ORIGINAL ARTICLE

Podoplanin is a novel myoepithelial cell markerin pleomorphic adenoma and other salivary gland tumorswith myoepithelial differentiation

Masayuki Tsuneki & Satoshi Maruyama &

Manabu Yamazaki & Ahmed Essa & Tatsuya Abé &

Hamzah Ali Babkair & Md Shahidul Ahsan & Jun Cheng &

Takashi Saku

Received: 11 September 2012 /Revised: 21 November 2012 /Accepted: 10 December 2012 /Published online: 22 December 2012# Springer-Verlag Berlin Heidelberg 2013

Abstract The expression of podoplanin, one of the rep-resentative immunohistochemical markers for lymphaticendothelium, is upregulated in various kinds of cancers.Based on our previous studies, we have developed ahypothesis that podoplanin plays a role in cell adhesionvia its association with extracellular matrix (ECM). Sincesalivary pleomorphic adenoma is histologically character-ized by its ECM-enriched stroma, we firstly wanted toexplore the expression modes of podoplanin in pleomor-phic adenoma and related salivary tumors by immunohis-tochemistry. In normal salivary gland, podoplanin wasspecifically localized in myoepithelial cells, which werealso positively labeled by antibodies against P63, of theintercalated duct as well as acini. In pleomorphic adeno-ma, podoplanin was colocalized with P63 and CD44 inbasal cells of glandular structures as well as in stellate/spindle cells in myxochondroid matrices, where perlecanand hyaluronic acid were enriched. The expression ofpodoplanin was confirmed at both protein and mRNAlevels in pleomorphic adenoma cell systems (SM-AP1and SM-AP4) by using immunofluorescence, westernblotting, and reverse transcription polymerase chain

reaction. Podoplanin was localized on the cell border aswell as in the external periphery of the cells. Moreover,podoplanin expression was also confirmed in tumor cellswith myoepithelial differentiation in myoepithelioma andintraductal papilloma. The results indicate that podoplanincan be regarded as a novel myoepithelial marker insalivary gland tumors and suggest that podoplanin’s com-munication with ECM molecules is essential to pheno-typic differentiation to myoepithelial cells.

Keywords Podoplanin . Myoepithelial cell . Pleomorphicadenoma . Salivary gland tumors . Extracellular matrix .

CD44 . Immunohistochemical marker . SM-AP cells

Introduction

Podoplanin, a sialomucin-like type I transmembrane gly-coprotein, which was originally identified in glomerularpodocytes [1], has been widely utilized as one of themost reliable markers for lymphatic endothelium [2].Recently, its expression has been documented in suchbenign and malignant tumors as thymoma [3], osteosar-coma [4], and laryngeal squamous cell carcinoma [5]. Wehave also immunolocalized podoplanin in odontogenictumors [6], as well as in carcinoma in situ (CIS) andsquamous cell carcinoma (SCC) of the oral mucosa [7,8]. In our previous studies, we suggested the function ofpodoplanin in communication with extracellular matrix(ECM) molecules. In addition, we were successful indemonstrating that podoplanin tethers oral SCC cells tohyaluronan-rich extracellular matrices in collaborationwith CD44 [8]. In our series of investigations, we have

M. Tsuneki : S. Maruyama :M. Yamazaki :A. Essa : T. Abé :H. A. Babkair :M. S. Ahsan : J. Cheng : T. Saku (*)Division of Oral Pathology, Department of Tissue Regenerationand Reconstruction, Niigata University Graduate School ofMedical and Dental Sciences, 2-5274 Gakkocho-dori, Chuo-ku,Niigata 951-8514, Japane-mail: tsaku@dent.niigata-u.ac.jp

S. Maruyama : T. Abé : T. SakuOral Pathology Section, Department of Surgical Pathology, NiigataUniversity Hospital, Niigata, Japan

Virchows Arch (2013) 462:297–305DOI 10.1007/s00428-012-1359-z

emphasized that podoplanin is expressed in neoplasticconditions for tumor cell adhesion to ECM, which maysecondarily affect cellular proliferation.

However, podoplanin, which was originally found innormal podocytes, has also been found in normal myoepi-thelial cells of the mouse submandibular glands [9]. Becauseof our investigations, the expression of podoplanin in myoe-pithelial cells seems to be reasonably accepted because thecells are located between salivary epithelial cells and thebasement membrane or extracellular milieu. Thus, we haveformulated the hypothesis that podoplanin should beexpressed in salivary tumor cells with myoepithelial differ-entiation, such as those in pleomorphic adenoma andmyoepithelioma.

As myoepithelial markers, actin, myosin, S-100 pro-tein, calponin, and P63 protein have been proposed forimmunohistochemical use [10]. However, the sensitivi-ties of antibodies against those molecules vary frommolecule to molecule, and it is not always clear wheth-er those molecules are localized due to either theirmyoepithelial character or their basal location. In thepresent study, we have attempted to investigate theexpression of podoplanin in pleomorphic adenoma,intraductal papilloma, and myoepithelioma, in whichtumor cells show myoepithelial differentiation, and toalso examine whether podoplanin could be a myoepi-thelial marker in salivary gland tumors and whether it isexpressed for the basal location or for the myoepithelialdifferentiation.

Materials and methods

Clinical samples

Ten cases of salivary gland pleomorphic adenoma and nor-mal submandibular gland, six of intraductal papilloma frompalatal minor salivary glands, and seven of myoepitheliomawere collected for the present study from the surgical pa-thology files of the Division of Oral Pathology, NiigataUniversity Graduate School of Medical and DentalSciences, during a 14-year period from 1998 to 2011 afterhistopathological review of those specimens. Of the tencases of pleomorphic adenoma, there were two from sub-mandibular gland, two from parotid gland, four from palatalminor salivary gland, and two from lip minor salivary gland.Of the nine cases of myoepithelioma, two were from sub-mandibular gland, one was from parotid gland, and six werefrom palatal minor salivary gland. Four types of histopath-ological varieties—epithelioid, clear, stellate, and spindletumor cells—were included in the nine cases of myoepithe-lioma. The surgical samples were fixed in 10 % formalinand routinely embedded in paraffin. Serial sections cut at

4 μm from paraffin blocks were used for hematoxylin andeosin (H-E) staining and immunohistochemistry. The exper-imental protocol for analyzing surgical materials wasreviewed and approved by the Ethical Board of the NiigataUniversity Graduate School of Medical and Dental Sciences(Oral Life Science).

Cell systems and reagents

Salivary gland pleomorphic adenoma cell systems (SM-AP1and SM-AP4) were established from pleomorphic adenomaarising in the parotid gland [11]. Pleomorphic adenoma cellswere cultured in Dulbecco’s modified Eagle medium(Gibco, Invitrogen Corporation, Carlsbad, CA, USA) whichcontained 10 % fetal bovine serum (Gibco), 50 μg/ml strep-tomycin, and 50 IU/ml penicillin (Gibco) [9]. They wereincubated at 37 °C in a humidified 5 % carbon dioxide/95 %air atmosphere.

Antibodies

Mouse monoclonal antibodies against human podoplanin(D2-40, IgG1), keratin (K) 19 (RCK108, IgG1), and p63protein (P63) (4A4, IgG2a) were obtained from Dako(Glostrup, Denmark). A rabbit monoclonal antibody againsthuman CD44 (EPR1013Y) was purchased from Epitomics,Inc. (Burlingame, CA, USA). Antibodies against the mousebasement membrane-type perlecan core protein were raisedin rabbits as described elsewhere [10] and have been shownto recognize human perlecan core protein epitopes [13, 14].A mouse monoclonal antibody to human hyaluronic acid(NDOG1, IgM) was purchased from AbD Serotec(Kidlington, UK). A mouse monoclonal antibody to humanβ-actin (mAbcam 8226, IgG1) was purchased from Abcamplc. (Cambridge, UK). Mouse and rabbit preimmuneIgGs for control experiments were also obtained fromDako.

Immunoperoxidase staining

Immunohistochemistry was performed using the ChemMateEnvision™ system (Dako) as described elsewhere [14]. ForK19, sections were pretreated with 0.2 % trypsin (type II,Sigma Chemical Co, St. Louis, MO, USA) in 0.01 M Tris–HCl (pH 7.6) containing 0.1 % CaCl2 for 30 min at 37 °C[14]. For P63, sections were autoclaved in citric acid buffer(pH 6.0) at 121 °C for 10 min according to the manufac-turer’s instructions. The sections were treated with 0.3 %hydrogen peroxide in methanol for 30 min at room temper-ature to block endogenous peroxidase activity and incubatedwith 5 % milk protein (Morinaga Milk Industry Co. Ltd,Tokyo, Japan) in 0.01-M phosphate-buffered saline (PBS,pH 7.4) containing 0.05 % Triton X-100 (T-PBS) for 1 h at

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room temperature to block nonspecific protein binding sites.They were then incubated overnight at 4 °C with the prima-ry antibodies diluted at 1:50 (anti-podoplanin and P63) and1:100 (anti-K19) in T-PBS. After overnight incubation, thesections were incubated with the Envision reagents for1 h at room temperature and treated with 0.02 % 3,3′-diaminobenzimine in 0.05-M Tris–HCl buffer (pH 7.6)containing 0.005 % hydrogen peroxide to visualize thereaction products [14]. Finally, the sections were coun-terstained with Dako REAL™ Hematoxylin (Dako). Forcontrol studies, the primary antibodies were replacedwith preimmune IgGs.

Immunofluorescence

Immunofluorescence experiments for pleomorphic adenomacell systems were performed using Lab-Tek™ II ChamberSlide system (Labtek, Scotts Valley, CA, USA). Cells were

plated at the concentration of 1.0×104 cells/well and culti-vated for 72 h. They were washed with PBS and fixed with4 % paraformaldehyde in 0.1-M phosphate buffer (pH 7.4)for 30 min on ice. To prevent nonspecific protein binding,they were incubated with 2 % normal goat serum (Dako) inT-PBS for overnight at 4 °C. The cells were then incubatedwith the primary antibody (anti-podoplanin, diluted at 1:50in T-PBS) and further with secondary antibody (AlexaFluor™ 568-conjugated goat anti-mouse IgG (H+L)(Invitrogen) diluted at 1:200 in T-PBS) for 1 h each at roomtemperature. Finally, cells were counterstained withCellstain™ Hoechst-33258 solution (Dojindo Laboratories,Kumamoto, Japan) diluted at 1:100 in PBS. Double-immunofluorescence studies were performed using paraffinsections from surgical specimens of pleomorphic adenoma.For podoplanin vs. CD44, sections were autoclaved in citricacid buffer (pH 6.0) at 120 °C for 10 min [8] and then blockedwith 5 % milk protein in PBS at room temperature for 1 h.

Fig. 1 Podoplanin expressionin normal submandibular gland.Hematoxylin and eosin (HE)(a) and immunoperoxidasestains for podoplanin (b, e),keratin (K) 19 (c), P63 (d, f),hematoxylin counterstain (a, c,e, f ×250; b, d ×300; scale bars,50 μm). In normal salivarygland (serous gland parts) (a),podoplanin was definitely lo-calized in the flat and spindle-shaped myoepithelial cells (b),which were simultaneouslypositive for P63 (d), of interca-lated ducts, inner epithelial cellsof which were contrastivelypositive for K19 (c). Also inacini and intercalated ducts ofmucous gland parts, myoepi-thelial cells were clearly dem-onstrated by their positivitiesfor podoplanin (e) and P63 (f)

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They were further incubated with the primary antibodiesdiluted in T-PBS (anti-podoplanin and anti-hyaluronan, 1:50;anti-CD44, 1:250; and anti-perlecan, 1:1000) overnight at 4 °C.The sections were then further incubated with the combinationsof the secondary antibodies as follows: for podoplanin vs.CD44 and podoplanin vs. perlecan, Alexa Fluor™ 568-conjugated goat anti-mouse IgG (H+L) and Alexa Fluor™488-conjugated goat anti-rabbit IgG (H+L) (Invitrogen); forpodoplanin vs. hyaluronan, Alexa Fluor™ 488-conjugated goatanti-mouse IgG (H+L) (Invitrogen) and goat anti-mouse IgMrhodamine (Tago Inc., Burlingame, CA, USA).

Reverse transcriptase-polymerase chain reaction

Total RNA was isolated from the cells using the ISOGENsystem (Nippon Gene Co., Ltd., Tokyo, Japan). First-strandcDNA was synthesized with the SuperScript™ III First-Strand Synthesis System (Invitrogen). Oligonucleotide pri-mers used in this study were as follows: podoplanin-forward, 5 ′-CCAAG CGCCA CAGCC TCAA-3 ′ ;podoplanin-reverse, 5 ′-GGCAC AGAGT CAGAAACGGT-3′; β-actin-forward, 5′-TCACC CACAC TGTGCCCATC TACGA-3′; and β-actin-reverse, 5′-CAGCGGAACC GCTCA TTGCC AATGG-3′ [8]. The thermocy-cling protocol during 28 amplification cycles was as fol-lows: denaturation at 94 °C for 1 min, annealing at 62 °C for1 min, extension at 72 °C for 1 min, and termination with afinal extension at 72 °C for 7 min. The amplified DNA

fragments were analyzed by 2 % agarose gel electrophoresis.Amplicon sizes for podoplanin and β-actin were 265 and295 bp, respectively.

Western blotting

Cell cultures in 60-mm dishes were lysed with 350 μl oflysis buffer (50 mM HEPES (pH 7.4), 150 mM NaCl,1 % Triton X-100, a protease inhibitor cocktail (NacalaiTesque, Kyoto, Japan), and 1 mM PMSF), and the su-pernatant of the cell lysates was recovered. After thetotal protein was determined, an aliquot of 10 μg ofproteins was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) underreducing conditions, and the gels were transferred topolyvinylidene difluoride (PVDF) membranes (Bio-RadLaboratories, Inc., Hercules, CA, USA). After incubationfor 1 h at room temperature with 0.5 % enhanced chemi-luminescence (ECL) blocking agent (GE Healthcare UKLtd., Buckinghamshire, UK) in 50 mM Tris-bufferedsaline (pH 7.4) containing 0.1 % Tween-20 (T-TBS),the PVDF membranes were further incubated overnightat 4 °C with primary antibodies diluted with T-TBS(anti-podoplanin, 1:100; anti-β-actin, 1:5,000). Followingwashing steps with T-TBS, the membranes were reactedwith secondary antibodies (ChemMate Envision™reagent, Dako, diluted at 1:1,000 in T-TBS) for 1 h atroom temperature [8]. Target protein bands were

Fig. 2 Immunohistochemicalprofiles for podoplanin inglandular structures ofpleomorphic adenoma. HE (a)and immunoperoxidase stainsfor podoplanin (b), keratin (K)19 (c), and P63 (d), hematoxy-lin counterstain (a, ×120; b–d,×250; scale bars, 100 μm). Inglandular structures of pleo-morphic adenoma (a), podopla-nin was characteristicallylocalized in the outer cells (b),which were also positive forP63 (d), indicating their myoe-pithelial differentiation. In con-trast, the inner cells werepositive for K19, indicatingtheir duct epithelium-like dif-ferentiation (c)

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visualized by ECL Plus™ western blotting detectionreagents (GE) according to the manufacturer’s instruction.

Results

Podoplanin localization in normal salivary gland

In normal submandibular gland (Fig. 1a), as well as inminor salivary gland (data not shown) obtained simulta-neously with salivary gland tumors in surgical specimens,podoplanin was characteristically localized in myoepithe-lial cells (Fig. 1b), which mantled K19+ duct epithelialcells of intercalated ducts (Fig. 1c) or acinar cells. Thepodoplanin localization was basically the same as that of

P63 protein, which has been regarded as one of the repre-sentative myoepithelial cell markers (Fig. 1d). Similar lo-calization profiles between podoplanin (Fig. 1e) and P63(Fig. 1f) in myoepithelial cells were confirmed in acini andintercalated ducts.

Podoplanin localization in pleomorphic adenoma

Histopathologically, two major types of tumor cell arrange-ments were distinguished in pleomorphic adenoma: one wasglandular structures which were composed of inner ductepithelium-like cells and outer myoepithelium-like cells(Fig. 2a), and the other was stellate/spindle-shaped cellsscattered in the background of myxochondroid stroma(Fig. 3a). In glandular structures (Fig. 2a), podoplanin

Fig. 3 Comparativeimmunohistochemical profilesbetween podoplanin, CD44,and hyaluronic acid inmyxochondroid stroma ofpleomorphic adenoma. HE (a)and immunoperoxidase stainsfor podoplanin withhematoxylin counterstain (b).Double immunofluorescencefor podoplanin (red) (c) vs.CD44 (green) (d) and theirmerge (e); for podoplanin (red)vs. perlecan (green) (f); and forpodoplanin (red) vs. hyaluronan(green) (g). (a, b ×120; c–e,×250; f, g ×300; scale bars,100 μm). In the myxochondroidstroma of pleomorphicadenoma, stellate- or spindle-shaped tumor cells werescarcely scattered (a). Podopla-nin (b) and CD44 (d) werecolocalized in those tumor cells(e), which were embedded inperlecan-rich (f) andhyaluronan-rich (g) extracellu-lar matrix

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(Fig. 2b) was distinctively localized in the outer cells, whichwere simultaneously positive for P63 (Fig. 2d) but not in theinner cells, which were positive for K19 (Fig. 2c). In themeantime, stellate/spindle-shaped tumor cells were definite-ly positive for podoplanin (Fig. 3b). Those podoplanin+stellate/spindle cells (Fig. 3c) were simultaneously positivefor CD44, one of the representative hyaluronan receptors(Fig. 3d, e). The myxochondroid stroma, in which podopla-nin+/CD44+ stellate/spindle-shaped cells were embedded,was positive for perlecan (Fig. 3f) as well as hyaluronic acid(Fig. 3g). The results indicated that podoplanin was specif-ically expressed in pleomorphic adenoma cells which weredifferentiated towards myoepithelium.

Podoplanin biosynthesis in pleomorphic adenoma cellsystems

The podoplanin expression both at gene and protein levelswas confirmed in SM-AP1 cells as well as in SM-AP4 cellsof pleomorphic adenoma-derived cell systems [9]. In immu-nofluorescence, podoplanin was characteristically localizedin the cell border as well as in the external periphery of thosetumor cells in culture, and its immunofluorescence modeswere stable in both low (Fig. 4a, left) and high (Fig. 4a,right) cell densities. In western blot analysis, smear patternsof protein bands with a molecular mass of around 40 kDawere obtained in similar fashions in the two cell systems(Fig. 4b), indicating glycosylation of podoplanin. RT-PCRrevealed a single band with 265 bp in SM-AP1 as well asSM-AP4 cells (Fig. 4c).

Podoplanin localization in the other salivary gland tumors

In intraductal papillomas (Fig. 5a), podoplanin was character-istically localized in the outer cells of glandular structures(Fig. 5b). Myoepitheliomas were histopathologically classifiedinto four major types: epithelioid cell (Fig. 5c), clear cell(Fig. 5e), plasmacytoid (Fig. 5g), and spindle cell (Fig. 5i).Podoplanin was demonstrated in any tumor cell types ofmyoepithelioma, though the staining intensity and stabilityvaried from area to area (Fig. 5b, d, f, h, j). The podoplaninpositivity in those tumor cells in varied shapes indicated that itcould possibly be a new molecular marker covering a widerange of myoepithelial differentiation in salivary gland tumors.

Discussion

In the present study, we have demonstrated for the first timethat podoplanin was a novel immunohistochemical markerfor myoepithelial cell differentiation in salivary glandtumors including pleomorphic adenoma, in which its char-acteristic hyaluronan-rich myxoid stroma seemed to be reg-ulated by podoplanin in collaboration with CD44 both attissue and cell levels. As far as we can ascertain from theliterature, this is the first trial to document podoplaninexpression profiles in salivary gland tumors.

Podoplanin has been utilized as one of the representativelymphatic endothelial cell markers [2]. In the meantime,various kinds of tumor cells have been shown to expresspodoplanin [3–5], and this fact has recently attracted theattention of cancer researchers. In the field of oral cancer,

Fig. 4 Gene and protein level expression of podoplanin in pleomor-phic adenoma cell systems. a Immunofluorescence for podoplanin(red) in SM-AP1 cell colonies with low (left) and high (right) densities,Hoechest-33258 (blue) counterstain. (Left, ×300; right, ×200; scalebars, 50 μm). b Western blot analysis for podoplanin (40 kDa) andβ-actin (42 kDa) in SM-AP1 and SM-AP4 cell lysates. c Reverse

transcriptase-polymerase chain reaction analysis for podoplanin(265 bp) and β-actin (295 bp) in SM-AP1 and SM-AP4 cells, pleo-morphic adenoma cell system. The podoplanin expression was con-firmed in SM-AP1 and SM-AP4 at both gene (c) and protein (b) levels.Morphologically, it was localized in the cell border or in the externalperiphery (a) of SM-AP1 cells

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Fig. 5 Immunohistochemicalprofiles for podoplanin insalivary gland tumor withmyoepithelial differentiation. a,b Intraductal papilloma; c–jmyoepithelioma: epithelioid (c,d), clear (e, f), plasmacytoid (g,h), and spindle (i, j) cell types.(a, c, e, g, i) HE stain; (b, d, f,h, j) immunoperoxidase stainfor podoplanin, hematoxylincounterstain (a–j, ×200; scalebar, 50 μm). In glandularstructures of intraductalpapilloma (a), podoplanin wasspecifically localized in theouter cells with myoepithelialcharacteristics (b). Also inmyoepithelioma, podoplaninwas demonstrated in all of thefour cell types (epithelioid (c,d), clear (e, f), plasmacytoid(g, h), and spindle (i, j)) ofmyoepithelioma

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podoplanin expression has been correlated with poor clini-cal outcomes in terms of survival rate [15–16], nodal me-tastasis [15], and malignant transformation of precancerouslesions [18], while opposite results have also been reported[8]. To elucidate molecular functions of podoplanin, wehave paid special attention to its association with ECMbecause podoplanin+ cells overlapped with Ki-67/PCNA-positive cells facing ECM deposits [19, 20] in oral CIS/SCC[7] and odontogenic tumors [6] in our morphological stud-ies. Our recent in vitro study revealed that podoplanincontributed with CD44 to adhesion of SCC cells ontohyaluronan-rich ECM [8]. From these lines of evidence,we have come to be interested in podoplanin expressionprofiles in pleomorphic adenoma because its colorful histo-logical feature is characterized by the accumulation of var-ious kinds of ECM molecules [21, 22]. In addition,podoplanin had already been localized in myoepithelial cellsof murine salivary glands [9], and thus we have hypothe-sized that podoplanin must be expressed also in neoplasticmyoepithelial cells.

In glandular structures of pleomorphic adenoma, podo-planin was specifically localized in the outer cells withP63 positivities, which were considered as myoepithelialcell characteristics. Podoplanin+ stellate/spindle-shapedcells in the myxochondroid stroma have been consideredas modified myoepithelial cells [22, 23]. In the presentstudy, we were also successful in demonstration of podo-planin expression in SM-AP cell systems of pleomorphicadenoma origin [11]. In addition to pleomorphic adenoma,podoplanin was also localized in outer cells of duct-likestructures of intraductal papilloma as well as in variouscell types of myoepithelioma [24–26]. It is thus possibleto conclude that podoplanin can be a new myoepithelialcell marker in salivary gland tumors with myoepithelialdifferentiation in addition to myosin [27], actin [26, 28],calponin [24, 28], P63 [25, 28], and S-100 protein [10,29].

Apart from the myoepithelial characteristics, the simulta-neous expression of podoplanin and CD44 in spindle/stellate-shaped cells embedded in hyaluronan-rich myxochondroidmatrix [21, 22] was noticeable because it indicates a possiblerole of podoplanin in ECM modulation in the neoplasticstroma [8, 22]. In CIS and epithelial dysplasia of the oralmucosa, we have emphasized a possibility that podoplaninplays a role in cell growth because of its overlapping locali-zation in Ki-67+ cell proliferating zones [7]. In our morerecent study on odontogenic tumors, we suggested anotherfunctional potential of podoplanin in cell adhesion because ofits expression profiles similar to those of fibronectin [6]. Lateron, we were able to show that podoplanin functions directly incell adhesion and eventually in cell proliferation using ZK-1cells of human tongue SCC origin [8]. Thus, these morpho-logical studies, as well as functional studies, have narrowed

the possible functions of podoplanin down to its com-munication with ECM. At this point, it would be pos-sible to consider that the significance of myoepithelialdifferentiation in some particular types of salivary tumorcells is related to their stromal formation, which aremost pronounced in pleomorphic adenoma [21, 22, 30]or adenoid cystic carcinoma [31, 32].

Taken together, our findings on podoplanin expressionprofiles may indicate that myoepithelial differentiation isrequired for tumor cells in their modulating ECM to survivein such poorly vascularized and hypoxic stroma. However,molecular mechanisms underlining the stromal induction ofsalivary tumors remain totally unknown and should be elu-cidated as the next step of our investigation.

Acknowledgments This work was supported in part by Grants-in-Aid for Scientific Research from the Japan Society for the Promotionof Science (JSPS) and for JSPS Fellows (M. Tsuneki), with additionalfunding from the Iwadare Scholarship Foundation, Japan.

Conflict of interest The authors declare that they have no conflict ofinterest.

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