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Engagement of αIIbβ3 (GPIIb/IIIa) with ανβ3 Integrin Mediates Interaction of Melanoma Cells with Platelets

A CONNECTION TO HEMATOGENOUS METASTASIS*
  • Anke S. Lonsdorf
    Correspondence
    Supported in part by Deutsche Forschungsgemeinschaft and the Olympia-Morata-Programme of the Ruprecht-Karls-University of Heidelberg. To whom correspondence may be addressed: Dept. of Dermatology, University Hospital, Ruprecht-Karls University Heidelberg, Voßstrasse 11, 69115 Heidelberg, Germany. Tel.: 49-6221-566364; Fax: 49-6221-561617;
    Affiliations
    Department of Dermatology, University Hospital, Ruprecht-Karls University Heidelberg, 69115 Heidelberg, Germany
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  • Björn F. Krämer
    Affiliations
    Department of Cardiovascular Medicine, University Hospital, Eberhard Karls-University Tübingen, 72076 Tübingen, Germany
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  • Manuela Fahrleitner
    Footnotes
    Affiliations
    Department of Cardiovascular Medicine, University Hospital, Eberhard Karls-University Tübingen, 72076 Tübingen, Germany
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  • Tanja Schönberger
    Footnotes
    Affiliations
    Department of Cardiovascular Medicine, University Hospital, Eberhard Karls-University Tübingen, 72076 Tübingen, Germany
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  • Stephan Gnerlich
    Footnotes
    Affiliations
    Department of Cardiovascular Medicine, University Hospital, Eberhard Karls-University Tübingen, 72076 Tübingen, Germany
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  • Sabine Ring
    Affiliations
    Department of Dermatology, University Hospital, Ruprecht-Karls University Heidelberg, 69115 Heidelberg, Germany
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  • Sarah Gehring
    Affiliations
    Department of Cardiovascular Medicine, University Hospital, Eberhard Karls-University Tübingen, 72076 Tübingen, Germany
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  • Stefan W. Schneider
    Footnotes
    Affiliations
    Experimental Dermatology and Department of Dermatology, Ruprecht-Karls University Heidelberg and Medical Faculty Mannheim, Mannheim 68167, Germany
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  • Michael J. Kruhlak
    Affiliations
    Experimental Immunology Branch, Center for Cancer Research, NCI, National Institutes of Health, Bethesda, Maryland 20892
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  • Sven G. Meuth
    Affiliations
    Neurological Clinic-Inflammatory Disorders of the Nervous System and Neurooncology/Institute of Physiology, University of Münster, 48149 Münster, Germany
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  • Bernhard Nieswandt
    Affiliations
    Department of Vascular Medicine and Rudolf Virchow Center, Deutsche Forschungsgemeinschaft Research Center for Experimental Biomedicine, University of Würzburg, Würzburg 97080, Germany
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  • Meinrad Gawaz
    Footnotes
    Affiliations
    Department of Cardiovascular Medicine, University Hospital, Eberhard Karls-University Tübingen, 72076 Tübingen, Germany
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  • Alexander H. Enk
    Affiliations
    Department of Dermatology, University Hospital, Ruprecht-Karls University Heidelberg, 69115 Heidelberg, Germany
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  • Harald F. Langer
    Correspondence
    To whom correspondence may be addressed: Dept. of Cardiovascular Medicine, University Hospital, Eberhard Karls-University Tübingen, Otfried-Müller Str. 10, 72076 Tübingen, Germany. Tel.: 49-7071-29-82887; Fax: 49-7071-29-5040;
    Footnotes
    Affiliations
    Department of Cardiovascular Medicine, University Hospital, Eberhard Karls-University Tübingen, 72076 Tübingen, Germany
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  • Author Footnotes
    * This work was supported, in whole or in part, by National Institutes of Health research career transition award (to A. S. L.). This work was also supported by the University of Tübingen (Center for Interdisciplinary Clinical Research, junior group program), the Volkswagen Foundation (Lichtenberg program), and Wilhelm Sander Foundation (to H. F. L.).
    This article contains supplemental Figs. 1–6 and additional references.
    2 Members of the Tuebingen Platelet Investigative Consortium funded by the Clinical Research Unit KFO 274 of the German Research Council.
    3 Supported by Deutsche Forschungsgemeinschaft Grant SFB/TR23.
Open AccessPublished:November 18, 2011DOI:https://doi.org/10.1074/jbc.M111.269811
      A mutual relationship exists between metastasizing tumor cells and components of the coagulation cascade. The exact mechanisms as to how platelets influence blood-borne metastasis, however, remain poorly understood. Here, we used murine B16 melanoma cells to observe functional aspects of how platelets contribute to the process of hematogenous metastasis. We found that platelets interfere with a distinct step of the metastasis cascade, as they promote adhesion of melanoma cells to the endothelium in vitro under shear conditions. Constitutively active platelet receptor GPIIb/IIIa (integrin αIIbβ3) expressed on Chinese hamster ovary cells promoted melanoma cell adhesion in the presence of fibrinogen, whereas blocking antibodies to aνβ3 integrin on melanoma cells or to GPIIb/IIIa significantly reduced melanoma cell adhesion to platelets. Furthermore, using intravital microscopy, we observed functional platelet-melanoma cell interactions, as platelet depletion resulted in significantly reduced melanoma cell adhesion to the injured vascular wall in vivo. Using a mouse model of hematogenous metastasis to the lung, we observed decreased metastasis of B16 melanoma cells to the lung by treatment with a mAb blocking the aν subunit of aνβ3 integrin. This effect was significantly reduced when platelets were depleted in vivo. Thus, the engagement of GPIIb/IIIa with aνβ3 integrin interaction mediates tumor cell-platelet interactions and highlights how this interaction is involved in hematogenous tumor metastasis.

      Introduction

      Tumor metastasis occurs through a multistep process (
      • Brooks S.A.
      • Lomax-Browne H.J.
      • Carter T.M.
      • Kinch C.E.
      • Hall D.M.
      Molecular interactions in cancer cell metastasis.
      ). The interaction of circulating tumor cells that have detached from the primary tumor with structures of the tissue microvasculature is a crucial step preceding the invasion of the target organ. The specific events determining tumor cell interactions with endothelial cells during hematogenous metastasis are well defined (
      • Sahai E.
      Illuminating the metastatic process.
      ), whereas the contribution of other cell types such as platelets in this process is less well understood.
      Besides their classical role in hemostasis and thrombosis, platelets have been implicated in various pathophysiological processes, including regeneration and inflammation or recently tumor metastasis (
      • Langer H.F.
      • Gawaz M.
      Platelets in regenerative medicine.
      ,
      • Gawaz M.
      • Langer H.
      • May A.E.
      Platelets in inflammation and atherogenesis.
      ,
      • Bambace N.M.
      • Holmes C.E.
      The platelet contribution to cancer progression.
      ,
      • Nachman R.L.
      • Rafii S.
      Platelets, petechiae, and preservation of the vascular wall.
      ,
      • Gay L.J.
      • Felding-Habermann B.
      Contribution of platelets to tumor metastasis.
      ,
      • Semple J.W.
      • Italiano Jr., J.E.
      • Freedman J.
      Platelets and the immune continuum.
      ,
      • Boilard E.
      • Nigrovic P.A.
      • Larabee K.
      • Watts G.F.
      • Coblyn J.S.
      • Weinblatt M.E.
      • Massarotti E.M.
      • Remold-O'Donnell E.
      • Farndale R.W.
      • Ware J.
      • Lee D.M.
      Platelets amplify inflammation in arthritis via collagen-dependent microparticle production.
      ). Both clinical and experimental evidence point to a role of platelets in the spread of cancer, as thrombocytopenia or anti-platelet treatments ameliorate experimental metastasis, and tumors provide a thrombogenic proinflammatory environment that promotes coagulation and endothelial cell activation (
      • Gasic G.J.
      • Gasic T.B.
      • Stewart C.C.
      Antimetastatic effects associated with platelet reduction.
      ,
      • Karpatkin S.
      • Pearlstein E.
      • Salk P.L.
      • Yogeeswaran G.
      Role of platelets in tumor cell metastases.
      ). Interestingly, patients with metastatic disease reveal increased platelet counts and significantly elevated numbers of activated platelets (
      • Wiesner T.
      • Bugl S.
      • Mayer F.
      • Hartmann J.T.
      • Kopp H.G.
      Differential changes in platelet VEGF, Tsp, CXCL12, and CXCL4 in patients with metastatic cancer.
      ). Depending on the type of tumor, various aspects of cancer progression may be affected by platelets, including tumor cell proliferation (
      • Wang Y.
      • Zhang H.
      Platelet-induced inhibition of tumor cell growth.
      ), tumor angiogenesis (
      • Zaslavsky A.
      • Baek K.H.
      • Lynch R.C.
      • Short S.
      • Grillo J.
      • Folkman J.
      • Italiano Jr., J.E.
      • Ryeom S.
      Platelet-derived thrombospondin-1 is a critical negative regulator and potential biomarker of angiogenesis.
      ), vessel stability within tumors (
      • Ho-Tin-Noé B.
      • Goerge T.
      • Cifuni S.M.
      • Duerschmied D.
      • Wagner D.D.
      Platelet granule secretion continuously prevents intratumor hemorrhage.
      ), or immune evasion (
      • Kopp H.G.
      • Placke T.
      • Salih H.R.
      Platelet-derived transforming growth factor-β down-regulates NKG2D thereby inhibiting natural killer cell antitumor reactivity.
      ,
      • Nieswandt B.
      • Hafner M.
      • Echtenacher B.
      • Männel D.N.
      Lysis of tumor cells by natural killer cells in mice is impeded by platelets.
      ). Moreover, platelets contribute to a specific gene expression profile of microvascular endothelial cells in the presence of tumor cells and consecutively to a permissive metastatic microenvironment (
      • Läubli H.
      • Spanaus K.S.
      • Borsig L.
      Selectin-mediated activation of endothelial cells induces expression of CCL5 and promotes metastasis through recruitment of monocytes.
      ). Integrins, a widely expressed family of transmembrane adhesion receptors, represent a central determinant for physiological platelet function. In particular, GPIIb/IIIa is involved in both cell-cell adhesion and thrombus formation at the vascular wall, establishing it as a therapeutic target in vascular diseases (
      • Gawaz M.
      • Geisler T.
      Coronary artery disease. Platelet activity: an obstacle for successful PCI.
      ). For heterotypic cell-cell interactions between platelets and other cells such as leukocytes involving GPIIb/IIIa and fibrinogen, the amino acids Arg-Gly-Asp (RGD) seem to be of particular importance, as treatment with synthetic RGD peptides was shown to block integrin binding (
      • Weber C.
      • Springer T.A.
      Neutrophil accumulation on activated, surface-adherent platelets in flow is mediated by interaction of Mac-1 with fibrinogen bound to αIIbβ3 and stimulated by platelet-activating factor.
      ,
      • Gawaz M.P.
      • Loftus J.C.
      • Bajt M.L.
      • Frojmovic M.M.
      • Plow E.F.
      • Ginsberg M.H.
      Ligand bridging mediates integrin αIIbβ3 (platelet GPIIB-IIIA)-dependent homotypic and heterotypic cell-cell interactions.
      ). Interestingly, pharmacological inhibition of GPIIb/IIIa has been demonstrated to reduce tumor cell metastasis, although the underlying mechanisms remain elusive (
      • Amirkhosravi A.
      • Mousa S.A.
      • Amaya M.
      • Blaydes S.
      • Desai H.
      • Meyer T.
      • Francis J.L.
      Inhibition of tumor cell-induced platelet aggregation and lung metastasis by the oral GpIIb/IIIa antagonist XV454.
      ). Similar to GPIIb/IIIa (
      • Puerschel W.C.
      • Gawaz M.
      • Worret W.I.
      • Ring J.
      Immunoreactivity of glycoprotein IIb is present in metastasized but not in nonmetastasized primary malignant melanoma.
      ), the integrin aνβ3 has been implicated as a potential target in tumor metastasis, as blockade of aνβ3 integrin on murine melanoma cells inhibits lung metastasis in vivo, in a time- and dose-dependent manner, which is mainly attributed to inhibition of angiogenesis (
      • Ramos O.H.
      • Kauskot A.
      • Cominetti M.R.
      • Bechyne I.
      • Salla Pontes C.L.
      • Chareyre F.
      • Manent J.
      • Vassy R.
      • Giovannini M.
      • Legrand C.
      • Selistre-de-Araujo H.S.
      • Crépin M.
      • Bonnefoy A.
      A novel α(v)β(3)-blocking disintegrin containing the RGD motive, DisBa-01, inhibits bFGF-induced angiogenesis and melanoma metastasis.
      ). Currently, the direct relevance of GPIIb/IIIa interacting with aνβ3 for melanoma metastasis has not been investigated. Collectively, these diverse observations reported in the literature have prompted us to investigate how platelets may participate in melanoma cell metastasis. We observed that platelets are relevant for melanoma cell adhesion and identified an interaction between the aforementioned integrins GPIIb/IIIa and ανβ3 as a relevant mechanism involved in adhesion events between platelets and melanoma cells.

      EXPERIMENTAL PROCEDURES

      Reagents

      RGD and RAD peptides were from Calbiochem; anti-mouse aν integrin (CD51) mAb was from Biolegend; anti-mouse CD61 mAb and rat IgG1k isotype control were from ebioscience; anti-mouse CD31 mAb from was Biolegend; rat anti-mouse GPIIb/IIIa mAb and rat anti-GPIb mAb were from Emfret Analytics, and corresponding rat anti-mouse IgG was from Rockland Immunochemicals. Rabbit anti-mouse thrombocyte hyperimmune serum and control serum were purchased from Accurate Chemical & Scientific Corp.; 5-carboxyfluorescein diacetate succinimidyl ester (DCF)
      The abbreviations used are: DCF
      2′,7′-dichlorofluorescein
      CMFDA
      5-chloromethylfluorescein diacetate.
      was from Molecular Probes; fibrinogen was from Sigma. For fixation of tissues, OCT mounting medium (Tissue Tek) was used. Abciximab/c7E3 was from Lilly Pharmaceuticals. Cy2-goat anti-hamster IgG, Cy3-goat anti-rat IgG, and DAPI were from Dianova; CMFDA was from Invitrogen.

      Mice

      6–12-Week-old C57BL/6J female mice were originally purchased from Charles River Laboratories (Sulzfeld, Germany) and housed at the central animal facilities of the University of Heidelberg or the University of Tuebingen, Germany. Animal care and experimental procedures were approved by the institutional review boards and performed in accordance with the institutional guidelines for animal welfare.

      Isolation of Murine and Human Platelets

      Human platelets were isolated as described previously (
      • Langer H.F.
      • Daub K.
      • Braun G.
      • Schönberger T.
      • May A.E.
      • Schaller M.
      • Stein G.M.
      • Stellos K.
      • Bueltmann A.
      • Siegel-Axel D.
      • Wendel H.P.
      • Aebert H.
      • Roecken M.
      • Seizer P.
      • Santoso S.
      • Wesselborg S.
      • Brossart P.
      • Gawaz M.
      Platelets recruit human dendritic cells via Mac-1/JAM-C interaction and modulate dendritic cell function in vitro.
      ,
      • Stellos K.
      • Langer H.
      • Gnerlich S.
      • Panagiota V.
      • Paul A.
      • Schönberger T.
      • Ninci E.
      • Menzel D.
      • Mueller I.
      • Bigalke B.
      • Geisler T.
      • Bültmann A.
      • Lindemann S.
      • Gawaz M.
      Junctional adhesion molecule A expressed on human CD34+ cells promotes adhesion on vascular wall and differentiation into endothelial progenitor cells.
      ,
      • Stellos K.
      • Langer H.
      • Daub K.
      • Schoenberger T.
      • Gauss A.
      • Geisler T.
      • Bigalke B.
      • Mueller I.
      • Schumm M.
      • Schaefer I.
      • Seizer P.
      • Kraemer B.F.
      • Siegel-Axel D.
      • May A.E.
      • Lindemann S.
      • Gawaz M.
      Platelet-derived stromal cell-derived factor-1 regulates adhesion and promotes differentiation of human CD34+ cells to endothelial progenitor cells.
      ). Briefly, venous blood was drawn from the antecubital vein of healthy volunteers and collected in acid/citrate/dextrose buffer. After centrifugation at 430 × g for 20 min, platelet-rich plasma was removed, added to Tyrodes/HEPES buffer (2.5 mmol/liter HEPES, 150 mmol/liter NaCl, 1 mmol/liter KCl, 2.5 mmol/liter NaHCO3, 0.36 mmol/liter NaH2PO4, 5.5 mmol/liter glucose, and 1 mg/ml BSA, pH 6.5), and centrifuged at 900 × g for 10 min. After removal of the supernatant, the resulting platelet pellet was resuspended in Tyrodes/HEPES buffer (pH 7.4 supplemented with 1 mmol/liter CaCl2 and 1 mmol/liter MgCl2). Murine platelets were isolated from pathogen-free C57BL/6J mice (Charles River Laboratories) as described previously (
      • Massberg S.
      • Konrad I.
      • Schürzinger K.
      • Lorenz M.
      • Schneider S.
      • Zohlnhoefer D.
      • Hoppe K.
      • Schiemann M.
      • Kennerknecht E.
      • Sauer S.
      • Schulz C.
      • Kerstan S.
      • Rudelius M.
      • Seidl S.
      • Sorge F.
      • Langer H.
      • Peluso M.
      • Goyal P.
      • Vestweber D.
      • Emambokus N.R.
      • Busch D.H.
      • Frampton J.
      • Gawaz M.
      Platelets secrete stromal cell-derived factor 1α and recruit bone marrow-derived progenitor cells to arterial thrombi in vivo.
      ).

      Cell Lines

      Murine B16/F1 melanoma cells (B16) and B16 cells sequentially transduced with cDNA encoding either CXCR4 (B16-CXCR4) in the pLNCX2 retroviral vector (Clontech) or pLNCX2 empty vector alone (B16-pLNCX2) or with cDNA encoding firefly (Photinus puralis) luciferase (B16-luc), as described previously (
      • Langer H.F.
      • Orlova V.V.
      • Xie C.
      • Kaul S.
      • Schneider D.
      • Lonsdorf A.
      • Fahrleitner M.
      • Choi E.Y.
      • Dutoit V.
      • Pellegrini M.
      • Grossklaus S.
      • Nawroth P.
      • Baretton G.B.
      • Santoso S.
      • Hwang S.T.
      • Arnold B.
      • Chavakis T.
      A novel function of junctional adhesion molecule-C in mediating melanoma cell metastasis.
      ), were the kind gifts from Dr. Sam Hwang (Medical College of Wisconsin). CHO cells expressing wild-type or mutated and thereby activated GPIIb/IIIa (αIIbβ3) integrin were a kind gift from Dr. Karl-Heinz Peter (Baker Heart Research Institute, Melbourne, Australia) (
      • Schwarz M.
      • Meade G.
      • Stoll P.
      • Ylanne J.
      • Bassler N.
      • Chen Y.C.
      • Hagemeyer C.E.
      • Ahrens I.
      • Moran N.
      • Kenny D.
      • Fitzgerald D.
      • Bode C.
      • Peter K.
      Conformation-specific blockade of the integrin GPIIb/IIIa. A novel antiplatelet strategy that selectively targets activated platelets.
      ,
      • Schwarz M.
      • Röttgen P.
      • Takada Y.
      • Le Gall F.
      • Knackmuss S.
      • Bassler N.
      • Büttner C.
      • Little M.
      • Bode C.
      • Peter K.
      Single-chain antibodies for the conformation-specific blockade of activated platelet integrin αIIbβ3 designed by subtractive selection from naive human phage libraries.
      ). Tumor cell lines, the murine endothelial cell line b.End.3, and the human melanoma cell line (MV3) were cultured as described previously (
      • Murakami T.
      • Maki W.
      • Cardones A.R.
      • Fang H.
      • Tun Kyi A.
      • Nestle F.O.
      • Hwang S.T.
      Expression of CXC chemokine receptor-4 enhances the pulmonary metastatic potential of murine B16 melanoma cells.
      ,
      • Kerk N.
      • Strozyk E.A.
      • Pöppelmann B.
      • Schneider S.W.
      The mechanism of melanoma-associated thrombin activity and von Willebrand factor release from endothelial cells.
      ) or grown as described by the supplier.

      Transmigration Assay

      Transmigration of B16 cells was performed as described previously (
      • Langer H.F.
      • Orlova V.V.
      • Xie C.
      • Kaul S.
      • Schneider D.
      • Lonsdorf A.
      • Fahrleitner M.
      • Choi E.Y.
      • Dutoit V.
      • Pellegrini M.
      • Grossklaus S.
      • Nawroth P.
      • Baretton G.B.
      • Santoso S.
      • Hwang S.T.
      • Arnold B.
      • Chavakis T.
      A novel function of junctional adhesion molecule-C in mediating melanoma cell metastasis.
      ,
      • Sachs U.J.
      • Andrei-Selmer C.L.
      • Maniar A.
      • Weiss T.
      • Paddock C.
      • Orlova V.V.
      • Choi E.Y.
      • Newman P.J.
      • Preissner K.T.
      • Chavakis T.
      • Santoso S.
      The neutrophil-specific antigen CD177 is a counter-receptor for platelet endothelial cell adhesion molecule-1 (CD31).
      ). Briefly, transmigration assays were performed using 6.5-mm transwells with 8-μm pore size (Costar, Bodenheim, Germany). b.End.3 cells were seeded on transwell filters 2 days prior to the assay and grown to confluence in the upper compartment in a humidified atmosphere (37 °C, 5% CO2). 600 μl of medium containing SDF-1 (200 ng/ml) or isolated platelets (1 × 107) was added to the lower compartment of the transwell system. 2 × 105 B16-CXCR4 or B16-pLNCX2 control cells, respectively, were added to the upper compartment on top of the endothelial monolayer in a total volume of 100 μl. After incubation for 3 h at 37 °C, cells from the upper chamber were removed with a cotton swab, and the filters were removed and stained with crystal violet. After washing the filters in distilled water, they were mounted on glass coverslips, and the number of transmigrated B16 cells was quantified on the lower side of the filter by cell counting using an inverted microscope (Zeiss Axiovert 200 M).

      Adhesion Assays

      Static Adhesion Assay

      Static adhesion assays and dynamic adhesion assays were performed as described previously (
      • Langer H.F.
      • Daub K.
      • Braun G.
      • Schönberger T.
      • May A.E.
      • Schaller M.
      • Stein G.M.
      • Stellos K.
      • Bueltmann A.
      • Siegel-Axel D.
      • Wendel H.P.
      • Aebert H.
      • Roecken M.
      • Seizer P.
      • Santoso S.
      • Wesselborg S.
      • Brossart P.
      • Gawaz M.
      Platelets recruit human dendritic cells via Mac-1/JAM-C interaction and modulate dendritic cell function in vitro.
      ,
      • Langer H.F.
      • Stellos K.
      • Steingen C.
      • Froihofer A.
      • Schönberger T.
      • Krämer B.
      • Bigalke B.
      • May A.E.
      • Seizer P.
      • Müller I.
      • Gieseke F.
      • Siegel-Axel D.
      • Meuth S.G.
      • Schmidt A.
      • Wendel H.P.
      • Müller I.
      • Bloch W.
      • Gawaz M.
      Platelet-derived bFGF mediates vascular integrative mechanisms of mesenchymal stem cells in vitro.
      ,
      • Langer H.
      • May A.E.
      • Daub K.
      • Heinzmann U.
      • Lang P.
      • Schumm M.
      • Vestweber D.
      • Massberg S.
      • Schönberger T.
      • Pfisterer I.
      • Hatzopoulos A.K.
      • Gawaz M.
      Adherent platelets recruit and induce differentiation of murine embryonic endothelial progenitor cells to mature endothelial cells in vitro.
      ). For static adhesion, 96-well polystyrene plates (Falcon) were coated with murine or human platelets (1 × 108/ml) for 2 h, washed with Tyrode's buffer to remove nonadherent platelets, and blocked for 30 min with 1% BSA in Tyrode's buffer. During the adhesion process, platelets become activated, which was verified using flow cytometry (supplemental Fig. 5). To this end, adherent platelets were incubated with a phycoerythrin-conjugated rat anti-mouse P-Selectin antibody (10 μg/ml). The platelets were then removed from the well using trypsin, washed, and analyzed using a FACSCalibur (BD Biosciences). In the case of human platelets, wells had been pre-coated with collagen (10 μg/ml). Subsequently, murine B16 melanoma cells (5 × 104/well) or human MV3 melanoma cells (5 × 104/well) were added and allowed to adhere for 40 min under cell culture conditions. After careful washing with PBS, adherent melanoma cells were counted and quantified per visual field. In some experiments, melanoma cells were preincubated for 30 min with blocking mAbs to murine CD51 (αν integrin), or platelets were preincubated with blocking mAbs to murine CD61 (β3 integrin) or to GPIIb/IIIa (JON/A (
      • Bergmeier W.
      • Schulte V.
      • Brockhoff G.
      • Bier U.
      • Zirngibl H.
      • Nieswandt B.
      Flow cytometric detection of activated mouse integrin αIIbβ3 with a novel monoclonal antibody.
      )) or control IgG, RGD peptide, or control RAD peptide as indicated in the figure legends. To exclude a difference in the number of adherent platelets by the treatment with JON/A or RGD, and as a consequence a reduction in melanoma cell adhesion, the number of adherent platelets after final washing was assessed using an automated whole blood analyzer (Sysmex Se 9000, Kobe, Japan; supplemental Fig. 6). Cells were collected from the well for analysis using trypsin.

      Flow Chamber Assay

      Interaction of B16 cells to platelets on murine endothelial cells (b.End.3 cells) was measured in a parallel plate flow chamber (Chromaphor, Ascheberg, Germany) at shear rates of 500 s−1, principally as described before (
      • Ring S.
      • Oliver S.J.
      • Cronstein B.N.
      • Enk A.H.
      • Mahnke K.
      CD4+CD25+ regulatory T cells suppress contact hypersensitivity reactions through a CD39, adenosine-dependent mechanism.
      ). Briefly, CMFDA-labeled B16 cells (2 × 105 cells/ml) were perfused over confluent b.End.3 monolayers and preincubated with freshly isolated murine platelets (1 × 108/ml) or Tyrode's buffer (control), respectively, for 4 h under cell culture conditions. In some experiments, platelets or melanoma cells were preincubated for 30 min with blocking mAbs to murine CD51 (aν integrin), murine CD61 (β3 integrin) or control IgG, RGD peptide or control RAD peptide as indicated in the figure legends. The number of adherent cells per visible field was visualized by video microscopy. Images were recorded and evaluated off-line for quantification at the indicated time points; adherent cells appear in yellow after merge of frames 50 and 55 in the off-line analysis at the respective time points after shear flow was started.

      Static Adhesion to CHO Cells Expressing Resting or Activated GPIIb/IIIa

      CHO cells (2 × 105/well) expressing wild-type or mutated and thereby activated GPIIb/IIIa integrin, as described previously (
      • Schwarz M.
      • Meade G.
      • Stoll P.
      • Ylanne J.
      • Bassler N.
      • Chen Y.C.
      • Hagemeyer C.E.
      • Ahrens I.
      • Moran N.
      • Kenny D.
      • Fitzgerald D.
      • Bode C.
      • Peter K.
      Conformation-specific blockade of the integrin GPIIb/IIIa. A novel antiplatelet strategy that selectively targets activated platelets.
      ,
      • Schwarz M.
      • Röttgen P.
      • Takada Y.
      • Le Gall F.
      • Knackmuss S.
      • Bassler N.
      • Büttner C.
      • Little M.
      • Bode C.
      • Peter K.
      Single-chain antibodies for the conformation-specific blockade of activated platelet integrin αIIbβ3 designed by subtractive selection from naive human phage libraries.
      ), were grown to confluency in 6-well plates (Nunc). In these cells, a constitutive high affinity state of GPIIb/IIIa was achieved by deleting the GFFKR region in the integrin α (GPIIIa) subunit (
      • Peter K.
      • Bode C.
      A deletion in the α subunit locks platelet integrin αIIbβ3 into a high affinity state.
      ). This region is highly conserved in the integrin protein family, and deleting the same region in other integrins (e.g. Mac-1) has resulted in a constitutive high affinity state (
      • Eisenhardt S.U.
      • Schwarz M.
      • Schallner N.
      • Soosairajah J.
      • Bassler N.
      • Huang D.
      • Bode C.
      • Peter K.
      Generation of activation-specific human anti-αMβ2 single-chain antibodies as potential diagnostic tools and therapeutic agents.
      ). Wells were washed with warm media to remove nonadherent CHO cells. B16 cells (5 × 104/well) were stained with CellTracker Orange (1:1000; Invitrogen) for 10 min to distinguish melanoma cells from CHO cells, washed, and added to the adherent CHO cells. In selected experiments CHO cells were preincubated with Abciximab/C7E3 inhibiting GPIIb/IIIa (10 μg/ml, Lilly) (
      • Schwarz M.
      • Meade G.
      • Stoll P.
      • Ylanne J.
      • Bassler N.
      • Chen Y.C.
      • Hagemeyer C.E.
      • Ahrens I.
      • Moran N.
      • Kenny D.
      • Fitzgerald D.
      • Bode C.
      • Peter K.
      Conformation-specific blockade of the integrin GPIIb/IIIa. A novel antiplatelet strategy that selectively targets activated platelets.
      ) or control IgG for 30 min. In some experiments, fibrinogen (300 μg/ml) was added to the assay system prior to studying adhesion of melanoma cells to CHO cells as indicated in the figure legends. After 45 min, nonadherent melanoma cells were removed by repeated washings, and adherent melanoma cells were quantified by direct counting using a Zeiss Axiovert fluorescence microscope. In all experiments, nonspecific binding to CHO cells was assessed and was subtracted to calculate specific binding.

      In Vitro [3H]Thymidine Proliferation Assay

      B16 cells, harvested in the exponential growth phase, were incubated at indicated numbers with freshly isolated and washed murine platelets (1 × 107/well) or cell culture media (control) in 96-well round-bottom plates (Falcon) under cell culture conditions. After 2 days, B16 proliferation was assayed by [3H]thymidine (5 μCi/ml) incorporation followed by scintillation counting (MicrobetaTriLux, PerkinElmer Life Sciences).

      Intravital Fluorescence Video Microscopy

      Intravital microscopy and induction of platelet accumulation to study platelet- melanoma cell interaction in vivo were performed principally as described before (
      • Stellos K.
      • Langer H.
      • Gnerlich S.
      • Panagiota V.
      • Paul A.
      • Schönberger T.
      • Ninci E.
      • Menzel D.
      • Mueller I.
      • Bigalke B.
      • Geisler T.
      • Bültmann A.
      • Lindemann S.
      • Gawaz M.
      Junctional adhesion molecule A expressed on human CD34+ cells promotes adhesion on vascular wall and differentiation into endothelial progenitor cells.
      ). Wild-type C57BL/6J mice (Charles River Laboratories) were anesthetized by intraperitoneal injection of a solution of midazolam (5 mg/kg body weight; Ratiopharm), medetomidine (0.5 mg/kg body weight; Pfizer), and fentanyl (0.05 mg/kg body weight, CuraMed Pharma GmbH). Polyethylene catheters (Portex) were implanted into the left jugular vein to administer platelet-depleting serum or control, respectively, and DCF-labeled B16 cells (2 × 105 cells/250 μl). The serum was given 30 min before the cells were injected to achieve sufficient platelet depletion. Platelet accumulation at the vascular wall was induced by temporary ligation of the supplying vessels for 60 min. Before and after induction of ischemia-reperfusion, the cell vascular wall interactions were visualized by in vivo video microscopy. In a control experiment to exclude thrombotic occlusion of the intestinal vessels, animals were treated similarly. Instead of staining melanoma cells with DCF, rhodamine-6G was injected to visualize any potential thrombus formation and thus exclude thrombi in this setting, which might interfere with melanoma cell adhesion. As a positive control, we treated mice locally with FeCl3 to induce extensive thrombus formation (supplemental Fig. 4). All images were recorded and evaluated off-line.

      In Vivo Metastasis Assay

      In vivo metastasis assays using B16-luc cells were performed principally as described previously (
      • Langer H.F.
      • Orlova V.V.
      • Xie C.
      • Kaul S.
      • Schneider D.
      • Lonsdorf A.
      • Fahrleitner M.
      • Choi E.Y.
      • Dutoit V.
      • Pellegrini M.
      • Grossklaus S.
      • Nawroth P.
      • Baretton G.B.
      • Santoso S.
      • Hwang S.T.
      • Arnold B.
      • Chavakis T.
      A novel function of junctional adhesion molecule-C in mediating melanoma cell metastasis.
      ,
      • Murakami T.
      • Maki W.
      • Cardones A.R.
      • Fang H.
      • Tun Kyi A.
      • Nestle F.O.
      • Hwang S.T.
      Expression of CXC chemokine receptor-4 enhances the pulmonary metastatic potential of murine B16 melanoma cells.
      ,
      • Fang L.
      • Lee V.C.
      • Cha E.
      • Zhang H.
      • Hwang S.T.
      CCR7 regulates B16 murine melanoma cell tumorigenesis in skin.
      ). Briefly, B16-luc cells in the exponential growth phase were harvested by trypsinization and washed twice with PBS before injection. Cell viability was >95% as determined by trypan blue dye exclusion. Platelet-depleting serum or control serum was administered intraperitoneally into C57BL/6 mice and randomly distributed into experimental groups as specified, 24 h before and 48 h after tumor inoculation. For footpad injections, cells (4 × 105 in 20 μl of PBS) were injected into the left hind footpads of mice. In this setting, a second experiment, for which platelets were continuously depleted over a longer period of 12 days, was conducted. Successful platelet depletion was monitored in the peripheral blood (data not shown). Tumor growth was measured with a caliper at the indicated time points after tumor inoculation. For B16-luc metastasis to the lungs, B16-luc cells (2 × 105 cells/200 μl of PBS) were injected into the lateral tail veins of mice. Mice were euthanized at the indicated time points for gross inspection and quantification of tumor burden in the lungs or the popliteal lymph nodes by using an in vitro bioluminescence system (MicroBeta TriLux Luminescence Counter, PerkinElmer Life Sciences) principally as described previously (
      • Langer H.F.
      • Orlova V.V.
      • Xie C.
      • Kaul S.
      • Schneider D.
      • Lonsdorf A.
      • Fahrleitner M.
      • Choi E.Y.
      • Dutoit V.
      • Pellegrini M.
      • Grossklaus S.
      • Nawroth P.
      • Baretton G.B.
      • Santoso S.
      • Hwang S.T.
      • Arnold B.
      • Chavakis T.
      A novel function of junctional adhesion molecule-C in mediating melanoma cell metastasis.
      ,
      • Murakami T.
      • Maki W.
      • Cardones A.R.
      • Fang H.
      • Tun Kyi A.
      • Nestle F.O.
      • Hwang S.T.
      Expression of CXC chemokine receptor-4 enhances the pulmonary metastatic potential of murine B16 melanoma cells.
      ). Figures show luciferase activity measurements in relative light units reflecting tumor burden.

      Immunofluorescence Microscopy

      Mice were intravenously injected with murine B16 melanoma cells (2 × 105 cells in 200 μl of PBS), and melanoma cells were allowed to circulate for 60 min. Before sacrificing mice and extracting the lungs, mice were perfused with PBS to efficiently remove circulating blood, including circulating platelets, thus allowing for the assessment of only endothelium-associated platelets or platelets permanently adherent to the endothelium of the lungs. Lung tissue was embedded into OTC (Tissue Tek®, Sakura) mounting medium and flash frozen at −80 °C. 6-μm tissue sections were air-dried, fixed in acetone for 5 min at −80 °C, rehydrated with PBS for 5 min at room temperature, and blocked with 3% skim milk in 5% goat serum for 2 h in a humidified chamber at 4 °C. Sections were then stained with hamster anti-mouse CD31 mAb (Serotec, 1:50), a rat anti-mouse GPIIb/IIIa antibody (JON/A, Emfret, 1:50), or the respective isotype control IgGs in blocking buffer with 0.3% Triton X (Neolab) for 1 h in a humidified chamber at 4 °C. After thorough washing in PBS, sections were incubated with Cy2-labeled goat anti-hamster IgG (Dianova) and Cy3-labeled goat anti-rat IgG (Dianova) at 1:250 and DAPI nuclear stain (Dianova) at 1:5000 in PBS for 30 min. After thorough washing in PBS, sections were covered with mounting medium, dried at room temperature overnight, and analyzed by immunofluorescence microscopy using a Leica microscope.

      Statistical Methods

      p values were based on unpaired two-sided Student's t tests unless otherwise specified. Significance was assumed at p < 0.05.

      RESULTS

      Here, we examined the functional relevance of platelets in hematogenous metastasis using a murine B16 melanoma cell line. When melanoma cells were injected intravenously into mice, platelets could be visualized adherent to the lung endothelium (supplemental Fig. 1). It is known that platelets express and secrete the CXC chemokine SDF-1α (CXCL12), which has recently been implicated in attracting circulating cells to the vascular wall (
      • Stellos K.
      • Langer H.
      • Daub K.
      • Schoenberger T.
      • Gauss A.
      • Geisler T.
      • Bigalke B.
      • Mueller I.
      • Schumm M.
      • Schaefer I.
      • Seizer P.
      • Kraemer B.F.
      • Siegel-Axel D.
      • May A.E.
      • Lindemann S.
      • Gawaz M.
      Platelet-derived stromal cell-derived factor-1 regulates adhesion and promotes differentiation of human CD34+ cells to endothelial progenitor cells.
      ,
      • Jin D.K.
      • Shido K.
      • Kopp H.G.
      • Petit I.
      • Shmelkov S.V.
      • Young L.M.
      • Hooper A.T.
      • Amano H.
      • Avecilla S.T.
      • Heissig B.
      • Hattori K.
      • Zhang F.
      • Hicklin D.J.
      • Wu Y.
      • Zhu Z.
      • Dunn A.
      • Salari H.
      • Werb Z.
      • Hackett N.R.
      • Crystal R.G.
      • Lyden D.
      • Rafii S.
      Cytokine-mediated deployment of SDF-1 induces revascularization through recruitment of CXCR4+ hemangiocytes.
      ,
      • Zernecke A.
      • Schober A.
      • Bot I.
      • von Hundelshausen P.
      • Liehn E.A.
      • Möpps B.
      • Mericskay M.
      • Gierschik P.
      • Biessen E.A.
      • Weber C.
      SDF-1α/CXCR4 axis is instrumental in neointimal hyperplasia and recruitment of smooth muscle progenitor cells.
      ). Expression of the cognate receptor for SDF-1α, CX-chemokine receptor 4 (CXCR4) has been shown to crucially support organ-selective hematogenous metastasis of melanoma cells to the lung (
      • Murakami T.
      • Maki W.
      • Cardones A.R.
      • Fang H.
      • Tun Kyi A.
      • Nestle F.O.
      • Hwang S.T.
      Expression of CXC chemokine receptor-4 enhances the pulmonary metastatic potential of murine B16 melanoma cells.
      ). Therefore, we considered that soluble signaling molecules secreted by platelets, such as SDF-1α, might contribute to the directional chemotaxis and transmigration properties of B16 melanoma cells. As shown in Fig. 1A, the presence of freshly isolated murine platelets did not enhance B16 melanoma transmigration. We observed only a minor increase in transmigrating B16 melanoma cells overexpressing CXCR4 (B16-CXCR4) in the presence of platelets, whereas the addition of high concentrations of recombinant SDF-1α to the lower chamber resulted in a significant increase in transmigrated tumor cells (Fig. 1A).
      Figure thumbnail gr1
      FIGURE 1Platelets mediate melanoma cell adhesion in vitro but have no effect on melanoma cell transmigration or proliferation. A, murine endothelial (b.End.3) cells were grown to confluence in transwell chambers. Murine B16 melanoma cells were added to the upper well and allowed to migrate toward the lower well containing buffer, platelets, or SDF-1 (200 ng/ml). B16 melanoma cells overexpressing CXCR-4 or control vector (B16-pLNCX2) were applied. No significant difference was observed regarding B16 transmigration in the absence or presence of platelets. *, p < 0.05 in the presence of SDF-1. B, static adhesion of B16 cells in the presence (PLT) or absence (ctrl.) of immobilized murine platelets (1 × 108/well). Data are representative of four experiments, each with three analyzed wells/condition (means ± S.D.). *, p < 0.05. C, proliferation of B16 cells was measured by [3H]thymidine incorporation after 48 h of co-culture with freshly isolated murine platelets (PLT) (2 × 107/well) at the indicated ratios or with Tyrode's buffer (ctrl.). Data from three experiments, each with three analyzed wells/condition (means ± S.D.), are shown. No significant difference was observed between groups. D, B16-luc tumor cell formation following subcutaneous inoculation of 4 × 105 B16-luc cells in 20 μl of PBS into the left footpad of C57BL/6 mice. Arrows indicate time points of intraperitoneal injection of 200 μl of anti-murine platelet-hyperimmune serum (1:10, closed circles) to deplete (depl.) platelets or control serum (open squares). Tumor volumes (mm3) were measured using a caliper at the indicated time points. Data are from two experiments (means ± S.D.) with 4 to 5 mice per group. No significant difference was observed between groups. E, B16-luc tumor metastasis to draining popliteal lymph nodes (LN). On day 21 following subcutaneous inoculation of B16-luc (4 × 105 cells/20 μl of PBS) into the left footpad of C57BL/6 mice, draining popliteal lymph nodes were processed to determine the metastatic tumor burden by bioluminescence measurements (in relative light units, RLU). Data are means ± S.D. from two experiments with 4 to 5 mice per group. No significant (n.s.) difference was observed between animals after platelet depletion (PLT-depl.) and control mice (ctrl.).
      Next, we investigated the influence of platelets on melanoma cell adhesion under static conditions. Platelets were allowed to adhere to the polystyrene surface of 96-well flat-bottom plates for 30 min before adding melanoma cells, and after careful washing, the amount of adherent melanoma cells was quantified. The presence of adherent platelets significantly increased melanoma cell adhesion, as shown for mouse platelets and murine B16 melanoma cells (Fig. 1B). Similarly, collagen-immobilized human platelets mediated adhesion of human melanoma cells (MV3) under static conditions (supplemental Fig. 2).
      After metastasizing cells have entered the target organ, they undergo subsequent proliferation. Although selection pressure from the host, in the form of growth factors, might play an important role in the formation of metastatic foci, the proliferation characteristics of [3H]thymidine-pulsed B16 melanoma cells remained unaltered when co-cultured with isolated murine platelets (Fig. 1C). In addition, we did not observe a significant alteration in the growth of subcutaneously implanted luciferase-expressing murine B16 melanoma tumors in the footpad of syngeneic C57BL/6J mice after in vivo depletion of platelets (Fig. 1D). Efficient platelet depletion was achieved with an intraperitoneal injection of rabbit anti-mouse platelet serum (depletion of over 97% at 24 h post-injection) as described previously (supplemental Fig. 3A) (
      • Carvalho-Tavares J.
      • Hickey M.J.
      • Hutchison J.
      • Michaud J.
      • Sutcliffe I.T.
      • Kubes P.
      A role for platelets and endothelial selectins in tumor necrosis factor-α-induced leukocyte recruitment in the brain microvasculature.
      ) and sustained for at least 72 h. Importantly, the number of circulating total leukocytes was unaltered by platelet depletion (supplemental Fig. 3B). Furthermore, ex vivo bioluminescence analysis of the harvested draining popliteal lymph nodes on day 21 after tumor inoculation revealed that the metastatic spread of tumor cells via the lymphatics was unaffected by changes in platelet counts of the host (Fig. 1E). Thus, although we found no significant platelet-mediated effect on the transmigrational and proliferative characteristics of B16 melanoma cells in vitro or the subcutaneous growth of tumors and lymphatic metastasis in vivo, platelets significantly increased the adhesion capacity of B16 melanoma cells.
      To determine the contribution of platelets to the adhesive capacity of tumor cells under flow conditions, we analyzed interactions of B16 cells with platelets on murine endothelial cells (b.End.3) in vitro by using a parallel plate flow chamber. The presence of platelets resulted in significantly more CMFDA-labeled B16 melanoma cells adhering to the endothelial monolayer under shear flow conditions compared with the absence of platelets (Fig. 2, A and B). Next, we monitored adhesion of circulating B16 melanoma cells to platelets under more physiological conditions in vivo using intravital microscopy. In the applied model, platelet accumulation at the vascular wall was provoked by ischemia-reperfusion injury induced by transient ligation of the mesenteric artery (
      • Stellos K.
      • Langer H.
      • Gnerlich S.
      • Panagiota V.
      • Paul A.
      • Schönberger T.
      • Ninci E.
      • Menzel D.
      • Mueller I.
      • Bigalke B.
      • Geisler T.
      • Bültmann A.
      • Lindemann S.
      • Gawaz M.
      Junctional adhesion molecule A expressed on human CD34+ cells promotes adhesion on vascular wall and differentiation into endothelial progenitor cells.
      ), and thus, platelet-melanoma cell interaction could be studied in vivo. Subsequently, fluorescently labeled B16 melanoma cells were injected into the jugular vein of syngeneic C57BL/6J mice after platelet depletion or control treatment. Adhesion of fluorescent tumor cells was directly assessed by real time intravital video microscopy of the mesenteric microvasculature. In line with the findings from our in vitro experiments, off-line video analysis revealed that the capacity of circulating tumor cells to adhere to the injured vascular wall was strongly diminished in the absence of platelets, indicating that circulating melanoma cells interact with platelets in vivo (Fig. 2, C and D). Thrombus formation, which might have influenced melanoma cell adhesion in this setting, was excluded (supplemental Fig. 4).
      Figure thumbnail gr2
      FIGURE 2Melanoma cells interact with platelets under flow conditions and in vivo. A and B, firm adhesion of CMFDA-labeled B16 cells (2 × 105/ml) was assessed after 3 and 5 min of flow at a shear rate of 500 s−1 on a monolayer of b.END.3 cells preincubated with freshly isolated murine platelets (PLT, 1 × 108/ml) or Tyrode's buffer (ctrl.). A, adherent cells appear in yellow after merge of frames 50 and 55 in the off-line analysis at the respective time points after the perfusion was started. B, quantification of B16 cell adhesion experiments to a monolayer of b.END.3 cells. Data are representative of four individual experiments with similar results. *, p < 0.05, means ± S.D. is depicted. C and D, intravital video microscopy demonstrating the in vivo kinetics of firm adhesion of i.v. injected DCF-labeled B16 cells (2 × 105 cells in 250 μl of PBS/mouse i.v.) to the endothelium of mesenteric vessels of C57BL/6J mice. Intravital video microscopy was performed before ligation of a jejunal branch of the superior mesenteric artery and at the indicated time points after reperfusion. Mice were depleted of platelets 30 min before tumor inoculation. Data are pooled from two experiments with 6–8 mice per group (means ± S.D.). *, p < 0.05 compared with control group. D, representative pictures of intravital video microscopy analysis before perfusion and at 60 min after reperfusion. Scale bar, 100 μm.
      In hemostasis, thrombosis, and inflammation, integrins are indispensable for platelet functions such as adhesion to the extracellular matrix and heterotypic cell-cell interaction with other cell types (
      • Gawaz M.
      • Langer H.
      • May A.E.
      Platelets in inflammation and atherogenesis.
      ). Both integrin expression and activation on tumor cells have been linked to tumor progression (
      • Chan B.M.
      • Matsuura N.
      • Takada Y.
      • Zetter B.R.
      • Hemler M.E.
      In vitro and in vivo consequences of VLA-2 expression on rhabdomyosarcoma cells.
      ,
      • Gosslar U.
      • Jonas P.
      • Luz A.
      • Lifka A.
      • Naor D.
      • Hamann A.
      • Holzmann B.
      Predominant role of α4-integrins for distinct steps of lymphoma metastasis.
      ,
      • Felding-Habermann B.
      • O'Toole T.E.
      • Smith J.W.
      • Fransvea E.
      • Ruggeri Z.M.
      • Ginsberg M.H.
      • Hughes P.E.
      • Pampori N.
      • Shattil S.J.
      • Saven A.
      • Mueller B.M.
      Integrin activation controls metastasis in human breast cancer.
      ). Platelets express several integrins, but the fibrinogen receptor GPIIb/IIIa is the most abundant. Once activated, it forms a bridge to proteins such as fibrinogen and mediates cell-cell and cell-matrix interactions (
      • Gawaz M.P.
      • Loftus J.C.
      • Bajt M.L.
      • Frojmovic M.M.
      • Plow E.F.
      • Ginsberg M.H.
      Ligand bridging mediates integrin αIIbβ3 (platelet GPIIB-IIIA)-dependent homotypic and heterotypic cell-cell interactions.
      ). The amino acid motif Arg-Gly-Asp (RGD) is essential for integrin-mediated cell-cell adhesion events, as it mediates fibrinogen binding and heterotypic integrin bridging (
      • Weber C.
      • Springer T.A.
      Neutrophil accumulation on activated, surface-adherent platelets in flow is mediated by interaction of Mac-1 with fibrinogen bound to αIIbβ3 and stimulated by platelet-activating factor.
      ). Indeed, we found that preincubation with RGD, but not control peptide, significantly reduced B16 melanoma cell adhesion to immobilized murine platelets (Fig. 3A). Control experiments showed that after adhesion platelets are activated as assessed by increased staining for the platelet activation marker P-Selectin (supplemental Fig. 5). Moreover, a strong reduction in platelet-mediated adhesion of B16 melanoma cells was achieved after preincubation of murine platelets with a blocking anti-CD61 (β3 integrin) mAb or an anti-GPIIb/IIIa Ab (JON/A) (Fig. 3, B and C) (
      • Bergmeier W.
      • Schulte V.
      • Brockhoff G.
      • Bier U.
      • Zirngibl H.
      • Nieswandt B.
      Flow cytometric detection of activated mouse integrin αIIbβ3 with a novel monoclonal antibody.
      ). To exclude that reduced melanoma cell adhesion was caused by a reduction in the number of present platelets after incubation with RGD or anti-GPIIb/IIIa Ab, the number of platelets in the well was analyzed and showed no difference between control and inhibitory protein (supplemental Fig. 6). Similarly, inhibition of the αν-integrin (CD51) subunit, highly expressed on the surface of B16 melanoma cells (data not shown) by a monoclonal antibody, reduced the increase in platelet-mediated adhesion of B16 melanoma cells (Fig. 3D). However, when we preincubated platelets with an anti-αν-integrin (CD51) antibody, we observed no difference in melanoma cell adhesion to platelets (Fig. 3E). To further investigate the relevance of the platelet fibrinogen receptor in platelet-B16 cell interactions, CHO cells expressing wild-type or mutated and thereby activated GPIIb/IIIa integrins were used to study B16 cell adhesion (
      • Schwarz M.
      • Meade G.
      • Stoll P.
      • Ylanne J.
      • Bassler N.
      • Chen Y.C.
      • Hagemeyer C.E.
      • Ahrens I.
      • Moran N.
      • Kenny D.
      • Fitzgerald D.
      • Bode C.
      • Peter K.
      Conformation-specific blockade of the integrin GPIIb/IIIa. A novel antiplatelet strategy that selectively targets activated platelets.
      ,
      • Schwarz M.
      • Röttgen P.
      • Takada Y.
      • Le Gall F.
      • Knackmuss S.
      • Bassler N.
      • Büttner C.
      • Little M.
      • Bode C.
      • Peter K.
      Single-chain antibodies for the conformation-specific blockade of activated platelet integrin αIIbβ3 designed by subtractive selection from naive human phage libraries.
      ). Our results indicate that both fibrinogen receptor activation and fibrinogen are necessary for adhesion of melanoma cells (Fig. 3F). When fibrinogen was added to CHO cells expressing the activated GPIIb/IIIa receptor, we observed a significant increase in melanoma cell adhesion. Equal amounts of added fibrinogen did not result in increased melanoma cell adhesion in the resting state of the GPIIb/IIIa receptor. The increase in CHO-melanoma cell adhesion could be reversed by inhibition of the GPIIb/IIIa receptor with abciximab (C7e3) (
      • Schwarz M.
      • Meade G.
      • Stoll P.
      • Ylanne J.
      • Bassler N.
      • Chen Y.C.
      • Hagemeyer C.E.
      • Ahrens I.
      • Moran N.
      • Kenny D.
      • Fitzgerald D.
      • Bode C.
      • Peter K.
      Conformation-specific blockade of the integrin GPIIb/IIIa. A novel antiplatelet strategy that selectively targets activated platelets.
      ), whereas control IgG did not reduce melanoma cell adhesion to the activated GPIIb/IIIa receptor (Fig. 3F). Along with these findings, exposure of B16 melanoma cells to RGD peptide substantially reduced the platelet-mediated adhesion of CMFDA-labeled melanoma cells to a endothelial monolayer (b.END.3 cells) under flow conditions, compared with control peptide or untreated B16 melanoma cells (Fig. 4A). Moreover, we observed that inhibition of CD61 blocked the increase of melanoma cell adhesion to platelets, when perfused over murine endothelial cells (Fig. 4B). Similarly, preincubation of melanoma cells with a blocking antibody to aν integrin (CD51) reduced the platelet-mediated increase of melanoma cell adhesion compared with control IgG (Fig. 4C).
      Figure thumbnail gr3
      FIGURE 3Inhibition of GPIIb/IIIa on platelets and ανβ3 on melanoma cells inhibits platelet-melanoma cell interaction. A–E, static adhesion of B16 cells in the presence or absence of immobilized murine platelets (PLT) (1 × 108/well). A, B16 cells were exposed to RGD peptide or RAD control peptide (10 μg/ml) prior to the assay. Data are representative of three experiments (means ± S.D. of three analyzed wells/condition). *, p < 0.05. B–D, similarly, adhesion of B16 cells to immobilized platelets was assessed after addition of blocking anti-CD61 mAb (10 μg/ml; or hamster IgG isotype control) (B), anti-GPIIb/IIIa mAb (JON/A, 10 μg/ml; or rat IgG isotype control) (C), and preincubation of B16 cells with anti-CD51 mAb (10 μg/ml; or rat IgG1 isotype control) (D). Data are representative of three experiments, each with three analyzed wells/condition (means ± S.D.). *, p < 0.05. E, furthermore, adhesion of B16 cells to immobilized platelets was assessed after preincubation of the immobilized platelets with blocking anti-CD51 mAb (10 μg/ml; or IgG1 isotype control (ctrl.)). Data are representative of three experiments, each with three analyzed wells/condition (means ± S.D.), and no significant difference was observed between groups. F, CHO cells bearing native or activated GPIIb/IIIa receptor (
      • Schwarz M.
      • Meade G.
      • Stoll P.
      • Ylanne J.
      • Bassler N.
      • Chen Y.C.
      • Hagemeyer C.E.
      • Ahrens I.
      • Moran N.
      • Kenny D.
      • Fitzgerald D.
      • Bode C.
      • Peter K.
      Conformation-specific blockade of the integrin GPIIb/IIIa. A novel antiplatelet strategy that selectively targets activated platelets.
      ,
      • Schwarz M.
      • Röttgen P.
      • Takada Y.
      • Le Gall F.
      • Knackmuss S.
      • Bassler N.
      • Büttner C.
      • Little M.
      • Bode C.
      • Peter K.
      Single-chain antibodies for the conformation-specific blockade of activated platelet integrin αIIbβ3 designed by subtractive selection from naive human phage libraries.
      ) were grown to confluency. B16 cells were labeled with a fluorescent dye, and adhesion to the CHO cells was assessed in the absence or presence of fibrinogen and a blocking antibody to the platelet fibrinogen receptor GPIIb/IIIa (c7E3) or IgG control. The number of adherent melanoma cells to surface-adherent CHO cells is presented. Nonspecific binding to CHO cells was assessed and was subtracted to calculate specific adhesion. The means ± S.D. (n = 6) is shown. *, p < 0.05.
      Figure thumbnail gr4
      FIGURE 4Inhibition of GPIIb/IIIa on platelets and ανβ3 on melanoma cells inhibits platelet-melanoma cell interaction under flow conditions. A–C, firm adhesion of CMFDA-labeled B16 cells (2 × 105/ml) on a monolayer of b.END.3 cells preincubated with freshly isolated murine platelets (PLT, 1 × 108/ml) or Tyrode's buffer (ctrl.) was assessed at shear rates of 500 s−1 at the indicated time points. A, B16 cells were exposed to RGD peptide or RAD control peptide (10 μg/ml) prior to the assay. Data are representative of four experiments with similar results (means ± S.D.). *, p < 0.05. B and C, similarly, adhesion of B16 cells to bEND.3 cells was assessed in the presence (PLT) or absence (ctrl.) of freshly isolated murine platelets after preincubation of platelets with blocking anti-CD61 (β3 integrin) mAb (10 μg/ml; or hamster IgG isotype control) (B), as well as preincubation of B16 cells with anti-CD51 (αν integrin) mAb (10 μg/ml; or rat IgG1 isotype control) (C). Data are representative of four experiments with similar results (means ± S.D.). *, p < 0.05.
      To assess the potential relevance of our findings for metastasis in vivo, we injected luciferase-transduced murine B16 melanoma cells intravenously and evaluated metastasis to the lung after 3 weeks by luminescence analysis of lung lysates ex vivo. Previous studies have shown that targeting ανβ3 integrin can reduce metastasis in vivo (
      • Ramos O.H.
      • Kauskot A.
      • Cominetti M.R.
      • Bechyne I.
      • Salla Pontes C.L.
      • Chareyre F.
      • Manent J.
      • Vassy R.
      • Giovannini M.
      • Legrand C.
      • Selistre-de-Araujo H.S.
      • Crépin M.
      • Bonnefoy A.
      A novel α(v)β(3)-blocking disintegrin containing the RGD motive, DisBa-01, inhibits bFGF-induced angiogenesis and melanoma metastasis.
      ,
      • Li X.
      • Regezi J.
      • Ross F.P.
      • Blystone S.
      • Ili D.
      • Leong S.P.
      • Ramos D.M.
      Integrin αvβ3 mediates K1735 murine melanoma cell motility in vivo and in vitro.
      ). Similarly, we observed decreased metastasis of B16 melanoma cells to the lung by treatment with a blocking anti-αν integrin (CD51) mAb (Fig. 5). However, this effect was significantly reduced in the absence of platelets in vivo (i.e. we observed no difference in tumor metastasis between mice treated with anti-CD51 mAb compared with control IgG, when mice were treated with anti-platelet serum). Overall, our results highlight the importance of the GPIIb/IIIa receptor on platelets for the interaction with melanoma cells through ανβ3 integrin.
      Figure thumbnail gr5
      FIGURE 5Effect of ανβ3 inhibition on tumor metastasis is reduced in absence of platelets in vivo. B16-luc cells (2 × 105 cells in 200 μl of PBS/animal) were injected i.v. into C57BL/6 mice, and lung tumor burden was assessed on day 21 by detection of luciferase activity ex vivo in lysates of the lung tissue by bioluminescence measurements (in relative light units, RLU). Animals had been injected intraperitoneally with anti-murine platelet-hyperimmune serum (open circles) for platelet (PLT) depletion or with control (ctrl.) serum (filled circles) 24 h before and 48 h after tumor cell inoculation. Where indicated, B16-luc cells were exposed to monoclonal anti-CD51 (10 μg/ml) or rIgG1k isotype control (10 μg/ml) for 30 min and washed with DMEM prior to i.v. injection. Data are pooled from two experiments with a total of 8–10 animals/group. *, p < 0.05, ns = nonsignificant compared with IgG control in the presence or absence of platelets.

      DISCUSSION

      Various molecular details responsible for the reciprocal relationship between metastasizing tumor cells and soluble components of the coagulation cascade have already been described (
      • Nierodzik M.L.
      • Karpatkin S.
      Thrombin induces tumor growth, metastasis, and angiogenesis. Evidence for a thrombin-regulated dormant tumor phenotype.
      ). However, the involvement of platelets, the cellular component of thrombi, in the pathogenesis of cancer metastasis is still poorly understood. The metastatic process is thought to occur as a cascade of distinct sequential steps, including tumor cell chemotaxis, adhesion, extravasation, and finally proliferation of the metastasizing cells. Although the factors involved in each step may vary, the mechanisms mediating the firm arrest of tumor cells to the vascular wall may be among the most important determinants. Here, we provide evidence that platelets interact with melanoma cells both in vitro and in vivo and that the interaction of integrin GPIIb/IIIa expressed on platelets and ανβ3 on melanoma cells contributes to the molecular interface between these two cells types, which may be of relevance for melanoma cell metastasis.
      Platelets in patients with newly diagnosed metastatic disease display an activated state measured by P-Selectin expression (
      • Wiesner T.
      • Bugl S.
      • Mayer F.
      • Hartmann J.T.
      • Kopp H.G.
      Differential changes in platelet VEGF, Tsp, CXCL12, and CXCL4 in patients with metastatic cancer.
      ), indicating that these cells are potentially an important factor in tumor metastasis. A study by Wang and Zhang (
      • Wang Y.
      • Zhang H.
      Platelet-induced inhibition of tumor cell growth.
      ) using various tumor cell lines suggested that platelets directly influence tumor cell proliferation in an MHC-independent manner. Furthermore, platelets are crucial regulators of tumor vessel stability and prevent intra-tumor hemorrhage thereby affecting tumor cell viability (
      • Ho-Tin-Noé B.
      • Goerge T.
      • Cifuni S.M.
      • Duerschmied D.
      • Wagner D.D.
      Platelet granule secretion continuously prevents intratumor hemorrhage.
      ). In turn, it has been reported that human melanoma cells can cause platelet aggregation (
      • Katagiri Y.
      • Hayashi Y.
      • Baba I.
      • Suzuki H.
      • Tanoue K.
      • Yamazaki H.
      Characterization of platelet aggregation induced by the human melanoma cell line HMV-I. Roles of heparin, plasma adhesive proteins, and tumor cell membrane proteins.
      ). In our experiments, local tumor progression was not affected by thrombocytopenia at least when platelets were depleted in the early phase after subcutaneous tumor cell implantation. Surprisingly, although platelets have been reported to provide chemotactic signals to various cell types, including leukocytes or stem cells (
      • Gawaz M.
      • Langer H.
      • May A.E.
      Platelets in inflammation and atherogenesis.
      ,
      • Massberg S.
      • Konrad I.
      • Schürzinger K.
      • Lorenz M.
      • Schneider S.
      • Zohlnhoefer D.
      • Hoppe K.
      • Schiemann M.
      • Kennerknecht E.
      • Sauer S.
      • Schulz C.
      • Kerstan S.
      • Rudelius M.
      • Seidl S.
      • Sorge F.
      • Langer H.
      • Peluso M.
      • Goyal P.
      • Vestweber D.
      • Emambokus N.R.
      • Busch D.H.
      • Frampton J.
      • Gawaz M.
      Platelets secrete stromal cell-derived factor 1α and recruit bone marrow-derived progenitor cells to arterial thrombi in vivo.
      ,
      • Langer H.
      • May A.E.
      • Daub K.
      • Heinzmann U.
      • Lang P.
      • Schumm M.
      • Vestweber D.
      • Massberg S.
      • Schönberger T.
      • Pfisterer I.
      • Hatzopoulos A.K.
      • Gawaz M.
      Adherent platelets recruit and induce differentiation of murine embryonic endothelial progenitor cells to mature endothelial cells in vitro.
      ,
      • von Hundelshausen P.
      • Weber K.S.
      • Huo Y.
      • Proudfoot A.E.
      • Nelson P.J.
      • Ley K.
      • Weber C.
      RANTES deposition by platelets triggers monocyte arrest on inflamed and atherosclerotic endothelium.
      ), we observed no effect of platelets on B16 melanoma transmigration. In contrast, we observed a robust increase in adhesivity of murine and human melanoma cells in the presence of platelets in various settings in vitro. Adherent platelets present potent adhesion receptors such as JAM-C, P-Selectin, GPIbα, or GPIIb/IIIa to other circulating cells providing a platform for a potent cellular bridging mechanism to the vascular wall (
      • Weber C.
      • Springer T.A.
      Neutrophil accumulation on activated, surface-adherent platelets in flow is mediated by interaction of Mac-1 with fibrinogen bound to αIIbβ3 and stimulated by platelet-activating factor.
      ,
      • Langer H.F.
      • Daub K.
      • Braun G.
      • Schönberger T.
      • May A.E.
      • Schaller M.
      • Stein G.M.
      • Stellos K.
      • Bueltmann A.
      • Siegel-Axel D.
      • Wendel H.P.
      • Aebert H.
      • Roecken M.
      • Seizer P.
      • Santoso S.
      • Wesselborg S.
      • Brossart P.
      • Gawaz M.
      Platelets recruit human dendritic cells via Mac-1/JAM-C interaction and modulate dendritic cell function in vitro.
      ,
      • Langer H.F.
      • Gawaz M.
      Platelet-vessel wall interactions in atherosclerotic disease.
      ,
      • Santoso S.
      • Sachs U.J.
      • Kroll H.
      • Linder M.
      • Ruf A.
      • Preissner K.T.
      • Chavakis T.
      The junctional adhesion molecule 3 (JAM-3) on human platelets is a counter-receptor for the leukocyte integrin Mac-1.
      ,
      • Ehlers R.
      • Ustinov V.
      • Chen Z.
      • Zhang X.
      • Rao R.
      • Luscinskas F.W.
      • Lopez J.
      • Plow E.
      • Simon D.I.
      Targeting platelet-leukocyte interactions. Identification of the integrin Mac-1-binding site for the platelet counter receptor glycoprotein Ibα.
      ,
      • Langer H.F.
      • Chavakis T.
      Leukocyte-endothelial interactions in inflammation.
      ,
      • Wagner D.D.
      • Frenette P.S.
      The vessel wall and its interactions.
      ). Recently, glycoprotein Ibα (GPIbα), the second most abundant receptor expressed on platelets, has been reported to be significantly involved in melanoma metastasis, as the functional absence of GPIbα correlated with a clear reduction in the number of lung metastatic foci in vivo (
      • Jain S.
      • Zuka M.
      • Liu J.
      • Russell S.
      • Dent J.
      • Guerrero J.A.
      • Forsyth J.
      • Maruszak B.
      • Gartner T.K.
      • Felding-Habermann B.
      • Ware J.
      Platelet glycoprotein Ibα supports experimental lung metastasis.
      ). In contrast, another report showed that GPIbα inhibition led to a significant increase in pulmonary metastasis, improved survival, and pulmonary arrest of tumor cells (
      • Erpenbeck L.
      • Nieswandt B.
      • Schön M.
      • Pozgajova M.
      • Schön M.P.
      Inhibition of platelet GPIb α and promotion of melanoma metastasis.
      ), presumably reflecting the complexity of tumor cell-platelet-endothelial interactions and manifesting the need for continued experimental studies. Here, we confirm by intravital microscopy that platelet depletion results in significantly decreased melanoma cell adhesion to the vascular wall in vivo, demonstrating an interaction of melanoma cells with platelets in vivo. Once adherent to the vessel wall, such as after endothelial injury, the platelet fibrinogen receptor GPIIb/IIIa becomes activated and contributes to thrombus formation and wound healing (
      • Langer H.F.
      • Gawaz M.
      Platelets in regenerative medicine.
      ,
      • Varga-Szabo D.
      • Pleines I.
      • Nieswandt B.
      Cell adhesion mechanisms in platelets.
      ). Interestingly, we observed that inhibition of GPIIb/IIIa resulted in both the reduced adhesion of melanoma cells to immobilized platelets and the reduced adhesion to endothelial cells under shear flow conditions in the presence of platelets. Similarly to GPIIb/IIIa, integrin ανβ3 can bind RGD proteins and mediate cell aggregation or cell adhesion in a homotypic or heterotypic fashion (
      • Gawaz M.P.
      • Loftus J.C.
      • Bajt M.L.
      • Frojmovic M.M.
      • Plow E.F.
      • Ginsberg M.H.
      Ligand bridging mediates integrin αIIbβ3 (platelet GPIIB-IIIA)-dependent homotypic and heterotypic cell-cell interactions.
      ,
      • Plow E.F.
      • Cierniewski C.S.
      • Xiao Z.
      • Haas T.A.
      • Byzova T.V.
      αIIbβ3 and its antagonism at the new millennium.
      ). Stable arrest and adhesion strengthening of circulating neutrophils to surface-adherent platelets in flow requires interactions of integrins with fibrinogen bound to platelet GPIIb/IIIa (
      • Weber C.
      • Springer T.A.
      Neutrophil accumulation on activated, surface-adherent platelets in flow is mediated by interaction of Mac-1 with fibrinogen bound to αIIbβ3 and stimulated by platelet-activating factor.
      ).
      It is clear that ανβ3 expressed on tumor cells regulates a broad range of cellular functions such as survival, apoptosis, migration, or angiogenesis contributing to tumorigenicity (
      • Nemeth J.A.
      • Nakada M.T.
      • Trikha M.
      • Lang Z.
      • Gordon M.S.
      • Jayson G.C.
      • Corringham R.
      • Prabhakar U.
      • Davis H.M.
      • Beckman R.A.
      α-v integrins as therapeutic targets in oncology.
      ,
      • Eliceiri B.P.
      • Cheresh D.A.
      The role of αv integrins during angiogenesis. Insights into potential mechanisms of action and clinical development.
      ). Regarding cell adhesion, expression of integrin ανβ3 promotes a metastatic phenotype in human melanoma by supporting specific adhesive properties of the tumor cells (
      • Felding-Habermann B.
      • Fransvea E.
      • O'Toole T.E.
      • Manzuk L.
      • Faha B.
      • Hensler M.
      Involvement of tumor cell integrin αv β3 in hematogenous metastasis of human melanoma cells.
      ). Using an RGD peptide and CHO cells expressing resting or constitutively active GPIIb/IIIa (
      • Schwarz M.
      • Meade G.
      • Stoll P.
      • Ylanne J.
      • Bassler N.
      • Chen Y.C.
      • Hagemeyer C.E.
      • Ahrens I.
      • Moran N.
      • Kenny D.
      • Fitzgerald D.
      • Bode C.
      • Peter K.
      Conformation-specific blockade of the integrin GPIIb/IIIa. A novel antiplatelet strategy that selectively targets activated platelets.
      ,
      • Schwarz M.
      • Röttgen P.
      • Takada Y.
      • Le Gall F.
      • Knackmuss S.
      • Bassler N.
      • Büttner C.
      • Little M.
      • Bode C.
      • Peter K.
      Single-chain antibodies for the conformation-specific blockade of activated platelet integrin αIIbβ3 designed by subtractive selection from naive human phage libraries.
      ), we demonstrated that melanoma cells adhere to platelets in an integrin-dependent fashion. These experiments furthermore indicate that both integrins interact with each other in the presence of fibrinogen.
      Furthermore, experiments using blocking antibodies to GPIIb/IIIa or ανβ3, respectively, revealed a significant involvement of these surface receptors in melanoma cell/platelet adhesion. These findings indicate an important potential mechanism, whereby interactions between GPIIb/IIIa on platelets and ανβ3 on tumor cells support metastatic spread.
      Besides contributing to cell-cell interactions, platelets may influence melanoma cell metastasis in a paracrine fashion. As platelets contain a very broad range of paracrine mediators (
      • Langer H.F.
      • Gawaz M.
      Platelets in regenerative medicine.
      ,
      • Gawaz M.
      • Langer H.
      • May A.E.
      Platelets in inflammation and atherogenesis.
      ,
      • Semple J.W.
      • Italiano Jr., J.E.
      • Freedman J.
      Platelets and the immune continuum.
      ,
      • Boilard E.
      • Nigrovic P.A.
      • Larabee K.
      • Watts G.F.
      • Coblyn J.S.
      • Weinblatt M.E.
      • Massarotti E.M.
      • Remold-O'Donnell E.
      • Farndale R.W.
      • Ware J.
      • Lee D.M.
      Platelets amplify inflammation in arthritis via collagen-dependent microparticle production.
      ), future studies will have to address the distinct contribution of soluble platelet-derived factors for hematogenous melanoma metastasis. Furthermore, it will be interesting to investigate, how circulating melanoma cells influence activation of platelets. In extension to our studies, it will have to be evaluated whether stability of platelet adhesion and platelet receptor binding capacity is an important factor for melanoma cell adhesion augmented by platelets.
      Our results demonstrate the relevance of the interaction between melanoma cells and platelets for hematogenous tumor cell metastasis and may indicate potential novel molecular targets for patients at risk for metastatic tumor spread. Although further detailed studies are needed to address this concept, in particular, the identification of novel adhesion receptors involved in platelet-melanoma cell cross-talk, such as the interaction of GPIIb/IIIa with ανβ3, may reveal promising new pharmacological approaches to block tumor metastasis.

      Acknowledgments

      We acknowledge Dr. Helmut Salih and Dr. Hans-Georg Kopp for critical revision of the manuscript and Sarah Gekeler for excellent technical assistance.

      Supplementary Material

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