Acta Neuropathol (2006) 111: 483–488
DOI 10.1007/s00401-006-0063-y

OR I G I NA L P AP E R

Kazuhiko Mishima · Yukinari Kato · Mika Kato Kaneko
Ryo Nishikawa · Takanori Hirose · Masao Matsutani

Increased expression of podoplanin in malignant astrocytic tumors
as a novel molecular marker of malignant progression

Received: 30 January 2006 / Revised: 22 February 2006 / Accepted: 6 March 2006 / Published online: 5 April 2006
 Springer-Verlag 2006

Abstract Podoplanin (aggrus) is a mucin-like transmembrane sialoglycoprotein that is expressed on lymphatic endothelial cells. Podoplanin is putatively involvedin cancer cell migration, invasion, metastasis, and malignant progression and may be involved in platelet aggregation. Previously, we showed upregulated expression of
podoplanin in central nervous system (CNS) germinomas, but not in non-germinomatous germ cell tumors,
except for parts of immature teratomas in limited numbers. However, little information exists about its role in
CNS astrocytic tumors. In this study, 188 astrocytic
tumors (30 diVuse astrocytomas, 43 anaplastic astrocytomas, and 115 glioblastomas) were investigated using
immunohistochemistry with an anti-podoplanin antibody,YM-1. In 11 of 43 anaplastic astrocytomas (25.6%) and in
54 of 115 glioblastomas (47.0%), podoplanin was expressedon the surface of anaplastic astrocytoma cells and glioblastoma cells, especially around necrotic areas andproliferating endothelial cells. However, the surrounding
brain parenchyma was not stained by YM-1. On theother hand, podoplanin expression was not observed in
diVuse astrocytoma (0/30: 0%). Furthermore, we investigated the expression of podoplanin using quantitative
real-time PCR and Western blot analysis in 54 frozen

K. Mishima · R. Nishikawa · M. Matsutani
Department of Neurosurgery, Saitama Medical School,
38 Morohongo Moroyama-machi Iruma-gun,
Saitama 350-0495, Japan
Y. Kato (&) · M. K. Kaneko
Research Center for Glycoscience,
National Institute of Advanced Industrial Science andTechnology (AIST), Open Space Laboratory C-2,
1-1-1 Umezono, Tsukuba, Ibaraki 305-8568, Japan
E-mail: yukinari-k@bea.hi-ho.ne.jp
Tel.: +81-29-8613197
Fax: +81-29-8613191
T. Hirose
Department of Pathology, Saitama Medical School,
38 Morohongo Moroyama-machi Iruma-gun,
Saitama 350-0495, Japan

astrocytic tumors (6 diVuse astrocytomas, 14 anaplastic
astrocytomas, and 34 glioblastomas). Podoplanin mRNAand protein expression were markedly higher in glioblastomas than in anaplastic astrocytomas. These data suggest that podoplanin expression might be associated withmalignancy of astrocytic tumors.
Keywords Podoplanin · Astrocytoma · Glioblastoma ·YM-1

Introduction
Astrocytic tumors are the most common tumors of thecentral nervous system (CNS) and are categorized into
diVuse astrocytomas (World Health Organization(WHO) Grade II), anaplastic astrocytomas (AA; WHO
Grade III) and glioblastomas (GBM; WHO Grade IV)[11]. Among them, GBMs are the most frequent andmost malignant type of astrocytic tumor. Despiteadvances in surgical techniques, radiation therapy, andadjuvant chemotherapy, their prognosis remains poor:
the median survival time for patients with GBMs is only
1 year [2]. Glioblastoma may occur de novo or may
result from progression of low-grade astrocytomas [4].
Molecular mechanisms of tumorigenesis and malignant
progression are associated with the inactivation of tumor
suppressor genes such as p53-Rb pathway or the overexpression of oncogenes such as epidermal growth factor
receptor [10]. However, the mechanisms of tumorigenesis
and progression of astrocytic tumors have not been
resolved. IdentiWcation of genes that are expressed diVerentially in high-grade astrocytomas, low-grade tumors,or normal brain tissues is important to elucidate themolecular mechanisms of tumorigenesis and to develop
novel therapeutic strategies.
Podoplanin was reported to be expressed in lymphatic
endothelium and in tumor-associated lymphangiogenesis;
also, podoplanin deWciency resulted in congenital lymphedema and impaired lymphatic vascular patterning[16]. Furthermore, expression of podoplanin has been

484

shown to be upregulated in skin squamous cell carcinoma[12], lung squamous cell carcinoma [9], malignant mesothelioma [14], Kaposi’s sarcoma, angiosarcoma [1],hemangioblastoma [15], testicular seminoma [7, 8], anddysgerminoma [17].
We have recently shown that podoplanin is overexpressed in CNS germinomas, but not in non-germinomatous germ cell tumors, except in a limited number of
immature teratomas with partial positive reactivity [13].
In adult non-neoplastic CNS, podoplanin was evident in
the subependymal areas, the leptomeninges, choroid
plexus, ependyma, and Purkinje cells [15, 17]. However,podoplanin expression in CNS astrocytic tumors has notbeen studied intensively. In this study, we investigatedpodoplanin expression in 188 astrocytic tumors.

Materials and methods
Tissue samples
Tumor specimens were obtained during surgery fromeight patients with diVuse astrocytomas, 14 patients withanaplastic astrocytomas, and 34 patients with glioblastomas. Informed consent had been obtained previously
from patients or their guardians. The tumor specimens
were routinely Wxed in 10% buVered formalin for 18–20 h
at room temperature and processed to paraYn. Sections(5 m thick) were cut and attached to poly-L-lysinecoated glass slides. Hematoxylin-eosin was used for routine staining. Tissue microarrays of 132 astrocytic
tumors (22 diVuse astrocytomas, 29 anaplastic astrocytomas, and 81 glioblastomas) were purchased from Cybrdi,
Inc. (Frederick, MD). The histology of these tissue samples was conWrmed by experienced neuropathologists.
Immunohistochemical analysis
Specimens were deparaVinized, rehydrated, and incubated
Wrst with YM-1 (1/100 diluted; Medical Biological Laboratories Co., Ltd, Nagoya, Japan) at room temperaturefor 1 h, then with biotin-conjugated secondary anti-rat
IgG antibody (DakoCytomation, Glostrup, Denmark)for 1 h, and Wnally 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 (DakoCytomation) for 3 min. Podoplanin
expression was assessed semi-quantitatively from thepercentage of tumor cells with cytoplasmic/membranestaining: 0, no staining; +, <10%; ++, 10–50%; and+++, >50%.
Western blot analysis
Tissues were solubilized with lysis buVer (25 mM Tris
(pH 7.4), 50 mM NaCl, 0.5% Na deoxycholate, 2% nonidet P-40, 0.2% SDS, 1 mM phenylmethylsulfonyl

Xuoride, and 50 mg/ml aprotinin). They were then electrophoresed under reducing conditions on 10–20% polyacrylamide gels (Daiichi Pure Chemicals Co., Ltd, Tokyo,
Japan). The separated proteins were transferred to anitrocellulose membrane. After blocking with 4% skim
milk in PBS, the membrane was incubated with YM-1
(1/500 diluted) or anti--actin antibody (1 g/ml; Sigma
Chemical Co., St. Louis, MO), and then with peroxidaseconjugated secondary antibodies (1/1,000 diluted; Amersham Pharmacia Biotech UK Ltd, Buckinghamshire,UK). The proteins were subsequently developed for 3 minusing ECL reagents (Amersham Pharmacia Biotech)using X-Omat AR Wlm (Eastman Kodak Co.).
Quantitative real-time PCR
Total RNAs were prepared from frozen sections thathave been obtained from astrocytic tumor patients,
employing an RNeasy mini prep kit (Qiagen, Inc.,
Hilden, Germany). The initial cDNA strand was synthesized using SuperScript III transcriptase (Invitrogen Co.,
Carlsbad, CA) by priming nine random oligomers andan oligo-dT primer according to the manufacturer’s
instructions. We performed PCR using oligonucleotides:
human podoplanin sense (5⬘-GGAAGGTGTCAGCT
CTGCTC-3⬘) and human podoplanin antisense (5⬘-CG
CCTTCCAAACCTGTAGTC-3⬘). Real-time 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, 72°C for 10 s. Subsequently, a melting curve program
was applied with continuous Xuorescence measurement.
A standard curve for podoplanin templates was generated through serial dilution of PCR products (1£108–
1£102 copies/l). The expression level of podoplanin was
normalized by total RNA weights. The statistical signiW
cance of podoplanin mRNA expression in astrocytic
tumor tissues was determined using paired t tests.

Results
Immunohistochemical staining for podoplanin
in malignant astrocytic tumors
The cellular distribution of podoplanin in astrocytic
tumors was examined immunohistochemically using
anti-podoplanin antibody, YM-1, which can strongly
recognize podoplanin [6, 13]. In this study, we used 56
surgical tissues (8 diVuse astrocytomas, 14 anaplastic
astrocytomas, and 34 glioblastomas). Podoplanin immunoreactivity was detected in 5 of 14 (35.7%) anaplastic
astrocytomas and in 18 of 34 (52.9%) glioblastomas;
staining was graded as +++ in 16 glioblastomas and as++ in two glioblastoma cases. We also stained otherastrocytic tumors of tissue microarrays. Podoplanin was
detected in 6 of 29 (21%) anaplastic astrocytomas and in
36 of 81 (44%) glioblastomas. In all, 11 of 43 anaplastic

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astrocytomas (25.6%) and 54 of 115 (47.0%) glioblastomas were stained using YM-1 (2, P<0.05; Table 1).
Representative staining for podoplanin in glioblastoma
samples is shown in Fig. 1. Immunostaining for podoplanin demonstrated predominantly cell surface patterns inglioblastoma cells (Fig. 1). In anaplastic astrocytoma, thetumor cell surface was stained using YM-1 (Fig. 1c, d). In
glioblastomas, podoplanin-positive tumor cells wereprominent around microvascular proliferations (Fig. 1e, f)
and necrotic tissues (Fig. 1g). Proliferating endothelial
cells were negative for podoplanin (Fig. 1e, f). Podoplanin was detected strongly in the plasma membrane ofhighly anaplastic multinucleated giant cells (Fig. 1h). In
non-neoplastic areas of the brain (Fig. 1a) and in diVuse
astrocytoma (Fig. 1b), podoplanin immunostaining wasabsent.
Podoplanin expression in malignant astrocytic tumors
using Western blot analysis
To conWrm immunohistochemical Wndings from astrocytic tumors, lysates of frozen tumor specimens from 54
patients (6 diVuse astrocytomas, 14 anaplastic astrocytomas, and 34 glioblastomas) were analyzed using Western
blotting. As shown in Fig. 2, an antibody to podoplanin,
YM-1, detected about 36-kDa proteins in extracts ofmalignant astrocytomas. Using YM-1, 6 of 14 anaplastic
astrocytomas (42.8%) and 22 of 34 glioblastomas (64.7%)
showed strong labeling (2, P<0.05), while all diVuse
astrocytomas were negative. Podoplanin expression
detected by Western blot analysis was closely correlatedwith the results of immunohistochemistry.
DiVerential expression of podoplanin mRNA in
astrocytic tumors
To quantify the expression of podoplanin mRNA in
human astrocytic tumors of diVerent grades, we performed quantitative real-time PCR analyses of astrocytic
tumors from 54 patients (6 diVuse astrocytomas, 14 anaplastic astrocytomas, and 34 glioblastomas). The relative
podoplanin mRNA expression levels of each tumor
grade are shown in Fig. 3. Average copies of podoplaninmRNA/g total RNA in diVuse astrocytomas (GradeII), anaplastic astrocytomas (Grade III), and glioblasto-

Table 1 Results of podoplanin
immunostaining in 188 patientswith astrocytic tumors

Tumor type

DiVuse astrocytoma
Surgical resection samples
Tissue microarray
Anaplastic astrocytoma
Surgical resection samples
Tissue microarray
Glioblastoma
Surgical resection samples
Tissue microarraymas (Grade IV) were 21.7§30.6, 16.1§35.4, and 411.2§
511.7, respectively. Podoplanin transcript levels were signiWcantly higher in glioblastomas than those in diVuse
astrocytomas, anaplastic astrocytomas, or non-neoplastic human brain tissues (P<0.01).

Discussion
Immunohistochemical, Western blot, and real-time PCR
analyses demonstrate that the expressions of podoplaninmRNA and protein are correlated with the malignant
progression from anaplastic astrocytoma (Grade III) to
glioblastomas (Grade IV). Of particular interest, 47.0%
of highly invasive glioblastomas express podoplanin,whereas 25.6% of anaplastic astrocytomas and 0% of theless invasive diVuse astrocytomas (Grade II) express
podoplanin by immunohistochemical staining on surgically resected and microarray tissues (Table 1). On theother hand, 6 of 14 anaplastic astrocytomas (42.8%) and
22 of 34 glioblastomas (64.7%) were strongly labeled byWestern blot analyses using YM-1 (Fig. 2). Of all 188
astrocytic tumors analyzed immunohistochemically, 132cases were derived from tissue microarrays whose tissue
spots are small and, therefore, the percentage of podoplanin-positive tumors might have been underestimated.
Furthermore, YM-1 detected podoplanin strongly byWestern blot analysis, as described previously [6]. For
these reasons, podoplanin-positive ratios in immunohistochemical analysis are inferred to be smaller than thoseof Western blot analysis, although the expression of
podoplanin detected by immunohistochemistry wasclosely correlated with that by Western blot or real-time
PCR analyses. The distribution of podoplanin-positive
tumor cells was prominent around necrotic tissue andproliferating endothelial cells in glioblastomas (Fig. 1).
Normal brain tissue surrounding the tumor bed was negative for podoplanin. Therefore, podoplanin expression
was correlated with high tumor grades and aggressive
histological behavior.
The biological functions of podoplanin remain largely
unknown. In vascular endothelial cells, overexpression of
T1/podoplanin induces elongated cell extensions andconsiderably increases cell adhesion, migration, and tube

No. of
cases
30
8
22
43
14
29
115
34
81

Podoplanin immunostaining

Positive
rate (%)

+++

++

+

¡

0
0
0
6
3
3
39
16
23

0
0
0
3
1
2
10
2
8

0
0
0
2
1
1
5
0
5

30
8
22
32
9
23
61
16
45

0
0
0
25.6
35.7
20.7
47
52.9
44

486

Fig. 1 Immunohistochemical detection of podoplanin in astrocytic
tumors. No staining is apparent in a normal brain (a, £200) and in
diVuse astrocytoma (b, £200). In anaplastic astrocytoma, the tumor
cell surface was stained positively (c, £200; d, £400). Accentuatedstaining is visible around an area of microvascular proliferation in

glioblastoma (e, £200; f, £400). Podoplanin immunostaining of
glioblastoma cells at the necrotic area (N) (g, £200) and in the plasma membrane of highly anaplastic multinucleated giant cells(h, £400). Bar=10 m

487
Fig. 2 Western blot analyses of
podoplanin expression in astrocytic tumors. Tissues from normal brain (a), diVuse
astrocytomas (b), anaplastic
astrocytomas (c), and glioblastomas (d) were solubilized andimmunoblotted using anti-human podoplanin monoclonal
antibody YM-1 (upper panel) or
anti--actin antibody (lower
panel)b diffuse astrocytoma

a normal brain
(kD)
45

1

2 3

(kD)
45

4

1

2 3

4

β -actin

6

Podoplanin

Podoplanin
30
45

5

30
45

β-actinc anaplastic astrocytoma
(kD)
45

1

2 3

4 5 6 7 8 9 10 11 12 13 14

Podoplanin3045

β -actind glioblastoma
(kD)
45

1

2

3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 2122 23 2425 26 2728 29 30 31 32 33 34

Podoplanin3045

β -actin

Podoplanin copies / µg total RNA

formation by promoting the rearrangement of the actin
cytoskeleton [16]. PA2.26/podoplanin was identiWed as acell surface protein induced in epidermal carcinogenesis
and skin remodeling [18, 19]. Expression of PA2.26/
podoplanin in pre-malignant keratinocytes induces afully transformed and metastatic phenotype. Furthermore, human PA2.26/podoplanin has been found in the2000

p < 0.01

1500

1000

N. S.
500

N. S.

N. S.

glioblastoma

anaplastic
astrocytoma

diffuseastrocytoma

normal0

Fig. 3 Quantitative real-time PCR analysis of podoplanin transcripts in astrocytic tumors. First-strand cDNA samples derived
from astrocytic tumor tissues of 54 patients (6 diVuse astrocytomas,
14 anaplastic astrocytomas, and 34 glioblastomas) and four normal
brain tissues were used as real-time PCR templates. The respective
expression levels of podoplanin were normalized to g or total
RNA, as described in Materials and methods

invasive front of oral squamous cell carcinomas, consistent with a role in tumor cell migration and invasion [12].
Moreover, a monoclonal antibody against gp44/aggrus/
podoplanin inhibits pulmonary metastasis of a highly
metastatic clone of mouse colon adenocarcinoma in vivo
[21, 22]. In this study, we showed upregulated expressionof podoplanin in CNS malignant astrocytic tumors.
Recently, Shibahara et al. [20] also reported podoplanin
expression in subsets of CNS tumors. However, theresults obtained so far showed only associations between
podoplanin expression and malignancy of astrocytic
tumors, while its direct biological function in malignant
astrocytomas remains to be established.
PA2.26/podoplanin was co-localized with ezrin,
radixin, moesin family proteins, which are concentrated
in cell surface projections, where they link the actin cytoskeleton to plasma membrane proteins [18]. Consistent
with the association of podoplanin with ezrin, the latter’s
immunoreactivity is also associated with increasing
malignancy of astrocytic tumors [3, 23]. The combinationof podoplanin and ezrin might thus represent a possible
tool for grading of astrocytic tumors.
Platelets play an important role in hemostasis andthrombosis and are also involved in tissue repair andtumor metastasis [5]. Glioblastoma is diVerentiated fromlow-grade astrocytomas based on the histological presence of tumor necrosis and associated microvascularproliferation [11]. Large necroses are attributable toinsuYcient blood supply and thrombosed tumor vessels
are often observed. We speculate that the local platelet
aggregation and thrombus formation might be increased
by podoplanin-expressing malignant astrocytic tumor
cells, resulting in tumor vessel obstruction and subsequentnecrosis. Indeed, our unpublished results suggest that
podoplanin expressed by glioblastoma cells induces
platelet aggregation in vitro (data not shown).

488

In conclusion, podoplanin expression was markedly
higher in glioblastomas than in anaplastic astrocytomas.
Furthermore, podoplanin expression was not observed in
diVuse astrocytoma. It will be intriguing to investigate thefunctional basis of the association between podoplanin
expression and malignant progression of astrocytomas.
Acknowledgments This study was supported in part by Kanae
Foundation for Life and Socio-medical Science (to Y.K.) and by
Osaka Cancer Research Foundation (to Y.K.). We thank Drs Fujita
and Tsuruo (University of Tokyo) for their great help, and Ms Kunita,
Mr Nakazawa (University of Tokyo), Ms Totake, and Ms Kobo
(Saitama Medical School) for their kind assistance. We thank
Dr Sugiyama (Hiroshima University) for providing us clinical samples.

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