MONOCLONAL ANTIBODIES IN IMMUNODIAGNOSIS AND IMMUNOTHERAPY
Volume 36, Number 6, 2017ª Mary Ann Liebert, Inc.
DOI: 10.1089/mab.2017.0046

Original Articles

Expression of Cat Podoplanin in Feline
Squamous Cell Carcinomas

Downloaded by Yukinari Kato from online.liebertpub.com at 12/21/17. For personal use only.

Shunsuke Itai,1,2,* Shinji Yamada,1,* Mika K. Kaneko,1 Hiroyuki Harada,2 Yumiko Kagawa,3Satoru Konnai,4 and Yukinari Kato1,5

Oral squamous cell carcinoma is an aggressive tumor in cats; however, molecular-targeted therapies against thistumor, including antibody therapy, have not been developed. Sensitive and specific monoclonal antibodies(mAbs) against highly expressed membrane proteins are needed to develop antibody therapies. Podoplanin, atype I transmembrane glycoprotein, is expressed in many human malignant tumors, including brain tumor,
esophageal cancer, lung cancer, mesothelioma, and oral cancer. Podoplanin binds to C-type lectin-like receptor2 (CLEC-2) and activates platelet aggregation, which is involved in cancer metastasis. Until now, we haveestablished several mAbs against podoplanin in humans, mice, rats, rabbits, dogs, cattle, and cats. We havereported podoplanin expression in canine melanoma and squamous cell carcinomas using an anti-dog podoplanin mAb PMab-38. In this study, we investigated podoplanin expression in 40 feline squamous cell carcinomas (14 cases of mouth floor, 13 of skin, 9 of ear, and 4 of tongue) by immunohistochemical analysis using
an anti-cat podoplanin mAb PMab-52, which we recently developed by cell-based immunization and screening(CBIS) method. Of the total 40 cases, 38 (95%) showed positive staining for PMab-52. In particular, 12 cases(30%) showed a strong membrane-staining pattern of squamous cell carcinoma cells. PMab-52 can be useful for
antibody therapy against feline podoplanin-expressing squamous cell carcinomas.
Keywords: cat podoplanin, immunohistochemistry, monoclonal antibody, squamous cell carcinoma

IntroductionO

ral squamous cell carcinoma is an aggressive tumor in cats,(1) for which no molecular targeted therapies have been developed. In particular, sensitive and specificmonoclonal antibodies (mAbs) should be established fordeveloping antibody-based therapies. Although many mAbs
against human membrane proteins have been developed, almost all mAbs do not cross-react with the feline ones.
Therefore, we need to develop sensitive and specific mAbs
that can be directed against feline membrane proteins, which
are overexpressed in feline cancers.
Podoplanin (PDPN/T1a/Aggrus), a type I transmembrane
glycoprotein,(2–6) activates platelet aggregation by binding to
the C-type lectin-like receptor-2 (CLEC-2) on the platelets.(7–10)
Podoplanin is expressed in normal cells, including renal
podocytes, pulmonary type I alveolar cells, and lymphatic

endothelial cells.(3) The interaction between podoplanin and
CLEC-2 facilitates lymphatic/blood vessel separation.(11) The
expression of human podoplanin has been reported in many
malignant tumors, including brain tumors,(12–15) oral cancers,(16) esophageal cancers,(17) lung cancers,(18) mesotheliomas,(19,20) testicular tumors,(21) osteosarcomas,(22–24) andchondrosarcomas.(23) Podoplanin expression is associated withcancer metastasis and malignant progression.(8,12,25)
We have established several mAbs against human,(26)mouse,(27) rat,(28) rabbit,(29) dog,(30) bovine,(31) and cat podoplanin.(32) Using these antipodoplanin mAbs, we have investigated podoplanin expression in cancer tissues. PMab-38, ananti-dog podoplanin mAb, reacted with 83% of squamous cell
carcinomas (15/18 cases)(33) and 90% of melanomas (9/10
cases).(34) Recently, we have established an anti-cat podoplanin mAb (clone: PMab-52), which is very useful in flow cytometry, Western blot, and immunohistochemical analyses.(32)1

Department of Antibody Drug Development, Tohoku University Graduate School of Medicine, Sendai, Miyagi, Japan.
Department of Oral and Maxillofacial Surgery, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University,
Tokyo, Japan.
3
North Lab, Hokkaido, Sapporo, Hokkaido, Japan.
4
Department of Disease Control, Faculty of Veterinary Medicine, Hokkaido University, Sapporo, Hokkaido, Japan.
5
New Industry Creation Hatchery Center, Tohoku University, Sendai, Miyagi, Japan.
*These authors contributed equally to this work.
2

243

Downloaded by Yukinari Kato from online.liebertpub.com at 12/21/17. For personal use only.

244

ITAI ET AL.

FIG. 1. Immunohistochemical analysis of podoplanin using PMab-52 in feline kidney. (A, C) Sections of feline kidneys
were autoclaved in citrate buffer (pH 6.0). After blocking, they were incubated with 1 mg/mL of PMab-52, followed bytreatment with EnVision+ kit. Color was developed using DAB, and the slides were counterstained with hematoxylin. (B, D)
H&E staining was performed against serial sections. Scale bar: 100 mm. DAB, 3, 3-diaminobenzidine tetrahydrochloride;
H&E, hematoxylin and eosin.

FIG. 2. Immunohistochemical analysis of podoplanin using PMab-52 in feline lungs. (A, C) Sections of feline lungs wereautoclaved in citrate buffer (pH 6.0). After blocking, they were incubated with 1 mg/mL of PMab-52, followed by treatment
with EnVision+ kit. Color was developed using DAB, and the slides were counterstained with hematoxylin. (B, D) H&E
staining was performed against serial sections. Scale bar: 100 mm.

ANTI-CAT PDPN MAB FOR SCC

In this study, we investigated podoplanin expression in felinesquamous cell carcinomas by immunohistochemical analysis
using PMab-52.
Materials and Methods
Feline tissues

Normal feline tissues were purchased from Zyagen (San
Diego, CA). Feline squamous cell carcinoma tissues wereobtained from North Lab (Hokkaido, Japan).

Downloaded by Yukinari Kato from online.liebertpub.com at 12/21/17. For personal use only.

Hybridoma production

In brief, BALB/c mice (CLEA Japan, Tokyo, Japan) were
immunized by intraperitoneal (i.p.) injection of cat
podoplanin-expressing CHO cells (CHO/cat podoplanin),which were produced by transfecting cat podoplanin into
CHO-K1 cells(32) [American Type Culture Collection(ATCC), Manassas, VA] along with Imject Alum (Thermo
Fisher Scientific, Inc., Waltham, MA). After several additional immunizations of CHO/cat podoplanin cells, a booster
injection of CHO/cat podoplanin cells was intraperitoneallyadministered 2 days before the spleen cells were harvested.
The spleen cells were fused with P3U1 cells (ATCC) using
PEG1500 (Roche Diagnostics, Indianapolis, IN). The hybridomas were grown in Roswell Park Memorial Institute(RPMI) 1640 medium (Nacalai Tesque, Inc., Kyoto, Japan)
supplemented with 10% heat-inactivated fetal bovine serum (Thermo Fisher Scientific, Inc.) and hypoxanthine,aminopterin, and thymidine selection medium supplement(Thermo Fisher Scientific, Inc.). The culture supernatants245

were screened using flow cytometry for binding to CHO/cat
podoplanin and CHO-K1.
Immunohistochemical analyses

First, 4-mm-thick histological sections were deparaffinizedin xylene and then rehydrated, followed by autoclavingin citrate buffer (pH 6.0; Agilent Technologies, Inc., Santa
Clara, CA) for 20 minutes. The sections were incubatedwith 1 mg/mL of PMab-52 for 1 hour at room temperature,
followed by treatment with Envision+ kit for 30 minutes(Agilent Technologies, Inc.). Color was developed using 3,3-diaminobenzidine tetrahydrochloride (Agilent Technologies, Inc.) for 2 minutes, and the sections were counterstained with hematoxylin (Wako Pure Chemical Industries,
Ltd., Osaka, Japan). The intensity of staining was evaluatedas 0, 1+, 2+, and 3+.
Results

In our recent studies, we immunized mice with CHO/cat
podoplanin cells and performed flow cytometry as the firstscreening. Using this cell-based immunization and screening(CBIS) method, we could obtain highly sensitive mAbs, which
are useful not only in flow cytometry but also in Western blot
and immunohistochemical analyses. Indeed, one of the established clones, PMab-52 (IgM, kappa), was sensitive andspecific against cat podoplanin in flow cytometry, Western blot,
and immunohistochemical analyses. PMab-52 reacted with only
cat podoplanin, whereas it did not react with human, mouse,rat, rabbit, dog, and bovine podoplanin in flow cytometry and

FIG. 3. Immunohistochemical analysis of podoplanin using PMab-52 in feline rectum. (A, C) Sections of feline rectum
were autoclaved in citrate buffer (pH 6.0). After blocking, they were incubated with 1 mg/mL of PMab-52, followed bytreating with EnVision+ kit. Color was developed using DAB, and the slides were counterstained with hematoxylin. (B, D)
H&E staining was performed against serial sections. Arrows: lymphatic endothelial cells. Scale bar: 100 mm.

246

ITAI ET AL.

Downloaded by Yukinari Kato from online.liebertpub.com at 12/21/17. For personal use only.

Table 1. Results of PMab-52 Immunostaining in 40 Feline Squamous Cell Carcinomas
No.

Site

Breed

Age (year)

Sex (reproductive status)

PMab-52 (IHC)

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40

Skin
Ear
Ear
TongueFloor of mouth
Floor of mouth
TongueFloor of mouth
Skin
Skin
Skin
Floor of mouth
Ear
Skin
Skin
Ear
Floor of mouth
Ear
Ear
Ear
Ear
Floor of mouth
Floor of mouth
Skin
Floor of mouth
TongueFloor of mouth
Floor of mouth
Skin
Skin
Skin
Floor of mouth
Skin
Floor of mouth
Skin
Skin
Floor of mouth
Floor of mouthEar
TongueMix
Mix
Mix
Scottish Fold
Mix
Mix
Mix
Mix
Mix
Mix
Mix
Mix
Mix
Mix
Mix
Mix
Mix
Mix
Mix
Mix
Mix
Mix
PersianMix
Maine Coon
Mix
American Shorthair
Mix
Mix
Mix
Mix
Persian
Mix
American Shorthair
Mix
Mix
Mix
PersianMix
Not specified

13
10
16
10
16
13
13
15
11
15
14
14
16
11
7
16
11
17
15
14
Not specified
13
12
14
Not specified
16
14
15
16
9
10
10
10
13
Not specified
12
16
13
11
15

F (spayed)
F (spayed)
F (spayed)
F (spayed)
F (spayed)
F (spayed)
F (spayed)
M (casted)
M (casted)
M (casted)
F (spayed)
F (spayed)
F (entire)
M (casted)
M (casted)
F (spayed)
F (spayed)
F (spayed)
F (spayed)
F (spayed)
F (entire)
F (spayed)
F (spayed)
F (entire)
Not specified
M (entire)
M (casted)
M (casted)
M (casted)
M (casted)
F (spayed)
M (casted)
F (spayed)
M (casted)F (entire)
F (spayed)
F (spayed)
M (entire)
F (spayed)
F (spayed)

1+
1+
3+
3+
1+
2+
1+
2+
3+
3+
3+
1+
1+
1+
2+
2+
2+
1+
2+
1+
0
2+
3+
2+
1+
2+
2+
0
3+
3+
2+
3+
3+
1+
1+
3+
1+
2+
2+
3+

Western blot analyses.(32) Although we have investigatedpodoplanin expression in human, rabbit, and canine cancers,
its expression in feline cancer remains to be clarified.
First, we investigated podoplanin expression in normal feline tissues, including the kidney, lung, and rectum, by immunohistochemical analysis. PMab-52 was bound to theBowman’s capsule and glomerular podocytes in feline kidneys(Fig. 1A, C). Also, it was bound to the type I alveolar cells of
feline lungs (Fig. 2A, C) and lymphatic endothelial cells(Fig. 3A, C), indicating that PMab-52 can detect cat podoplaninin normal tissues because a similar expression pattern has beenreported in other species, including human and mouse.(35)
Next, we investigated cat podoplanin expression in various
squamous cell carcinomas because its expression has been

Table 2. Summary of PMab-52 Immunostaining in 40 Feline Squamous Cell Carcinomas
PMab-52 immunostaining
Site
Floor of mouth
Skin
Ear
TongueTotal

No. of cases

3+

2+

1+

0

No. of positive cases

Positive rate (%)

14
13
9
4
40

2
7
1
2
12

6
3
3
1
13

5
3
4
1
13

1
0
1
0
2

13/14
13/13
8/9
4/4
38/40

92.9
100
88.9
100
95.0

ANTI-CAT PDPN MAB FOR SCC

noma cells in a membrane-staining pattern (Fig. 4). All
staining patterns are depicted in Supplementary Figure S1and S2 (Supplementary Data are available online at www.liebertpub.com/mab). In total, 95% (38/40) of squamous cell
carcinomas were detected using PMab-52 (Table 2).

Downloaded by Yukinari Kato from online.liebertpub.com at 12/21/17. For personal use only.

reported in many previous studies with other species.(18,33)
All specimens of squamous cell carcinomas used in this study
are listed in Table 1. Among the 40 cases analyzed, 14 werefrom mouth floor, 13 from skin, 9 from ear, and 4 from tongue(Table 2). PMab-52 was observed in squamous cell carci-

247

FIG. 4. Immunohistochemical analysis of cat podoplanin using PMab-52 in squamous cell carcinomas. (A, C, E, G)
Sections of feline squamous cell carcinomas were autoclaved in citrate buffer (pH 6.0). After blocking, they were incubatedwith 1 mg/ml of PMab-52, followed by treatment with EnVision+ kit. Color was developed using DAB, and the slides werecounterstained with hematoxylin. (B, D, F, H) H&E staining was performed against serial sections. (A–D) case 9; (E–H)
case 11. Arrows: lymphatic endothelial cells; arrowheads: vascular endothelial cells. Scale bar: 100 mm.

248

PMab-52 also detected lymphatic endothelial cells aroundthe squamous cell carcinoma cells, but it could not detectvascular endothelial cells (Fig. 4C). PMab-52 very weakly
detected normal lymphatic endothelial cells (Fig. 3), indicating that the expression level of podoplanin is different
between normal and cancerous tissues.

Downloaded by Yukinari Kato from online.liebertpub.com at 12/21/17. For personal use only.

Discussion

In this study, we first investigated podoplanin expressionin feline normal tissues. PMab-52 strongly bound to feline
kidneys (Fig. 1) and lungs (Fig. 2). These results are consistent with our previous study regarding podoplanin expression in rabbit normal tissues.(29) In contrast, anti-dog
podoplanin mAb PMab-38 reacted with canine kidney, but
not with canine lungs.(30) PMab-52 was weakly bound tonormal lymphatic endothelial cells (Fig. 3), although podoplanin is known to be a specific marker for lymphatic endothelial cells. Likewise, PMab-38 did not detect normal
lymphatic endothelial cells of canine normal tissues.(30) NZ1, an anti-human podoplanin mAb, detects lymphatic endothelial cells but not those of the lung and kidney.(26) Moreover, cancer-specific mAbs LpMab-2(15) and LpMab-23(36)do not react with human podoplanin in all normal tissues.
Taken together, two main reasons can explain the different
reaction patterns of anti-podoplanin mAbs: (1) the expression
level of podoplanin is different among tissues and (2) the
epitopes recognized by antipodoplanin mAbs vary due tomany reasons, including tissue-specific posttranslational
modification or cancer-specific glycosylation.
We investigated cat podoplanin expression in 40 squamouscell carcinoma tissues. PMab-52 strongly detected lymphatic
endothelial cells around squamous cell carcinoma cells(Fig. 4C), whereas it weakly detected normal lymphatic endothelial cells (Fig. 3C), indicating that the expression levelof podoplanin differs between normal and cancer tissues. We
have previously reported that podoplanin expression can be
induced by transforming growth factor-b,(37) which might
explain the different expression levels of podoplanin incancer tissues. Cancer-associated fibroblasts were also detected in some cases (case 14; Supplementary Fig. S1), whichhave been observed in many previous studies using antipodoplanin mAbs in human cancer tissues(38–40) and using
PMab-38 in canine squamous cell carcinomas.(33) Targeted
therapy against cancer-associated fibroblasts is also important because cancer-associated fibroblasts have been associated with malignancy.(38–40)
In conclusion, PMab-52 can be useful for clarifying thepathophysiological function of podoplanin in feline squamous cell carcinomas. Furthermore, it is a potential candidatefor developing antibody-based therapies against felinesquamous cell carcinomas.
Author Disclosure Statement

Y.K. received research funding from Ono PharmaceuticalCo., Ltd.
Acknowledgments

We thank Takuro Nakamura, Miyuki Yanaka, Noriko
Saidoh, Saori Handa, and Yoshimi Nakamura for excellent
technical assistance. This work was supported, in part, by the

ITAI ET AL.

Basic Science and Platform Technology Program for Innovative Biological Medicine from Japan Agency for Medical Research and Development, AMED (Y.K.), by Projectfor utilizing glycans in the development of innovative drug
discovery technologies from AMED (Y.K.), and by Platform
Project for Supporting Drug Discovery and Life Science
Research (Basis for Supporting Innovative Drug Discovery
and Life Science Research [BINDS]) from AMED.
References

1. Gardner DG: Spontaneous squamous cell carcinomas of theoral region in domestic animals: A review and consideration of their relevance to human research. Oral Dis 1996;
2:148–154.
2. Breiteneder-Geleff S, Matsui K, Soleiman A, Meraner P,
Poczewski H, Kalt R, Schaffner G, and Kerjaschki D: Podoplanin, novel 43-kd membrane protein of glomerularepithelial cells, is down-regulated in puromycin nephrosis.
Am J Pathol 1997;151:1141–1152.
3. Breiteneder-Geleff S, Soleiman A, Kowalski H, Horvat R,
Amann G, Kriehuber E, Diem K, Weninger W, Tschachler
E, Alitalo K, and Kerjaschki D: Angiosarcomas express
mixed endothelial phenotypes of blood and lymphatic
capillaries: Podoplanin as a specific marker for lymphatic
endothelium. Am J Pathol 1999;154:385–394.
4. Kato Y, Fujita N, Kunita A, Sato S, Kaneko M, Osawa M,
and Tsuruo T: Molecular identification of Aggrus/T1alphaas a platelet aggregation-inducing factor expressed in colorectal tumors. J Biol Chem 2003;278:51599–51605.
5. Kaneko MK, Kato Y, Kitano T, and Osawa M: Conservation of a platelet activating domain of Aggrus/podoplanin as a platelet aggregation-inducing factor. Gene
2006;378:52–57.
6. Martin-Villar E, Scholl FG, Gamallo C, Yurrita MM,
Munoz-Guerra M, Cruces J, and Quintanilla M: Characterization of human PA2.26 antigen (T1alpha-2, podoplanin), a small membrane mucin induced in oral squamouscell carcinomas. Int J Cancer 2005;113:899–910.
7. Suzuki-Inoue K, Kato Y, Inoue O, Kaneko MK, Mishima
K, Yatomi Y, Yamazaki Y, Narimatsu H, and Ozaki Y:
Involvement of the snake toxin receptor CLEC-2, in
podoplanin-mediated platelet activation, by cancer cells. J
Biol Chem 2007;282:25993–26001.
8. Kato Y, Kaneko MK, Kunita A, Ito H, Kameyama A,
Ogasawara S, Matsuura N, Hasegawa Y, Suzuki-Inoue K,
Inoue O, Ozaki Y, and Narimatsu H: Molecular analysis of
the pathophysiological binding of the platelet aggregationinducing factor podoplanin to the C-type lectin-like receptor CLEC-2. Cancer Sci 2008;99:54–61.
9. Kaneko MK, Kunita A, Abe S, Tsujimoto Y, Fukayama M,
Goto K, Sawa Y, Nishioka Y, and Kato Y: Chimeric antipodoplanin antibody suppresses tumor metastasis throughneutralization and antibody-dependent cellular cytotoxicity.
Cancer Sci 2012;103:1913–1919.
10. Nagae M, Morita-Matsumoto K, Kato M, Kaneko MK,
Kato Y, and Yamaguchi Y: A Platform of C-Type lectinlike receptor CLEC-2 for binding O-glycosylated podoplanin and nonglycosylated rhodocytin. Structure 2014;22:
1711–1721.
11. Bertozzi CC, Schmaier AA, Mericko P, Hess PR, Zou Z,
Chen M, Chen CY, Xu B, Lu MM, Zhou D, Sebzda E,
Santore MT, Merianos DJ, Stadtfeld M, Flake AW, Graf T,
Skoda R, Maltzman JS, Koretzky GA, and Kahn ML:

Downloaded by Yukinari Kato from online.liebertpub.com at 12/21/17. For personal use only.

ANTI-CAT PDPN MAB FOR SCC

Platelets regulate lymphatic vascular development through
CLEC-2-SLP-76 signaling. Blood 2010;116:661–670.
12. Mishima K, Kato Y, Kaneko MK, Nishikawa R, Hirose T,
and Matsutani M: Increased expression of podoplanin inmalignant astrocytic tumors as a novel molecular marker ofmalignant progression. Acta Neuropathol (Berl) 2006;111:
483–488.
13. Mishima K, Kato Y, Kaneko MK, Nakazawa Y, Kunita A,
Fujita N, Tsuruo T, Nishikawa R, Hirose T, and Matsutani
M: Podoplanin expression in primary central nervous system germ cell tumors: A useful histological marker for the
diagnosis of germinoma. Acta Neuropathol (Berl) 2006;
111:563–568.
14. Kato Y, Vaidyanathan G, Kaneko MK, Mishima K, Srivastava N, Chandramohan V, Pegram C, Keir ST, Kuan
CT, Bigner DD, and Zalutsky MR: Evaluation of antipodoplanin rat monoclonal antibody NZ-1 for targeting
malignant gliomas. Nucl Med Biol 2010;37:785–794.
15. Kato Y, and Kaneko MK: A cancer-specific monoclonal
antibody recognizes the aberrantly glycosylated podoplanin. Sci Rep 2014;4:5924.
16. Ochoa-Alvarez JA, Krishnan H, Pastorino JG, Nevel E,
Kephart D, Lee JJ, Retzbach EP, Shen Y, Fatahzadeh M,
Baredes S, Kalyoussef E, Honma M, Adelson ME, Kaneko
MK, Kato Y, Young MA, Deluca-Rapone L, Shienbaum
AJ, Yin K, Jensen LD, and Goldberg GS: Antibody andlectin target podoplanin to inhibit oral squamous carcinoma
cell migration and viability by distinct mechanisms. Oncotarget 2015;6:9045–9060.
17. Schoppmann SF, Jesch B, Riegler MF, Maroske F,
Schwameis K, Jomrich G, and Birner P: Podoplanin expressing cancer associated fibroblasts are associated withunfavourable prognosis in adenocarcinoma of the esophagus. Clin Exp Metastasis 2013;30:441–446.
18. Kato Y, Kaneko M, Sata M, Fujita N, Tsuruo T, and Osawa
M: Enhanced expression of Aggrus (T1alpha/podoplanin),a platelet-aggregation-inducing factor in lung squamouscell carcinoma. Tumor Biol 2005;26:195–200.
19. Kimura N, and Kimura I: Podoplanin as a marker for mesothelioma. Pathol Int 2005;55:83–86.
20. Abe S, Morita Y, Kaneko MK, Hanibuchi M, Tsujimoto Y,
Goto H, Kakiuchi S, Aono Y, Huang J, Sato S, Kishuku M,
Taniguchi Y, Azuma M, Kawazoe K, Sekido Y, Yano S,
Akiyama S, Sone S, Minakuchi K, Kato Y, and Nishioka Y:
A novel targeting therapy of malignant mesothelioma using
anti-podoplanin antibody. J Immunol 2013;190:6239–6249.
21. Kato Y, Sasagawa I, Kaneko M, Osawa M, Fujita N, andTsuruo T: Aggrus: A diagnostic marker that distinguishes
seminoma from embryonal carcinoma in testicular germ
cell tumors. Oncogene 2004;23:8552–8556.
22. Kaneko MK, Oki H, Ogasawara S, Takagi M, and Kato Y:
Anti-podoplanin monoclonal antibody LpMab-7 detects
metastatic legions of osteosarcoma. Monoclon. Antib Immunodiagn Immunother 2015;34:154–161.
23. Ariizumi T, Ogose A, Kawashima H, Hotta T, Li G, Xu Y,
Umezu H, Sugai M, and Endo N: Expression of podoplaninin human bone and bone tumors: New marker of osteogenic
and chondrogenic bone tumors. Pathol Int 2010;60:193–202.
24. Kunita A, Kashima TG, Ohazama A, Grigoriadis AE, andFukayama M: Podoplanin is regulated by AP-1 and promotes platelet aggregation and cell migration in osteosarcoma. Am J Pathol 2011;179:1041–1049.
25. Kunita A, Kashima TG, Morishita Y, Fukayama M, Kato Y,
Tsuruo T, and Fujita N: The platelet aggregation-inducing

249

factor aggrus/podoplanin promotes pulmonary metastasis.
Am J Pathol 2007;170:1337–1347.
26. Kato Y, Kaneko MK, Kuno A, Uchiyama N, Amano K,
Chiba Y, Hasegawa Y, Hirabayashi J, Narimatsu H, Mishima K, and Osawa M: Inhibition of tumor cell-induced
platelet aggregation using a novel anti-podoplanin antibody reacting with its platelet-aggregation-stimulating
domain. Biochem Biophys Res Commun 2006;349:1301–
1307.
27. Yamada S, Kaneko MK, Nakamura T, Ichii O, Konnai S,
and Kato Y: Development of mPMab-1, a mouse-rat chimeric antibody Against Mouse Podoplanin. Monoclon
Antib Immunodiagn Immunother 2017;36:77–79.
28. Oki H, Honma R, Ogasawara S, Fujii Y, Liu X, Takagi M,
Kaneko MK, and Kato Y: Development of Sensitive
Monoclonal Antibody PMab-2 Against Rat Podoplanin.
Monoclon Antib Immunodiagn Immunother 2015;34:
396–403.
29. Honma R, Fujii Y, Ogasawara S, Oki H, Liu X, Nakamura
T, Kaneko MK, Takagi M, and Kato Y: Establishment of anovel monoclonal antibody PMab-32 against rabbit podoplanin. Monoclon Antib Immunodiagn Immunother2016;35:41–47.
30. Honma R, Kaneko MK, Ogasawara S, Fujii Y, Konnai
S, Takagi M, and Kato Y: Specific detection of dog podoplanin expressed in renal glomerulus by a novel
monoclonal antibody PMab-38 in immunohistochemistry. Monoclon Antib Immunodiagn Immunother 2016;35:
212–216.
31. Honma R, Ogasawara S, Kaneko M, Fujii Y, Oki H, Nakamura T, Takagi M, Konnai S, and Kato Y: PMab-44
detects bovine podoplanin in immunohistochemistry.
Monoclon Antib Immunodiagn Immunother 2016;35:186–
190.
32. Yamada S, Itai S, Nakamura T, Yanaka M, Saidoh N,
Chang YW, Handa S, Harada H, Kagawa Y, Ichii O,
Konnai S, Kaneko MK, and Kato Y: PMab-52: Specific andsensitive monoclonal antibody against cat podoplanin for
immunohistochemistry. Monoclon Antib Immunodiagn
Immunother 2017;36:224–230.
33. Kaneko MK, Honma R, Ogasawara S, Fujii Y, Nakamura
T, Saidoh N, Takagi M, Kagawa Y, Konnai S, and Kato Y:
PMab-38 recognizes canine podoplanin of squamous cell
carcinomas. Monoclon Antib Immunodiagn Immunother2016;35:263–266.
34. Ogasawara S, Honma R, Kaneko MK, Fujii Y, Kagawa Y,
Konnai S, and Kato Y: Podoplanin expression in canine
melanoma. Monoclon Antib Immunodiagn Immunother2016;35:304–306.
35. Kaji C, Tsujimoto Y, Kato Kaneko M, Kato Y, and Sawa
Y: Immunohistochemical examination of novel rat monoclonal antibodies against mouse and human podoplanin.
Acta Histochem Cytochem 2012;45:227–237.
36. Yamada S, Kaneko MK, and Kato Y: LpMab-23: A cancerspecific monoclonal antibody against human podoplanin.
Monoclon Antib Immunodiagn Immunother 2017;36:
72–76.
37. Suzuki H, Kato Y, Kaneko MK, Okita Y, Narimatsu H, andKato M: Induction of podoplanin by transforming growth
factor-beta in human fibrosarcoma. FEBS Lett 2008;582:
341–345.
38. Takahashi A, Ishii G, Kinoshita T, Yoshida T, Umemura S,
Hishida T, Yoh K, Niho S, Goto K, Ohmatsu H, Ohe Y,
Nagai K, and Ochiai A: Identification of prognostic im-

250

Downloaded by Yukinari Kato from online.liebertpub.com at 12/21/17. For personal use only.

munophenotypic features in cancer stromal cells of highgrade neuroendocrine carcinomas of the lung. J Cancer Res
Clin Oncol 2013;139:1869–1878.
39. Shindo K, Aishima S, Ohuchida K, Fujino M, Mizuuchi
Y, Hattori M, Ohtsuka T, Tokunaga S, Mizumoto K,
Tanaka M, and Oda Y: Podoplanin expression in thecyst wall correlates with the progression of intraductal
papillary mucinous neoplasm. Virchows Arch 2014;465:
265–273.
40. Ono S, Ishii G, Nagai K, Takuwa T, Yoshida J, Nishimura
M, Hishida T, Aokage K, Fujii S, Ikeda N, and Ochiai A:
Podoplanin-positive cancer-associated fibroblasts could
have prognostic value independent of cancer cell phenotype
in stage I lung squamous cell carcinoma: Usefulness of
combining analysis of both cancer cell phenotype andcancer-associated fibroblast phenotype. Chest 2013;143:
963–970.

ITAI ET AL.

Address correspondence to:
Yukinari Kato
Department of Antibody Drug Development
Tohoku University Graduate School of Medicine
New Industry Creation Hatchery Center
2-1 Seiryo-machi
Aoba-ku
Sendai
Miyagi 980-8575
JapanE-mail: yukinari-k@bea.hi-ho.ne.jp;
yukinarikato@med.tohoku.ac.jp
Received: September 14, 2017
Accepted: October 2, 2017