An anti-podoplanin monoclonal antibody LpMab-7 detects metastatic
legions of osteosarcoma
Mika K. Kaneko1,3, Hiroharu Oki1,2,3, Satoshi Ogasawara1,3, Michiaki Takagi2, Yukinari Kato1
1

Department of Regional Innovation, Tohoku University Graduate School of Medicine, 2-1 Seiryo-machi,
Aoba-ku, Sendai, Miyagi 980-8575, Japan; 2Department of Orthopaedic Surgery, Yamagata University
Faculty of Medicine, 2-2-2 Iida-nishi, Yamagata 990-9585, Japan

Corresponding author: Yukinari Kato
Department of Regional Innovation, Tohoku University Graduate School of Medicine, 2-1 Seiryo-machi,
Aoba-ku, Sendai, Miyagi 980-8575, Japan
E-mail: yukinarikato@med.tohoku.ac.jp; or, yukinari-k@bea.hi-ho.ne.jp
TEL/FAX: +81-22-717-8207
3
Theses authors contributed equally to this work.
Key words: osteosarcoma, metastasis, monoclonal antibody, podoplanin, CasMab
Osteosarcoma is the most common primary malignant bone tumor, and is highly metastatic to thelungs. Therefore, the development of a novel molecular targeting therapy against metastasis ofosteosarcoma is necessary. A platelet aggregation-inducing factor, podoplanin/aggrus, is involved intumor metastasis. Furthermore, podoplanin expression was reported to be involved in the poor
prognosis of osteosarcoma patients. However, the association between podoplanin expression andmetastasis of osteosarcoma remains unclear because of the lack of highly-sensitive anti-podoplanin
monoclonal antibodies (mAbs). In this study, we used a novel anti-podoplanin mAb, LpMab-7,
which is more sensitive than well-known anti-podoplanin mAbs in immunohistochemistry.
Immunohistochemical analysis using LpMab-7 showed that podoplanin expression at primarylesions is observed in 15 out of 16 (93.8%) cases. Furthermore, podoplanin expression at metastatic
lesions was higher compared with primarily lesions in 3 out of 4 (75%) cases with lung metastasis.
Because LpMab-7 has high sensitivity against podoplanin, LpMab-7 is expected to be useful formolecular targeting therapy for osteosarcomas.
podoplanin has been reported in many tumors.(6,Several studies have reported that
osteosarcoma tissues and cell lines such HOS,
U-2 OS, and MG63 express podoplanin.(17)Moreover, podoplanin expression was reported to
be involved in poor prognosis of osteosarcoma
patients. However, the association between
podoplanin expression and metastasis ofosteosarcoma remains to be clarified because of
the lack of high-sensitive anti-podoplanin
monoclonal antibodies (mAbs). Although manyanti-podoplanin mAbs have been developed,
almost all anti-podoplanin mAbs react with theplatelet aggregation-inducing (PLAG) domain ofhuman podoplanin.(12, 20-24) Rabbit polyclonal
antibodies produced by immunizing recombinant
rat podoplanin also recognize PLAG domains.(25)Recently, we developed several anti-podoplaninmAbs against a non-PLAG domain, including
LpMab-7.(26) In this study, we investigated theusefulness of an anti-podoplanin mAb, LpMab-7

Introduction
Osteosarcoma is the most common primarymalignant bone tumor, and possesses a high rateof systemic spread especially to the lungs.(1)Survival rate was improved by the advance of
aggressive systemic chemotherapy. Patients withno metastatic disease, approximately 70% areconsidered to be long-time survivors.(2) In contrast,
approximately 14% of the patients experiencelung metastases at diagnosis.(3) Primary metastasis
is one of the risk factors, and increase theMoreover,multidrugmortality
rate.(4)combination chemotherapy for osteosarcoma
causes ototoxicity, cardiac toxicity, and secondary
malignancies.(5) Therefore, a novel moleculartargeting therapy against metastatic osteosarcoma
should be established.
Podoplanin (PDPN/Aggrus/T1α) is a platelet
aggregation-inducing type Ⅰ transmembranesialoglycoprotein, which is involved in tumor
invasion and metastasis.(6, 7) Expression of

8-19)

1

in immunohistochemistry.

was observed. These results indicated that a novelanti-podoplanin mAb, LpMab-7 is much more
useful in immunohistochemistry of osteosarcomas
than previously established anti-podoplaninmAbs.

Materials and Methods
Osteosarcoma tissues
This study examined 16 osteosarcoma patients
who underwent surgery at Yamagata University
Hospital.(27) The ethical committee of the
Yamagata University Faculty of Medicine
approved our study. Informed consent for
obtaining samples and for subsequent data
analyses was obtained from each patient or thepatient’s guardian. The pathological diagnosis ofall specimens in this study was confirmed by apathologist(Prof.
Mitsunori
Yamakawa,
Yamagata University Faculty of Medicine).(27)

Immunohistochemical
analysisagainstmetastatic osteosarcomas
LpMab-7 shows much higher sensitivity
against podoplanin in immunohistochemistry;
therefore, we next compared the podoplanin
expression in both primary osteosarcomas andtheir metastatic lesions. We obtained 4 sets ofprimary lesions and metastatic lesions from thesame patients in this study (Table 2). In OS1,
podoplanin expression was observed only in thenormal osteocytes (Fig. 3A; right), not in
osteosarcoma cells (Fig. 3A and 3C); in contrast,
podoplanin in the metastatic lesion was detectedby LpMab-7 (Fig. 3B and 3D). Of interest, theintensity of podoplanin expression at metastatic
lesions was higher than primarily lesions in 3 outof 4 cases (OS1, OS2, and OS5) with lungmetastasis (Table 2). Because podoplanin
expression at both primary and metastatic lesionsof OS14 is high, the difference of intensity by
LpMab-7 staining was not observed. In contrast,
NZ-1 and D2-40 signals were very weak inmetastatic lesions (Fig. 4). These results indicatethat LpMab-7 is very useful for detecting
podoplanininmetastatic
lesionsofosteosarcomas.

Immunohistochemical analyses
Podoplanin protein expression was detected
immunohistochemically in paraffin-embedded
tumor specimens. Briefly, 4-μm-thick histologic
sections were deparaffinized in xylene andrehydrated. Then, they were autoclaved in citratebuffer (pH 6.0; Dako, Glostrup, Denmark) for 20
min. Sections were incubated with 5 μg/ml ofprimary antibodies overnight at 4 °C followed bytreatment with an LSAB+ kit or Envision+ kit(Dako). Color was developed using 3,3-diaminobenzidine tetrahydrochloride (DAB;
Dako) for 10 min, and then the sections werecounterstained with hematoxylin (Wako Pure
Chemical Industries, Ltd., Osaka, Japan). Staining
was assessed semi-quantitatively from thepercentage
of
tumor
cells
with
membranous/cytoplasmic staining: 0, no staining;+, <10%; ++, 10–50%; and +++, >50%, andstaining intensity: -, no staining; +, weak; ++,medium; +++, strong.

Discussion
We previously developed NZ-1 by immunizing
rats with PLAG domain of podoplanin to inhibit
platelet aggregation and cancer metastasis byblocking the association between podoplanin and
CLEC-2.(12, 28, 29) NZ-1 possesses very highbinding-affinity, which was clarified by several
methods including Scatchard analysis (KD=9.8 ×
10-10 M) and BIAcore (KD=1.2 × 10-10 M).(19, 30)
Furthermore, NZ-1 was internalized into glioma
cell lines and also accumulated efficiently into
tumors in vivo.(19) Rat-human chimericanti-podoplanin antibody (NZ-8), which was
producedfrom
NZ-1,
possesses
antibody-dependent cellular cytotoxicity (ADCC)
and complement-dependent cytotoxicity (CDC)
against podoplanin-expressing glioblastoma or
malignant mesothelioma cell lines.(15, 29)
Unexpectedly, the intensity of NZ-1 was very
weak in metastatic lesions of osteosarcomas inthis study, probably because i) the NZ-1 epitope

Results
Immunohistochemical analysis against primaryosteosarcomas
Immunohistochemical analysis showed that
LpMab-7 detected podoplanin more sensitively
than NZ-1 (Table 1, Fig. 1). LpMab-7 showedthat
podoplanin
expression
atprimaryosteosarcomas is observed in 15 out of 16(93.8%) cases. In contrast, NZ-1 detected
podoplanin in 12 out of 16 (75%) cases. In almost
all cases, the intensity of LpMab-7 was also
higher than NZ-1. Furthermore, the intensity of
LpMab-7 is much higher than that of D2-40 (Fig.
2). The membranous/cytosolic staining pattern2

(42-47 amino acids)(20) was blocked by theattachment of glycan complex to podoplanin; ii)
CLEC-2 or other counterparts inhibited the NZ-1
binding to PLAG domain; iii) the conformation of
podoplanin at metastatic lesions is different fromthat at primary lesions. In contrast, a novelanti-podoplaninmAb,
LpMab-7,detects
podoplanin not only at primary lesions but also atmetastatic lesions of osteosarcomas, although thebinding affinity of LpMab-7 (KD=5.7 × 10-10 M in
ELISA and KD=2.0 × 10-8 M in flow cytometry) is
lower than NZ-1.(26) In this report, we mainlycompared the reactivity of NZ-1 (rat IgG2a,
lambda) and LpMab-7 (mouse IgG1, kappa). To
remove the possibility that the difference ofsensitivity in immunohistochemistry occurred bysecondary antibodies, we also examined thedifference of sensitivity between D2-40 (mouse
IgG1, kappa) and LpMab-7. D2-40 iscommercially available and the most used mAbagainst human podoplanin in pathology or
histology because D2-40 is also known as alymphatic endothelial marker.(20) However, theintensity of LpMab-7 is much higher than that of
D2-40
inimmunohistochemistryofosteosarcomas, demonstrating that LpMab-7
sensitivity is not dependent on the species and thesubclass. Because the concentration of D2-40 is
unknown, we could not calculate thebinding-affinity of D2-40. The epitope of D2-40
in PLAG domain(20) is different from that of
LpMab-7 in non-PLAG domain;(26) therefore, theepitope of LpMab-7 might be more critical for thehigh-sensitivity in immunohistochemistry. Indeed,
there are many immunohistochemical methods
suitable for each mAb; therefore, we should
further consider the other methods for comparing
those anti-podoplanin mAbs. Another grouppreviously reported that human podoplanin,
detected by NZ-1, is highly expressed inosteosarcomas using 133 osteosarcoma tissues.(17)
Although 33 metastatic lesions were investigatedin that study, the signal intensity was notdiscussed. In contrast, we discussed both signal
intensity
andpercentage
inimmunohistochemistry
using
three
anti-podoplanin mAbs. Furthermore, 4 sets ofprimary lesions and metastatic lesions from thesame patients were used in this study, andpodoplanin upregulation was observed in themetastatic lesions using LpMab-7. Further studies
are necessary to clarify that podoplanin issignificantly upregulated in the metastatic lesions
compared with primary lesions of osteosarcomas.

Because LpMab-7 detected metastatic lesions ofosteosarcomas, it is expected to be useful formolecular targeting therapy for osteosarcomas.
Acknowledgements
We thank Yuta Tsujimoto, Takuro Nakamura,
Kanae Yoshida, and Noriko Saidoh for their
excellent technical assistance. This work was
supported in part by the Platform for Drug
Discovery, Informatics, and Structural Life
Science (PDIS) from the Ministry of Education,
Culture, Sports, Science and Technology (MEXT)
of Japan (Y.K.); by the Basic Science andPlatform Technology Program for Innovative
Biological Medicine from MEXT of Japan(Y.K.); by the Regional Innovation Strategy
Support Program from MEXT of Japan (Y.K.);
and by a Grant-in-Aid for Scientific Research (C)(M.K.K., Y.K.) and a Grant-in-Aid for Young
Scientists (B) (S.O.) from MEXT of Japan.
Author Disclosure Statement
The authors have no financial interests todisclose.
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Figure legends
Fig. 1. Immunohistochemical analysis by
LpMab-7 and NZ-1 against osteosarcoma
tissues. Sections were incubated with 5 μg/ml of
LpMab-7 (A) and NZ-1 (B), followed bybiotin-labeled anti-mouse IgG and anti-rat IgG,
respectively. Then, LSAB+ kit was used, andcolor was developed using DAB andcounterstained with hematoxylin. Original
magnification: ×200.
Fig. 2. Immunohistochemical analysis by
LpMab-7 and D2-40 against osteosarcoma
tissues. Sections were incubated with 5 μg/ml of
LpMab-7 (A) and 1/200 diluted D2-40 (B),followed by EnVision+. Color was developed
using DAB and counterstained with hematoxylin.
Original magnification: ×200.
Fig. 3. Immunohistochemical analysis against
primary and metastatic osteosarcomas using
LpMab-7. Sections of primary (A, C) andmetastatic (B, D) osteosarcomas (OS1) wereincubated with 5 μg/ml of LpMab-7, followed byEnvision+ kit. Color was developed using DAB
and counterstained with hematoxylin. Original

5

magnification: ×100 (A, B); ×200 (C, D). Scalebar: 100 µm.

and anti-rat IgG, respectively. Then, LSAB+ kit
was used, and color was developed using DAB
and counterstained with hematoxylin. (B)Sections were incubated with 5 μg/ml of
LpMab-7 and 1/200 diluted D2-40, followed by
EnVision+. Color was developed using DAB andcounterstained with hematoxylin. Original
magnification: ×200.	
 Fig. 4. Immunohistochemical analysis against
metastatic
osteosarcomas
using
three
anti-podoplanin mAbs. (A) Sections of andmetastatic osteosarcomas (OS1, OS2, OS5, OS14)
were incubated with 5 μg/ml of LpMab-7 and
NZ-1, followed by biotin-labeled anti-mouse IgG

6