Project Summary Traditionally viewed as the bandaids of the blood, the contribution of platelets to the progression of malignancy is emerging as a compelling focus for therapeutic intervention. Complex interactions between tumor cells, and circulating platelets play an important role in tumor growth and dissemination, and a growing body of data supports a role for platelet activation and release of chemokines in metastases and neovascularization. Supporting this concept is the evidence that elevated platelet counts (thrombocytosis) at time of diagnosis with malignancy is a harbinger of an aggressive cancer with a poor prognosis. One very interesting and provocative connection between cancer and platelets is the increasing evidence that tumor cells hijack platelets to promote a more pro-malignant phenotype to drive disease progression. Our laboratories have been instrumental in establishing the pro-malignant role of platelets in metastasis and neovascularization. We have recently discovered that tumor cells can instruct platelets to release CCL5, a known driver of tumor cell invasion and metastasis, and have expanded the role of CCL5 not only as a regulator of metastasis but also as a central controller of platelet production. Despite this progress, how tumor cells instruct megakaryocytes to increase platelet production, and how malignancy reprograms megakaryocytes and manipulates platelet phenotype to support tumor growth and metastasis remains an enigma. Since platelets play a central role in driving cancer, this proposal will address three interrelated while independent Specific Aims focused on the cell biological and molecular pathways by which tumor cells hijack megakaryocytes and platelets to promote cancer growth. Specific Aim 1 will determine the role of the chemokine CCL5 as a major driver of thrombocytosis in malignancy via upregulation of megakaryocyte maturation and proplatelet production. Specific Aim 2 will determine how malignancy reprograms megakaryocytes to produce a more pro-malignant platelet phenotype, with upregulation of factors essential to neovascularization and metastasis. Specific Aim 3 will determine the clinical significance of CCL5 driven platelet production and megakaryocyte reprogramming by elucidating the impact of each in breast cancer patients. This work is innovative because it enters unchartered territory and takes a multifaceted experimental approach to understanding how tumor cells hijack platelets by uniting work on platelet production with cancer biology. Taken together, we expect that this investigation will demonstrate the molecular mechanisms by which malignancy can subvert normal platelet biology by manipulating the megakaryocyte to promote cancer growth and metastasis, and lay the foundation for the development of novel therapeutic modalities.