Anat Aharon1,2, Anni Rebibo Sabbah1,2, Liron Issman3, Hila Berkovich1,2, Reem Copty1,2, Yeshayahu Talmon3, Benjamin Brenner1,2. 1. Department of Hematology and Bone Marrow Transplantation, Rambam Health Care Campus, Haifa, Israel. 2. Bruce Rappaport Faculty of Medicine, Technion-Israel Institute of Technology, Haifa, Israel. 3. Department of Chemical Engineering, The Russell Berrie Nanotechnology Institute (RBNI), Israel Institute of Technology, Haifa, Israel.
Abstract
BACKGROUND: The involvement of extracellular vesicles (EVs) in cancer-associated thrombosis (CT) is unclear. This study aimed to explore the properties of EVs derived from breast cancer (BC) cells following exposure to high- or low-dose chemotherapeutic agents and evaluate thrombogenic effects of these EVs on endothelial cells (ECs). METHODS: EVs were isolated from BC cell lines (non-metastatic MCF7, high-metastatic MDA-MB-231), pre-exposed to serum-free medium (control), with or without increasing doses of doxorubicin or paclitaxel. EV structure and size were studied using electron microscopy and Nano-sight. Antigen levels were measured by fluorescence-activated cell sorting (FACS). EV effects on EC thrombogenicity were assessed using FACS, factor Xa chromogenic assay and RT-PCR. RESULTS: Serum-free medium BC cell resulted in EV shedding that additionally increased when MDA-MB-231 cells were exposed to high doses of both agents. Tissue factor (TF) levels were similarly low (9-13%) in all EVs compared with the high expression on their parental MDA-MB-231 cells (76-83%). EVs derived from MDA-MB-231 cells stimulated with high-dose doxorubicin demonstrated significantly (fivefold; p < 0.001) elevated levels of negatively charged phospholipids, a 97% decrease in TF pathway inhibitor (TFPI) levels and a sixfold increase (p < 0.001) in procoagulant activity. These EVs also enhanced EC thrombogenicity. Effects of EVs originating from MCF7 cells were less pronounced. CONCLUSION: These findings suggest that thrombogenic properties of BC-derived EVs may depend on the type and dose of the applied chemotherapy agent and may also be affected by the cell metastatic nature. Schattauer GmbH Stuttgart.
BACKGROUND: The involvement of extracellular vesicles (EVs) in cancer-associated thrombosis (CT) is unclear. This study aimed to explore the properties of EVs derived from breast cancer (BC) cells following exposure to high- or low-dose chemotherapeutic agents and evaluate thrombogenic effects of these EVs on endothelial cells (ECs). METHODS: EVs were isolated from BC cell lines (non-metastatic MCF7, high-metastatic MDA-MB-231), pre-exposed to serum-free medium (control), with or without increasing doses of doxorubicin or paclitaxel. EV structure and size were studied using electron microscopy and Nano-sight. Antigen levels were measured by fluorescence-activated cell sorting (FACS). EV effects on EC thrombogenicity were assessed using FACS, factor Xa chromogenic assay and RT-PCR. RESULTS: Serum-free medium BC cell resulted in EV shedding that additionally increased when MDA-MB-231 cells were exposed to high doses of both agents. Tissue factor (TF) levels were similarly low (9-13%) in all EVs compared with the high expression on their parental MDA-MB-231 cells (76-83%). EVs derived from MDA-MB-231 cells stimulated with high-dose doxorubicin demonstrated significantly (fivefold; p < 0.001) elevated levels of negatively charged phospholipids, a 97% decrease in TF pathway inhibitor (TFPI) levels and a sixfold increase (p < 0.001) in procoagulant activity. These EVs also enhanced EC thrombogenicity. Effects of EVs originating from MCF7 cells were less pronounced. CONCLUSION: These findings suggest that thrombogenic properties of BC-derived EVs may depend on the type and dose of the applied chemotherapy agent and may also be affected by the cell metastatic nature. Schattauer GmbH Stuttgart.