Journal of Cancer Therapy, 2013, 4, 1273-1282
http://dx.doi.org/10.4236/jct.2013.48150 Published Online October 2013 (http://www.scirp.org/journal/jct)
1273
Usefulness of Immuno-Magnetic Beads Conjugated with
Anti-EpCAM Antibody for Detecting Endometrial
Cancer Cells*
Yoshikatsu Koga1, Satoshi Katayose2, Nobuko Onoda2, Takahiro Kasamatsu3, Tomoyasu Kato3,
Shunichi Ikeda3, Mitsuya Ishikawa3, Ken Ishitani4, Yasuo Hirai4, Hideo Matsui4,
Yasuhiro Matsumura1
1Division of Developmental Therapeutics, Research Center for Innovative Oncology, National Cancer Center Hospital East, Kashiwa,
Japan; 2R & D Department Unit 1, JSR Life Sciences Corporation, Tsukuba, Japan; 3Department of Gynecology, National Cancer
Center Hospital, Tokyo, Japan; 4Department of Obstetrics and Gynecology, Tokyo Women’s Medical University, Tokyo, Japan.
Received July 26th, 2013; revised August 24th, 2013; accepted September 1st, 2013
Copyright © 2013 Yoshikatsu Koga et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
ABSTRACT
A simple and non-invasive method for detecting endometrial cancer in women with abnormal uterine bleeding is re-
quired. For this purpose, we prepared immuno-magnetic beads conjugated with anti-human EpCAM rat monoclonal
antibody (mAb) for isolating exfoliated endometrial cells including endometrial cancer cells in vaginal discharge. The
affinities of the anti-human EpCAM rat mAbs were analyzed by flow cytometry and immunocytochemistry and then
magnetic beads were conjugated with the mAbs. The rate of retrieval of endometrial cells using the immuno-magnetic
beads was calculated. Endometrial cells were isolated using the immuno-magnetic beads from the vaginal discharges of
22 patients with endometrial cancer and 16 non-malignant controls. The isolated cells were stained using endometrial
cancer specific-mAbs and analyzed by flow cytometry and imaging cytometry. The immuno-magnetic beads conjugated
with high-affinity mAb (clone 1456) appeared to have very low auto-fluorescence. Sufficient enrichment of Ep-CAM-
positive cells using immuno-magnetic beads was observed in both simulation and clinical samples. The overall sensi-
tivities of flow cytometry and imaging cytometry to detect endometrial cancer cells were 72.7% and 45.5%, respec-
tively. Meanwhile, the overall specificities of flow cytometry and imaging cytometry for healthy controls were 75.0%
and 81.3%, respectively. Our immuno-magnetic beads have very low auto-fluorescence, so they could be useful for
fluorescent analysis, such as fluorescent immunochemical staining. In the future, these novel immuno-magnetic beads
could be used for cytological study.
Keywords: Immuno-Magnetic Beads; Auto-Fluorescence; Endometrial Cancer; Cancer Screening
1. Introduction
Endometrial cancer is one of the most common malig-
nancies of the female genital tract globally [1]. In the
USA, approximately 80% of patients with cancer of the
uterus were diagnosed as having endometrial cancer [2]
and the number of patients with endometrial cancer has
been increasing in Japan [3]. Regarding cancer screening
of female genital cancers, cervical cancer of the uterus
can be diagnosed more simply than endometrial cancer
[4]. However, there is no screening method for endo-
metrial cancer at present [5]. Abnormal uterine bleeding
is a common symptom of endometrial cancer; however,
endometrial cancer is diagnosed in only 10% of women
with abnormal uterine bleeding [6,7] and no organic
cause is found in 60% to 70% of these women [8]. For
many years, dilatation followed by curettage has been the
standard method for detecting endometrial cancer in
women with abnormal uterine bleeding. In the last dec-
ade, several screening methods for women with abnor-
mal uterine bleeding, such as transvaginal sonography
[9,10], endometrial cytology [10], suction endometrial
curettage [10], and endometrial sampling [10,11], were
reported to reduce the cost and invasiveness. However,
these screening methods are still invasive, time-con-
*Disclosures: The authors declare that no actual or potential conflicts o
f
interest exist.

Usefulness of Immuno-Magnetic Beads Conjugated with Anti-EpCAM Antibody
for Detecting Endometrial Cancer Cells
1274
suming, and expensive, and above all, inconvenient for
women. Therefore, a simple, economic, and non-invasive
method of detecting cancer through cancer screening for
patients with abnormal uterine bleeding is required.
Recently, we reported a cytological analysis for the
detection of endometrial cancer cells combined with en-
dometrial cancer-specific monoclonal antibodies (mAbs)
and imaging cytometry using exfoliated endometrial cells
[12]. Exfoliated endometrial cancer cells and normal
endometrial cells were observed in the vaginal discharge
of patients with endometrial cancer. In addition, to detect
endometrial cancer cells, mAbs against cysteine-rich
with EGF-like domain 1 (CRELD1), G-protein-coupled
receptor kinase 5 (GRK5), solute carrier family 25 mem-
ber 27 (SLC25A27), and stanniocalcin 2 (STC2) were
used. However, almost all tampon-retrieved cells were
granulocytes and normal squamous cells from the vagina,
and the rate of endometrial cells in all tampon-retrieved
cells was less than 1% [12].
Meanwhile, to isolate exfoliated colonocytes from fe-
ces, we previously reported immuno-magnetic beads
conjugated with anti-human epithelial cell adhesion
molecule (EpCAM) mouse mAb (Magnosphere MC290/
anti-EpCAM mouse IgG, JSR Life Sciences, Tsukuba,
Japan) [13]. The same as for fecal samples, exfoliated
endometrial cells could be isolated using immuno-mag-
netic beads. The capturing mAbs (anti-human EpCAM
mAbs conjugated to Magnosphere) and the detecting
mAbs (endometrial cancer-specific mAbs) are both
mouse monoclonal antibodies; thus, the secondary anti-
body for immunochemical staining cross-reacted with the
mAbs for capture and those for detection. To resolve this
issue, we have established anti-human EpCAM rat mAb
and immuno-magnetic beads with very low auto-fluo-
rescence for the isolation of exfoliated endometrial cells
in the present study. Then, the exfoliated endometrial
cells collected using immuno-magnetic beads were sub-
jected to cellular analysis.
2. Materials and Methods
2.1. Establishment of Anti-Human EpCAM Rat
Monoclonal Antibodies
A recombinant human EpCAM/Fc chimera (R & D Sys-
tems, Minneapolis, MN) was used as an immunogen. 0.1
mg of the antigen was mixed with complete Freund’s
adjuvant (Difco, Detroit, MI) and injected intraperito-
neally into Wistar rat (Japan SLC, Shizuoka, Japan). The
ELISA-positive hybridoma cells were cloned by limiting
dilution in 96-well culture plates and established as sta-
ble hybridoma cells.
Immunoglobulin G was separately purified from each
ascites fluid sample using protein G affinity chromatog-
raphy (GE Healthcare Life Science, Piscataway, NJ). The
purified IgG fractions were used for further characteriza-
tion and were evaluated for their reactivity.
2.2. Flow Cytometry of Anti-Human
EpCAM mAb
HT-29 cells and UMUC-3 cells were used as EpCAM-
positive and -negative cells, respectively. 2 × 105 tryp-
sinized cells were put into a 2-mL tube and incubated
with 0.2 μg of each mAb for 30 min at 4˚C. After rinsing
with PBS containing 0.5% bovine serum albumin (BSA)
and 2 mM ethylenediaminetetraacetic acid (EDTA) (B. E.
PBS), the cells were incubated with 0.1 μg of DyLight
649-conjugated donkey anti-rat IgG secondary antibody
(Jackson ImmunoResearch, West Grove, PA) for 30 min
at 4˚C. Finally, the cells were rinsed with B. E. PBS and
nuclear-stained with propidium iodide (PI) solution (In-
vitrogen, Eugene, OR). The stained cells were analyzed
by flow cytometry using a Guava easyCyte.
2.3. Immunocytochemistry of Anti-Human
EpCAM mAb
EpCAM-positive and -negative cells were pre-cultured
on BD Falcon culture slides (BD Biosciences, Bedford,
MA). The cells were fixed in 4% paraformaldehyde
(Wako, Osaka, Japan) for 30 min at 4˚C and rinsed with
ultrapure water. Endogenous peroxidase was blocked
with a 3% hydrogen peroxide solution in 100% methanol
for 30 min at room temperature, followed by rinsing with
PBS. Nonspecific protein binding was blocked with 5%
skim milk in PBS for 30 min at room temperature. After
draining off the skim milk solution, the cells were incu-
bated with 4 μg of each anti-human EpCAM rat mAb for
1 hr at room temperature. After rinsing with PBS, the
cells were incubated with 0.1 μg of HRP-conjugated
donkey anti-rat IgG secondary antibody (Jackson Immu-
noResearch) for 30 min at room temperature. After rins-
ing with PBS, the cells were incubated with the 3,3’-dia-
minobenzidine tetrahydrochloride (DAB+) liquid system
(Dako, Glostrup, Denmark) for 5 min at room tempera-
ture. Finally, the cells were rinsed and counter-stained
with hematoxylin solution.
2.4. Immuno-Magnetic Beads Conjugated with
Anti-Human EpCAM Rat mAb
Magnetic beads of 3.0 μm diameter, Magnosphere
MC290/Tosyl (JSL Life Sciences), were prepared in this
study. The magnetic beads were directly conjugated with
anti-human EpCAM rat mAb. The sizes of the obtained
magnetic beads were determined to be 3.0 μm based on
electron microscopic observation. Also, commercially
available immuno-magnetic beads conjugated with anti-

Usefulness of Immuno-Magnetic Beads Conjugated with Anti-EpCAM Antibody
for Detecting Endometrial Cancer Cells
1275
human EpCAM mouse mAb, Dynabeads Epithelial En-
rich (Dynal, Oslo, Norway) and Magnosphere MC290/
anti-EpCAM mouse IgG (JSR Life Sciences), were used
in the present study.
2.5. Simulation Analysis for Retrieving Cells
Using Immuno-Magnetic Beads
In the present study, the immuno-magnetic beads were
used for the isolation of endometrial cells from the sam-
ples of abnormal uterine bleeding or menstrual bleeding;
thus, EpCAM-positive cells added to peripheral blood
were used in the simulation study. 1 × 106 HT-29 cells
with 3 mL of peripheral blood (approximately 1 × 107
granulocytes) were prepared in the simulation study. 30
mL of a retrieval PBS buffer containing 0.1% BSA and 2
mM EDTA was added to the sample. The EpCAM-
positive cells were captured using 40 μL of immuno-
magnetic beads, and the mixtures were incubated for 30
min under gentle rolling at room temperature. Then, the
mixtures on the magnet were incubated on a shaking
platform for 15 min at room temperature. Subsequently,
the supernatant was removed and the retrieved cells were
fixed in 4% paraformaldehyde for 30 min at 4˚C. In the
clinical simulation study, menstrual blood from a healthy
volunteer was subjected to the same method as described
above.
To analyze the efficiency of immuno-magnetic beads,
the cells retrieved using the immuno-magnetic beads
were stained with the anti-human EpCAM goat poly-
clonal Ab (R & D Systems) and assessed using an imag-
ing cytometer, CELAVIEW (Olympus, Tokyo, Japan).
The retrieved cells were plated into each well of flat-
bottomed 96-well tissue culture plates (Corning). The
plates were centrifuged at 30 g for 5 min at 4˚C and the
supernatant was removed, followed by drying over-night
at room temperature. The cells were permeabilized by
incubation in PBS containing 0.2% Triton X-100 for 20
min at room temperature. Non-specific protein binding
was blocked with 5% skim milk in PBS for 30 min at
room temperature. After draining off the skim milk solu-
tion, the cells were incubated with 1 μg of commercially
available anti-human EpCAM goat polyclonal Ab, fol-
lowed by incubation for 1 hr at room temperature. After
rinsing with PBS, the sections were incubated with 0.1
μg of DyLight 488-conjugated bovine anti-goat IgG sec-
ondary antibody (Jackson ImmunoResearch) for 30 min
at room temperature.
2.6. Patients with Endometrial Cancer or
Non-Malignant Controls
From January 2012 to December 2012, 22 patients with
histologically confirmed endometrial cancer and 16 non-
malignant controls before menopause were enrolled in
this study (Ta bl e 1). All the patients had undergone
surgical resection of their primary cancer at the National
Cancer Center Hospital, Tokyo, Japan. The endometrial
cancer patients were slightly older than the non-malig-
nant controls. All patients and controls received detailed
information about the study and gave written consent to
participate in the study, which was approved by the Insti-
tutional Review Board of the National Cancer Center,
Japan, and Tokyo Women’s Medical University.
2.7. Retrieval of Naturally Exfoliated
Endometrial Cells Using
Immuno-Magnetic Beads
The participants in this study inserted a small tampon (3
× 1 cm, Unicharm, Tokyo, Japan) into their vagina and
took the tampon out after about 3 hrs [12]. The tampon
was then placed in a centrifuge tube (Corning) containing
30 mL of retrieval buffer and was shipped immediately
to our laboratory at 4˚C. To retrieve the exfoliated cells
from the tampon, it was pressed thoroughly and all
Table 1. Characteristics of endometrial cancer patients and
non-malignant controls.
Patients (N = 22) Controls (N = 16)
Age [median (range)] 60 (47 - 75) 41 (29 - 52)
EpCAM + cells FCM
[median (range)] 17 (1 - 2322) 113 (2 - 1964)
EpCAM + cells ICM
[median (range)] 17 (0 - 1893) 82 (0 - 2014)
Histology [Number (%)]
Endometrioid
adenocarcinoma 18 (81.8%)
Mixed adenocarcinoma 2 (9.1%)
Serous adenocarcinoma 1 (4.5%)
Carcinosarcoma 1 (4.5%)
FIGO classification
[Number (%)]
stage I 14 (63.6%)
stage II 4 (18.2%)
stage III 3 (13.6%)
stage IV 1 (4.5%)
Tumor size
[mm, median (range)] 37 (9 - 82)
Patients: patients with endometrial cancer, Controls: non-malignant patients
without malignancy in the uterus, EpCAM + cells FCM: positively stained
cells using EpCAM Ab counted by flow cytometry, EpCAM + cells ICM:
positively stained cells using EpCAM Ab counted by imaging cytometry,
FIGO: International Federation of Gynecology and Obstetrics.

Usefulness of Immuno-Magnetic Beads Conjugated with Anti-EpCAM Antibody
for Detecting Endometrial Cancer Cells
1276
obtained fluid was collected in another new centrifuge
tube. The exfoliated endometrial cells were captured by
adding 40 μL of immuno-magnetic beads into the sample
solution, and the mixtures were incubated for 30 min
under gentle rolling conditions at room temperature. The
mixtures on the magnet were incubated on a shaking
platform for 15 min at room temperature. Then, the
supernatant was removed and the retrieved cells were
fixed in 4% paraformaldehyde for 30 min at 4˚C. The
cells were rinsed with PBS and stored in 1 mL of PBS-
based storage buffer containing 0.1% BSA, 2 mM EDTA,
and 0.1% NaN3 at 4˚C until analysis.
2.8. Cellular Analysis Using Flow Cytometry and
Imaging Cytometry
For cellular analysis using flow cytometry, the exfoliated
endometrial cells retrieved using the immuno-magnetic
beads were put into a 2-mL tube and incubated with 4 μg
of anti-CRELD1 (clone 2D1E12I), GRK5 (clone 2F11C3),
SLC25A27 (clone 3A8B14), and STC2 (clone 2D4C4)
mouse mAbs (ACTGen, Nagano, Japan) as well as 1 μg
of anti-human EpCAM goat polyclonal antibody for 1 hr
at room temperature. After rinsing with retrieval buffer,
the cells were incubated with 0.1 μg of DyLight 488-
conjugated bovine anti-goat IgG secondary antibody and
0.1 μg of DyLight 649-conjugated donkey anti-mouse
IgG secondary antibody for 30 min at room temperature.
Finally, the cells were rinsed with retrieval buffer and
nuclear-stained with PI solution. The stained cells were
analyzed using flow cytometry with a Guava easyCyte
(Millipore, Billerica, MA).
For cellular analysis using imaging cytometry, the
exfoliated endometrial cells retrieved using immuno-
magnetic beads were prepared as described in the simu-
lation experiment.
3. Results
3.1. Evaluation of Anti-Human EpCAM Rat
Monoclonal Antibodies
Mean intensities of EpCAM positive cells with isotype
control (negative control), clone B8-4 (positive control),
clone 118, clone 533, clone 572, clone 787, clone 1097,
clone 1286, clone 1456, and clone 1468 were 3.33, 1683,
1823, 810, 1865, 1693, 1571, 1531, 1670, and 861, re-
spectively (Figure 1(a)). Mean intensities of EpCAM-
negative cells with the same mAbs were 3.54, 3.75, 5.00,
3.52, 3.92, 12.9, 5.41, 6.33, 3.58, and 3.24, respectively
(Figure 1(b)). The affinities to EpCAM-positive cells
using two mAbs (clones 533 and 1468) were lower than
that to clone B8-4. Meanwhile, non-specific reactions to
EpCAM-negative cells were observed for four mAbs
(clones 118, 787, 1097, and 1286). From the results of
flow cytometry, two mAbs (clones 572 and 1456) were
selected as candidates for the conjugation. In the immu-
nocytochemical staining, EpCAM-positive cells were
stained positively by clone B8-4 and clone 1456 mAbs
(Figure 1(c)). However, these cells could not be stained
by clone 572 mAb. Thus, clone 1456 was used as anti-
human EpCAM rat mAb in the following experiments.
3.2. Low Auto-Fluorescence of
Immuno-Magnetic Beads Conjugated with
Anti-Human EpCAM Rat mAb
The levels of auto-fluorescence of commercially avail-
able immuno-magnetic beads (Dynabeads Epithelial En-
rich and MC290/EpCAM mouse IgG) and our new im-
muno-magnetic beads (MC290/EpCAM rat IgG) were
compared (Figure 2(a)). Fluorescent intensities were
assessed at wavelengths of excitation (Ex) 488 nm/emis-
sion (Em) 525 nm (green fluorescence), Ex 488 nm/Em
583 nm (yellow fluorescence), Ex 488 nm/Em 680 nm
(red fluorescence), and Ex 640 nm/Em 661 nm (red2
fluorescence). Mean intensities of Dynabeads Epithelial
Enrich in terms of green, yellow, red, and red2 fluores-
cence were 13.7, 41.5, 44.3, and 10.8, respectively. Cor-
responding mean intensities of MC290/EpCAM mouse
IgG were 3.01, 4.61, 4.05, and 6.03, respectively. In ad-
dition, mean intensities of MC290/EpCAM rat IgG were
2.82, 4.20, 3.29, and 6.35, respectively. Consequently,
MC290 beads showed low auto-fluorescence and Dyna-
beads showed high auto-fluorescence. The binding of
Dynabeads and MC290 beads to EpCAM-positive cells
negative control
clone B8-4
clone 118
clone 533
clone 572
clone 787
clone 1097
clone 1286
clone 1456
clone 1468
EpCAM
A) B)
clone B8-4clone 572clone 1456
EpCAM
positive cells
EpCAM
negative cells
C)
(a)
(c)
(b)
Figure 1. Evaluation of anti-human EpCAM rat mono-
clonal antibodies. (a) Flow cytometry using EpCAM-posi-
tive cells. Seven clones of anti-human EpCAM rat mAbs
were compared to a positive control (anti-human EpCAM
mouse mAb; clone B8-4). (b) Flow cytometry using Ep-
CAM-negative cells. (c) Immunocytochemistry of EpCAM-
positive and -negative cells using clone B8-4, clone 1456,
and clone 572. Scale bar represents 100 μm.

Usefulness of Immuno-Magnetic Beads Conjugated with Anti-EpCAM Antibody
for Detecting Endometrial Cancer Cells
1277
is shown in Figure 2(b). EpCAM-positive cells were
retrieved by using both Dynabeads Epithelial Enrich and
MC290 beads; however, Dynabeads showed auto-fluo-
rescence. Meanwhile, MC290 had the lowest level of
auto-fluorescence. Thus, our MC290/EpCAM rat IgG
appeared to be useful for fluorescent immunochemistry.
3.3. Cell Isolation Using Immuno-Magnetic
Beads Conjugated with Anti-Human
EpCAM Rat mAb
In the simulation experiment, EpCAM-positive cells
were added to granulocytes at a ratio of approximately
1:10. The actual proportion of EpCAM-positive cells was
8.8% (EpCAM-positive cells/all cells: 659/7508) (Figures
3(a) and (e)). After the isolation using the beads, the rate
of EpCAM-positive cells was 48.5% (306/634) (Figures
3(b) and (f)). In the clinical sample, menstrual blood of a
healthy volunteer was used. The rate of EpCAM-positive
cells in the clinical subject was 3.0% (361/12,112) (Fig-
ures 3(c) and (g)). After the isolation using the beads, the
rate of EpCAM-positive cells in the clinical subject was
61.9% (224/394) (Figures 3(d) and (h)). Recovery rate
of EpCAM-positive cells using immuno-magnetic beads
was 50% to 67%. Thus, EpCAM-positive cells before
cell isolation were more than that after cell isolation. All
cells were stained by DAPI (blue) and EpCAM-positive
cells were stained by anti-EpCAM mAb (green) and
DAPI (blue). After cell isolation, many particles without
staining as shown in the Figures 3(f) and (h) were im-
muno-magnetic beads. Sufficient enrichment of EpCAM-
positive cells using MC290/EpCAM rat IgG was thus
observed in both the simulation experiment and the cli-
nical samples.
3.4. Clinical Evaluation of Cellular Analysis
The median numbers of EpCAM-positive cells observed
in endometrial cancer patients using flow cytometry and
imaging cytometry were 17 (range 1 - 2322) and 17 (0 -
1893), respectively. Meanwhile, the median numbers of
EpCAM-positive cells observed in healthy controls using
flow cytometry and imaging cytometry were 113 (range
2 - 1964) and 82 (0 - 2014), respectively. The samples
with less than 10 EpCAM-positive cells were not appli-
cable in this study. Samples with over 10 EpCAM-posi-
tive cells were examined and for practical purpose, more
than 20% positive by the cancer detecting mAbs were
designated “positive”. The sensitivities and specificities
of cellular analyses using flow cytometry and imaging
cytometry are shown in Table 2. In the flow cytometry,
immuno-staining-positive cells using CRELD1, GRK5,
SLC25A27, and STC2 mAbs were observed in 40.9%,
18.2%, 18.2%, and 27.3% of endometrial cancer patients,
blight field + fluorostainingEpCAM/DAPI/auto-fluorescence
DynabeadsMC290
B)
Ex 488/Em 525 nmEx 488/Em 583 nmEx 488/Em 680 nmEx 640/Em 661 nm
fluorescent intensity
cell count
A)
(a)
Dynabeads Epithelial Enrich
MC290/anti-EpCAMmouse IgG
MC290/anti-EpCAM rat IgG
(b)
Figure 2. Auto-fluorescence of each bead. (a) Auto-fluorescence of Dynabeads Epithelial Enrich, MC290/EpCAM mouse IgG
and MC290/EpCAM rat IgG. Fluorescent intensities were assessed at wavelengths of excitation (Ex) 488 nm/emission (Em)
525 nm, Ex 488 nm/Em 583 nm, Ex 488 nm/Em 680 nm, and Ex 640 nm/Em 661 nm. (b) Binding abilities of Dynabeads and
MC290 beads to EpCAM-positive cells. EpCAM-positive cells were retrieved by using both Dynabeads Epithelial Enrich and
MC290 beads. Dynabeads exhibited some auto-fluorescence, while MC290 exhibited less. Scale bar represents 100 μm.

Usefulness of Immuno-Magnetic Beads Conjugated with Anti-EpCAM Antibody
for Detecting Endometrial Cancer Cells
1278
(a) (b)
(c) (d)
)
)
(e) (f) (g) (h)
Figure 3. Cell isolation using immuno-magnetic beads tagged with anti-human EpCAM rat mAb. (a) Imaging cytometry of
simulation study before cell isolation. The rate of EpCAM-positive cells in the simulation subject before bead isolation was
8.8% (EpCAM-positive cells/all cells: 659/7508). (b) Imaging cytometry of simulation study after cell isolation. The rate of
EpCAM-positive cells in the simulation subject after bead isolation was 48.5% (306/634). (c) Imaging cytometry of clinical
simulation study before cell isolation. The rate of EpCAM-positive cells in the clinical subject before bead isolation was 3.0%
(361/12,112). (d) Imaging cytometry of clinical simulation study after cell isolation. The rate of EpCAM-positive cells in the
clinical subject after bead isolation was 61.9% (224/394). (e) Immunocytochemistry of simulation study before cell isolation.
(f) Immunocytochemistry of simulation study after cell isolation. (g) Immunocytochemistry of clinical simulation study before
cell isolation. (h) Immunocytochemistry of clinical simulation study after cell isolation. EpCAM protein was stained with Dy-
Light 488 (green) and the nucleus was stained with DAPI (blue). Scale bar represents 100 μm.

Usefulness of Immuno-Magnetic Beads Conjugated with Anti-EpCAM Antibody
for Detecting Endometrial Cancer Cells
1279
Table 2. Sensitivities and specificities of cellular analyses using flow cytometry and imaging cytometry.
Endometrial cancer patients (N = 22)
Flow cytometry Imaging cytometry
No. Sensitivity (%, 95% CI) No. Sensitivity (%, 95% CI)
Combined marker 16 72.7% (49.8 - 89.3) 10 45.5% (24.4 - 67.8)
CRELD1 9 40.9% (20.7 - 63.7) 4 18.2% (5.2 - 40.3)
GRK5 4 18.2% (5.2 - 40.3) 4 18.2% (5.2 - 40.3)
SLC25A27 4 18.2% (5.2 - 40.3) 2 9.1% (1.1 - 29.2)
STC2 6 27.3% (10.7 - 50.3) 2 9.1% (1.1 - 29.2)
Non-malignant controls (N = 16)
Flow cytometry Imaging cytometry
No. Specificity (%, 95% CI) No. Specificity (%, 95% CI)
Combined marker 12 75.0% (47.7 - 92.7) 13 81.3% (54.3 - 96.0)
CRELD1 14 87.5% (61.7 - 98.5) 15 93.8% (69.8 - 99.8)
GRK5 15 93.8% (69.8 - 99.8) 16 100% (79.4 - 100)
SLC25A27 14 87.5% (61.7 - 98.5) 15 93.8% (69.8 - 99.8)
STC2 15 93.8% (69.8 - 99.8) 14 87.5% (61.7 - 98.5)
CRELD1: cysteine-rich with EGF-like domain 1 (clone 2D1E12I), GRK5: G-protein-coupled receptor kinase 5 (clone 2F11C3), SLC25A27: solute carrier
family 25 member 27 (clone 3A8B14), STC2: stanniocalcin 2 (clone 2D4C4), 95% CI: 95% confidence interval.
respectively. In the imaging cytometry, immuno-stain-
ing-positive cells using CRELD1, GRK5, SLC25A27,
and STC2 mAbs were observed in 18.2%, 18.2%, 9.1%,
and 9.1% of endometrial cancer patients, respectively.
Approximately 90% of non-malignant controls were
negative in both flow cytometry and imaging cytometry
using these cancer-specific mAbs. The overall sensitivi-
ties of flow cytometry and imaging cytometry to detect
endometrial cancer cells were 72.7% [95% confidence
interval (CI), 49.8 - 89.3] and 45.5% (95% CI, 24.4 -
67.8), respectively. On the other hand, the overall speci-
ficities of flow cytometry and imaging cytometry for
non-malignant controls were 75.0% (95% CI, 47.7 - 92.7)
and 81.3% (95% CI, 54.3 - 96.0), respectively.
4. Discussion
The tampon retrieval sample contained several kinds of
exfoliated cell, such as granulocytes, normal squamous
cells, and endometrial cells [12]. Thus, a cell isolation
method using immuno-magnetic beads was important for
the isolation of exfoliated endometrial cells from tampon.
There were two problems regarding the fluorescent im-
munochemical staining of exfoliated endometrial cells
retrieved using immuno-magnetic beads. One was that
the fluorescent secondary antibody cross-reacted to both
the capturing mAb and the detecting mAb because both
were mouse mAbs. To resolve this issue, we succeeded
in developing anti-human EpCAM rat IgG (clone 1456),
the affinity of which was as high as that of anti-human
EpCAM mouse IgG (clone B8-4). We established clone
B8-4 as high affinity mAb in the previous study [13].
Clone B8-4 was useful for immunohistochemistry, flow
cytometry, and cell isolation. Thus, we used clone B8-4
as a positive control in this study. We think that clone
572 may recognize protein structure of EpCAM antigen
on living cells, thus, this mAb was positive for flow cy-
tometry but negative for immunochemical staining. The
other problem to be resolved was the auto-fluorescence
of immuno-magnetic beads. The fluorescent intensity
cannot be calculated correctly if the immuno-magnetic
beads exhibit auto-fluorescence. Polyurethanes are fre-
quently used in biomedical applications because of their
excellent biocompatibility [14,15]. However, polyure-
thanes are known to emit auto-fluorescence [16]. Be--
cause the commercially available immuno-magnetic beads
(Dynabeads Epithelial Enrich) are coated with polyure-
thane emitting auto-fluorescence, these beads cannot be
used in our cellular analysis. The beads prepared by us
exhibited very low auto-fluorescence because the sur-
faces of our immuno-magnetic beads are coated with a
poly-glycidyl methacrylate layer that does not emit auto-
fluorescence.

Usefulness of Immuno-Magnetic Beads Conjugated with Anti-EpCAM Antibody
for Detecting Endometrial Cancer Cells
1280
In the simulation study, our newly developed immuno-
magnetic beads could retrieve EpCAM-positive cells
efficiently. Moreover, the beads could isolate the endo-
metrial cells from various types of cell adsorbed to a
tampon. Both endometrial cancer cells and normal en-
dometrial cells were EpCAM-positive. Almost EpCAM-
positive cells from non-malignant controls were exfoli-
ated normal endometrial cells. Meanwhile, EpCAM-
positive cells from endometrial cancers were consisted of
endometrial cancer cells and normal endometrial cells.
Not only endometrial cancer cells but also tubal cancer,
cervical cancer, or vaginal cancer cells may be exfoliated
into a vaginal discharge. There is, therefore, potential to
detect these cells in a same sample. In this study, mag-
netic beads conjugated with anti-human EpCAM mAb
(EpCAM-beads) were used for isolation of exfoliated
endometrial cancer cells from vaginal discharge. Since
exfoliated cancer cells from squamous cell carcinoma
(most of cervical cancer and vaginal cancer) are EpCAM-
negative, they were not isolated by EpCAM-beads. On
the other hand, exfoliated cancer cells from adenocarci-
noma (most of tubal cancer and a few of cervical cancer)
were isolated by EpCAM-beads because they were Ep-
CAM-positive. Unfortunately, we do not have a mAb
which can distinguish cervical cancer cells from normal
squamous cells. In our previous study, most tampon-
retrieved cells were granulocytes and normal squamous
cells [12] and the rate of endometrial cells in menstrual
blood was only 3% in this study. In particular, few en-
dometrial cells were obtained from among tampon-re-
trieved cells of the non-malignant control in a non-men-
strual period. The major symptom of endometrial cancer
is abnormal uterine bleeding; thus, subjects with abnor-
mal uterine bleeding or normal menstrual blood were
enrolled in this clinical study as controls. A lot of Ep-
CAM-positive endometrial cells were obtained from
controls with menstrual period but the least EpCAM-
positive cells were obtained from controls with non-men-
strual period. Thus, menstrual blood was used for non-
malignant control in this study. The median EpCAM-
positive cells of controls were over 100. Meanwhile,
major symptom of endometrial cancer patient was ab-
normal uterine bleeding. However, almost patients with
endometrial cancer were in menopause and their endo-
metria had changed to be atrophic. Therefore, a few ex-
foliated EpCAM-positive cells were obtained from pa-
tients with endometrial cancer but most of them appeared
to be malignant endometrial cells. Immunomagnetic beads
that could retrieve the endometrial cells from a sample of
uterine blood appeared to be useful.
In this study, the detecting mAbs were the same as in
our previous study [12]. Recently, several mAbs against
EpCAM, EphA2, MMP2, survivin, and podoplanin were
investigated for endometrial cancer treatment and the
detection of lymphovascular invasion [15,17-19]. How-
ever, these mAbs insufficient to detect endometrial can-
cer cells, so further development of endometrial cancer-
specific mAbs should be performed. Meanwhile, several
microRNAs (miRNAs) that were highly expressed in
endometrial cancer tissue were reported [20-22]. The
miRNA expression test of exfoliated endometrial cells
might become an alternative endometrial cancer screen-
ing method in the future.
The present study showed that the sensitivity to detect
endometrial cancer patients and the specificity for non-
malignant controls with flow cytometry analysis were
73% and 75%, respectively. Meanwhile, the sensitivity
and the specificity by imaging cytometry analysis were
46% and 81%, respectively. Generally, cancer screening
is performed for average-risk population to reduce the
cancer mortality in the group. Thus, cancer screening is
performed annually and should be low-cost and non-
(less)-invasive. Although sufficient sensitivity of cancer
screening is 50% to 75%, specificity is needed over 95%.
In this context, we have to admit that specificity of our
cellular analysis was low. We are, therefore, developing
further specific mAbs to increase the specificity. Recent-
ly serum human epididymis protein 4 (HE4) has been
used for the detection of endometrial cancer [23-27]. An-
gioli et al. reported that the sensitivity and specificity of
serum HE4 test were 59% and 100%, respectively [27].
Serum (plasma) protein analyses (containing HE4, CEA
or CA125) were useful for detection of cancer relapse or
therapeutic effect of chemotherapy. Therefore, these are
used for so-called tumor markers as other cancers. For
example, in colorectal cancer, CEA is useless for the
early stage but is available as a tumor marker because
serum CEA level in patients with early stage of colorec-
tal cancer is not higher than that in healthy controls.
Therefore, fecal occult blood test is generally used in
colorectal cancer screening. With the same reason, serum
protein was not useful for endometrial cancer screening
and proteins derived from endometrial cancer cells were
contained more in a vaginal discharge. It is then specu-
lated that the vaginal discharge containing exfoliated
cells is useful for endometrial cancer screening. These
findings are still insufficient for endometrial cancer
screening, so further investigations are needed.
Sensitivity and specificity of our cellular analysis are
less than those of traditional methods, such as curettage.
Histological diagnosis using endometrial cytology or bio-
psy is used for final diagnosis. However, examinees suf-
fer physical and psychological pain with curettage. In
addition, current screening methods including curettage
are time consuming and expensive and the medical ex-
amination is inconvenient for women. Our novel cyto-

Usefulness of Immuno-Magnetic Beads Conjugated with Anti-EpCAM Antibody
for Detecting Endometrial Cancer Cells
1281
logical or histological methods are non-invasive com-
pared to former cytology or histology. Cellualr analysis
using immuno-magnetic beads and fluorostaining could
be performed without human’s eye; thus, it could reduce
the cost. Moreover, the procedures of cellular analysis
can be performed automatically using immuno-magnetic
beads and magnet. In this method, the women only col-
lect vaginal discharge using a tampon and send the sam-
ple to a laboratory at suitable storage condition. Although
both the sensitivity and specificity should be enhanced,
we think that this method may become convenient and
cheap, and the new mAbs sets detecting endometrial
cancer should be developed. In this study, the number of
clinical samples was small. Thus, many issues remain to
be resolved in order to improve these cellular analyses
for the detection of endometrial cancer cells using im-
muno-magnetic beads and flow cytometry. Our immuno-
magnetic beads have very low auto-fluorescence, so they
should be useful for fluorescent analysis, such as fluo-
rescent immunochemical staining. In the future, these
novel materials could lead to the next-generation meth-
odology for cellular diagnosis.
5. Acknowledgements
We thank Ms. Noriko Abe, Ms. Masae Ohmaru, Ms.
Miyuki Miura and Ms. Yuka Nishina for their technical
assistance and Ms. Kaoru Shiina for her secretarial assis-
tance.
This work was supported by the Advanced Research
for Medical Products Mining Programme of the National
Institute of Biomedical Innovation (NIBIO) of Japan (Y.
Koga); the Innovation Promotion Program from the New
Energy and Industrial Technology Development Organi-
zation (NEDO) of Japan (Y. Matsumura); the Japan So-
ciety for the Promotion of Science (JSPS) through the
Funding Program for World-Leading Innovative R & D
on Science and Technology (FIRST Program), initiated
by the Council for Science and Technology Policy
(CSTP) (Y. Matsumura); and the National Cancer Center
Research and Development Fund (Y. Matsumura).
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