by Ludovic Durrieu, Alamelu Bharadwaj, and David M. Waisman Analysis of the thrombotic and fibrinolytic activities of tumor cell–derived extracellular vesicles by Ludovic Durrieu, Alamelu Bharadwaj, and David M. Waisman BloodAdv Volume 2(10):1054-1065 May 22, 2018 © 2018 by The American Society of Hematology

Ludovic Durrieu et al. Blood Adv 2018;2:1054-1065 © 2018 by The American Society of Hematology

Purification of exosomes and MVs from tumor cells. Purification of exosomes and MVs from tumor cells. Representative schema for the purification of exosomes and MVs by differential centrifugation. Tumor cells were seeded in dishes (1) and cultured for 5 days (2). The conditioned media were collected (3) and centrifuged at 500g (4) to eliminate the cells (5), and the supernatant was collected (6) and centrifuged at 2000g (7) to eliminate the cell debris (8). To isolate MVs, the supernatant was collected (9a) and centrifuged at 20 000g (10a). This pellet was used as a source of “MVs” (11a). To isolate the exosomes, the supernatant was collected (9b) and centrifuged at 10 000g (10b) to eliminate the large particles (11b). Then, the supernatant was collected (12b) and centrifuged at 100 000g (13b). The pellet was used as a source of “exosomes” (14b). Ludovic Durrieu et al. Blood Adv 2018;2:1054-1065 © 2018 by The American Society of Hematology

Characterization of exosomes and MVs from tumor cells. Characterization of exosomes and MVs from tumor cells. Exosomes and MVs, collected by differential centrifugation, were characterized by TEM (A) and western blot (C). The size of MVs was determined by TEM on 25 measurements from MB-MDA-231 cells and 25 measurements from MCF7 cells. (B) The size of exosomes was determined from 20 measurements from MD-MBA-231 cells, 15 measurements from MCF7 cells, and 15 measurements from A549 cells. Results are presented as mean ± SEM. (C) Cell lysates, exosomes, and MVs (30 μg) from each cell line were analyzed by western blotting for TSG101, CD81, and actinin-4. One representative blot is shown for each cell line. Each blot was repeated 3 times with different samples. ****P ≤ .0001, Mann-Whitney U test (1 tailed). Ludovic Durrieu et al. Blood Adv 2018;2:1054-1065 © 2018 by The American Society of Hematology

Thrombotic activity of exosomes and MVs from MDA-MB-231 cell line. Thrombotic activity of exosomes and MVs from MDA-MB-231 cell line. The thrombotic activity of exosomes and MVs was tested using pure fibrin clots or clots produced in platelet-poor plasma. A total of 30 μg of exosomes or MVs was added to pure fibrinogen or platelet-poor plasma. The OD was measured at 350 nm every 2 minutes for 1 hour at 37°C. α-Thrombin was added to the fibrinogen or platelet-poor plasma as a positive control. Thrombotic activity in the presence of fibrinogen is shown for MVs (Ai) and exosomes (Bi) from MDA-MD-231 cells. The thrombotic activity with human plasma is shown for MVs (Aii) and exosomes (Bii) from MDA-MD-231 cells. Representative photographs of the experiment are presented in the lower panels. *P ≤ .05, **P ≤ .01, ***P ≤ .001, Mann-Whitney U test (1 tailed). Ludovic Durrieu et al. Blood Adv 2018;2:1054-1065 © 2018 by The American Society of Hematology

Fibrinolytic activity of exosomes and MVs from cancer cells on a fibrin clot. Fibrinolytic activity of exosomes and MVs from cancer cells on a fibrin clot. The analysis was performed on MDA-MB-231 cells (A-B), MCF7 cells (C-F), and NB4 cells (G-J) cell lines. Each experiment was repeated with 3 independent EV isolations. A fibrin clot was formed by the incubation of thrombin with pure fibrinogen. Exosomes and MVs (30 μg of protein) were added to the top of clot, followed by the addition of plasminogen (0.15 μM), with or without inhibitor (100 mM ε-AcA or 2.2 μM Apro). For a positive control, plasminogen (0.15 μM) and tPA (25 nM) were added to the fibrin clot. OD was measured at 350 nm every 20 minutes for 18 hours at 37°C. Results are represented as the mean ± SEM (n = 3 independent samples). The percentage of clot retention is shown for MVs (Ai) and exosomes (Bi) from MDA-MB-231 cells, MVs (Ci) and exosomes (Di) from MCF7 cells, MVs (Ei) and exosomes (Fi) from MCF7-PMA cells, MVs (Gi) and exosomes (Hi) from NB4 cells, and MVs (Ii) and exosomes (Ji) from NB4-ATRA cells. A representative photograph of the experiment is shown for MVs (Aii) and exosomes (Bii) from MDA-MB-231 cells, MVs (Cii) and exosomes (Dii) from MCF7 cells, MVs (Eii) and exosomes (Fii) from MCF7-PMA cells, MVs (Gii) and exosomes (Hii) from NB4 cells, and MVs (Iii) and exosomes (Jii) from NB4-ATRA cells. *P ≤ .05, **P ≤ .01, ****P ≤ .0001, Mann-Whitney U test (1 tailed). Ludovic Durrieu et al. Blood Adv 2018;2:1054-1065 © 2018 by The American Society of Hematology

Fibrinolytic activity of exosomes and MVs from cancer cells on a fibrin clot. Fibrinolytic activity of exosomes and MVs from cancer cells on a fibrin clot. The analysis was performed on MDA-MB-231 cells (A-B), MCF7 cells (C-F), and NB4 cells (G-J) cell lines. Each experiment was repeated with 3 independent EV isolations. A fibrin clot was formed by the incubation of thrombin with pure fibrinogen. Exosomes and MVs (30 μg of protein) were added to the top of clot, followed by the addition of plasminogen (0.15 μM), with or without inhibitor (100 mM ε-AcA or 2.2 μM Apro). For a positive control, plasminogen (0.15 μM) and tPA (25 nM) were added to the fibrin clot. OD was measured at 350 nm every 20 minutes for 18 hours at 37°C. Results are represented as the mean ± SEM (n = 3 independent samples). The percentage of clot retention is shown for MVs (Ai) and exosomes (Bi) from MDA-MB-231 cells, MVs (Ci) and exosomes (Di) from MCF7 cells, MVs (Ei) and exosomes (Fi) from MCF7-PMA cells, MVs (Gi) and exosomes (Hi) from NB4 cells, and MVs (Ii) and exosomes (Ji) from NB4-ATRA cells. A representative photograph of the experiment is shown for MVs (Aii) and exosomes (Bii) from MDA-MB-231 cells, MVs (Cii) and exosomes (Dii) from MCF7 cells, MVs (Eii) and exosomes (Fii) from MCF7-PMA cells, MVs (Gii) and exosomes (Hii) from NB4 cells, and MVs (Iii) and exosomes (Jii) from NB4-ATRA cells. *P ≤ .05, **P ≤ .01, ****P ≤ .0001, Mann-Whitney U test (1 tailed). Ludovic Durrieu et al. Blood Adv 2018;2:1054-1065 © 2018 by The American Society of Hematology

Fibrinolytic activity of exosomes and MVs on a plasma clot. Fibrinolytic activity of exosomes and MVs on a plasma clot. The analysis was performed on MVs from MDA-MB-231 cells (A) and exosomes from MDA-MB-231 cells (B). Each experiment was repeated 3 times with different samples. The plasma clot was formed by incubating human platelet-poor plasma with thrombin. Then, exosomes and MVs (30 μg of protein) were added to the top of clot, followed by plasminogen (0.15 μM), with or without inhibitor (ε-AcA at 100 mM or Apro at 2.2 μM). A positive control was included, in which plasminogen (0.15 μM) and tPA (25 nM) were added directly to the freshly formed clot. The OD was measured at 350 nm every 20 minutes for 18 hours at 37°C. Results are presented as mean ± SEM (n = 3 independent samples). The percentage of clot retention is shown for MVs (Ai) and exosomes (Bi) from MDA-MD-231 cells. A representative photograph of the experiment is shown for MVs (Aii) and exosomes (Bii) from MDA-MD-231 cells. ****P ≤ .0001, Mann-Whitney U test (1 tailed). Ludovic Durrieu et al. Blood Adv 2018;2:1054-1065 © 2018 by The American Society of Hematology

Exosomes from MDA-MB-231 cells can increase the plasmin generation of tumor cells. Exosomes from MDA-MB-231 cells can increase the plasmin generation of tumor cells. Exosomes were collected from MDA-MB-231 cells and incubated with MCF7 and A549 cells for 2 hours. Subsequently, tumor cells were seeded at 50 000 cells per well for 3 days. Cells were tested for plasmin generation with plasminogen at 0.5 μM final concentration and the substrate S2251 at 0.5 μM final concentration. OD was read at 405 and 600 nm for 4 hours every 4 minutes at 37°C. The experiment was repeated 3 times in triplicate. Results show the rate of plasmin generation per 1 million cells. Cells with no incubation of exosomes are represented by the red bars, and cells with incubation of exosomes are represented by blue bars. *P < .05, Mann-Whitney U test (1 tailed). Ludovic Durrieu et al. Blood Adv 2018;2:1054-1065 © 2018 by The American Society of Hematology