INTERNATIONAL JOURNAL OF ONCOLOGY 43: 57-62, 2013
Abstract. Malignant fibrous histiocytoma (MFH), a high-
grade, undifferentiated sarcoma, is highly aggressive,
resistant to radiochemotherapy and associated with poor
prognosis. There are no specific immunohistochemical
markers for its diagnosis. The MFH cell line SFT7913 served
as and immunogen for the generation of the FU3 monoclonal
antibody in our laboratory. FU3 reacted strongly with MFH
cells and with perivascular mesenchymal cells. In this study,
we demonstrated that the antigen recognized by FU3 was
identical to aminopeptidase N (APN/CD13) using FU3
immunoaffinity chromatography and N-terminal amino acid
sequencing. Frequent (80%) and high-grade (>50% of cells)
expression of APN/CD13 was observed in MFH, although
low-grade expression was seen in some other sarcomas.
Moreover, small interfering RNA (siRNA) that specifically
targets APN/CD13 significantly suppressed MFH cell inva-
sion in vitro. The newly developed monoclonal antibody
FU3 specifically recognizes CD13 on MFH cells. Decreased
expression of CD13, mediated by siRNA-mediated knock-
down, attenuated the invasive capacity of MFH cells. Thus,
results indicate that APN/CD13 could be an important diag-
nostic biomarker and therapeutic target for MFH.
Malignant fibrous histiocytoma (MFH), also called high grade
undifferentiated sarcoma, collectively represent the most
common types of sarcoma in the fifth and sixth decades of life.
The overall incidence among adults approximates to 1-2 cases
per 100,000 patients. Most MFH occur in the extremities and
deep soft tissue (1). Approximately 5% of patients have metas-
tases at presentation and MFH is aggressive with an overall
5-year survival probability of 50-60% (1). Surgical removal is
presently the sole effective treatment and the goal of surgery is
complete resection with negative margin.
Histopathologically, MFH shows a wide variety of morpho-
logical patterns. MFH commonly presents with marked
cytological and nuclear pleomorphism, often with bizarre
tumor giant cells, admixed with spindle cells and frequently
with rounded histiocyte-like cells in varying proportions (2).
MFH shows no evidence of true monocyte/macrophage/histio-
cytic differentiation. Several hypotheses suggest MFH arises
from fibroblasts or primitive mesenchymal cells but current
research does not show a definable line of differentiation. The
diagnosis is controversial, but various cases are eligible for
consideration as MFH (high grade undifferentiated sarcoma).
At present, there are no useful immunohistochemical markers
for the diagnosis of MFH; therefore, it is difficult to search for
the origin of MFH.
Our laboratory generated monoclonal antibody FU3 using
an MFH cell line as an immunogen. FU3 reacted strongly with
the surface membrane of cultured MFH cells and with perivas-
cular mesenchymal cells in frozen tissue sections. Accordingly,
MFH may share common antigenicity with perivascular
mesenchymal cells (3). Immuno-electron-microscopic studies
demonstrated FU3-positive reactivity on the surface of cell
membranes, which suggests that FU3 recognizes cell surface
The aim of this study was to identify the antigen recog-
nized by FU3 antibody. Furthermore, we examined whether
the antigen could be effectively applied to diagnosis and treat-
ment of MFH.
Materials and methods
Cell culture. The MFH cell line SFT8503 was established in our
laboratory, as described previously (6). The cell line was main-
tained in growth medium, Dulbecco's modified Eagle's medium/
Ham's F-12 (Wako, Japan), supplemented with 10% fetal calf
serum, streptomycin (50 µg/ml) and penicillin G (50 U/ml).
Identification of APN/CD13 as the target antigen of FU3,
a human monoclonal antibody that recognizes
malignant fibrous histiocytoma
MIKIKO AOKI, KAZUKI NABESHIMA, HIROYUKI HAYASHI,
MAKOTO HAMASAKI and HIROSHI IWASAKI
Department of Pathology, Fukuoka University School of Medicine and Hospital, Jonan-ku,
Fukuoka 814-0180, Japan
Received February 27, 2013; Accepted April 22, 2013
Correspondence to: Professor K. Nabeshima, Department of
Pathology, Fukuoka University School of Medicine and Hospital,
7-45-1 Nanakuma, Jonan-ku, Fukuoka 814-0180, Japan
Key words: aminopeptidase N, APN/CD13, human monoclonal
antibody FU3, malignant fibrous histiocytoma
AOKI et al: IDENTIFICATION OF APN/CD13 AS THE TARGET ANTIGEN OF FU3
Protein extraction and western blotting. The cultured cells
were lysed in RIPA lysis buffer (50 mM Tris-HCl, pH 7.4,
150 mM NaCl, 1 mM EDTA, 1% NP-40; Millipore, Bedford,
MA) and the lysed cells were sonicated on ice for 5 min
three times and centrifuged at 15,000 rpm for 20 min at 4˚C.
The resultant supernatants were subjected to sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). After
electrophoresis, the proteins were transferred electrophoreti-
cally to Immobilon membrane (Millipore). Non-specific sites
were blocked with 5% dry fat milk in Tris-buffered saline (TBS)
at 37˚C for 1 h and the membrane was incubated overnight
at 4˚C with monoclonal FU3 antibody or with commercially
available CD13 antibody (Clone 38C12, ThermoScientific,
Cheshire, UK). After washing with TBS-T (TBS containing
0.05% Tween-20), the membrane was incubated for 1 h with
peroxidase-conjugated anti-mouse IgG. Color was developed
with chemiluminescence reagents according to the instruc-
tions supplied by the manufacturer (DuPont New England
Nuclear, Boston, MA).
Immunoaffinity chromatography. Monoclonal antibody FU3,
which was generated against MFH cells using a mouse
hybridoma technique as previously described (3,4), was
used to prepare an affinity column (7). We used cyanogen
bromide CNBr-activated Sepharose 4B (GE Healthcare Ltd.,
UK) as an immunoaffinity matrix coupling to mouse FU3
antibody. The column was washed with phosphate buffered
saline (PBS). Antigens bound to the matrix were then eluted
from the column with 0.2 M glycine (pH 2.3). The eluted
material was immediately neutralized with PBS and stored at
4˚C. The eluted protein fraction of the column was purified
by SDS-PAGE and immunoblotting. The purified protein was
subjected to N-terminal amino acid sequencing (Takara Bio
Inc. Otsu, Japan).
Tissue samples. Our study included formalin-fixed, paraffin-
embedded sections from 25 MFH [10 men, 15 women; age
range, 26-81 (mean, 71) years], 9 synovial sarcoma [2 men,
7 women; age range, 20-88 (mean, 43) years], 10 liposarcoma
[4 men, 6 women; age range, 20-74 (mean, 55) years], 10 leio-
myosarcoma (4 men, 6 women; age range, 25-83 (mean, 62)
years], 11 chondrosarcoma [7 men, 4 women; age range, 12-63
(mean, 42) years] and 5 osteosarcoma [5 women; age range,
14-57 (mean, 56) years], diagnosed at the Department of
Pathology, Fukuoka University, Japan.
Immunohistochemistry. Immunohistochemical staining was
performed using 4-µm thick paraffin-embedded sections, were
deparaffinized and heated in a microwave oven (700 W) for
10 min to expose antigens in 10 mM Na-citrate buffer (pH 6.0;
for CD13) or 1 mM EDTA/10 mM Tris-HCl buffer (pH 9.0;
for FU3). The exposed antigen was detected using the labeled
streptavidin-biotin method. The reaction was identified with
naphthol AS-BI phosphate and counterstained with Mayer's
The staining results were evaluated semiquantitatively by
two independent observers. Immunostaining was considered
negative if stained tumor cells were <10%. In specimens consid-
ered positive, staining of the tumor was quantitated on a scale
from 1-4 based on the percentage of positive tumor cells. The
scale was structured as follows: 1+, 10-25% of cells positive; 2+,
25-50% of cells positive; 3+, 50-75% of cells positive; 4+, >75%
of cells positive. In specimens considered high expression cases,
staining of the tumor was quantified on scales 3+ and 4+.
Small interfering RNA (siRNA). SFT8503 cells were grown
to sub-confluence and treated with small interfering RNA
(siRNA) for CD13 (Smart Pool, Dharmacon, Chicago, IL) or
control siRNA (B-Bridge International, Sunnyvale, CA) using
Lipofeamine 2000 (Invitrogen, Carlsbad, CA) accroding to the
In vitro invasion assay. In vitro Matrigel invasion assay was
performed by using 24-well Chemotaxicell chambers (pore
size, 8 µm, Kubota Co., Tokyo, Japan) on 24-well culture plate.
The upper and lower side of each chamber was coated with
Matrigel (25 µg/filter, BD Biosciences). Hepatocyte growth
factor (HGF; Peprotech Inc. Rocky Hill, NJ) was used as a
chemoattractant. After incubation of cells for 72 h, the filters
were fixed with formalin and stained with hematoxylin and
the total number of cells that had invaded the Matrigel-coated
filter were counted.
Immunoaffinity chromatography using FU3 antibody.
Extracted proteins from SFT8503 MFH cells were purified
using immunoaffinity chromatography with FU3 antibody,
followed by immunoblotting. The N-terminal amino acid
sequencing of the 150-kDa band (Fig. 1, arrow) revealed
A-K-G-F-Y-I-S-K-S-L, which is identical to that of aminopep-
tidase N (APN)/CD13, known as an important zinc-dependent
The FU3 recognizing antigen is possibly aminopeptidase N
(CD13). To confirm the identity of FU3-reactive antigen as
APN/CD13 we used immunoblotting and siRNA methods.
First, by immunoblotting SFT8503 MFH whole cell extracts
and an eluted protein from immunoaffinity chromatography
Figure 1. Western immunoblot analysis of whole ectracts (lane 1), flow
through (lane 2), washings (lane 3) and elutes (lanes 4-10) immunostained
with monoclonal antibody FU3 and showing the 150-kDa band.
INTERNATIONAL JOURNAL OF ONCOLOGY 43: 57-62, 2013
columns, both CD13 and FU3 antibodies recognized an
identical 150-kDa band (Fig. 2a).
Second, CD13-specific siRNA treatment (0.01 pmol/µl)
of SFT8503 MFH cells caused significant downregulation
of both CD13 and FU3-reactive 150 kDa proteins (Fig. 2b.).
In view of the evidence, we considered that the FU3 reactive
antigen was identical to APN/CD13.
CD13 expression in normal skin. Immunohistochemically,
CD13 antibody stained perivascular mesenchymal cells,
represented by small spindle or polygonal cells around small
blood vessels, in normal skin (Fig. 3). A small number of
fibroblasts also reacted with CD13 antibody; however, the
stratified squamous epithelium and endothelial cells were not
CD13 antibody-reactive. These findings were similar to those
of FU3 reactive cells (3-6), supporting of the identification of
FU3 antigen as APN/CD13.
Figure 2. FU3 recognizing antigen corresponds to the aminopeptidase N (CD13). (a) Western immunoblot analysis of whole cell extracts and immunoaffinity
column-purified proteins. Both CD13 and FU3 recognizes identical 150-kDa protein. (b) CD13 siRNA (0.01 pmol/µl) treatment causes downregulation of both
CD13 and FU-3-reactive protein at 72 h. NT, non-treated; cont, control siRNA.
Figure 3. Reactivity of normal skin cells with CD13. Perivascular mesen-
chymal cells in the normal dermis show staining, whereas epidermal cells
and endothelial cells remain unstained. CD13-positive fibroblasts are also
noted in part.
Figure 4. Immunohistochemical expression of CD13 in MFH. Representative cases of perivascular (a and b) and diffuse (c and d) patterns. The perivascular
type shows strong cytoplasmic staining along the membrane (b). The diffuse type shows moderate cytoplasmic reactivity (d). Original magnifications (a and c),
x40; (b and d), x200.
AOKI et al: IDENTIFICATION OF APN/CD13 AS THE TARGET ANTIGEN OF FU3
CD13 expression in MFH and soft tissue tumors. Expression
of CD13 was examined immunohistochemically in 70 soft
tissue tumors wich included 25 MFH and 45 other soft tissue
tumors (Fig. 4 and Table I). Twenty cases of MFH (80%)
were positive for CD13 and of these, there were 17 (68%)
high expression cases. In MFH, CD13 expression pattern
were classified into two types; perivascular (Fig. 4a and b)
and diffuse types (Fig. 4c and d). Tissues of the perivascular
type exhibited intense cytoplasmic and membrane staining of
CD13 (Fig. 4b). In the diffuse type, almost all MFH cells had
positively stained cytoplasms (Fig. 4d). Positive reactivity with
CD13 was observed in several synovial sarcoma, leiomyosar-
coma and osteosarcoma, but high-expression was found only
in two cases of leiomyosarcoma (Table I).
Inhibition of MFH (SFT8503) cell invasion by CD13 siRNA.
To investigate a biological role for APN/CD13 in MFH, we
examined effect of CD13 siRNA treatment on MFH cell
invasion. We used HGF as a chemoattractant factor because
overexpression of HGF has been reported in MFH (8,9).
Treatment of SFT8503 MFH cells with CD13 siRNA (0.01
and 0.02 pmol/µl) downregulated CD13 expression in a dose-
dependent manner at 48 h (data not shown) and at 72 h (Fig. 5a)
after transfection. HGF (100 ng/ml) induced greater SFT8503
cell invasion than serum-free medium. In the presence of
CD13-targeted siRNA, HGF-stimulated MFH cell invasion
was significantly attenuated as compared with that observed
with control siRNA (Fig. 5b).
Development of an MFH recognizing FU3 antibody provided
some important findings, especially on the cellular origin of
MFH (4-6). The antibody, however, has not been widely used,
one of the reasons may be that FU3 antibody is available
only on frozen tissue sections but not on paraffin-embedded
specimens. In this study, we demonstrated that the FU3 anti-
body recognizies an antigen identical to APN/CD13 using
immunoaffinity chromatography and direct N-terminal amino
acid sequencing. Immunohistochemically, greater amounts of
APN/CD13 were observed more frequently in MFH tissues as
compared with that observed in other sarcomas in paraffin-
embedded specimens. Moreover, MFH cell invasion was
significantly suppressed by transfection of APN/CD13 siRNA.
The results from this study may point toward the use of APN/
CD13, the FU3 antigen, as an important biomarker in the diag-
nosis and treatment of patients with MFH.
APN/CD13, a 150-kDa metalloproteinase, is a multifunc-
tional cell surface aminopeptidase. The human APN gene has
been mapped to chromosome 15q25-26. APN/CD13 expres-
sion has been reported in hematopoietic cells of myeloid origin,
fibroblasts, synaptic membranes in the central nervous system
and epithelial cells of liver, kidney and intestine (10,11). High
expression levels of APN/CD13 has been detected in various
epithelial tumors and its expression correlates with increased
clinical malignant behavior in pancreatic carcinoma and in
colon and non-small lung cancer (12-14).
There is little information on the expression of APN/CD13
and its role in MFH. An immunohistochemical study of MFH
demonstrated a positive reaction to APN/CD13 in six of ten
cases (15). Another report showed four cell lines derived
from an MFH expressed APN/CD13, using flow cytometric
analysis (16). Immunohistochemically, APN/CD13 antibody
showed strong reactivity with perivascular mesenchymal cells
in the normal skin tissue and with MFH cells, to a similar
extent seen with FU3. These findings lend support to our result
that the FU3 recognizing antigen is identical to APN/CD13.
Although we can not directly compare our results with those
Figure 5. Expression of CD13 in SFT8503 cells and effect of CD13 siRNA on tumor invasion. (a) CD13 protein expression level by western blotting. Treatment
with CD13 siRNA downregulated the level more severely at 0.02 pmol/µl than 0.01 pmol/µl at 72 h. (b) Tumor cell invasion of Matrigel was significantly
suppressed by transfection of CD13 siRNA (0.02 pmol/µl) at 72 h. NT, non-treated; cont, control si RNA. *P<0.01 by Student's t-test.
Table I. CD13 expression in soft tissue tumors.
No. of Positive
cases cases (%) cases (>50% of cells)
Positive cases, 1+ to 4+; high expression cases, >3+.
INTERNATIONAL JOURNAL OF ONCOLOGY 43: 57-62, 2013
of previous results of APN/CD13 expression (15), owing to
the differences in assessment methods, our findings were
roughly consistent with previous studies on other soft tissues
sarcomas. All our five cases of osteosarcoma were positive
for APN/CD13, but no high expression cases were observed.
Only two cases of leiomyosarcoma showed high expression.
APN/CD13 immunostaining may be applicable to narrow the
differential diagnosis of soft tissue sarcomas to MFH.
Our immunohistochemical study in normal skin demon-
strated APN/CD13 expression in the perivascular cells and in
some dermal fibroblasts. APN/CD13 expression in perivascular
cells has also been reported in lung tissue; the majority of these
CD13-positive cells were slender perivascular fibroblastic cells
(17). Dermal fibroblasts express APN/CD13 to a relatively
great degree in vitro (18,19). On the basis of immunoreactivity,
MFH cells may have intimate relationship with perivascular
cells and fibroblasts.
APN/CD13 might participate in tumor progression by
regulating processes such as tumor invasion and angiogen-
esis (12,20-24). Our study, however, is the first to show that
downregulation of APN/CD13 expression leads to marked
suppression of invasion by MFH cells, although this is based
on only in vitro data. In a previous study with osteosarcoma
cell lines, CD13 siRNA treatment caused reduced cellular
attachment to and increased proteolytic degeneration of
the extracellular matrix (25). Anti-APN/CD13 antibody
reduced the migratory activity of human dermal fibroblasts
(19). These reduced activities may also occur in MFH cells
and these possibilities are now under investigation in our
Since the discovery in 1976 of the first APN enzymatic
inhibitor bestatin, many APN inhibitors have been developed
(26). Bestatin is already used clinically for the treatment of
adult acute non-lymphocytic leukemia via peroral adminis-
tration. Bestatin-mediated suppression of APN/CD13 activity
in an APN/CD13-expressing ovarian carcinoma cells led to
reduced migration, proliferation and peritoneal dissemination
of tumor cells in a mouse model, which resulted in prolonged
survival (27). Bestatin may also represent a new approach
for improving the therapeutic efficacy of radiotherapy for
uterine cervical carcinoma (28) and for enhancing paclitaxel
chemosensitivity in ovarian carcinoma (29). Recently, some
novel potent APN/CD13 inhibitors have also been reported,
several of which show better inhibitory activity than bestatin
against APN on human carcinoma cells (30,31).
In conclusion, our study indicates that APN/CD13 may
be useful for diagnosing MFH and importantly, might
serve as a new molecular target for therapy for patients
We acknowledge the expert technical assistance of Ms.
M. Onitsuka, M. Ishiguro and C. Fujita in immunohisto-
chemical staining and in vitro studies.
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