Platelet-tumour cell interaction in vitro: the effects of anastrozole and anti-platelet therapy

Abstract

Thromboembolic disorders are the second leading cause of mortality in cancer patients. The relationship between cancer and thrombosis is well established with reciprocal interactions between tumour cells and platelets, facilitating thrombosis and tumour progression. Nevertheless, there remains a lack of knowledge regarding therapeutic interventions. Breast cancer is the most commonly diagnosed cancer among women, with hormone-dependant breast cancer, treated with hormone-therapy, the most commonly diagnosed subtype. In cardiovascular disease antiplatelet therapy has been efficacious, thus it could be effective against cancer-related thromboembolic disorders. In this study we investigated the effects of combined Anastrozole (hormone-therapy) with antiplatelet drugs (Aspirin and Clopidogrel cocktail/ Atopaxar), on biological markers associated with thrombosis and tumour progression in vitro using breast cancer cell lines exposed to vascular components. The cytotoxicity of the treatments over 24 hours on hormone-dependant breast cancer cell lines (MCF7 and T47D) were first established by testing various concentration effects on membrane integrity and corresponding cellular morphology. Having determined an optimal concentration, effects on proliferation and migration were assessed. Thereafter, whole blood collected from six healthy female volunteers, was co-incubated with treated breast cancer cells and reciprocal interactions on hypercoagulation and tumour progression assessed. Hypercoagulation was examined by studying thrombin activity and platelet activation was determined using flow cytometry and corresponding morphology. In breast cancer cells, mRNA levels of genes (TGFβ1, vimentin, E-cadherin, N-cadherin) which facilitate epithelial mesenchymal-transition (EMT) and secretion of inflammatory cytokines which mediate growth and angiogenesis, were assessed.Our findings indicated that combined antiplatelet and Anastrozole treatment induced low but insignificant levels of collective cell migration in both cell lines. Additionally, MCF7 cells had a higher proliferation rate than T47D cells highlighting the heterogeneous nature of the cell lines. Treatment did not prevent breast cancer cells from inducing hypercoagulation as shown by thrombin activity and platelet activation. Gene expression analysis revealed that combined therapy facilitated the maintenance of an epithelial-like phenotype in MCF7 cells and may have induced a partial-EMT in T47D cells. Cytokine secretion revealed elevated levels of growth factors (TGFβ isoforms) and angiogenic factors (PDGF isoforms and VEGF). Thus, collectively these findings show that combined Anastrozole and antiplatelet therapy did not prevent hypercoagulation, and may promote tumour survival by elevating the secretion of inflammatory mediators