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// Rafaela Erices 1, 2 , Sofia Cubillos 2 , Raul Aravena 2, 3 , Felice Santoro 2 , Monica Marquez 2 , Renan Orellana 1, 15 , Carolina Ramirez 2 , Pamela Gonzalez 2, 4 , Patricia Fuenzalida 2 , Maria Loreto Bravo 2, 4, 5 , Barbara Oliva 2, 4 , Sumie Kato 1 , Carolina Ibanez 5, 6, 7 , Jorge Branes 1 , Erasmo Bravo 8 , Catalina Alonso 8 , Karen Garcia 7, 9 , Clemente Arab 10 , Vicente A. Torres 11, 16 , Alejandro S. Godoy 2, 12 , Jaime Pereira 6 , Galdo Bustos 13 , Julio Cesar Cardenas 13, 14 , Mauricio A. Cuello 1 , Gareth I. Owen 2, 4, 5, 7, 16 1 Division of Obstetrics and Gynecology, Faculty of Medicine, Pontificia Universidad Catolica de Chile, Santiago, Chile 2 Department of Physiological Sciences, Faculty of Biological Sciences, Pontificia Universidad Catolica de Chile, Santiago, Chile 3 Universidad Santo Tomas, Santiago, Chile 4 Biomedical Research Consortium of Chile, Santiago, Chile 5 Millennium Institute on Immunology and Immunotherapy, Pontificia Universidad Catolica de Chile, Santiago, Chile 6 Hematology and Oncology Department, Faculty of Medicine, Pontificia Universidad Catolica de Chile, Santiago, Chile 7 Center UC Investigation in Oncology, Pontificia Universidad Catolica de Chile, Santiago, Chile 8 Hospital Gustavo Fricke, Vina de Mar, Santiago, Chile 9 Hospital Sotero del Rio, Santiago, Chile 10 Hospital Luis Tisne, Santiago, Chile 11 Institute for Research in Dental Sciences, Faculty of Dentistry, Universidad de Chile, Santiago, Chile 12 Department of Urology, Roswell Park Cancer Institute, Buffalo, NY, USA 13 Anatomy and Developmental Biology, Institute of Biomedical Science, Geroscience Center for Brain Health and Metabolism, University of Chile, Santiago, Chile 14 Buck Institute for Research on Aging, Novato, CA, USA 15 Universidad Bernardo OHiggins, Facultad de Salud, Departamento de Ciencias Quimicas y Biologicas, General Gana, Santiago, Chile 16 Advanced Center for Chronic Diseases (ACCDiS), Faculty of Medicine, Universidad de Chile, Santiago, Chile Correspondence to: Gareth I. Owen, email: gowen@bio.puc.cl Keywords: thrombocytosis, hemostasis, EA.hy926, SKOV3, UCI101 Received: July 27, 2016 Accepted: January 27, 2017 Published: February 15, 2017 ABSTRACT Clinical studies have suggested a survival benefit in ovarian cancer patients with type 2 diabetes mellitus taking metformin, however the mechanism by which diabetic concentrations of metformin could deliver this effect is still poorly understood. Platelets not only represent an important reservoir of growth factors and angiogenic regulators, they are also known to participate in the tumor microenvironment implicated in tumor growth and dissemination. Herein, we investigated if diabetic concentrations of metformin could impinge upon the previously reported observation that platelet induces an increase in the tube forming capacity of endothelial cells (angiogenesis) and upon ovarian cancer cell aggressiveness. We demonstrate that metformin inhibits the increase in angiogenesis brought about by platelets in a mechanism that did not alter endothelial cell migration. In ovarian cancer cell lines and primary cultured cancer cells isolated from the ascitic fluid of ovarian cancer patients, we assessed the effect of combinations of platelets and metformin upon angiogenesis, migration, invasion and cancer sphere formation. The enhancement of each of these parameters by platelets was abrogated by the present of metformin in the vast majority of cancer cell cultures tested. Neither metformin nor platelets altered proliferation; however, metformin inhibited the increase in phosphorylation of focal adhesion kinase induced by platelets. We present the first evidence suggesting that concentrations of metformin present in diabetic patients may reduce the actions of platelets upon both endothelial cells and cancer cell survival and dissemination.

Background An increase in circulating platelets, or thrombocytosis, is recognized as an independent risk factor of bad prognosis and metastasis in patients with ovarian cancer; however the complex role of platelets in tumor progression has not been fully elucidated. Platelet activation has been associated with an epithelial to mesenchymal transition (EMT), while Tissue Factor (TF) protein expression by cancer cells has been shown to correlate with hypercoagulable state and metastasis. The aim of this work was to determine the effect of platelet-cancer cell interaction on TF and “Metastasis Initiating Cell (MIC)” marker levels and migration in ovarian cancer cell lines and cancer cells isolated from the ascetic fluid of ovarian cancer patients. Methods With informed patient consent, ascitic fluid isolated ovarian cancer cells, cell lines and ovarian cancer spheres were co-cultivated with human platelets. TF, EMT and stem cell marker levels were determined by Western blotting, flow cytometry and RT-PCR. Cancer cell migration was determined by Boyden chambers and the scratch assay. Results The co-culture of patient-derived ovarian cancer cells with platelets causes: 1) a phenotypic change in cancer cells, 2) chemoattraction and cancer cell migration, 3) induced MIC markers (EMT/stemness), 3) increased sphere formation and 4) increased TF protein levels and activity. Conclusions We present the first evidence that platelets act as chemoattractants to cancer cells. Furthermore, platelets promote the formation of ovarian cancer spheres that express MIC markers and the metastatic protein TF. Our results suggest that platelet-cancer cell interaction plays a role in the formation of metastatic foci. Electronic supplementary material The online version of this article (doi:10.1186/s12885-015-1304-z) contains supplementary material, which is available to authorized users.