Preferential Killing of Breast Tumor Initiating Cells by N,N-Diethyl

CancerTherapy: Preclinical
Preferential Killing of Breast Tumor Initiating Cells by N,N-Diethyl-2[4-(Phenylmethyl)Phenoxy]Ethanamine/Tesmilifene
Tao Deng,1 Jeff C. Liu,1 Kathleen I. Pritchard,2,3 Andrea Eisen,2,3 and Eldad Zacksenhaus1,3,4,5
Abstract
Purpose: N,N-Diethyl-2-[4-(phenylmethyl)phenoxy]ethanamine (DPPE; tesmilifene) is thought
to potentiate the antineoplastic effect of cytotoxic drugs. In a phase III randomized trial for
metastatic breast cancer using doxorubicin with or without DPPE, addition of the latter resulted
in a significant improvement in overall survival and a trend toward a difference in progression-free
survival but, paradoxically, no difference in objective tumor response. Here we tested the
hypothesis that DPPE targets breast tumor-initiating cells (TICs).
Experimental Design: Human breast TICs from pleural effusions were identified as
CD44+:CD24-/low cells by flow cytometry and functionally by their ability to form nonadherent
spheres in culture. Mouse mammary TICs from two different models of breast cancer were
identified as cells capable of initiating spheres in culture and secondary tumors following
transplantation into the mammary gland of syngeneic mice.
Results: We show that at physiologically attainable concentrations, treatment with DPPE alone
reduced tumorsphere formation and viability of CD44+:CD24-/low breast cancer cells.The kinetics
of killing varied for the different breast tumor cells and required continuous exposure to the drug.
Whereas doxorubicin killed CD44+:CD24-/low and CD44 -:CD24+ cells equally well, DPPE
induced apoptosis preferentially in CD44+:CD24-/low cells. Treatment of Her2/Neu+ mammary
tumor cells with DPPE in vitro efficiently killed TICs, as determined by flow cytometry and
transplantation assays; DPPE further cooperated with doxorubicin to completely eradicate
tumorigenic cells.
Conclusions: Our results show that continuous treatment with DPPE alone directly targets
breast TICs, and provide rationale to test for cooperation between DPPE and known drugs with
efficacy toward breast cancer subtypes.
Breast
(e.g., doxorubicin) and the taxanes (e.g., docetaxel; ref. 3).
However, at best, only 50% of women treated with these agents
will respond to therapy (4). Thus, there is an urgent need for
novel approaches to combat metastatic breast cancer.
There is growing evidence that cancer is organized in
a hierarchy in which relatively rare tumor-initiating cells
(TICs; also known as cancer stem cells) are capable of
disseminating cancer, whereas the majority of tumor cells have
lost this potential (5 – 9). This hierarchy has been first
documented in acute myeloid leukemia (10, 11). More
recently, TICs (cancer stem cells) were identified in breast
(12) and brain (13) cancers as well as other tumor types
(14 – 21). Breast TICs express early epithelial markers such as
the cell surface marker CD44, whereas nontumorigenic cells
express CD24 (12). CD44+:CD24-/low cells, but not CD24+
cells, sorted from malignant pleural effusions give rise to new
tumors when injected into the fat pad of immunocompromised
NOD/SCID mice (12).
Mouse models of cancer provide the power of mouse
genetics, the availability of ample samples of genetically
defined tumors and syngeneic transplantation assays. Several
groups have recently identified TICs in mouse models of breast
cancer (22 – 25). Our group has shown that TICs derived from
Her2/Neu+ as well as Wnt1-induced mammary tumors are
capable of forming spheres under nonadherent conditions in a
defined medium containing epidermal growth factor and
fibroblast growth factor (23). The tumorsphere-forming units
cancer mortality is declining in Western countries
primarily due to improvements in breast cancer screening and
in the adjuvant treatment of early stage disease (1, 2).
Metastatic breast cancer remains an incurable illness for
which few treatments have actually been shown to prolong
survival. The most active chemotherapeutic agents in the
treatment of metastatic breast cancer include the anthracyclines
Authors’ Affiliations: 1 Toronto General Research Institute-University Health
Network ; 2 Sunnybrook Odette Cancer Centre ; and 3 Medicine, 4 Medical
Biophysics, and 5Laboratory Medicine and Pathobiology, University of Toronto,
Toronto, Ontario, Canada
Received 7/3/08; revised 9/8/08; accepted 9/9/08.
Grant support:YM Biosciences, Inc., a matching grant from the Canadian Institute
for Health Research, the National Cancer Institute of Canada-Terry Fox Breast
Cancer Group Grant 13005, and the Ontario Institute for Cancer Research.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
Note: Supplementary data for this article are available at Clinical Cancer Research
Online (http://clincancerres.aacrjournals.org/).
Requests for reprints: Eldad Zacksenhaus, Division of Cell and Molecular
Biology,Toronto General Research Institute-University Health Network, 67 College
Street, Room 407,Toronto, Ontario, Canada M5G 2M1. Phone: 416-340-4800, ext.
5106; Fax: 416-340-3453; E-mail: eldad.zacksenhaus@ utoronto.ca. Andrea Eisen,
Preventive Oncology,Toronto Sunnybrook Regional Cancer Centre, 2075 Bayview
Avenue, Toronto, ON M4N 3M5, Canada. Phone: 416-480-4617; E-mail: andrea.
eisen@ sunnybrook.ca.
F 2009 American Association for Cancer Research.
doi:10.1158/1078-0432.CCR-08-1708
www.aacrjournals.org
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CancerTherapy: Preclinical
prednisone in the treatment of hormone refractory prostate
cancer (39). Understanding the mechanism by which DPPE
affects chemotherapy and overall survival may lead to
improved therapy (39). Using four different breast cancer
models, we now show that treatment with DPPE alone
preferentially kills TICs.
Translational Relevance
Previous models implicated N,N-diethyl-2-[4-(phenylmethyl)phenoxy]ethanamine (DPPE; tesmilifene) in the
potentiation of cytotoxicity by antineoplastic drugs. In a
phase III randomized trial for metastatic breast cancer using
doxorubicin with or without DPPE, addition of the latter
resulted in a significant improvement in overall survival
and a trend toward a difference in progression-free survival
but, paradoxically, no difference in objective tumor response. We have now shown that continuous exposure to
pharmacologically attainable doses of DPPE effectively
kills tumor-initiating cells in four different models of breast
cancer. Our results suggest that DPPE-based therapies
should involve continuous or frequent administration of
the drug. Furthermore, more potent DPPE derivatives
with better specificity may be developed using the tumorinitiating cell ^ specific assays presented in our study.
Finally, using these assays, combinatorial drug regimens
with DPPE plus other antineoplastic agents with efficacy
toward breast cancer subtypes should be assessed.
Materials and Methods
Patient recruitment and consent. Subjects with metastatic breast
cancer and malignant pleural effusions were recruited from the
clinical practices of a large, tertiary care cancer center under an Research
Ethics Board – approved protocol. All patients provided written
informed consent. Pleural fluid samples were obtained by standard
thoracocentesis.
Enrichment of lineage-depleted breast cancer cells from pleural
effusions. Pleural effusions were obtained and processed within 2 to
4 h after thoracocentesis. The samples were centrifuged at 1,000 rpm
for 10 min and cell pellets were washed with HBSS plus 2%
heat-inactivated fetal bovine serum (HIBS) and 2 mmol/L EDTA.
Enrichment of epithelial cells was achieved using a negative selection kit
StemSep (StemCell Technologies, Inc.). Briefly, 100 AL per 5 107 cells
of a cocktail containing biotinylated antibodies against human
hematopoietic cells (CD2, CD14, CD16, CD38, CD45, and CD66)
and glycophorin A were added. After 30-min incubation on ice, 60 AL
of magnetic colloid were added and incubated for another 30 min on
ice. Finally, the cells were passed trough magnetic columns and the
supernatant containing epithelial cells was collected. The purified cells
were cultured either as adherent monolayer with DMEM plus 10% fetal
bovine serum or under nonadherent conditions (see below) plus
penicillin/streptomycin.
Mice and PCR-based genotyping. MMTV-Wnt1 (40) and MMTV-Neu
(41) transgenic mice, both on pure FvB background, were maintained
in accordance with the Canadian Animal Care Council. The mice were
genotyped by PCR analysis of DNA extracted from tail biopsies
using the following primers: Wnt1, forward 5¶-GGACTTGCTTCTCTTCTCATAGCC and reverse 5¶-CCACACAGGCATAGAGTGTCTGC; Neu,
forward 5¶-CTAGGCCACAGAATTGAAAGATCT and reverse 5¶-gTAGGTGGAAATTCTAGCATCATCC. Mice were sacrificed when tumors were
0.5 to 1 cm in diameter.
Enrichment of lineage-depleted epithelial cells from mouse mammary
tumors. Mammary tumors were resected and minced by sterile razor
blades, washed with HBSS plus 2% HIBS, and digested with 100 units/
mL collagenase/hyaluronidase (Stem Cell Technologies) for 30 min at
37jC. The resulting samples were passed through a 40-Am cell strainer
(BD falcon) and centrifuged at 450 g for 5 min. The cell pellets
were washed with HBSS + 2% HIBS, and lineage-negative (Lin-)
epithelial cells were isolated using a negative selection EasySep Kit
(StemCell Technologies). Briefly, 10 AL of FcR blocker were added
per 1 108 cells to prevent nonspecific interaction. Biotinylated
antibodies (0.5 Ag/106 cells) against hematopoietic (anti-CD45 and
anti-TER119; StemCell Technologies), stromal (anti-CD140a; Biosciences), and endothelial (anti-CD31; BD PharMingen) cells were added
and incubated for 15 min at room temperature. Antibody conjugation
to magnetic nanoparticles was achieved through 15-min incubation
with EasySep Biotin Selection cocktail followed by 10 min with EasySep
Magnetic Nanoparticles. An EasySep Magnetic device was used for
5 min to separate the Lin+ cells that attached to the tube surface from
the Lin- epithelial cells that remained in the supernatant.
In vitro differentiation and immunocytostaining. Sorted
CD44+:CD24-/low cells were plated on collagen 1 – coated coverslips
(BD Biocoat) in growth medium (DMEM/F-12, 10% fetal bovine
serum, 5 Ag/mL insulin, 1 Ag/mL hydrocortisone, 5 ng/mL epidermal
growth factor, and penicillin/streptomycin) for 5 d and then
differentiated for 24 h in medium containing DMEM/F-12, 5 Ag/mL
insulin, 1 Ag/mL hydrocortisone, 3 Ag/mL prolactin, and penicillin/
(TFU) were indistinguishable by several criteria from TICs.
Specifically, TFUs and TICs were identified in the same fraction
of sorted cells, and cultured tumorspheres retained the
differentiation capacity, cell surface markers, and tumorigenic
potential of freshly isolated TICs. Thus, TFUs can be used as a
surrogate for TICs in vitro.
Implicit in the cancer stem cell model is that targeted killing
of TICs, rather than the bulk of nontumorigenic cells, may be
curative (5, 6, 26). However, TICs are not only rare but, in some
instances, are also shown to be more resistant to radiation and
cytotoxic therapy (27 – 31). The identification of agents that
enhance the specificity of antineoplastic drugs toward TICs or
that preferentially kill TICs is therefore of great interest.
The tamoxifen analogue N,N-diethyl-2-[4-(phenylmethyl)phenoxy]ethanamine (DPPE; tesmilifene) has been implicated
in the inhibition of histamine binding to CYP 3A4, a P450
isozyme that metabolizes several antineoplastic agents, as well
as in the inhibition of p-glycoproteins involved in multidrug
resistance (32 – 34). It is therefore thought that DPPE exerts its
effect by potentiating the cytotoxicity of various antineoplastic
drugs (35 – 37). The exact mechanism of action and the
biological targets of DPPE are not known.
In a phase III randomized trial for metastatic breast cancer
using doxorubicin with or without DPPE, it was observed that
the addition of the latter resulted in a significant improvement
in overall survival and a trend toward a difference in
progression-free survival but, paradoxically, no difference in
objective tumor response (38). The effect of adding DPPE was
not reproduced in the most recent trial of epirubicin plus
cyclophosphamide +/- DPPE in naBve, previously untreated
patients with metastatic breast cancer, possibly because of the
excellent response by the EC arm alone,6 although results from
long-term follow-up are not yet available. Another trial with
docetaxel +/- DPPE is ongoing (YM Biosciences, Inc.). DPPE
has also been shown to have activity with mitoxantrone and
6
YM Biosciences, unpublished.
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Preferential Killing of Breast TICs by DPPE/Tesmilifene
Colorimetric reading at 570 nm was done using a microplate reader
(Molecular Devices). For MTT assay of tumorsphere cells, the protocol
was modified as follows. Briefly, MTT was added to sphere cells and
incubated for 2 h at 37jC. The supernatant including spheres was
transferred to tubes and centrifuged at 500 g for 5 min. After
aspirating the supernatant, the sphere cells were solubilized by adding
100 AL DMSO to liberate the dye.
Transplantation. Dissociated tumorsphere cells were resuspended
in 10 AL serum-free DMEM/F-12 Ham medium, mixed at 1:1 ratio with
10 AL Matrigel (BD Bioscience), placed on ice, and then injected into #
4 mammary glands of syngeneic FvB mice or immunocompromised
Rag1-/- mice anesthetized with isoflurane. Sites of needle injection and
incision were sealed with liquid bandage (NewSkin).
Statistical analysis. All data are represented as mean F SD.
Two-tailed distribution Student’s t test was carried out. P < 0.05 (*)
and P < 0.01 (**) were considered statistically significant.
streptomycin. After fixation in 3.5% paraformaldehyde, cells were
permeabilized in OPAS buffer (100 mmol/L PIPES, 1 mmol/L EGTA,
100 mmol/L KOH, 4% polyethylene glycol 8000, 0.1% Triton
X-100). Rabbit anti – smooth muscle actin (1:200 dilution; Novus
Biologics), mouse anti – keratin 18 (1:200 dilution; Fitzgerald), rabbit
anti – keratin 14 (1:200 dilution; Panomics), and secondary goat
anti-rabbit Alexa 488 (green; 1:200 dilution; Molecular Probes) or goat
anti-mouse Alexa 568 (red, 1:200 dilution, Molecular Probes)
were used for immunocytostaining. Nuclei were visualized with
4¶,6¶-diamidino-2-phenylindole (Sigma).
Drug treatment. A stock solution of DPPE in sodium acetate was
obtained from YM Biosciences. Doxorubicin (Sigma) was dissolved in
N,N-dimethylformamide at 1 mg/mL. Before experiments, drugs were
further diluted in serum-free DMEM/F-12 Ham culture medium. For
adherent cultures, 50,000 MCF7 or pleural effusion cells were seeded
onto 24-well plates. For nonadherent cultures, dissociated cells were
seeded onto 24-well ultra-low attachment plates (Corning, Costar) at a
cell density of 50,000 per well.
Tumorsphere formation assay. Enriched tumor cells from pleural
effusions or MCF7 cells were seeded onto 6- or 24-well ultra-low
attachment plates (Corning, Costar) in DMEM/F-12 Ham medium
(Sigma) containing 10 ng/mL basic fibroblast growth factor (Sigma),
20 ng/mL epidermal growth factor (Sigma), 5 Ag/mL insulin (Sigma),
and 0.4% bovine serum albumin and cultured at 37jC, 5% CO2. For
mouse tumorspheres, Lin- mammary epithelial cells were plated on
ultra-low attachment plates (Corning, Costar) in DMEM/F-12 Ham
medium containing 20 ng/mL basic fibroblast growth factor (Sigma),
20 ng/mL epidermal growth factor, 4 Ag/mL heparin (Sigma), and B-27
supplement (1:50 dilution; Life Technologies). Medium was refreshed
twice a week. Spheres were mechanically and enzymatically dissociated
every 2 wk in 1 (0.25%) trypsin-EDTA solution (Life Technologies)
for 1 min at room temperature, followed by passing through 25-gauge
needles seven times. The dissociated tumorsphere cells were added to
24-well ultra-low attachment plates at a cell density of 50,000 per well
and treated as indicated in each figure.
Flow cytometry analysis and sorting. Human tumor cells were
washed, counted, and resuspended in 50 AL (per 5 105 cells) of
HBSS plus 2% HIBS. Ten microliters of anti-CD44 [allophycocyanin
(APC)] and antihuman CD24 (FITC; both from BD Biosciences) were
added for 30 min on ice followed by two washes with HBSS/2% HIBS.
The stained cells were washed again and resuspended in HBSS/2% HIBS
containing the viability dye 7-amino-actinomycin D (BD Pharmingen)
and passed through a cell strainer. For mouse mammary tumors,
we used anti-CD49f conjugated to R-phycoerythrin (CD49f-PE) and
anti-CD24 conjugated to FITC (CD24-FITC; BD Pharmingen). Flow
cytometry analysis was done on a FACSCalibur (Becton Dickinson) and
cell sorting was done on a FACSAria-13 color sorter (Becton Dickinson)
at 30 psi, gating on the 7-amino-actinomycin D – negative (live) cells.
Annexin V and cell cycle analysis. Apoptosis was detected using
PE-Annexin V (BD Biosciences). For pleural effusion cells, triple
staining was carried out by first adding CD44-APC and CD24-FITC
for 20 min on ice. The cells were washed with HBSS/2% HIBS and
resuspended in Annexin V binding buffer. Annexin V-PE was added and
incubated for 15 min at room temperature followed by flow cytometry.
Cell cycle analysis was done on cells stained with propidium iodide/
RNase (BD Pharmingen). One-milliliter aliquots of cell suspensions
were pelleted at 400 g for 5 min and fixed in 70% ethanol at 4jC for
at least 60 min, and then f1 105 cells were washed with 3 mL PBS
(PBS) and stained with 500 AL propidium iodide/RNase. Samples were
incubated for 15 min in the dark at room temperature and then
analyzed by flow cytometry.
3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
assay. Cell viability was measured by 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide (MTT) assay. For MCF7 monolayer
cells, a total of 1,000 cells per well were seeded into 96-well plates and
grown overnight in duplicate. After drug treatment, MTT (1 mg/mL)
was added for 2 h at 37jC, followed by 100 AL DMSO overnight.
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Results
DPPE inhibits the growth of MCF7 cells when cultured
as tumorspheres but not as monolayer, whereas doxorubicin
kills both. We first tested the effect of DPPE on the breast
cancer cell line MCF7. The cells were plated under adherent
conditions; treated with DPPE, doxorubicin, or both drugs; and
the effect of cell viability was assessed by the colorimetric
MTT assay, which measures mitochondrial reductase activity.
Exposure of MCF7 cells to clinically attainable concentration of
DPPE (5 Amol/L; refs. 42, 43) had only a minor effect, with a
20% reduction in total cell survival relative to cells treated with
vehicle alone (dimethyformamide; Fig. 1A). Under these
conditions, clinically relevant concentrations of doxorubicin
(0.05 and 0.1 Ag/mL) led to about 45% and 75% reductions in
live MCF7 cells, respectively. Loss of viability was not enhanced
by combined treatment with DPPE and doxorubicin. In this
and other experiments, treatment with vehicle alone (dimethyformamide) did not have any significant effect on cell viability
relative to no treatment (data not shown).
We next tested the effect of DPPE on the formation of MCF7
tumorspheres cultured in ultra-low attachment plates in
defined medium containing epidermal growth factor and
fibroblast growth factor. Under these nonadherent conditions,
tumorspheres are formed by stem cells or TICs, whereas
more differentiated cells tend to die by anoikis (44). Thus,
MCF7 cells were trypsinized to obtain single cells, plated under
nonadherent conditions, and treated with the indicated drugs.
The number of spheres in each culture was determined 7 days
later. In contrast to the modest effect on MCF7 cells grown as
monolayer (Fig. 1A, top), DPPE (5 Amol/L) treatment reduced
tumorsphere formation by 79.5 F 3.5% (mean F SD) relative
to vehicle-treated cultures (Fig. 1A, bottom). Treatment with
both DPPE and doxorubicin had a moderate additive effect,
leading to 88.6 F 0.86% suppression. To test whether the
inhibition of MCF7 tumorsphere formation was cytostatic or
cytotoxic, the cells were treated for 2 weeks under nonadherent
conditions with the various drugs. The tumorspheres were then
counted, trypsinized, and replated under the same conditions
without drugs. As shown in Fig. 1B, the number of tumorspheres treated with DPPE continuously (four doses) dropped
to 2 F 1.4% after 2 weeks of treatment (top) and the remaining
cells did not form spheres when further cultured in drug free
media (bottom). Thus, DPPE seems to exert a cytotoxic effect on
TFUs. Notably, continuous exposure to DPPE was critical. A
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Fig. 1. Specific killing of MCF7 TFUs by DPPE. A, MCF7 cells seeded under adherent (top) or nonadherent (bottom) conditions were treated with doxorubicin (Dox), DPPE, or
combinations of both drugs for 7 d.The number of live adherent cells was assessed by the MTT assay (top) and the number of tumorspheres in the nonadherent cultures was
counted microscopically (bottom). B, MCF7 were plated in tumorsphere conditions and treated with single or four doses (Cont.; twice per week) of the indicated drugs for
14 d.The number of spheres formed after 2 wk is indicated (top).The cultures were trypsinized and reseeded in the absence of drugs.The number of spheres formed after
2 additional weeks is presented (bottom). Columns, mean number of tumorspheres from an experiment done in duplicate; bars, SD. Similar results were obtained in three
independent experiments. *, P < 0.05, compared with dimethyformamide (DMF) control. C, CD24-CD44 flow cytometry profiles of MCF7 cells grown as monolayer andT11
pleural effusion cells cultured as monolayer for1wk or under nonadherent conditions for 2 wk. Note that under nonadherent conditions, about half the population became
CD44+:CD24+.T9 pleural effusion cells cultured as monolayer over an 8-wk period. Note the gradual conversion of CD44+:CD24-/low cells into CD44+:CD24+ cells.
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Preferential Killing of Breast TICs by DPPE/Tesmilifene
However, the rare TFUs that formed tumorspheres in nonadherent conditions were highly sensitive to DPPE.
CD44+:CD24-/low breast tumor cells from pleural effusions give
rise to nonadherent tumorspheres. The analysis of MCF7 breast
cancer cells is limited by the low percentage of TICs in the
culture (Fig. 1C) and the fact that the cells have been clonally
maintained in culture for an extended period of time. To
corroborate our results, we analyzed the effect of DPPE on
primary metastatic breast tumor cells from pleural effusions.
TICs were previously identified in pleural effusions at a
frequency of 1/100 to 1/1,000 in sorted CD44+:CD24-/low cells
but at <1/10,000 in the CD44+:CD24+ fraction (12). We
single dose treatment reduced the number of spheres by only
55 F 2.8%, compared with 98 F 2.1% following continuous
treatment.
The number of MCF7 tumorspheres formed in untreated
cultures under nonadherent conditions was f1/500 to 1/1,000
plated cells. This low percentage of tumorsphere-forming
cells coincided with the CD24-CD44 profile of adherent
MCF7 cells. As shown in Fig. 1C, the percentage of
CD44+:CD24-/low cells in MCF7 cells was 0.82%, with the vast
majority of the cells (99.11 F 1.2%) being CD44-:CD24+. Thus,
DPPE may have little discernable effect on MCF7 cells grown as
monolayer because of the low frequency of TFUs in this culture.
Fig. 2. Sorted CD44+:CD24-/low, but not CD24+, pleural effusion cells form spheres under nonadherent conditions. A, flow cytometry profile of hematopoietic-depleted
human pleural effusion cells and the gating for cell sorting (left), post-sorting profile (middle), and the flow cytometry profile of the sorted cells 2 wk after seeding onto
adherent plates (right). Note that the sorting (left and middle) and the flow cytometry profiles (right) were done on different flow cytometry machines. The sorted
CD44+:CD24-/low cells gave rise to CD24+/- cells expressing intermediate levels of CD24, whereas the sorted CD24+ cells did not convert to CD44+:CD24-/low. B, spheres
were formed after culturing the sorted CD44+:CD24-/low cells in ultra-low attachment plates; no spheres were formed from the CD44+:CD24+ population.
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Fig. 3. DPPE treatment inhibits tumorsphere formation and preferentially targets the CD44+:CD24-/low population in pleural effusion cells. A, tumorsphere formation assay
(top) and cell survival MTTassay (bottom) were carried out on nonadherent hematopoietic-depleted pleural effusion cells after 2 wk of continuous (two doses per week)
treatment with doxorubicin (0.05) Ag/mL, DPPE (5 Amol/L), DPPE plus doxorubicin, or dimethyformamide. Columns, mean number of tumorspheres from an experiment
done in duplicate; bars, SD. Similar results were obtained in three independent experiments. B, pleural effusion cells (1 104) were seeded onto 24-well plates as adherent
cultures overnight, treated with a single dose of the indicated drugs for 2 d, and then processed for flow cytometry with FITC-CD24 and APC-CD44. Representative
data of three independent experiments. C, left, average of three experiments done as in B and presented as the ratio of CD44+:CD24-/low in drug-treated relative to
dimethyformamide-treated cultures. Bars, SD. Right, average of three experiments done as in B and presented as the absolute number of CD44+:CD24-/low cells. D, DPPE
induces apoptotic cell death in the CD44+:CD24-/low cell population. Plural effusion cells were treated with the indicated drugs for 2 d and then processed for triple flow
cytometry analysis with FITC-CD24, APC-CD44, and PE-Annexin V. The CD44+:CD24-/low and CD44+:CD24+ cells were first gated as indicated and then the percentage of
Annexin V+ cells in the gated fractions was determined (n = 3 per treatment). Note that doxorubicin induced apoptosis in both cell types whereas DPPE preferentially
targeted the CD44+:CD24-/low population. *, P < 0.05; **, P < 0.001, compared with dimethyformamide control.
which were larger in size, represented a more homogenous
population of tumor epithelial cells (Supplementary Fig. S1B).
Following purification, tumor cells were cultured in adherent or
nonadherent conditions. Five of the 12 samples had high RBC
content, and attempts to recover tumor epithelial cells were
unsuccessful. Of the remaining seven samples, two samples did
not survive in culture; three samples (ER+) survived as tumorspheres for about 8 to 12 weeks and were used to establish
conditions for cell sorting (not shown); and two samples, T9
(ER+) and T11 (ER-), gave rise to monolayer cultures that
survived for several months and were further analyzed as
obtained pleural effusions from 11 breast cancer patients with
metastatic disease (12 samples in total; Supplementary Table
S1). The samples varied in their content of RBC, hematopoietic
cells, and tumor epithelial cells. The hematopoietic fraction
varied from f60% to >95% in different pleural effusion
samples. To remove the hematopoietic cells, samples were
passed through StemSep columns (StemCell Technologies)
containing antihuman antibodies to the following antigens:
CD2, CD14, CD16, CD38, CD45, CD66, and glycophorin A.
Flow cytometry analysis revealed efficient depletion of hematopoietic cells (Supplementary Fig. S1A); the remaining cells,
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Preferential Killing of Breast TICs by DPPE/Tesmilifene
Flow cytometry analysis of the T9 and T11 primary
pleural effusions grown as monolayer cultures revealed that
both contained mostly CD44+:CD24-/low cells (Fig. 1C).
When plated in ultra-low attachment plates, the cells formed
described below. Ponti et al. (45) reported similar results
with plural effusions; they managed to propagate tumorspheres
from only 3 [all estrogen receptor positive (ER+)] of 16
breast tumors.
Fig. 4. DPPE suppresses growth of tumorspheres derived from MMTV-Wnt1and MMTV-Neu mouse mammary tumor cells. A and B, tumorsphere formation assays (top)
for MMTV-Wnt1or MMTV-Neu tumor cells treated with the indicated drugs for 2 wk. The tumorspheres were dissociated and replated in the absence of drugs for additional
2 wk and then counted (middle) or subjected to MTTcell survival assays (bottom). Representative data of three independent experiments done in duplicates. *, P < 0.05,
relative dimethyformamide control. C and D, micrographs of MMTV-Wnt1or MMTV-Neu spheres (arrows) 2 wk after replating in drug free media. Original magnification, 20.
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Fig. 5. DPPE induces apoptotic death in MMTV-Wnt1and MMTV-Neu mouse mammary tumor cells. A, dose response of MMTV-Neu tumorsphere formation following
treatment with the indicated doses and drug concentrations. Representative data of three independent experiments done in duplicates. B, representative flow cytometry
profiles of Annexin V ^ stained MMTV-Neu tumorspheres treated for 2 wk with the indicated drugs. C, kinetics of apoptotic response of MMTV-Neu tumor cells to the
indicated drugs. Primary MMTV-Neu tumorspheres were dissociated into single cells, seeded onto ultra-low attachment plates, and continuously treated with the indicated
drugs. After 24 h, 72 h, 1wk, or 2 wk, the treated cells were dissociated, stained with 7-amino-actinomycin D (7AAD) and Annexin V, and analyzed by flow cytometry.
Points, mean AnnexinV staining of three independent experiments (n = 3) for each drug and time point; bars, SD. Relative AnnexinV was calculated by normalizing the data to
the levels of intrinsic apoptosis in dimethyformamide-treated cells. D, average levels of Annexin V staining following 2-wk treatment of Wnt1tumor cells with the indicated
drugs. Note moderate effect of DPPE on Wnt1tumor cells compared with Neu tumors (B and C). E, representative CD24-FITC and CD49f-PE flow cytometry profiles of
MMTV-Wnt1tumor cells treated with the indicated drugs for 2 wk under adherent conditions. F, average of three experiments showing the percentage of CD24+:CD49f+ cells
after 2-wk treatment with the indicated drugs. *, P < 0.05, relative to dimethyformamide control.
Clin Cancer Res 2009;15(1) January 1, 2009
126
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Preferential Killing of Breast TICs by DPPE/Tesmilifene
Fig. 6. Targeted killing of TICs by DPPE. Representative FvB female mice, 3 mo after injections with 250 live Her2/Neu tumorsphere cells pretreated for 2 wk with the
indicated drugs. Arrows, secondary tumors.
tumorspheres that consisted primarily of CD44+:CD24-/low
cells. Following extended culturing under adherent conditions,
there was a gradual loss of CD44+:CD24-/low cells and a
concomitant increase in the CD44-/low:CD24+ population; by
2 to 4 weeks, both cell populations were observed, and by
8 weeks (passage 4), most PE-T9 cells became CD44-:CD24+
cells (Fig. 1C). To directly test the relationship between
CD44+:CD24-/low and CD24+ cells and identify the cells
capable of forming spheres, T9 and T11 primary cells grown
as monolayer were isolated by fluorescence-activated cell
sorting on the basis of CD24 and CD44 expression and then
plated under adherent or nonadherent conditions (Fig. 2A).
The sorted CD44+:CD24-/low cells grew well as monolayer
cultures and formed spheres in nonadherent conditions
(Fig. 2B). In contrast, sorted CD24+ cells grew poorly under
either conditions and did not form spheres. Similar observations with immortalized human mammary epithelial cells have
recently been reported (46). Importantly, flow cytometry
analysis done 1 week after plating the sorted cells under
adherent conditions revealed that the CD44+:CD24-/low cells
gave rise to CD44+:CD24-/low as well as CD44low:CD24+/- cells,
expressing intermediate levels of CD24 (Fig. 2A, right). By
contrast, the CD44+:CD24+ cell population lost viability
and remained CD24+. Finally, to test the differentiation
capacity of the sorted CD44+:CD24-/low tumor cells, they were
seeded onto collagen 1 – coated coverslips, induced to differentiate, and immunostained with antibodies against smooth
muscle actin, cytokeratin 14 (myoepithelial markers), and
cytokeratin 18 (luminal marker). In four different sorting/
differentiation experiments, the CD44+:CD24-/low fractions
differentiated to express one or more of these markers in
>50% of the tumor cells (Supplementary Fig. S2). Thus, the
CD44+:CD24-/low cell population exhibited limited self-renewal
capacity under the conditions we used, gave rise to CD44+:
CD24+ cells, formed tumorspheres in culture, and had the
ability to differentiate into distinct cell types.
DPPE suppresses CD44 + :CD24 -/low breast tumor cells
from pleural effusions by inducing apoptotic death. We next
investigated the effect of DPPE on the T9 and T11 primary
pleural effusion cultures described above. The tumor cells
www.aacrjournals.org
were trypsinized to obtain single-cell suspension, plated in
ultra-low attachment plates, and treated with vehicle alone
(dimethyformamide) or physiologic attainable concentrations
of doxorubicin (0.05 Ag/mL), DPPE (5 Amol/L), or both.
Two weeks later, tumorsphere number and cell survival
(MTT assay) were determined. Doxorubicin treatment reduced
tumorsphere growth relative to vehicle-treated cultures by only
2-fold (Fig. 3A). In contrast, cultures treated with DPPE or
DPPE plus doxorubicin exhibited a 6-fold reduction in sphere
formation and viability.
To determine which cell population was targeted by DPPE,
the cultures were treated with the various drugs for 2 days
and stained with fluorescent-conjugated CD24 and CD44
antibodies. Flow cytometry analysis revealed that doxorubicin
Table 1. Summary of transplantation experiments
with MMTV-Wnt1 and MMTV-Neu tumorspheres
Treatment
Tumor/injections
250 cells
100 cells
3 mo
5 mo
3 mo
5 mo
MMTV-Wntl
DMF
Dox
DPPE
DPPE/Dox
2/6
1/6
1/6
0/6
3/6
1/6
2/6
0/6
3/6
0/6
0/6
0/6
4/6
1/6
2/6
0/6
MMTV-Neu
DMF
Dox
DPPE
DPPE/Dox
12/13
4/12
0/12
0/12
13/13
4/12
5/12
0/12
3/9
0/8
0/8
0/8
4/7
0/6
0/6
0/6
NOTE: Dissociated tumorsphere cells were seeded onto ultra-low
attachment plates and treated with the indicated drugs for 2 wk
(four changes). The cultures were then trypsinized, and live cells
were counted by trypan blue exclusion assay. The indicated
number of live cells was mixed with Matrigel and transplanted
into the #4 inguinal mammary glands of recipient female mice.
Incidence of tumors after 3 and 5 mo is indicated.
Abbreviations: DMF, dimethyformamide; Dox, doxorubicin.
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Clin Cancer Res 2009;15(1) January 1, 2009
CancerTherapy: Preclinical
treatment had only a moderate effect on the CD44+:CD24-/low
cell populations [1.2-fold reduction (P > 0.01) relative to
dimethyformamide control group; Fig. 3B and C]. By contrast,
DPPE reduced the ratio of CD44+:CD24-/low to CD24+ by 3.3fold (P < 0.01) and the absolute number of CD44+:CD24-/low
cell population by 3.8-fold (P < 0.01), indicating that DPPE
preferentially targets the CD44+:CD24-/low cell population.
To investigate the mechanisms by which DPPE suppressed
cell viability, the cultures were again treated with the various
drugs for 2 days, and then unfixed cells were stained with FITCCD24 and APC-CD44 as well as PE-Annexin V. Annexin V
detects phosphatidylserine residues that translocate to the outer
cell membrane during early stages of apoptosis. The intrinsic
level of cell death for the CD44+:CD24-/low cells (13.96 F
0.48%) was slightly lower than that for the CD44+:CD24+ cells
(18.06 F 3.99%; Fig. 3D). Doxorubicin treatment increased
apoptotic cell death by f2-fold in both cell populations. In
contrast, DPPE increased the rate of apoptosis in CD44+:CD24-/
low
cells nearly 2-fold (26.38 F 5.01%; Fig. 3D, right) but did
not increase apoptosis above background level in the CD24+
population (Fig. 3D, left). Treatment with DPPE plus doxorubicin slightly increased overall cell death in the CD44+:CD24-/
low
cell population (29.84 F 1.77%). Thus, DPPE induces
apoptotic cell death in CD44+:CD24-/low cells.
DPPE inhibits survival and cell cycle progression of Her2/neu
and Wnt1 mammary tumor cells. We next analyzed mammary
tumor cells derived from MMTV-Wnt1 and MMTV-Her2/neu
transgenic mice, in which TICs can be functionally analyzed
by transplantation into syngeneic mice (23). We first tested
the effect of DPPE on tumorsphere formation, apoptosis,
and cell cycle progression in vitro. Primary tumors from
MMTV-Wnt1 and MMTV-Neu were dissociated into single-cell
suspension by enzymatic treatment and then immunodepleted
for hematopoietic (CD45/Ter119 antibodies), endothelial
(CD31 antibody), and stromal (CD140a antibody) cells
(Supplementary Fig. S1C). The Lin- tumor cells were then
seeded into ultra-low attachment plates as described (23).
Primary tumorspheres from each tumor type were dissociated
by light trypsinization and replated in the presence of drugs
or vehicle alone. After 2 weeks of continuous treatment
(four doses), the number of tumorspheres in each culture was
assessed. As shown in Fig. 4A and B (top), DPPE treatment
alone reduced the number of tumorspheres in both the Neu
and WNt1 cultures. The effect of DPPE was more pronounced
in the Neu-induced tumors (<2 F 0.71% relative to dimethyformamide control) than in the Wnt1-induced tumors (39.5 F
1.41%). The tumorspheres were then dissociated and replated
in the absence of drugs; 2 weeks later, tumorspheres were either
counted or subjected to MTT assay to measure viability. Very
few tumorspheres arose in the DPPE-treated Neu cultures
whereas f23% of Wnt1 tumorspheres survived (relative to
vehicle-treated cultures). Similar inhibition of tumorsphere cell
viability was observed with the MTT assay (Fig. 4A and B,
bottom). Examples of the cultures treated with the various drugs
are shown in Fig. 4C and D.
We next determined the sensitivity of Neu tumorspheres
to different doses and concentrations of DPPE over a
2-week period. Continuous treatment with physiologically
relevant concentration of DPPE (i.e., four changes over
2 weeks; 5 Amol/mL) produced the strongest effect (Fig. 5A).
At a higher concentration (10 Amol/mL), DPPE suppressed
Clin Cancer Res 2009;15(1) January 1, 2009
tumorsphere formation even more efficiently (not shown). At
lower drug concentrations or doses, the effect of DPPE on
tumorsphere formation was significantly reduced. To determine
whether DPPE induces apoptotic cell death in mouse
mammary tumor cells as it does in human breast tumor cells
from pleural effusions (Fig. 3), tumorspheres were dissociated,
replated under nonadherent conditions, and treated with the
indicated drugs for different time periods. The cells were then
collected and stained with 7-amino-actinomycin D, which is
excluded by live cells, plus Annexin V, which specifically labels
apoptotic cells, and analyzed by flow cytometry. Continuous
exposure to DPPE induced massive cell death in Her2/neu
tumors, whereas doxorubicin, at the concentrations we
used, had only a moderate effect (Fig. 5B). Kinetic analysis
revealed that the induction of apoptosis by DPPE was modest
(50 F 5.8%) in the first 3 days of treatment and dramatically
increased >5-fold after 2 weeks of treatment (Fig. 5C).
In parallel experiments with Wnt1-induced tumor cells,
DPPE treatment for 2 weeks increased the level of apoptosis
by only 2.2-fold, compared with 1.5-fold induced by doxorubicin (F0.19; Fig. 5D). In MMTV-Wnt1 – induced mammary
tumors, TICs and TFUs are found in the CD24+:CD49f+ doublepositive cell population (23, 24).7 To test the effect of DPPE on
tumor cells by marker analysis, monolayer MMTV-Wnt1 tumor
cells were treated for 2 weeks with the various drugs and then
subjected to flow cytometry analysis with CD24 and CD49f.
The percentage of CD24+:CD49f+ cells in primary tumors
was initially f38% (data not shown) but was reduced to f3%
after 2 weeks in monolayer culture (Fig. 5E), indicating strong
selection against TICs. Treatment with doxorubicin increased
the percentage of CD24+:CD49f+ cells by f4-fold, suggesting
that this drug preferentially kills the CD24-:CD49f- non-TIC
population. By contrast, treatment with DPPE reduced the
percentage of CD24+:CD49f+ cells by 2-fold (Fig. 5E and F).
Thus, the tumorsphere formation assay, Annexin V staining,
and marker analysis indicate that (a) Neu tumor cells are more
sensitive than Wnt1 tumor cells to DPPE; (b) both Neu and
Wnt1 tumor cells are significantly more sensitive to DPPE than
to doxorubicin; and (c) continuous treatment with DPPE is
required for targeted killing of TFUs/TICs.
To test the effect of DPPE on cell cycle progression,
tumorspheres treated for 1 week with the indicated drugs were
stained with propidium iodide to label DNA content and
analyzed by flow cytometry. The percentage of cells in S/G2/M
phases was f15.92 F 2.94% and 8.96 F 0.21% for Wnt1 and
Neu tumorspheres, respectively (Supplementary Fig. S3).
Exposure to 0.05 Ag/mL doxorubicin had little effect on cell
cycle distribution of either culture. In contrast, treatment with
DPPE suppressed the S/G2/M phases by 33 F 3.35% and 59 F
13.43% in Wnt1 and Neu tumorspheres, respectively, suggesting that DPPE preferentially eliminates cycling cells.
Direct killing of Neu and Wnt1 TICs by DPPE. Whereas
tumorsphere formation can be used as a surrogate for tumor
initiation, the ultimate test for TICs is their ability to form
secondary tumors after transplantation into recipient mice.
Thus, to directly test the effect of DPPE on TICs, Neu or Wnt1
tumorspheres were dissociated and replated in the presence of
the various drugs under nonadherent conditions. After 2 weeks,
7
128
Our unpublished data.
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Preferential Killing of Breast TICs by DPPE/Tesmilifene
MMTV-Wnt1 mammary tumors as tested by both in vitro and
in vivo assays. In aggregate, our data suggest that DPPE directly
targets TICs. This model may explain the significant improvement in overall survival and progression-free survival observed
in the doxorubicin plus DPPE arm despite no objective
difference in tumor response (38), as targeted killing of rare
TICs may reduce additional metastasis and improve overall
survival without significantly affecting the bulk of metastatic
tumors.
The most parsimonious model for the cooperation between
DPPE and doxorubicin is that their individual effect on TICs is
simply accumulative. Each drug kills TICs, albeit with different
efficacy, and combined treatment with both drugs has an
additive effect. Another level of cooperation is suggested from
data in Table 1 showing that treatment with DPPE increases
the latency for tumor formation by 2.6-fold relative to
doxorubicin. The late onset of these tumors may also be
explained if the cancer stem cell hierarchy is not as stable as is
commonly thought (i.e., if non-TICs can revert to TICs by
genetic or epigenetic changes after a prolonged period of time
following transplantation; ref. 50). Thus, DPPE and doxorubicin may cooperate by targeting different cells: DPPE
preferentially targets TICs; doxorubicin preferentially kills
non-TICs (with the potential to revert); and combined
treatment eliminates both cell types, hence any tumorigenic
potential. Additional analysis will be required to address the
stability of the cancer stem cell hierarchy and its effect on
chemotherapy in the contexts of drugs, such as DPPE, that
target TICs.
We have shown that continuous exposure to high, pharmacologically attainable doses of DPPE (5 Amol/L) effectively kills
TICs in vitro. The need for continuous exposure may be the
hallmark of drugs that target TICs as these cells are thought to
undergo slow self-renewal. Our results suggest that DPPE-based
therapies should involve continuous or frequent administration
of the drug. This is notably a challenge given the side effect of
the drugs at the current dose (38), but may be overcome with a
better understanding of the pharmacokinetics of DPPE.
Alternatively, more potent DPPE derivatives with better
specificity may be developed. The identification of the cellular
target(s) of DPPE and the affected biological process/pathway
are therefore of paramount importance.
The variation in tumor response to DPPE likely reflects both
the percentage of TICs in a given tumor and the biology of TICs
from each individual breast cancer subtype. Breast carcinomas
represent several subtypes including ER+, Her2/Neu+, and
Basal-like (Her2/Neu-:PR-:ER-; ref. 51). We have found that
the metastatic breast tumors from pleural effusions and HER2/
Neu+ mammary tumors were highly sensitive to DPPE, Wnt1
TICs were less sensitive, whereas MCF7 sensitivity depended on
culture conditions (very low as monolayer; high as spheres).
The high sensitivity of HER2/Neu+ mammary tumors to DPPE
is consistent with the high frequency of TICs in this tumor
subtype (23, 31, 52). Consistent with the differential sensitivity
of different tumor types to DPPE, stratification of the clinical
results from the National Cancer Institute of Canada MA.19
trial revealed that ER-negative tumors responded better than the
entire tumor set (33). Given the high sensitivity of the Neu+
tumor cells to DPPE (Figs. 4 – 6; Table 1), it would be of interest
to determine the overall survival difference for Her2/Neu+
breast cancer patients treated with DPPE. In addition, the effect
the cultures were trypsinized and counted by trypan blue
exclusion assay; 50, 100, or 250 live cells were mixed with
Matrigel and injected into the mammary glands (#4) of
syngeneic FvB female mice. Tumor incidence after 3 and
5 months is depicted in Figure 6 and Table 1. Treatment
of Wnt1 tumorspheres with DPPE or doxorubicin reduced
tumor incidence to 4/12 and 2/12, respectively, relative to
vehicle-treated cultures (7/12 tumors after injections of 250 or
100 cells). Treatment with both drugs completely abolished
tumor initiation (0/12; Table 1).
With Neu tumorsphere cells, doxorubicin treatment alone
reduced but did not completely eliminate tumor incidence
3 months after transplantation (4/18 total injections of 250 or
50 cells) as compared with vehicle-treated cells (15/20). In
contrast, DPPE-treated tumorsphere cells did not give rise
to tumors at this stage (0/18; Table 1). Figure 6 shows
representative recipient mice 3 months after transplantation
with Neu tumor cells pretreated with the indicated drugs. After
2 additional months (i.e., 5 months posttransplantation), five
tumors appeared in the DPPE-treated cells. The latency of
tumor formation following DPPE treatment was 2.6-fold
longer than following doxorubicin treatment. Specifically, the
latencies for tumor formation in the dimethyformamide-,
doxorubicin-, and DPPE-treated groups were 63.6 F 13.7,
51.7 F 3.5, and 137.2 F 11.7 days, respectively. Combined
treatment of the Neu tumor cells with DPPE plus doxorubicin
completely abolished TICs (0/18 injections in total). We
conclude that DPPE effectively kills TICs and that combined
treatment with DPPE plus doxorubicin cooperates to eradicate
both Wnt1 and Neu TICs.
Discussion
Here we show that DPPE (tesmilifene) can act as a single
agent to target TICs. It induces apoptotic death in TICs when
administered alone and, in certain contexts, cooperates with
doxorubicin to further kill these tumorigenic cells. Our
observation that DPPE can directly target TICs questions
previous models that implicated DPPE in the potentiation of
cytotoxicity by antineoplastic drugs (33). These models were
strengthened by the observation that the molecular structure of
DPPE includes a tertiary amine, phenyl rings, and carbonyl
group often found in p-glycoprotein inhibitors (47 – 49).
However, DPPE also enhances cytotoxicity of multidrug
resistance – negative tumor cells, and the enhancement (or
cooperation) with other drugs is not accompanied by increased
intracellular concentration of these drugs (42). In fact, it
was hypothesized that DPPE may activate, rather than inhibit,
p-glycoproteins (33). We propose that the effect of DPPE as
a single agent has been overlooked in previous experiments
because TICs were not assayed directly. As we observed, DPPE
alone had a minor effect on MCF7 cells grown as monolayer, in
which only a very small fraction of the cells are TICs. By
testing for TICs directly, for example by quantifying the ability
of MCF7 breast tumor cells to form tumorspheres under
nonadherent conditions, we were able to show efficient and
direct killing of TICs by DPPE. In addition to MCF7 cells,
specific killing of TICs by DPPE was observed with two
independent metastatic breast tumors from breast cancer
patients with malignant pleural effusions. Finally, we showed
that DPPE specifically killed TICs from MMTV-Neu and
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129
Clin Cancer Res 2009;15(1) January 1, 2009
CancerTherapy: Preclinical
of DPPE on HER2+ and ER+ tumors may be tested in
combinations with anthracyclin plus herceptin, lapatinib plus
capecitabine, or hormonal therapies, respectively.
In conclusion, using four different breast tumor models,
we showed that DPPE directly kills TICs and, in certain
contexts, cooperates with doxorubicin to further eliminate
these tumorigenic cells. A continuous exposure to 5 Amol/L
concentration of the drug is required for optimal results. Thus,
DPPE may serve as a prototype TIC-specific inhibitor with
available clinical data.
Disclosure of Potential Conflicts of Interest
K.I. Pritchard is a member of the speakers bureau of Aventis, Pfizer, AstraZeneca,
and Pharmacia. She is a consultant for Aventis, Roche, Pharmacia, Ortho-Biotech,
Pfizer, GSK Abraxis,YM Biosciences, and Biomera.
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