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Dendritic Cell Subsets in the Peritoneal Fluid
and Peripheral Blood of Women Suffering
from Ovarian Cancer
I. Wertel,
1
*G. Polak,
1
W. Bednarek,
1
B. Barczyn
´ski,
1
J. Rolin
´ski,
2
and J. Kotarski
1
1
1st Department of Gynaecology, University School of Medicine, Lublin, Poland
2
Department of Clinical Immunology, University School of Medicine, Lublin, Poland
Background: Evaluation of immature myeloid and lymphoid dendritic cells (DCs) in the peritoneal fluid
(PF) and peripheral blood (PB) mononuclears of women with ovarian carcinoma (n547) and benign
ovarian tumors (n537).
Methods: Mononuclear cells were isolated from PF and PB, stained with monoclonal antibodies
(mAbs) against DC antigens (anti-BDCA-1, anti-BDCA-2), and estimated using flow cytometry.
Results: The percentage of PF myeloid DC (MDC) in mononuclears was significantly lower in patients
with ovarian cancer in comparison to the group of nonmalignant ovarian tumors (0.65% and 6.95%).
The percentage of PF lymphoid DCs (LDCs) was higher in patients with ovarian cancer than in the refer-
ence group (0.64% and 0.09%). The percentage of PB MDCs and LDCs did not differ significantly
between studied groups. In women suffering from ovarian cancer the percentage of both MDCs and LDCs
was higher in the PF than in the PB. In the reference group the percentage of MDCs was higher but that
of LDCs was lower in the PF than in the PB. In women with ovarian cancer, PF MDCs/LDCs ratio was
lower in comparison to patients with serous cystadenoma. In PB the ratio of MDCs to LDCs did not differ
significantly between studied groups.
Conclusions: We concluded that MDCs population may be affected by the presence of malignant dis-
ease. LDC subsets may have influence on the local immune response in the PF of women with malignant
tumors of the ovary. q2008 Clinical Cytometry Society
Key terms: myeloid DCs; lymphoid DCs; peritoneal fluid; cystadenocarcinoma
How to cite this article: Wertel I, Polak G, Bednarek W, Barczyn
´ski B, Rolin
´ski J, Kotarski J. Dendritic cell subsets
in the peritoneal fluid and peripheral blood of women suffering from ovarian cancer. Cytometry Part B 2008;
74B: 251–258.
INTRODUCTION
Epithelial ovarian carcinoma (EOC) is the main cause
of death in women with gynecological malignancies. At
the time of diagnosis, 75% of patients with EOC have
advanced intraperitoneal disease (1). Standard treatment
includes surgery, combined chemotherapy or/and radia-
tion, with 90% of the patients developing a recurrence
(2). The 5-year survival rate in these patients, according
to the different data, does not exceed 15% (1).
It has been well documented that multiple mechanisms
may be involved in the development and progression of a
tumor, including a defect in the immune system at the
induction and/or effector phases of antitumor immune
responses, such as antigen presentation in the absence of
major histocompatibility complex (MHC) Class II mole-
cules or costimulatory molecules. The majority of tumor
antigens (Ags) are nonmutated self-antigens to which the
immune system is tolerant. As a result, tumor Ags may
not be effectively presented to the immune system, or T
cells responding to tumor Ags may be anergized (3,4).
One of the most promising strategies to correct such
*Correspondence to: I. Wertel, 1st Department of Gynaecology, Uni-
versity School of Medicine, ul. Staszica 16, 20-081 Lublin, Poland.
E-mail: iwonawertel@gmail.com
Grant sponsor: State Committee for Scientific Research (Warsaw,
Poland); Grant number: 2 PO5E 120 27.
Received 22 October 2007; Accepted 20 January 2008
Published online 26 February 2008 in Wiley InterScience (www.
interscience.wiley.com).
DOI: 10.1002/cyto.b.20410
Cytometry Part B (Clinical Cytometry) 74B:251–258 (2008)
q2008 Clinical Cytometry Society
defects is the enhancement of tumor antigen presentation
with the help of dendritic cells (DCs), the most powerful
inducers of tumor-specific (CD8
1
) cytotoxic T-lymphocyte
responses. Unlike other antigen-presenting cells (APCs),
such as B cells and macrophages, DCs are capable of ini-
tiating not only secondary immune responses but also pri-
mary immune responses (such as activation of the naive
T cells) that are directed against specific antigens (5–8).
The DC system represents a heterogeneous group of
APC differing at the level of precursor cells, factors influ-
encing growth and maturation, phenotype, and APC
function (5,6,8). Circulating DC precursors home to
lymphoid and nonlymphoid tissues where they reside as
immature cells. At this stage, DCs are well equipped to
acquire antigens but nevertheless express low levels of
the requisite MHC and costimulatory molecules needed
for T-lymphocyte stimulation. Following antigen engulf-
ment and processing, DCs migrate to secondary lymph-
oid organs where they mature and become APCs that
are able to select and activate naive Ag-specific T cells
and induce an Ag-specific immune response (5,8).
Morphologically and phenotypically distinct DCs,
which are present at many different anatomical sites, are
derived from two lineages: myeloid or lymphoid (9,10).
Myeloid DCs (DC-1) are a major subpopulation of human
peripheral blood (PB) DCs, which are CD4
1
, Lin
2
,
CD11c
bright
, CD123
dim
, CD45RO
1
, and CD2
1
. They
express myeloid markers (CD13, CD33) as well as Fc
receptors (CD32, CD64, FceRI). Myeloid DCs (MDCs)
also express the blood DC antigen-BDCA-1 (CD1c) that
is specific for immature PB MDCs (6,11).
Lymphoid DCs (DC-2) have been described recently in
human PB (12), lymphoid tissue (13), and in the perito-
neal fluid (PF) (14,15). DC-2 cells express a specific
BDCA-2 (CD303) marker. Immunophenotyping of BDCA-
2
1
DC characterizes these cells as being CD4
1
, Lin
2
,
CD11c
2
, CD123
bright
, CD45RA
1
, CD2
2
, and expressing
neither myeloid lineage markers nor Fc receptors (11).
Almand et al. (16) detected that in the PB of patients
with breast, head and neck, and lung cancers the num-
ber of DCs was dramatically reduced. Similar results
were found by Yanagimoto et al. (17) in patients with
pancreatic cancer. The decrease of DCs in the PB from
these patients closely correlated with the stage and dura-
tion of the disease (16). However, there is a lack of
detailed information relating to the presence and a role
of DC subsets in the microenvironment of nonmalignant
and malignant body fluids.
The purpose of our study was to establish if the imma-
ture MDCs and lymphoid DCs (LDCs) are present in the
PF and PB of patients with ovarian cancer.
METHODS
Patients
Of 84 women, 47 patients, aged 30–85 years, were
found to have ovarian epithelial cancer. In the study
group, the grade was I in one case, II in 18 cases, and
III in 28 cases. The levels of preoperative Ca-125 marker
ranged from 45.71 to 11,179.00 with the median
1,747.72 U/ml. The patient profiles are presented in Ta-
ble 1. None of the women received chemotherapy
before operation.
Because sterilization is forbidden by Polish law, there
was no possibility to obtain PF from healthy, fertile
women. Thus we decided to choose the reference group
of the 37 patients with serous cystadenoma. The study
was approved by the Lublin University School of Medi-
cine Ethics Committee.
Cell Preparation
PF and PB were taken into heparinized tubes (sodium
heparin). In the noncancer group, all visible PF was aspi-
rated during surgical procedure from the anterior and
posterior cul-de-sacs, under direct vision to avoid blood
contamination. All women had venous blood samples
collected before the surgical procedure. PF and PB
mononuclear cells were isolated by density gradient cen-
trifugation on Lymphoprep (Nycomed, Norway) for
25 min at 600gat room temperature. Interphase cells
were collected and washed twice in phosphate-buffered
saline (PBS). The cells were counted in a Neubauer
chamber. The results were expressed as the number of
mononuclear cells per milliliter of PF and PB.
The cell surface antigens were determined on fresh
cells at the time of sample submission. Isolated cells
(1 310
6
) were incubated for 20 min at 48C with mono-
clonal antibodies (mAbs) specified against DC surface
antigens and washed twice in PBS containing 0.2 mM
ethylene-diaminetetraacetic acid and 0.5% bovine serum
albumin. The following directly conjugated mAbs were
used: anti-BDCA-1 (CD1c) FITC, anti-BDCA-2 (CD303)
FITC (Miltenyi Biotec, Bergisch Gladbach, Germany), and
anti-CD19 CyChrome, anti-CD123 PE (Pharmingen, San
Diego, CA). Mouse anti-human IgG
2a
isotype control was
used for anti-CD1c staining. Mouse anti-human IgG
1
iso-
type control was used for anti-BDCA-2 staining.
Flow Cytometric Analysis
Flow cytometric analysis of stained samples was per-
formed on FacsCanto instrument (Becton Dickinson, San
Jose, CA). A total of 300,000 events were acquired and an-
alyzed using FacsDiva software. Cell debris and dead cells
were excluded from the analysis based on scatter signals.
In our study, we identified immature MDCs as BDCA-1
(CD1c)
1
CD19
2
cells. BDCA-1 (CD1c) marker is also
expressed on a subpopulation of CD19
1
small resting B
lymphocytes. Therefore, the mononuclear cell analysis
Table 1
Clinical Characteristic of the Tumors
Histologic type Number of patients
Serous cystadenocarcinoma 21
Undifferentiated carcinoma 14
Mucinous cystadenocarcinoma 8
Clear cell carcinoma 4
Serous cystadenoma 37
252 WERTEL ET AL.
Cytometry Part B: Clinical Cytometry
region was analyzed for BDCA-1 (CD1c) and CD19 stain-
ing and BDCA-1 (CD1c)
1
B cells were excluded from
CD1c
1
DCs by counterstaining for CD19. Next, the
mononuclear cell analysis region was analyzed for BDCA-
2 and CD123 antigens. BDCA-2
1
CD123
1
cells were
counted as immature LDCs as described by Dzionek
et al. (11). The identification of DCs by flow cytometry
in the PF is presented in Figure 1.
We expressed the results as a percentage of MDC and
LDC in mononuclear cells. In addition, we calculated the
MDCs/LDCs ratio (M/L ratio) and the concentration of
DCs per milliliter of PB and PF.
Statistical Analysis
Statistical analysis was performed using nonparametric
tests. All data were presented as medians with the inter-
quartile ranges (25th to 75th). Wilcoxon paired test was
used to compare the results of PF and PB. Mann–Whit-
ney U test was applied to the results of statistical com-
parison between the studied groups. Statistical compari-
sons among more than two groups were performed
using Kruskal–Wallis test for independent samples.
Spearman’s rank test was used to assess the relationship
between concentrations of mononuclear cells and con-
centrations of DCs. Pvalue less than 0.05 was consid-
ered statistically significant.
RESULTS
The concentration of mononuclears and DC subsets in
the PF and PB of patients with ovarian tumors are pre-
sented in Figure 2.
Concentration of Mononuclears and DCs in the PF and PB
of Women with Ovarian Cancer and Serous Cystadenoma
The concentration of mononuclear cells was signifi-
cantly higher in the PF of patients with ovarian cancer
(P<0.001) than in women with serous cystadenoma.
The concentration of MDCs was significantly lower in
the PF of patients with ovarian cancer (P<0.001) than
in women with benign tumors (Figure 2). In contrary,
the concentration of LDCs was higher (P<0.001) in
the PF of patients with malignant disease than in the ref-
erence group. The concentration of mononuclears,
MDCs, and LDCs in the PB did not differ significantly
between studied groups (Figure 2).
The Percentage of DCs in the PF and PB of Women with
Ovarian Cancer
The percentage of MDC and LDC subsets in mononu-
clears was significantly higher in the PF than in the PB
(Figure 3).
The MDC to LDC ratio in patients with ovarian cancer
did not differ significantly between PF (median, 1.0;
interquartile range, 0.45–2.46) and PB (median, 1.0;
interquartile range, 0.60–2.03).
The Percentage of DCs in the PF and PB
of Women with Serous Cystadenoma
The percentage of MDCs in the PF was 6.95% (inter-
quartile range, 3.56–13.32%) and was significantly higher
than in the PB (0.23%; interquartile range, 0.15–0.26%).
However, the percentage of LDCs was significantly
higher in the PB (0.20%; interquartile range, 0.13–0.27%)
FIG. 1. The identification of PF dendritic cells by flow cytometry
(P1). The mononuclear cell analysis region applied to light scatters.
The P1-gated events were analyzed for BDCA-1 (CD1c) and CD19
staining, and BDCA-11CD192cells were counted as immature MDCs
(P2). The P1-gated events were then analyzed for BDCA-2 and CD123,
and BDCA-21CD1231cells were counted as LDCs (P3).
253DENDRITIC CELLS IN OVARIAN CANCER PATIENTS
Cytometry Part B: Clinical Cytometry
compared with PF (0.09%; interquartile range, 0.05–
0.17%).
The MDC to LDC ratio in women with benign ovarian
tumors was significantly higher in the PF (median, 71.2;
interquartile range, 33.12–129.41) than in the PB (me-
dian, 1.0; interquartile range, 0.71–1.37).
The Percentage of DCs in the PF and PB of Women with
Ovarian Cancer and Benign Tumors
The percentages of PF mononuclear cells that were
identified as MDC and LDC in women with ovarian
tumors are presented in Figures 3a and 3b.
The percentage of MDCs was significantly lower in
the PF of patients with ovarian cancer (0.65%; interquar-
tile range, 0.24–1.36%) than in women with benign
tumors (6.95%; interquartile range, 3.56–13.32%). In con-
trary, the percentage of LDCs was higher in the PF of
patients with malignant disease (0.64%; interquartile
range, 0.29–1.43%) than in the reference group (0.09%;
interquartile range, 0.05–0.17%). We also found that the
ratio of MDCs to LDCs in the PF was different between
women with malignant (1.0%; interquartile range, 0.45–
2.46) and benign (71.2; interquartile range, 33.12–
129.41) ovarian tumors.
The percentage of MDCs and LDCs and the ratio of
MDCs to LDCs in the PB did not differ significantly
between studied groups (1.0; interquartile range, 0.60–
2.03 vs. 1.0; interquartile range, 0.71–1.37).
FIG. 2. The concentration (310
6
per ml) of mononuclears (a) and dendritic cells (b,c) in the peritoneal fluid (PF) and peripheral blood (PB) of
patients with epithelial ovarian cancer (EOC) and serous cystadenoma.
254 WERTEL ET AL.
Cytometry Part B: Clinical Cytometry
The Percentage of DC in Patients with Different Histologic
Type of Ovarian Cancer
The percentages of PF and PB MDC and LDC in
women grouped by histologic type of ovarian cancer are
presented in Figure 4.
The percentage of MDC and LDC in patients with se-
rous carcinoma (sCa), undifferentiated carcinoma
(udCa), and mucinous cystadenocarcinoma (mCa) was
significantly higher (P<0.05) in the PF than in the PB.
The percentage of MDC and LDC in patients with clear
cell carcinoma (ccCa) did not differ significantly
between PF and PB. The MDC to LDC ratio in patients
with different histologic type of ovarian cancer did not
differ significantly between PF and PB.
The percentage of PB and PF MDCs and LDCs did not
differ significantly among patients with sCa, udCa, mCa,
and ccCa (Fig. 4).
The percentage of PF MDCs in patients with sCa,
udCa, mCa, and ccCa was significantly lower but that of
LDCs was significantly higher in comparison to patients
with serous cystadenoma (Fig. 4).
The MDC to LDC ratio in the PF of patients with sCa
(1.0; interquartile range, 0.45–2.46), udCa (1.02; inter-
quartile range, 0.46–1.56), mCa (1.32; interquartile
range, 0.63–2.03), and ccCa (1.32; interquartile range,
0.04–5.07) was significantly lower than in women with
benign ovarian tumors (71.2; interquartile range, 33.12–
129.41).
The percentage of MDC and LDC and the ratio of
MDCs to LDCs in the PB did not differ significantly
between women with different histologic types of ovar-
ian cancer and the reference group.
Correlation Between Concentration of Mononuclear Cells
and DC Subsets
There was a positive correlation between concentra-
tions of PF and also PB mononuclear cells and both MDC
and LDC in patients with malignant and benign ovarian
tumors. The statistical data are detailed in Table 2.
DISCUSSION
Most cases of ovarian malignancies are advanced at
the time of diagnosis. Therefore, ovarian cancer is one
of the leading causes of death among gynecologic malig-
nancies with a 5-year survival rate of about 15% (1). The
early clinical development of ovarian neoplasm is associ-
ated with nonrecognition of cancer by the immune sys-
tem. The fact that ovarian tumors, which differ so signifi-
cantly from the normal ovarian tissue, are not recog-
nized as foreign and not eliminated remains unclear. It
should be appreciated that the resistance of neoplastic
tissue can reflect either antigenic similarity of neoplastic
and normal tissue or aberrant immune response, includ-
ing nonrecognition of foreign cancer antigens by APCs
(3,4).
DC are the most potent antigen-presenting cells play-
ing a key role in the induction of protective immune
responses and maintenance of immunological memory.
Their exceptional ability to instruct naive T cells to initi-
ate immune responses is critically beneficial for the host
defense against neoplastic cells (6). Furthermore, a large
body of literature demonstrates a close relationship
between the presence of DCs within various malignant
tumors and prognosis (18–21). On the other hand, sev-
eral investigators have described the defective function
of DCs in human suffering from cancer, such as the lack
of expression of costimulatory molecules, consistent
with the phenotype of immature and nonactivated DCs
(16,22–24).
Ovarian cancer differs from others malignancies of
genital tract because of the spread throughout the peri-
toneal cavity. Serous ovarian neoplasms spread along the
peritoneum even in the case of relatively small tumors
of the ovary. It has been suggested that immune altera-
tion may be responsible for this phenomenon. There-
fore, our study was undertaken not only to investigate
the PB DCs but also to examine DCs in the local PF
immune system of women with ovarian cancer.
For identification of MDC and LDC subsets we chose
relatively novel markers: BDCA-1 and BDCA-2. With
regard to the presented, currently insurmountable diffi-
culties in obtaining normal PF, we selected the PF from
FIG. 3. The percentage of PF and PB MDC (a) and LDC (b)inthe
mononuclears of patients with epithelial ovarian cancer (EOC) and se-
rous cystadenoma (S.C.).
255DENDRITIC CELLS IN OVARIAN CANCER PATIENTS
Cytometry Part B: Clinical Cytometry
women with serous cystadenoma as a reference fluid
developing in the absence of neoplastic process.
Our results show that PF environment in women with
both malignant and nonmalignant ovarian tumors con-
tains considerably more DCs than PB. This observation
appears to support our previous hypothesis that the PB
DCs are specifically recruited into peritoneal cavity (14).
The reason for the accumulation of DCs in the PF has
not been clearly explained yet. It has been demonstrated
that many tumor types attract macrophages and DC
through the direct or indirect production of chemoat-
tractant factors (25,26). APC recruitment is expected to
contribute to the development of antitumoral immune
response, and such an effect has been demonstrated in a
number of animal studies. However, a number of studies
have demonstrated that the infiltration of tumors by APC
contributed to their aggressive phenotype by supplying
factors necessary for tumor cell proliferation and inva-
siveness, such as growth and angiogenic factors, as well
as proteolytic enzymes (25,26). Experimental studies
confirmed that MDCs, contrary to LDCs, are highly mi-
gratory cells with remarkable capacity for transmigrating
FIG. 4. The percentages of PB (a) and PF (b) MDC and LDC in women grouped by histologic type of ovarian cancer and in the reference group, se-
rous cystadenoma (S.C.).
Table 2
Correlation Between Concentration of Mononuclear Cells and Myeloid (M) and Lymphoid (L) DC Subsets
in the Peritioneal Fluid (PF) and Peripheral Blood (PB) of Patients
Patients Correlation between
RSpearman rank
correlation coefficient t(N-2) P
Ovarian cancer (n547) PF mononuclear cells and MDC 0.745 6.988 <0.00l
PF mononuclear cells and LDC 0.597 4.656 <0.001
PB mononuclear cells and MDC 0.668 5.607 <0.001
PB mononuclear cells and LDC 0.591 4.577 <0.001
Serous Cystadenoma (n537) PF mononuclear cells and MDC 0.701 5.648 <0.001
PF mononuclear cells and LDC 0.481 3.159 <0.05
PB mononuclear cells and MDC 0.762 5.893 <0.001
PB mononuclear cells and LDC 0.700 4.913 <0.001
256 WERTEL ET AL.
Cytometry Part B: Clinical Cytometry
across endothelium in response to chemotactic agents
without requiring endothelial support (27). But migra-
tion of DCs from blood is not the only explanation for
finding high concentration of DCs in PF. The other possi-
bilities may be: accumulation of DCs in the peritoneal
cavity by the breakdown of their journey to the lymph-
oid tissues, local generation from precursors, or nonphy-
siological proliferation of immature DCs.
The comparison of DC subsets between studied
groups demonstrate that the concentration and the per-
centage of PF MDCs in mononuclear cells was signifi-
cantly lower in patients with ovarian cancer in compari-
son to the group of nonmalignant ovarian tumors. These
results suggest that PF MDCs population may be affected
by the presence of the malignant disease and might con-
tribute to diminished acquired immune responses
observed in these women.
In contrast, the concentration and the percentage of PF
LDCs was significantly higher in patients with ovarian
cancer than in the reference group. This fact may be im-
portant for understanding of the mechanism of tumor
immune escape, because LDCs are expected to induce
tolerance rather than immunity. In the study by Zou et al.
(28) it was shown that LDCs infiltrating ovarian carci-
noma inhibited tumor-specific immunity by suppressing T-
cell activation. The investigation by Curiel et al. (15)
shows comparable results in tumor ascites of women
with ovarian carcinomas. They demonstrated that numer-
ous functional LDCs accumulate in tumor ascites and in-
hibit antitumor immunity. The same authors found that
LDCs produce high levels of the angiogenic cytokines
(TNF-aand IL-8) and induce potent neovascularization in
vivo. Thus, tumors may manipulate DCs distribution and
alter DCs function to support tumor angiogenesis.
In addition, we have calculated MDCs/LDCs ratio
which expresses the equilibrium between presentation
of antigens for induction of an effective immune
response or stimulation of T-cell tolerance (29,30). We
found that in the PF of patients with ovarian cancer the
BDCA-1/BDCA-2 DCs ratio was significantly lower in
comparison to patients with benign disease. These data
may suggest that LDCs subsets have influence on the
local immune response in the PF of patients with malig-
nant disease. Interestingly, these cells markedly outnum-
bered MDCs in the PF but not in the PB. PF LDCs may
actively suppress the Ag-specific T-cell response and thus
could be involved in immunosuppression. However the
precise role of PF LDCs still needs to be evaluated.
Some studies documented a significant dysfunction of
type 1 T-cell responses in tumor-bearing hosts, suggest-
ing that tumor progression might be associated with a
preferential type 2 T-cell response (31). However, factors
which influence the Th2 predominance in malignant tu-
mor patients still remain enigmatic. The available data
indicate that BDCA-1 and BDCA-2 DCs were claimed to
stimulate Th1 and Th2 types of immune responses,
respectively (6,11).
In this study we have demonstrated a significant accu-
mulation of immature lymphoid (BDCA-2 positive ) DCs
in the malignant PF in comparison with PF from women
with benign disease. Therefore, we concluded that PF
LDCs in patients with ovarian cancer may favor Th2 lym-
phocyte differentiation and/or the induction of immuno-
logical tolerance which is now considered as one of the
important mechanisms of tumor escape from immune
system control.
Further studies on the DCs’ function are necessary for
the complete understanding of the influence of tumor
microenvironment on DCs and the importance of this
fact for circumventing the immune system by cancer. It
should be stressed that PF as an area where both
immune and tumors cells coexist is an excellent clinical
model for studying local immune response. Detailed
investigation on the role of defective DCs in antitumor
immunity may provide new insights into the develop-
ment of novel therapeutic strategies for malignancies.
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