Heliyon 5 (2019) e02063

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PMab-235: A monoclonal antibody for immunohistochemical analysis
against goat podoplanin
Yoshikazu Furusawa a, b, c, Shinji Yamada a, Takuro Nakamura a, Masato Sano a, Yusuke Sayama a,
Shunsuke Itai a, d, Junko Takei a, d, Hiroyuki Harada d, Masato Fukui c, Mika K. Kaneko a,
Yukinari Kato a, b, *a

Department of Antibody Drug Development, Tohoku University Graduate School of Medicine, 2-1 Seiryo-machi, Aoba-ku, Sendai, Miyagi, 980-8575, JapanNew Industry Creation Hatchery Center, Tohoku University, 2-1 Seiryo-machi, Aoba-ku, Sendai, Miyagi, 980-8575, JapancZENOAQ RESOURCE CO., LTD., 1-1 Tairanoue, Sasagawa, Asaka-machi, Koriyama, Fukushima, 963-0196, Japand
Department of Oral and Maxillofacial Surgery, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, 1-5-45, Yushima, Bunkyo-ku,
Tokyo, 113-8510, Japanb

A R T I C L E I N F O

A B S T R A C T

Keywords:
Lymphatic endothelial cells
PMab-235
Goat podoplanin
PDPN

Sensitive and speciﬁc monoclonal antibodies (mAbs) against not only human but also mouse, rat, rabbit, dog, cat,bovine, pig, and horse podoplanins (PDPNs) have been established in our previous studies. However, anti-goat
PDPN (gPDPN) has not been established yet. PDPN has been utilized as a lymphatic endothelial cell marker
especially in pathological diagnoses; therefore, mAbs for immunohistochemical analyses using formalin-ﬁxed
parafﬁn-embedded tissues are needed. Although we recently demonstrated that an anti-bovine PDPN mAb,
PMab-44 cross-reacted with gPDPN, PMab-44 did not detect lymphatic endothelial cells in immunohistochemistry. In this study, we immunized mice with gPDPN-overexpressing Chinese hamster ovary (CHO)–K1 (CHO/
gPDPN) cells, and screened mAbs against gPDPN using ﬂow cytometry. One of the mAbs, PMab-235 (IgG1,
kappa), speciﬁcally detected CHO/gPDPN cells by ﬂow cytometry. Furthermore, PMab-235 strongly detected lungtype I alveolar cells, renal podocytes, and lymphatic endothelial cells of colon by immunohistochemistry. Theseﬁndings suggest that PMab-235 may be useful as a lymphatic endothelial cell marker for goat tissues.

1. Introduction
Podoplanin (PDPN)/T1alpha/Aggrus is a type I transmembrane sialoglycoprotein, which is expressed in many cell types, such as pulmonary
type I alveolar cells, renal podocytes, mesothelial cells, and epithelial
cells or lymphatic endothelial cells of many organs [1]. PDPN could
induce platelet aggregation by binding to the endogenous receptor of
PDPN, C-type lectin-like receptor-2 (CLEC-2) [2]. PDPN has been used to
distinguish lymphatic endothelial cells from vascular endothelial cells inpathophysiological studies [3]. The PDPN-CLEC-2 interaction facilitates
the separation of lymphatic vessels and embryonic blood [4]. Our previous analyses of glycopeptides produced by Edman degradation andmass spectrometry demonstrated that the disialyl-core1 (NeuAcα2-3Gal
β1-3(NeuAcα2-6)GalNAcα1-O-Thr) structure was attached to a glycosylation site at residue Thr52 of human PDPN (hPDPN) [5]. Sialic
acid-deﬁcient PDPN recovered its activity after additional sialylation,
indicating that the sialylated core1 of Thr52 is critical for PDPN-induced

platelet aggregation.
The expression of hPDPN has been reported in many malignant tumors, including malignant oral squamous cell carcinomas [6], lungsquamous cell carcinomas [7], esophageal squamous cell carcinomas [8],malignant mesotheliomas [9], osteosarcomas [10], chondrosarcomas[11], brain tumors [12], and seminomas of testicular tumors [13]. Our
previous studies demonstrated that PDPN expression in Chinese hamster
ovary (CHO)–K1 cells promoted pulmonary metastasis in both anexperimental and a spontaneous mouse model [14]. No differences in thesize of metastatic foci or in primary tumor growth were found in either
set of mice. PDPN-expressing cells, which were covered with platelets,
were found to be arrested in the lung microvasculature 30 minutes after
injection. Furthermore, lung metastasis resulting from PDPN expression
decreased the survival of the mice. We showed that point mutation at theplatelet aggregation-stimulating (PLAG) domain of hPDPN lost bothplatelet aggregation and metastasis. Inhibition of platelets with aspirin
reduced the formation of PDPN-promoted metastasis, indicating that

* Corresponding author.
E-mail addresses: yukinarikato@med.tohoku.ac.jp, yukinari-k@bea.hi-ho.ne.jp (Y. Kato).
https://doi.org/10.1016/j.heliyon.2019.e02063
Received 18 February 2019; Received in revised form 10 May 2019; Accepted 5 July 20192405-8440/© 2019 The Author(s). Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

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Heliyon 5 (2019) e02063

2. Materials and methods

Lipofectamine LTX with Plus Reagent (Thermo Fisher Scientiﬁc Inc.,
Waltham, MA, USA). Stable transfectants were selected by limiting
dilution and cultivation in a medium containing 0.5 mg/ml of G418
(Nacalai Tesque, Inc., Kyoto, Japan).
The P3U1, CHO–K1, CHO/gPDPN, CHO/hPDPN [33], CHO/mouse
PDPN (mPDPN) [33], CHO/rat PDPN (rPDPN) [16], CHO/rabbit PDPN
(rabPDPN) [17], CHO/dPDPN [19], CHO/bovPDPN [18], CHO/cPDPN[20], CHO/pPDPN [21], CHO/horPDPN [22], CHO/tigPDPN [25],CHO/aPDPN [24], CHO/bPDPN [26], CHO/tasPDPN [23], CHO/sheep
PDPN (sPDPN) [34], and CHO/whale PDPN (wPDPN) [35] were cultured
in a Roswell Park Memorial Institute (RPMI) 1640 medium (Nacalai
Tesque, Inc.), supplemented with 10% of heat-inactivated fetal bovine
serum (FBS; Thermo Fisher Scientiﬁc Inc.), 100 units/mL of penicillin,
100 μg/mL of streptomycin, and 25 μg/mL of amphotericin B (Nacalai
Tesque, Inc.). Ch 1 Es was cultured in E-MEM with Earle's Salts, L-Gln and
Non-Essential Amino Acids (Nacalai Tesque, Inc.), supplemented with
10% of heat-inactivated FBS (Thermo Fisher Scientiﬁc Inc.), 100
units/mL of penicillin, and 100 μg/mL of streptomycin (Nacalai Tesque,Inc.). The cells were grown in an incubator at 37  C with humidity and
5% CO2 and 95% air atmosphere.

2.1. Cell lines

2.2. Hybridoma production

Ch 1 Es, a ﬁbroblastic cell line from normal fetal goat esophagus was
obtained from the Japanese Collection of Research Bioresources Cell
Bank (Osaka, Japan). P3X63Ag8U.1 (P3U1) and CHO–K1 cells wereobtained from the American Type Culture Collection (ATCC; Manassas,
VA, USA). Synthesized DNA (Euroﬁns Genomics KK, Tokyo, Japan)
encoding gPDPN (accession No.: XM_005690821.3) plus an N-terminal
MAP16 tag (PGTGDGMVPPGIEDKI), which is recognized by an antiMAP16 tag mAb (PMab-1: the same mAb against MAP tag [32]),
was subcloned into a pCAG-Neo vector (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan). Plasmids were transfected using

Female BALB/c mice (6 weeks of age) were purchased from CLEA
Japan (Tokyo, Japan). The animals were housed under speciﬁc pathogenfree conditions. The Animal Care and Use Committee of Tohoku University approved all animal experiments. We used a Cell-Based Immunization and Screening (CBIS) method [21, 22, 23, 24, 25] to develop
mAbs against gPDPN. Brieﬂy, one BALB/c mouse was immunized with
CHO/gPDPN cells (1  108) intraperitoneally (i.p.) together with the
Imject Alum (Thermo Fisher Scientiﬁc Inc.). The procedure included
three additional immunizations, followed by a ﬁnal booster injection
administered i.p. 2 days prior to the harvest of spleen cells. Subsequently,

PDPN contributes to the establishment of metastasis by promoting
platelet aggregation without affecting subsequent growth.
We have developed monoclonal antibodies (mAbs) against not only
human [12], mouse [15], rat [16], rabbit [17], but also bovine [18], dog[19], cat [20], pig [21], horse [22], Tasmanian devil [23], alpaca [24],tiger [25], and bear [26] PDPNs. About anti-hPDPN mAbs, we established cancer-speciﬁc mAbs (CasMabs), such as LpMab-2 [27] and
LpMab-23 [28,29]. An anti-dog PDPN (dPDPN), PMab-38 also showedcancer-speciﬁcity [19, 30, 31]. PMab-44 (an anti-bovine PDPN
(bovPDPN) mAb), PMab-52 (an anti-cat PDPN (cPDPN) mAb), PMab-213(an anti-pig PDPN (pPDPN) mAb), PMab-219 (an anti-horse PDPN
(horPDPN) mAb), PMab-233 (an anti-Tasmanian devil PDPN (tasPDPN)mAb), PMab-225 (an anti-alpaca PDPN (aPDPN) mAb), PMab-231 (ananti-tiger PDPN (tigPDPN) mAb), and PMab-247 (an anti-bear PDPN
(bPDPN) mAb) have been shown to be useful for ﬂow cytometry, Western
blot, and immunohistochemical analyses [18, 20, 21, 22, 23, 24, 25, 26].
In this study, we immunized mice with CHO/goat PDPN (gPDPN) cells
and established mAbs against gPDPN.

Fig. 1. Schematic illustration of the Cell-Based Immunization and Screening (CBIS) method. Stable transfectants expressing the protein of interest are used as an
immunogen with no puriﬁcation procedure. The selection of hybridomas secreting speciﬁc mAbs is performed by ﬂow cytometry using parental and transfectant cells.
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2.5. Immunohistochemical analyses

these spleen cells were fused with P3U1 cells using PEG1500 (Roche
Diagnostics, Indianapolis, IN, USA), and the hybridomas were grown inan RPMI medium supplemented with hypoxanthine, aminopterin, andthymidine (HAT) for selection (Thermo Fisher Scientiﬁc Inc.). The culture supernatants were screened by ﬂow cytometry.

Goat tissues were collected in NIPPON ZENYAKU KOGYO CO., LTD.
(Fukushima, Japan), ﬁxed in 4%-Paraformaldehyde Phosphate Buffer
Solution (Nacalai Tesque, Inc.). Histological sections (4-μm thick) weredirectly autoclaved in a citrate buffer (pH 6.0; Nichirei Biosciences, Inc.,Tokyo, Japan) for 20 min, and were blocked using the SuperBlock T20(PBS) Blocking Buffer (Thermo Fisher Scientiﬁc Inc.), incubated with
PMab-235 (1 μg/mL) or PMab-44 (1 μg/mL) for 1 h at the room temperature, and then treated with the Envision þ Kit for mouse (Agilent
Technologies Inc. Santa Clara, CA, USA) for 30 min. Color was developed
using 3,30 -diaminobenzidine tetrahydrochloride (DAB; Agilent Technologies Inc.) for 2 min, and counterstaining was performed using hematoxylin (FUJIFILM Wako Pure Chemical Corporation).

2.3. Flow cytometry
The cells were harvested following a brief exposure to 0.25% trypsin
and 1 mM ethylenediaminetetraacetic acid (EDTA; Nacalai Tesque, Inc.).
The cells were washed with 0.1% bovine serum albumin (BSA) in
phosphate-buffered saline (PBS) and treated with primary mAbs for 30
min at 4  C. Thereafter, the cells were treated with Alexa Fluor 488-conjugated anti-mouse IgG (1:2000; Cell Signaling Technology, Inc., Danvers, MA, USA) or Oregon Green anti-rat IgG (1:2000; Thermo Fisher
Scientiﬁc Inc.). Then, ﬂuorescence data were collected using the SA3800
Cell Analyzer (Sony Corp., Tokyo, Japan).

3. Results
One mouse was immunized with CHO/gPDPN cells (Fig. 1). The
developed hybridomas were seeded into 96-well plates and cultivated for
10 days. Wells positive for CHO/gPDPN and negative for CHO–K1 wereselected by ﬂow cytometry. The screening approach identiﬁed strong
signals from CHO/gPDPN cells and weak or no signals from CHO–K1
cells in 29 of the 480 wells (6.0%). PMab-235 (IgG1, kappa) was ﬁnallyselected via ﬂow cytometry against Ch 1 Es, a ﬁbroblastic cell line fromnormal fetal goat esophagus (Fig. 2). PMab-235 recognized CHO/gPDPN
cells, but showed no reaction with CHO–K1 cells, as assessed by ﬂow
cytometry (Fig. 2). As a positive control, an anti-MAP16 tag (clone PMab-2.4. Determination of binding afﬁnity by ﬂow cytometry
CHO/gPDPN was suspended in 100 μL of serially diluted PMab-235.
Then, Alexa Fluor 488-conjugated anti-mouse IgG (1:200; Cell Signaling
Technology, Inc.) was added. Fluorescence data were collected using theEC800 Cell Analyzer (Sony Corp.). The dissociation constant (KD) was
calculated by ﬁtting the binding isotherms to built-in one-site binding
models in the GraphPad PRISM 6 (GraphPad Software, Inc., La Jolla, CA,
USA).

Fig. 2. Detection of gPDPN by ﬂow cytometry using PMab-235. CHO/gPDPN, CHO–K1, and Ch 1 Es cells were treated with PMab-235 (red line) or anti-MAP16 tag(PMab-1; blue line) at a concentration of 1 μg/mL or 0.1% BSA in PBS (gray) for 30 min, followed by incubation with secondary antibodies.
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Heliyon 5 (2019) e02063

Fig. 3. Cross-reaction of PMab-235 to PDPNs of the other species by ﬂow cytometry. CHO–K1 cells transfected with PDPNs of the other species were treated with
PMab-235 (red line) or each positive control (blue line) at a concentration of 1 μg/mL or 0.1% BSA in PBS (gray) for 30 min, followed by incubation with secondary antibodies.

4Y. Furusawa et al.

Heliyon 5 (2019) e02063(GPGDDMVNPGLEDRIE) mAb; clone LpMab-17 as an anti-LP tag(NSVTGIRIEDLPTSES) mAb; clone LpMab-7 as an anti-RIEDL tag (RIEDL)
mAb; and clone PMab-44 as an anti-BAP tag (EKTTLGVEDYTTTPAA)
mAb. All positive control mAbs showed high reactivity for each
PDPN-overexpressed CHO cell lines, such as human, mouse, rat, rabbit,
dog, bovine, cat, pig, horse, Tasmanian devil, sheep, alpaca, tiger, whale,or bear PDPNs by ﬂow cytometry (Fig. 3). PMab-235 crossreacted with
only bovine PDPN, and did not react with human, mouse, rat, rabbit, dog,
cat, pig, horse, Tasmanian devil, sheep, alpaca, tiger, whale, or bear
PDPNs by ﬂow cytometry (Fig. 3).
In addition, kinetic analysis conducted by ﬂow cytometry was
employed to assess the interaction of PMab-235 with CHO/gPDPN cells.
KD of PMab-235 for CHO/gPDPN cells was determined to be 1.5  1081) reacted with CHO/gPDPN.
We next investigated the crossreactivity of PMab-235 with PDPNs of
the other species. PDPN expression levels in CHO cell lines wereconﬁrmed by positive controls, such as anti-PDPN mAbs or anti-peptide
tag mAbs. We used clone LpMab-12 [36] as an anti-hPDPN mAb; clone
PMab-1 [15] as an anti-mPDPN mAb; clone PMab-2 [16] as ananti-rPDPN mAb; clone PMab-32 [17] as an anti-rabPDPN mAb; clone
PMab-38 [19] as an anti-dPDPN mAb; clone PMab-44 [18] as ananti-bovPDPN mAb; clone PMab-52 [20] as an anti-cPDPN mAb; clone
PMab-210 [35] as an anti-pPDPN mAb; and clone PMab-202 [37] as ananti-horPDPN mAb. We also used clone NZ-1 as an anti-PA16 tag(GLEGGVAMPGAEDDVV) mAb; clone PMab-2 as an anti-RAP14 tag(DMVNPGLEDRIEDL) mAb; clone PMab-2 as an anti-RAP16 tag

Fig. 4. Immunohistochemical analyses for goat lung. Lung sections were incubated with 1 μg/mL of PMab-235 (A, B), 1 μg/mL of PMab-44 (C, D) or with blocking
buffer (E, F), followed by that with the Envision þ Kit. (G, H) Hematoxylin and eosin staining. Scale bar ¼ 100 μm.
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4. Discussion

M, indicating a moderate afﬁnity of PMab-235 for CHO/gPDPN cells.
The immunohistochemical analyses revealed that PMab-235 strongly
stained type I alveolar cells of lung (Fig. 4A and B), podocytes of kidney(Fig. 5A and B), and lymphatic endothelial cells of colon (Fig. 6A and B).
We used PMab-44 as a positive control to stain gPDPN because PMab-44
was shown to crossreact with gPDPN in our previous study [38].
Although PMab-44 strongly stained type I alveolar cells of lung (Fig. 4C
and D), it did not stain podocytes of kidney (Fig. 5C and D) and lymphatic
endothelial cells of colon (Fig. 6C and D). No staining was observed
without primary antibodies against type I alveolar cells of lung (Fig. 4E
and F), podocytes of kidney (Fig. 5E and F), and lymphatic endothelial
cells of colon (Fig. 6E and F).

In 1979, Magari et al. reported morphological studies on liver lymphatics in human, pig, calf, dog, rabbit, and goat [39]. They studied form,
distribution, and structure of liver lymphatics using both light andelectron microscopes. Further, Ezeasor et al. reported the distribution andcharacteristics of lymph vessels in caprine (goat) hemal nodes after
glutaraldehyde ﬁxation and epoxy resin embedding [40]. Because
anti-gPDPN mAbs, which are useful for immunohistochemical analysis todetect lymphatic endothelial cells, have not been reported, speciﬁc
detection of lymphatic endothelial cells was difﬁcult. Previously, we
immunized mice with a synthesized peptide of gPDPN, such as
which is corresponding to platelet
37-KNEQTTLGVEDYMT-49,

Fig. 5. Immunohistochemical analyses for goat kidney. Kidney sections were incubated with 1 μg/mL of PMab-235 (A, B), 1 μg/mL of PMab-44 (C, D) or with blocking
buffer (E, F), followed by that with the Envision þ Kit. (G, H) Hematoxylin and eosin staining. Scale bar ¼ 100 μm.
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Heliyon 5 (2019) e02063

Fig. 6. Immunohistochemical analyses for goat colon. Colon sections were incubated with 1 μg/mL of PMab-235 (A, B), 1 μg/mL of PMab-44 (C, D) or with blocking
buffer (E, F), followed by that with the Envision þ Kit. (G, H) Hematoxylin and eosin staining. Scale bar ¼ 100 μm.

(Fig. 4), podocytes of kidney (Fig. 5), and lymphatic endothelial cells ofcolon (Fig. 6), indicating that PMab-235 is useful for the detection ofgPDPN by immunohistochemistry. PMab-235 cross-reacted with bovine
PDPN not only in ﬂow cytometry (Fig. 3) but also in immunohistochemistry (data not shown). Unfortunately, PMab-235 did not react with
gPDPN in western blot analysis (data not shown). In conclusion, weestablished PMab-235 against gPDPN, which is suitable for use in ﬂow
cytometry and immunohistochemical analyses using CBIS method. The
epitope of PMab-235 needs further investigation to clarify the sensitivity
and speciﬁcity of PMab-235 against gPDPN.

aggregation-stimulating (PLAG) domain. Unfortunately, we could notestablish speciﬁc mAbs for immunohistochemical analysis against
gPDPN (data not shown). Although an anti-bovine PDPN mAb PMab-44
crossreacted with gPDPN in immunohistochemistry for goat lung tissues,
it did not react with lymphatic endothelial cells of goat tissues [38]. In
the present study, we employed the CBIS method to develop sensitive andspeciﬁc mAbs against gPDPN to facilitate the immunohistochemical
analysis of parafﬁn-embedded tissue sections. Established PMab-235
reacted with endogenous gPDPN of a ﬁbroblastic goat cell line as well
as CHO/gPDPN cells (Fig. 2). The immunohistochemical analyses
revealed that PMab-235 strongly stained type I alveolar cells of lung7

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Author contribution statement
Yoshikazu Furusawa: Performed the experiments; Wrote the paper.
Shinji Yamada, Takuro Nakamura: Performed the experiments.
Masato Sano, Shunsuke Itai, Junko Takei: Analyzed and interpreted
the data.
Hiroyuki Harada, Masato Fukui: Contributed reagents, materials,
analysis tools or data.
Mika K. Kaneko, Yukinari Kato: Conceived and designed the experiments; Wrote the paper.
Funding statement
Yukinari Kato was supported in part by AMED (Grant numbers:
JP18am0101078, JP18am0301010, and JP18ae0101028). Yukinari
Kato was supported by JSPS KAKENHI (Grant Number 19K07705). Mika
K.Kaneko was supported by JSPS KAKENHI (Grant Number 17K07299).
Competing interest statement
Yukinari Kato received research funding from ZENOAQ RESOURCE
CO., LTD.
Additional information
No additional information is available for this paper.
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