Download Side Population Is Enriched in Tumorigenic, Stem

Research Article
Side Population Is Enriched in Tumorigenic, Stem-Like Cancer
Cells, whereas ABCG2+ and ABCG2 Cancer Cells Are
Similarly Tumorigenic
1
1
1
1
Lubna Patrawala, Tammy Calhoun, Robin Schneider-Broussard, Jianjun Zhou,
1
1,2
Kent Claypool, and Dean G. Tang
1
Department of Carcinogenesis, Science Park-Research Division, The University of Texas M.D. Anderson Cancer Center, Smithville, Texas
and 2Program in Environmental and Molecular Carcinogenesis, Graduate School of Biomedical Sciences, Houston, Texas
Abstract
Introduction
Recently, several human cancers including leukemia and
breast and brain tumors were found to contain stem-like
cancer cells called cancer stem cells (CSC). Most of these CSCs
were identified using markers that identify putative normal
stem cells. In some cases, stem-like cancer cells were
identified using the flow cytometry-based side population
technique. In this study, we first show that f30% of cultured
human cancer cells and xenograft tumors examined (f30 in
total) possess a detectable side population. Purified side
population cells from two cell lines (U373 glioma and MCF7
breast cancer) and a xenograft prostate tumor (LAPC-9) are
more tumorigenic than the corresponding non–side population cells. These side population cells also possess some
intrinsic stem cell properties as they generate non–side
population cells in vivo, can be further transplanted, and
preferentially express some ‘‘stemness’’ genes, including Notch1 and b-catenin. Because the side population phenotype is
mainly mediated by ABCG2, an ATP-binding cassette halftransporter associated with multidrug resistance, we subsequently studied ABCG2+ and ABCG2 cancer cells with respect
to their tumorigenicity in vivo. Although side population cells
show increased ABCG2 mRNA expression relative to the non–
side population cells and all cancer cells and xenograft tumors
examined express ABCG2 in a small fraction (0.5-3%) of the
cells, highly purified ABCG2+ cancer cells, surprisingly, have
very similar tumorigenicity to the ABCG2 cancer cells.
Mechanistic studies indicate that ABCG2 expression is
associated with proliferation and ABCG2+ cancer cells can
generate ABCG2 cells. However, ABCG2 cancer cells can
also generate ABCG2+ cells. Furthermore, the ABCG2 cancer
cells form more and larger clones in the long-term clonal
analyses and the ABCG2 population preferentially expresses
several ‘‘stemness’’ genes. Taken together, our results suggest
that (a) the side population is enriched with tumorigenic
stem-like cancer cells, (b) ABCG2 expression identifies mainly
fast-cycling tumor progenitors, and (c) the ABCG2 population contains primitive stem-like cancer cells. (Cancer Res 2005;
65(14): 6207-19)
Note: J. Zhou is currently at the Dermatology Branch, National Cancer Institute,
NIH, Building 10, Room 12N262, 10 Center Drive, MSC 1908, Bethesda, MD 20892-1908.
Requests for reprints: Dean Tang, Department of Carcinogenesis, Science ParkResearch Division, The University of Texas M.D. Anderson Cancer Center, 1808 Park
Road 1C, Smithville, TX 78957. Phone: 512-237-9575; Fax: 512-237-2475; E-mail: dtang@
mdanderson.org.
I2005 American Association for Cancer Research.
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Stem cells, which have now been found in multiple adult tissues
and organs, have several fundamental properties. First, they are
generally very rare. For example, the long-term hematopoietic stem
cells (LT-HSC) in mouse bone marrow constitute f0.02% and the
short-term HCSs (ST-HSC) f0.1% of the total cells (1). Second,
stem cells in their normal microenvironment (i.e., niche) rarely
divide, although they possess tremendous proliferative potential
(2). Third, stem cells can self-renew; that is, they can regenerate
themselves when they divide to give rise to progenitor cells (2).
Fourth, stem cells possess multipotential, oligopotential, or
unipotential differentiation ability (3). Many adult stem cells also
seem to have the ability to trans-differentiate into other cell types,
although this phenomenon is still being hotly debated (3, 4). Finally,
stem cells may express unique markers or properties that can allow
their enrichment and identification.
Indeed, many stem cells are identified by their expression of
unique markers. For example, mouse LT-HSCs, ST-HSCs, and
multipotent progenitors can be identified and separated by their
marker phenotypes, c-kithiSca-1hiThy1.1loLin /loFlk , c-kithiSca1hiThy1.1loLin /loFlk+, and c-kithiSca-1hiThy1.1 Lin /loFlk+, respectively (1). Unfortunately, most organ-restricted stem cells or
progenitors lack unique and specific markers. One way to identify
them relies on the slow-cycling properties of stem cells. When
pulsed by bromodeoxyuridine (BrdU) for a period of time followed
by ‘‘chasing’’ for longer time intervals, slow-dividing stem cells will
be identified as label-retaining cells (LRC; refs. 2, 5). The LRCs
purified from the stem cell niche (i.e., the bulge in the hair follicle)
in transgenic animals possess many of the expected stem cell
properties (2, 5). Another way to identify putative adult stem cells
was pioneered by Goodell et al. (6), who observed that when
bone marrow–derived cells are incubated with Hoechst dye 33342
and then analyzed by dual-wavelength flow cytometry, a small
population of cells does not accumulate an appreciable amount of
dye and is thus identified as a Hoechstlo side population.
Remarkably, the side population is highly enriched in HSCs (6).
Since its initial application in bone marrow HSCs, the side
population technique has been adapted to identify putative stem
cells and progenitors in multiple tissues/organs including umbilical cord blood (7), skeletal muscle (8–10), mammary glands
(11, 12), lung (13–15), liver (16), epidermis (17, 18), forebrain (19),
testis (20, 21), heart (22), kidney (23), limbal epithelium (24), and
prostate (25).
The side population–enriched stem cells are rare (f0.01-5%;
refs. 18, 23, 26) and heterogeneous, varying with tissue types, stages
of development, and methods of preparation (10, 27, 28). For
example, the bone marrow side population cells contain not only
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HSCs but also mesenchymal stem cells (29) and do not capture all
HSCs (30) but only a subset of long-term repopulating HSCs (31).
The skeletal muscle side population cells are composed of mostly
bone marrow–derived cells (8, 9) with only a minor population of
resident muscle stem/progenitor cells (i.e., satellite cells). The lung
side population cells are also heterogeneous comprising both
CD45+ (i.e., bone marrow derived) and CD45 cells (13–15).
Similarly, the testis side population cells may be enriched in
spermatogonial, germinal, as well as mesenchymal (i.e., Leydig)
stem cells (20, 21). Although the side population cells in most cases
seem enriched in primitive stem cells, there are also reports that
suggest that the side population cells do not identify stem cells
(17, 18, 32).
The side population phenotype is mediated by the ABC family of
transporter proteins. One of the major mediators seems to be
ABCG2 or BCRP (33), which was initially identified in drugselected MCF7 breast cancer cells and later found to efflux
multiple chemotherapeutic drugs and xenobiotics (34). The
strongest evidence linking ABCG2 and the side population
phenotype comes from the nearly complete loss of the bone
marrow side population phenotype in abcg / mice (35). Other
supporting evidence is that side population cells preferentially
express ABCG2 (13, 16, 18, 20, 22, 24, 36, 37) and that ABCG2
expression is detected in known stem/progenitor cells such as
HSCs (33), nestin-positive islet-derived progenitors (36), hepatic
oval cells (16), limbal basal cells (24), and neural stem cell (38). On
the other hand, it should be noted that only a fraction of the side
population cells expresses ABCG2 (e.g., ref. 24) and that both side
population and known stem/progenitor cells also express other
ABC transporters such as MDR-1 (i.e., ABCB1 or P-glycoprotein),
MRP-1 (ABCC1), and ABCA2 (36, 37, 39), suggesting that these
latter molecules may also be involved in mediating the side
population phenotype. In support, enforced expression of MDR-1
in murine bone marrow cells leads to the expansion of the side
population (40).
Recently, several human cancers including leukemia (1, 41)
and breast (42) and brain (37, 43–46) tumors were found to
contain stem-like cells (SLC) called cancer stem cell (CSCs;
ref. 47). Most of these tumorigenic SLCs were identified using
markers that identify putative normal stem cells. Interestingly,
SLCs have also been identified in immortalized cell lines (39),
long-term cultured cancer cells (37, 48), or patient tumor
samples (37) using the side population technique. These
observations suggest that even long-term cultured (cancer) cells
may retain SLCs and that the side population approach
represents a valid marker-independent method to identify such
cells. In a recent study,3 we have also provided independent
evidence for the existence of SLCs in multiple human tumor cell
cultures as well as xenograft tumors. In the current study, we
first seek to confirm the utility of the side population technique
to identify tumorigenic SLCs in cultured human cancer cells and
xenograft tumors. Then we focus on the question whether the
higher tumorigenicity associated with the side population might
be related to the expression of ABCG2, a major mediator of the
side population phenotype in bone marrow cells. Our results
surprisingly show that in contrast to the tumorigenic differences
3
C. Jeter et al. Stem-like cancer cells in culture and xenograft tumors: expression
and roles of stemness genes, submitted for publication.
Cancer Res 2005; 65: (14). July 15, 2005
between the side population and non–side population cells, the
ABCG2+ and ABCG2 cancer cells show very similar tumorigenicities in vivo.
Materials and Methods
Cells, reagents, and animals. Various cancer cell lines including
those of prostate (PPC-1, Du145, LNCaP, and PC3 cells), breast (T47D,
MCF-7, MDA-MB231, MDA-MB435, and MDA-MB468), colon (COLO 320,
DLD-1, RKO, and HCT116), bladder (RT4, UC14, UC1, and UC3), glioma
(D54, U87, U251, and U373), melanoma (WM266-4 and WM562-4), cervix
(HeLa), and ovary (SKOV-3) were obtained from American Type Culture
Collection (Manassas, MA) or collaborators and cultured in the
recommended medium containing 5% to 10% of heat-inactivated fetal
bovine serum (FBS). Xenograft human prostate tumors LAPC-4 and
LAPC-9 were kindly provided by C. Sawyers (Department of Medicine,
University of California, Los Angeles, CA) (49) and maintained in
nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice.
Du145 xenograft tumors were established in our lab using early-passage
cells and maintained in NOD/SCID mice. All animals were obtained from
The Jackson Laboratory (Bar Harbor, ME) and maintained in standard
conditions according to the Institutional guidelines. The monoclonal antiABCG2, FITC-, and PE-conjugated anti-ABCG2 monoclonal antibodies,
isotype control antibody, and secondary antibodies were all obtained
from Chemicon (Temecula, CA). Monoclonal antibodies against Ber-EP4
and CD31 were obtained from DakoCytomation, Inc. (Carpinteria, CA) and
BD PharMingen (San Diego, CA), respectively. All chemicals were obtained
from Sigma (St. Louis, MO) unless specified otherwise.
Side population analysis. The protocol was based on Goodell et al. (6)
with slight modifications. Briefly, cells (1 106 cells/mL) were incubated in
prewarmed DMEM/5% FBS containing freshly added Hoechst 33342 (5 Ag/
mL final concentration) for 90 minutes at 37jC with intermittent mixing. In
some experiments, cells were incubated with the Hoechst dye in the
presence of verapamil (50 Amol/L) or reserpine (100 Amol/L). At the end of
incubation, cells were spun down in the cold and resuspended in ice-cold
PBS. 7-AAD (2 Ag/mL final concentration) was added for 5 minutes before
fluorescence-activated cell sorting (FACS) analysis, which allows for the
discrimination of dead versus live cells. As positive controls, we used HL60
promyelocytic leukemia cells selected by chronic exposure to doxorubicin.
Samples were analyzed on a Coulter Epics flow cytometer. The Hoechst dye
was excited with the UV laser at 351 to 364 nm and its fluorescence
measured with a 515-nm side population filter (Hoechst blue) and a 608
EFLP optical filter (Hoechst red). A 540 DSP filter was used to separate the
emission wavelengths.
Indirect immunofluorescence, flow cytometry analysis of ABCG2
expression, and fluorescence-activated cell sorting of ABCG2+ cells.
For fluorescence microscopy (50), 3 to 10 103 cells were plated on glass
coverslips. Next day, cells were fixed in 4% paraformaldehyde (10 minutes
at room temperature) and blocked in 10% goat whole serum. Coverslips
were incubated sequentially with monoclonal antibody anti-ABCG2,
biotinylated goat anti-mouse IgG, and FITC-conjugated streptavidin with
washings in between. Generally 1,200 to 1,500 cells were counted for each
cell type to quantify the ABCG2+ cells. For flow cytometry, cells were gently
dissociated with Accutase (Innovative Cell Technologies, Inc., San Diego,
CA) and washed (twice) in serum-free medium. Cells were stained live in
the staining solution containing bovine serum albumin and insulin and
FITC- or PE-conjugated monoclonal anti-ABCG2 (15 minutes at 4jC).
Samples were analyzed on a Coulter flow cytometer. A minimum of
500,000 viable cells was measured per sample, and cell debris and cell
aggregates were electronically gated out. For FACS, 2 to 4 107 cells were
similarly stained for ABCG2 and used to sort out ABCG2+ and ABCG2
cells. For the positive population, only the top 10% most brightly stained
cells were selected. For the negative population, only the bottom 5% to 10%
most dimly stained cells were selected. The purity of ABCG2+ cells, as
determined by both post-sorting flow analyses as well as restaining followed
by fluorescence microscopy analyses, was z98% and the purity of the
ABCG2 cells was z99%.
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Side Population, ABCG2, and CSC/Progenitor Cells
Xenograft tumor experiments and in vivo tumorigenicity. Xenograft
prostate (Du145, LAPC-4, and LAPC-9) and glioma (U373) tumors were
aseptically dissected out from animals and minced into f1 mm3 pieces in
DMEM supplemented with 10% FBS. After rinsing in the same medium
(twice) followed by PBS to wash out serum, tumor tissues were incubated
with 1 Accumax (1,200-2,000 units/mL proteolytic activity containing
collagenase and DNase; Innovative Cell Technologies) at 10 mL/1 g tissue in
DPBS for 20 minutes at 37jC under rotating conditions. At the end, residual
tissues were allowed to precipitate to the bottom of tubes and dissociated
cells collected from the supernatant. When necessary, the residual tumor
pieces were subjected to one or more rounds of Accumax digestion and
dissociated cells pooled. A single cell suspension was obtained by filtering the
supernatant through a 40-Am cell strainer (BD Falcon, Bedford, MA). Upon
viability count using erythrosin B (American Type Culture Collection), cell
suspension was gently loaded onto a layer of Histopaque-1077 gradient
(Sigma-Aldrich, St. Louis, MO) and centrifuged at 400 g for 30 minutes at
room temperature. RBC, dead cells, and debris were removed from the
bottom of the tube and live nucleated cells collected at the interface. Then the
cell mixture was depleted of lineage-positive host cells using the MACS
Lineage Cell Depletion Kit (Miltenyi Biotec, Auburn, CA). Briefly, cells were
first incubated (10 minutes at 4jC) in the staining solution [PBS (pH 7.2), 0.5%
FBS, 0.5 Ag/mL insulin] containing biotinylated antibodies against a panel of
lineage antigens (CD5, CD45R, CD11b, anti-Ly-6G, 7-4, and Ter-119). Cells
were then incubated with the anti-biotin Microbeads (15 minutes at 4jC)
and the Lin cells were eluted using the MS columns. The eluted prostate
cancer cells were all human epithelial cells as confirmed by their expression of
Ber-EP4, a surface marker unique to human epithelial cells, indicating that we
had obtained highly purified human tumor cells using this protocol.
For tumor experiments, various numbers of cells, either unsorted or
sorted populations (i.e., side population, non–side population, ABCG2+, and
ABCG2 ) were injected in 40 AL of medium/Matrigel (1:1) s.c. into either
male or female NOD/SCID mice (4-8 weeks old). MCF7 cells were injected
into female mice. Tumor development was monitored starting from the
second week. The primary tumor sizes were measured with a caliper on a
weekly basis and approximate tumor weights determined using the formula
0.5ab 2, where b is the smaller of the two perpendicular diameters.
Tumorigenicity was measured mainly by tumor incidence (i.e., the number
of tumors/number of injections) and latency (i.e., time from injection to
detection of palpable tumors). All animals were terminated at 6 to 9 months
after tumor cell injection. Tumors harvested were fixed in formalin and
paraffin sections were made for H&E staining or immunohistochemistry for
CD31.
Relationship between ABCG2 expression and cell proliferation. Two
sets of experiments were done. In one, cells undergoing mitotic division
were determined in ABCG2+ and ABCG2 populations of cells. In the other,
purified ABCG2+ and ABCG2 cancer cells, cultured for various time
intervals, were pulsed with BrdU (2.5 Amol/L 3 hours) and processed for
BrdUrd staining (50).
In vitro and in vivo self-renewal and clonal analyses. Purified ABCG2+
and ABCG2 cancer cells were plated at clonal density (i.e., 100-400 cells per
well; depending on cell type) in the flat-bottomed 6-well culture dishes. Cells
were cultured for different time periods. The percentage of cells that
initiated a clone was presented as cloning efficiency. The clone sizes (i.e., the
number of cells/clone) were determined for some time points. Triplicate
samples were run for each cell type and experiments repeated when feasible.
For in vivo self-renewal experiments, tumor cells purified from the tumors
derived from unsorted, ABCG2+, or ABCG2 cells were stained for ABCG2
and used to quantify the ABCG2-expressing cells by flow cytometry.
Preparation of mouse bone marrow and newborn mouse keratinocytes. To prepare bone marrow, we sacrificed C57BL/6 mice (6-8 weeks old)
by cervical dislocation and removed the skin covering the femurs. The
bones were removed by cutting below the knee and cutting at the hip. The
muscle was then cleaned from both femurs. The bone marrow cells were
flushed out of the femur using a 27-gauge needle in 50 mL of PBS, washed
once, and used in the side population analysis. For keratinocytes, newborn
pups were cleaned and anesthetized on ice for at least 30 minutes. The tail
and limbs were removed and discarded. The skin was removed and floated
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on 2 mL trypsin (0.25%) solution with dermal side down overnight at 4jC.
Epidermis was removed from dermis, placed in Waymouth’s Medium (Life
Technologies, Gaithersburg, MD) containing 10% FBS and 1% penicillinstreptomycin and minced. The minced epidermis was gently stirred for
20 minutes in a sterile glass beaker. The resulting solution was filtered
through a 30-Am mesh and plated at 3 106 cells per 35-mm dish for
2.5 hours. The medium was changed to Keratinocyte Growth Medium - 2
(KGM-2; Cambrex BioScience Walkersville, Inc., Walkersville, MD) with
0.5 mmol/L calcium. Cells were either used freshly or cultured for a short
period of time (to expand) and used in the side population analysis.
Reverse transcription-PCR analysis. Total RNA was isolated using the
Absolutely RNA Nanoprep Kit (Stratagene, La Jolla, CA) and used in
semiquantitative reverse transcription-PCR (RT-PCR) analysis (50). The
PCR primers included ABCG2 (sense, 5V-CTGAGATCCTGAGCCTTTGG-3V;
antisense, 5V-TGCCCATCACAACATCATCT-3V); Notch-1 (sense, 5V-ATCGGGCACCTGAACGTGGCG-3; antisense, 5V-CACGTCTGCCTGGCTCGG CTC-3V);
h-catenin (sense, 5V-ACTGGCAGCAACAGTCTTACC-3V; antisense, 5V-TTTGAAGGCAGTCTGTC GTAAT-3V); SMO (sense, 5V-ATCTCCACAGGAGAGACTGGTTCGG-3V; antisense, 5V-AAAGTGGG GCCTTGGGAACATG-3V);
Oct-4 (sense, 5V-GTGGAGGAAGCTGCAAACAATGAAA-3V; antisense, 5VGACCGAGGAGTTACAGTGCAGTGAAG-3V); and glyceraldehyde-3-phosphate dehydrogenase (sense, 5V-ACCACAGTCCATGC CATCAC-3V; antisense,
5V-TCCACCACCCTGTTGCTGTA-3V).
Results
Some cultured human cancer cells and xenograft tumors
have a side population. Several articles have recently reported the
presence of stem cell–enriched side population in long-term
cultured mouse C2C12 myogenic cells (39), rat C6 glioma cells (48),
or human brain tumor (i.e., glioma and medulloblastoma; ref. 37)
cells. We first sought to determine whether this phenomenon is
generally applicable to other human tumor cells in culture, in
particular, the human epithelial cancer cells. To that end, we first
established the side population protocol on our flow cytometer
using as experimental controls the HL60-Dox cells (courtesy of
Dr. M. Andreef, Department of Blood and Marrow Transplantation,
UT MD Anderson Cancer Center, Houston, TX); i.e., HL60 leukemia
cells chronically exposed to a low concentration of doxorubicin.
The HL60-Dox cells overexpressed the multidrug resistance
proteins that allowed them to efflux various drugs and xenobiotics
including the Hoechst 33342 dye.4 As shown in Fig. 1, unselected
HL60 cells did not show a side population (A) whereas >90% of the
HL60-Dox cells were in the side population (B), which presented as
a distinct ‘‘tail’’ on the histogram and was completely inhibited by
either verapamil (C) or reserpine (D), chemicals previously shown
to block the side population phenotype (e.g., refs. 6, 33). To
determine the sensitivity of our system, we titrated the HL60-Dox
cells into HL60 cultures and then did side population analysis. The
results (Fig. 1E-H) revealed that our system could reliably detect a
side population of f0.01%. Indeed, using these experimental
conditions, we reliably detected f0.01% and 0.5% side population
in mouse bone marrow cells (Fig. 1I) and newborn mouse
keratinocytes (Fig. 1L), respectively, and the side population
phenotypes in these cells could also be blocked by verapamil or
reserpine (Fig. 1J and K; data not shown).
Using the above protocol, we surveyed f30 cultured human
tumor cell lines of the prostate, breast, colon, glioma, bladder,
ovary, cervix, glioma, and melanoma and we reliably detected a
side population (0.04-0.2%) in f30% of the cell lines (Table 1; data
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Unpublished observations.
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Figure 1. Side population (SP ) analysis.
A-H, determination of the sensitivity of the
side population protocol using HL60-Dox
cells. Vera, verapamil; Res, reserpine.
E-H, HL60-Dox cells were added to HL60
cells at the concentrations indicated. I-K,
mouse bone marrow (BM ) cells were used
in side population analysis in the absence
(I ) or presence of verapamil (J ) or
reserpine (K). L, newborn mouse
keratinocytes were used in side population
analysis.
not shown). We also analyzed side population in tumor cells freshly
purified from three xenograft human prostate tumors (i.e., Du145,
LAPC-4, and LAPC-9), and we only detected a distinct side
population (0.07%) in the LAPC-9 tumor (Table 1; data not shown).
These results suggest that only some cultured human cancer cells
and tumors cells freshly purified from xenograft tumors contain a
detectable side population and that most cultured human cancer
cells may have a side population too small (i.e., <0.01%) to be
reliably detected.
Side population cells are more tumorigenic than the non–
side population cells. The side population cells isolated from the
rat C6 glioma have been shown to be more tumorigenic than the
non–side population cells (48). To determine whether the side
population cells we identified in human cancer cells might also be
more tumorigenic, we did several small-scale tumor experiments.
Cancer Res 2005; 65: (14). July 15, 2005
We first purified the side population cells from the U373 glioma
cells, which had f0.1% side population (Table 1; Fig. 2A, a-b).
When 1,000 U373 side population cells were injected into the NOD/
SCID mice, a prominent tumor arose within about 1 month
(Table 2; Fig. 2A, c). The side population tumor histologically
resembled clinical samples in that it had palisades-like structures
(Fig. 2A, d) and was highly vascularized (Fig. 2A, e). In contrast, no
tumor was observed in 7 months when 50,000 non–side population
U373 cells were injected (Table 2).
We similarly isolated side population cells from MCF7 breast
cancer cells, which had f0.2% side population (Table 1;
Fig. 2B, a). When injected into the NOD/SCID mice, we observed
a cell number–dependent tumor development (Table 2). For
example, we observed a 17% (one of six) tumor incidence with
1,000 side population cells injected and 50% (three of six) tumor
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Side Population, ABCG2, and CSC/Progenitor Cells
incidence with 10,000 side population cells injected (Table 2).
Tumor latency was also reduced with increased numbers of side
population cells injected (Table 2). In contrast to the side
population MCF7 cells, the non–side population MCF7 at 1,000
cells did not give rise to any tumors (zero of six). With 10,000
cells injected, we observed one tumor with six injections
(Table 2). Even with 250,000 cells injected, we only observed
one of two tumor incidence (Table 2). In addition, the tumors
from the non–side population MCF7 cells arose with longer
latencies (Table 2).
Finally, we purified from the LAPC-9 prostate xenograft tumors
the side population cells, which constituted f0.07% of the total
tumor (Table 1; Fig. 2C, a-b). When injected into the NOD/SCID
mice, as few as 100 cells generated a tumor (25% incidence; Table 2).
With 1,000 side population cells injected, we observed a tumor
incidence of 75% (three of four; Table 2). With 1,500 side population
cells injected, we observed two of two tumor formation with shorter
latencies (Table 2). Tumors derived from the side population cells
histologically resembled the unsorted LAPC-9 tumors (data not
shown). In contrast, no tumor generation was observed with 1,500
(zero of six) or 15,000 (zero of four) non–side population LAPC-9
cells injected. Even 150,000 non–side population LAPC-9 cells
injected did not give rise to tumors within 9 months, although
tumor did arise with 300,000 non–side population cells (Table 2).
These results suggest that the side population LAPC-9 tumor cells
are probably >100 times more tumorigenic than the non–side
population LAPC-9 cells.
Side population cells have some stem cell properties. To
determine whether the higher tumorigenicity in the side population cells might be associated with some of the intrinsic stem cell
properties, we first used the purified side population and non–side
population U373 cells in a clonogenic assay, which partially
measures the self-renewal capacity of the cells. As shown in
Fig. 2A, f, whereas f10% of the side population U373 cells could
sustain a clonal growth, <0.01% of the non–side population U373
cells were clonogenic. Then we asked whether the side population
cell-generated tumors contain non–side population cells and can
be further passaged in vivo. To that end, we took the U373 side
population tumor obtained from the preceding experiments
(above) and purified tumor cells out and did side population
analysis. The results revealed a side population of f0.1% (data not
shown), which was similar to the percentage of side population
cells in the first-generation tumor (Table 1; Fig. 2A, a). These
observations suggest that the side population cells can give rise to
non–side population cells in vivo. When injected into the NOD/
SCID mice, 100 side population cells generated tumors with f60%
(7 of 11) efficiency and 1,000 side population U373 cells also
generated a tumor (Table 2). By contrast, no tumors were observed
with 1,000 non–side population cells injected (zero of six). Even
200,000 non–side population U373 cells injected did not generate a
Table 1. Side population and ABCG2 in human tumor cells
Cells
Prostate tumor cells
LNCaP
LAPC9
Du145
PC3
PPC-1
Breast tumor cells
MCF7
T47D
MDA-MB468
MDA-MB231
MDA-MB435
Colon tumor cells
COLO 320
DLD-1
RKO
HCT116
Glioma cells
D54
U87
U251
U373
Others
WM266-4
WM562-4
HeLa
SKOV3
c
Characteristics
Side population*
ABCG2
AR+, low tumorigenicity; low metastatic
AR+, intermediate tumorigenicity; metastatic
AR , intermediate tumorigenicity; metastatic
AR , high tumorigenicity; highly metastatic
AR , high tumorigenicity; highly metastatic
UD
0.07
UD
UD
UD
1.9
1.2
0.8
2.0
0.2
ER+, low tumorigenicity
ER+, low tumorigenicity
ER , intermediate tumorigenicity
ER , tumorigenic and metastatic
ER , tumorigenic and metastatic
0.2
ND
ND
UD
UD
0.6
1.3
0.1
0.6
0.7
tumorigenic
tumorigenic and invasive
highly tumorigenic; metastatic
highly tumorigenic; metastatic
ND
ND
ND
ND
1.3
0.2
3.7
3.1
highly
highly
highly
highly
0.1
0.05
0.04
0.1
0.7
2.0
1.9
2.5
UD
UD
UD
0.05
2.3
0.6
0.5
0.7
tumorigenic
tumorigenic
tumorigenic
tumorigenic
and
and
and
and
invasive
invasive
invasive
invasive
melanoma
melanoma
cervical carcinoma
ovarian adenocarcinoma; metastatic
Abbreviations: UD, undetectable; ND, not determined.
*The numbers indicate the % and represent the mean values of three to seven independent experiments.
cThe numbers represent the mean % derived from two to five independent immunostaining and/or flow cytometry analyses.
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Figure 2. Side population (SP) cells are more tumorigenic and have some stem cell properties. A, U373 side population analysis (a-b) and the side population
cell-initiated tumor (c ) analyzed for histology (d; 100) or CD31 staining (e; 100). Clonogenic assays using side population and non–side population cells
(f ). B, MCF7 side population analysis (a ) or RT-PCR assays (b). C, LAPC-9 side population analysis (a-b ) and RT-PCR assays (c ).
tumor (Table 2), suggesting that the U373 side population cells
possess self-renewal capacities in vitro and in vivo and they are
probably >200 more tumorigenic than the non–side population
cells.
To determine whether the side population cells have some other
intrinsic properties of stem cells such as preferential expression of
‘‘stemness’’ genes, which are important for stem cell self-renewal,
proliferative capacity, or fate determination (2, 47, 51),3 we did
semiquantitative RT-PCR analysis of Notch-1, h-catenin (a crucial
molecule in the Wnt signaling pathway), and Smoothened (SMO;
the activating receptor for the Hedgehog signaling pathway). As
shown in Fig. 2B, b, the side population MCF7 cells expressed
higher levels of Notch-1 and h-catenin mRNAs than the non–side
population MCF7 cells although both populations expressed
similar levels of SMO mRNA.
Taken together, these results suggest that the side population
cancer cells have some intrinsic properties of stem cells, similar to
observations in various normal stem cell populations (see
Introduction).
Expression of ABCG2 in a small subset of cancer cells:
association of ABCG2 with cell proliferation. Three considerations made us to turn our attention to ABCG2. First, ABCG2
is one of the primary mediators of the side population
phenotype in mouse bone marrow and some other cells (35).
Cancer Res 2005; 65: (14). July 15, 2005
Second, the majority of the cancer cells we have studied do not
possess a detectable side population under our experimental
conditions therefore an alternate ‘‘marker’’ is needed to identify
the rare tumorigenic cells. Third, one of the potential concerns
in the side population analysis is that the chronic accumulation
of the low levels of the Hoechst dye in the non–side population
cells might be toxic to these cells, although our post-sort viability
analysis as well as culture of multiple non–side population cells
did not reveal such cytotoxicities (data not shown). Regardless, if
ABCG2 could be used as a surrogate marker for the side
population cells, it would completely avoid the potential
cytotoxicity problem. With these considerations, we first
examined the relationship between side population cells and
ABCG2 expression. As expected, the side population MCF7 cells
expressed higher levels of ABCG2 mRNA than the non–side
population cells (Fig. 2B, b). We then examined the ABCG2
protein expression, using both immunofluorescence staining and
flow cytometry, in the same spectrum of human tumor cell lines
and xenograft tumor-derived cells. In every case, we detected
ABCG2 expression in a small percentage (0.1-4%) of the cells
(Table 1; Fig. 3). A similar ABCG2 expression pattern (i.e., 0.1-2%)
was also observed in several bladder cell lines (Fig. 3E, d; data
not shown) as well as in LAPC-4 and LAPC-9 xenograft tumors
(data not shown).
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Side Population, ABCG2, and CSC/Progenitor Cells
Table 2. Side population is enriched with tumorigenic cells
Cell type
Cell injected (n)
U373-SP-lj*
U373-NSP-1j
U373-SP-2jc
U373-NSP-2j
MCF7-SP*
MCF7-NSP
LAPC9-SPc
LAPC9-NSP
1,000
50,000
100
1,000
50,000
200,000
1,000
10,000
10,000
250,000
100
1,000
1,500
150,000
300,000
Tumor incidence (latency)
1/1 (33 d)
0/1 (terminated in 7
7/11 (34-48 d)
1/1 (39 d)
0/1 (terminated in 7
0/1 (terminated in 7
1/6 (160 d)
3/6 (130-150 d)
1/6 (194 d)
1/2 (190 d)
2/8 (52 and 108 d)
3/4 (108 d)
2/2 (41 and 72 d)
0/1 (terminated in 9
1/1 (92 d)
mo)
mo)
mo)
mo)
NOTE: Tumor incidence refers to the number of tumors/the number
of injections. Latency refers to the time from tumor cell injection to
the appearance of a palpable tumor.
*Cultured cells: 1j and 2j refer to the first and second-generation
tumors, respectively.
cXenograft tumor-derived cells.
Interestingly, we observed that many of the ABCG2+ cancer
cells were in the process of cell division (Fig. 4). Overall, we
observed that f30% of the ABCG2+ cancer cells were mitotic,
whereas only f1% of the ABCG2 cells were mitotic. In clonal
analyses, we found that the majority of divided cells that had
completed or were about to complete cytokinesis equally
distributed ABCG2 to both daughter cells (Fig. 4G-I and J-L).
However, in f1% of the dividing cells undergoing cytokinesis
ABCG2 seemed to segregate asymmetrically to mainly one
daughter cell (e.g., Fig. 4M-O). These observations suggest that
ABCG2 might preferentially mark proliferating cells and that some
ABCG2-expressing cancer cells might be undergoing asymmetrical
cell division, a cardinal feature of stem cells. To determine
whether ABCG2 expression might be associated with cell
proliferation in general, we prospectively purified ABCG2+ and
ABCG2 cancer cells to near homogeneity and used them in a
BrdU-labeling experiment. As shown in Fig. 5, acutely purified
ABCG2+ Du145 prostate (A) and MDA-MB435 breast (B) cancer
cells that had been BrdU-pulsed and cultured for only 3 hours
showed significantly more proliferation than the corresponding
ABCG2 cells.
ABCG2+ and ABCG2 tumor cells are similarly tumorigenic.
Next, we purified ABCG2+ and ABCG2 cells and did tumor
experiments to determine whether the ABCG2+ cancer cells might
be more tumorigenic. Much to our surprise, when the two
populations of U373 cells were injected into the NOD/SCID mice,
we did not observe any major differences with respect to their
tumorigenicities (Table 3). In fact, the ABCG2 cells tended to
generate tumors slightly faster than the ABCG2+ or unsorted cells
(Table 3).
To determine whether the lack of correlation between ABCG2
expression and tumorigenicity may be a cell type–restricted
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phenomenon, we carried out similar tumor experiments using
purified ABCG2+ and ABCG2 cells from prostate (Du145),
breast (MDA-MB435), and colon (HCT116) cancer cell cultures.
In every case, we failed to observe any significant difference in
tumor incidence or latency periods between the two populations
(Table 3; data not shown). We also compared the ABCG2+ and
ABCG2 cells purified from Du145 xenograft tumors and again
did not observe any major differences in their tumorigenicities
(Table 3).
Evidence that ABCG2 expression marks proliferating
tumor progenitors whereas ABCG2 population contains
primitive cancer stem cells. If the ABCG2+ cells proliferate
faster than the ABCG2 cancer cells, why are they not more
tumorigenic? One possibility is that ABCG2+ cells are mostly fast
proliferating tumor progenitors (i.e., transit amplifying cells)
rather than primitive, slow-cycling CSCs. To test this possibility,
we first determined whether the ABCG2+ tumor cells could
generate ABCG2 cells and, more importantly, whether ABCG2
cells could generate ABCG2+ cells. As shown in Table 4, tumors
derived from the ABCG2+ cancer cells all contained only a
fraction of ABCG2+ cells (Table 4), suggesting that ABCG2+ cells
generated ABCG2 tumor cells in vivo. Except for one tumor
derived from the 1,000 ABCG2+ Du145 cells, we detected only
small percentages of ABCG2+ cells in all other tumors derived
from the ABCG2+ tumor cells (Table 4). On the other hand,
tumors derived from the ABCG2 cells also contained a small
fraction of ABCG2+ cells (Table 4), suggesting that some
ABCG2 cells also have the ability to generate ABCG2+ tumor
cells.
Next, we did a series of clonal analyses to determine the
relationship between ABCG2+ and ABCG2 cancer cells. Although
freshly purified ABCG2+ Du145 (Fig. 5A; 3 hours) or MDA-MB435
(Fig. 5B; 3 hours) cells proliferated faster than their corresponding
ABCG2 counterparts, culture for as short as 1 day eliminated or
reduced this proliferative difference. Continued cultures of these
cells revealed increasing proliferating (i.e., BrdU+) cells in the
ABCG2 populations (Fig. 5A-B). These results suggest the
possibility that, with time in culture, the ABCG2+ tumor
progenitors gradually lose their proliferative capacity whereas
primitive CSCs in the ABCG2 population give rise to highly
proliferative ABCG2+ tumor progenitors. In support, immunostaining revealed the emergence of ABCG2+ cells from the starting
ABCG2 cancer cells within 1 week (data not shown), consistent
with the ability of some ABCG2 tumor cells to generate ABCG2+
cells in vivo (Table 4).
Consistent with the BrdU incorporation assays, clonal analyses
revealed that at earlier time points the ABCG2+ tumor cells had
higher cloning efficiency; that is, more cells had the ability to
establish a clone (Fig. 5C-D). However, at later time points, the
ABCG2 tumor cells picked up and formed similar or higher
(Fig. 5C-D) percentages of clones. Subsequently, we carried out
differential clonal analyses in which purified ABCG2+ and
ABCG2 tumor cells were plated at clonal density and clonal
sizes were quantified at a shorter and a longer time point. As
shown in Fig. 5E, 7 days after plating, more ABCG2+ Du145
cells formed larger clones. However, at 14 days post plating,
there were significantly more large clones derived from the
ABCG2 Du145 cells (Fig. 5G). The average clonal sizes (cells/
clone) of the ABCG2+ versus ABCG2 Du145 cells were 101 and
60 cells at 7 days versus 4,716 and 5,658 cells at 14 days,
respectively (P < 0.01 in both cases, ANOVA). Similarly, more
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Figure 3. ABCG2 expression in human cancer cells. Cultured glioma (A), or prostate (B), breast (C ), colon (D ), or other (E ; see Table 1) cancer cells were stained for
ABCG2 using a monoclonal antibody. Original magnifications, 100.
ABCG2+ MDA-MB435 cells formed larger clones than the
ABCG2 MDA-MB435 cells at 7 days after plating (Fig. 5F).
However, by 11 days after plating, there were significantly more
large clones (i.e., >500 cells per clone) derived from the ABCG2
MDA-MB435 cells (Fig. 5H). The average clonal sizes of the
ABCG2+ versus ABCG2 MDA-MB435 cells were 30 and 16 cells
at 7 days (P < 0.01) versus 346 and 356 cells at 11 days,
respectively. Together, the proliferation (Fig. 5A-B) and clonal
(Fig. 5C-H) analyses provide strong evidence that the ABCG2+
tumor cells likely represent fast-proliferating tumor progenitor
cells, whereas the ABCG2 population contains slow-cycling,
primitive CSC cells that, with time, could establish robust clonal
growth and also generate fast-cycling progenitor cells.
Finally, we did RT-PCR analysis to assess the mRNA expression
of several stemness genes (Fig. 6A-B). Consistent with the concept
that the ABCG2 population contains primitive, stem-like cancer
cells, we found that purified ABCG2 tumor cells expressed higher
mRNA levels of Notch-1, h-catenin, and SMO (Fig. 6A-B). Even Oct-4,
a transcription factor essential for embryonic stem cell self-renewal
Cancer Res 2005; 65: (14). July 15, 2005
(51) and recently shown to be expressed in some adult stem cells
(52), also showed preferential expression in the ABCG2 cells in
three of four cell types (Fig. 6A). Interestingly, the Notch-1 mRNA
was detected nearly exclusively in the ABCG2 tumor cells (Fig. 6A).
Similarly, the h-catenin mRNA was observed only in the ABCG2
Du145 cells (Fig. 6B). These RT-PCR results provide strong support
for the existence of primitive CSC in the ABCG2 tumor cell
population.
Discussion
A long-time puzzle to tumor biologists is the observations that
even with long-term cultured cancer cells, in general sufficient
numbers of cells have to be injected to initiate an orthotopic
tumor in recipient animals (reviewed in refs. 1, 47), suggesting
that even in long-term tumor cell cultures, not all cells are equal
and only a small population of cells is tumorigenic. Indeed, when
multiple human cancer cells, which have been in culture under
different conditions for years or even decades are assessed for
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Side Population, ABCG2, and CSC/Progenitor Cells
their clonal growth and clonogenic potentials, we find that only
a small percentage of cells possesses such potentials.3 Furthermore, when tumor cell–derived spheres or xenograft tumors
in situ are pulsed with BrdU followed by extended chase, only a
very minor population of the cells manifests as the long-term
LRCs,3 which are known to preferentially identify stem cells (2, 5).
In further support, long-term cultured rat C6 glioma cells (48)
and some human brain tumor (i.e., glioma and medulloblastoma;
ref. 37) cells are found to contain a side population, which is
known to be enriched in stem cells (see Introduction).
Importantly, the C6 side population cells are more tumorigenic
than and can also generate the non–side population cells (48),
providing the first direct evidence for a population of more
tumorigenic cells in long-term tumor cell cultures. It is these
observations (37, 48)3 that have prompted us to first determine
whether the side population technique can be generally applied
Figure 4. Association of ABCG2 expression with cell division. B-C and E-F, cells that have just undergone nuclear division but have not undergone cytokinesis
(arrows ). By contrast, cells in (G-I, J-L , and M-O ) have just undergone cytokinesis. Cells in (D-O ) were clonal cultures. Original magnifications, 400.
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to other cultured human tumor cells, in particular, the human
epithelial cancer cells.
Our results reveal that f30% cultured human cancer cell lines
and xenograft tumor-derived cells possess a side population that
can be reliably detected under the current experimental
conditions. Several notable points are worthy of mention. First,
in most literature reports, the side population is identified on
either a MoFlo or FACS Vantage flow cytometer as a continuous
tail of the non–side population. Therefore, the discrimination of
the side population from the non–side population is, to a certain
extent, arbitrary and can vary significantly from experiment to
experiment. In contrast, using a Coulter Epics flow cytometer
and our modified protocol, we have in most cases identified the
side population as a distinct ‘‘side’’ population separate from the
main non–side population. This could potentially give us
relatively pure side population cells. Second, in support of this
possibility, our system seems to identify the side population cells
in a more stringent manner and the percentages of the side
population cells may be more representative of putative CSCs in
the cultures or xenograft tumors. Therefore, only f30% cultured
human cancer cell lines and xenograft tumor-derived cells
possess a side population of 0.04% to 0.2%. These percentages
Figure 5. Proliferative and self-renewal properties of ABCG2+
and ABCG2- tumor cells. Purified ABCG2+ and ABCG2- Du145 (A)
or MDA-MB435 (B ) cells were plated for either 3 hours or cultured
for the time periods indicated. Cells were pulsed with BrdUrd for
3 hours before the end of each time point. Columns, mean %
BrdUrd+ cells from two experiments with 500 to 1,200 total cells
counted; bars, FSE. *, P < 0.001 (t test). , P < 0.01 (A) or <0.05
(B). Purified ABCG2+ and ABCG2- Du145 (C ) or MDA-MB435
(D) cells were plated at clone density (400 cells per well) and
cultured for the times indicated. At the end, the numbers of clones
were quantified. % Cloning efficiency. *, P < 0.01. Purified ABCG2+
and ABCG2- Du145 (E and G ) or MDA-MB435 (F and H) cells
were plated at clone density (100 cells per well) and cultured for
7 (E and F ), 14 (G), or 11 (H ) days. At the end, the numbers of
cells in each clone were counted and clones were grouped
according to their sizes. On average 100 to 200 clones were
randomly counted for each condition. Representative of two
independent experiments. *, P < 0.05.
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Side Population, ABCG2, and CSC/Progenitor Cells
side population cells in the non–side population as epithelial
cancer cells are very ‘‘sticky’’.
The major focus of the current study is to address whether the
higher tumorigenicity associated with the side population cells is
related to ABCG2 expression. Because the side population
phenotype in some cell types is mainly mediated by ABCG2, the
side population cells (e.g., in MCF7) express higher levels of
ABCG2 mRNA, ABCG2 is expressed only in a small subset of
cancer cells, and ABCG2 expression in cancer cells seems
associated with cell proliferation, it stands to reason that the
ABCG2+ cancer cells might or should be more tumorigenic than
the ABCG2 tumor cells. Surprisingly, however, highly purified
ABCG2+ cells from several types of tumor cells are not more
tumorigenic than the corresponding ABCG2 cells. Further
proliferation assays, clonal analyses, self-renewal, and molecular
studies suggest a model in which the ABCG2 population
contains primitive stem-like cancer cells with higher self-renewal
(because of higher levels of stemness genes) and proliferative
potentials but are normally slow cycling (Fig. 6C). These cells then
give rise to ABCG2+ tumor progenitor cells that are more actively
proliferating but possess reduced self-renewal and long-term
proliferative capacities (Fig. 6). The ABCG2+ tumor progenitor
cells eventually give rise to ABCG2 , partially or even fully
differentiated tumor cells that constitute the bulk of tumor cell
mass (Fig. 6).
The side population has been shown to be very heterogeneous
(10, 27, 28). Therefore, the side population detected in cancer cells
might contain several subsets of cells, one of which expresses
ABCG2 thus explaining the increased ABCG2 expression in the side
population. The higher tumorigenicity associated with the side
population might be conferred by combined effects of several other
subpopulations of cells in addition to the ABCG2+ cells. For
example, cells expressing other ABC family members might also
contribute to the cancer cell side population phenotype. Indeed, it
has been shown that only a fraction of side population cells
expresses ABCG2 (24) and that both side population and known
stem/progenitor cells also express other ABC transporters such as
MDR-1 (i.e., ABCB1 or P-glycoprotein), MRP-1 (ABCC1), and
Table 3. ABCG2 and tumorigenesis
Samples
Cell
injected (n)
Incidence
Latency
(median), d
U373-unsorted
1,000
10,000
1,000
10,000
1,000
10,000
100,000
100
1,000
10,000
100,000
100
1,000
100
1,000
10,000
100
1,000
100
1,000
10,000
100
1,000
10,000
100
1,000
100
1,000
10,000
3/3
4/4
8/8
2/2
4/4
3/4
4/4
3/5
4/6
5/6
4/4
3/8
2/6
2/6
1/6
2/6
2/6
1/2
2/6
2/6
1/4
4/6
4/6
2/4
7/8
3/4
6/8
3/4
5/6
26
19
30
26
19
19
19
77
53
45
35
81
80
73
63
55
101
63
78
54
54
67
64
61
57
47
57
57
52
U373-ABCG2+
U373-ABCG2
Du145-unsorted
Du145-ABCG2+
Du145-ABCG2
Du145-ABCG2+*
Du145-ABCG2
MDA-MB435-unsorted
MDA-MB435-ABCG2+
MDA-MB435-ABCG2
*Cells in this experiment were purified from the Du145 xenograft
tumors. Cells in all other experiments were purified from cultured
cells.
are similar to the side population of multiple normal stem cell
or progenitor cell populations [i.e., 0.01-5%; refs. 18, 23, 26; e.g.,
mouse bone marrow (0.01%; Fig. 1), newborn mouse keratinocyte
progenitors (0.5%; Fig. 1), and human bone marrow (f0.03%;
ref. 8)]. The majority of the cancer cell lines or xenograft tumors
examined seems to possess too small a side population to be
reliably detected.
Importantly, the side population cells purified from two cell
lines and one xenograft tumor are more tumorigenic than
the non–side population counterparts. Furthermore, the side
population cells are found to possess several intrinsic properties
of stem cells: self-renewal, preferential expression of some
stemness genes, and an ability to give rise to non–side population
cells. These results thus extend the others studies on rat C6
glioma cells (48) and support the concept that the side
population is indeed enriched in stem-like tumorigenic cells. It
should be noted that with higher numbers of the non–side
population epithelial cancer cells (i.e., MCF7 and LAPC-9)
injected, we also observed tumor development. These results
may suggest that the non–side population also contains a very
small percentage of tumorigenic cells, although the results might
have stemmed from the contamination of small numbers of the
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Table 4. ABCG2+ and ABCG2 cells in tumors
Tumors derived from*
Du145
ABCG2+ (100 cells)
ABCG2+ (1,000 cells)
ABCG2 (100 cells)
ABCG2 (10,000 cells)
MDA-MB435
ABCG2+ (100 cells)
ABCG2+ (1,000 cells)
ABCG2 (100 cells)
ABCG2 (1,000 cells)
ABCG2 (10,000 cells)
ABCG2+ cells (%)
n
0.2,
0.9,
0.1,
3.8,
0.2
37.4
0.3, 5.1
1.1
2
2
3
2
0.8, 0.6
0.2, 0.1
0.8
0.1
0.5, 5.7, 6.7
2
2
1
1
3
*Xenograft tumors derived from either unsorted or sorted (i.e.,
ABCG2+ or ABCG2 ) cells were harvested to prepare single-cell
human tumor cell suspension (see Materials and Methods), which was
then used in ABCG2 staining and flow cytometry analysis.
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Figure 6. ABCG2- cancer cells express higher
levels of stemness genes. A-B, RT-PCR analysis
of stemness genes using purified ABCG2+ (+)
and ABCG2- ( ) cancer cells. Negative controls
(Neg. CTL) refer to RT using H2O and positive
control (Pos. CTL ) in (H ) refers to unsorted Du145
cells previously shown to express h-catenin.3
C, a hypothetical model. See text for more
discussion.
ABCA2 (36, 37, 39). In addition, enforced expression of MDR-1 in
murine bone marrow cells is sufficient to expand the side
population (40). We are currently using cultured cancer cells as
well as xenograft and primary human tumor samples to determine
the molecular basis of the higher tumorigenicity associated with
the side population (e.g., amplification of oncogenes and/or
mutations of specific tumor suppressors), dissect different
subpopulations of the side population with respect to their
tumorigenic potentials, and elucidate the interrelationship between
the side population and several other tumorigenic populations we
have identified.
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Acknowledgments
Received 2/21/2005; revised 4/16/2005; accepted 4/28/2005.
Grant support: NIH grants CA90297, AG023374, and P30 CA16672; NIEHS grant
ES07784; American Cancer Society grant RSG MGO-105961; Department of Defense
grant DAMD17-03-1-0137; Prostate Cancer Foundation; and M.D. Anderson Cancer
Center (PCRP and IRG).
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.
We thank C. Conti, T-J. Liu, M. Andreef, and C. Sawyers for providing cells; C. Carter
for assistance in preparing mouse bone marrow cells; the Histology Core for excellent
assistance in tissue processing and immunohistochemistry; the Animal Facility Core
for help in tumor experiments; E. Richie for her insights; and members of the Tang lab
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Cancer Res 2005; 65: (14). July 15, 2005
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Side Population Is Enriched in Tumorigenic, Stem-Like
Cancer Cells, whereas ABCG2 + and ABCG2− Cancer Cells
Are Similarly Tumorigenic
Lubna Patrawala, Tammy Calhoun, Robin Schneider-Broussard, et al.
Cancer Res 2005;65:6207-6219.
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