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Biochemical and Biophysical Research Communications xxx (2008) xxx–xxx

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Biochemical and Biophysical Research Communications
journal homepage: www.elsevier.com/locate/ybbrc

Podocalyxin expression in malignant astrocytic tumors
Norihito Hayatsu a, Mika Kato Kaneko b, Kazuhiko Mishima c, Ryo Nishikawa c,
Masao Matsutani c, Janet E. Price b, Yukinari Kato b,*a

Graduate School of Medicine, Kyoto University, Yoshida-konoe-cho, Sakyo-ku, Kyoto 606-8501, Japan
Department of Cancer Biology, Unit 173, University of Texas M.D. Anderson Cancer Center, 1515 Holcombe, Houston, TX 77030, USAcSaitama Medical University International Medical Center, 1397-1 Yamane Hidaka-shi, Saitama 350-1298, Japanba r t i c l ei n f oArticle history:
Received 4 July 2008
Available online xxxx
Keywords:
Podocalyxin
Keratan sulfate
Proteoglycan
Astrocytic tumor
Glioblastomaa b s t r a c t
Podocalyxin is an anti-adhesive mucin-like transmembrane sialoglycoprotein that has been implicated in
the development of aggressive forms of cancer. Podocalyxin is also known as keratan sulfate (KS) proteoglycan. Recently, we revealed that highly sulfated KS or another mucin-like transmembrane sialoglycoprotein podoplanin/aggrus is upregulated in malignant astrocytic tumors. The aim of this study is toexamine the relationship between podocalyxin expression and malignant progression of astrocytictumors. In this study, 51 astrocytic tumors were investigated for podocalyxin expression using immunohistochemistry, Western blot analysis, and quantitative real-time PCR. Immunohistochemistry detected
podocalyxin on the surface of tumor cells in six of 14 anaplastic astrocytomas (42.9%) and in 17 of 31 glioblastomas (54.8%), especially around proliferating endothelial cells. In diffuse astrocytoma, podocalyxin
expression was observed only in vascular endothelial cells. Podocalyxin might be associated with themalignant progression of astrocytic tumors, and be a useful prognostic marker for astrocytic tumors.
Ó 2008 Elsevier Inc. All rights reserved.

Astrocytic tumors are the most common tumors of the central
nervous system (CNS) and are categorized into diffuse astrocytomas (World Health Organization (WHO) Grade II), anaplastic astrocytomas (WHO Grade III), and glioblastomas (WHO Grade IV) [1].
Glioblastoma may occur de novo or may result from progressionof low-grade astrocytomas [2]. Molecular mechanisms of malignant progression are associated with inactivation of tumor suppressor genes such as p53-Rb pathway or overexpression of
oncogenes such as epidermal growth factor receptor (EGFR) [3].
However, the mechanisms of malignant progression of astrocytic
tumors have not been resolved completely. Identiﬁcation of genesthat are expressed differentially in high-grade or low-grade astrocytomas is important to elucidate the molecular mechanisms ofmalignant progression and to develop novel therapeutic strategies.
Podocalyxin is a type I transmembrane sialoglycoprotein, which
belongs to the CD34 family. Podocalyxin is expressed on thesurface of various normal cells, including kidney podocytes, vascular endothelial cells, hematopoietic stem cells, and platelets [4–7].
The physiological function of podocalyxin is as an anti-adhesion
molecule that maintains open ﬁltration pathways between neighboring podocyte foot processes through the charge-repulsive
Abbreviations: KS, keratan sulfate; CNS, central nervous system; WHO, World HealthOrganization.
* Corresponding author. Fax: +81 29 861 3191.
E-mail address: yukinari-k@bea.hi-ho.ne.jp (Y. Kato).

effects of its large, highly sialylated and sulfated extracellular domain [8]. In canine kidney (MDCK) cells, podocalyxin overexpression leads to the inhibition of cell–cell interaction as shown bydecreased cell–cell adhesion, decreased tight junction-dependenttransepithelial resistance, and redistribution of cell junction proteins [9]. Decreased cell–cell interaction is a prominent feature ofcancer cells that display a metastatic phenotype, suggesting a possible role for anti-adhesive molecules, such as podocalyxin, in cancer progression. The role of podocalyxin in cancer remains unclear,although its expression has been reported in breast, liver, pancreas,kidney, prostate, testis, and blood cell cancers [10–16]. However,podocalyxin has been implicated in the development of aggressive
forms of cancer. Increased podocalyxin protein expression is correlated with poor outcome in breast carcinomas, and is implicated in
more aggressive forms of prostate cancer [10,14]. Furthermore,
podocalyxin variants were found to be associated with both therisk of prostate cancer and prostate tumor aggressiveness. Recently, podocalyxin was reported to increase the aggressive phenotype of breast and prostate cancer in vitro through its interaction
with ezrin [17]. Thus, podocalyxin is a candidate for playing a critical role in cancer aggressiveness and malignancy. Podocalyxin isalso reported to be useful to differentiate pancreatic ductal adenocarcinomas from adenocarcinomas of the biliary and gastrointestinal tracts [12].
Recently, we showed that the expression of highly sulfatedkeratan sulfate (KS) recognized by 5D4 antibody is increased in0006-291X/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved.
doi:10.1016/j.bbrc.2008.07.049

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parallel with increasing malignancy of astrocytic tumors [18,19].
KS expression is induced by high expression of ﬁve glycogenes involved in KS synthesis. However, in addition to high expression
levels of glycogenes involved in KS synthesis, core proteins of KS
proteoglycan also might contribute to the high expression of KS.
Podocalyxin was recently identiﬁed as a KS proteoglycan [20]. In
this study, 51 astrocytic tumors (six diffuse astrocytomas, 14 anaplastic astrocytomas, and 31 glioblastomas) were investigatedusing immunohistochemistry and Western blot with an anti-podocalyxin antibody. Furthermore, we investigated the podocalyxintranscript levels using quantitative real-time PCR in 51 frozen
astrocytic tumors.

Materials and methods
Tissue samples. Tumor specimens were obtained during surgery
from six patients with diffuse astrocytomas, 14 patients with anaplastic astrocytomas, and 31 patients with glioblastomas [19,21].
Informed consent had been obtained previously from patients ortheir guardians. The histology of these tissue samples was conﬁrmed by experienced neuropathologists.
Immunohistochemical analysis. Specimens were deparafﬁnized,
rehydrated and incubated ﬁrst with goat anti-human podocalyxin(2 lg/ml) at 4 °C for 18 h, then with biotin-conjugated secondaryanti-goat IgG antibody (Dako, Glostrup, Denmark) for 1 h, and ﬁnally with peroxidase-conjugated biotin-streptavidin complex(Vectastain ABC Kit; Vector Laboratories Inc., Burlingame, CA) for
1 h. Color was developed using 3, 3-diaminobenzidine tetrahydrochloride tablet sets (Dako) for 3 min. KS expression was assessedsemi-quantitatively from the percentage of tumor cells with cytoplasmic/membrane staining: 0, no staining; +, <10%; ++, 10–50%;
and +++, >50%.
Western blot analysis. The tissues were lysed with lysis buffer(25 mM Tris (pH 7.4), 50 mM NaCl, 0.5% Na deoxycholate, 2% Nonidet P-40, 0.2% SDS, 1 mM phenylmethylsulfonyl ﬂuoride, and50 mg/ml aprotinin) [19,21]. Samples of the supernatant fraction
were collected after centrifuging at 15,000g for 30 min. Four
micrograms of the proteins were electrophoresed under reducing
conditions on 10% polyacrylamide gel (Atto Bioscience, Tokyo, Japan). The separated proteins were transferred to a PVDF membrane. After blocking with 3% skim milk in PBS with 0.05% Tween
20, the membrane was incubated with goat anti-human podocalyxin (0.1 lg/ml; R&D Systems, Minneapolis, MN) or anti-b-actin antibody (1/5000 dilution; Sigma, St. Louis, MO), andsubsequently with peroxidase-conjugated anti-goat or anti-mouse
antibodies (1/5000 dilution; Bio-Rad Laboratories Inc., Hercules,CA). It was then developed for 1 min with ECL reagents (AmershamPharmacia Biotech Inc.) using Amersham Hyperﬁlm ECL (Amersham Pharmacia Biotech Inc.).
Quantitative real-time PCR analysis. Total RNAs were preparedfrom 51 astrocytic tumors (six diffuse astrocytomas, 14 anaplastic
astrocytomas, and 31 glioblastomas) using an RNeasy mini prep kit(Qiagen Inc., Hilden, Germany). The initial cDNA strand was synthesized using SuperScript III transcriptase (Invitrogen Corp., Carlsbad, CA) by priming an oligo-dT primer according to themanufacturer’s instructions. We performed PCR using oligonucleotides: human podocalyxin sense (50 -acaggaaacaccctctgtgc-30 ) andhuman podocalyxin antisense (50 -gaaggtggctttgactgctc-30 ). Realtime PCR was carried out using the QuantiTect SYBR Green PCR(Qiagen Inc.). The PCR conditions were 95 °C for 15 min (1 cycle),followed by 40 cycles of 94 °C for 15 s, 53 °C for 20 s, and 72 °C
for 10 s. Subsequently, a melting curve program was applied withcontinuous ﬂuorescence measurement. A standard curve for podocalyxin templates was generated through serial dilution of PCRproducts (1  108 to 1  102 copies/ ll). The expression level ofpodocalyxin was normalized by total RNA weights. The statisticalsigniﬁcance of podocalyxin mRNA expression in astrocytic tumor
tissues was determined using paired t tests.
Statistical analyses. Results are expressed as the mean ± standard deviation. Student’s t-test was used to determine signiﬁcanceamong the groups. A value of p < 0.05 was considered signiﬁcant.
Results
Immunohistochemical staining for podocalyxin in malignant astrocytictumors
The cellular distribution of podocalyxin in astrocytic tumors
was examined immunohistochemically using goat anti-humanpodocalyxin polyclonal antibody. This polyclonal antibody wasproduced in goats immunized with recombinant human podocalyxin extracellular domain (23–425 a.a.), and puriﬁed using
podocalyxin afﬁnity chromatography. In many recent studies,
this antibody was applied to the immunohistochemistry, Western blot, and immunoprecipitation, indicating that this antibody is speciﬁc to human podocalyxin [20,22,23]. Herein, weused 51 surgical tissue samples (six diffuse astrocytomas: GradeII, 14 anaplastic astrocytomas: Grade III, and 31 glioblastomas:Grade IV) for immunohistochemistry. Podocalyxin immunoreactivity was detected in six of 14 (42.9%) anaplastic astrocytomas
and in 17 of 31 (54.8%) glioblastomas; staining was graded as+++ in seven glioblastoma and as ++ in three glioblastoma cases(Table 1). Podocalyxin was not detected on tumor cell surfaces
in diffuse astrocytomas, yet was observed in vascular endothelial cells in these specimen (Fig. 1A and B). Representative
staining for podocalyxin in astrocytic tumor samples is shownin Fig. 1. Immunostaining for podocalyxin demonstrated predominantly cell-surface patterns. In anaplastic astrocytoma, thetumor cell surface was stained using anti-podocalyxin (Fig. 1Cand D). In glioblastomas, podocalyxin-positive tumor cells wereprominent around microvascular proliferations (Fig. 1E and F).
Proliferating endothelial cells were also positive for podocalyxin(Fig. 1F).
Analysis of podocalyxin expression using Western blot in astrocytictumors
To conﬁrm the podocalyxin expression in astrocytic tumors, lysates of frozen tumor specimens from 51 patients were analyzed
using Western blot analysis with anti-podocalyxin antibody. As
shown in Fig. 2, podocalyxin was highly detected in extracts ofanaplastic astrocytoma and glioblastoma. Samples of one of 14
anaplastic astrocytomas (7.1%) and seven of 31 glioblastomas(22.6%) showed relatively high levels of podocalyxin, while expression in ﬁve of 14 anaplastic astrocytomas (35.7%) and 11 of 31 glioblastomas (35.5%) was more moderate. The other astrocytic
tumors including diffuse astrocytic tumors produced weak immunoreactive bands, since podocalyxin is expressed in all vascularendothelial cells (Fig. 1).

Table 1Results of podocalyxin immunostaining in 51 patients with astrocytic tumors
Tumor type

Diffuse astrocytoma(grade II)
Anaplastic astrocytoma(grade III)
Glioblastoma (grade IV)

No. of cases

Podocalyxin

Positiverate++++++



6000

60%

1401

58

42.9%

3173

714

54.8%

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3Fig. 1. Immunohistochemical detection of podocalyxin in astrocytic tumors. Podocalyxin was not detected on tumor cell surfaces in diffuse astrocytomas; however,podocalyxin staining was observed in vascular endothelial cells, although as shown in this ﬁgure few endothelial cells are usually detected in diffuse astrocytomas (A: 100,B: 400). In anaplastic astrocytoma, the tumor cell surface was stained positively (C: 100, D: 400). Accentuated staining is visible around an area of microvascularproliferation in glioblastoma (E: 100, F: 400). Bar, 10 lm.

Differential expression of the podocalyxin mRNA in astrocytic tumors
To quantify the expression of podocalyxin mRNA in humanastrocytic tumors of different grades, we performed quantitative
real-time PCR analyses of astrocytic tumors from 51 patients. The
relative podocalyxin mRNA expression levels of each tumor grade
are shown in Fig. 3. Average copy numbers of podocalyxin
mRNA/lg total RNA in diffuse astrocytomas, anaplastic astrocytomas, and glioblastomas were 535 ± 289, 918 ± 595, and3670 ± 2916, respectively. Podocalyxin transcript levels were signiﬁcantly higher in glioblastomas than in diffuse astrocytomas or
anaplastic astrocytomas (p < 0.01).
Discussion
Podocalyxin expression has been reported to increase theaggressive phenotype of breast and prostate cancer, and be correlated with their poor prognosis [10,14,17]. Recently, we showedthat highly sulfated keratan sulfate detected by 5D4 antibody isupregulated in accordance with malignancy of astrocytic tumors[19]. We speculated that expression of core proteins of KS proteoglycan is upregulated in malignant astrocytic tumors. Podocalyxin
was recently identiﬁed as a keratan sulfate (KS) proteoglycan: podocalyxin has the keratan sulfate antigens TRA-1-60 and TRA-1-81,

which are also known as human pluripotent stem cell markers, inembryonal carcinoma [20]. Furthermore, podocalyxin expression
correlates with tumor aggressiveness or malignancy; therefore,
we herein investigated the podocalyxin expression in brain
tumors, focusing on astrocytic tumors of different grades.
In this study, we ﬁrst investigated the podocalyxin expression
by immunohistochemistry (Fig. 1), and showed signiﬁcantly different podocalyxin expression between anaplastic astrocytomas(Grade III; 42.9%) and glioblastoma (Grade IV; 54.8%; p < 0.05).
Using Western blot analysis and real-time PCR, we conﬁrmed thisresult (Figs. 2 and 3). Taken together, these results indicate that
podocalyxin expression might be associated with the malignant
progression of astrocytic tumors. We previously investigated theexpression of podoplanin, another mucin-like sialoglycoprotein,which is associated with malignant progression of astrocytic tumors [21,24,25]. Podoplanin was detected on the cell surface in
27% of anaplastic astrocytomas and 47% of glioblastomas. In contrast, podoplanin expression was not observed in diffuse astrocytomas. Because both podocalyxin and podoplanin are associated withthe malignancy of astrocytic tumors, we investigated whether podocalyxin expression is correlated with podoplanin; however, wefound no correlation between the expression levels of the two
sialoglycoproteins (data not shown). We might be able to predictthe malignancy of astrocytic tumors if we appropriately combine

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Fig. 2. Western blot analysis of podocalyxin expression in astrocytic tumors. Tissues from diffuse astrocytomas (A: lane 1–6), anaplastic astrocytomas (B: lane 7–20), andglioblastomas (C: lane 21–51) were solubilized, and 4 lg of the proteins were electrophoresed under reducing conditions on 10% polyacrylamide gel. The separated proteins
were transferred to a PVDF membrane. After blocking with 3% skim milk in PBS, the membrane was incubated with anti-podocalyxin (upper panel) or anti-b-actin antibody(lower panel).

(Y. Kato), the Toyama Foundation (Y. Kato), the Inoue Foundationfor Science (Y. Kato), and Osaka Cancer Research Foundation(M.K. Kaneko).
References

Fig. 3. Quantitative real-time PCR analysis of podocalyxin in astrocytic tumors. The
transcript levels for a podocalyxin gene in 51 astrocytic tumors (six diffuse
astrocytomas, 14 anaplastic astrocytomas, and 31 glioblastomas) were measured
using real-time PCR. Values normalized to the level of total RNA are presented.
**p < 0.01, *p < 0.05.

the expression level of these several molecules. Further study isneeded to clarify the pathophysiological function of podocalyxin
in astrocytic tumors, such as invasiveness or angiogenesis, using
more clinical samples.
In summary, we investigated the expression of podocalyxin in
51 astrocytic tumors using immunohistochemistry, Western blot,
and real-time PCR analyses. Podocalyxin expression was not observed in diffuse astrocytoma except in vascular endothelial cells.
Furthermore, podocalyxin mRNA and protein expression weremarkedly higher in glioblastomas than in anaplastic astrocytomas.
These data suggest that podocalyxin expression is associated withmalignancy of astrocytic tumors, and might be useful as a prognostic factor.
Acknowledgments
This study was supported in part by Mitsubishi Pharma
Research Foundation (Y. Kato), the YASUDA Medical Foundation[1] P. Kleihues, P.C. Burger, V.P. Collines, E.W. Newcomb, H. Ohagi, W.K. Cavenee,
Astrocytic Tumors, Glioblastma, International Agency for Research on Cancer
Press, Lyons, France, 2000. pp. 29–39.
[2] A. Giese, R. Bjerkvig, M.E. Berens, M. Westphal, Cost of migration: invasion ofmalignant gliomas and implications for treatment, J. Clin. Oncol. 21 (2003)
1624–1636.
[3] P. Kleihues, H. Ohgaki, Primary and secondary glioblastomas: from concept toclinical diagnosis, Neuro-oncology 1 (1999) 44–51.
[4] D. Kerjaschki, D.J. Sharkey, M.G. Farquhar, Identiﬁcation and characterizationof podocalyxin-the major sialoprotein of the renal glomerular epithelial cell, J.
Cell Biol. 98 (1984) 1591–1596.
[5] J.E. Schnitzer, C.P. Shen, G.E. Palade, Lectin analysis of common glycoproteinsdetected on the surface of continuous microvascular endothelium in situ and inculture: identiﬁcation of sialoglycoproteins, Eur. J. Cell Biol. 52 (1990) 241–251.
[6] T. Hara, Y. Nakano, M. Tanaka, K. Tamura, T. Sekiguchi, K. Minehata, N.G.
Copeland, N.A. Jenkins, M. Okabe, H. Kogo, Y. Mukouyama, A. Miyajima,
Identiﬁcation of podocalyxin-like protein 1 as a novel cell surface marker forhemangioblasts in the murine aorta-gonad-mesonephros region, Immunity 11(1999) 567–578.
[7] A. Miettinen, M.L. Solin, J. Reivinen, E. Juvonen, R. Vaisanen, H. Holthofer,Podocalyxin in rat platelets and megakaryocytes, Am. J. Pathol. 154 (1999)
813–822.
[8] R. Doyonnas, D.B. Kershaw, C. Duhme, H. Merkens, S. Chelliah, T. Graf, K.M.
McNagny, Anuria, omphalocele, and perinatal lethality in mice lacking theCD34-related protein podocalyxin, J. Exp. Med. 194 (2001) 13–27.
[9] T. Takeda, W.Y. Go, R.A. Orlando, M.G. Farquhar, Expression of podocalyxin
inhibits cell–cell adhesion and modiﬁes junctional properties in Madin–Darby
canine kidney cells, Mol. Biol. Cell 11 (2000) 3219–3232.
[10] A. Somasiri, J.S. Nielsen, N. Makretsov, M.L. McCoy, L. Prentice, C.B. Gilks, S.K.
Chia, K.A. Gelmon, D.B. Kershaw, D.G. Huntsman, K.M. McNagny, C.D. Roskelley,
Overexpression of the anti-adhesin podocalyxin is an independent predictor ofbreast cancer progression, Cancer Res. 64 (2004) 5068–5073.
[11] X. Chen, J. Higgins, S.T. Cheung, R. Li, V. Mason, K. Montgomery, S.T. Fan, M. vande Rijn, S. So, Novel endothelial cell markers in hepatocellular carcinoma, Mod.
Pathol. 17 (2004) 1198–1210.
[12] J.T. Ney, H. Zhou, B. Sipos, R. Buttner, X. Chen, G. Kloppel, I. Gutgemann,
Podocalyxin-like protein 1 expression is useful to differentiate pancreatic
ductal adenocarcinomas from adenocarcinomas of the biliary andgastrointestinal tracts, Hum. Pathol. 38 (2007) 359–364.
[13] P. Stanhope-Baker, P.M. Kessler, W. Li, M.L. Agarwal, B.R. Williams, The Wilms
tumor suppressor-1 target gene podocalyxin is transcriptionally repressed byp53, J. Biol. Chem. 279 (2004) 33575–33585.
[14] G. Casey, P.J. Neville, X. Liu, S.J. Plummer, M.S. Cicek, L.M. Krumroy, A.P. Curran,
M.R. McGreevy, W.J. Catalona, E.A. Klein, J.S. Witte, Podocalyxin variants andrisk of prostate cancer and tumor aggressiveness, Hum. Mol. Genet. 15 (2006)
735–741.
[15] W.M. Schopperle, D.B. Kershaw, W.C. DeWolf, Human embryonal carcinomatumor antigen, Gp200/GCTM-2, is podocalyxin,Biochem. Biophys. Res.
Commun. 300 (2003) 285–290.
[16] T.W. Kelley, D. Huntsman, K.M. McNagny, C.D. Roskelley, E.D. Hsi, Podocalyxin:a marker of blasts in acute leukemia, Am. J. Clin. Pathol. 124 (2005) 134–142.

Please cite this article in press as: N. Hayatsu et al., Podocalyxin expression in malignant astrocytic tumors, Biochem. Biophys. Res. Commun. (2008), doi:10.1016/j.bbrc.2008.07.049

ARTICLE IN PRESS
N. Hayatsu et al. / Biochemical and Biophysical Research Communications xxx (2008) xxx–xxx[17] S. Sizemore, M. Cicek, N. Sizemore, K.P. Ng, G. Casey, Podocalyxin increases theaggressive phenotype of breast and prostate cancer cells in vitro through its
interaction with ezrin, Cancer Res. 67 (2007) 6183–6191.
[18] N. Hayatsu, S. Ogasawara, M.K. Kaneko, Y. Kato, H. Narimatsu, Expression ofhighly sulfated keratan sulfate synthesized in human glioblastoma cells,
Biochem. Biophys. Res. Commun. 368 (2008) 217–222.
[19] Y. Kato, N. Hayatsu, M.K. Kaneko, S. Ogasawara, T. Hamano, S. Takahashi, R.
Nishikawa, M. Matsutani, K. Mishima, H. Narimatsu, Increased expression ofhighly sulfated keratan sulfate synthesized in malignant astrocytic tumors,
Biochem. Biophys. Res. Commun. 369 (2008) 1041–1046.
[20] W.M. Schopperle, W.C. DeWolf, The TRA-1-60 and TRA-1-81 humanpluripotent stem cell markers are expressed on podocalyxin in embryonalcarcinoma, Stem Cells 25 (2007) 723–730.
[21] K. Mishima, Y. Kato, M.K. Kaneko, R. Nishikawa, T. Hirose, M. Matsutani,
Increased expression of podoplanin in malignant astrocytic tumors as a novel
molecular marker of malignant progression, Acta Neuropathol. (Berlin) 111(2006) 483–488.

5[22] J. Achenbach, M. Mengel, I. Tossidou, I. Peters, J. K. Park, M. Haubitz, J. H.
Ehrich, H. Haller, M. Schiffer, Parietal epithelia cells in the urine as a marker ofdisease activity in glomerular diseases, Nephrol. Dial. Transpl., in press.
[23] A.B. Choo, H.L. Tan, S.N. Ang, W.J. Fong, A. Chin, J. Lo, L. Zheng, H. Hentze, R.J.
Philp, S.K. Oh, M. Yap, Selection against undifferentiated human embryonicstem cells by a cytotoxic antibody recognizing podocalyxin-like protein-1,Stem Cells 26 (2008) 1454–1463.
[24] Y. Kato, M.K. Kaneko, A. Kunita, H. Ito, A. Kameyama, S. Ogasawara, N.
Matsuura, Y. Hasegawa, K. Suzuki-Inoue, O. Inoue, Y. Ozaki, H. Narimatsu,
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 (2008) 54–61.
[25] Y. Kato, M.K. Kaneko, A. Kuno, N. Uchiyama, K. Amano, Y. Chiba, Y. Hasegawa, J.
Hirabayashi, H. Narimatsu, K. Mishima, M. Osawa, Inhibition of tumor cellinduced platelet aggregation using a novel anti-podoplanin antibody reactingwith its platelet-aggregation-stimulating domain, Biochem. Biophys. Res.
Commun. 349 (2006) 1301–1307.

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