Müllerian inhibiting substance preferentially inhibits
stem/progenitors in human ovarian cancer cell lines
compared with chemotherapeutics
Xiaolong Weia,1, David Dombkowskib, Katia Meirellesa, Rafael Pieretti-Vanmarckea, Paul P. Szoteka, Henry L. Changa,
Frederic I. Prefferb, Peter R. Muellerc, Jose Teixeirad, David T. MacLaughlina, and Patricia K. Donahoea,1
aPediatric Surgical Research Laboratories, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02114;bFlow Cytometry Laboratory,
Department of Pathology and Center for Regenerative Medicine, Massachusetts General Hospital, Boston, MA 02114; andcAbdominal Imaging and
Intervention Division anddVincent Center for Reproductive Biology, Massachusetts General Hospital, Boston, MA 02114
Contributed by Patricia K. Donahoe, September 2, 2010 (sent for review July 2, 2010)
somatic stem cells, are thought to be capable of unlimited self-
renewal and, when stimulated, proliferation and differentiation.
initially selected from 95 human cell surface antigens as each was
shared among human ovarian primary cancers, ovarian cancer cell
lines, and normal fimbria. A total of 150 combinations of markers
were reduced to a panel of three—CD44, CD24, and Epcam—which
selected, in three ovarian cancer cell lines, those cells which best
formedcolonies. Cells expressing CD44,CD24,and Epcamexhibited
stem cell characteristics of shorter tumor-free intervals in vivo after
Also, doxorubicin, cisplatin, and paclitaxel increased this enriched
population which, conversely, was significantly inhibited by Mülle-
rian inhibiting substance (MIS) or the MIS mimetic SP600125. These
findings demonstrate that flow cytometry can be used to detect
a population which shows differential drug sensitivity, and imply
that treatment of patients can be individualized to target both
stem/progenitor cell enriched and nonenriched subpopulations.
The findings also suggest that this population, amenable to isola-
tion by flow cytometry, can be used to screen for novel treatment
paradigms, including biologic agents such as MIS, which will im-
prove outcomes for patients with ovarian cancer.
a stem cell disease (1–4), it is becoming increasingly im-
portant to be able to identify cancer stem/progenitor cells and to
develop treatment modalities that specifically target the stem cell
enriched population, coupled with treatments effective against
the larger population not enriched for stem cells. The concept of
cancer stem cells has opened new areas of research in carcino-
genesis, but has the more immediate translational potential of
uncovering new treatment targets.
We previously identified somatic label-retaining cells with stem
cell features in ovarian surface epithelium (5), and with others (6,
7), postulate that somatic stem cells or their immediate progeni-
tors can revert to cancer stem cells (8). It is also possible that the
stem cells may remain the same, but that signals which control the
stem/progenitor cell activity may change. Several recent studies
have demonstrated that cancer stem cells may confer chemo-
therapeutic resistant ovarian tumor growth and metastasis (2, 9).
Ovarian cancer is diagnosed in approximately 25,000 new
cases per year in the United States and is associated with a 50%
mortality rate (10, 11); more than 90% of cases are epithelial in
origin (12, 13). Epithelial ovarian cancers fall into four main
subtypes: mucinous, endometrioid, clear cell, and serous (14).
Serous ovarian carcinomas (15, 16) account for the overall high
ovarian cancer-related mortality. Fewer than 25% are detected
s evidence is accumulating to indicate that cancer could be
at an early stage; thus, there is an urgent need for better pre-
dictive molecular markers that characterize early oncologic
transformation (17) to permit earlier detection, to uncover ad-
ditional therapeutic targets, and to change therapeutic protocols.
“Side population” (SP) cells identified by Hoechst 33342 dye ex-
clusion in a wide range of cancers were found to be enriched for
cancer stem cells (1, 18, 19). We found that SP cells form larger
tumors and have higher tumorigenic propensity than do non-SP
(NSP) cells, but did so in mouse ovarian cancer cell lines (MOV-
CAR7). These stem/progenitor cells were inhibited by Müllerian
inhibiting substance (MIS), whereas the lipophilic chemotherapeu-
tic agent doxorubicin more significantly inhibited the NSP cells (4).
These findings predict that chemotherapeutic agents and MIS may
differentially affect populations in human ovarian cancer that are
relatively chemoresistant and demonstrate stem cellcharacteristics.
In the present study, we performed flow cytometry and FACS
to screen for stem cell markers and identified stem/progenitor
cellenrichedpopulations asdefinedby functional assaysofcolony
formation and invasion in vitro, and by limiting dilution implan-
attempt to define other markers specific to enriched stem/pro-
genitor cell populations in these epithelial cancers for later use in
screening human patients, 130 markers were screened and those
amenable to flow cytometry and FACS (n = 95) (Fig. 1A), and
conserved between patient primary ovarian cancer cells in ascites,
human ovarian cancer cell lines, and the epithelium of normal
human fimbria, were selected for further study (Fig. 1A). Eight
surface markers conserved in all three sources were tested in
various combinations and, of these, CD44+CD24+Epcam+most
consistently enriched for a population capable of colony growth.
These “triple-positive” (3+) cells also had a shorter tumor-free
interval in vivo when xenotransplanted by limiting dilution, and
migrated better in invasion assays in vitro than did triple-negative
(3−) cells. Further, we assessed the effects of chemotherapeutic
agents and MIS on survival of these ovarian cancer cell pop-
ulations enriched or not enriched for stem-like cells. From these
studies,wedemonstrated that, althoughchemotherapeutic agents
significantly inhibited viable nonenriched populations, they con-
sistently enhanced stem/progenitor cell enriched populations.
By contrast, MIS and its anthrapyrazolone small molecule ago-
nist, SP600125, preferentially inhibited the stem/progenitor cell-
enriched population. These differential results suggest that che-
motherapeutic drugs and MIS should in the future be studied to
determine if they can function in rationally selected combinations
Author contributions: X.W., F.I.P., J.T., D.T.M., and P.K.D. designed research; X.W., D.D.,
K.M., R.P.-V., P.P.S., and H.L.C. performed research; D.D., F.I.P., P.R.M., and J.T. contrib-
uted new reagents/analytic tools; X.W., D.D., K.M., R.P.-V., P.P.S., H.L.C., D.T.M., and P.K.D.
analyzed data; and X.W. wrote the paper.
The authors declare no conflict of interest.
1To whom correspondence may be addressed. E-mail: email@example.com or pdonahoe@
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
| November 2, 2010
| vol. 107
| no. 44www.pnas.org/cgi/doi/10.1073/pnas.1012667107
to assure that both populations are effectively targeted (20, 21).
Identification of a readily definable stem cell like population that
is amenable to isolation by flow cytometry will aid in the de-
velopment of future therapeutic strategies.
In Fig. 1B, the left arm indicates that unseparated cells were
cultured and treated with chemotherapeutic agents, MIS, or ve-
hicle alone for 3 d. Flow cytometry analyzed 50,000 cells for via-
bility and for percent of cells in side population relative to NSP
or expressing Epcam+, CD44+, and CD24+relative to those not
expressing these three markers.
The right arm reflects cells separated by FACS and analyzed
after using 150 combinations of eight markers. Combinations
with the most robust colony growth in all cell lines were chosen
for functional testing in vitro by invasion and proliferation assays,
and by in vivo assays to determine tumor-free interval after in-
jection of cells subjected to limiting dilutions.
SP of Ovarian Cancer Cell Line Is Enhanced After Treatment with
Chemotherapeutic Agents. The verapamil-sensitive SP of human
ovarian cancer cell lines was selected by exclusion of the DNA-
binding Hoechst 33342 dye (Fig. S1). SKOV-3, OVCAR-5, and
IGROV-1 cells, treated for 3 d with doxorubicin, showed an in-
creased percentage of SP cells (Fig. S2 A and C) and a decreased
percentage of NSP cells. OVCAR-5 and IGROV-1 cells treated
with cisplatin also significantly increased the percentage of SP
cells in OVCAR-5 (Fig. S2 B and D) and in IGROV-1 while de-
percentage of SP cells in OVCAR-5 and SKOV-3 cells (Fig. S2 B
and D). Chemotherapeutic agents dose-dependently reduced the
total number of cells after 3 d of treatment (Table S1).
MIS and Its Anthrapyrazolone Agonist, SP600125, Diminish SP Cells.
The receptor-mediated ligand MIS, compared with vehicle alone,
decreased the percentage of SP cells (Fig. S3 A and C) in the
human ovarian cancer cell lines known to be responsive to MIS in
vitro or in vivo, namely OVCAR-5 (20) and IGROV-1 (21, 22).
SP600125, an anthrapyrazolone, discovered in a small molecule
screen to act as an MIS agonist in MIS type II receptor (23, 24),
reduced the percentage of SP cells in OVCAR-5 and IGROV-1
(Fig. S3BandDandTableS1).SP600125 alsodecreased the NSP
of IGROV-1 cells, but at higher doses (Fig. S3D). Thus, different
from chemotherapeutic agents, MIS and its agonist SP600125,
after 3 d of treatment in culture, significantly reduce the ratio of
viable SP cells of a number of human ovarian cancer cell lines.
Enrichment of Cancer Stem Cell Populations in Human Ovarian Cancer
Cell Lines by Selection with a Marker Panel Compatible with Flow
Cytometry. Primary ascites from three patients with ovarian cancer
were screened in flow cytometry for relative binding of 95 fluo-
rescently labeled surface markers. After negative selection for
CD45 and CD31 cells, the panel was narrowed to 24 cell surface
markers reconfirmed in five ovarian cancer cell lines (Fig. 1A). A
panel of eight surface markers consistently found in both primary
cancers and cancer cell lines, as well as normal Fallopian tube in-
fundibular cells (Fig. 1A), was further tested in more than 50 dif-
ferent combinations (Table S2) for their ability to form colonies
(25) in each of three cell lines, OVCAR-5, IGROV-1, or SKOV-3
cells (Fig. 1B, right arm). The CD44+CD24+Epcam+population
formed morelarge colonies after 14d thandidothercombinations
CD44+CD24+Epcam+population, and found that the 3+ cell
there is an overlap between SP and CD44+CD24+Epcam+cells.
Triple-Positive Cells Invade Matrigel More Effectively than Do 3−
Cells. CD44+CD24+Epcam+cells in serum-free media invade
through an extracellular matrix-coated membrane insert after in-
cubation for an additional 72 h in serum containing media. When
stained and then quantitated by ImageJ software (National Insti-
tutes of Health), 3+ cells showed significantly more invasion
Triple Positive Cells Injected into the Right Flank of NOD/SCID Mice
Grew Tumors Earlier than Did Triple Negative Left Flank Cells.
CD44+CD24+Epcam+cells grew tumors earlier at comparable
cell dilutions (103and 102) than did 3− cells. Tumor-free inter-
vals were then subjected to Kaplan-Meier analysis and log-rank
(Mantel–Cox) and Gehan–Breslow–Wilcoxon tests for signifi-
cance. The tumor free interval was shorter (P = 0.015 for 103
cells, P = 0.049 for 102cells; n = 5 animals for each) for the 3+
cells compared with 3− cells (Fig. 2C).
Marker Panel-Selected Cells Are Diminished by MIS and SP600125.
After treatment with chemotherapeutic agents or MIS, remaining
viable populations of OVCAR-5 cells were selected by flow
markers conserved between
primary ascites from ovarian
cell lines, and normal human
Fallopian tube fimbria. Com-
binations of eight conserved
ina flowchart of experimental
(A) Selection of hu-
Wei et al. PNAS
| November 2, 2010
| vol. 107
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cytometry for 3+ markers (Fig. 1B, left arm), and the remaining
cell numbers determined and compared with those cells that were
3−. There was a dramatic expansion of the ratio of 3+ OVCAR-5
cells (Table S4, right column) when treated with doxorubicin
S4) whereas the total number of viable cells slightly decreased. By
comparison, both the total numbers of viable unseparated cells
and 3+ separated cells decreased significantly when treated with
MIS or SP600125 (Fig. 3 C and D and Table S4).
Effects of Chemotherapeutic Agents and MIS and Its Agonist on Cell
Survival of 3+ and 3− Cells. We tested the effect of chemothera-
peutic agents and MIS on the survival of 3+ and 3− populations
in 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
(MTT) assays. Separated OVCAR-5 cells plated at 2,000 cells
per well were treated with different doses of doxorubicin for 3 d.
Doxorubicin slightly inhibited the survival of 3+ cells (P < 0.05),
but more strongly inhibited 3− cells (P < 0.01) (Fig. 4A, Upper),
as did cisplatin (Fig. 4A, Lower). Similar results were seen on
cells separated by FACS into NSP (Fig. S5A), which were more
inhibited than SP by doxorubicin and cisplatin. These re-
sults indicate that chemotherapeutic agents preferentially inhibit
3− and NSP (Fig. S5A), and that 3+ and SP cells show relative
resistance to chemotherapeutic agents.
The 3+ and 3− cells from OVCAR-5 cells were treated with
3+ cells at lower doses than those required to inhibit 3− cells, al-
7 d). SP600125 also significantly inhibited growth of 3+ cells (P <
0.01), but required higher doses to inhibit 3− cells, although both
were significant (Fig. 4B, Lower, and Fig. S5D). Survival of SP cells
It is essential to identify ovarian cancer populations enriched
for tumor-initiating cells and, at the same time, to elucidate the
molecular mechanisms that regulate normal self-renewal or dif-
ferentiation, and to determine how such mechanisms can be co-
opted or changed to contribute to transformation and drug re-
sistance and whether modulation of these mechanisms will affect
patient outcome. SP cells have been described as being enriched
for cancer stem cells and characterized in several mammalian
tissues or cancers by efflux of lipophilic substrates, including the
cell-permeable DNA-specific bisbenzimidazole dye Hoechst 33342
and some chemotherapeutic agents (1, 4, 5, 19, 26–28). Having
identified SP cells in mouse ovarian cancer cell lines (4) and shown
that they were more tumorigenic than NSP cells, we now show
tumor free interval in vivo. (A and B) Colony growth of OVCAR-5 (A) and SKOV-3 (B) in six-well plates. CD44/CD24/Epcam 3+ (Upper) gave optimal colony
growth compared with 3− (Lower), as quantified on the right. (B) The 3+ cells isolated from SKOV-3 and plated on a membrane insert coated with ECM
invaded to the undersurface of the membrane better than did 3− cells (*P < 0.05; n = 3). (C) The 3+ cells sorted from OVCAR-5 and serially diluted and injected
s.c. into 5-wk-old female NOD/SCID mice (n = 5 mice for each group) grew tumors more readily than did 3− cells. (Bars indicate SD.)
Characterization of populations enriched for stem/progenitor cells in human ovarian cancer cell lines by colony growth and invasion in vitro and by
| www.pnas.org/cgi/doi/10.1073/pnas.1012667107 Wei et al.
that SP populations existing in human ovarian cancer cell lines
(OVCAR-5, IGROV-1, and SKOV-3) form more colonies than
NSP cells and are resistant to chemotherapeutic agents (doxoru-
bicin, cisplatin, and paclitaxel), but more sensitive to MIS and its
To consider other therapeutic agents that can target SP cells
agents and tested the effects of MIS, as mouse ovarian surface
epithelium expresses MIS RII receptors (21, 29, 30), as do human
ovarian cancer cell lines and primary ovarian cancer ascites cells
(20). We found that MIS and its small molecule MISRII receptor-
dependent agonist, SP600125 (23), significantly decreased SP cell
percentages in OVCAR-5, IGROV-1, and SKOV-3.
As little is known about markers more specific for the stem cell
population of human ovarian cancer, we performed a screen of
95 flow cytometry-compatible cell surface markers that were
conserved in human primary ovarian cancer ascites and in human
ovarian cancer cell lines (OVCAR-3, OVCAR-5, OVCAR-8,
SKOV-3, and IGROV-1; Fig. 1A). Eight of these (Epcam, CD24,
CD44, CD90, CD105, CD133, E-cadherin, and SP) were pre-
served in normal human fimbria from 16 patients undergoing
excision for benign disease, as fimbria removed prophylactically
with ovaries in patients with familial breast cancer were found
to be the site of occult tumors bearing characteristics of serous
cystadenocarcinoma of the ovary (31–33). Normal infundibulum
as a surrogate for ovarian surface epithelium contained a signifi-
cant SP after enzymatic digestion of the human normal fimbria,
and therefore was used to screen cancer stem cell surface markers
that could be subsequently used in human patient ascites.
enriched stem cell populations in human ovarian cancer cell lines. OVCAR-5
cells were treated with increasing doses of doxorubicin (A), cisplatin (B), MIS
(C), or SP600125 (D) for 3 d and stained with a combination of CD44, CD24,
and Epcam antibodies. A total of 50,000 cells were tested by flow analysis for
viability. Percentages of marker panel selected populations were determined
and fold changes calculated (n = 3 separate experiments performed in tripli-
cate at each dose for each drug).
MIS and its agonist SP600125 decrease the percentage of marker-
marker-enriched stem cell populations. CD44/CD24/Epcam 3+ and 3− cells
were sorted from OVCAR-5 cells by FACS and tested in MTT assays. (A)
Doxorubicin treatment (10, 30, 60 nM) and cisplatin treatment (0.5, 1, 2 μM)
inhibited the proliferation of 3− cells compared with 3+ cells (*P < 0.05; **P <
0.01). (B) MIS treatment (37.5, 112.5, 225 nM) and SP600125 treatment (5, 10,
16 μM), by contrast, inhibited proliferation of the 3+ population significantly
more than the 3− population (*P < 0.05; **P < 0.01). (Bars indicate SD.)
Chemotherapeutic agents and MIS differentially affect survival of
Wei et al.PNAS
| November 2, 2010
| vol. 107
| no. 44
Several of these markers have been identified in human cancer
(34–41). For example, Epcam (CD326) is a glycosylated type I
membrane adhesion molecule expressed in a variety of human
epithelial tissues, cancers, and stem cells (39). CD44 is the re-
37). Epcam/CD44 expression profiles are significantly higher in
colorectal primary tumors than in normal colonic tissues, and
tumors originating from Epcam+/CD44+cells generated the full
morphologic and phenotypic heterogeneity of their parental
lesions (36). CD133 (prominin-1) originally used to isolate he-
matopoietic and endothelial progenitor cells, was subsequently
found to be a marker of tumor-initiating cells in a number of
other human cancers (9, 35, 42–44). CD24, a mucin-like adhesion
molecule, is highly expressed in a large variety of human cancers
and contributes to tumor growth and metastases by binding to
platelet-selectin (45, 46). Like CD44+cells (47), CD24+cells
(48) from human ovarian tumors were recently reported to be
enriched in cancer stem cells and to form tumor xenografts in
nude mice, whereas CD24−cells were nontumorigenic. Stem cells
from different cancers may have different phenotypes, i.e., breast
cancer stem cells are CD44+CD24lowand colorectal cancer-
derived cell lines are CD44+CD24+(49). Our findings demon-
strate that a phenotype of CD44+CD24+Epcam+is enriched for
stem/progenitor cells in human ovarian cancer cell lines.
E-cadherin (CD324), a ligand for integrin α-E/β-7, encodes
a glycoprotein with five extracellular cadherin repeats. Loss of
E-cadherin is associated with epithelial-to-mesenchymal trans-
formation and cancer progression by increasing proliferation,
invasion, and/or metastasis (50, 51).
After detecting verapamil-sensitive SP cells in OVCAR-3,
SKOV-3, and IGROV-1, in multiple replicates, we used SP for
comparison while developing a panel of surface markers that
would allow us to recover live cells in which to examine molec-
ular mechanisms and to observe the response of separated cells
to drugs, for later use in human patients.
Colony formation assays used to screen the eight marker panel
on three human ovarian cancer cell lines showed that the combi-
nation of Epcam+, CD24+, and CD44+formed more colonies
than other marker combinations. It was necessary to use this 3+
panel in combination, as each marker alone was not sufficiently
selective (Table S3).
As chemotherapeutic agents have been shown in other cancers
to contribute to resistance (37, 52) and to enrich for stem cell
characteristics (37), we examined the effects of chemotherapeutic
agents and MIS and demonstrated that chemotherapeutic agents
preferentially inhibited the population not enriched for tumor
initiating cells; in contrast, receptor-mediated MIS and its agonist
SP600125 inhibited significantly the populations enriched for tu-
mor-initiating cells. The fact that these enriched populations
showed resistance to chemotherapeutic agents, but maintained
sensitivity to MIS and to SP600125, indicates that there is exper-
imental rationale to use this combination of markers to study
for stem-like cells. Furthermore, there is a need to design in-
dividualized treatment which specifically addresses the patient-
specific population enriched for tumor initiating cells. This ap-
proach will hopefully lead to improved outcomes for patients with
ovarian cancer. In addition, the panel-selected epithelial cells can
now be used as an experimental tool to examine for variations
(53, 54) that highlight mechanistic differences that could serve
as future therapeutic targets. Furthermore, this marker panel is
immediately translatable to the clinic to select patient-specific
Materials and Methods
Cell Lines, Chemotherapeutic Agents, and MIS. Human ovarian cancer cell
lines—OVCAR-5 (55, 56), SKOV-3 (57, 58), and IGROV-1 (59) cells—were
maintained in the pediatric surgical research laboratories as previously de-
scribed (4, 21). Cells were treated with doxorubicin, cisplatin, paclitaxel
(all from NovaPlus), MIS, or SP600125 (Sigma) (23). MIS was purified in
our laboratory, and its bioactivity assessed in embryonic Müllerian duct
regression assays (60, 61). The treatment doses were selected (21) around the
IC50for each drug for each cell line (10–60 nM for doxorubicin, 0.5–2 μM for
cisplatin, 0.5–4 nM for paclitaxel, 37.5–225 nM for MIS, and 5–16 μM for
Harvesting of Primary Human Ovarian Cancer Ascites. Primary ascites were
removed therapeutically from patients with ovarian cancer at the Massachu-
setts General Hospital [institutional review board (IRB) approval 2007-P-
001918], centrifuged at 450 × g for 20 min, resuspended in DMEM/F-12 me-
dium, filtered using a sterile cell strainer (70-μm nylon mesh; Fisher Scientific),
recentrifuged at 225 × g for 5 min, resuspended with ammonium chloride
solution to lyse red blood cells, diluted with DMEM/F-12 medium, and recen-
trifuged. Cells were then washed twice in the same media, stained with
antibodies for 20 min at 4 °C, washed again, and resuspended in PBS solution
for immediate flow analysis.
Digestion of Normal Fimbria Epithelium. Normal human Fallopian tube fimbria
removed from sixteen patients undergoing surgery for benign uterine dis-
ease at the Massachusetts General Hospital (IRB approval 2007-P-001918/4) or
at Dana Farber Cancer Institute (IRB Legacy 02–0251) were enzymatically
digested at 37 °C in 0.2% (wt/vol) with collagenase type II (type II in DMEM;
Gibco-BRL) for 30 to 45 min, followed by dilution with DMEM/F-12 medium.
Normal epithelial cells, released by scraping the fimbria, were collected,
centrifuged at 225 × g for 5 min, resuspended in DMEM/F-12 medium, fil-
tered through a sterile cell strainer (70 μm nylon mesh), and treated in the
same manner as the ascites cells before flow cytometric analysis.
Flow Cytometry and Cell Number After Treatment of Cell Lines. Cells were
plated in T75 flasks at 10% to 15% confluency (Fig. 1B, left arm), then treated
with increasing doses of doxorubicin, cisplatin, paclitaxel, MIS, SP600125, or
vehicle alone for 3 d. Flow cytometry was performed in the Massachusetts
General Hospital Department of Pathology Flow Cytometry Laboratory us-
ing a custom designed high-resolution 7-laser LSR (BD Science) (4, 62). Cells
were stained with Hoechst dye 33342 (Invitrogen) for 90 min at 37 °C, or
antibodies to the markers for 30 min at 4 °C. A total of 50,000 cells were
tested by flow analysis for viability using 7-AAD (Sigma) and for verapamil-
sensitive (50–100 μg/mL; Invitrogen) exclusion of Hoechst dye or for binding
of optimal marker combinations (as detailed later).
Colony Formation to Select Optimal Combination of Surface Markers. Single
and stained with a variety of combinations of antibodies to 95 markers to
choose eight markers conserved in human ovarian cancer cell lines, primary
ascites, and primary human fimbria epithelial cells and also detected on other
cancer stem cells: anti-human CD24-PE (eBioscience), anti-mouse/human
CD44-APC/Cy7 or CD44-Alexa Fluor–700 (BioLegend), anti-human CD90-APC
(BioLegend), anti-human CD105-PE (Invitrogen), anti-human CD133-APC
(BioLegend), anti-human Epcam-Alexa Fluor–647 or Epcam-APC (BioLegend),
and anti-human E-cadherin-FITC (CD324; BioLegend; Fig. 1B, right arm).
(For additional antibodies tested, see link to Stem Progenitor Cell Marker
Panel at www.massgeneral.org/children/research/researchers/wei_xiaolong.
aspx.) Cells sorted from the three cell lines were separated by various markers
and selected for viability, plated on six-well plates (500, 1,000, or 2,000 cells/
well), and incubated for 14 d at 37 °C, with a media change at day 7. At
day 14, cells were washed, fixed with methanol, stained with Giemsa, and
analyzed with ImageJ (National Institutes of Health).
Effects of Treatment on Cell Survival. The SP and NSP populations and the 3+
and 3− populations, after sorting, were tested in a cell survival tetrazole
MTT assay as previously described (21) at 2,000 cells per well in 200 μL of
media per well after treatment for 3 d with different doses of chemother-
apeutic agents (doxorubicin, cisplatin, paclitaxel), MIS, and the MIS agonist
SP600125, then compared with an appropriate vehicle as control.
Cell Invasion Assay. In vitro cell invasion assay was performed according to the
manufacturer’s protocols of an ECM invasion assay kit (Millipore), comparing
3+ versus 3− populations.
Tumor-Free Interval or Time to Appearance of Xenotransplanted Tumors After
Limiting Dilution. The 3+ and 3− cell populations were isolated from OVCAR-5
cell lines by FACS, serially diluted (105, 103, 102cells), resuspended in 1:1 PBS/
Matrigel (BD Biosciences) at 4 °C in 200 μL, and injected s.c. into the right
flank and 3− into the left flank of 4- to 6-wk-old female NOD.CB17-Prkdc/
| www.pnas.org/cgi/doi/10.1073/pnas.1012667107Wei et al.
Scid/J mice (Jackson Laboratory) under a protocol approved by the Massa-
chusetts General Hospital Institutional Animal Care and Use Committee (IRB
approval 2009 N000033/1). Mice were monitored weekly for tumor forma-
tion, and time of appearance was recorded after euthanasia by CO2 in-
halation. Tumors were measured and then fixed or frozen for further study.
Statistical Analysis. Univariate two-tailed t tests were used (4, 21) to com-
pare MIS and drugs with vehicle as a control. Kaplan-Meier and log-rank
(Mantel–Cox) and Gehan–Breslow–Wilcoxon analyses were used to compare
differences of time to tumor appearance. Analyses were performed using
Prism 5 software for Mac OSX (version 5.0a; GraphPad Software).
ACKNOWLEDGMENTS. We thank Caroline Coletti for excellent editorial
support. This work was supported by a Massachusetts General Hospital
Executive Committee on Research Award for Medical Discovery (to X.W.),
National Institutes of Health Grant R01 CA17393 (to P.K.D. and D.T.M.),
Harvard Stem Cell Institute (P.K.D.), Ovarian Cancer Foundation (D.T.M.),
and gifts from Commons Development, the McBride Family Foundation, the
Austen Foundation, and the Surdna-Gar Foundation.
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Wei et al.PNAS
| November 2, 2010
| vol. 107
| no. 44