Title

Evaluation of immunohistochemical staining with PMab-38, an anti-dog podoplanin monoclonal antibody, in various
canine tumor tissues

Author(s)Kiname, Kohei; Yoshimoto, Sho; Kato, Daiki; Tsuboi, Tsuboi; Tanaka, Yuiko; Yoshitake, Ryohei; Eto, Shotaro;
Shinada, Masahiro; Chambers, James; Saeki, Kohei; Kinoshita, Ryohei; Yamada, Shinji; Uchida, Kazuyuki; Kaneko,
Mika K.; Nishimura, Ryohei; Kato, Yukinari; Nakagawa, Takayuki

Citation

Japanese Journal of Veterinary Research, 67(1): 25-34

Issue Date2019-02

Doc URL

http://hdl.handle.net/2115/72729

Type

File Information

bulletin (article)

p025-034 Daiki Kato.pdf

Instructions for use

Hokkaido University Collection of Scholarly and Academic Papers : HUSCAP

Japanese Journal of Veterinary Research 67(1): 25-34, 2019

REGULAR PAPER

Experimental Research

Evaluation of immunohistochemical staining with
PMab-38, an anti-dog podoplanin monoclonal
antibody, in various canine tumor tissues
Kohei Kiname1,†), Sho Yoshimoto1,†), Daiki Kato1,＊), Masaya Tsuboi2),
Yuiko Tanaka1), Ryohei Yoshitake1), Shotaro Eto1), Masahiro Shinada1),
James Chambers2), Kohei Saeki1), Ryohei Kinoshita1), Shinji Yamada3),
Kazuyuki Uchida2), Mika K. Kaneko3), Ryohei Nishimura1),
Yukinari Kato3, 4) and Takayuki Nakagawa1)
1)

Laboratory of Veterinary Surgery, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1, Yayoi,
Bunkyo-ku, Tokyo 113-8657, Japan
2)
Laboratory of Veterinary Pathology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1,
Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan3)
Department of Antibody Drug Development, Tohoku University Graduate School of Medicine, 2-1, Seiryo-machi, Aoba-ku,
Sendai, 980-8575, Japan
4)
New Industry Creation Hatchery Center, Tohoku University, 2-1, Seiryo-machi, Aoba-ku, Sendai, 980-8575, Japan
Received for publication, March 6, 2018; accepted, June 29, 2018


Abstract
Podoplanin (PDPN) is a type I transmembrane sialoglycoprotein with O-glycosylation and a highcontent of sialic acid. PDPN has been reported to be expressed in various human tumors and promotetumor progression, epithelial-mesenchymal transition, and distant metastasis. PDPN is also expressedin cancer-associated fibroblasts (CAFs), which promote tumor growth. We have developed novel tumor
specific anti-PDPN antibodies. PMab-38, which is an anti-dog PDPN monoclonal antibody, recognized
PDPN expression in canine squamous cell carcinoma (SCC) and malignant melanoma. However, there
has been no research into PMab-38 recognition of other types of tumors and systemic normal tissue.
The objective of this study was to evaluate the staining positivity of PMab-38 by immunohistochemical
staining of various paraffin-embedded canine tumor and systemic normal tissues. Immunohistochemical
analysis revealed that PMab-38 positively stained tumor cells in 9/11 (82%) SCC and 9/11 (82%)
pulmonary adenocarcinoma tissues. In the tumor stroma, large spindle-shaped mesenchymal cells,
which were suspected to be CAFs, were stained by PMab-38 in almost all tumor types: 9/10 (90%) for
anal sac adenocarcinoma and 11/12 (92%) for transitional cell carcinoma tissues. Almost all normal
tissues were negatively stained with PMab-38 except part of the kidney glomerulus. Taken together,
our findings provide evidence that various types of tumor cells were strongly stained by PMab-38, but
most normal cells were not stained. These results indicate that PDPN recognized by PMab-38 might bea target for tumor antigen targeting therapy. Further studies are required to investigate the antitumor effect in various canine tumors by antibody therapy using PMab-38.

Key Words: podoplanin, PMab-38, canine cancer†Kohei Kiname and Sho Yoshimoto equally contributed as first author.
*Corresponding author: Daiki Kato, Laboratory of Veterinary Surgery, Graduate School of Agricultural and Life
Sciences, The University of Tokyo, 1-1-1, Yayoi, Bunkyo-ku, Tokyo, 113-8657, Japan
Fax: +81-3-5841-5405. E-mail: feetkato@yahoo.co.jp
doi: 10.14943/jjvr.67.1.25

26

Immunohistochemical staining with PMab-38

Introduction
Podoplanin (PDPN) is a type I transmembranesialoglycoprotein with O-glycosylation and a highcontent of sialic acid27). It has been reported that
it is expressed in renal podocytes, lymphatic
endothelial cells (LECs), and pulmonary typeI alveolar cells4,25,28). It has been widely used
as a specific marker for LECs because it isstrongly expressed in them25). Since PDPN has
essential functions in maintaining lymph node
microarchitecture, PDPN deficient mice showedmalformation of lymphatic nodules3,33). It has also
been reported that PDPN interacts with C-type
lectin-like receptor 2 (CLEC2) in platelets andinduces PDPN-mediated platelet aggregation4,33).
In human medicine, PDPN was reported to
be expressed in various human tumors, such as
squamous cell carcinoma (SCC), malignant
melanoma (MM), malignant mesothelioma,
hemangiosarcoma (HSA), osteosarcoma (OSA),
germinoma, and malignant astrocytic2,10,21).
Increasing evidence suggests that PDPN promoted
tumor progression, epithelial-mesenchymaltransition, and distant metastasis by facilitatingtumor cell migration and inducing platelet
aggregation35). Currently, it is understood that thetumor stroma contributes to tumor progression
by interacting with tumor cells16,18). Cancerassociated fibroblasts (CAFs), one of the major
components of the tumor stroma, were reported
to over express PDPN14,15,30,38). As CAFs correlatewith poor prognosis in human patients withtumors, it has been suggested that PDPN
expressed in CAFs helps tumor progression andlocal invasion22).
Recently, an increasing number of monoclonal
antibodies (mAb) have been developed fortherapeutic treatment of tumors26). Tumor antigen
targeting cytotoxic antibodies, such as anti-CD20
and anti-human epidermal growth factor receptor
2 (HER2), have significantly improved theclinical outcome of patients with malignant
tumors19,34). The best tumor antigens for cytotoxic
antibody therapy are expressed only on the tumor

cell’s membrane but not on normal tissues’ cell’smembrane. However, only a few tumor antigens
specifically expressed on tumor cells weredeveloped. Although PDPN is strongly expressedin human tumor cells, it is also expressed invarious normal tissue types. Therefore, PDPN
has been thought to be an unsuitable target forcytotoxic antibody therapy.
It is well known that aberrant glycosylationof glycoproteins is a common feature of neoplastictransformation32). We focused on tumor specificmodification of PDPN and developed novel tumor
specific anti-PDPN antibodies, which targeted a
specific epitope in various species12). Of these, theanti-dog PDPN (dPDPN) monoclonal antibody,PMab-38, recognized dPDPN expression in canine
SCC and MM but not in pulmonary type I cells
and LECs as in previous studies7,9,23). However,there has been no research screening for
PMab-38 recognition in various types of tumorsand systemic normal tissues. The objective of thisstudy was to evaluate the staining positivity ofPMab-38 by immunohistochemical analysis ofvarious paraffin-embedded canine tumor andsystemic normal tissues and to investigate thepossibility of applying PMab-38 to canine
patients with tumors.

Materials and methods
Specimens: A total of 134 paraffin-embedded
tumor tissues were evaluated. These tissues were
surgically removed from dogs at the Veterinary
Medical Center of the University of Tokyo
between 2012 and 2016. Permission for resected
tissue collection and usage for this study was
obtained from the owners. The tissue samples
were diagnosed at the Department of Veterinary
Pathology at the University of Tokyo. All samples
were fixed in 10% neutral buffered formalin,embedded in paraffin wax, and cut into
4-μm-thick serial sections. Tumor types included
mammary adenocarcinoma (MAC, n ＝ 13), SCC(n ＝ 11), pulmonary adenocarcinoma (PAC, n ＝Kohei Kiname et al.

2711), MM (n ＝ 10), fibrosarcoma (FSA, n ＝ 10),OSA (n ＝ 10), anal sac adenocarcinoma (ASAC,
n ＝ 11), peripheral nerve sheath tumor (PNST,n ＝ 8), thyroid carcinoma/adenocarcinoma (TC/AC, n ＝ 9), thymoma (THY, n ＝ 9), hepatocellular
carcinoma (HCC, n ＝ 10), and HSA (n ＝ 11).
Healthy dog tissues were obtained from healthy
beagles euthanized for other experiments. Allprocedures were approved by the Animal Careand Use Committee at the University of Tokyo.

The specimens were considered positive forPMab-38 if histological evidence of cell staining
was present. Positive rates of each tumor types
were investigated with PMab-38 on tumor cells
and stroma cells separately. Staining specificityof PMab-38 was assured by the lack of staining
in the negative control sections.

Immunohistochemistry: Paraffin-embedded tumor
sections were dewaxed and rehydrated in xylene
and graded ethanol followed by antigen retrieval
using Dako Target Retrieval Solution, pH 9.0(Agilent Technologies, Santa Clara, CA, USA),at 100°C for 30 min in boiling water. Afterwashing with tris buffered saline- 0.1% tween 20(TBS-t), endogenous peroxidase was blockedwith 3% hydrogen peroxide in methanol for
10 min. Specimens were then washed with TBS-t
and incubated in 8% skimmed milk for 1 h atroom temperature to reduce non-specific binding
before overnight incubation with primary
antibodies including mouse IgG1 anti-dPDPN(PMab-389)) antibodies, anti-pan-dPDPN (PMab4837)) antibodies, or the anti-α-smooth muscle
actin (α-SMA) antibody (clone: 1A4, Agilent
Technologies) at 4°C in a humidified chamber. A
negative control was incubated with the purified
mouse IgG1, κ isotype antibody (Clone: MG1-45,
BioLegend, San Diego, CA, USA), under identicalconditions. For PMab-38 immunostaining, asection of canine cutaneous SCC was used as the
positive control9,11). After washing with TBS-t,
sections were incubated with a horseradish
peroxidase (HRP)-conjugated anti-mouse antibody(Envision＋ System-HRP Labelled polymer;
K4001; Agilent Technologies) for 30 min at room
temperature. Thereafter, the sections were washedwith TBS-t, incubated with 3,3’ diaminobenzidine(DAB; Dojindo Laboratories, Rockville, MD, USA)
solution, and counterstained with Mayer’s
hematoxylin. For the negative control, sections
were subjected to the same procedures.

1. Evaluation of PMab-38 staining in various
types of canine tumor tissues
Immunohistochemical analysis using
PMab-38 was performed in 134 specimens ofvarious tumor types. The positive staining ratio
for tumor cells varied among the tumor types. In
evaluated tumor tissues, PMab-38 positively
stained tumor cells in 9/11 (82%) SCC, 9/11 (82%)
PAC, 4/10 (40%) FSA, 2/13 (15%) MAC, 1/10
(10%) OSA, and 1/10 (10%) ASAC. The ratio of
positive tumor cells in the tumor tissues variedamong samples. More than 50% of the tumor
cells were stained in some samples of SCC and
PAC but not in other samples, where less than
10% of tumor cells were stained. No staining was
detected in the other six types of tumors, MM,
TCC, PNST, TC/AC, THY, HCC, and HSA(Table 1).
In tumor stroma, large spindle-shaped
mesenchymal cells were stained by PMab-38 inalmost all of the tumor types and the positive
ratios in the analyzed specimens were 9/11 (82%)
for SCC, 6/11 (55%) for PAC, 3/13 (20%) for MAC,
9/10 (90%) for ASAC, 11/12 (92%) for TCC, 2/8(25%) for PNST, 2/9 (22%) for TC/AC, 2/9 (22%)
for THY, and 1/10 (10%) for HCC (Table 1). In
FSA and OSA samples, we did not evaluate thecells separately because we could not distinguishtumor from stroma cells (Fig. 1). Surrounding
normal tissues were not stained by PMab-38 in
all cases.

Results

28

Immunohistochemical staining with PMab-38

Table 1. Evaluation of PMab-38 staining in various types of canine tumor tissues
Tumor types*a

SamplesTumor cells (%)

Stroma (%)*b

SCC

11

9 (82)

9 (82)PAC

11

9 (82)

6 (55)

MAC13

2 (15)

3 (20)FSA

10

4 (40)

ND

OSA

10

1 (10)

ND

ASAC

10

1 (10)

9 (90)

TCC

12

0 (0)

11 (92)

PNST

8

0 (0)

2 (25)

TC/AC

9

0 (0)

2 (22)

THY

9

0 (0)

2 (22)

HCC

10

0 (0)

1 (10)

HSA

11

0 (0)

0 (0)

MM

10

0 (0)

0 (0)

We analyzed the positive ratio of PMab-38 staining of tumor and stroma cell separately in eachtumor type. The positive ratio of tumor cells was high in SCC and PAC, but tumor cells of sometumor types were not stained by PMab-38. However, stroma cells of almost all tumor types were
stained by PMab-38, and stroma cells of SCC, PAC, ASAC, and TCC had a high positive ratio.
a
SCC: Squamous cell carcinoma, PAC: Pulmonary adenocarcinoma, MAC: Mammary adenocarcinoma,
FSA: Fibrosarcoma, OSA: Osteosarcoma, ASAC: Anal sac adenocarcinoma, TCC: Transitional cell
carcinoma, PNST: Peripheral nerve sheath tumor, TC/AC: Thyroid carcinoma/adenocarcinoma,
THY: Thymoma, HCC: Hepatocellular cell carcinoma, HSA: Hemangiosarcoma, MM: Malignant
melanoma.
b
ND: Not-determined

Fig. 1. Evaluation of the staining pattern of PMab-38. Representative staining patterns of PMab-38 in PAC,
SCC, and FSA are shown. Tumor cell membranes of SCC and PAC were stained by PMab-38. In this SCC sample,
the unstained area consisted of normal cells, and all tumor cells were stained. In PAC tissue, invasive tumor cells
in the bronchial tube are shown, and most of them were stained by PMab-38. In FSA samples, some cells were
stained, but we could not distinguish tumor cells from stroma cells.

Kohei Kiname et al.

29

2. Evaluation of the staining pattern of PMab-38
in tumor cells
Cell membranes of tumor cells were strongly
stained by PMab-38 in positively stained samples(Fig. 1). Positively stained tumor cells showedvarious staining patterns, including diffuse andfocal staining, but we did not find any specificpattern in accordance with tumor types. The
intensity of PMab-38 staining of tumor cells was
equivalent in all positively stained samples. In
some PAC samples, invasive tumor cells in the
bronchial tube were also positively stained with
PMab-38.
3. Evaluation of PMab-38 staining in tumor
stroma cells
Since large spindle-shaped mesenchymal
cells were stained by PMab-38 in tumor stroma,these cells were suspected to be CAFs, which
express PDPN in some types of human tumors.
In this study, we analyzed whether canine CAFs
were recognized by PMab-38 according theirmorphology and α-SMA staining, which is
generally utilized to detect CAFs36,39).
Positively stained cells by α-SMA staining
and PMab-38 staining were compared in thisstudy36) (Fig. 2). Parts of α-SMA positive cells were
stained by PMab-38. PMab-38-positive CAFs
tended to be located adjacent to tumor nests.
CAF membranes were strongly stained, and theircytoplasm was weakly stained by PMab-38.
Staining intensity and the ratio of PMab-38
positive CAFs were especially high in TCC
samples compared to other tumor types.
4. Evaluation of PMab-38 staining in systemic
normal tissues
To explore the PMab-38 reaction against
normal tissues, we examined PMab-38 stainingof systemic normal tissues including skin,adipose, mammary gland, muscle, patella, brain,
tonsil, thyroid, lymph node, esophagus, lung,heart muscle, liver, pancreas, spleen, kidney,large intestine, urinary bladder, and uterus
tissues. Although normal tissues were positively

Fig. 2. Evaluation of PMab-38 staining in tumor
stroma cells. 
Representative staining pattern for
PMab-38 and α-SMA in TCC are shown. Large spindleshaped mesenchymal cells in the tumor stroma wereclearly stained by α-SMA and part of the α-SMA
positive cells were stained by PMab-38. CAFs stainedby PMab-38 tended to be located adjacent to the tumor
nests.

stained with anti-pan-dPDPN antibody, mostnormal tissues were not positively stained with
PMab-38 (Fig. 3, Fig. S1). Lung and lymphatic
nodules, which were reported to express
PDPN5,25), were also negatively stained by PMab38. Although the intensity was weaker thanSCC, part of the normal glomerulus in thekidney, which might be podocytes, was positively
stained by PMab-38 as previously reported9)(Fig. S2).

Discussion
This study demonstrated that cell
membranes of tumor cells were stained by
PMab-38 in more than half of the SCC and PAC
samples and a portion of the FSA, MA, OSA, and30

Immunohistochemical staining with PMab-38

Fig. 3. Evaluation of PMab-38 staining in systemic normal tissues. Systemic normal tissues were stained by
PMab38. SCC tissue was used as a positive control for PMab-38 staining. Most of the normal tissues were notpositively stained with PMab-38. Only part of the kidney glomerulus was stained, but the lung and lymph nodes
were not stained by PMab-38.

ASAC samples. In human medicine, it wasreported that PDPN was expressed in manytumors, such as SCC, MM, HSA, OSA, malignant
mesothelioma, Kaposi’s sarcoma, testicularseminoma, dysgerminoma, and glioblastoma.
However, it was reported that human PAC did
not express PDPN2,10.21). Interestingly, in thisstudy, most canine PAC was positively stainedwith PMab-38. In contrast to the human report,
all canine HSA samples were not stained.
Although there are discrepancies between dog
and human tumors, there is a correlation between
tumor types that express PDPN in humans anddogs. Our findings indicated that PMab-38 might
be used for evaluating the prognosis or in
antibody therapy for various types of tumors
positively stained by PMab-38.
In this study, we did not detect positive

staining of PMab-38 in any of the MM samples(n ＝ 10). Previous research reported that 9 of 10
amelanotic MM samples were positively stainedby PMab-3823). This difference may be related tothe histlogical differences of canine MM because
all of our samples were melanotic MM. As
evaluated, sample numbers were small in bothstudies, and therefore, further investigation forthis heterogeneity might be needed.
In this study, both oral and tonsillar SCC
were stained by PMab-38. Oral SCC, which
accounts for 20-30% of canine oral tumors, islocally invasive and systemic therapy is needed
when the lesion is unresectable29). Tonsillar
SCC has characteristics of rapid growth with
infiltration of underlying tissues, early invasionof regional lymph nodes, and frequent metastasis
to the lungs and other distant organs6). Since,

Kohei Kiname et al.

responses to chemotherapy are poor for thesetumors, new therapies are desired to improve
clinical outcomes for dogs with SCC. A cytotoxic
antibody therapy using PMab-38 might be a goodcandidate therapeutic option for dogs with SCC.
It has been reported that PDPN expression
is related to tumor malignancy, such as tumor
invasion and metastasis. In human keratinocytes
and MCF7 breast tumor cells, the forced
expression of PDPN led to a drastic change
in morphology and promoted cell spreading,migration, and invasion35). PDPN expression was
also reported to be related to lymph node and lungmetastasis by introducing lymphangiogenesis
and platelet aggregation1,8,13,17). In PAC tissues inthis study, tumor cells which invaded bronchialtubes were stained by PMab-38. This finding
suggests the possibility that PDPN function incanine tumor cell invasion is similar to humanreports. Furthermore, inhibition of dPDPN
expressing tumor cells using PMab-38 may show
anti-tumor effects via suppression of invasive
and metastatic tumor cells.
We found that canine CAFs were stained by
PMab-38 in almost all types of tumors, similar toreports in humans2,10,21). Both staining intensity
and the ratio of positive CAFs in tumor tissues
were higher than other tumor types, especially in
TCC samples. In the other tumor types, thestaining pattern of PMab-38 was heterogenic
within tumor types. Although the underlying
mechanism of PDPN expression in CAFs remains
to be investigated, it was reported that fibroblasts
had clonal heterogeneity and the protein
expression pattern differed among clones31), which
could explain the heterogenetic staining patternof PMab-38.
Several reports have shown that human andcanine CAFs played an important role in tumor
progression and dissemination by secreting
cytokines and growth factors16,39). Moreover, it
was reported that human melanoma with PDPNpositive CAFs showed significantly increased
lymphatic invasion and sentinel lymph nodemetastasis10). The evidence regarding the negative

31

function of CAFs has propelled the developmentof CAF-targeting therapies. Ohshio et al. reported
that CAFs targeting therapy with antifibrotic
agent enhanced anti-tumor immune responses
with a dendric cell-based vaccine therapy insyngeneic mouse models24). Mertensers et al.
demonstrated a cytotoxic drug for CAFs
suppressed tumor growth and improved hostsurvival through the mechanism of CAF apoptosis
in a synergistic rat model of cholangiocarcinoma20).
These preclinical studies may bring about a novelanti-tumor strategy in dogs, such as cytotoxic
antibody therapy of chimeric PMab-38 thatrecognizes dPDPN expression in CAFs.
Although various normal tissues expressed
PDPN, all normal tissues were negatively stainedwith PMab-38 except part of the kidney
glomerulus. This staining difference between
tumor tissues and normal tissues confirmed that
PMab-38 recognized the dPDPN epitope, which
was expressed in tumor tissues but not normal
tissues. The mechanism of tumor specific epitope
expression is unknown and further study to
uncover the mechanism is needed. Positive
staining in part of the glomerulus had the same
pattern as the previous report. However, thestaining pattern of the normal glomerulus was
weaker compared to that of tumor tissues. Thisfinding suggests that cytotoxic antibody therapy
using PMab-38 will be less damaging to normalorgans.
To the best of our knowledge, this is the firstreport of PMab-38 reactivity against various
canine tumors and systemic normal tissues. Cell
membranes of tumor cells were strongly stainedby PMab-38 in SCC and PAC, but most normal
cells were not stained. Remarkably, CAFs ofmost TCC and ASAC samples were extensively
stained by PMab-38. These results ofimmunohistochemical staining with PMab-38
indicate that dPDPN recognized by PMab-38
might be a target for tumor antigen targetingtherapy.

32

Immunohistochemical staining with PMab-38

Acknowledgements
This research was supported in part by AMED
under Grant Numbers: JP17am0301010 (Y.K.),
JP17am0101078 (Y.K.), and JP17ae0101028 (Y.K.),
and by JSPS KAKENHI Grant Number 17K07299(M.K.K.) and Grant Number 16K10748 (Y.K.).

Supplemental data
Supplemental data associated with thisarticle can be found, in the online version, at
http://dx.doi.org/10.14943/jjvr.67.1.25

References
1) Adachi Y, Nakamura H, Kitamura Y,
Taniguchi Y, Araki K, Shomori K, Horie Y,
Kurozawa Y, Ito H, Hayashi K. Limphatic
vessel density in pulmonary adenocarcinoma
immunohistochemically evaluated with
anti-podoplanin or anti-D2-40 antibody iscorrelated with lymphatic invasion or lymph
node metastases. Pathol Int 57, 171-177,
20072) Ariizumi T, Ogose A, Kawashima H, Hotta T,
Li G, Xu Y, Umezu H, Sugai M, Endo N.
Expression of podoplanin in human bone andbone tumors: New marker of osteogenic andchondrogenic bone tumors. Pathol Int 60,
193-202, 2010
3) Astarita JL, Cremasco V, Fu J, Darnell MC,
Peck JR, Nieves-Bonilla JM, Song K, Kondo
Y, Woodruff MC, Gogineni A, Onder L,
Ludewig B, Weimer RM, Carroll MC,
Mooney DJ, Xia L, Turley SJ. The CLEC-2podoplanin axis controls the contractility offibroblastic reticular cells and lymph node
microarchitecture. Nat Immunol 16, 75-84,
20154) 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,
Kahn ML. Platelets regulate lymphatic
vascular development through CLEC-2-SLP-76
signaling. Blood 116, 661-670, 2010
5) Breiteneder-Geleff S, Matsui K, Soleiman A,

Meraner P, Poczewski H, Kalt R, Schaffner
G, Kerjaschki D. Podoplanin, Novel 43-kd
Membrane Protein of Glomerular Epithelial
Cells, is Down-Regulated in Puromycin
Nephrosis. Am J Pathol 151, 1141-1152, 1997
6) Brooks MB, Matus RE, Leifer CE, Alfieri
AA, Patnaik AK. Chemotherapy versus
chemotherapy plus radiotherapy in thetreatment- of tonsillar squamous cell
carcinoma in the cog. J Vet Intern Med 2,
206-211, 1988
7) Chang YW, Yamada S, Kaneko MK, Kato Y.
Epitope mapping of monoclonal antibody
PMab-38 against dog podoplanin. Monoclon
Antib Immunodiagn Immunother 36, 291295, 20178) Cueni LN, Hegyi I, Shin JW, Albinger-Hegyi
A, Gruber S, Kunstfeld R, Moch H, Detmar
M. Tumor lymphangiogenesis and metastasis
to lymph nodes induced by cancer cell
expression of podoplanin. Am J Pathol 177,
1004-1016, 20109) Honma R, Kaneko MK, Ogasawara S, Fujii
Y, Konnai S, Takagi M, Kato Y. Specificdetection of dog podoplanin expressed inrenal glomerulus by a novel monoclonal
antibody PMab-38 in immunohistochemistry.
Monoclon Antib Immunodiagn Immunother35, 212-216, 201610) Kan S, Konishi E, Arita T, Ikemoto C,
Takenaka H, Yanagisawa A, Katoh N, Asai J.
Podoplanin expression in cancer-associatedfibroblasts predicts aggressive behavior inmelanoma. J Cutan Pathol 41, 561-567,
201411) Kaneko MK, Honma R, Ogasawara S, Fujii
Y, Nakamura T, Saidoh N, Takagi M,
Kagawa Y, Konnai S, Kato Y. PMab-38
recognizes canine podoplanin of squamouscell carcinomas. Monoclon Antib Immunodiagn
Immunother 35, 263-266, 201612) Kato Y, Kaneko MK. A cancer-specificmonoclonal antibody recognizes theaberrantly glycosylated podoplanin. Sci Rep
4, 5924, 201413) Kato Y, Kaneko MK, Kunita A, Ito H,
Kameyama A, Ogasawara S, Matsuura N,
Hasegawa Y, Suzuki-Inoue K, Inoue O,
Ozaki Y, Narimatsu H. Molecular analysis of
the pathophysiological binding of the platelet
aggregation-inducing factor podoplanin to
the C-type lectin-like receptor CLEC-2.
Cancer Sci 99, 54-61, 2008
14) Kawase A, Ishii G, Nagai K, Ito T, Nagano T,
Murata Y, Hishida T, Nishimura M, YoshidaJ, Suzuki K, Ochiai A. Podoplanin expression

Kohei Kiname et al.

by cancer associated fibroblasts predicts poor
prognosis of lung adenocarcinoma. Int J
Cancer 123, 1053-1059, 2008
15) Kitano H, Kageyama S, Hewitt SM, Hayashi
R, Doki Y, Ozaki Y, Fujino S, Takikita M,
Kubo H, Fukuoka J. Podoplanin expression incancerous stroma induces lymphangiogenesis
and predicts lymphatic spread and patient
survival. Arch Pathol Lab Med 134, 15201527, 201016) Król M, Pawłowski KM, Szyszko K,
Maciejewski H, Dolka I, Manuali E, Jank M,
Motyl T. The gene expression profiles ofcanine mammary cancer cells grown withcarcinoma-associated fibroblasts (CAFs) as a
co-culture in vitro. BMC Vet Res 8, 35, 201217) Kunita A, Kashima TG, Morishita Y,
Fukayama M, Kato Y, Tsuruo T, Fujita N.
The platelet aggregation-inducing factor
aggrus/podoplanin promotes pulmonary
metastasis. Am J Pathol 170, 1337-1347,
200718) Liotta LA, Kohn EC. The microenvironmentof the tumour-host interface. Nature 411,
375-379, 200119) McLaughlin P, Grillo-López AJ, Link BK,
Levy R, Czuczman MS, Williams ME,
Heyman MR, Bence-Bruckler I, White CA,
Cabanillas F, Jain V, Ho AD, Lister J, Wey
K, Shen D, Dallaire BK. Rituximab chimericanti-CD20 monoclonal antibody therapy forrelapsed indolent lymphoma: half of patients
respond to a four-dose treatment program. J
Clin Oncol 16, 2825-2833, 199820) Mertens JC, Fingas CD, Christensen JD,
Smoot RL, Bronk SF, Werneburg NW,
Gustafson MP, Dietz AB, Roberts LR,
Sirica AE, Gores GJ. Therapeutic effects ofdeleting cancer-associated fibroblasts incholangiocarcinoma. Cancer Res 73, 897-907,
201321) Mishima K, Kato Y, Kaneko MK, Nakazawa
Y, Kunita A, Fujita N, Tsuruo T, Nishikawa
R, Hirose T, Matsutani M. Podoplanin
expression in primary central nervous systemgerm cell tumors: A useful histologicalmarker for the diagnosis of germinoma. Acta
Neuropathol 111, 563-568, 200622) Neri S, Ishii G, Hashimoto H, Kuwata T,
Nagai K, Date H, Ochiai A. Podoplaninexpressing cancer-associated fibroblasts leadand enhance the local invasion of cancer cells
in lung adenocarcinoma. Int J Cancer 137,
784-796, 201523) Ogasawara S, Honma R, Kaneko MK, Fujii
Y, Kagawa Y, Konnai S, Kato Y. Podoplanin33

expression in canine melanoma. Monoclon
Antib Immunodiagn Immunother 35, 304306, 201624) Ohshio Y, Teramoto K, Hanaoka J, Tezuka
N, Itoh Y, Asai T, Daigo Y, Ogasawara K..
Cancer-associated fibroblast-targeted strategy
enhances antitumor immune responses indendritic cell-based vaccine. Cancer Sci 106,134-142, 201525) Ordóñez NG. Podoplanin: A novel diagnostic
immunohistochemical marker. Adv Anat
Pathol13, 83-88, 2006
26) Redman JM, Hill EM, AlDeghaither D,
Weiner LM. Mechanisms of action oftherapeutic antibodies for cancer. Mol
Immunol 67, 28-45, 201527) Retzbach EP, Sheehan SA, Nevel EM, Batra
A, Phi T, Nguyen TP, Kato Y, Baredes S,
Fatahzadeh M, Shienbaum AJ, Goldberg GS.
Podoplanin emerges as a functionally relevant
oral cancer biomarker and therapeutic target.
Oral Oncol 78, 126-136, 201828) Schacht V, Dadras SS, Johnson LA, Jackson
DG, Hong YK, Detmar M. Up-regulation of
the lymphatic marker podoplanin, a mucintype transmembrane glycoprotein, in humansquamous cell carcinomas and germ celltumors. Am J Pathol 166, 913-921, 200529) Schmidt BR, Glickman NW, DeNicola DB, deGortari AE, Knapp DW. Evaluation of
piroxicam for the treatment of oral squamouscell carcinoma in dogs. J Am Vet Med Assoc
218, 1783-1786
30) Schoppmann SF, Berghoff A, Dinhof C,
Jakesz R, Gnant M, Dubsky P, Jesch B,
Heinzl H, Birner P. Podoplanin-expressing
cancer-associated fibroblasts are associatedwith poor prognosis in invasive breast cancer.
Breast Cancer Res Treat 134, 237-244, 201231) Schor SL, Schor AM. Clonal heterogeneityin fibroblast phenotype: implications forthe control of epithelial-mesenchymal
interactions. Bioessays 7, 200-204, 198732) Tuccillo FM, de Laurentiis A, Palmieri C,
Fiume G, Bonelli P, Borrelli A, Tassone P,
Scala I, Buonaguro FM, Quinto I, Scala G.
Aberrant glycosylation as biomarker forcancer: focus on CD43. Biomed Res Int 2014,
742831, 2014
33) Uhrin P, Zaujec J, Breuss JM, Olcaydu D,
Chrenek P, Stockinger H, Fuertbauer E,
Moser M, Haiko P, Fässler R, Alitalo K,
Binder BR, Kerjaschki D. Novel function
for blood platelets and podoplanin indevelopmental separation of blood andlymphatic circulation. Blood 115, 3997-4005,

34

Immunohistochemical staining with PMab-38

201034) Vogel CL, Franco SX. Clinical experience
with trastuzumab (Herceptin). Breast J 9,
452-462, 2003
35) Wicki A, Lehembre F, Wick N, Hantusch B,
Kerjaschki D, Christofori G. Tumor invasionin the absence of epithelial-mesenchymaltransition: Podoplanin-mediated remodelingof the actin cytoskeleton. Cancer Cell 9,261-272, 200636) Xing F, Saidou J, Watabe K. Cancer
associated fibroblasts (CAFs) in tumor
microenvironment. Front Biosci (LandmarkEd) 15, 166-179, 201037) Yamada S, Itai S, Kaneko MK, Kato Y.
PMab-48 recognizes dog podoplanin oflymphatic endothelial cells. Monoclon AntibImmunodiagn Immunother 37, 63-66, 201838) Yamanashi T, Nakanishi Y, Fujii G, AkishimaFukasawa Y, Moriya Y, Kanai Y, Watanabe
M, Hirohashi S. Podoplanin expression
identified in stromal fibroblasts as a favorable
prognostic marker in patients with colorectal
carcinoma. Oncology 77, 53-62. 200939) Yoshimoto S, Hoshino Y, Izumi Y, Takagi S.
α-Smooth muscle actin expression in cancerassociated fibroblasts in canine epithelial
tumors. Jpn J Vet Res 65, 135-144, 2017