Pro-metastatic human breast cancer cell line, MDA-MB-231 (MDA) and platelet interactions in an in vitro aggregation assay

ALBERTO MF; BERMEJO EI; CALDERAZZO JC; MESCHENGIESER SS; LAZZARI MA; SÁNCHEZ LUCEROS A

Amsterdam

Congreso; XXIV Congress of the International Society on Thrombosis and Haemostasis; 2013

International Society on Thrombosis and Haemostasis

Metastasis remains the major cause of cancer treatment failure. Platelets play a key role in hematogenous metastasis and contribute to this process by both thrombin-dependent and -independent mechanisms. Aims: Our aim was to characterize interactions between pro-metastatic human breast cancer cell MDA and platelets, and compare the effectiveness of antiplatelet therapies to define strategies to study the platelet role in metastasis. Methods: MDA were cultured at 37º and 5%CO2 in CaDMEM containing glucose and supplemented with 1% glutamine, 1% penicilin/streptomycin and 10% heat-inactivated fetal bovine serum. Cells were scraped, washed and suspended in Ca2+ Mg2+ Tyrode?s buffer 3 days after being passed. Blood was collected from healthy volunteers who had not taken any drugs known to affect platelet function for at least 14 days prior to the study. Washed platelets (WP) in Ca2+ Mg2+ Tyrode?s buffer were prepared at 2.5 9 108 platelets/mL. The interactions between WP and MDA were measured by light aggregometry. WP were placed in a lumi-aggregometer (Chronolog), incubated for 15 min at 37 °C, stirring at 1000 rpm, with increasing MDA cells with or without citrated plasma (1/800 final dilution). The effects of heparin (1 UI/mL) and three antiplatelet drugs (100 mM aspirin, 1 nM PGE1 and NO donnor 10 lM sodium nitroprusside) were assayed. The time to produce 50% aggregation of WP was registered (T50%). Results: MDA cells were tested for their ability to induce WP aggregation. MDA cells (1.3 105?6.5 105 cells/mL) did not produce WP aggregation when they were incubated in the aggregometer for 15 min at 37 °C in absence of citrated plasma. However when citrated plasma was added, in a very low dilution (1/800) to avoid fibrin formation, MDA cells induced WP aggregation in a concentration-dependent manner (1.3 102; 1.3 103;
1.3 104 cells/mL). A linear relationship was obtained when T50% was plotted vs. MDA cell concentration in linear-log mode. Heparin at 1 UI/mL completely inhibited WP aggregation suggesting a mechanism mediated by thrombin generation. When WP were treated with asprin or PGE1, T50% increased a 45% (n: 4, P = 0.03) and a 76% (n: 4, P = 0.03) respectively for 1.3 104 MDA cells/mL, keeping the same linear-log relationship for lower MDA cell concentration. When NO donor sodium nitroprusside was assayed, WP aggregation was completely inhibited at 1.3 102 and 1.3 103 cells/mL and T50% was increased a 280% (n: 4, P = 0.01) at 1.3 104 cells/mL. Conclusion: In our in vitro study, MDA-MB-231 (distinguished by their invasive phenotype) were capable of producing WP aggregation in a thrombin-dependent manner at very low concentrations of clotting factors and tumour cells, revealing their thrombogenic power. Only the antiplatelet therapy with NO donor sodium nitroprusside was effective, this finding may help to define strategies to study the platelet role in metastasis in in vivo models. Impairment of platelet function within the tumour microenvironment may provide a clinically useful approach to inhibit metastasis.REACH THE WORLD GRANT