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• W2128585791 abstract "Tumor cell-induced platelet aggregation has been reported to facilitate hematogenous metastasis. Aggrus/podoplanin is a platelet aggregation-inducing factor that is up-regulated in a number of human cancers and has been implicated in tumor progression. We studied herein the role of Aggrus in tumor growth, metastasis, and survival in vivo. Aggrus expression in Chinese hamster ovary cells promoted pulmonary metastasis in both an experimental and a spontaneous mouse model. No differences in the size of metastatic foci or in primary tumor growth were found in either set of mice. Aggrus-expressing cells, which were covered with platelets, arrested in the lung microvasculature 30 minutes after injection. In addition, lung metastasis resulting from Aggrus expression decreased the survival of the mice. By generating several Aggrus point mutants, we revealed that point mutation at the platelet aggregation-stimulating domain of Aggrus (Thr34 and Thr52) obliterated both platelet aggregation and metastasis. Furthermore, administration of aspirin to mice reduced the number of metastatic foci. These results indicate that Aggrus contributes to the establishment of metastasis by promoting platelet aggregation without affecting subsequent growth. Thus, Aggrus could serve as an ideal therapeutic target for drug development to block metastasis. Tumor cell-induced platelet aggregation has been reported to facilitate hematogenous metastasis. Aggrus/podoplanin is a platelet aggregation-inducing factor that is up-regulated in a number of human cancers and has been implicated in tumor progression. We studied herein the role of Aggrus in tumor growth, metastasis, and survival in vivo. Aggrus expression in Chinese hamster ovary cells promoted pulmonary metastasis in both an experimental and a spontaneous mouse model. No differences in the size of metastatic foci or in primary tumor growth were found in either set of mice. Aggrus-expressing cells, which were covered with platelets, arrested in the lung microvasculature 30 minutes after injection. In addition, lung metastasis resulting from Aggrus expression decreased the survival of the mice. By generating several Aggrus point mutants, we revealed that point mutation at the platelet aggregation-stimulating domain of Aggrus (Thr34 and Thr52) obliterated both platelet aggregation and metastasis. Furthermore, administration of aspirin to mice reduced the number of metastatic foci. These results indicate that Aggrus contributes to the establishment of metastasis by promoting platelet aggregation without affecting subsequent growth. Thus, Aggrus could serve as an ideal therapeutic target for drug development to block metastasis. Metastasis is the major cause of death from cancer, yet the optimal strategy against it remains uncertain. The pathogenesis of metastasis is dynamic and consists of the following steps: 1) cellular transformation and tumor growth, 2) angiogenesis, 3) detachment and local invasion of the host stroma, 4) entry into the bloodstream, 5) transport along circulation, 6) arrest in the capillary, 7) extravasation, and 8) proliferation within the foreign tissue.1Fidler IJ The pathogenesis of cancer metastasis: the ‘seed and soil’ hypothesis revisited.Nat Rev Cancer. 2003; 3: 453-458Crossref PubMed Scopus (3479) Google Scholar However, even if tumor cells successfully proceed through steps 1 to 3 and get into the circulation, almost all circulating tumor cells are rapidly destroyed by the shear forces or are attacked by the immune system, and less than 0.01% of these cells survive to produce metastasis.2Fidler IJ Metastasis: quantitative analysis of distribution and fate of tumor emboli labeled with 125I-5-iodo-2′-deoxyuridine.J Natl Cancer Inst. 1970; 45: 773-782PubMed Google Scholar Thus, a key to success in metastasis relies on tumor survival in the bloodstream. One of the strategies by which tumor cells achieve this is platelet aggregation, ie, tumor cell-induced platelet aggregation (TCIPA).Platelet aggregation is believed to protect tumor cells from immunological assault in the circulation. Indeed, it has been shown that platelets protect tumors from tumor necrosis factor α-mediated cytotoxicity.3Philippe C Philippe B Fouqueray B Perez J Lebret M Baud L Protection from tumor necrosis factor-mediated cytolysis by platelets.Am J Pathol. 1993; 143: 1713-1723PubMed Google Scholar Another survival advantage is the tendency for the large tumor-platelet aggregate to embolize the microvasculature at a new extravasation site.4Malik AB Pulmonary microembolism.Physiol Rev. 1983; 63: 1114-1207PubMed Google Scholar Platelets also facilitate the adhesion of tumor cells to the vascular endothelium5Mehta P Potential role of platelets in the pathogenesis of tumor metastasis.Blood. 1984; 63: 55-63PubMed Google Scholar (step 6) and release a number of growth factors that promote tumor cell growth. Mice without platelets, because of genetic elimination of Nf-E2, showed marked protection against metastasis.6Camerer E Qazi AA Duong DN Cornelissen I Advincula R Coughlin SR Platelets, protease-activated receptors, and fibrinogen in hematogenous metastasis.Blood. 2004; 104: 397-401Crossref PubMed Scopus (318) Google Scholar Recently, it has been reported that platelets contribute to tumor-induced angiogenesis by releasing angiogenic growth factors, such as vascular endothelial growth factor.7Verheul HM Hoekman K Lupu F Broxterman HJ van der Valk P Kakkar AK Pinedo HM Platelet and coagulation activation with vascular endothelial growth factor generation in soft tissue sarcomas.Clin Cancer Res. 2000; 6: 166-171PubMed Google Scholar, 8Salven P Orpana A Joensuu H Leukocytes and platelets of patients with cancer contain high levels of vascular endothelial growth factor.Clin Cancer Res. 1999; 5: 487-491PubMed Google Scholar, 9Salgado R Vermeulen PB Benoy I Weytjens R Huget P Van Marck E Dirix LY Platelet number and interleukin-6 correlate with VEGF but not with bFGF serum levels of advanced cancer patients.Br J Cancer. 1999; 80: 892-897Crossref PubMed Scopus (158) Google Scholar There are several mechanisms involved in TCIPA, and these can vary among different tumor cells. For example, tumor cells can activate platelets by tumor cell-induced thrombin generation through a coagulation pathway,10Nierodzik ML Plotkin A Kajumo F Karpatkin S Thrombin stimulates tumor-platelet adhesion in vitro and metastasis in vivo.J Clin Invest. 1991; 87: 229-236Crossref PubMed Scopus (207) Google Scholar, 11Esumi N Fan D Fidler IJ Inhibition of murine melanoma experimental metastasis by recombinant desulfatohirudin, a highly specific thrombin inhibitor.Cancer Res. 1991; 51: 4549-4556PubMed Google Scholar releasing adenosine 5′-diphosphate (ADP),12Camez A Dupuy E Bellucci S Calvo F Bryckaert MC Tobelem G Human platelet-tumor cell interactions vary with the tumor cell lines.Invasion Metastasis. 1986; 6: 321-334PubMed Google Scholar thromboxane A2,13Honn KV Steinert BW Moin K Onoda JM Taylor JD Sloane BF The role of platelet cyclooxygenase and lipoxygenase pathways in tumor cell induced platelet aggregation.Biochem Biophys Res Commun. 1987; 145: 384-389Crossref PubMed Scopus (38) Google Scholar matrix metalloproteinase 2,14Jurasz P Sawicki G Duszyk M Sawicka J Miranda C Mayers I Radomski MW Matrix metalloproteinase 2 in tumor cell-induced platelet aggregation: regulation by nitric oxide.Cancer Res. 2001; 61: 376-382PubMed Google Scholar and the membrane protein Aggrus.15Kato Y Fujita N Kunita A Sato S Kaneko M Osawa M Tsuruo T Molecular identification of Aggrus/T1alpha as a platelet aggregation-inducing factor expressed in colorectal tumors.J Biol Chem. 2003; 278: 51599-51605Crossref PubMed Scopus (246) Google ScholarAggrus/podoplanin is a type I transmembrane sialomucin-like glycoprotein that consists of an extracellular domain with abundant serine and threonine residues as potential O-glycosylation sites, a single transmembrane portion, and a short cytoplasmic tail with putative sites for protein kinase C and cAMP phosphorylation.16Martín-Villar E Scholl FG Gamallo C Yurrita MM Munoz-Guerra M Cruces J Quintanilla M Characterization of human PA2.26 antigen (T1alpha-2, podoplanin), a small membrane mucin induced in oral squamous cell carcinomas.Int J Cancer. 2005; 113: 899-910Crossref PubMed Scopus (239) Google Scholar Because Aggrus/podoplanin is expressed on the lymphatic endothelium but not on blood vessel endothelium, it is also widely used in histopathology as a specific marker for lymphatic endothelium and lymphangiogenesis.17Breiteneder-Geleff S Soleiman A Kowalski H Horvat R Amann G Kriehuber E Diem K Weninger W Tschachler E Alitalo K Kerjaschki D Angiosarcomas express mixed endothelial phenotypes of blood and lymphatic capillaries: podoplanin as a specific marker for lymphatic endothelium.Am J Pathol. 1999; 154: 385-394Abstract Full Text Full Text PDF PubMed Scopus (931) Google Scholar Aggrus expression has been shown to be up-regulated in a number of different cancers, including squamous cell carcinomas (oral cavity,16Martín-Villar E Scholl FG Gamallo C Yurrita MM Munoz-Guerra M Cruces J Quintanilla M Characterization of human PA2.26 antigen (T1alpha-2, podoplanin), a small membrane mucin induced in oral squamous cell carcinomas.Int J Cancer. 2005; 113: 899-910Crossref PubMed Scopus (239) Google Scholar lung,18Kato Y Kaneko M Sata M Fujita N Tsuruo T Osawa M Enhanced expression of Aggrus (T1alpha/podoplanin), a platelet-aggregation-inducing factor in lung squamous cell carcinoma.Tumour Biol. 2005; 26: 195-200Crossref PubMed Scopus (190) Google Scholar skin,19Schacht V Dadras SS Johnson LA Jackson DG Hong YK Detmar M Up-regulation of the lymphatic marker podoplanin, a mucin-type transmembrane glycoprotein, in human squamous cell carcinomas and germ cell tumors.Am J Pathol. 2005; 166: 913-921Abstract Full Text Full Text PDF PubMed Scopus (526) Google Scholar and head and neck20Yuan P Temam S El-Naggar A Zhou X Liu DD Lee JJ Mao L Overexpression of podoplanin in oral cancer and its association with poor clinical outcome.Cancer. 2006; 107: 563-569Crossref PubMed Scopus (255) Google Scholar), granulosa cell tumors,19Schacht V Dadras SS Johnson LA Jackson DG Hong YK Detmar M Up-regulation of the lymphatic marker podoplanin, a mucin-type transmembrane glycoprotein, in human squamous cell carcinomas and germ cell tumors.Am J Pathol. 2005; 166: 913-921Abstract Full Text Full Text PDF PubMed Scopus (526) Google Scholar mesotheliomas,21Kimura N Kimura I Podoplanin as a marker for mesothelioma.Pathol Int. 2005; 55: 83-86Crossref PubMed Scopus (172) Google Scholar testicular seminomas,22Kato Y Sasagawa I Kaneko M Osawa M Fujita N Tsuruo T Aggrus: a diagnostic marker that distinguishes seminoma from embryonal carcinoma in testicular germ cell tumors.Oncogene. 2004; 23: 8552-8556Crossref PubMed Scopus (135) Google Scholar central nervous system tumors,23Shibahara J Kashima T Kikuchi Y Kunita A Fukayama M Podoplanin is expressed in subsets of tumors of the central nervous system.Virchows Arch. 2006; 448: 493-499Crossref PubMed Scopus (93) Google Scholar, 24Mishima K Kato Y Kaneko MK Nishikawa R Hirose T Matsutani M Increased expression of podoplanin in malignant astrocytic tumors as a novel molecular marker of malignant progression.Acta Neuropathol. 2006; 111: 483-488Crossref PubMed Scopus (207) Google Scholar, 25Mishima K Kato Y Kaneko MK Nakazawa Y Kunita A Fujita N Tsuruo T Nishikawa R Hirose T Matsutani M Podoplanin expression in primary central nervous system germ cell tumors: a useful histological marker for the diagnosis of germinoma.Acta Neuropathol. 2006; 111: 563-568Crossref PubMed Scopus (119) Google Scholar and lobular breast cancers,26Wicki A Lehembre F Wick N Hantusch B Kerjaschki D Christofori G Tumor invasion in the absence of epithelial-mesenchymal transition: podoplanin-mediated remodeling of the actin cytoskeleton.Cancer Cell. 2006; 9: 261-272Abstract Full Text Full Text PDF PubMed Scopus (453) Google Scholar suggesting that increased expression of Aggrus is associated with tumor malignancy and poor clinical outcome.20Yuan P Temam S El-Naggar A Zhou X Liu DD Lee JJ Mao L Overexpression of podoplanin in oral cancer and its association with poor clinical outcome.Cancer. 2006; 107: 563-569Crossref PubMed Scopus (255) Google Scholar We previously showed expression of Aggrus-induced platelet aggregation with no requirement for plasma components.15Kato Y Fujita N Kunita A Sato S Kaneko M Osawa M Tsuruo T Molecular identification of Aggrus/T1alpha as a platelet aggregation-inducing factor expressed in colorectal tumors.J Biol Chem. 2003; 278: 51599-51605Crossref PubMed Scopus (246) Google Scholar We also identified the segment of EDxxVTPG in the extracellular domain as the platelet aggregation-stimulating (PLAG) domain, which is critical for the platelet-aggregating activity of Aggrus.15Kato Y Fujita N Kunita A Sato S Kaneko M Osawa M Tsuruo T Molecular identification of Aggrus/T1alpha as a platelet aggregation-inducing factor expressed in colorectal tumors.J Biol Chem. 2003; 278: 51599-51605Crossref PubMed Scopus (246) Google Scholar Aggrus contains three tandem repeats of the PLAG domain.27Kaneko MK Kato Y Kitano T Osawa M Conservation of a platelet activating domain of Aggrus/podoplanin as a platelet aggregation-inducing factor.Gene. 2006; 378: 52-57Crossref PubMed Scopus (118) Google Scholar In addition, these PLAG domains were highly conserved among Aggrus homologues from human, mouse, rat, dog, and hamster.27Kaneko MK Kato Y Kitano T Osawa M Conservation of a platelet activating domain of Aggrus/podoplanin as a platelet aggregation-inducing factor.Gene. 2006; 378: 52-57Crossref PubMed Scopus (118) Google Scholar However, it is yet to be elucidated whether its platelet-aggregating activity is directly involved in the in vivo metastasis-forming activity.In this study, we investigated the role of human Aggrus in tumor growth, metastasis, and survival. We established Chinese hamster ovary (CHO) cells, which had been stably transfected with wild-type (WT) Aggrus or its PLAG domain mutants. We discovered that Aggrus expression promoted pulmonary metastasis in both experimental and spontaneous metastasis models and decreased survival of the mice. Platelet aggregation-inducing activity of Aggrus is directly associated with metastasis formation because introducing a point mutation into the PLAG domains or administration of aspirin to mice decreased the formation of pulmonary metastasis.Materials and MethodsCell Culture ConditionsCHO cells were cultured in RPMI 1640 medium (Nissui, Tokyo, Japan) supplemented with 10% heat-inactivated fetal bovine serum (Sigma, St. Louis, MO), 2 mmol/L l-glutamine (Life Technologies, Inc., Grand Island, NY), and 100 μg/ml kanamycin at 37°C in a humidified atmosphere of 5% CO2 and 95% air.AnimalsFemale BALB/c mice (5 weeks old) and BALB/c-nu/nu mice (5 weeks old) were purchased from Charles River Japan, Inc. (Kanagawa, Japan). Animals were housed under pathogen-free conditions. The Animal Care and Use Committee of the Institute of Molecular and Cellular Biosciences approved the animal experiments described herein.Establishment of CHO Cells Stably Expressing WT or Mutant Aggrus ProteinsThe pcDNA3 vector containing WT human Aggrus cDNA was established in our laboratory, as described previously.15Kato Y Fujita N Kunita A Sato S Kaneko M Osawa M Tsuruo T Molecular identification of Aggrus/T1alpha as a platelet aggregation-inducing factor expressed in colorectal tumors.J Biol Chem. 2003; 278: 51599-51605Crossref PubMed Scopus (246) Google Scholar Substitution of the appropriate threonine codons to alanine codons in human Aggrus cDNA was accomplished using the QuickChange site-directed mutagenesis kit (Stratagene, La Jolla, CA). CHO cells were then transfected with the plasmids using LipofectAMINE 2000 reagent (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. Stable transfectants were selected by cultivating the cells in the medium containing 1 mg/ml Geneticin (G418; Sigma). We used two independent clones of each Aggrus-expressing clones. The expression level of human Aggrus was confirmed by Western blot analysis.Western Blot AnalysisCultured cell pellets or mice tumors were solubilized with lysis buffer (25 mmol/L Tris, pH 7.4, 50 mmol/L NaCl, 0.5% sodium deoxycholate, 2% Nonidet P-40, 0.2% sodium dodecyl sulfate, 1 mmol/L phenylmethyl sulfonyl fluoride, and 50 mg/ml aprotinin), electrophoresed, and blotted onto a nitrocellulose membrane. The membranes were incubated with an anti-human Aggrus YM-1 monoclonal antibody (hybridoma culture supernatant)28Kaneko M Kato Y Kunita A Fujita N Tsuruo T Osawa M Functional sialylated O-glycan to platelet aggregation on Aggrus (T1alpha/podoplanin) molecules expressed in Chinese hamster ovary cells.J Biol Chem. 2004; 279: 38838-38843Crossref PubMed Scopus (83) Google Scholar or an anti-β-actin antibody (Sigma). Membranes were subsequently washed and incubated with horseradish peroxidase-conjugated secondary antibody. After washing, the membranes were developed with an enhanced chemiluminescence system, according to the manufacturer's instructions (GE Health Care UK Ltd., Buckinghamshire, UK).Flow CytometryThe cell surface expression of WT-Aggrus and Aggrus point mutants was confirmed by flow cytometric analysis. Cells were harvested by brief exposure to trypsin. After washing with phosphate-buffered saline, cells were treated with YM-1 monoclonal antibody for 1 hour at 4°C, and then cells were incubated with Oregon Green 488-conjugated antibody (Invitrogen Molecular Probes, Eugene, OR) for 30 minutes at 4°C. Flow cytometric analysis was performed using a Cytomics FC500 flow cytometry system (Beckman-Coulter, Miami, FL).ImmunohistochemistrySpecimens were deparaffinized, rehydrated, and incubated with YM-1 monoclonal antibody at 23°C for 2 hours. Then, the specimens were incubated with biotin-conjugated secondary anti-rat IgG antibody (DakoCytomation, Glostrup, Denmark) for 30 minutes followed by incubation with peroxidase-conjugated biotin-streptavidin complex (Vectastain ABC kit; Vector Laboratories, Peterborough, UK) for 30 minutes. Color was developed with 3,3-diaminobenzidine tetrahydrochloride tablet sets (DakoCytomation).Cell Proliferation AssaysThe in vitro growth of stable transfectants was assessed using Cell Counting Kit-8 (Dojin Laboratories, Kumamoto, Japan). Briefly, 1 × 103 cells were seeded into a 96-well plate. The cells were allowed to grow for 1 to 4 days. Then, the cells were incubated with 10 μl of the water-soluble tetrazolium salt-8 reagent for 2 hours. The optical density was measured at 450 nm, with a 655-nm reference, using a microplate reader (model 550; Bio-Rad, Hercules, CA).To examine three-dimensional proliferation of stable clones, 1 × 103 cells were seeded into a 96-well plate (Sumilon Celltight Spheroid; Sumilon, Tokyo, Japan). The spheroids were fed every other day by carefully replacing half of the spent medium with fresh medium. To calculate mean size of the spheroids, diameters were measured every other day. The cells were allowed to grow for 6 days.In Vivo Detection of Platelets Associated with Aggrus-Expressing Cells and Cell SurvivalThe mock-transfected or WT-Aggrus-transfected CHO cells (CHO/control or CHO/WT-Aggrus, respectively) were stained with PKH67 using a green fluorescent cell linker kit (Sigma), according to the manufacturer's instruction, and resuspended in Hanks' balanced salt solution (HBSS) without calcium or magnesium to a final concentration of 2.5 × 106 cells/ml. Female BALB/c-nu/nu mice were injected intravenously with 200 μl of the labeled cells (5 × 105 cells/mouse), and lungs were removed 30 minutes and 6 hours after injection. The number of arrested cells was determined using fluorescence microscopy on 12 images at ×200 magnification from at least two mice at each time point per group. To detect association of CHO/WT-Aggrus cells with platelets, frozen mouse lung sections were stained 30 minutes after injection with an anti-mouse CD41 antibody (Becton Dickinson, San Jose, CA) followed by incubation with a biotinylated second antibody (DakoCytomation) and avidin R-phycoerythrin (Biomeda Corporation, Foster City, CA). The extent of platelet association with CHO/WT-Aggrus cells was quantitated by evaluating sections using conventional epifluorescence. Images were captured on a fluorescence microscopy at ×200 magnification. At least eight independent images for each lung were analyzed. In some experiments, mice were injected intraperitoneally with 75 mg/kg aspirin (Sigma) for 3 days until the day of intravenous injection of the stable clones (5 × 105 cells/mouse) into lateral tail vein of female BALB/c-nu/nu mice. Thirty minutes after cell injection, blood was drawn for testing platelet aggregation, and lungs were removed to count the arrested cells.Experimental Lung Metastasis and Animal SurvivalCHO/control, CHO/WT-Aggrus (clones 3 and 5), and CHO cells that were stably transfected with plasmid containing T34A-human Aggrus (CHO/T34A-Aggrus clones 4 and 5) or T52A-human Aggrus (CHO/T52A-Aggrus clones 10 and 48), and were harvested, washed, and resuspended in HBSS (2.5 × 106 cells/ml). Then, the stable clones (5 × 105 cells/mouse) were inoculated intravenously into lateral tail vein of female BALB/c-nu/nu mice. After 17 days, the mice were euthanized, and surface lung metastatic foci were counted and measured. The lung tissues from CHO/control and CHO/WT-Aggrus-bearing mice were processed for hematoxylin and eosin (H&E) and elastica-van Gieson (EVG) staining and immunohistochemical analysis to confirm the expression of human Aggrus in the metastatic foci. To investigate the effect of aspirin on metastasis formation, female BALB/c-nu/nu mice were injected intraperitoneally with aspirin (75 mg/kg) or phosphate-buffered saline (PBS) for 3 days until the day of intravenous injection of the stable clones (2.5 × 105 cells/mouse). Additional injections of aspirin or PBS were given daily until day 17. After 17 days of cell injection, lung foci were counted and measured. PBS was used as a control. For the survival study, the stable clones (5 × 105 cells/mouse) were inoculated intravenously into lateral tail vein of female BALB/c-nu/nu mice. The survival of the mice was checked daily for 50 days.Tumorigenicity and Spontaneous Metastasis AssaysCHO/control and CHO/WT-Aggrus cells were harvested, washed, and resuspended in HBSS (5 × 107 cells/ml). Cells (5 × 106) in 0.1 ml of HBSS were injected subcutaneously into the back, close to the neck, of female BALB/c-nu/nu mice. Tumors were measured with calipers at 13, 15, 18, 20, 24, and 27 days after injection. Tumor volume was calculated by the following formula: volume = W2Fidler IJ Metastasis: quantitative analysis of distribution and fate of tumor emboli labeled with 125I-5-iodo-2′-deoxyuridine.J Natl Cancer Inst. 1970; 45: 773-782PubMed Google Scholar × L/2, where W = short diameter and L = long diameter. Mice were euthanized 30 days after injection. Lungs and primary tumor tissues were harvested for H&E and EVG staining. Small portions of each tumor tissues were analyzed to confirm the human Aggrus expression by Western blot analysis.Platelet Aggregation AssayPlatelet aggregation was monitored by measuring electric impedance29Cardinal DC Flower RJ The electronic aggregometer: a novel device for assessing platelet behavior in blood.J Pharmacol Methods. 1980; 3: 135-158Crossref PubMed Scopus (623) Google Scholar using a whole-blood aggregometer (model 560; Chronolog, Havertown, PA). Heparinized blood was drawn from BALB/c mice or aspirin-pretreated BALB/c-nu/nu mice by cardiac puncture. Whole blood was then diluted with an equal amount of normal saline. The sample was placed in a plastic cuvette containing a magnetic stir bar and was kept at 37°C for 10 minutes before analysis. The platelet aggregation was then initiated by adding CHO/WT-Aggrus cells (5 × 106 cells/sample) or 10 μmol/L ADP (final concentration) and monitored for up to 20 minutes. In some experiments, whole blood was pretreated with appropriate concentrations of aspirin for 5 minutes at 37°C before stimulation.Statistical AnalysisAll data are shown as means ± SEM, except for cell proliferation assay data that is shown by means ± SD. Student's t-test, Mann-Whitney U-test, and one-way analysis of variance followed by Tukey-Kramer multiple comparisons were performed, where appropriate. The mouse survival assay was evaluated by Kaplan-Meier analysis and the log rank test. P values less than 0.05 were considered statistically significant. All statistical tests were two-sided.ResultsAggrus Induced Platelet Aggregation and Enhanced Cell Survival in VivoOur previous in vitro studies suggested that Aggrus was associated with TCIPA.15Kato Y Fujita N Kunita A Sato S Kaneko M Osawa M Tsuruo T Molecular identification of Aggrus/T1alpha as a platelet aggregation-inducing factor expressed in colorectal tumors.J Biol Chem. 2003; 278: 51599-51605Crossref PubMed Scopus (246) Google Scholar To confirm further the role of Aggrus in in vivo TCIPA and metastasis formation, we injected intravenously green fluorescent (PKH67)-labeled CHO/control or CHO/WT-Aggrus cells into mice and analyzed their pulmonary retention. Several reports suggested that TCIPA was observed 30 minutes after tumor cell injection.30Kim YJ Borsig L Varki NM Varki A P-selectin deficiency attenuates tumor growth and metastasis.Proc Natl Acad Sci USA. 1998; 95: 9325-9330Crossref PubMed Scopus (382) Google Scholar, 31Borsig L Wong R Feramisco J Nadeau DR Varki NM Varki A Heparin and cancer revisited: mechanistic connections involving platelets, P-selectin, carcinoma mucins, and tumor metastasis.Proc Natl Acad Sci USA. 2001; 98: 3352-3357Crossref PubMed Scopus (584) Google Scholar Thus, we examined the pulmonary retention of the Aggrus-expressing cells after 30 minutes and 6 hours of cell injection. The number of PKH67-labeled CHO/WT-Aggrus cells retained in the lung microvasculature was significantly higher than that of CHO/control cells (Figure 1B, P < 0.001). Pulmonary retention of CHO/WT-Aggrus cells, but not CHO/control cells, was also observed 6 hours after injection (Figure 1, A and B). Thus, Aggrus could be associated with the initial arrest and the survival of CHO/WT-Aggrus cells in vivo. To detect Aggrus-induced platelet aggregation in vivo, frozen lung sections were stained with an antibody to CD41, a defined marker for platelets in mouse.30Kim YJ Borsig L Varki NM Varki A P-selectin deficiency attenuates tumor growth and metastasis.Proc Natl Acad Sci USA. 1998; 95: 9325-9330Crossref PubMed Scopus (382) Google Scholar, 31Borsig L Wong R Feramisco J Nadeau DR Varki NM Varki A Heparin and cancer revisited: mechanistic connections involving platelets, P-selectin, carcinoma mucins, and tumor metastasis.Proc Natl Acad Sci USA. 2001; 98: 3352-3357Crossref PubMed Scopus (584) Google Scholar At 30 minutes after cell injection, extensive platelet decoration (red) around CHO/WT-Aggrus cells (green) was observed in 38.8% of the cells, whereas only 7.6% of CHO/control cells were covered with platelets (Figure 1, C and D; P < 0.05). These findings suggest that Aggrus facilitates initial arrest in the lung microvasculature by inducing platelet aggregation and contributes to initial survival of the tumor cells to promote metastasis formation.Threonine-34 (T34) and Threonine-52 (T52) in Human Aggrus Are Critical Sites for Platelet Aggregation-Inducing AbilityWe previously showed that human Aggrus had one PLAG domain containing T52 (in the PLAG3 domain), that mouse Aggrus had one domain containing T34 (in the PLAG1 domain), and that these threonine residues were critical for their platelet aggregation-inducing activities.15Kato Y Fujita N Kunita A Sato S Kaneko M Osawa M Tsuruo T Molecular identification of Aggrus/T1alpha as a platelet aggregation-inducing factor expressed in colorectal tumors.J Biol Chem. 2003; 278: 51599-51605Crossref PubMed Scopus (246) Google Scholar Detailed alignment of the Aggrus homologues revealed that Aggrus contained three tandem repeats of the PLAG domains (PLAG1 to 3) and that the threonine residues in the PLAG1 and the PLAG3 seem to be critical for the activity.27Kaneko MK Kato Y Kitano T Osawa M Conservation of a platelet activating domain of Aggrus/podoplanin as a platelet aggregation-inducing factor.Gene. 2006; 378: 52-57Crossref PubMed Scopus (118) Google Scholar However, the significance of the T34 in the PLAG1 domain of human Aggrus has not been determined yet. Therefore, we confirmed the importance of T34 in platelet-aggregating activity of human Aggrus by establishing stable transfectants. We established stable CHO clones expressing WT human Aggrus (CHO/WT-Aggrus clones 3 and 5; WT-3 and WT-5), human Aggrus containing a T34A mutation (CHO/T34A-Aggrus clones 4 and 5; T34A-4 and -5), human Aggrus containing a T52A mutation (CHO/T52A-Aggrus clones 10 and 48; T52A-10 and -48), and vector alone (CHO/control; control). Western blot analysis showed all clones, with the exception of the CHO/control, expressed Aggrus protein (Figure 2A). Flow cytometric analysis suggested that WT and point-mutated Aggrus proteins were trafficking to the cell surface properly (Figure 2B). The stable clones grew at similar rate throughout 72 hours (Figure 2C), suggesting that Aggrus expression did not affect CHO cell growth in vitro. To examine anchorage-independent growth of the clones, three-dimensional spheroid growth was compared. Spheroids were measured for 6 days. We could not see any difference in anchorage-independent growth (Figure 2D). These cells were further tested for their ability to induce platelet aggregation using whole blood aggregometer. Both CHO/WT-Aggrus clones (WT-3 and WT-5) induced platelet aggregation (Figu" @default.
• W2128585791 created "2016-06-24" @default.
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• W2128585791 date "2007-04-01" @default.
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• W2128585791 title "The Platelet Aggregation-Inducing Factor Aggrus/Podoplanin Promotes Pulmonary Metastasis" @default.
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• W2128585791 doi "https://doi.org/10.2353/ajpath.2007.060790" @default.
• W2128585791 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/1829466" @default.
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