MONOCLONAL ANTIBODIES IN IMMUNODIAGNOSIS AND IMMUNOTHERAPY
Volume 36, Number 4, 2017ª Mary Ann Liebert, Inc.
DOI: 10.1089/mab.2017.0020

Downloaded by Yukinari Kato from online.liebertpub.com at 08/15/17. For personal use only.

Antipodocalyxin Antibody chPcMab-47 Exerts
Antitumor Activity in Mouse Xenograft Modelsof Colorectal Adenocarcinomas
Mika K. Kaneko,1,* Akiko Kunita,2,* Shinji Yamada,1 Takuro Nakamura,1 Miyuki Yanaka,1 Noriko Saidoh,1
Yao-Wen Chang,1 Saori Handa,1 Satoshi Ogasawara,3 Tomokazu Ohishi,4 Shinji Abe,5,6 Shunsuke Itai,1,7
Hiroyuki Harada,7 Manabu Kawada,4 Yasuhiko Nishioka,6 Masashi Fukayama,2 and Yukinari Kato1,8

Podocalyxin (PODXL) is expressed in several cancers, including brain tumors and colorectal cancers. PODXL
overexpression is an independent predictor of progression, metastasis, and poor outcome. We recently immunized mice with recombinant human PODXL, which was produced using LN229 glioblastoma cells, andproduced a clone PcMab-47 that could be used for investigating PODXL expression by flow cytometry andimmunohistochemical analysis. Herein, we produced a human–mouse chimeric PcMab-47 (chPcMab-47)
and investigated its antitumor activity against PODXL-expressing tumors. chPcMab-47 reacted with LN229,
LN229/PODXL, and Chinese hamster ovary (CHO)/PODXL cells, but it did not react with CHO-K1 or
PODXL-knockout LN229 cell line (PDIS-13). chPcMab-47 exerted antitumor activity against a mouse xenograft model using CHO/PODXL. Furthermore, chPcMab-47 was reactive with colorectal cancer cell lines suchas HCT-15, Caco-2, HCT-8, and DLD-1. chPcMab-47 also exhibited antitumor activity against a mouse
xenograft model using HCT-15. These results suggest that chPcMab-47 could be useful for antibody therapy
against PODXL-expressing cancers.
Keywords: podocalyxin, PODXL, chimeric antibody

Introduction

P

odocalyxin (PODXL) is a type I transmembraneprotein with a molecular weight of 150,000–200,000,
which was first found in podocytes of the rat kidney,(1–4) and
its homologs have been reported in humans.(5,6) PODXL is ahighly glycosylated sialomucin with N-glycan, O-glycan, andkeratan sulfate(7) and is similar to CD34, which is known as ahematopoietic stem cell marker.(8)PODXL is expressed in the kidney, heart, pancreas, andbreast tissues and plays an important role in the developmentof several tissues.(9) PODXL acts as an adhesive molecule tobind to platelets or vascular endothelial cells(10,11); however,it also functions as an antiadhesive molecule through its
negatively charged mucin domain, including sialic acid andkeratan sulfate, and for the formation and maintenance of thefiltration slits between the podocyte processes in the kidney.(12) Furthermore, PODXL regulates cell morphology byassociating with actin cytoskeletal proteins such as Na+/H+
exchanger regulatory factor-1/2 and ezrin.(13,14)
PODXL is known as one of the pluripotent stem cellmarkers. The antigen of monoclonal antibodies (mAbs) suchas TRA-1-60 and TRA-1-81 is keratan sulfate, which is attached to PODXL.(4) PODXL is also a diagnostic marker anda prognostic indicator in several cancers, including brain
tumors(7,15) and colorectal cancers,(16–19) indicating that theoverexpression of PODXL in cancer is a potential target for
antibody therapy. Recently, we established the anti-PODXL
mAb, PcMab-47, for use in flow cytometry and immunohistochemistry.(20) In this study, we produced a human–

1

Department of Antibody Drug Development, Tohoku University, Graduate School of Medicine, Sendai, Japan.
Department of Pathology, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan.
3
Department of Chemistry, Graduate School of Science, Chiba University, Chiba, Japan.
4
Institute of Microbial Chemistry (BIKAKEN), Numazu, Microbial Chemistry Research Foundation, Shizuoka, Japan.
Departments of 5Clinical Pharmacy Practice Pedagogy and 6Respiratory Medicine and Rheumatology, Graduate School of Biomedical
Sciences, Tokushima University, Tokushima, Japan.
7
Department of Oral and Maxillofacial Surgery, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University,
Tokyo, Japan.
8
New Industry Creation Hatchery Center, Tohoku University, Sendai, Japan.
*These authors contributed equally to this work.
2

157

158

mouse chimeric PcMab-47 (chPcMab-47) and investigatedits antitumor activity against PODXL-expressing tumors.
Materials and Methods

Downloaded by Yukinari Kato from online.liebertpub.com at 08/15/17. For personal use only.

Cell lines

LN229, Caco-2, HCT-8, and Chinese hamster ovary(CHO)-K1 cell lines were obtained from the American Type
Culture Collection (Manassas, VA). HCT-15 and DLD-1 celllines were obtained from Cell Resource Center for Biomedical Research Institute of Development, Aging, and Cancer(Tohoku University, Miyagi, Japan). CHO-S cell line waspurchased from Thermo Fisher Scientific, Inc. (Waltham,MA). LN229 and CHO-K1 cells were transfected with
PODXL plasmids, which included the full length of PODXL,
using Lipofectamine 2000 (Thermo Fisher Scientific, Inc.),
according to the manufacturer’s instructions. LN229/PODXLknockout (KO) cells (PDIS-13) were produced using CRISPR/
Cas9 plasmids (Target ID: HS0000056763) against human
PODXL (Sigma-Aldrich Corp., St. Louis, MO).(21) PDIS-13 isavailable from the Cell Bank of Kato’s Lab (www.med-tohokuantibody.com/topics/001_paper_cell.htm) in Tohoku University(Miyagi, Japan).
CHO-K1 and CHO-K1/PODXL cells were cultured in
Roswell Park Memorial Institute (RPMI) 1640 medium containing l-glutamine (Nacalai Tesque, Inc., Kyoto, Japan).
LN229, LN229/PODXL, PDIS-13, Caco-2, HCT-8, HCT-15,
and DLD-1 cells were cultured in Dulbecco’s modified
Eagle’s medium (DMEM) containing l-glutamine (Nacalai
Tesque, Inc.) supplemented with 10% heat-inactivated fetal
bovine serum (Thermo Fisher Scientific, Inc.) at 37C in ahumidified atmosphere of 5% CO2 and 95% air. G418
(0.5 mg/mL; Wako Pure Chemical Industries Ltd., Osaka,
Japan) was added to the CHO-K1/PODXL and LN229/
PODXL cultures. 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.

KANEKO ET AL.

primary mAbs for 30 minutes at 4C, followed by treatment
with fluorescein isothiocyanate (FITC)-labeled goat antihuman IgG (Thermo Fisher Scientific, Inc.). Fluorescence data
were collected using the Cell Analyzer EC800 (Sony Corp.,
Tokyo, Japan).
Determination of the binding affinity
using flow cytometry

LN229/PODXL (2 · 105 cells) cells were resuspended in
100 mL of serially diluted PcMab-47 or chPcMab-47 (0.6–
10 mg/mL), followed by the addition of secondary antimouse
IgG or antihuman IgG (Thermo Fisher Scientific, Inc.).
Fluorescence data were collected using a cell analyzer

Antibodies

PcMab-47, a mouse anti-PODXL mAb (IgG1, kappa), wasdeveloped as previously described.(21) Human IgG was purchased from Sigma-Aldrich Corp.. To generate the human–
mouse chimeric anti-PODXL (chPcMab-47), appropriate VH
and VL cDNAs of mouse PcMab-47 and CH and CL of human
IgG1 were subcloned into pCAG-Ble and pCAG-Neo vectors(Wako Pure Chemical Industries, Ltd.), respectively. Antibody expression vectors were transfected into CHO-S cells
using the Lipofectamine LTX reagent (Thermo Fisher Scientific, Inc.). Stable transfectants of CHO-S/chPcMab-47
cells were selected by cultivating the transfectants in a medium containing 0.5 mg/mL of both geneticin and zeocin(InvivoGen, San Diego, CA). CHO-S/chPcMab-47 cells
were cultivated in CHO-S-SFM II medium (Thermo Fisher
Scientific, Inc.). chPcMab-47 was purified using Protein GSepharose (GE Healthcare Bio-Sciences, Pittsburgh, PA).
Flow cytometry

Cell lines were harvested by brief exposure to 0.25% trypsin/1 mM ethylenediaminetetraacetic acid (EDTA) (Nacalai
Tesque, Inc.). After washing with 0.1% bovine serum albuminin phosphate-buffered saline (PBS), cells were treated with

FIG. 1. Specific detection of PODXL by chPcMab-47
using flow cytometry. (A) LN229 and LN229/PODXL cells
were treated with chPcMab-47 (1 mg/mL; red) or control
PBS (black) for 30 minutes at 4C, followed by treatment
with FITC-labeled antihuman IgG. (B) PDIS-13 (LN229/
PODXL-knockout cells) was treated with chPcMab-47(1 mg/mL; red) or control PBS (black) for 30 minutes at 4C,
followed by treatment with FITC-labeled antihuman IgG.
PBS, phosphate-buffered saline; PODXL, podocalyxin.

chPcMab-47 AGAINST PODXL(EC800; Sony Corp.). The dissociation constants (KD) wereobtained by fitting the binding isotherms using the built-in
one-site binding models in GraphPad PRISM 6 (GraphPad
software, Inc., La Jolla, CA).

159
Statistical analyses

All data were expressed as mean – standard error of themean. Two-way analysis of variance was performed as appropriate. p values <0.05 were considered to be statisticallysignificant. All statistical tests were two sided.

Downloaded by Yukinari Kato from online.liebertpub.com at 08/15/17. For personal use only.

Antitumor activity of anti-PODXL antibodies

CHO/PODXL cells and HCT-15 cells were trypsinized
and washed with PBS. The cell density was adjusted using
PBS to 5.0 · 107 cells/mL, and 100 mL per animal of the cellsuspension was subcutaneously inoculated into BALB/cnude mice. After 1 day, 100 mL of 1 mg/mL of chPcMab-47
and human IgG was injected into the peritoneal cavity ofmice. Additional antibodies were injected once a week forseveral weeks. Human natural killer (NK) cells (8.0 · 105
cells; Takara Bio, Inc., Shiga, Japan) were injected aroundthe tumors several times. The tumor diameter was measured
every 3–7 days and was calculated using the following formula: volume = W2 · L/2, where W is the short diameter and L
is the long diameter. The mice were euthanized 30 (CHO/
PODXL) or 29 days (HCT-15) after cell implantation.

FIG. 2. Determination of the binding affinity of chPcMab-47
and PcMab-47 using flow cytometry. LN229/PODXL cells(2 · 105 cells) were resuspended in 100 mL of serially diluted
chPcMab-47 (A) or PcMab-47 (B) (0.6–10 mg/mL), followedby addition of secondary antihuman IgG or antimouse IgG.
Fluorescence data were collected using a cell analyzer.

FIG. 3. Antitumor activity of chPcMab-47 against CHO/PODXL. (A) CHO-K1 and CHO/PODXL cells were treatedwith chPcMab-47 (1 mg/mL; red) or control PBS (black) for 30
minutes at 4C, followed by treatment with FITC-labeled antihuman IgG. (B) CHO/PODXL cells (5 · 106 cells/100 mL)
were subcutaneously inoculated into BALB/c nude mice. After
1 day, 100 mg of chPcMab-47 or control human IgG was injected into the peritoneal cavity of the mice. The antibodies
were further injected four times (days 1, 4, 11, and 18; control
group: n = 6; chPcMab-47 group: n = 6). NK cells were injected around the tumor on days 4 and 11. The tumor diameter
was measured at 3- to 4-day intervals and was calculated using
the following formula: tumor volume = W2 · L/2, where W isshort diameter and L is long diameter. *p < 0.05, ****p < 0.001
with two-way ANOVA. (C) Comparison of the tumor size(day 30). ANOVA, analysis of variance; CHO, Chinese hamster
ovary; NK, natural killer.

160

KANEKO ET AL.

Results

Downloaded by Yukinari Kato from online.liebertpub.com at 08/15/17. For personal use only.

Production of human–mouse chimericantibody, chPcMab-47

We first checked the sensitivity and specificity of chPcMab47 using flow cytometry. As depicted in Figure 1A, chPcMab47 recognized endogenous PODXL, which is expressed in
LN229 cells (a glioblastoma cell line), whereas it did not reactwith LN229/PODXL-KO cells (PDIS-13) (Fig. 1B), indicatingthat chPcMab-47 is specific against PODXL. The reaction ofchPcMab-47 against LN229/PODXL was higher than thatagainst LN229 (Fig. 1A). We next performed a kinetic analysisof the interaction of chPcMab-47 or PcMab-47 with LN229/
PODXL using flow cytometry. As shown in Figure 2, KD ofchPcMab-47 was determined to be 1.1 · 10-8 M, indicatingthat chPcMab-47 possesses higher affinity against PODXL
than PcMab-47 (KD: 2.9 · 10-8 M).
Antitumor activity of chPcMab-47 against CHO/PODXL

chPcMab-47 reacted with CHO/PODXL but did not reactwith CHO-K1 (human PODXL-negative cells) (Fig. 3A). To
investigate the antitumor activity of chPcMab-47 on primarytumor growth in vivo, CHO/PODXL cells were subcutaneously
implanted into the flanks of nude mice. chPcMab-47 andcontrol human IgG were injected four times (on days 1, 4, 11,
and 18 after cell injections) into the peritoneal cavity of mice,and human NK cells were injected twice (on days 4 and 11)
around the tumors. Tumor formation was observed in micefrom the control and treated groups. However, chPcMab-47
significantly reduced tumor development compared with control human IgG on day 30 (Fig. 3B and C).
Antitumor activity of chPcMab-47
against colorectal cancers

PODXL is expressed in several cancers such as colorectal
cancers(16–19) and breast cancers.(22–24) We performed flow
cytometry using colorectal cancer cells, which revealed that
chPcMab-47 reacted with the colorectal cancer cell lines
HCT-15, Caco-2, HCT-5, and DLD-1 (Fig. 3). We selected
HCT-15 because of its tumorigenicity in vivo.(25–27) To study
the antitumor activity of chPcMab-47 on primary tumor
growth in vivo, HCT-15 cells were subcutaneously implanted
into the flanks of nude mice. chPcMab-47 and control human
IgG were injected four times into the peritoneal cavity of‰
FIG. 4. Antitumor activity of chPcMab-47 against HCT-15
cells. (A) HCT-15, Caco-2, HCT-8, and DLD-1 cells weretreated with chPcMab-47 (1 mg/mL; blue) or control PBS(black) for 30 minutes at 4C, followed by treatment with FITClabeled antihuman IgG. (B) HCT-15 cells (5 · 106 cells/100 mL)
were subcutaneously inoculated into BALB/c nude mice. After
1 day, 100 mg of chPcMab-47 or control human IgG was injected into the peritoneal cavity of the mice. The antibodies
were further injected four times (days 1, 7, 14, and 21; control
group: n = 7; chPcMab-47 group: n = 7). NK cells were injectedaround the tumor on days 7 and 14. The tumor diameter wasmeasured at 3- to 4-day intervals and was calculated using thefollowing formula: tumor volume = W2· L/2, where W is short
diameter and L is long diameter. *p < 0.05 with two-way
ANOVA. (C) Comparison of the tumor size (day 29).

mice, and human NK cells were injected twice around thetumors. Tumor formation was observed in mice from thecontrol and treated groups. However, chPcMab-47 significantly reduced tumor development compared with control
human IgG on day 29 (Fig. 4B and C).
Discussion

In our previous study, we immunized mice with recombinant PODXL, which was purified from the culture supernatant

Downloaded by Yukinari Kato from online.liebertpub.com at 08/15/17. For personal use only.

chPcMab-47 AGAINST PODXLof LN229/ectodomain-PODXL.(21) One clone, PcMab-47(mouse IgG1, kappa) was successfully produced. PcMab-47
reacted with CHO/PODXL and LN229 cells (a glioblastoma
cell line) but did not react with CHO-K1 cells (human
PODXL-negative cells) and LN229/PODXL-KO cells (PDIS13), indicating that PcMab-47 is specific against PODXL.
PcMab-47 is also useful for immunohistochemical analysis
against human colorectal adenocarcinomas and breast cancers.(21) In this study, we produced a human–mouse chimericantibody from PcMab-47 and investigated whether chPcMab47 possesses antitumor activity.
We investigated whether chPcMab-47 reacts with colorectal cancer cell lines. As shown in Figure 4, chPcMab-47
reacted with HCT-15, Caco-2, HCT-5, and DLD-1 cells at alow concentration of 1 mg/mL. We further checked the reactivity of chPcMab-47 against other colorectal cancer celllines. It was observed that chPcMab-47 showed a moderate
reaction with LST147T and SW1116 cells, a slight reaction
with HT29 and HCT-116 cells, and no reaction with colo201
and colo205 cells, indicating that 80% (8/10) of cells weremore or less detected by chPcMab-47 (data not shown). We
searched the literature to find out whether those cell lines
grow subcutaneously in BALB/c nude mice, and we discovered that HCT-15 is the best cell line for investigating
antitumor activity.(25–27) In this study, we injected humanNK cells around the tumors because chPcMab-47 needs to
induce ADCC activity.(28,29) Although chPcMab-47 significantly reduced tumor development compared with control
human IgG on day 29 (Fig. 4B), the antitumor activity of
chPcMab-47 was not adequate for antibody-based target
therapy. This result suggests that we need to combine
chPcMab-47 with anticancer drugs or apply it to novel antitumor reagents, including T cells and viruses, to exert antitumor activity against cancer cells.
Taken together, chPcMab-47 could be useful for antibody
therapy against PODXL-expressing cancers. PODXL isknown to be expressed in the kidney, heart, pancreas, andbreast tissues and plays an important role in these tissues,(9)indicating that we need to develop cancer-specific mAbs inthe near future.
Acknowledgments

The authors are grateful to Kimiko Takeshita for thetechnical assistance. This work was supported, in part, by theTranslational Research Network Program from Japan
Agency for Medical Research and development, AMED
(Y.K.), by the Basic Science and Platform Technology Program for Innovative Biological Medicine from AMED
(Y.K.), by the Platform for Drug Discovery, Informatics,
and Structural Life Science (PDIS)/the Platform Project for
Supporting Drug Discovery and Life Science Research 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 Numbers 26440019 and 17K07299(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 of161

Medical Science, the University of Tokyo. The authors would
like to thank Enago (www.enago.jp) for English language
review.
Author Disclosure Statement

No competing financial interests exist.
References

1. 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.
2. 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.
3. Schopperle WM, Kershaw DB, and DeWolf WC: Humanembryonal carcinoma tumor antigen, Gp200/GCTM-2, is podocalyxin. Biochem Biophys Res Commun 2003;300:285–290.
4. 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.
5. 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.
6. 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.
7. 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.
8. 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.
9. 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.
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 intercellular communication 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 aDownloaded by Yukinari Kato from online.liebertpub.com at 08/15/17. For personal use only.

162

subset of the CD34 family of stem cell surface proteins.
Stem Cells 2006;24:1150–1161.
15. 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 inglioblastoma multiforme stem-like cells and is associatedwith poor outcome. PLoS One 2013;8:e75945.
16. 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 podocalyxinlike protein is an independent factor of poor prognosis in colorectal cancer. Br J Cancer 2011;105:666–672.
17. Kaprio T, Fermer C, Hagstrom J, Mustonen H, Bockelman C,
Nilsson O, and Haglund C: Podocalyxin is a marker of poor
prognosis in colorectal cancer. BMC Cancer 2014;14:493.
18. 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.
19. 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 andepidermal growth factor receptor expression in colorectal
cancer. J Transl Med 2016;14:128.
20. Ogasawara S, Kaneko MK, Yamada S, Honma R, Nakamura T, Saidoh N, Yanaka M, Yoshida K, Fujii Y, andKato Y: PcMab-47: novel antihuman podocalyxin monoclonal antibody for immunohistochemistry. Monoclon Antib Immunodiagn Immunother. 2017;36:50–56.
21. 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.
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

KANEKO ET AL.

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 cellsurface mucin podocalyxin regulates collective breast
tumor budding. Breast Cancer Res 2016;18:11.
25. Hosono T, Fukao T, Ogihara J, Ito Y, Shiba H, Seki T, andAriga T: Diallyl trisulfide suppresses the proliferation andinduces apoptosis of human colon cancer cells throughoxidative modification of beta-tubulin. J Biol Chem 2005;
280:41487–41493.
26. Ho AS, Chen CH, Cheng CC, Wang CC, Lin HC, Luo TY,
Lien GS, and Chang J: Neutrophil elastase as a diagnostic
marker and therapeutic target in colorectal cancers. Oncotarget 2014;5:473–480.
27. Guan SS, Chang J, Cheng CC, Luo TY, Ho AS, Wang CC,
Wu CT, and Liu SH: Afatinib and its encapsulated polymeric micelles inhibits HER2-overexpressed colorectal
tumor cell growth in vitro and in vivo. Oncotarget 2014;5:
4868–4880.
28. Abe S, Kaneko MK, Tsuchihashi Y, Izumi T, Ogasawara S,
Okada N, Sato C, Tobiume M, Otsuka K, Miyamoto L,
Tsuchiya K, Kawazoe K, Kato Y, and Nishioka Y: Antitumor effect of novel anti-podoplanin antibody NZ-12
against malignant pleural mesothelioma in an orthotopic
xenograft model. Cancer Sci 2016;107:1198–1205.
29. 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.

Address correspondence to:
Dr. Yukinari Kato
New Industry Creation Hatchery Center
Tohoku University
2-1 Seiryo-machi, Aoba-ku
Miyagi, Sendai 980-8575
JapanE-mail: yukinari-k@bea.hi-ho.ne.jp;
yukinarikato@med.tohoku.ac.jp
Received: March 16, 2017
Accepted: April 20, 2017