Blood platelets and cancer: the involvement of platelet-derived extracellular vesicles in tumour progression.

In addition to their roles in haemostasis and thrombosis, blood platelets have been extensively implicated in the progression of cancer. During metastasis, circulating tumour cells (CTCs) induce platelet activation and aggregation, that are exploited to increase cancer dissemination though multiple mechanisms [1, 2]. Platelet-derived extracellular vesicles (PEVs) are shed from platelet membrane in response to extracellular stimuli and carry important biological signals to recipient cells. In vitro, cancer cells induce the release of PEVs and, accordingly, cancer patients display increased levels of circulating PEVs [3, 4]. PEVs are known mediators of cell-to-cell communication and gaining growing attention as potential mediators of the platelet-cancer interplay [5]. Nevertheless, PEVs ability to regulate target cancer cells is largely unexplored. This study aims to investigate the effects of PEVs on breast cancer cells in vitro. Four breast cancer cell lines MDA-MB-231, SKBR3, MCF-7, and BT474, characterized by different metastatic potential, were used. PEVs were purified by differential centrifugation from thrombin-stimulated platelets. Breast cancer cells were co-cultured with PEVs and different aspects were analysed. PEVs specifically bind to different breast cancer cells and elicit cell-specific functional responses. We found that PEVs massively interact with the metastatic cell lines MDA-MB-231 and SKBR3 and the ductal carcinoma cell line BT474, but not with MCF-7 cell line. Confocal microscopy analysis demonstrated that PEVs are internalized by the three cell lines, although with different efficiency. PEVs internalization was associated to the acquisition of the specific platelet marker integrin αIIb, as demonstrated by several approaches, such as immunofluorescence microscopy and immunoblotting analyses. Within 48 hours, in SKBR3 and BT474 cell lines, PEVs induced minor alteration in the different phases of the cell cycle without affecting cell viability. Conversely, PEVs potently stimulated migration and invasion of MDA-MB-231, without affecting the progression of cell cycle. PEVs triggered a sustained increase of intracellular Ca2+ concentration in MDA-MB-231, SKBR3 and BT474 cells, indicating that these microvesicles activate signalling responses that may influence the behaviour of recipient cells. Although in all the analysed breast cancer cells PEVs evoked intracellular Ca2+ mobilization, only in MDA-MB-231 cells the event was associated to the stimulation of selected signalling proteins implicated in migration, including p38MAPK and myosin light chain 2 (MLC2). Importantly, the inhibition of myosin light chain phosphorylation by a Rho kinase inhibitor prevented PEVs-stimulated migration of MDA-MB-231 cells. Our results indicate that different breast cancer cells can interact and internalize PEVs, which induce intercellular calcium movements. Moreover, PEVs are versatile regulators of cancer cell behaviour and elicit a variety of different responses depending on the specific breast cancer cell subtype. References 1. Gay, L.J. and B. Felding-Habermann, Contribution of platelets to tumour metastasis. Nat Rev Cancer, 2011. 11(2): p. 123-34. 2. Bambace, N.M., J.E. Levis, and C.E. Holmes, The effect of P2Y-mediated platelet activation on the release of VEGF and endostatin from platelets. Platelets, 2010. 21(2): p. 85-93. 3. Zarà, M., et al., Release of Prometastatic Platelet-Derived Microparticles Induced by Breast Cancer Cells: A Novel Positive Feedback Mechanism for Metastasis. TH Open, 2017. 1(2): p. e155-e163. 4. Chaari, M., et al., Impact of breast cancer stage, time from diagnosis and chemotherapy on plasma and cellular biomarkers of hypercoagulability. BMC Cancer, 2014. 14: p. 991. 5. Mezouar, S., et al., Involvement of platelet-derived microparticles in tumor progression and thrombosis. Semin Oncol, 2014. 41(3): p. 346-58.