MONOCLONAL ANTIBODIES IN IMMUNODIAGNOSIS AND IMMUNOTHERAPY
Volume 36, Number 2, 2017
Mary Ann Liebert, Inc.
DOI: 10.1089/mab.2017.0008

PcMab-47:
Novel Antihuman Podocalyxin Monoclonal
Antibody for Immunohistochemistry
Satoshi Ogasawara,1,* Mika K. Kaneko,1,2,* Shinji Yamada,1,2 Ryusuke Honma,1 Takuro Nakamura,1,2
Noriko Saidoh,1,2 Miyuki Yanaka,1,2 Kanae Yoshida,1 Yuki Fujii,1 and Yukinari Kato1–3

Podocalyxin (PODXL) is a CD34-related sialomucin and a well-known marker of embryonic stem cells.
PODXL is expressed in many types of tumors including colorectal cancers, breast cancers, and brain tumors.
Overexpression of PODXL is an independent predictor of progression, metastasis, and poor outcome. PODXL
is also expressed in many normal cells such as renal podocytes and endothelial cells (ECs). However, highsensitive and high-specific anti-PODXL monoclonal antibodies (mAbs) have not been established. Herein, we
immunized mice with recombinant human PODXL, which was produced using LN229 glioblastoma cells. The
anti-PODXL mAb, PcMab-47, reacted with endogenous PODXL-expressing cancer cell lines and normal cells
independently of glycosylation in flow cytometry. Immunohistochemical analysis showed that PcMab-47 detected PODXL-expressing normal cells such as podocytes of kidney or ECs. Furthermore, PcMab-47 stained
PODXL-expressing cancer cells of colon or breast cancers. These results suggest that PcMab-47 could be usefulfor investigating the expression and function of PODXL in cancers and normal tissues.
Keywords: podocalyxin, PODXL, monoclonal antibody, immunohistochemistry
mucin domain, including sialic acid and keratan sulfate, for theformation and maintenance of the filtration slits between the
podocyte processes in the kidney.(12) In addition, PODXL
regulates cell morphology by associating with actin cytoskeletal proteins, such as Na+/H+ exchanger regulatory factor(NHERF)-1/2 and ezrin.(13,14) PODXL is also known as one ofthe pluripotent stem cell markers. The antigen of the monoclonal antibodies (mAbs), TRA-1-60 and TRA-1-81, is keratan
sulfate, which is attached to PODXL.(3) The ligand of the recombinant N-terminal domain of the lectin BC2L-C from
Burkholderia cenocepacia (rBC2LCN) is also reported as Oglycan of PODXL.(15)PODXL is known as a diagnostic marker and prognostic
indicator in several cancers, including brain tumors,(6,16)
prostate cancers,(17) testicular tumors,(2) renal cancers,(18) oral
cancers,(19) thyroid cancers,(20) bladder cancers,(21) breast
cancers,(22–24) ovarian cancers,(25) colorectal cancers,(26–29)
pancreatic cancers,(30,31) and gastric cancers.(32) The glycans on PODXL bind to P-/E-/L-selectin expressed onplatelets, endothelium, and leukocytes, respectively.(33–35)

Introduction

P

odocalyxin (PODXL), which is also known as PCLP,
MEP21, Gp200, Gp135, thrombomucin, GCTM2, TRA1-60 antigen, or TRA-1-81 antigen, is a type I transmembraneprotein with a molecular weight of 150,000–200,000.(1–3)
PODXL is expressed in normal tissues, including kidney,
heart, pancreas, and breast tissues, as well as in neurons,
ductal luminal cells, podocytes, endothelial cells (ECs),
oviductal luminal cells, and mesothelial cells; PODXL also
plays an important role in the development of tissues.(4)PODXL is similar to CD34, which is known as a hematopoietic stem cell marker.(5) A sialomucin, PODXL is highly
glycosylated with N-glycan, O-glycan, and keratan sulfate.(6)PODXL was first found in rat podocytes of the kidney,(7) and
its homologues have been reported in humans.(8,9)
PODXL has different functions in various tissues. PODXL
acts as an adhesive molecule to bind to platelets or vascularendothelial cells (VECs)(10,11); in contrast, PODXL functions
as an antiadhesive molecule through its negatively charged

Departments of 1Regional Innovation and 2Antibody Drug Development, Tohoku University Graduate School of Medicine, Sendai,
Japan.
3
New Industry Creation Hatchery Center, Tohoku University, Sendai, Japan.
*Contributed equally to this work.
ª Satoshi Ogasawara et al., 2017; Published by Mary Ann Liebert, Inc. This Open Access article is distributed under the terms of theCreative Commons License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction inany medium, provided the original work is properly credited.

50

PCMAB-47 AGAINST PODXL

These interactions enhance the formation of platelet–tumor–
leukocyte aggregates and tumor cell arrest in the microvasculature.(36) Therefore, the overexpression of PODXL in cancer isa potential target for antibody therapy.
In this study, we established the anti-PODXL mAb, PcMab47, for use in flow cytometry and immunohistochemistry.

51
Materials and Methods
Cell lines

LN229, Caco-2, MDA-MB-468, HEK-293T, Chinese
hamster ovary (CHO)-K1, glycan-deficient CHO cell lines(Lec1, Lec2, and Lec8), and P3U1 were obtained from the

FIG. 1. Specific detection of PODXL by PcMab-47 using flow cytometry. (A) CHO-K1 and CHO-K1/PODXL were treatedwith PcMab-47 (1 mg/mL; red) or control PBS (black) for 30 minutes at 4C, followed by treatment with antimouse IgG-Oregon
green. (B) LN229, LN229/PODXL, and PDIS-13 (LN229/PODXL-knockout cells) were treated with PcMab-47 (1 mg/mL; red)or control PBS (black) for 30 minutes at 4C, followed by treatment with antimouse IgG-Oregon green. (C) Caco-2, MDA-MB468, HEK-293T, and vascular endothelial cells were treated with PcMab-47 (1 mg/mL; red) or control PBS (black) for 30
minutes at 4C, followed by treatment with antimouse IgG-Oregon green. (D) Lec1 (N-glycan-deficient CHO)/PODXL, Lec2
(sialic acid-deficient CHO)/PODXL, Lec8 (galactose-deficient CHO)/PODXL, and Lec13 (fucose-deficient CHO)/PODXL
were treated with PcMab-47 (1 mg/mL; red) or control PBS (black) for 30 minutes at 4C, followed by treatment with antimouse
IgG-Oregon green. (E) PDIS-12 (N-glycan-deficient HEK-293T), PDIS-22 (sialic acid-deficient HEK-293T), PDIS-18(galactose-deficient HEK-293T), and PDIS-20 (N-glycan/sialic acid/galactose-deficient HEK-293T) were treated with PcMab47 (1 mg/mL; blue) or control PBS (black) for 30 minutes at 4C, followed by treatment with antimouse IgG-Oregon green.
Fluorescence data were collected using the Cell Analyzer EC800. PBS, phosphate buffered saline; PODXL, podocalyxin.

52

American Type Culture Collection (Manassas, VA). Human VECs were purchased from Cambrex (Walkersville,
MD). Lec13 was provided by Dr. Pamela Stanley.
LN229, Lec1, Lec2, Lec8, and Lec13 were transfectedwith PODXL plasmids, which included the ectodomain or
full length of PODXL, using Lipofectamine 2000 (Thermo
Fisher Scientific, Inc., Waltham, MA) according to the
manufacturer’s instructions. LN229/hPODXL-knockout(KO) cells (PDIS-13) were produced using CRISPR/Cas9
plasmids (Target ID: HS0000056763) against human
PODXL (Sigma-Aldrich, St. Louis, MO).
The cell lines HEK-293T/GnT-1-KO (PDIS-12), HEK293T/SLC35A1-KO (PDIS-22), HEK-293T/SLC35A2-KO(PDIS-18), and HEK-293T/GnT-1/SLC35A1/SLC35A2-KO(PDIS-20) were generated by transfecting TALEN or
CRISPR/Cas9 plasmids, which target hsMgat1 (Wako
Pure Chemical Industries Ltd., Osaka, Japan), SLC35A1(Target ID: HS0000168432; Sigma-Aldrich), and SLC35A2(Target ID: HS0000062603; Sigma-Aldrich), respectively,
using a Gene Pulser Xcell electroporation system, and werescreened using lectin profiling. These glycan-deficient celllines are available from Cell Bank of Kato’s Lab (www.medtohoku-antibody.com/topics/001_paper_cell.htm) in Tohoku
University (Miyagi, Japan).
CHO-K1, Lec1, Lec2, Lec8, Lec13, CHO-K1/PODXL,
Lec1/PODXL, Lec2/PODXL, Lec8/PODXL, Lec13/PODXL,and P3U1 were cultured in RPMI 1640 medium, including lglutamine (Nacalai Tesque, Inc., Kyoto, Japan). l-Proline(0.04 mg/mL) was added for Lec1, Lec2, Lec8, and Lec13.
LN229, LN229/PODXL, LN229/ectodomain-PODXL, PDIS13, HEK-293T, Caco-2, MDA-MB-468, PDIS-12, PDIS-22,
PDIS-18, and PDIS-20 were cultured in Dulbecco’s modified
Eagle’s medium, including l-glutamine (Nacalai Tesque,Inc.), supplemented with 10% heat-inactivated fetal bovine

OGASAWARA ET AL.

serum (FBS; Thermo Fisher Scientific, Inc.) at 37C in a humidified atmosphere of 5% CO2 and 95% air. G418 (0.5 mg/mL;
Wako Pure Chemical Industries Ltd.) was added for CHOK1/PODXL, Lec1/PODXL, Lec2/PODXL, Lec8/PODXL,
Lec13/PODXL, LN229/PODXL, and LN229/ectodomainPODXL. VECs were cultured in EC medium EGM-2 MV
supplemented with 5% FBS (Cambrex Corp.). Antibiotics,
including 100 U/mL of penicillin, 100 mg/mL of streptomycin, and 25 mg/mL of amphotericin B (Nacalai Tesque, Inc.),
were added to all media.
Hybridoma production

Four-week-old female BALB/c mice (CLEA, Tokyo,
Japan) were immunized by intraperitoneal (i.p.) injection of
the purified ectodomain of human PODXL (100 mg) together
with Imject Alum (Thermo Fisher Scientific, Inc.). Afterseveral additional immunizations, a booster i.p. injection of
LN229/PODXL was given 2 days before the mice were euthanized by cervical dislocation, and spleen cells were harvested. The spleen cells were fused with P3U1 cells using
PEG1500 (Roche Diagnostics, Indianapolis, IN). Hybridomas
were grown in RPMI 1640 medium including l-glutaminewith hypoxanthine, aminopterin, and thymidine selection
medium supplement (Thermo Fisher Scientific, Inc.). Culture
supernatants were screened using enzyme-linked immunosorbent assay (ELISA) for binding to the purified ectodomain
of PODXL. Proteins were immobilized on Nunc Maxisorp 96well immunoplates (Thermo Fisher Scientific, Inc.) at 1 mg/mL
for 30 minutes. After blocking with 1% bovine serum albumin(BSA) in 0.05% Tween20/phosphate buffered saline (PBS;
Nacalai Tesque, Inc.), the plates were incubated with culture
supernatant followed by 1:2000 diluted peroxidase-conjugatedantimouse IgG (Agilent Technologies, Inc., Santa Clara, CA).

FIG. 2. Immunohistochemical analysis of human kidney by PcMab-47. A section of kidney was incubated with 1 mg/mL of
PcMab-47, followed by the Envision+ kit (A, B). Color was developed using 3, 3-diaminobenzidine tetrahydrochloride, andcounterstained with hematoxylin. Another section of kidney was also stained using hematoxylin and eosin (C, D). Scale bar: 100 mm.

PCMAB-47 AGAINST PODXL

53

The enzymatic reaction was produced with a 1-Step Ultra
TMB-ELISA (Thermo Fisher Scientific, Inc.). The opticaldensity was measured at 655 nm using an iMark microplate
reader (Bio-Rad Laboratories Inc., Hercules, CA).

for 30 minutes at 4C, followed by treatment with Oregon
Green 488 goat antimouse IgG (Thermo Fisher Scientific,Inc.). Fluorescence data were collected using the Cell Analyzer EC800 (Sony Corp., Tokyo, Japan).

Flow cytometry

Immunohistochemical analyses

Cell lines were harvested by brief exposure to 0.25%
Trypsin/1 mM EDTA (Nacalai Tesque, Inc.). After washingwith 0.1% BSA in PBS, cells were treated with primary mAbs

Human normal tissues and cancer tissues were purchasedfrom BioChain Institute, Inc. (Newark, CA) and US Biomax,
Inc. (Rockville, MD), respectively. Four-micrometer-thick

FIG. 3. Immunohistochemical analysis of human cancers by PcMab-47. Sections of colon cancer (A, B) and breast cancer(C, D) were incubated with 10 mg/mL of PcMab-47, followed by the Envision+ kit. Color was developed using 3,3-diaminobenzidine tetrahydrochloride and counterstained with hematoxylin. Colon cancer (E, F) and breast cancer (G, H)
were also stained using hematoxylin and eosin. Arrows: endothelial cells. Scale bar: 100 mm.

54

histological sections were deparaffinized in xylene and rehydrated. After the antigen retrieval procedure (autoclave
using citrate buffer, pH 6.0; Agilent Technologies, Inc.),
sections were incubated with 1 or 10 mg/mL of PcMab-47 for
1 or 3 hours at room temperature followed by treatment withEnvision+ kit (Agilent Technologies, Inc.) for 30 minutes.
Color was developed using 3, 3-diaminobenzidine tetrahydrochloride (Agilent Technologies, Inc.) for 5 minutes,
and then the sections were counterstained with hematoxylin(Wako Pure Chemical Industries Ltd.).
Results and Discussion

In our previous study, we developed the original technology
for production of cancer-specific monoclonal antibodies(CasMabs) against membranous proteins.(37) We successfully produced the antipodoplanin (PDPN) CasMab clone
LpMab-2 that specifically recognizes cancer-type PDPN in
tumor tissues and not in normal-type PDPN, which is expressed in lymphatic vessels.(37) Moreover, the CasMab
technology is useful for generating antiglycopeptide mAbs
(GpMabs). We have produced the following anti-PDPN
GpMabs: LpMab-3, LpMab-9, LpMab-12, LpMab-19,
and LpMab-21.(38–40) In addition, the CasMab technology
can generate mAbs that bind to various novel epitopes of
PDPN. We have produced the following anti-PDPN mAbs:
LpMab-7, LpMab-10, and LpMab-17, which recognize
novel epitopes.(37,40–42) This study describes the production of sensitive and specific anti-PODXL mAbs using the
CasMab technology.
Herein, we immunized mice with recombinant PODXL,
which was purified from the culture supernatant of LN229/
ectodomain-PODXL. A booster i.p. injection of LN229/
PODXL was administered. The culture supernatants werescreened using ELISA for binding to purified PODXL. As asecond screening, we performed flow cytometry for reaction
with LN229 and LN229/PODXL. A stronger reaction against
LN229/PODXL than against LN229 was necessary, and one
clone, PcMab-47 (mouse IgG1, kappa), was produced after
limiting dilution.
PcMab-47 reacted with CHO-K1/PODXL, and did not reactwith CHO (human PODXL-negative cell) in flow cytometry(Fig. 1A). PcMab-47 recognized endogenous PODXL, whichis expressed in LN229 (a glioblastoma cell line), whereas it did
not react with LN229/PODXL-KO cells (PDIS-13) (Fig. 1B),
indicating that PcMab-47 is specific against PODXL. The reaction of PcMab-47 against LN229/PODXL was higher thanthat against LN229 (Fig. 1B).
PODXL is expressed in many cancers, such as breast
cancers(22–24) and colorectal cancers.(26–29) We performed
flow cytometry using several types of cancer cells and normal
cells. PcMab-47 reacted with the cancer cell lines, such as
Caco-2 (colon adenocarcinoma) and MDA-MB-468 (breast
cancer) (Fig. 1C). PODXL is also expressed in normal tissuesof kidney and ECs.(4) PcMab-47 reacted with the normal celllines, including HEK-293T (renal epithelial cell) and with
vascular VECs (Fig. 1C). These results show the usefulnessof PcMab-47 for detecting PODXL in flow cytometry.
We next investigated whether the epitope of PcMab-47
includes N-glycan or O-glycan because PODXL is highly
glycosylated (Fig. 1D, E). As shown in Figure 1D, PcMab-47
reacted with all glycan-deficient PODXL-expressing CHO

OGASAWARA ET AL.

cells, specifically Lec1/PODXL (N-glycan-deficient), Lec2/
PODXL (sialic acid-deficient), Lec8/PODXL (galactosedeficient), and Lec13/PODXL (fucose-deficient). Furthermore, PcMab-47 reacted with all glycan-deficient HEK-293T
cells such as PDIS-12 (N-glycan-deficient), PDIS-22 (sialic
acid-deficient), PDIS-18 (galactose-deficient), and PDIS-20(N-glycan/sialic acid/galactose-deficient), indicating thatglycans are not included in the PcMab-47 epitope (Fig. 1E).
Next, we investigated the immunohistochemical utility of
PcMab-47 in normal tissues or cancers. As shown in Figure 2,
PcMab-47 stained podocytes or ECs of kidney (Fig. 2A, B).
PcMab-47 reacted with colon adenocarcinoma, in which
membrane/cytoplasmic-staining pattern was observed (Fig. 3A,
B). Strong expression of PODXL was also observed in ECs incolon adenocarcinoma (Fig. 3B). In contrast, weak expressionof PODXL was observed in cancer cells, but stronger expression was detected in ECs in breast cancer (Fig. 3C, D). These
results indicate that PcMab-47 is very useful for immunohistochemical detection of both cancer cells and normal cells that
express PODXL.
Therefore, PcMab-47 may facilitate investigation of theexpression and function of PODXL in cancer and normal
tissues. Future investigations should include production ofdifferent epitope-possessing anti-PODXL mAbs to elucidate
the function of PODXL.
Acknowledgments

This work was supported, in part, by the Basic Scienceand Platform Technology Program for Innovative Biological Medicine from Japan Agency for Medical research anddevelopment, AMED (Y.K.), by the Translational Research
Network Program from AMED (Y.K.), by the Platform for
Drug Discovery, Informatics, and Structural Life Science(PDIS) from AMED (Y.K.), by project for utilizing glycans
in the development of innovative drug discovery technologies from AMED (Y.K.), by the Regional Innovation
Strategy Support Program from the Ministry of Education,
Culture, Sports, Science and Technology (MEXT) of Japan(Y.K.), and by JSPS KAKENHI Grant Number 26440019(M.K.K.) and 16K10748 (Y.K.). This work was performed,
in part, under the Cooperative Research Program of Institutefor Protein Research, Osaka University, CR-16-05, and by
the Grant for Joint Research Project of the Institute ofMedical Science, the University of Tokyo. The authors
would like to thank Takeshi Murata (Chiba University),
Hiroaki Uchida (The University of Tokyo), and Hideaki
Tahara (The University of Tokyo) for their specialized advice, and also thank Enago (www.enago.jp) for the English
language review.
Author Disclosure Statement

No competing financial interests exist.
References

1. McNagny KM, Pettersson I, Rossi F, Flamme I, Shevchenko
A, Mann M, and Graf T: Thrombomucin, a novel cell surface
protein that defines thrombocytes and multipotent hematopoietic progenitors. J Cell Biol 1997;138:1395–1407.
2. Schopperle WM, Kershaw DB, and DeWolf WC: Humanembryonal carcinoma tumor antigen, Gp200/GCTM-2, isPCMAB-47 AGAINST PODXL

podocalyxin. Biochem Biophys Res Commun 2003;300:
285–290.
3. Schopperle WM, and DeWolf WC: The TRA-1-60 and
TRA-1-81 human pluripotent stem cell markers are expressed on podocalyxin in embryonal carcinoma. StemCells 2007;25:723–730.
4. Doyonnas R, Kershaw DB, Duhme C, Merkens H, Chelliah
S, Graf T, and McNagny KM: Anuria, omphalocele, andperinatal lethality in mice lacking the CD34-related protein
podocalyxin. J Exp Med 2001;194:13–27.
5. Berenson RJ, Andrews RG, Bensinger WI, Kalamasz D,
Knitter G, Buckner CD, and Bernstein ID: Antigen CD34+
marrow cells engraft lethally irradiated baboons. J Clin
Invest 1988;81:951–955.
6. Hayatsu N, Kaneko MK, Mishima K, Nishikawa R, Matsutani M, Price JE, and Kato Y: Podocalyxin expression inmalignant astrocytic tumors. Biochem Biophys Res Commun 2008;374:394–398.
7. Kerjaschki D, Sharkey DJ, and Farquhar MG: Identificationand characterization of podocalyxin—the major sialoprotein of the renal glomerular epithelial cell. J Cell Biol
1984;98:1591–1596.
8. Kerjaschki D, Poczewski H, Dekan G, Horvat R, Balzar E,
Kraft N, and Atkins RC: Identification of a major sialoprotein in the glycocalyx of human visceral glomerularepithelial cells. J Clin Invest 1986;78:1142–1149.
9. Kershaw DB, Beck SG, Wharram BL, Wiggins JE, Goyal
M, Thomas PE, and Wiggins RC: Molecular cloning andcharacterization of human podocalyxin-like protein. Orthologous relationship to rabbit PCLP1 and rat podocalyxin. J Biol Chem 1997;272:15708–15714.
10. Larrucea S, Butta N, Rodriguez RB, Alonso-Martin S,
Arias-Salgado EG, Ayuso MS, and Parrilla R: Podocalyxin
enhances the adherence of cells to platelets. Cell Mol LifeSci 2007;64:2965–2974.
11. Larrucea S, Butta N, Arias-Salgado EG, Alonso-Martin
S, Ayuso MS, and Parrilla R: Expression of podocalyxin
enhances the adherence, migration, and intercellularcommunication of cells. Exp Cell Res 2008;314:2004–
2015.
12. Takeda T, McQuistan T, Orlando RA, and Farquhar MG:
Loss of glomerular foot processes is associated with uncoupling of podocalyxin from the actin cytoskeleton. J Clin
Invest 2001;108:289–301.
13. Takeda T: Podocyte cytoskeleton is connected to the integral membrane protein podocalyxin through Na+/H+exchanger regulatory factor 2 and ezrin. Clin Exp Nephrol
2003;7:260–269.
14. Tan PC, Furness SG, Merkens H, Lin S, McCoy ML,
Roskelley CD, Kast J, and McNagny KM: Na+/H+ exchanger regulatory factor-1 is a hematopoietic ligand for asubset of the CD34 family of stem cell surface proteins.
Stem Cells 2006;24:1150–1161.
15. Tateno H, Matsushima A, Hiemori K, Onuma Y, Ito Y,
Hasehira K, Nishimura K, Ohtaka M, Takayasu S, Nakanishi M, Ikehara Y, Nakanishi M, Ohnuma K, Chan T,
Toyoda M, Akutsu H, Umezawa A, Asashima M, andHirabayashi J: Podocalyxin is a glycoprotein ligand of thehuman pluripotent stem cell-specific probe rBC2LCN.
Stem Cells Transl Med 2013;2:265–273.
16. Binder ZA, Siu IM, Eberhart CG, Ap Rhys C, Bai RY,
Staedtke V, Zhang H, Smoll NR, Piantadosi S, Piccirillo
SG, Dimeco F, Weingart JD, Vescovi A, Olivi A, Riggins
GJ, and Gallia GL: Podocalyxin-like protein is expressed in55

glioblastoma multiforme stem-like cells and is associatedwith poor outcome. PLoS One 2013;8:e75945.
17. Casey G, Neville PJ, Liu X, Plummer SJ, Cicek MS,
Krumroy LM, Curran AP, McGreevy MR, Catalona WJ,
Klein EA, and Witte JS: Podocalyxin variants and risk of
prostate cancer and tumor aggressiveness. Hum Mol Genet
2006;15:735–741.
18. Hsu YH, Lin WL, Hou YT, Pu YS, Shun CT, Chen CL, Wu
YY, Chen JY, Chen TH, and Jou TS: Podocalyxin EBP50
ezrin molecular complex enhances the metastatic potentialof renal cell carcinoma through recruiting Rac1 guanine
nucleotide exchange factor ARHGEF7. Am J Pathol
2010;176:3050–3061.
19. Lin CW, Sun MS, and Wu HC: Podocalyxin-like 1 is associated with tumor aggressiveness and metastatic gene
expression in human oral squamous cell carcinoma. Int J
Oncol 2014;45:710–718.
20. Yasuoka H, Tsujimoto M, Hirokawa M, Tori M, Nakahara
M, Miyauchi A, Kodama R, Sanke T, and Nakamura Y:
Podocalyxin expression in undifferentiated thyroid carcinomas. J Clin Pathol 2008;61:1228–1229.
21. Boman K, Larsson AH, Segersten U, Kuteeva E, Johannesson H, Nodin B, Eberhard J, Uhlen M, Malmstrom
PU, and Jirstrom K: Membranous expression of podocalyxinlike protein is an independent factor of poor prognosis inurothelial bladder cancer. Br J Cancer 2013;108:2321–2328.
22. Somasiri A, Nielsen JS, Makretsov N, McCoy ML, Prentice
L, Gilks CB, Chia SK, Gelmon KA, Kershaw DB, Huntsman
DG, McNagny KM, and Roskelley CD: Overexpression of
the anti-adhesin podocalyxin is an independent predictor ofbreast cancer progression. Cancer Res 2004;64:5068–5073.
23. Snyder KA, Hughes MR, Hedberg B, Brandon J, Hernaez
DC, Bergqvist P, Cruz F, Po K, Graves ML, Turvey ME,
Nielsen JS, Wilkins JA, McColl SR, Babcook JS, Roskelley
CD, and McNagny KM: Podocalyxin enhances breast tumor
growth and metastasis and is a target for monoclonal antibody therapy. Breast Cancer Res 2015;17:46.
24. Graves ML, Cipollone JA, Austin P, Bell EM, Nielsen JS,
Gilks CB, McNagny KM, and Roskelley CD: The cell surface mucin podocalyxin regulates collective breast tumor
budding. Breast Cancer Res 2016;18:11.
25. Cipollone JA, Graves ML, Kobel M, Kalloger SE, Poon T,
Gilks CB, McNagny KM, and Roskelley CD: The antiadhesive mucin podocalyxin may help initiate the transperitoneal metastasis of high grade serous ovarian carcinoma.
Clin Exp Metastasis 2012;29:239–252.
26. Larsson A, Johansson ME, Wangefjord S, Gaber A, Nodin
B, Kucharzewska P, Welinder C, Belting M, Eberhard J,
Johnsson A, Uhlen M, and Jirstrom K: Overexpression of
podocalyxin-like protein is an independent factor of poor
prognosis in colorectal cancer. Br J Cancer 2011;105:666–
672.
27. Kaprio T, Fermer C, Hagstrom J, Mustonen H, Bockelman
C, Nilsson O, and Haglund C: Podocalyxin is a marker ofpoor prognosis in colorectal cancer. BMC Cancer 2014;
14:493.
28. Kaprio T, Hagstrom J, Fermer C, Mustonen H, Bockelman
C, Nilsson O, and Haglund C: A comparative study of two
PODXL antibodies in 840 colorectal cancer patients. BMC
Cancer 2014;14:494.
29. Larsson AH, Lehn S, Wangefjord S, Karnevi E, Kuteeva E,
Sundstrom M, Nodin B, Uhlen M, Eberhard J, Birgisson H,
and Jirstrom K: Significant association and synergistic adverse prognostic effect of podocalyxin-like protein and56epidermal growth factor receptor expression in colorectal
cancer. J Transl Med 2016;14:128.
30. Heby M, Elebro J, Nodin B, Jirstrom K, and Eberhard J:
Prognostic and predictive significance of podocalyxin-likeprotein expression in pancreatic and periampullary adenocarcinoma. BMC Clin Pathol 2015;15:10.
31. Saukkonen K, Hagstrom J, Mustonen H, Juuti A, Nordling
S, Fermer C, Nilsson O, Seppanen H, and Haglund C:
Podocalyxin is a marker of poor prognosis in pancreatic
ductal adenocarcinoma. PLoS One 2015;10:e0129012.
32. Laitinen A, Bockelman C, Hagstrom J, Kokkola A, Fermer
C, Nilsson O, and Haglund C: Podocalyxin as a prognostic
marker in gastric cancer. PLoS One 2015;10:e0145079.
33. Amo L, Tamayo-Orbegozo E, Maruri N, Eguizabal C,
Zenarruzabeitia O, Rinon M, Arrieta A, Santos S, Monge J,
Vesga MA, Borrego F, and Larrucea S: Involvement ofplatelet-tumor cell interaction in immune evasion. Potential
role of podocalyxin-like protein 1. Front Oncol 2014;4:245.
34. Thomas SN, Schnaar RL, and Konstantopoulos K: Podocalyxinlike protein is an E-/L-selectin ligand on colon carcinoma
cells: Comparative biochemical properties of selectin ligands in host and tumor cells. Am J Physiol Cell Physiol
2009;296:C505–C513.
35. Dallas MR, Chen SH, Streppel MM, Sharma S, Maitra A,
and Konstantopoulos K: Sialofucosylated podocalyxin is afunctional E- and L-selectin ligand expressed by metastatic
pancreatic cancer cells. Am J Physiol Cell Physiol 2012;
303:C616–C624.
36. Laubli H, and Borsig L: Selectins promote tumor metastasis. Semin Cancer Biol 2010;20:169–177.
37. Oki H, Kaneko MK, Ogasawara S, Tsujimoto Y, Liu X,
Sugawara M, Takakubo Y, Takagi M, and Kato Y: Characterization of monoclonal antibody LpMab-7 recognizing
Non-PLAG domain of podoplanin. Monoclon Antib Immunodiagn Immunother 2015;34:174–180.
38. Oki H, Ogasawara S, Kaneko MK, Takagi M, Yamauchi M,
and Kato Y: Characterization of monoclonal antibody
LpMab-3 recognizing sialylated glycopeptide of podoplanin. Monoclon Antib Immunodiagn Immunother 2015;34:
44–50.

OGASAWARA ET AL.

39. Kaneko MK, Oki H, Hozumi Y, Liu X, Ogasawara S,
Takagi M, Goto K, and Kato Y: Monoclonal antibody
LpMab-9 recognizes O-glycosylated N-terminus of human
podoplanin. Monoclon Antib Immunodiagn Immunother2015;34:310–317.
40. Kato Y, Ogasawara S, Oki H, Goichberg P, Honma R, Fujii
Y, and Kaneko MK: LpMab-12 established by CasMab
technology specifically detects sialylated O-Glycan on
Thr52 of platelet aggregation-stimulating domain of human
podoplanin. PLoS One 2016;11:e0152912.
41. Ogasawara S, Oki H, Kaneko MK, Hozumi Y, Liu X,
Honma R, Fujii Y, Nakamura T, Goto K, Takagi M, andKato Y: Development of monoclonal antibody LpMab-10
recognizing non-glycosylated PLAG1/2 domain including
Thr34 of human podoplanin. Monoclon Antib Immunodiagn
Immunother 2015;34:318–326.
42. Kato Y, Ogasawara S, Oki H, Honma R, Takagi M, Fujii Y,
Nakamura T, Saidoh N, Kanno H, Umetsu M, Kamata S,
Kubo H, Yamada M, Sawa Y, Morita K, Harada H, Suzuki
H, and Kaneko MK: Novel monoclonal antibody LpMab17 developed by CasMab technology distinguishes human
podoplanin from monkey podoplanin. Monoclon AntibImmunodiagn Immunother 2016;35:109–116.

Address correspondence to:
Dr. Yukinari Kato
New Industry Creation Hatchery Center
Tohoku University
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: February 2, 2017
Accepted: March 9, 2017