Quake, J.M. Brown, I.L. Weissman,
M.F. Clarke, Association of reactive oxygen species levels and radioresistance in
cancer stem cells, Nature 458 (2009) 780–783.
[12] S. Vinogradov, X. Wei, Cancer stem cells and drug resistance: the potential of na-
nomedicine, Nanomedicine 7 (2012) 597–615.
[13] X.Y. Ke, V.W. Lin Ng, S.J. Gao, Y.W. Tong, J.L. Hedrick, Y.Y. Yang, Co-delivery of
thioridazine and doxorubicin using polymeric micelles for targeting both cancer
cells and cancer stem cells, Biomaterials 35 (2014) 1096–1108.
[14] S. Krishnamurthy, V.W. Ng, S. Gao, M.H. Tan, J.L. Hedrick, Y.Y. Yang, Codelivery of
dual drugs from polymeric micelles for simultaneous targeting of both cancer cells
and cancer stem cells, Nanomedicine 10 (2015) 2819–2832.
[15] Y. Zhang, H. Zhang, X. Wang, J. Wang, X. Zhang, Q. Zhang, The eradication of
breast cancer and cancer stem cells using octreotide modified paclitaxel active
targeting micelles and salinomycin passive targeting micelles, Biomaterials 33
(2012) 679–691.
[16] K. Kataoka, A. Harada, Y. Nagasaki, Block copolymer micelles for drug delivery:
design, characterization and biological significance, Adv. Drug Deliv. Rev. 47
(2001) 113–131.
[17] H. Cabral, K. Kataoka, Progress of drug-loaded polymeric micelles into clinical
studies, J. Control. Release 190 (2014) 465–476.
[18] H. Kinoh, Y. Miura, T. Chida, X. Liu, K. Mizuno, S. Fukushima, Y. Morodomi,
N. Nishiyama, H. Cabral, K. Kataoka, Nanomedicines eradicating cancer stem-like
cells in vivo by pH-triggered intracellular cooperative action of loaded drugs, ACS
Nano 10 (2016) 5643–5655.
[19] M. Harada, I. Bobe, H. Saito, N. Shibata, R. Tanaka, T. Hayashi, Y. Kato, Improved
anti-tumor activity of stabilized anthracycline polymeric micelle formulation, NC-
6300, Cancer Sci. 102 (2011) 192–199.
[20] A. Takahashi, Y. Yamamoto, M. Yasunaga, Y. Koga, J. Kuroda, M. Takigahira,
J. Zhang et al.
M. Harada, H. Saito, T. Hayashi, Y. Kato, T. Kinoshita, N. Ohkohchi, I. Hyodo,
Y. Matsumura, NC-6300, an epirubicin-incorporating micelle, extends the anti-
tumor effect and reduces the cardiotoxicity of epirubicin, Cancer Sci. 104 (2013)
920–925.
[21] H. Mukai, T. Kogawa, N. Matsubara, Y. Naito, M. Sasaki, A. Hosono, A first-in-
human Phase 1 study of epirubicin-conjugated polymer micelles (K-912/NC-6300)
in patients with advanced or recurrent solid tumors, Investig. New Drugs 35 (2017)
307–314.
[22] H. Cabral, Y. Matsumoto, K. Mizuno, Q. Chen, M. Murakami, M. Kimura, Y. Terada,
M.R. Kano, K. Miyazono, M. Uesaka, N. Nishiyama, K. Kataoka, Accumulation of
sub-100 nm polymeric micelles in poorly permeable tumours depends on size, Nat.
Nanotechnol. 6 (2011) 815–823.
[23] L. Tang, X. Yang, Q. Yin, K. Cai, H. Wang, I. Chaudhury, C. Yao, Q. Zhou, M. Kwon,
J.A. Hartman, I.T. Dobrucki, L.W. Dobrucki, L.B. Borst, S. Lezmi, W.G. Helferich,
A.L. Ferguson, T.M. Fan, J. Cheng, Investigating the optimal size of anticancer
nanomedicine, Proc. Natl. Acad. Sci. 111 (2014) 15344–15349.
[24] M.R. Kano, Y. Bae, C. Iwata, Y. Morishita, M. Yashiro, M. Oka, T. Fujii, A. Komuro,
K. Kiyono, M. Kaminishi, K. Hirakawa, Y. Ouchi, N. Nishiyama, K. Kataoka,
K. Miyazono, Improvement of cancer-targeting therapy, using nanocarriers for in-
tractable solid tumors by inhibition of TGF-beta signaling, Proc. Natl. Acad. Sci. U.
S. A. 104 (2007) 3460–3465.
[25] H. Nakano, S. Omura, Chemical biology of natural indolocarbazole products: 30
years since the discovery of staurosporine, J. Antibiot. 62 (2009) 17–26.
[26] M.H. Wright, A.M. Calcagno, C.D. Salcido, M.D. Carlson, S.V. Ambudkar,
L. Varticovski, Brca1 breast tumors contain distinct CD44+/CD24 − and CD133 +
cells with cancer stem cell characteristics, Breast Cancer Res. 10 (2008) R10.
[27] S. Ricardo, A.F. Vieira, R. Gerhard, D. Leitao, R. Pinto, J.F. Cameselle-Teijeiro,
F. Milanezi, F. Schmitt, J. Paredes, Breast cancer stem cell markers CD44, CD24 and
ALDH1: expression distribution within intrinsic molecular subtype, J. Clin. Pathol.
64 (2011) 937–946.
[28] A.M. Calcagno, C.D. Salcido, J.-P. Gillet, C.-P. Wu, J.M. Fostel, M.D. Mumau,
M.M. Gottesman, L. Varticovski, S.V. Ambudkar, Prolonged drug selection of breast
cancer cells and enrichment of cancer stem cell characteristics, J. Natl. Cancer Inst.
102 (2010) 1637–1652.
[29] C.M. Seynaeve, M.G. Kazanietz, P.M. Blumberg, E.A. Sausville, P.J. Worland,
Differential inhibition of protein kinase C isozymes by UCN-01, a staurosporine
analogue, Mol. Pharmacol. 45 (1994) 1207–1214.
[30] W.L. Tam, H. Lu, J. Buikhuisen, B.S. Soh, E. Lim, F. Reinhardt, Z.J. Wu, J.A. Krall,
B. Bierie, W. Guo, X. Chen, X.S. Liu, M. Brown, B. Lim, R.A. Weinberg, Protein
kinase C α is a central signaling node and therapeutic target for breast cancer stem
cells, Cancer Cell 24 (2013) 347–364.
[31] R. Behera, V. Kumar, K. Lohite, S. Karnik, G.C. Kundu, Activation of JAK2/STAT3
signaling by osteopontin promotes tumor growth in human breast cancer cells,
Carcinogenesis 31 (2010) 192–200.
[32] L.L. Marotta, V. Almendro, A. Marusyk, M. Shipitsin, J. Schemme, S.R. Walker,
135
Journal of Controlled Release 264 (2017) 127–135
N. Bloushtain-Qimron, J.J. Kim, S.A. Choudhury, R. Maruyama, Z. Wu, M. Gonen,
L.A. Mulvey, M.O. Bessarabova, S.J. Huh, S.J. Silver, S.Y. Kim, S.Y. Park, H.E. Lee,
K.S. Anderson, A.L. Richardson, T. Nikolskaya, Y. Nikolsky, X.S. Liu, D.E. Root,
W.C. Hahn, D.A. Frank, K. Polyak, The JAK2/STAT3 signaling pathway is required
for growth of CD44(+)CD24(−) stem cell-like breast cancer cells in human tumors,
J. Clin. Invest. 121 (2011) 2723–2735.
[33] H.E. Lee, J.H. Kim, Y.J. Kim, S.Y. Choi, S.W. Kim, E. Kang, I.Y. Chung, I.A. Kim,
E.J. Kim, Y. Choi, H.S. Ryu, S.Y. Park, An increase in cancer stem cell population
after primary systemic therapy is a poor prognostic factor in breast cancer, Br. J.
Cancer 104 (2011) 1730–1738.
[34] K.J. Harrington, S. Mohammadtaghi, P.S. Uster, D. Glass, A.M. Peters, R.G. Vile,
et al., Effective targeting of solid tumors in patients with locally advanced cancers
by radiolabeled pegylated liposomes, Clin. Cancer Res. 7 (2001) 243–254.
[35] R.K. Ramanathan, R.L. Korn, N. Raghunand, J.C. Sachdev, R.G. Newbol,
G. Jameson, G.J. Fetterly, J. Prey, S.G. Klinz, J. Kim, J. Cain, B.S. Hendriks,
D.C. Drummond, E. Bayever, J.B. Fitzgerald, Correlation between ferumoxytol
uptake in tumor lesions by MRI and response to nanoliposomal irinotecan in pa-
tients with advanced solid tumors: a pilot study, Clin. Cancer Res. 23 (2017)
3638–3648.
[36] H. Lee, A.F. Shields, B.A. Siegel, K.D. Miller, I. Krop, C.X. Ma, P.M. LoRusso,
P.N. Munster, K. Campbell, D.F. Gaddy, S.C. Leonard, E. Geretti, S.J. Blocker,
D.B. Kirpotin, V. Moyo, T.J. Wickham, B.S. Hendriks, 64Cu-MM-302 positron
emission tomography quantifies variability of enhanced permeability and retention
of nanoparticles in relation to treatment response in patients with metastatic breast
cancer, Clin. Cancer Res. (2017), http://dx.doi.org/10.1158/1078-0432.CCR-16-
3193.
[37] B.B. Zhou, H. Zhang, M. Damelin, K.G. Geles, J.C. Grindley, P.B. Dirks, Tumour-
initiating cells: challenges and opportunities for anticancer drug discovery, Nat.
Rev. Drug Discov. 8 (2009) 806–823.
[38] A. Mayati, A. Moreau, M. Le Vée, B. Stieger, C. Denizot, Y. Parmentier, O. Fardel,
Protein kinases C-mediated regulations of drug transporter activity, localization and
expression, Int. J. Mol. Sci. 18 (2017) 764.
[39] K.F. Ejendal, C.A. Hrycyna, Multidrug resistance and cancer: the role of the human
ABC transporter ABCG2, Curr. Protein Pept. Sci. 3 (2002) 503–511.
[40] A. Gomez-Cabrero, W. Wrasidlo, R.A. Reisfeld, IMD-0354 targets breast cancer stem
cells: a novel approach for an adjuvant to chemotherapy to prevent multidrug re-
sistance in a murine model, PLoS One 8 (2013) e73607.
[41] X. Yang, S.K. Sarvestani, S. Moeinzadeh, X. He, E. Jabbari, Three-dimensional-en-
gineered matrix to study cancer stem cells and tumorsphere formation: effect of
matrix modulus, Tissue Eng. A 19 (2012) 669–684.
[42] E. Charafe-Jauffret, C. Ginestier, F. Iovino, J. Wicinski, N. Cervera, P. Finetti,
M.H. Hur, M.E. Diebel, F. Monville, J. Dutcher, M. Brown, P. Viens, L. Xerri,
F. Bertucci, G. Stassi, G. Dontu, D. Birnbaum, M.S. Wicha, Breast cancer cell lines
contain functional cancer stem cells with metastatic capacity and a distinct mole-
cular signature, Cancer Res. 69 (2009) 1302–1313.