# Improved Recovery of Exfoliated Colonocytes from Feces Using Newly

Authors: Yoshikatsu Koga; Masahiro Yasunaga; Satoshi Katayose; Yoshihiro Moriya; Takayuki Akasu; Shin Fujita; Seiichiro Yamamoto; Hideo Baba; Yasuhiro Matsumura
Journal: Gastroenterology Research and Practice
 (2008)

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## Abstract

We demonstrated the feasibility of a new methodology for isolating colonocytes from feces. To reduce costs and improve the recovery rate of colonocytes from feces, we attempted to develop new immunomagnetic beads. Several sizes of magnetic beads were prepared and tagged with a monoclonal antibody against EpCAM. We made several new monoclonal antibodies against EpCAM, and each monoclonal antibody was tagged to the magnetic beads. In the simulation, the most efficient recovery of HT-29 cells was obtained using the smallest size of beads. Also, beads tagged with a monoclonal antibody with a higher affinity against EpCAM had a higher recovery rate. Similar results were obtained when the smallest size of beads with the highest-affinity monoclonal antibody was applied to clinical samples. The newly developed immunomagnetic beads may be useful for isolating colorectal cancer cells from feces, enabling the cytological or molecular biological diagnosis of CRC.


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## Body

## 1. Introduction

Colorectal cancer  (CRC) is one of
the most common malignancies worldwide. CRC is the third leading  cause of cancer-related
mortality and the second leading cause of cancer-related incidence. Nevertheless,
the survival rate of patients with CRC is high if this cancer can be diagnosed
and surgically resected at an early stage [1]. Thus, to
reduce the mortality rate associated with CRC, the development of a screening test
by which early-stage cancers can be detected is necessary.The
fecal occult blood test has been used widely as a screening test for CRC [2–4]. However, three recent large-scale studies have shown that the sensitivity
of a fecal occult blood test was not very high when a total colonoscopy in all
subjects was used as a reference standard [5–7]. Therefore, several new systems have been developed for
diagnosing colorectal cancer based on the detection of mutated DNA in feces [8–19]. However,
these methods are time consuming and are not sufficiently sensitive. The major
reason for this inaccuracy is the fact that nucleic acids in feces are derived
from an enormous number and a variety of bacteria and normal cells.
Accordingly, the proportion of genes derived from cancer cells in feces is typically
as low as 1%, at most [9]. This makes
the clinical application of gene-detecting methods difficult.Previously,
we reported that cancer cells remain viable in the feces and can be isolated
from naturally evacuated feces using an immunomagnetic bead methods that we developed
[20, 21]. After
extracting DNA from cells isolated from feces, CRC-related gene alterations
were examined and a diagnostic sensitivity of 71% (82/116) and a specificity of
88% (73/83) were obtained [21].These figures were relatively satisfactory, but
the numbers of cells isolated from the feces were not very high and, in
addition, commercially available immunomagnetic beads are relatively expensive.
In this context, to improve the function of immunomagnetic beads for retrieving
colonocytes from naturally evacuated feces and to reduce the cost performance
of this method, we attempted to develop new immunomagnetic beads and to
evaluate their usefulness.


## 2. Materials and Methods

### 2.1. Immunomagnetic Beads

First, we prepared three different sizes of
magnetic beads (3.0, 4.9, and 5.9μm). Beads with each different size were
tagged with commercially available antihuman EpCAM monoclonal antibody (mAb),
VU-1D9 (AbD Serotec, Oxford,
UK) (see Table 1). Briefly, magnetic beads for cell separation were prepared from polymer microspheres
and supermagnetic iron oxide extracted from magnetic fluid. To make magnetic
beads with different sizes, uniform polymer microspheres with diameters of 1.8,
3.3, or 3.9 μm were coated with the iron oxide using mechanical shearing
stress. The composite beads were then overcoated with a hydrophilic polymer
layer through the polymerization of glycidyl methacrylate on the bead. After
the hydrolysis of the poly glycidyl methacrylate layer, a tosyl group was
introduced to the layer as an active group for antibody conjugation. The sizes
of the obtained magnetic beads were determined to be 3.0, 4.9, and 5.9 μm based
on electron microscopic observation (see Figure 1(a)).Table 1
New immunomagnetic beads.
BeadsSize (μm)Antihuman EpCAM antibodiesA3.0VU-1D9B4.9VU-1D9C5.9VU-1D9D3.0Clone1-2E3.0Clone B8-4F3.0Clone B8-7VU-1D9, commercially available EpCAM antibody; clone1-2, clone B8-4, and clone B8-7 were developed by us in the present study.Simulation to evaluate the recovery rates of colonocytes using several sizes of beads. (a) Electron microscopy images show 3 different sizes of magnetic beads prepared by us. The sizes are 3.0, 4.9, and 5.9μm. Scale bar, 50 μm. (b) Comparison of cell recovery rates. The cell recovery rates using beads A (smallest, 3.0 μm), beads B (medium, 4.9 μm), and beads C (largest, 5.9 μm) are 65.9 ± 1.37 (%, mean ± SD), 61.1 ± 0.98, and 57.1 ± 0.75, respectively. The columns show the cell retrieval rates and the bars show the standard deviations. Significant differences are showed by an asterisk (*, P  =  .0001) or a double asterisk (**, P  <  .0001). (c) Papanicolaou staining of HT-29 cells captured using immunomagnetic beads. The arrowheads show the cell-bead complexes. The beads A have adhered to the HT-29 cells with the highest densely. Scale bar, 50 μm.
(a)(b)(c)Immobilization of the antibody onto the beads
was carried out using a 2-step reaction. First, an antimouse IgG mAb was
coupled to the bead using the amino groups of the antibody. One hundred
micrograms of the goat antimouse IgG (Fc; Millipore Corporation, Billerica, Ma, USA) were added
to 1 mL of 1 wt% bead suspension in 0.1 M borate buffer, pH 9.5, and reacted for
24 hours at 37°C. After the elimination of uncoupled antibodies through
repeated washing with TBS-T (25 mM Tris-buffered saline, pH 7.2, containing
0.01% Tween 20), anti-EpCAM mAb was immobilized on the bead as a second step.
Twenty micrograms of commercially available anti-EpCAM mAb, VU-1D9, were added
to 1 mL of 1 wt% bead in TBS-T and mixed for 1 hour at room temperature. After
the reaction, uncaptured antibody was washed away with TBS-T and stored at 4°C
until use.Next, we developed new antihuman EpCAM mAbs in
order to obtain an antibody with higher affinity against EpCAM. A recombinant
human EpCAM/Fc chimera (R&D Systems, Minneapolis,
Minn, USA)
was used as an immunogen. The antigen (0.1 mg) was mixed with complete Freund's
adjuvant (Difco, Detroit, Mich, USA) and injected intraperitoneally (IP) into BALB/c mice (Charles
River Japan, Shizuoka, Japan). Subsequent injections were made using an RIBI
adjuvant system, MPL  +  DM emulsion (Corixa, Seattle, Wash, USA) every three weeks for a total
of 7 times. Three weeks later, the mice were given an intravenous (i.v.) booster
injection of 0.1 mg of the antigen in phosphate buffered saline. Three days
later, spleen cells from the immunized mice were fused with myeloma cells
(P3X63Ag8.653) at a ratio of 7:1 in 50% polyethylene glycol 4000 (Sigma, St.
Louis, Mo, USA) in RPMI 1640 at room temperature for 1 minute. After
centrifugation, the cells were pelleted, washed, resuspended in RPMI 1640
containing 10% NCTC 109, 20% FCS, and 50 ng/L of mouse IL-6 (R&D Systems),
and plated in flat-bottomed 96-well tissue culture palates (Costar Corning, Corning,
NY, USA). Following overnight incubation in a humidified 5% CO2 atmosphere at 37°C, hypoxanthine-aminopterin-thymidine
(HAT) medium was added to start HAT selection. Hybridoma clones were cultured
for an additional 8 days, and then the culture media were assayed for specific antibody
production using the ELISA method on Maxisorp microtiter plates (Nunc,  Roskilde, Denmark)
coated with the antigen. Briefly, an aliquot of undiluted hybridoma culture supernatant
was added to the antigen coated wells. After 1 hour of incubation and a subsequent
wash cycle, an appropriate dilution of peroxidase-conjugated rabbit IgG (Bethyl
Laboratories, Montgomery, Ala, USA)
to mouse IgG reagent was added. The enzyme-linked IgG binding was detected
using an H2O2 and o-phenylenediamine substrate solution. The color
intensity was measured automatically using a SpectraMax Plus microplate
spectrophotometer (Molecular Devices, Sunnyvale, Calif, USA).
The ELISA positive hybridoma cells were cloned by limiting dilution in 96-well
culture plates and established as stable hybridoma cells.The isotype of the mAb was determined using an MONO Ab-ID KIT (Zymed,
San Francisco, Calif, USA)
according to the manufacturer's instructions. Ascites fluids were obtained from
pristine-primed CD1-Foxn1nu mice (Charles River Japan) injected with each mAb
producing hybridoma clone. Immunoglobulin G was separately purified from each
ascites fluid sample using protein G affinity chromatography (GE Healthcare
Life Science, Piscataway, NJ, USA).
The purified IgG fractions were used for further characterization and were evaluated
for their reactivity. The immunoglobulin concentration was determined by
measuring the absorbance at 280 nm using an extinction coefficient of 1.38 for
mouse IgG.The affinities to EpCAM of the newly developed antihuman
EpCAM mAbs were analyzed using flow cytometry with a BD FACSCalibur (BD, Franklin Lakes,
NJ, USA).
HT-29 cells and UMUC-3 cells were used as an EpCAM positive control and
negative control, respectively. The antihuman EpCAM mAbs and nonspecific mouse
IgG1 were directly labeled using Zenon mouse IgG labeling kits (Molecular
Probes, Eugene, Ore, USA) according to the manufacturer's instructions.Finally, the new antihuman EpCAM mAbs were conjugated
to the optimal size of magnetic beads chosen in the present study (see Table1).


### 2.2. Simulation

A
simulation was conducted to determine the optimal bead conditions for the recovery
of HT-29 colorectal cancer cells using a previously established method [21]. Briefly, 2-gram
fecal samples were homogenized in a Hanks-HEPES-FBS buffer (40 mL) consisting of
Hanks solution, 10% fetal bovine serum (FBS), and 25 mM HEPES buffer (pH 7.35)
at 200 times per minute for 1 minute using a Stomacher (Seward, Thetford, UK). A
total of 1  ×  105 HT-29 cells were added to the homogenized solution and
filtered through a nylon filter (pore size: 512 μm). The HT-29 cells were
retrieved using 80 μL of several immunomagnetic beads, and the mixtures were
incubated for 30 minutes under gentle rolling conditions at room temperature.
The mixtures on the magnet were incubated on a shaking platform for 15 minutes
at room temperature. Then, the supernatant was removed, and the retrieved cells
were counted using a NucleoCounter (ChemoMetec A/S, Allerød, Denmark).Finally, to determine the optimal immunomagnetic beads, HT-29
colorectal cancer cells were retrieved using these immunomagnetic beads, and
then the cell-bead complexes were fixed and stained using papanicolaou stain.


### 2.3. Patients with Colorectal Cancer

From
March 2007 to July 2008, 40 patients with histologically confirmed colorectal
cancer were enrolled in the present study. Nineteen of the 40 patients were enrolled
in a study to analyze the optimal bead size from March 2007 to September 2007. Then,
21 of the 40 patients were enrolled in a study to evaluate the quality of the antibody
from October 2007 to July 2008. All the patients had undergone surgical
resection of their primary cancer at the National Cancer Center Hospital, Tokyo, Japan.
All patients were thoroughly
informed of the content of the study, and provided their written
consent to participate in
the study. The study was approved by the Institutional Review Board of the National
Cancer Center, Japan.


### 2.4. Clinical Evaluation

Before surgical resection, naturally evacuated fecal samples were
obtained from patients with colorectal cancer. Each fecal sample was divided
into 2-gram samples for use in the evaluation of several immunomagnetic beads
with different sizes and different affinities of the EpCAM antibody. The fecal
samples were prepared as described in the simulation section. Colonocytes isolated
from the feces were stored at −80°C until genomic DNA extraction.Genomic
DNA was extracted from colonocytes isolated from feces using an Allprep mini
kit (QIAGEN, Valencia, Calif,
USA)
according to the manufacturer's instructions.For
the genomic DNA analysis, we targeted a consensus sequence of human Alu repeats.
The sequences for the Alu primers and probe used in this study were as follows:
forward primer, 5′-TAGTAGAGACGGGGTTTCACCTTG-3′; reverse primer, 5′-AGCTTGCAGTGAGCCGAGAT-3′;
probe, 5′-GAGAATGGCGTGAA-3′. The reporter dye at the 5′-end of the probe was
FAM, and the quencher dye at the 3′-end was MGB.The
reaction mixture for the genomic DNA analysis consisted of 4μL of a template
DNA, 10 μL of TaqMan Fast Universal PCR Master Mix (Applied Biosystems, Foster, Calif,
USA), 500 nM of
forward and reverse primers, and 250 nM of probe in a total reaction volume of
20 μL. Real-time PCR amplification was performed using precycling heat activation at 95°C
for 20 seconds, followed by 25 cycles of denaturation at 95°C
for 3 seconds, and annealing/extension at 62°C for 30 seconds in an Applied Biosystems 7500 Fast Real-Time PCR
System (Applied Biosystems). The absolute quantification of genomic DNA in each
sample was determined using a standard curve with serial dilutions (10 ng to 100 fg) of TaqMan control genomic DNA (Applied Biosystems). A negative control
(without template) was run in each reaction plate.


### 2.5. Statistical Analysis

The
cell retrieval rate for each group was analyzed using a Tukey-Kramer multiple
comparisons test. Statistical differences in the cell retrieval abilities of the
new immunomagnetic beads were determined using a two-sided Mann-Whitney U test.
Statistical analyses were performed using StatView Ver. 5 for Windows (Abacus
Concepts, Berkeley, Calif, USA).
Values ofP  <  .05 were considered statistically significant.


## 2.1. Immunomagnetic Beads

First, we prepared three different sizes of
magnetic beads (3.0, 4.9, and 5.9μm). Beads with each different size were
tagged with commercially available antihuman EpCAM monoclonal antibody (mAb),
VU-1D9 (AbD Serotec, Oxford,
UK) (see Table 1). Briefly, magnetic beads for cell separation were prepared from polymer microspheres
and supermagnetic iron oxide extracted from magnetic fluid. To make magnetic
beads with different sizes, uniform polymer microspheres with diameters of 1.8,
3.3, or 3.9 μm were coated with the iron oxide using mechanical shearing
stress. The composite beads were then overcoated with a hydrophilic polymer
layer through the polymerization of glycidyl methacrylate on the bead. After
the hydrolysis of the poly glycidyl methacrylate layer, a tosyl group was
introduced to the layer as an active group for antibody conjugation. The sizes
of the obtained magnetic beads were determined to be 3.0, 4.9, and 5.9 μm based
on electron microscopic observation (see Figure 1(a)).Table 1
New immunomagnetic beads.
BeadsSize (μm)Antihuman EpCAM antibodiesA3.0VU-1D9B4.9VU-1D9C5.9VU-1D9D3.0Clone1-2E3.0Clone B8-4F3.0Clone B8-7VU-1D9, commercially available EpCAM antibody; clone1-2, clone B8-4, and clone B8-7 were developed by us in the present study.Simulation to evaluate the recovery rates of colonocytes using several sizes of beads. (a) Electron microscopy images show 3 different sizes of magnetic beads prepared by us. The sizes are 3.0, 4.9, and 5.9μm. Scale bar, 50 μm. (b) Comparison of cell recovery rates. The cell recovery rates using beads A (smallest, 3.0 μm), beads B (medium, 4.9 μm), and beads C (largest, 5.9 μm) are 65.9 ± 1.37 (%, mean ± SD), 61.1 ± 0.98, and 57.1 ± 0.75, respectively. The columns show the cell retrieval rates and the bars show the standard deviations. Significant differences are showed by an asterisk (*, P  =  .0001) or a double asterisk (**, P  <  .0001). (c) Papanicolaou staining of HT-29 cells captured using immunomagnetic beads. The arrowheads show the cell-bead complexes. The beads A have adhered to the HT-29 cells with the highest densely. Scale bar, 50 μm.
(a)(b)(c)Immobilization of the antibody onto the beads
was carried out using a 2-step reaction. First, an antimouse IgG mAb was
coupled to the bead using the amino groups of the antibody. One hundred
micrograms of the goat antimouse IgG (Fc; Millipore Corporation, Billerica, Ma, USA) were added
to 1 mL of 1 wt% bead suspension in 0.1 M borate buffer, pH 9.5, and reacted for
24 hours at 37°C. After the elimination of uncoupled antibodies through
repeated washing with TBS-T (25 mM Tris-buffered saline, pH 7.2, containing
0.01% Tween 20), anti-EpCAM mAb was immobilized on the bead as a second step.
Twenty micrograms of commercially available anti-EpCAM mAb, VU-1D9, were added
to 1 mL of 1 wt% bead in TBS-T and mixed for 1 hour at room temperature. After
the reaction, uncaptured antibody was washed away with TBS-T and stored at 4°C
until use.Next, we developed new antihuman EpCAM mAbs in
order to obtain an antibody with higher affinity against EpCAM. A recombinant
human EpCAM/Fc chimera (R&D Systems, Minneapolis,
Minn, USA)
was used as an immunogen. The antigen (0.1 mg) was mixed with complete Freund's
adjuvant (Difco, Detroit, Mich, USA) and injected intraperitoneally (IP) into BALB/c mice (Charles
River Japan, Shizuoka, Japan). Subsequent injections were made using an RIBI
adjuvant system, MPL  +  DM emulsion (Corixa, Seattle, Wash, USA) every three weeks for a total
of 7 times. Three weeks later, the mice were given an intravenous (i.v.) booster
injection of 0.1 mg of the antigen in phosphate buffered saline. Three days
later, spleen cells from the immunized mice were fused with myeloma cells
(P3X63Ag8.653) at a ratio of 7:1 in 50% polyethylene glycol 4000 (Sigma, St.
Louis, Mo, USA) in RPMI 1640 at room temperature for 1 minute. After
centrifugation, the cells were pelleted, washed, resuspended in RPMI 1640
containing 10% NCTC 109, 20% FCS, and 50 ng/L of mouse IL-6 (R&D Systems),
and plated in flat-bottomed 96-well tissue culture palates (Costar Corning, Corning,
NY, USA). Following overnight incubation in a humidified 5% CO2 atmosphere at 37°C, hypoxanthine-aminopterin-thymidine
(HAT) medium was added to start HAT selection. Hybridoma clones were cultured
for an additional 8 days, and then the culture media were assayed for specific antibody
production using the ELISA method on Maxisorp microtiter plates (Nunc,  Roskilde, Denmark)
coated with the antigen. Briefly, an aliquot of undiluted hybridoma culture supernatant
was added to the antigen coated wells. After 1 hour of incubation and a subsequent
wash cycle, an appropriate dilution of peroxidase-conjugated rabbit IgG (Bethyl
Laboratories, Montgomery, Ala, USA)
to mouse IgG reagent was added. The enzyme-linked IgG binding was detected
using an H2O2 and o-phenylenediamine substrate solution. The color
intensity was measured automatically using a SpectraMax Plus microplate
spectrophotometer (Molecular Devices, Sunnyvale, Calif, USA).
The ELISA positive hybridoma cells were cloned by limiting dilution in 96-well
culture plates and established as stable hybridoma cells.The isotype of the mAb was determined using an MONO Ab-ID KIT (Zymed,
San Francisco, Calif, USA)
according to the manufacturer's instructions. Ascites fluids were obtained from
pristine-primed CD1-Foxn1nu mice (Charles River Japan) injected with each mAb
producing hybridoma clone. Immunoglobulin G was separately purified from each
ascites fluid sample using protein G affinity chromatography (GE Healthcare
Life Science, Piscataway, NJ, USA).
The purified IgG fractions were used for further characterization and were evaluated
for their reactivity. The immunoglobulin concentration was determined by
measuring the absorbance at 280 nm using an extinction coefficient of 1.38 for
mouse IgG.The affinities to EpCAM of the newly developed antihuman
EpCAM mAbs were analyzed using flow cytometry with a BD FACSCalibur (BD, Franklin Lakes,
NJ, USA).
HT-29 cells and UMUC-3 cells were used as an EpCAM positive control and
negative control, respectively. The antihuman EpCAM mAbs and nonspecific mouse
IgG1 were directly labeled using Zenon mouse IgG labeling kits (Molecular
Probes, Eugene, Ore, USA) according to the manufacturer's instructions.Finally, the new antihuman EpCAM mAbs were conjugated
to the optimal size of magnetic beads chosen in the present study (see Table1).


## 2.2. Simulation

A
simulation was conducted to determine the optimal bead conditions for the recovery
of HT-29 colorectal cancer cells using a previously established method [21]. Briefly, 2-gram
fecal samples were homogenized in a Hanks-HEPES-FBS buffer (40 mL) consisting of
Hanks solution, 10% fetal bovine serum (FBS), and 25 mM HEPES buffer (pH 7.35)
at 200 times per minute for 1 minute using a Stomacher (Seward, Thetford, UK). A
total of 1  ×  105 HT-29 cells were added to the homogenized solution and
filtered through a nylon filter (pore size: 512 μm). The HT-29 cells were
retrieved using 80 μL of several immunomagnetic beads, and the mixtures were
incubated for 30 minutes under gentle rolling conditions at room temperature.
The mixtures on the magnet were incubated on a shaking platform for 15 minutes
at room temperature. Then, the supernatant was removed, and the retrieved cells
were counted using a NucleoCounter (ChemoMetec A/S, Allerød, Denmark).Finally, to determine the optimal immunomagnetic beads, HT-29
colorectal cancer cells were retrieved using these immunomagnetic beads, and
then the cell-bead complexes were fixed and stained using papanicolaou stain.


## 2.3. Patients with Colorectal Cancer

From
March 2007 to July 2008, 40 patients with histologically confirmed colorectal
cancer were enrolled in the present study. Nineteen of the 40 patients were enrolled
in a study to analyze the optimal bead size from March 2007 to September 2007. Then,
21 of the 40 patients were enrolled in a study to evaluate the quality of the antibody
from October 2007 to July 2008. All the patients had undergone surgical
resection of their primary cancer at the National Cancer Center Hospital, Tokyo, Japan.
All patients were thoroughly
informed of the content of the study, and provided their written
consent to participate in
the study. The study was approved by the Institutional Review Board of the National
Cancer Center, Japan.


## 2.4. Clinical Evaluation

Before surgical resection, naturally evacuated fecal samples were
obtained from patients with colorectal cancer. Each fecal sample was divided
into 2-gram samples for use in the evaluation of several immunomagnetic beads
with different sizes and different affinities of the EpCAM antibody. The fecal
samples were prepared as described in the simulation section. Colonocytes isolated
from the feces were stored at −80°C until genomic DNA extraction.Genomic
DNA was extracted from colonocytes isolated from feces using an Allprep mini
kit (QIAGEN, Valencia, Calif,
USA)
according to the manufacturer's instructions.For
the genomic DNA analysis, we targeted a consensus sequence of human Alu repeats.
The sequences for the Alu primers and probe used in this study were as follows:
forward primer, 5′-TAGTAGAGACGGGGTTTCACCTTG-3′; reverse primer, 5′-AGCTTGCAGTGAGCCGAGAT-3′;
probe, 5′-GAGAATGGCGTGAA-3′. The reporter dye at the 5′-end of the probe was
FAM, and the quencher dye at the 3′-end was MGB.The
reaction mixture for the genomic DNA analysis consisted of 4μL of a template
DNA, 10 μL of TaqMan Fast Universal PCR Master Mix (Applied Biosystems, Foster, Calif,
USA), 500 nM of
forward and reverse primers, and 250 nM of probe in a total reaction volume of
20 μL. Real-time PCR amplification was performed using precycling heat activation at 95°C
for 20 seconds, followed by 25 cycles of denaturation at 95°C
for 3 seconds, and annealing/extension at 62°C for 30 seconds in an Applied Biosystems 7500 Fast Real-Time PCR
System (Applied Biosystems). The absolute quantification of genomic DNA in each
sample was determined using a standard curve with serial dilutions (10 ng to 100 fg) of TaqMan control genomic DNA (Applied Biosystems). A negative control
(without template) was run in each reaction plate.


## 2.5. Statistical Analysis

The
cell retrieval rate for each group was analyzed using a Tukey-Kramer multiple
comparisons test. Statistical differences in the cell retrieval abilities of the
new immunomagnetic beads were determined using a two-sided Mann-Whitney U test.
Statistical analyses were performed using StatView Ver. 5 for Windows (Abacus
Concepts, Berkeley, Calif, USA).
Values ofP  <  .05 were considered statistically significant.


## 3. Results

### 3.1. Recovery Rates of Colonocytes Using Several Sizes of Magnetic Beads in the Simulation Study

We
succeeded in preparing three different sizes of magnetic beads. The sizes of
beads A, beads B, and beads C were 3.0, 4.9, and 5.9μm, respectively (see Table 1 and Figure 1(a)). To determine the best size of magnetic beads for cell recovery,
various sizes of magnetic beads were tagged with a commercially available mAb,
VU-1D9 (see Table 1). In the simulation, the cell
recovery rates using beads A, B, and C were 65.9±1.37 (%, mean ± SD), 61.1±0.98,
and 57.1±0.75, respectively. The recovery rate using beads A (the smallest size)
was significantly higher than those using beads B (P  =  .0001) and beads
C (P  <  .0001) (see Figure 1(b)). Microscopic observation also showed
that the HT-29 cells were captured using the newly developed immunomagnetic
beads, and that more HT-29 cells were bound to beads A than to the larger beads
B and beads C (see Figure 1(c)).


### 3.2. Newly Developed Anti-EpCAM Monoclonal Antibodies

We developed three new anti-EpCAM mAbs, named clone 1-2, clone
B8-4, and clone B8-7. Flow cytometry analysis using HT-29 cells (positive for
the EpCAM antigen) showed that the affinity of clone 1-2 was 3 times higher than
that of nonspecific mouse IgG1, while the affinities of clones B8-4 and B8-7 were
500 times higher than that of nonspecific mouse IgG1 (see Figure2(a)). The
affinity intensities of clones 1-2, B8-4, and B8-7 to UMUC-3 cells (negative for
EpCAM antigen) were almost identical to that of nonspecific mouse IgG1 to
UMUC-3 cells. These results show that clone 1-2 was a low-affinity mAb and that
clones B8-4 and B8-7 were high-affinity mAb (see Figure 2(a)). Each antibody
was then conjugated to the optimal size (3.0 μm) of magnetic beads (see Table 1).A simulation experiment for the recovery rates of colonocytes depending on the affinity of mAbs. (a) The affinities of newly developed antihuman EpCAM mAbs are analyzed using flow cytometry. HT-29 cells, which are positive for EpCAM antigen, and UMUC-3 cells, which are negative for EpCAM antigen, are used. The white peak, black peak, dark-gray peak, and light-gray peak show nonspecific mouse IgG1 mAb, clone 1-2, clone B8-4, and clone B8-7, respectively. (b) Simulation examining the recovery of the colonocytes. The cell recovery rates using beads D (tagged with a lower affinity mAb, Clone 1-2), beads E (tagged with a higher affinity mAb, Clone B8-4), and beads F (tagged with a higher affinity mAb, Clone B8-7) are 4.5 ± 0.98 (%, mean ± SD), 73.5 ± 1.96, and 71.2 ± 3.39, respectively. The columns show the cell retrieval rates and the bars show the standard deviations. Significant differences are showed by an asterisk (*,P  <  .0001).
(a)(b)


### 3.3. Recovery Rate of Colonocytes Depending on the Affinity of Monoclonal Antibodies Against EpCAM in the Simulation Study

In the simulation, the cell recovery
rates using beads D, E, and F were4.5±0.98 (%, mean ± SD), 73.5±1.96, and 71.2±3.39, respectively. The recovery rate using beads D was significantly lower than
those using beads E (P  <  .0001) and beads F (P  <  .0001) (see
Figure 2(b)).


### 3.4. Cell Retrieval Ability of Newly Developed Immunomagnetic Beads in Clinical Samples

In
the clinical study examining cell retrieval ability according to bead size, the
median amount of DNA from 2-gram stool using beads A, B, and C was 1.50 ng (range 0.09–8.12), 0.58 ng
(range 0–4.23), and 0.25 ng
(range 0–4.62),
respectively (see Figure3(a)). The amount of extracted DNA using beads A (the smallest beads) was not significantly different from that using beads B (P  =  .09). However, the amount of extracted DNA using beads A was significantly higher than that using beads C (P  =  .02).
Meanwhile, in the clinical study
examining cell retrieval ability according to antibody affinity, the median
amount of DNA from 2-gram stool using beads D, E, and F was 0.75 ng (range 0.03–3.65), 1.66 ng
(range 0.12–5.74), and 0.99 ng
(range 0.08–5.67),
respectively (see Figure 3(b)). The amount of extracted DNA using beads D was
significantly less compared with that using beads E (P  =  .01).Cell retrieval ability of newly developed immunomagnetic beads in clinical samples. (a) Cell retrieval ability using several sized immunomagnetic beads in clinical samples. The median amounts  of DNA from 2-gram stool using beads A (smallest, 3.0μm), beads B (medium, 4.9 μm), and beads C (largest, 5.9 μm) are 1.50 ng (range; 0.09–8.12), 0.58 ng (range; 0–4.23), and 0.25 ng (range; 0–4.62), respectively. The horizontal bar is a median amount of extracted DNA from isolated colonocytes from 2-gram stool; the upper vertical bar is a maximal amount of extracted DNA; the lower vertical bar is a minimal amount of extracted DNA; and the column contained 50% of these populations. (b) Cell retrieval ability using immunomagnetic beads tagged with several affinities mAbs in clinical samples. The median amounts of DNA from 2-gram stool using beads D (tagged with a lower affinity mAb, Clone 1-2), beads E (tagged with a higher affinity mAb, Clone B8-4), and beads F (tagged with a higher affinity mAb, Clone B8-7) are 0.75 ng (range; 0.03–3.65), 1.66 ng (range; 0.12–5.74), and 0.99 ng (range; 0.08–5.67), respectively. The horizontal bar is a median amount of extracted DNA from isolated colonocytes from 2-gram stool; the upper vertical bar is a maximal amount of extracted DNA; the lower vertical bar is a minimal amount of extracted DNA; and the column contained 50% of these populations.
(a)(b)These results clarified that the smaller immunomagnetic beads and the
beads that were conjugated with higher-affinity Abs were more efficient at
retrieving colonocytes from feces.


## 3.1. Recovery Rates of Colonocytes Using Several Sizes of Magnetic Beads in the Simulation Study

We
succeeded in preparing three different sizes of magnetic beads. The sizes of
beads A, beads B, and beads C were 3.0, 4.9, and 5.9μm, respectively (see Table 1 and Figure 1(a)). To determine the best size of magnetic beads for cell recovery,
various sizes of magnetic beads were tagged with a commercially available mAb,
VU-1D9 (see Table 1). In the simulation, the cell
recovery rates using beads A, B, and C were 65.9±1.37 (%, mean ± SD), 61.1±0.98,
and 57.1±0.75, respectively. The recovery rate using beads A (the smallest size)
was significantly higher than those using beads B (P  =  .0001) and beads
C (P  <  .0001) (see Figure 1(b)). Microscopic observation also showed
that the HT-29 cells were captured using the newly developed immunomagnetic
beads, and that more HT-29 cells were bound to beads A than to the larger beads
B and beads C (see Figure 1(c)).


## 3.2. Newly Developed Anti-EpCAM Monoclonal Antibodies

We developed three new anti-EpCAM mAbs, named clone 1-2, clone
B8-4, and clone B8-7. Flow cytometry analysis using HT-29 cells (positive for
the EpCAM antigen) showed that the affinity of clone 1-2 was 3 times higher than
that of nonspecific mouse IgG1, while the affinities of clones B8-4 and B8-7 were
500 times higher than that of nonspecific mouse IgG1 (see Figure2(a)). The
affinity intensities of clones 1-2, B8-4, and B8-7 to UMUC-3 cells (negative for
EpCAM antigen) were almost identical to that of nonspecific mouse IgG1 to
UMUC-3 cells. These results show that clone 1-2 was a low-affinity mAb and that
clones B8-4 and B8-7 were high-affinity mAb (see Figure 2(a)). Each antibody
was then conjugated to the optimal size (3.0 μm) of magnetic beads (see Table 1).A simulation experiment for the recovery rates of colonocytes depending on the affinity of mAbs. (a) The affinities of newly developed antihuman EpCAM mAbs are analyzed using flow cytometry. HT-29 cells, which are positive for EpCAM antigen, and UMUC-3 cells, which are negative for EpCAM antigen, are used. The white peak, black peak, dark-gray peak, and light-gray peak show nonspecific mouse IgG1 mAb, clone 1-2, clone B8-4, and clone B8-7, respectively. (b) Simulation examining the recovery of the colonocytes. The cell recovery rates using beads D (tagged with a lower affinity mAb, Clone 1-2), beads E (tagged with a higher affinity mAb, Clone B8-4), and beads F (tagged with a higher affinity mAb, Clone B8-7) are 4.5 ± 0.98 (%, mean ± SD), 73.5 ± 1.96, and 71.2 ± 3.39, respectively. The columns show the cell retrieval rates and the bars show the standard deviations. Significant differences are showed by an asterisk (*,P  <  .0001).
(a)(b)


## 3.3. Recovery Rate of Colonocytes Depending on the Affinity of Monoclonal Antibodies Against EpCAM in the Simulation Study

In the simulation, the cell recovery
rates using beads D, E, and F were4.5±0.98 (%, mean ± SD), 73.5±1.96, and 71.2±3.39, respectively. The recovery rate using beads D was significantly lower than
those using beads E (P  <  .0001) and beads F (P  <  .0001) (see
Figure 2(b)).


## 3.4. Cell Retrieval Ability of Newly Developed Immunomagnetic Beads in Clinical Samples

In
the clinical study examining cell retrieval ability according to bead size, the
median amount of DNA from 2-gram stool using beads A, B, and C was 1.50 ng (range 0.09–8.12), 0.58 ng
(range 0–4.23), and 0.25 ng
(range 0–4.62),
respectively (see Figure3(a)). The amount of extracted DNA using beads A (the smallest beads) was not significantly different from that using beads B (P  =  .09). However, the amount of extracted DNA using beads A was significantly higher than that using beads C (P  =  .02).
Meanwhile, in the clinical study
examining cell retrieval ability according to antibody affinity, the median
amount of DNA from 2-gram stool using beads D, E, and F was 0.75 ng (range 0.03–3.65), 1.66 ng
(range 0.12–5.74), and 0.99 ng
(range 0.08–5.67),
respectively (see Figure 3(b)). The amount of extracted DNA using beads D was
significantly less compared with that using beads E (P  =  .01).Cell retrieval ability of newly developed immunomagnetic beads in clinical samples. (a) Cell retrieval ability using several sized immunomagnetic beads in clinical samples. The median amounts  of DNA from 2-gram stool using beads A (smallest, 3.0μm), beads B (medium, 4.9 μm), and beads C (largest, 5.9 μm) are 1.50 ng (range; 0.09–8.12), 0.58 ng (range; 0–4.23), and 0.25 ng (range; 0–4.62), respectively. The horizontal bar is a median amount of extracted DNA from isolated colonocytes from 2-gram stool; the upper vertical bar is a maximal amount of extracted DNA; the lower vertical bar is a minimal amount of extracted DNA; and the column contained 50% of these populations. (b) Cell retrieval ability using immunomagnetic beads tagged with several affinities mAbs in clinical samples. The median amounts of DNA from 2-gram stool using beads D (tagged with a lower affinity mAb, Clone 1-2), beads E (tagged with a higher affinity mAb, Clone B8-4), and beads F (tagged with a higher affinity mAb, Clone B8-7) are 0.75 ng (range; 0.03–3.65), 1.66 ng (range; 0.12–5.74), and 0.99 ng (range; 0.08–5.67), respectively. The horizontal bar is a median amount of extracted DNA from isolated colonocytes from 2-gram stool; the upper vertical bar is a maximal amount of extracted DNA; the lower vertical bar is a minimal amount of extracted DNA; and the column contained 50% of these populations.
(a)(b)These results clarified that the smaller immunomagnetic beads and the
beads that were conjugated with higher-affinity Abs were more efficient at
retrieving colonocytes from feces.


## 4. Discussion

CRC develops from the colorectal mucosa and therefore is in continuous  contact with feces from an early stage. Previously, we
proposed that colorectal cancer cells exfoliated into the feces might survive
for a considerable period in the feces, since feces do not inhibit cancer
expansion towards the colorectal lumen [20–22]. Meanwhile, we predicted that normal colorectal mucous cells in
contact with the feces would be in an apoptotic stage and would probably be
exfoliated into the feces. To date, therefore, we have tried to develop a
method by which exfoliated colorectal cancer cells can be isolated from
naturally evacuated feces and then utilized in cytological or molecular
biological diagnosis [21]. In the
present study, we developed new immunomagnetic beads and evaluated their
efficiencies in terms of the recovery of exfoliated colorectal cancer cells in
feces because we had recognized an urgent need to improve the accuracy of this
method of isolating colonocytes from feces and to reduce the cost performance.Intuitively, larger magnetic beads seem more likely to be
attracted to magnetic body, compared with smaller magnetic beads, since their
larger size corresponds to a greater number of iron particles inside the bead.
Therefore, we initially thought that more cell-bead complexes using the larger
magnetic beads would adhere to the magnetic body, enabling more cells to be
collected. However, in a study to determine the optimal bead size, the smaller
beads adhered to the HT-29 cells more densely than the larger beads (see Figure1(c)). Thus, the total number of cell-bead complexes attracted to the magnetic
body was significantly higher when the smaller beads were used. The cell
retrieval rate using the smaller beads was significantly higher than those
using the larger beads (see Figure 1(b)). The results of an experiment using
clinical samples were similar to those obtained in the simulation (see Figure 3(a)).
Consequently, the smallest beads appeared to collect colonocytes from feces
more efficiently than the larger beads.In our preliminary experiments using a
flow cytometry, it was found that EpCAM antigen was expressed strongly in human
colorectal cancer cell lines, DLD-1, HCT116, HCT-15, HT-29, LoVo, and SW480. We
also analyzed the positivity of cancer tissue and normal mucosal tissue using immunohistochemistry.
Both the cancer tissues and the normal mucosal tissues were strongly positive
(data not shown). We then made several mAbs against
human EpCAM and consequently obtained mAbs with either a high affinity or a low
affinity to the EpCAM antigen (see Figure2(a)). Each antibody was conjugated
to the optimum (the smallest) beads, and these immunomagnetic beads were compared
with each other. In a simulation using feces seeded with 1  ×  105 HT-29
cells, the cell retrieval rate using immunomagnetic beads tagged with a high-affinity
antibody was superior to that of the beads tagged with a low-affinity antibody (see
Figure 2(b)). This result indicated that the higher-affinity antibody tagged to
the magnetic beads was useful for retrieving the cells present in fecal samples.
In addition, the results of a clinical study were similar to the results of the
simulation (see Figure 3(b)).Previously, we demonstrated the feasibility of a new methodology
for isolating colonocytes from naturally evacuated feces, followed by
cytological or molecular biological analysis of the colonocytes to detect
colorectal cancer [21]. An
improvement in the immunomagnetic beads was the most important issue for detecting
colorectal cancer using our diagnostic method. Dynabeads Epithelial Enrich,
immunomagnetic beads for isolating free circulating cancer cells from serum, was
used in our original method. However, immunomagnetic beads developed for the
collection of colorectal cancer cells from stool solutions, which contain large
amounts of residual substances, were not available. Therefore, we decided to
develop immunomagnetic beads designed especially for the isolation of
colorectal cancer cells from feces.


## 5. Conclusions

We succeeded in developing suitable immunomagnetic beads for the isolation
of colonocytes from feces. The new immunomagnetic beads, which were 3.0μm in
size and were conjugated with a new monoclonal antibody possessing a higher
affinity to EpCAM, could effectively isolate colonocytes from feces. This
result is promising for the future of CRC diagnosis.



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*Source: 605273-2008-12-22.xml*