・Rahadiani N, Ikeda JI, Makino T, Tian T, Qiu Y, Mamat S, Wang Y, Doki Y, Aozasa K, Morii E.
Tumorigenic Role of Podoplanin in Esophageal Squamous-Cell Carcinoma.

Ann Surg Oncol. 2010 Jan 12. [Epub ahead of print]

BACKGROUND: Podoplanin, a mucin-type transmembrane glycoprotein, is thought to be one of the cancer stem cell markers for squamous-cell carcinoma of the vulva. The objectives of the present study were to examine the role of podoplanin in esophageal squamous-cell carcinoma (ESCC). METHODS: Expression of podoplanin was examined immunohistochemically in 61 cases of ESCC that had not been treated with chemotherapy or radiotherapy before surgery. Because cancer stem-cell quantities have been reported to increase with chemotherapy and radiotherapy, cases in patients who did not receive such prior therapies were included in this study. Cases with >10% tumor cells showing signals for podoplanin were categorized as podoplanin high, and the others were classified as podoplanin low. The effects of podoplanin on the behavior of cancer cells were evaluated in ESCC cell lines in which podoplanin expression was knocked down. RESULTS: To examine whether podoplanin could be used as a cancer stem cell marker for ESCC, podoplanin-positive and podoplanin-negative fractions were sorted separately from the ESCC cell line and cultured. Podoplanin-positive ESCC cells yielded both podoplanin-positive and podoplanin-negative cells, whereas few cells were obtained from podoplanin-negative ESCC cells. When podoplanin expression was knocked down, ESCC cell lines became vulnerable to anticancer drugs and showed defective invasion and tumorigenic activities. Nineteen (31.1%) of 61 cases were categorized as podoplanin high. Podoplanin-high cases were correlated with T category, stage of disease, lymphatic and vascular invasion, recurrence, and prognosis of patients. Podoplanin-low cases showed better overall and disease-free survival. CONCLUSIONS: There is a role for podoplanin in tumorigenesis and malignant progression in ESCC.

・Barth K, Blasche R, Kasper M.
T1alpha/podoplanin shows raft-associated distribution in mouse lung alveolar epithelial E10 cells.

Cell Physiol Biochem. 2010;25(1):103-12. Epub 2009 Dec 22.

AIMS: T1alpha/(podoplanin) is abundantly expressed in the alveolar epithelial type I cells (ATI) of rodent and human lungs. Caveolin-1 is a classical primary structural protein of plasmalemal invaginations, so-called caveolae, which represent specialized lipid rafts, and which are particularly abundant in ATI cells. The biological functions of T1alpha in the alveolar epithelium are unknown. Here we report on the characteristics of raft domains in the microplicae/microvillar protrusions of ATI cells, which contain T1alpha. METHODS: Detergent resistant membranes (DRMs) from cell lysates of the mouse epithelial ATI-like cell line E10 were prepared using different detergents followed by flotation in a sucrose gradient and tested by Western and dot blots with raft markers (caveolin-1, GM1) and nonraft markers (transferrin receptor, PDI and beta-Cop). Immunocytochemistry was employed for the localization of T1alpha in E10 cells and in situ in rat lungs. RESULTS: Our biochemical results showed that the solubility or insolubility of T1alpha and caveolin-1 differs in Triton X-100 and Lubrol WX, two distinct non-ionic detergents. Caveolin-1 was unsoluble in both detergents, whereas T1alpha was Triton X-100 soluble but Lubrol WX insoluble. Immunofluorescence double stainings revealed that both proteins were colocalized with GM1, while caveolin-1 and T1alpha were not colocalized in the plasma membrane. Cholesterol depletion modified the segregation of T1alpha in Lubrol WX DRMs. Cellular processes in ultrathin sections of cultured mouse E10 cells were immunogold positive. Immunoelectron microscopy (postembedding) of rat lung tissue revealed the preferential localization of T1alpha on apical microvillar protrusions of ATI cells. CONCLUSION: We conclude that T1alpha and caveolin-1 are located in distinct plasma membrane microdomains, which differ in their protein-lipid interactions. The raft-associated distribution of T1alpha may have an impact on a specific, not yet clarified function of this protein in the alveolar epithelium.

・Uhrin P, Zaujec J, Breuss JM, Olcaydu D, Chrenek P, Stockinger H, Fuertbauer E, Moser M, Haiko P, Fassler R, Alitalo K, Binder BR, Kerjaschki D.
Novel function for blood platelets and podoplanin in developmental separation of blood and lymphatic circulation.

Blood. 2010 Jan 28. [Epub ahead of print]

＊我々の3報の論文（Kato et al., JBC2003, Kaneko et al., JBC2004, Suzuki-Inoue et al., JBC2007）が引用されている。

＊リンパ管の発生にポドプラニンが重要な役割を果たしていることを示す論文が、podoplaninを命名したグループらにより発表された。これまで＜ポドプラニンの機能は不明＞と言い続けてきたが、ようやく明確に＜ポドプラニンの機能は、血小板凝集を起こすことによりリンパ管の発生を制御する＞ということができる。

During embryonic development lymph sacs form from the cardinal vein, and sprout centrifugally to form mature lymphatic networks. Separation of the lymphatic from the blood circulation by a hitherto unknown mechanism is essential for the homeostatic function of the lymphatic system. O-glycans on the lymphatic endothelium have recently been suggested to be required for establishment and maintenance of distinct blood and lymphatic systems, primarily by mediating proper function of podoplanin. Here we show that this separation process critically involves platelet activation by podoplanin. We found that platelet aggregates build up in wild-type embryos at the separation zone of podoplanin positive lymph-sacs and cardinal veins, but not in podoplanin(-/-) embryos. Thus, podoplanin(-/-) mice develop a "non-separation" phenotype, characterized by a blood-filled lymphatic network after ~E13.5, which however partially resolves in postnatal mice. The same embryonic phenotype is also induced by treatment of pregnant mice with acetyl salicylic acid, podoplanin blocking antibodies, or by inactivation of the kindlin-3 gene required for platelet aggregation. Therefore, interaction of endothelial podoplanin of the developing lymph-sac with circulating platelets from the cardinal vein is critical for separating the lymphatic from the blood vascular system.

・Rodrigo JP, Garcia-Carracedo D, Gonzalez MV, Mancebo G, Fresno MF, Garcia-Pedrero JM.
Podoplanin expression in the development and progression of laryngeal squamous cell carcinomas.

Mol Cancer. 2010 Mar 2;9(1):48. [Epub ahead of print]

＊我々の論文3報（Kato et al., Tumor Biol. 2005, Kaneko et al., Gene2006, Kunita et al.,AJP2007）が引用されている。

BACKGROUND: : Podoplanin expression is attracting interest as a marker for cancer diagnosis and prognosis. We therefore investigated the expression pattern and clinical significance of podoplanin during the development and progression of laryngeal carcinomas. RESULTS: : Podoplanin expression was determined by immunohistochemistry in paraffin-embedded tissue specimens from 84 patients with laryngeal premalignancies and 53 patients with laryngeal squamous cell carcinomas. We found podoplanin expression extending from the basal to the suprabasal layer of the epithelium in 37 (44%) of 84 dysplastic lesions, whereas normal epithelium showed negligible expression. Patients carrying podoplanin-positive lesions had a higher laryngeal cancer incidence than those with negative expression, that was borderline to statistical significance (51% versus 30%, P = .071). Podoplanin expression in laryngeal carcinomas exhibited two distinct patterns. 20 (38%) cases showed diffuse expression in most tumor cells and 33 (62%) focal expression at the proliferating periphery of tumor nests. High podoplanin expression was inversely correlated with T classification (P = .033), disease stage (P = .006), and pathological grade (P = .04). There was a trend, although not significant, towards reduced disease-specific survival for patients with low podoplanin levels (P = .31) and diffuse expression pattern (P = .08). CONCLUSIONS: : Podoplanin expression increases in the early stages of laryngeal tumourigenesis and it seems to be associated with a higher laryngeal cancer risk. Podoplanin expression in laryngeal squamous cell carcinomas, however, diminishes during tumour progression. Taken together, these data support a role for podoplanin expression in the initiation but not in the progression of laryngeal cancers.

・Ariizumi T, Ogose A, Kawashima H, Hotta T, Li G, Xu Y, Umezu H, Sugai M, Endo N.
Expression of podoplanin in human bone and bone tumors: New marker of osteogenic and chondrogenic bone tumors.

Pathol Int. 2010 Mar;60(3):193-202.

＊我々の論文11報（Kato,Cancer Sci2008; Kaneko, JBC2004; Kato, JBC2003; Kaneko, FEBS lett2007; Suzuki-Inoue,JBC2007; Kato, BBRC2006; Kunita, AJP2007; Kato,TumorBiol2005; Mishima,ANP2006a; Suzuki,FEBSlett2008; Mishima, ANP2006b）が引用されている。

Abstract Podoplanin is known as a lymphatic marker because its expression is detected in lymphatic but not vascular endothelium. Podoplanin is also expressed in several normal tissues including osteocytes or osteoblasts. A systematic examination of the podoplanin expression was conducted in normal skeletal tissues and some bone tumor cell lines, and the diagnostic value determined in primary bone tumors. Podoplanin mRNA was expressed at a high level in bone marrow tissue and cartilage, and was upregulated with differentiation to osteoblasts in bone marrow cells. Strong podoplanin expression was seen in osteocytes, chondrocytes, and osteoblasts on immunohistochemistry. Podoplanin mRNA was expressed at a high level in several osteosarcoma and chondrosarcoma cell lines, whereas podoplanin was expressed at a low level in a Ewing's/primitive neuroectodermal tumor cell line. In the clinical samples, osteosarcomas (22/26) expressed podoplanin at various levels. In small cell osteosarcomas (2/2), podoplanin was expressed strongly, although the tissue samples included few diagnostic osteoids. Chondrosarcomas (10/10) expressed podoplanin strongly, and chondroblastomas (5/5) expressed podoplanin moderately, while podoplanin was absent or expressed at low levels in Ewing's sarcomas (0/5), chordomas (0/6) and giant cell tumors of bone (1/7). Therefore, podoplanin may be a sensitive immunohistochemical marker of osteogenic and chondrogenic bone tumors.

・Margaritescu C, Raica M, Pirici D, Simionescu C, Mogoanta L, Stinga AC, Stinga AS, Ribatti D.
Podoplanin expression in tumor-free resection margins of oral squamous cell carcinomas: an immunohistochemical and fractal analysis study.

Histol Histopathol. 2010 Jun;25(6):701-11.

＊我々の論文1報（Kaneko et al, Gene2006）が引用されている。

Abstract Podoplanin is involved in tumorigenesis and cancer progression in head and neck malignancies and its expression is not restricted to lymphatic vessel endothelium. The aim of this study was to establish podoplanin expression in the tumor-free resection margins of oral squamous cell carcinomas (OSCCs) and to evaluate the geometric complexity of the lymphatic vessels in oral mucosa by utilizing fractal analysis. As concerns the podoplanin expression in noncancerous tissue, forty tumor-free resection margins from OSCCs were investigated utilizing immunohistochemistry for D2-40 antibody and image densitometry analysis. Podoplanin expression was extremely low in basal cells, especially in resection margins of OSCCs developed in the lower lip regions. However, a highly variable D2-40 expression in tumor-free resection margins associated with hyperplastic or dysplastic lesions was identified. Moreover, podoplanin expression also extended to the basal layer of the lower lip skin appendages, the myoepithelial cells of acini and ducts of minor salivary glands, and other structures from the oral cavity. As concerns the study of the density and complexity of oral lymphatic vessels architecture by means of immunohistochemistry (D2-40, CD31 and Ki-67 antibodies) and fractal analysis, we demonstrated that in normal oral mucosa the geometry of the lymphatic vessels was less complex at the level of the lower lip compared to the anterior part of the oral floor mucosa or the tongue. A comparative analysis between the normal and pathological aspects revealed statistically significant differences between the fractal dimension (FD) of the vessels' outline, especially in the tongue. Fractal analysis proved an increasing lymphatic network complexity from normal to premalignant oral mucosal lesions, providing additional prognostic information in oral malignant tumors.

・Bertozzi CC, Schmaier AA, Mericko P, Hess PR, Zou Z, Chen M, Chen CY, Xu B, Lu MM, Zhou D, Sebzda E, Santore MT, Merianos DJ, Stadtfeld M, Flake AW, Graf T, Skoda R, Maltzman JS, Koretzky GA, Kahn ML.
Platelets regulate lymphatic vascular development through CLEC-2-SLP-76 signaling.

Blood. 2010 Apr 2. [Epub ahead of print]

＊我々の論文2報（Inoue-Suzuki et al, JBC2007, Kunita et al.,AJP2007）が引用されている。

Abstract Although platelets appear by E10.5 in the developing mouse, an embryonic role for these cells has not been identified. The SYK-SLP-76 signaling pathway is required in blood cells to regulate embryonic blood-lymphatic vascular separation, but the cell type and molecular mechanism underlying this regulatory pathway is not known. In the present study we demonstrate that platelets regulate lymphatic vascular development by directly interacting with lymphatic endothelial cells through CLEC-2 receptors. PODOPLANIN (PDPN), a transmembrane protein expressed on the surface of lymphatic endothelial cells, is required in non-hematopoietic cells for blood-lymphatic separation. Genetic loss of the PDPN receptor CLEC-2 ablates PDPN binding by platelets and confers embryonic lymphatic vascular defects like those seen in animals lacking PDPN or SLP-76. PF4-Cre mediated deletion of Slp-76 is sufficient to confer lymphatic vascular defects, identifying platelets as the cell type in which SLP-76 signaling is required to regulate lymphatic vascular development. Consistent with these genetic findings, we observe SLP-76-dependent platelet aggregate formation on the surface of lymphatic endothelial cells in vivo and ex vivo. These studies identify a non-hemostatic pathway in which platelet CLEC-2 receptors bind lymphatic endothelial PDPN and activate SLP-76 signaling to regulate embryonic vascular development.

・Suzuki-Inoue K, Inoue O, Ding G, Nishimura S, Hokamura K, Eto K, Kashiwagi H, Tomiyama Y, Yatomi Y, Umemura K, Shin Y, Hirashima M, Ozaki Y.
Essential in vivo roles of the c-type lectin receptor CLEC-2: Embryonic/neonatal lethality of CLEC-2-deficient mice by blood/lymphatic misconnections and impaired thrombus formation of CLEC-2-deficient platelets.

J Biol Chem. 2010 Jun 4. [Epub ahead of print]

＊我々の論文2報（Kato,Cancer Sci2008; Suzuki-Inoue,JBC2007）が引用されている。

Abstract CLEC-2 has been recently described as playing crucial roles in thromobosis/hemostasis, tumor metastasis, and lymphangiogenesis based on the following findings. The snake venom rhodocytin is known as a strong platelet activator, and this effect has been shown to be mediated by CLEC-2 by us. Podoplanin, which is expressed on the surface of tumor cells, is an endogenous ligand for CLEC-2 and facilitates tumor metastasis by inducing platelet aggregation. Mice deficient in podoplanin, which is also expressed on the surface of lymphatic endothelial cells, show abnormal patterns of lymphatic vessel formation. In this study, we, for the first time, report on the generation and phenotype of CLEC-2-deficient mice. These mice are lethal at the embryonic and neonatal stage associated with disorganized and blood-filled lymphatic vessels and severe edema. Moreover, using transplantation of fetal liver cells from the CLEC-2-/- or CLEC-2+/+ embryos, we were able to demonstrate that CLEC-2 is involved in thrombus stabilization in vitro and in vivo, possibly through homophilic interactions without apparent increase in bleeding tendency. We propose that CLEC-2 could be an ideal novel target protein for an anti-platelet drug, which inhibits pathological thrombus formation, but not physiological hemostasis.

・Cortez MA, Nicoloso MS, Shimizu M, Rossi S, Gopisetty G, Molina JR, Carlotti C Jr, Tirapelli D, Neder L, Brassesco MS, Scrideli CA, Tone LG, Georgescu MM, Zhang W, Puduvalli V, Calin GA.
miR-29b and miR-125a regulate podoplanin and suppress invasion in glioblastoma.

Genes Chromosomes Cancer. 2010 Jul 27. [Epub ahead of print]

＊我々の論文3報（Kato, JBC2003; Kato,Tumor Biol2005; Mishima, ANP2006a）が引用されている。

Abstract Glioblastoma is the most frequent and malignant brain tumor, characterized by an elevated capacity for cellular proliferation and invasion. Recently, it was demonstrated that podoplanin membrane sialo-glycoprotein encoded by PDPN gene is over-expressed and related to cellular invasion in astrocytic tumors; however the mechanisms of regulation are still unknown. MicroRNAs are noncoding RNAs that regulate gene expression and several biological processes and diseases, including cancer. Nevertheless, their roles in invasion, proliferation, and apoptosis of glioblastoma are not completely understood. In this study, we focused on miR-29b and miR-125a, which were predicted to regulate PDPN, and demonstrated that these microRNAs directly target the 3' untranslated region of PDPN and inhibit invasion, apoptosis, and proliferation of glioblastomas. Furthermore, we report that miR-29b and miR-125a are downregulated in glioblastomas and also in CD133-positive cells. Taken together, these results suggest that miR-29b and miR-125a represent potential therapeutic targets in glioblastoma. (c) 2010 Wiley-Liss, Inc.

・Idrees M, Saxena R, Cheng L, Ulbright TM, Badve S.
Podoplanin, a novel marker for seminoma: A comparison study evaluating immunohistochemical expression of podoplanin and OCT3/4.

Ann Diagn Pathol. 2010 Oct;14(5):331-6.

＊我々の論文1報（Kato, Oncogene2004）が引用されている。

Abstract

Podoplanin, a 38-kd membrane glycoprotein of podocytes, is the human homologue of mouse protein, aggrus, a 44-kd sialoglycoprotein; the latter has recently been reported in testicular seminomas but not in embryonal carcinomas, suggesting that it may be a specific marker for seminomas. The physiologic role of podoplanin has yet to be determined and its function in tumors is not well characterized. In this study we investigate the utility of podoplanin expression in the diagnosis of testicular germ cell tumors. Sixty-eight cases of testicular mixed germ cell tumors were analyzed using a polyclonal antibody and compared to the results for OCT3/4. Stained sections were graded semiquantitatively as follows: negative (no expression), 1+ (mild intensity), 2+ (moderate intensity), 3+ (intense staining). Diffuse cytoplasmic expression of podoplanin with membrane accentuation was seen in the seminoma component of all cases. Lymphocytes and interstitial cells were negative. In mixed germ cell tumors, podoplanin identified small clusters of seminoma cells lying adjacent to nonseminomatous components. Focal staining was present in one third of cases of choriocarcinoma. There was no significant staining of any nontrophoblastic, nonseminomatous elements. In contrast to OCT3/4, podoplanin does not stain embryonal carcinoma. The availability of a marker for seminoma will enable better recognition and distinction from embryonal carcinoma, particularly in nongonadal sites. The identification by podoplanin of seminoma cells lying adjacent to foci of embryonal carcinoma gives credence to the hypothesis that the latter arise from seminomas as part of a process of "differentiation."

・Evaluation of anti-podoplanin rat monoclonal antibody NZ-1 for targeting malignant gliomas.

第69回日本癌学会学術総会（大阪）にて加藤が発表しました。来年の癌学会（名古屋）までには、NZ-1の前臨床試験の終了を目指します。

Abstract

Podoplanin/Aggrus is a mucin-like sialoglycoprotein that is highly expressed in malignant gliomas. The purpose of this study is to determine whether an anti-podoplanin antibody is suitable to target radionuclides to malignant gliomas. NZ-1 was radioiodinated with 125I using Iodogen [125I-NZ-1(Iodogen)] or N-succinimidyl 4-guanidinomethyl 3-[131I]iodobenzoate ([131I]SGMIB-NZ-1), and paired-label internalization assays of NZ-1 were performed. The tissue distribution of 125I-NZ-1(Iodogen) and that of [131I]SGMIB-NZ-1 were then compared in athymic mice bearing glioblastoma xenografts. Paired-label internalization assays in LN319 glioblastoma cells indicated that [131I]SGMIB-NZ-1 resulted in higher intracellular retention of radioactivity (26.3 ± 0.8%) compared to that from the 125I-NZ-1(Iodogen) (10.0 ± 0.1%). Likewise, tumor uptake of [131I]SGMIB-NZ-1 (39.9 ± 8.8%ID/g) in athymic mice bearing D2159MG xenografts in vivo was significantly higher than that of 125I-NZ-1(Iodogen) (29.7 ± 6.1%ID/g). The overall results suggest that an anti-podoplanin antibody NZ-1 warrants further evaluation for antibody-based therapy against glioblastoma.

ポドプラニン関連の演題としては、我々の演題の他、国立癌研究センター東病院のグループと、群馬大学のブループから、1演題ずつ発表がありました。
国立癌センターのグループは、これまでポドプラニンを肺癌の癌幹細胞マーカーとして提唱しており、いくつかの論文を発表しています。肺癌細胞株A431から抗ポドプラニン抗体18H5を使って細胞を分離し、それらの集団が癌幹細胞としての性質を持つことを証明しています。一方で、MDアンダーソンのグループは、我々の樹立した抗ポドプラニン抗体NZ-1を使って、脳腫瘍の組織から癌幹細胞の分画を分離しています。MDアンダーソンのグループによると、18H5では癌幹細胞を分離することができないとしており、この違いが癌腫の違いによるのか、それとも、実験材料の違い（細胞株か腫瘍組織）なのか、いろいろと議論が必要なところです。
群馬大学のグループからの発表では、ポドプラニンを導入した中皮腫の細胞株が、抗がん剤によるアポトーシスに耐性になっており、さらに浸潤能なども亢進していることを報告しています。

・Shintaku M, Honda T, Sakai T.
Expression of podoplanin and calretinin in meningioma: an immunohistochemical study.

Brain Tumor Pathol. 2010 Apr;27(1):23-7. Epub 2010 Apr 28.

＊我々の論文2報（Mishima, ANP2006a, Mishima, ANP2006b）が引用されている。

Abstract

Meningioma is derived from arachnoid cells covering the surface of the brain and spinal cord, and it has characteristics shared by mesothelioma arising from the pleura and peritoneum in that it takes an epithelioid appearance despite its mesenchymal origin. We examined the immunohistochemical expression of podoplanin and calretinin, both of which are well-known markers of mesothelioma, in 24 surgical cases of meningioma of various types. In most cases, a linear immunoreactivity for podoplanin was found along the cell surface of most neoplastic cells. An intracytoplasmic, finely granular, or diffuse immunoreactivity was also noted in some cells. These findings corresponded well to immunoreactivity for epithelial membrane antigen (EMA), but immunoreactivity for podoplanin was more crisp or sharply delineated and clear compared with that for EMA. These findings indicate that podoplanin can be used as an immunohistochemical marker that is equivalent to EMA in the differential diagnosis of meningioma. However, most meningiomas did not contain calretinin-immunoreactive cells, a finding that differs from the diffuse immunoreactivity seen in mesothelioma.

・Braun N, Alscher DM, Fritz P, Edenhofer I, Kimmel M, Gaspert A, Reimold F, Bode-Lesniewska B, Ziegler U, Biegger D, Wuthrich RP, Segerer S.
Podoplanin-positive cells are a hallmark of encapsulating peritoneal sclerosis.

Nephrol Dial Transplant. 2010 Aug 13. [Epub ahead of print]

＊我々の論文1報（Ogasawara, Hybridoma2008）が引用されている。

Abstract

BACKGROUND: Encapsulating peritoneal sclerosis (EPS) and simple peritoneal sclerosis are important complications of long-term peritoneal dialysis (PD). Podoplanin is expressed by mesothelial cells and lymphatic vessels, which are involved in inflammatory reactions in the peritoneal cavity. METHODS: We studied 69 peritoneal biopsies from patients on PD (n = 16), patients with EPS (n = 18) and control biopsies taken at the time of hernia repair (n = 15) or appendectomy (n = 20). Immunohistochemistry was performed to localize podoplanin. Additionally, markers of endothelial cells, mesothelial cells, myofibroblasts (smooth muscle actin), proliferating cells, and double labelling for smooth muscle actin/podoplanin were used on selected biopsies. RESULTS: Podoplanin was present on the endothelium of lymphatic vessels in the submesothelial fibrous tissue and on mesothelial cells. In patients on PD and in biopsies with appendicitis, the mesothelial cells demonstrated a cuboidal appearance and circumferential podoplanin staining, with gaps between the cells. The number of lymphatic vessels was variable, but prominent at sites of fibrosis. In patients with EPS, a diffuse infiltration of podoplanin-positive cells with a fibroblastic appearance was present in 15 out of 18 biopsies. This pattern was focally present in 3 out of 16 on PD and none in the 35 controls. The podoplanin-positive cells did not express the endothelial marker or the mesothelial marker (calretinin). CONCLUSIONS: EPS is characterized by a population of podoplanin and smooth muscle actin double-positive cells. Podoplanin might be a suitable morphological marker supporting the diagnosis and might be involved in the pathogenesis of EPS.

・Oe S, Hasegawa K, Nagase S, Kato R, Torii Y, Udagawa Y.
Expression of podoplanin in epithelial ovarian carcinomas and its potential as a marker for clear cell adenocarcinoma.

Int J Gynecol Pathol. 2010 Sep;29(5):405-10.

＊我々の論文3報（Kato, JBC2003; Kato,Oncogene2004; Kaneko, JBC2004）が引用されている。

Abstract Podoplanin is a 43-kd mucin-type transmembrane glycoprotein that is a candidate marker for the pathologic diagnosis of mesothelioma and lymphatic endothelial cells and lymphangiogenesis. The aim of this study was to investigate podoplanin expression in epithelial ovarian carcinomas. Immunohistochemistry was performed on the paraffin-embedded tissues from 78 patients with epithelial ovarian carcinomas consisting of serous adenocarcinoma (SA), endometrioid adenocarcinoma (EM), mucinous adenocarcinoma (MA), and clear cell adenocarcinoma (CCC) cases. Only 36.8% (7/19) of SA, 33.3% (6/18) of EM, and 15.8% (3/19) of MA cases were positive for podoplanin expression, whereas 54.5% (12/22) of CCC samples were positive. Immunohistochemical scores (mean+/-SD) were 1.2+/-1.5, 1.9+/-2.6, 0.8+/-1.6 and, 3.6+/-4.0 in SA, EM, MA, and CCC, respectively. Podoplanin expression was significantly stronger in CCC than in other histologic types. However, no significant correlation was observed between its expression and FIGO stage, the presence of endometriosis, lymph node metastasis, or recurrence. There was also no correlation between podoplanin expression and overall survival. We confirmed the expression of podoplanin in epithelial ovarian carcinomas, particularly in CCC. Podoplanin might have utility as a marker for CCC in pathologic diagnosis. Further investigation is needed to clarify the relationship between podoplanin expression and the biologic characteristics of CCC.

・Cueni LN, Chen L, Zhang H, Marino D, Huggenberger R, Alitalo A, Bianchi R, Detmar M.
Podoplanin-Fc reduces lymphatic vessel formation in vitro and in vivo and causes disseminated intravascular coagulation when transgenically expressed in the skin.

Blood. 2010 Aug 17. [Epub ahead of print]

＊我々の論文4報（Kato, JBC2003; Kaneko, JBC2004, Suzuki-Inoue, JBC2007, Kato, Cancer Sci2008）が引用されている。

Abstract Podoplanin is a small transmembrane protein required for development and function of the lymphatic vascular system. To investigate the effects of interfering with its function, we produced an Fc fusion protein of its ectodomain. We found that podoplanin-Fc inhibited several functions of cultured lymphatic endothelial cells and also specifically suppressed lymphatic vessel growth, but not blood vessel growth, in mouse embryoid bodies in vitro and in mouse corneas in vivo. Using a keratin 14 (K14) expression cassette, we created transgenic mice that overexpressed podoplanin-Fc in the skin. No obvious outward phenotype was identified in these mice, but surprisingly, podoplanin-Fc - although produced specifically in the skin - entered the blood circulation and induced disseminated intravascular coagulation (DIC), characterized by microthrombi in most organs and by thrombopenia, occasionally leading to fatal hemorrhage. These findings reveal an important role of podoplanin in lymphatic vessel formation and indicate the potential of podoplanin-Fc as an inhibitor of lymphangiogenesis. These results also demonstrate the ability of podoplanin to induce platelet aggregation in vivo, which likely represents a major function of lymphatic endothelium. Finally, K14 podoplanin-Fc mice represent a novel genetic animal model of DIC.

・Cueni LN, Hegyi I, Shin JW, Albinger-Hegyi A, Gruber S, Kunstfeld R, Moch H, Detmar M.
Tumor lymphangiogenesis and metastasis to lymph nodes induced by cancer cell expression of podoplanin.

Am J Pathol. 2010 Aug;177(2):1004-16. Epub 2010 Jul 8

＊我々の論文4報（Kato, JBC2003; Kato,Tumor Biol2005; Mishima, ANP2006a; Kunita, AJP2007）が引用されている。

Abstract The membrane glycoprotein podoplanin is expressed by several types of human cancers and might be associated with their malignant progression. Its exact biological function and molecular targets are unclear, however. Here, we assessed the relevance of tumor cell expression of podoplanin in cancer metastasis to lymph nodes, using a human MCF7 breast carcinoma xenograft model. We found that podoplanin expression promoted tumor cell motility in vitro and, unexpectedly, increased tumor lymphangiogenesis and metastasis to regional lymph nodes in vivo, without promoting primary tumor growth. Importantly, high cancer cell expression levels of podoplanin correlated with lymph node metastasis and reduced survival times in a large cohort of 252 oral squamous cell carcinoma patients. Based on comparative transcriptional profiling of tumor xenografts, we identified endothelin-1, villin-1, and tenascin-C as potential mediators of podoplanin-induced tumor lymphangiogenesis and metastasis. These unexpected findings identify a novel mechanism of tumor lymphangiogenesis and metastasis induced by cancer cell expression of podoplanin, suggesting that reagents designed to interfere with podoplanin function might be developed as therapeutics for patients with advanced cancer.

・Martin-Villar E, Fernandez-Munoz B, Parsons M, Yurrita MM, Megias D, Perez-Gomez E, Jones GE, Quintanilla M.
Podoplanin Associates with CD44 to Promote Directional Cell Migration.

Mol Biol Cell. 2010 Oct 20. [Epub ahead of print]

＊我々の論文3報（Kato, Cancer Sci 2008, Nakazawa Blood 2008; Kunita, AJP 2007）が引用されている。

Abstract

Podoplanin is a transmembrane glycoprotein up-regulated in different human tumors, especially those derived from squamous stratified epithelia (SCCs). Its expression in tumor cells is linked to increased cell migration and invasiveness; however, the mechanisms underlying this process remain poorly understood. Here we report that CD44, the major hyaluronan (HA) receptor, is a novel partner for podoplanin. Expression of the CD44 standard isoform (CD44s) is coordinately up-regulated together with that of podoplanin during progression to highly aggressive SCCs in a mouse skin model of carcinogenesis, and during epithelial-mesenchymal transition (EMT). In carcinoma cells, CD44 and podoplanin colocalise at cell surface protrusions. Moreover, CD44 recruitment promoted by HA-coated beads or cross-linking with a specific CD44 antibody induced corecruitment of podoplanin. Podoplanin-CD44s interaction was demonstrated both by coimmunoprecipitation experiments and, in vivo, by fluorescence resonance energy transfer/fluorescence lifetime imaging microscopy (FRET/FLIM), the later confirming its association on the plasma membrane of cells with a migratory phenotype. Importantly, we also show that podoplanin promotes directional persistence of motility in epithelial cells, a feature that requires CD44, and that both molecules cooperate to promote directional migration in SCC cells. Our results support a role for CD44-podoplanin interaction in driving tumor cell migration during malignancy.

・Huber GF, Fritzsche FR, Zullig L, Storz M, Graf N, K Haerle S, Jochum W, Stoeckli SJ, Moch H.
Podoplanin expression correlates with sentinel lymph node metastasis in early squamous cell carcinomas of the oral cavity and oropharynx.

Int J Cancer. 2010 Nov 23. [Epub ahead of print]

＊我々の論文4報（Kato, Cancer Sci 2008; Kunita, AJP 2007; Mishima, ANP 2006, Chaipan, RV 2010）が引用されている。

Abstract

In patients with early head and neck squamous-cell carcinoma (HNSCC), occult lymph node metastasis is difficult to predict by clinical or pathological parameters. Such parameters however are necessary to select patients either for elective neck dissection or the sentinel lymph node procedure. The membrane glycoprotein podoplanin is normally expressed in lymphatic endothelial cells. Recently, expression of podoplanin by cancer cells was demonstrated to promote tumor cell motility and tumor lymphangiogenesis in vitro. The value of cancer cell-expressed podoplanin was to be determined as a predictive marker for sentinel lymph node (SLN) metastasis in early HNSCC of the oral cavity and oropharynx. 120 patients with HNSCC of the oral cavity and oropharynx undergoing a SLN biopsy were enrolled in this prospective clinical trial of sentinel lymph node biopsy. Cancer cell-expressed podoplanin was determined by immunohistochemistry using tissue microarrays. Podoplanin expression was quantified by the Intensity Reactivity Score (IRS) and categorized into expression and non-expression. SLN examination revealed occult metastasis in 45 patients (37.5%). Twenty nine of 120 (24.2%) primary HNSCC showed podoplanin expression. Podoplanin expression correlated significantly with sentinel lymph node metastasis (p=0.029) and remained a significant predictor for lymph node status even after controlling for tumor stage (p=0.028). As a predictive marker for SLN metastasis however, podoplanin expression reached a sensitivity of a mere 36% and a specificity of 83%. Podoplanin expression is associated with metastasis to lymph nodes in vivo. Podoplanin immunohistochemistry in early HNSCC of the oral cavity and oropharynx may help to select patients for the SLN procedure and to identify patients with increased risk for presence of occult lymph node metastasis in the neck.

・Suzuki H, Onimaru M, Yonemitsu Y, Maehara Y, Nakamura S, Sueishi K.
Podoplanin in cancer cells is experimentally able to attenuate prolymphangiogenic and lymphogenous metastatic potentials of lung squamoid cancer cells.

Mol Cancer. 2010 Oct 31;9(1):287. [Epub ahead of print]

＊我々の論文3報（Suzuki-Inoue, JBC 2007, Nakazawa Blood 2008; Kunita, AJP 2007）が引用されている。抗ポドプラニン抗体として、AngioBioから販売されているマウス抗ポドプラニン抗体（クローン：D2-40）を使用。（AngioBioからは正式に公表されていないが、AngioBioから販売されているマウス抗ポドプラニン抗体（Cat No.:11-003）はD2-40である。）

Abstract

BACKGROUND: : Podoplanin, a mucin-like transmembrane glycoprotein, is reportedly expressed in a variety of malignant cells and is generally regarded as a factor for promoting tumor progression in conventional studies. By contrast, a clinicopathologically conflicting role for podoplanin, namely as a favorable prognostic factor for patients with lung/cervical squamous cell carcinoma (SCC), has recently been reported. Here, we investigated the role of podoplanin expressed in lung squamoid cancer cells (LSCCs) in experimental tumor progression. RESULTS: : Using EBC-1 cells, a lung SCC cell line without podoplanin expression and with lymphogenous metastatic potential, stable transformants with or without an exogenous human podoplanin gene were established and applied to a mouse tumor implantation model. In vivo examinations revealed that exogenous podoplanin had no influence on tumor growth, whereas it significantly restrained axillary lymph node metastasis associated with the suppression of lymphangiogenesis but not angiogenesis and with the downregulation of EBC-1-derived VEGF-C but not other lymphangiogenesis-related factor mRNAs in implanted tumor tissue. In vitro examinations to clarify the mechanisms underlying the in vivo phenomena revealed that exogenous podoplanin significantly suppressed the expression of VEGF-C mRNA and of the protein, and also increased the level of phosphorylated c-jun N terminal kinase (JNK) in EBC-1 cells. The former effect of exogenous podoplanin was impaired by treatment with either JNK inhibitor sp600125 or podoplanin-siRNA, and the latter effect was impaired by treatment with podoplanin-siRNA, suggesting that podoplanin was able to activate JNK, thereby downregulating VEGF-C gene expression in LSCCs (podoplanin-JNK-VEGF-C axis). Furthermore, supporting evidence in regard to the axis present in LSCCs was obtained from similar experiments using H157 cells, another lung SCC cell line expressing endogenous podoplanin. CONCLUSIONS: : Our findings suggested that LSCC-associated podoplanin was functional and could attenuate the potential for lymph node metastasis, possibly based on the suppression of tumor lymphangiogenesis; thus, podoplanin in cancer cells may become a useful biomarker to measure the malignancy of lung SCC.

Wang Y, Sun J, Gu Y, Zhao S, Groome LJ, Alexander JS.
D2-40/podoplanin expression in the human placenta.

Placenta. 2010 Nov 20. [Epub ahead of print]

Abstract

Placental tissue expresses many lymphatic markers. The current study was undertaken to examine if D2-40/podoplanin, a lymphatic endothelial marker, was expressed in the human placenta, and how it is altered developmentally and pathologically. We examined D2-40/podoplanin and VEGFR-3 expressions in placentas from normotensive pregnancies at different gestational ages and in placentas from women with clinically defined preeclampsia. D2-40 expression in systemic lymphatic vessel endothelium served as a positive control. Protein expression for D2-40, VEGFR-3, and β-actin was determined by Western blot in placentas from normotensive (n = 6) and preeclamptic (n = 5) pregnancies. Our results show that D2-40/podoplanin was strongly expressed in the placenta, mainly as a network plexus pattern in the villous stroma throughout gestation. CD31 was limited to villous core fetal vessel endothelium and VEGFR-3 was found in both villous core fetal vessel endothelium and trophoblasts. D2-40/podoplanin expression was significantly decreased, and VEGFR-3 significantly increased in preeclamptic placental tissues compared to normotensive placental controls. Placental villous stroma is a reticular-like structure, and the localization of D2-40 to the stroma suggests that a lymphatic-like conductive network may exist in the human placenta. D2-40/podoplanin is an O-linked sialoglycoprotein. Although little is known regarding biological functions of sialylated glycoproteins within the placenta, placental D2-40/podoplanin may support fetal vessel angiogenesis during placenta development and reduced D2-40/podoplanin expression in preeclamptic placenta may contribute to altered interstitial fluid homeostasis and impaired angiogenesis in this pregnancy disorder.