Journal of Cancer Therapy, 2010, 1, 165-173
doi:10.4236/jct.2010.14026 Published Online December 2010 (http://www.scirp.org/journal/jct)
Expression Compilation of Several Putative Cancer
Stem Cell Markers by Primary Ovarian Carcinoma
Jiabo Di1, Refika Yigit2, Carl G. Figdor1, Tjitske Duiveman-de Boer1, Leon F. A. G. Massuger2,
Ruurd Torensma1
1Departments of Tumor Immunology, Nijmegen Centre for Molecular Life Sciences, Radboud University Nijmegen Medical Centre,
Nijmegen, the Netherlands；2Department of Obstetrics and Gynecology, Nijmegen Centre for Molecular Life Sciences, Radboud
University Nijmegen Medical Centre, Nijmegen, the Netherlands.
Received July 23rd, 2010; revised July 26th, 2010; accepted August 3rd, 2010.
ABSTRACT
Cancer stem cells (CSCs) or tumor initiating cells are rare cells that are able to establish a tumor or metastasis. Identi-
fication of those CSCs is, however, cumbersome even in established cell lines. Several cancer stem cell markers were
reported to be expressed by ovarian cancer. Those cancer stem cells are gifted with lower vulnerability to irradiation
and cytostatic drug s explaining the high in cidence of recurrence after treatmen t. A variety of different cancer stem cell
markers were described for epithelia l tumors. Also, cancer cell lines were assessed for stem cell markers with no com-
mon denominator. The expression of CD24, CD44, CD117, CD133, ABCG2, ALDH was determined for cells from 22
patients. Ovarian cancer cells were collected from ascites. Part of the tumor cells were analyzed immediately and
stained for the above mentioned cancer stem cell markers. The remainder of the cells was cultured for several weeks
using standard stem cell cu lture conditions. We observed a larg e variety in expression of putative stem cell markers for
primary tumors. After two weeks of culture spheres were seen in several cultures, indicative for cancer stem cells,
though not all patients’ cells were able to form spheres. Our data show for the first time the heterogeneity in marker
display in primary tumors. Also for the cu ltured cells stem cell markers were determined. None of the stem cell markers
was expressed by all patients’ cells. No correlation with tumor type was demonstrated. The complexity of expression
challenges the isolation of cancer stem cells.
Keywords: Ovarian Cancer, Cancer Stem Cells, Stem cell markers
1. Introduction
Ovarian cancer is the fifth leading cause of death from
cancer in women and the most lethal gynecological ma-
lignancy. It has a poor prognosis due to late patient
presentation because early symptoms are not evident or
vague and confused with more common gastrointestinal
diseases. The incidence of ovarian cancer is approxi-
mately 1 in 60 women. When diagnosed early the sur-
vival is far better than at advanced stages of the disease,
at which point the 5-year survival is only 30%. Moreover,
the development of a chemoresistant tumor after surgery
and chemotherapy aggravates the situation. Novel treat-
ment modalities are needed to increase the survival rate.
Targeting the cancer stem cell (CSC) has been put
forward as such a new treatment modality [1,2]. How-
ever, the CSC concept is heavily debated. The CSC is a
relatively rare cancer cell that has the ability to
self-renewal giving rise to another malignant stem cell as
well as commit to terminal differentiation into multiple
lineages of more mature cancer cells [3-6]. The differen-
tiated cells constitute the bulk of the tumor, but they lack
self-renewal capacity and have limited proliferation po-
tential.
Increasing experimental evidences suggest that CSCs
may play a decisive role in the initiation and progression
of tumors [7]. CSCs were originally identified in leukemia,
Bonnet and co-workers demonstrated that human leuke-
mias are driven by a small population of leukemic stem
cells capable of transferring the disease to NOD/SCID
mice [8]. This concept was extended to solid epithelial
tumors by Al-Hajj and co-workers, who demonstrated that
a small population of cells within breast cancer with stem
cell properties, bearing the surface marker CD24lowCD44high.
Subsequently, CSCs are identified and prospectively iso-
This study was supported by a grant from the Netherlands Institute for
Regenerative Medicine.

Expression Compilation of Several Putative Cancer Stem Cell Markers by Primary Ovarian Carcinoma 166
lated from a variety of epithelial cancers, including pan-
creas, colon and prostate cancers [5,9-15]. The cancer
stem cell hypothesis has recently also been explored in
ovarian cancer. In 2008, Zhang and co-colleagues claimed
that epithelial ovarian cancers derive from a subpopulation
of CD44+CD117+ cells [16]. Recently, Ferrandina and
Curley independently found that CD133 expression de-
fines a tumor initiating subpopulation of cells in human
ovarian cancer [17,18]. Moreover, Gao and co-workers
reported that CD24 could be utilized as a surface marker
to enrich ovarian CSCs [19]. Ovarian CSCs were also
detected in the so-called side populations, which are tu-
morigenic and chemoresistant [20-22]. The hypothesis that
CSCs are responsible for tumor recurrence and metastasis
of ovarian cancer gets more and more support. If indeed
CSCs are the targets to treat ovarian cancer, novel strate-
gies have to be developed to target specifically those cells.
Current chemotherapeutic drugs do not kill CSCs because
those stem cells express several pumps, for instance
ABCG2, that expel cytostatic drugs out of the cell [22,23].
In the current study, we tested the expression of the
described putative epithelial CSC markers in primary
ovarian tumor ascites, in order to assess whether they can
be utilized as general markers to identify and isolate ova-
rian CSCs. Our data shows that the expression of those
CSC markers is very diverse and is patient-dependent,
and no correlation was found between pattern of surface
marker display and tumor histologicalsubtype. These
suggest that ovarian tumors are heterogeneous, and a
more general marker is needed to prospectively isolate or
target ovarian CSCs for elimination.
2. Materials and Methods
2.1. Primary Tumor Sphere Culture
Freshly-isolated ascites fluid was received from the De-
partment of Obstetrics and Gynecology, Radboud Uni-
versity Nijmegen Medical Centre. Following filtration
through a 100 μm cell strainer (BD FalconTM), viable
ascites-derived mononuclear cells were isolated by cen-
trifugation over LymphoprepTM (Axis-Shield). The as-
cites-derived mononuclear cells were suspended in
GIBCO™ Defined Keratinocyte-SFM (1x; Invitrogen)
supplemented with recombinant human EGF (20 ng/ml;
R&D systems) and plated at a density of 2 × 106 tumor
cells/24 well plate with Ultra-Low Attachment surface
(Corning Incorporated). Cells were grown at 37°C with
5% CO2 [24].
2.2. Aldefluor Assay
ALDEFLUOR assay was performed with the ALDE-
FLUOR kit from Stem Cell Technologies. Prior to treat-
ment, cells were suspended in aldefluor assay buffer (1 ×
106cells/ml). aldefluor assay reagent containing the ALDH
substrate BODIPY-aminoacetaldehyde (BAAA) was added
to both control and test samples (5 µl/ 1 × 106cells). Be-
fore addition of BAAA, the negative control sample was
treated with a specific inhibitor of ALDH, diethylaminoben-
zaldehyde (DEAB, 5 µl/ 5x105cells). Then the samples were
incubated for 45minutes at 37oC to allow conversion of
the substrate BAAA to the fluorescent product
BODIPY-aminoacetate (BAA). The amount of intracellu-
lar fluorescent product was then measured using a
CyAnTM ADP flow cytometer (Beckman Coulter).
2.3. Immunofluorescence Staining and
Microscopy
ALDEFLUOR treated cells were rinsed in PBS, and
then cells were adhered to poly-L-lysine-coated cover
slips and fixated with 2% PFA in PBS for 15 minutes.
Cover slips were mounted onto glass slides with Mo-
wiol. Subsequently, cells were examined by fluores-
cence microscopy (Olympus).
2.4. Flow Cytometric Analysis of Cancer Stem
Cell Markers
Cells were isolated from ascitic fluid and cultured for
two weeks in stem cell medium. To stain for cell surface
markers, washed with PBA (PBS/0.1% BSA/0.1% so-
dium azide) and incubated with the following antibodies
at 4°C for 30 minutes: CD24-PE (Beckman Coulter),
CD44-APC (BD PharmingenTM), c-Kit-FITC (CD117)
(Santa Cruz Biotechnology), CD133-PE (Miltenyi Bio-
tec), ABCG2-PE (R&D Systems). Before flow cytomet-
ric analysis the cells were washed and resuspended in
PBA solution. Cells were then examined using the
CyAnTM ADP flow cytometer. The exitation wavelenght
for FITC was 488 nm and emission wavelenght 530 nm,
for phycoerythrin excitation at 561 nm and emission at
585 nm and for allophycocyanin excitation at 643 nm
and emission at 665 nm.
3. Results
3.1. Self-renewing Spheres Are Not Formed in
All Patients with Ovarian Cancer
One key determinant of stem cells is the capability for
extensive proliferation. To demonstrate whether cells
with distinct proliferation abilities were present in human
ovarian tumors, we established cultures with as-
cites-derived tumor cells from 22 ovarian cancer patients.
(Table 1). To assess the formation of tumor spheres,
freshly isolated as well as frozen tumor cells were cul-
tured under conditions proved to favor the growth of
stem cells [16,25-27]. By this approach, we found that
not all of the ascites yielded tumor spheres during pri-

Expression Compilation of Several Putative Cancer Stem Cell Markers by Primary Ovarian Carcinoma 167
Table 1. Summary of patient population.
Ptn
No. Age FIGO stage Histological subtype Sphere formation
4 61 III Serous Yes
10 85 III Adenocarcinoma* Yes
14 81 III Adenocarcinoma* No
15 62 Ic Endometrioid Yes
17 57 III Serous No
19 33 III Mucinous No
20 73 III Serous Yes
21 64 III Serous No
23 57 IV Serous No
30 46 III Adenocarcinoma* Yes
31 67 IV Serous No
33 84 III Serous Yes
35 95 III Sarcoma No
36 77 IV Serous No
37 69 III Serous No
41 36 III Mucinous No
44 69 III Serous No
45 77 III Serous Yes
50 58 III Adenocarcinoma* No
51 52 Ic Mucinous Yes
52 63 III Adenocarcinoma* No
55 63 III Serous Yes
*: Histological subtype undefined; Ptn No.: patient number
mary culture for 2 weeks. Regardless of histological sub-
types, only the cells from 9 out of the 22 patients were
capable of generating sphere-like clusters (Table 2).
3.2. Expression of Putative Ovarian CSC
Markers Are Diverse Among Patients
Ovarian CSCs were identified and prospectively isolated
by several cell surface markers, including CD44+CD117+,
CD133+ and CD24+ [16,27]. To investigate whether
these markers are generally functional to isolate CSCs
from all ovarian cancer patients, we cultured the as-
cites-derived tumor cells for 2 weeks and analyzed CSC
marker expression by flow cytometry (Figure 1). Re-
markably, we found that the expression pattern of these
CSC markers was different among patients, and the ex-
pression of those markers was not associated with histo-
logical subtype of the patients. Moreover, we found no
correlation between expressions of any of these CSC
markers with the ability of tumor sphere formation.
We found cells from only 2 out of 11 patients expressed
CD133, of which one patient had 73.3% while the other
had only 3.7% CD133+ cells. Both tumors were classified
as serous adenocarcinoma. We also found that cells from
all the 13 analyzed patients expressed CD24, although the
CD24+ cells ranged from 3.2% to 86.7%. Similarly, CD44
was also widely expressed, ranging from 2.2% to 88.2%.
Table 2. Statistics of patient population (n = 22).
Age 33-95 64 (median)
Ic 2
III 17
FIGO stage
IV 3
Serous 12
Mucinous 3
Endometrioid 1
Adenocarcinoma* 5
Histologic
subtype
Sarcoma 1
Included Ptn No.: 4, 10, 14, 15, 17, 19, 20, 21, 23, 30, 31, 33, 35, 36, 37, 41,
44, 45, 50, 51, 52, 55; *: Histological subtype undefined
Cells from only 1 in 13 patients had no CD44 expression
and 2 out of 13 patients had very low CD44 expression.
Interestingly, 2 of the 3 patients were diagnosed with
mucinous adenocarcinoma while the other patient was
difficult to be classified into histological subtype. In
contrast to CD24 and CD44, the rare population of
CD117+ cells was detected in 7 out of 11 patients, rang-
ing from 2.9% to 11.2%. We detected a low percentage
(<2%) of CD44+CD117+ cells in 3 out of 6 patient ascites
fluids. In addition to the CD44+CD117+ cell population,
we also observed patients with CD44+CD117- cells as well
as patients with CD44-CD117+ cell populations in the same
histological subtype. Moreover, ABCG2 has been found
over-expressed in CD133+ sphere-forming cells in ovarian
cancer patients [16], and is believed to be associated with
chemo-resistance. We therefore examined whether it is
up-regulated in all ovarian cancer patients. We stained as-
cites-derived tumor cells after primary culture and test
with the expression of surface markers, ABCG2 was also
not detected in all the patients. We only observed a slight
increase of ABCG2 expression (< 1%) in 1 out of the 13
patients.
Table 3. Overview of ALDH expression.
Percentage of gated ALDH+ cells in Region 2
Before culture After primary culture
Ptn
No.
With
DEAB
Without
DEAB
With
DEAB
Without
DEAB
4 0.02 1.18 0.03 0.44
9 0.02 0.65 0.02 0.17
10 0.02 6.99 0.02 0.18
17 0.01 1.32 - -
19 - - 0.01 2.54
20 0.02 0.38 0.02 0.05
21 - - 0.02 0.54
23 0.03 3.93 0.03 0.85
30 0.01 2.74 0.01 0.01
33 - - 0.02 1.07
35 0.01 1.65 - -
36 0.03 3.00 0.03 0.08
42 - - 0.03 0.05
45 0.01 1.39 0.01 0.76
50 0.03 0.18 0.03 1.29
51 0.01 4.86 0.01 0.01
52 0.02 0.72 0.02 0.08
-: Data not available;Data displayed in percentage of positive cells; Ptn No.:
patient number

Expression Compilation of Several Putative Cancer Stem Cell Markers by Primary Ovarian Carcinoma 168
(a)
(b)
Figure 1. Expression of putative ovarian CSC markers. A. Isotype controls are indicated by the grey lines. B. Double staining
of CD44 and CD117 for several patients tumor cells.

Expression Compilation of Several Putative Cancer Stem Cell Markers by Primary Ovarian Carcinoma 169
(a)
(b)
Figure 2. ALDH-positive cells are reduced during primary culture. A. Representative flow cytometry analysis of ascites-derived
ovarian tumor cells using the ALDEFLUOR assay. Cells were incubated with ALDEFLUOR substrate BAAA and the specific in-
hibitor of ALDH, DEAB, for ~45 min. Gates were set according to DEAB control (ALDH+ cells ≤ 0.03%). B. ALDH 1 staining of
ascites-derived tumor cells before culture. ALDH 1 positive cells were very bright comparing to DEAB treated cells. ALDH 1 stain-
ing of ascites-derived tumor cells after primary culture for 2weeks. The signal intensity of ALDH 1 positive cells was reduced.

Expression Compilation of Several Putative Cancer Stem Cell Markers by Primary Ovarian Carcinoma 170
3.3. Aldehyde Dehydrogenase 1 (ALDH1)
Expression/activity Is Changed in Ovarian
Cancer Patient upon Culturing.
ALDH1 is expressed in stem and progenitor cells of sev-
eral tissue types, including CSCs. ALDH1+ cells are en-
riched in colon, breast and ovarian cancers. It predicts
poor clinical outcome in breast cancer while correlates
with poor and favorable prognosis in ovarian cancer
[28,29]. So we wondered whether ALDH1 can be ap-
plied to identify ovarian CSC in cells isolated from as-
cites. We utilized the ALDEFLUOR assay to assess the
presence and expression of the population with ALDH1
enzymatic activity in ascites-derived tumor cells before
and after primary culture of 2 weeks. Analysis of freshly
isolated as well as frozen tumor cells from different pa-
tients showed an average of 2% (2.2% ± 2.0, n = 13)
ALDH1-positive population. However, after 2 weeks of
culture the ALDH1-positive cells were reduced to 1%
(0.5% ± 0.7 n = 15) (Figure 2, Table 4). Among the 15
patients tested after primary culture, 14 of them showed
significant decrease in ALDH1 expression comparing to
fresh cells, while only 1 patient showed up-regulation of
ALDH1. Moreover, we also found that ALDH1 signal
intensity was reduced upon culturing under confocol
microscopy, (Figure 2). From these results we conclude
that ALDH1 cannot be used as a functional marker to
enrich ovarian CSCs.
4. Discussion
The CSC hypothesis has fundamental implications for
cancer biology in addition to clinical implications for
cancer elimination. The development of novel cancer
therapeutics requires targeting this important CSC popu-
lation, to prevent tumor relapse. The success of these
new approaches is based on defining the ‘real’ CSCs.
Several criteria have been established to identify, isolate
and characterize CSCs, including self-renewal tumor
sphere formation and expression of distinct cell surface
antigens, permitting consistent isolation. And based on
these criteria, putative ovarian CSCs have been identified
and prospectively isolated from patient ascites, ovarian
cancer cell lines, primary tumor tissues as well as mouse
cell lines. They all proved that only a small subpopula-
tion of tumor cells bearing certain surface makers are
capable of self-renewal, and are capable of initiating and
supporting the malignant tumor growth. We did a large
study with ascites derived from 22 ovarian cancer pa-
tients and for the first time revealed that, due to hetero-
geneity of patients, expression of the putative ovarian
CSC makers are not applicable to isolate CSCs from all
patients.
Firstly, our study showed that not cells from all pa-
tients are capable of anchorage-independent growth and
three-dimensional tumor sphere formation in stem cell
culture media. This sphere formation difference could be
explained by the origin of CSCs in the patients. There are
two hypothesis elucidate the origin of CSCs, one claims
that CSCs are generated from transformed normal stem
cells, so that they can make use of the already active
self-renewal machinery. The other claims that CSCs are
generated from progenitor cells by regaining the self-renew
pathway. We envisage that normal stem cell-derived CSCs
are capable of sphere formation, whereas progeni-
tor-derived ones are not. Moreover, it is also possible
that the amount of malignant CSCs in some patients was
not sufficient to form tumor spheres, and the tumorigenic
capacity of the CSCs from each patient is different.
However, we cannot exclude that current culture condi-
tions based on other epithelial tumors are not optimal yet
for the proliferation of ovarian CSCs from all patients.
Interestingly, we found cells from patients of FIGO stage
I could form tumor spheres, indicating the existent of
CSCs at the initiating stage of malignant tumor growth.
Such a concept has important ramifications. It lends
support to the hierarchy model of tumor heterogeneity,
meaning that only a small subpopulation of malignant
cells is responsible for tumor initiation.
Secondly, we demonstrated that those putative makers
are also not applicable to isolate CSCs from all primary
ovarian tumors, since their expression differs dramatically
among patients. For instance, we showed that CD133
was only detected in 2 patients; we found that CD24 and
CD44 were expressed by almost all the patients analyzed,
whereas the cell populations bearing CD24 or CD44 in
most patients were too large to be considered as malig-
nant stem cells; we also revealed that CD44+CD117+
populations were detectable in only 3 out of 7 patients.
Consistently, recent studies using ovarian tumor cell
lines as well as primary tumor specimens revealed that
putative CSC markers, including ALDH are displayed in
a complex pattern [30]. In one study using the NCI60
tumor cell line panel , it is found that expression of
CD44, CD24, and CD133 varied greatly even between
cell lines of the same tumor type [31]. In the same study,
they demonstrated cell populations with ALDH activity
ranged widely, from 0 up to 38.8%. In order to increase
understanding of how to target and ultimately eradicate
an ovarian CSC population, researchers make use of dif-
ferent ovarian tumor materials to isolate those cells from
the bulk of cells. However, our results suggest that ovar-
ian cancers are very heterogeneous, study material has a
profound influence on the putative markers identified.
The complexity of marker expression suggests that the-
rapeutic targeting utilize of different tumor materials
could result in different putative CSC populations bear-

Expression Compilation of Several Putative Cancer Stem Cell Markers by Primary Ovarian Carcinoma 171
ing distinct surface markers. Future studies aiming to
isolate CSCs have to take this into account.
Moreover, we found that expression of putative ovar-
ian CSC markers neither correlate with clonogenic ca-
pacity of ascites-derived tumor cells, nor associate with
patient histosubtypes. Studies have shown that a small
percentage of CD24+, or CD133+ cells or CD44+CD117+
cells isolated from ovarian tumor specimens or ovarian
tumor cell lines were considered ovarian cancer-initiating
cell populations. They proved that only a small percent-
age of isolated cells bearing one of those markers were
able to form colonies in vitro, indicating that tumors are
composed of biologically and functionally distinct cell
populations. And they demonstrated that cells within the
tumorigenic clones bear stem cell properties and are ca-
pable of transferring the disease into nude mice[19].
However, we found that CD24 was expressed in cells
from all tested patients. 2 out of 13 patients were bearing
rare populations of CD24+ cells (< 10%), of which one
patient had tumor sphere formation in vitro while the
other had not. Whereas 4 out of 13 patients were with
extremely large CD24+ populations (> 60%), of which
cells from 2 patients formed in vitro spheres but the oth-
ers did not. Moreover, in our study CD133 expression
was detected in cells from 2 out of 11 analyzed patient
ascites, belonging to the serous subtype. One of the 2
patients with large CD133+ population (73.3%) formed
tumor sphere while the other patient with 3.7% CD133+
cells did not. And in our hands only 3 patients were con-
taining CD44+CD117 + cells are restricted to FIGO stage
III serous adenocarcinoma, although the patient with
lowest CD44+CD117+ cell population did not generate
tumor spheres. Collectively, these results again indicate
that ovarian cancers are very heterogeneous, and even
tumors with the same phenotypes might originate from
distinct populations of CSCs. These observations indeed
indicate that those putative markers cannot be used as a
general target to enrich for ovarian CSCs from all histo-
logical distinct patients.
In addition to the surface markers, we showed that
ABCG2 expression was only detectable in 1 out of 11
analyzed patient ascites, suggesting that ABCG2 is not
necessarily the efflux transporter functional in all the
ovarian cancer patients; we cannot exclude the existence
and functions of other membrane transporters in che-
mo-resistance.
Finally, we showed that ALDH1 expression or its ac-
tivity was changed after primary culture, indicating that
it is not a valid marker for ovarian CSC. However, the
reason why ALDH1 expression was reduced upon cul-
turing remains unclear. It has been show that ALDH1 is
a predictor of favorable prognosis in ovarian cancer by
immunohistochemical staining of ALDH1 expression in
a large number of primary ovarian carcinomas [29]. In
contrast, another study reported that higher ALDH1 ex-
pression is significantly associated with poor clinical
outcome in serous ovarian cancer.[28] Our study again
suggests that different histological type ovarian epithelial
tumors have remarkably distinct molecular background.
Therefore, the prognostic value of ALDH1 in ovarian
cancer still needed to be further investigated.
Here we studied primary cells obtained from ascites of
ovarian cancer patients. It remains to be proven that the
cells present in the ascites are a reflection of the cells
present in the tumor mass. Since stem cells are found in
so-called niches and cells in the ascites lost contact with
their niche it cannot be excluded that cells in the ascites
fluid are not equivalent to the tumor cells in the solid
tumor [32]. However, tumor spread in the abdomen of
the patients is likely to proceed from ascites cells pre-
dicting the presence tumor initiating cells in ascites.
Taken together, our study revealed that ovarian cancer
is a heterogonous disease [33] and all the reported puta-
tive ovarian CSC markers are not applicable to isolate
CSCs from all patients. This distinct marker expression
may due to the difference between tumor cell lines and
primary tumor materials. Thus, a universal target to iso-
late ovarian CSCs is required to facilitate tumor elimina-
tion, using ‘representative’ patient materials.
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