Research Article
Tumor Biol 2005;26:195–200
DOI: 10.1159/000086952

Received: November 27, 2004
Accepted after revision: February 11, 2005
Published online: July 7, 2005

Enhanced Expression of Aggrus (T1alpha/
Podoplanin), a Platelet-Aggregation-Inducing
Factor in Lung Squamous Cell Carcinoma
Yukinari Katoa, c Mika Kanekoa Makoto Satab Naoya Fujitac
Takashi Tsuruoc, d Motoki OsawaaaDepartment of Experimental and Forensic Pathology and b First Department of Internal Medicine,
Yamagata University School of Medicine, Yamagata, and c Institute of Molecular and Cellular Biosciences,
University of Tokyo, and d Cancer Chemotherapy Center, Japanese Foundation for Cancer Research,
Tokyo, Japan

Key Words
Aggrus  Lung cancer  Platelet aggregation 
Squamous cell carcinoma  Tumor marker

Abstract
Aggrus (T1/podoplanin, known as a specific marker for
type I alveolar cells or lymphatic endothelial cells) is atransmembrane sialoglycoprotein that aggregates platelets. Previously, we showed that upregulated expressionof Aggrus occurs in colorectal tumors or testicular tumorsand could be associated with platelet-aggregating activity and metastatic ability. In testicular tumors, Aggrus isspecifically expressed in seminoma. The present study
investigates Aggrus expression in human primary lungcancer tissues of different types. Microarray analysis
demonstrated that aggrus was significantly expressedin squamous cell carcinoma (10/15; 66.7%). Immunohistochemical analysis also showed that the incidence of
positive staining in sections of squamous cell carcinoma(7/8; 87.5%) was higher than that in adenocarcinoma(2/13; 15.4%). Furthermore, Aggrus expression was detected in a squamous cell carcinoma cell line, NCI-H226,
by real-time PCR. These findings indicated that overexpression of Aggrus occurred in squamous cell carcinomaof the lung. Therefore, Aggrus could be a useful diagnostic marker for squamous cell carcinoma of the lung.
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Introduction

Lung cancer is the leading cause of cancer deathworldwide, accompanied by a continuing increase of patients [1]. The major classiﬁcation of carcinomas consists of small cell lung carcinoma (SCLC) or non-SCLC(NSCLCs). Furthermore, NSCLCs are divided histologically into adenocarcinoma, squamous cell carcinoma,
and large cell carcinoma. The NSCLCs must be diagnosed precisely at the time of surgical resection as distinct
from SCLC because prognosis depends on the histopathological types. Therefore, speciﬁc diagnostic markers
have been sought to evaluate types and malignancy.
We have previously established several clones possessing different platelet-aggregation-inducing capabilities
and metastatic abilities from a mouse colon adenocarcinoma 26 cell line [2]. The expression level of Aggrus, aplatelet-aggregating factor, was correlated with the platelet-aggregating activity and metastatic ability in a mousecolon adenocarcinoma 26 cell line [3, 4]. Cloning of
cDNA revealed that Aggrus was identical to a separately
isolated protein of mouse T1 antigen (also known as
gp38P/OTS-8) [5–7]. In experiments using the 8F11 antibody that neutralized platelet aggregation, the plateletaggregation-stimulating (PLAG) domain was identiﬁed
to a portion spanning amino acid sequence 28–39 ofmouse Aggrus molecules [5]. Amino acid sequences of
PLAG domains are highly conserved in orthologues ofMika Kaneko, PhD, Department of Developmental Neuroscience
Tokyo Metropolitan Institute for Neuroscience
2-6 Musashidai, Fuchu, Tokyo 183-8526 (Japan)
Tel. +81 42 325 3881, Fax +81 42 321 8678
E-Mail mkaneko@tmin.ac.jp or mikanekok@hotmail.com

mouse Aggrus and human Aggrus (also known as hT12/gp36/podoplanin) despite a relatively low overall amino acid identity (39%) [5, 8]. Residual mutagenesis tothreonine in the domain abolished PLAG activities ofboth human and mouse Aggrus. It was also evident thatoverexpression of the aggrus gene occurred in human intestinal cancer tissues or testicular tissues, which was displayed on cancer proﬁling arrays [5, 9].
Aggrus expression was also found in lymphatic endothelium [10], alveolar type I cells in rat lung [11], choroid
plexus, ciliary epithelium of the eye, intestine, kidney,
thyroid and esophagus of the fetal rat [12] and in MC3T3E1 mouse osteoblast cells [7]. Scholl et al. [13] reported
that PA2.26/Aggrus is concentrated in actin-rich microvilli and plasma membrane projections, such as ﬁlopodia,
lamellipodia and rufﬂes, where it colocalizes with members of the ERM (ezrin, radixin, moesin) protein family,
indicating that PA2.26/Aggrus is involved in cell migration. Ramirez et al. [14] generated T1/aggrus null micecontaining a targeted mutation in the T1/aggrus gene
locus. Homozygous null mutant mice die at birth of respiratory failure. Recently, we indicated that sialylated
core 1 structures were critical for the platelet aggregation
activity [15, 16].
This study uses real-time PCR and immunohistochemical analysis to investigate expression of Aggrus inclinical specimens of NSCLCs and cells of four lung cancer cell lines.

Quantitative Real-Time PCR
The ﬁrst-strand cDNA samples of patients 3, 20 and 21 werepurchased from BD Biosciences. Total RNAs were prepared fromfour lung cell lines employing an RNeasy mini prep kit (Qiagen,Hilden, Germany). First-strand cDNAs were synthesized by MMLV reverse transcriptase (USB, Cleveland, Ohio, USA) via priming nine random oligomers according to the manufacturer’s instructions. The primer set for aggrus was as follows: the forward primer
was 5-GGAAGGTGTCAGCTCTGCTC-3 and the reverse primer was 5-CGCCTTCCAAACCTGTAGTC-3. The primer set for-actin was: 5-ACTCTTCCAGCCTTCCTTCCTG-3 and 5ATCTCCTTCTGCATCCTGTCGG-3. Real-time PCR was performed using the QuantiTect SYBR Green PCR (Qiagen). The
PCR conditions were 95 ° C for 15 min for 1 cycle, followed by 40
cycles of 94 ° C for 15 s, 55 ° C for 25 s, 72 ° C for 20 s, and 0.2 ° C s–1
to 40 ° C to perform a melting curve analysis. Standard curves fortemplates of aggrus, and an endogenous control, -actin, were generated by serial dilution of the PCR products (1 ! 108 copies/l to
1 ! 103 copies/l).
Immunohistochemistry
A lung tissue array was purchased from Ambion (Austin, Tex.,
USA). Anti-human Aggrus polyclonal antibody (TT679) was prepared in rabbits by immunization of a synthetic oligopeptide,
CEGGVAMPGAEDDVV, corresponding to amino acids 38–51 of
the human Aggrus sequence plus N-terminal cysteine. Afﬁnity puriﬁcation was performed using a gel column, which contained thechemically linked peptide [5]. The monoclonal antibody to humancytokeratin 19 (BA17) was obtained from Research Diagnostics(Flanders, N.J., USA). The antibodies were incubated with deparafﬁnized and rehydrated sections at a concentration of 1 g/ml at
23 ° C for 4 h. The DAKO CSA system (DakoCytomation, Glostrup,
Denmark) was employed for subsequent development, in which
procedures were performed according to the manufacturer’s instructions.

Materials and Methods
Cell Culture Conditions
Human lung cancer cell lines, A549, NCI-H23, NCI-H226 and
NCI-H522 [17–20] were cultured in RPMI 1640 supplemented
with 10% fetal bovine serum at 37 ° C in humidiﬁed air containing
5% CO2.
Cancer Proﬁling Array Analysis
Cancer Proﬁling Arrays I and II were purchased from BD Biosciences (Braintree, Mass., USA). Cancer Proﬁling Array I consistsof 241 cDNA pairs and Array II consists of 160 cDNA pairs, synthesized from human tumorigenic and corresponding normal tissues. Each pair was independently normalized based on the expression of four housekeeping genes and immobilized in separate dots.
The full-length 32P-labeled human aggrus cDNA was used for hybridization; the radioactivity of each dot was visualized and quantiﬁed with a BAS1000 Bio-Imaging analyzer (Fuji Film, Tokyo,
Japan). The expression level of human aggrus mRNA in individual patients was normalized by measuring the radioactivity of each
dot after hybridization with 32P-labeled ubiquitin cDNA [5]. Statistical signiﬁcance of aggrus mRNA expression in squamous cell
carcinoma and adenocarcinoma tissues was performed using pairedt test.

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Tumor Biol 2005;26:195–200

Results

Expression of aggrus in Lung Squamous Cell
Carcinoma Tissues
Expression of the aggrus gene was estimated for humanlung cancer tissues displayed in the Cancer Proﬁling Arrays I and II. In these arrays, cDNA pairs from primarytumor tissues and corresponding normal tissues from individual patients were immobilized on a nylon membrane. We revealed here the increase in aggrus mRNA
expression in squamous cell carcinoma of the lung compared with adenocarcinoma in Cancer Proﬁling Array I(ﬁg. 1a). Results of Cancer Proﬁling Array II have been
described previously [5]. Signal intensities were detected
semiquantitatively after hybridization with 32P-labeled
cDNA probes of human aggrus and ubiquitin (ﬁg. 1a).
The level of aggrus expression was represented as a relative value to that of ubiquitin expression. The T/N ratio
was more than 1.5 in 10 of 15 squamous cell carcinoma

Kato/Kaneko/Sata/Fujita/Tsuruo/Osawa

N T

N T

a

Aggrus6
Average
5

4
T/N ratioin squamous cell carcinoma of lung. Human aggrus mRNA expression in tumor (T)and normal (N) tissues in the same patient
was assessed by hybridizing 32P-labeled human aggrus cDNA to Cancer Proﬁling Arrays I and II. Radioactivity of each dot was
visualized in Proﬁling Array I (a) and quantiﬁed in Proﬁling Arrays I and II (b). b The
relative aggrus mRNA expression level inthe each dot was normalized by measuring
radioactivity of each dot after hybridization
with 32P-labeled ubiquitin cDNA. The T/Nratio was calculated by dividing the normalized radioactivity of tumor samples withthat of normal tissues in individual patients. Horizontal columns = Average T/Nratio in either adenocarcinoma or squamous cell carcinoma.

Patient No.

Fig. 1. Increased aggrus mRNA expression

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21

3

2

1

0
Adenocarcinoma(n = 12)

Ubiquitinb

Squamous cell
carcinoma(n = 15)

cled in ﬁg. 1a) and performed quantitative real-time PCR
to conﬁrm array analysis results (ﬁg. 2). Consistent withthe array analysis, quantitative PCR analysis revealed
that human aggrus mRNA expression was markedly enhanced in squamous cell carcinoma derived from patients 3 and 20, but not from patient 21.

specimens (67%), in contrast to lower ratios exhibited byadenocarcinoma specimens. As shown in ﬁgure 1b, themean value in squamous cell carcinoma was signiﬁcantly
higher than that in adenocarcinoma (p ! 0.01).
We could obtain cDNA prepared from only 3 squamous cell carcinoma patients (patients 3, 20 and 21 cir-Expression of Aggrus Protein in Lung Squamous Cell
Carcinoma Tissues
Previously, we generated polyclonal antibody to human Aggrus (TT679) since the 8F11 antibody, which canbind to mouse Aggrus protein and neutralize mouse Aggrus-induced platelet aggregation, could not recognize human Aggrus [5]. The TT679 antibody speciﬁcally recognized the 36-kDa human Aggrus protein and immunohistochemically reacted with colorectal tumors or seminoma[5, 9]. TT679 also stained lymphatic vessels in colon tissues like several anti-podoplanin antibodies [9]. In thisstudy, we stained 21 lung cancer specimens (8 squamouscell carcinomas and 13 adenocarcinomas). Strong staining was observed in almost all squamous cell carcinoma
specimens (ﬁg. 3a, c), but specimens from adenocarcinoma patients did not stain (ﬁg. 3e). Furthermore, nostaining was observed in normal lung (data not shown).
Seven of 8 squamous cell carcinoma patients (87.5%)
showed positive staining of Aggrus, whereas only 2 of 13
adenocarcinoma patients (15.4%) showed positive staining (p ! 0.01). Subsequently, we compared the TT679
reactivity with anti-human cytokeratin 19 monoclonal

Enhanced Expression of Aggrus

Tumor Biol 2005;26:195–200

Fig. 2. Quantitative analysis of aggrus transcripts in lung tumorsand corresponding normal tissues by real-time PCR. The ﬁrststrand cDNA samples derived from tumorigenic and corresponding normal tissues of patients 3, 20 and 21 were used as templates
for real-time PCR. The respective expression levels of aggrus werenormalized to that of the -actin transcript. Aggrus/-actin = Aggrus mRNA/-actin mRNA ratio.

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Fig. 3. Aggrus protein was strongly ex-pressed in squamous cell carcinoma of thelung. Immunostaining was done by TT679(a, c, e) or BA17 (b, d, f) with a DAKO CSA
System. Squamous cell carcinoma from patient 31 (a, b), squamous cell carcinoma
from patient 42 (c, d) and adenocarcinoma from patient 35 (e, f) are shown. Bars =10 m.

antibody (BA17), which is used as a tumor marker ofsquamous cell carcinoma (ﬁg. 3b, d, f) [21]. Five of 8 squamous cell carcinoma patients (62.5%) showed positive
staining of cytokeratin 19 by BA17. Interestingly, squamous cell carcinoma specimens from patients 42 and 51
were stained only by TT679, not by BA17, indicating that
TT679 is highly reactive with squamous cell carcinoma
compared with BA17. Of 13 adenocarcinoma specimens,
2 adenocarcinoma specimens (patients 49 and 52) were
stained by both TT679 and BA17, while the others neither stained by TT679 nor by BA17.
Expression of aggrus in Culture Cells Derived from
Lung Cancer
Expression of aggrus was examined further in four established cell lines of human lung cancer. Total mRNA

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Tumor Biol 2005;26:195–200

was prepared from cultured cells of one squamous cell
carcinoma cell line (NCI-H226) and three adenocarcinoma cell lines (NCI-H23, NCI-H522 and A549). In realtime RT-PCR analysis, a high level of aggrus expression
was evident only in NCI-H226 cells, but not in the others(ﬁg. 4).

Discussion

Human Aggrus was expressed in colorectal tumors [5]and testicular tumors [9]. In testicular tumors, Aggrus
expression was high in seminomas, but its expression was
not observed in embryonal carcinomas [9]. On the other
hand, aggrus expression in lung tumors varied from patient to patient in Cancer Proﬁling Array II [5]. Therefore,

Kato/Kaneko/Sata/Fujita/Tsuruo/Osawathis study estimated the human aggrus expression in human lung cancer of different histological types. As shownin ﬁgure 1, we revealed the increase in aggrus mRNAlevel in squamous cell carcinoma of the lung comparedwith corresponding normal lung tissues in Cancer Proﬁling Array. Furthermore, the increase was not seen in adenocarcinoma of the lung, indicating that overexpressionof aggrus is speciﬁc for squamous cell carcinoma. In immunohistochemical analysis, human Aggrus was also speciﬁcally detected in squamous cell carcinoma tissues(ﬁg. 3). Human Aggrus is also known as hT1-2, a speciﬁc marker of type I alveolar cells [8]; furthermore, it wasalso expressed in bronchial epithelium (data not shown).
Squamous cell carcinoma arises in the larger, more central bronchi, while adenocarcinoma are usually more peripherally located. From now on, the reason why Aggrus
was speciﬁcally overexpressed in squamous cell carcinoma should be clariﬁed, although both squamous cell carcinomas and adenocarcinomas were known to be derived
from the bronchial epithelium.
We investigated the expression of aggrus in cell lines
derived from squamous cell carcinoma or adenocarcinoma. As shown in ﬁgure 4, aggrus was speciﬁcally detectedin NCI-H226, a squamous cell carcinoma cell line, by
real-time PCR analysis. Previously, we clariﬁed the expression of Aggrus on NCI-H226 by ﬂow cytometry andshowed the platelet-aggregation-inducing ability of NCIH226 [15]. In our previous studies, we observed the expression of mouse aggrus in a mouse colon adenocarcinoma cell line (NL-17) and a melanoma cell line (B16-

F10) that have platelet-aggregation-inducing capability.
Furthermore, their pulmonary metastatic abilities werehighly associated with their platelet-aggregation-inducing
capabilities [5]. Therefore, NCI-H226 cells might also
have metastatic ability in vivo. Since we could not obtain
sufﬁcient clinical information about patients examinedin this study, we could not discuss the relationship of theexpression level of Aggrus with the clinical prognosis ofsquamous cell carcinoma patients. Further studies, including careful clinical follow-ups of squamous cell carcinoma patients, are needed to evaluate the signiﬁcanceof Aggrus with respect to prognosis. However, it is intriguing to speculate that overexpression of Aggrus in lungsquamous cell carcinoma might be associated with its migrating activitiy [13].
NSCLCs are a heterogeneous group of tumors withvariable clinical courses [1]. However, most studies thathave investigated treatment efﬁcacy for NSCLCs havegrouped squamous cell carcinoma and adenocarcinomaof the lung together as NSCLCs, despite likely biologicaldifferences between the two histological subtypes. Indeed, clinical prognosis is better for squamous cell carcinoma than for adenocarcinoma. Therefore, the availability of a reliable tumor marker that would reﬂect the change
in tumor burden would be helpful to: specify treatment
protocols; determine which patients need more aggressive surgery, adjuvant chemotherapy, or radiotherapy;
monitor treatment efﬁcacy, and to assist in decision-making for further therapy in NSCLCs. For the detection ofchanges in tumor behavior of NSCLCs, several tumor
markers have been evaluated, including carcinoembryonic antigen, squamous cell carcinoma antigen and soluble cytokeratin 19 fragment (CYFRA21-1) [22–24]. Somestudy results have revealed that these markers may contribute to diagnosis, staging, monitoring, and prognosis
of NSCLCs to some extent, but that they are not yet ideal, especially in squamous cell carcinoma [22–24]. Sensitivity of CYFRA21-1, which seems to have the highest
sensitivity in squamous cell carcinoma of lung thus far,is 57% for squamous cell carcinoma and 27% for adenocarcinoma of the lung [24], whereas those of Aggrus are
67 and 0%, respectively, when the cutoff values of the
T/N ratio were deﬁned as 1.5 in Cancer Proﬁling Array.
Regarding immunostaining of Aggrus by TT679, 87.5%
of squamous cell carcinomas and 15.4% of adenocarcinomas showed positive staining. These data indicate a higher sensitivity and speciﬁcity of Aggrus for lung squamouscell carcinoma than CYFRA21-1. The detection of Aggrus protein in this study was limited to the tissues fromlung cancer patients. Therefore, we should produce theEnhanced Expression of Aggrus

Tumor Biol 2005;26:195–200

Fig. 4. Expression of aggrus in the squamous cell carcinoma cellline (SCC). Total RNAs were prepared from the indicated humanlung cancer cell lines. The respective expression levels of aggrus
were normalized to that of the -actin transcript. AC = Adenocarcinoma; aggrus/-actin = aggrus mRNA/-actin mRNA ratio.

199

ELISA system that can detect the serum Aggrus and compare its speciﬁcity with other tumor markers of squamouscell carcinoma.
In conclusion, Aggrus is overexpressed in squamouscell carcinoma of the lung, and NCI-H226 cells, which
expressed Aggrus on the cell membrane, could possess
platelet-aggregation-inducing ability. Therefore, Aggrus
may be associated with platelet-aggregating activity andmetastatic abilities of squamous cell carcinoma of thelung. Furthermore, Aggrus could be a novel sensitive andspeciﬁc tumor marker for patients with lung squamouscell carcinoma. In the future, Aggrus will be useful as atumor marker if it can be detected in the serum of patients.

Acknowledgments
This study was supported in part by a special grant for Advanced
Research on Cancer from the Ministry of Education, Culture,
Sports, Science, and Technology, Japan, by the Public Trust Nishi
Cancer Research Fund (to Y.K.), and by the Kato Memorial Bioscience Foundation (to N.F.).

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