T. N. Augustine1, K. Pather1, T. Dix-Peek2, R. Duarte2 1University Of The Witwatersrand,School Of Anatomical Sciences, Faculty Of Health Sciences,Johannesburg, SOUTH AFRICA, South Africa 2University Of The Witwatersrand,Department Of Internal Medicine, School Of Clinical Medicine, Faculty Of Health Sciences,Johannesburg, SOUTH AFRICA, South Africa
Introduction
Cancer is associated with hypercoagulability, with therapies including hormone-therapy linked to an increased risk of thrombotic complications. Notwithstanding the contribution of other hematological processes and components, platelets are implicated in contributing to a hypercoagulable state, with breast cancer patients undergoing Tamoxifen treatment at a greater risk for thrombotic complications. Nevertheless, laboratory studies show that Tamoxifen prevents platelet activation. Therein lies the controversy – we postulate that experimental design and methodological approaches to this question have resulted in disparate conclusions. We thus assessed the effects of Tamoxifen on breast cancer cell induced platelet activation, and further assessed whether alterations in estrogen receptor (ER) profiles were associated with platelet induction capacity.
Materials and Methods
MCF7 and T47D cells were treated with 2μM Tamoxifen for 24 hours at 37°C and 5% CO2 prior to exposure to whole blood. Peripheral whole blood from healthy female volunteers (n=5, age 19 – 30 years old with specific exclusion criteria) was collected in 3.2% sodium citrate vacuette coagulation tubes (Human Research Ethics Committee, University of the Witwatersrand Approval #M160826). The first 2ml of blood drawn was discarded to exclude the effect of mechanically activated platelets. Cells were exposed to blood for 2.5min followed by sample preparation for platelet activation (CD41+CD62p+) using flow cytometry, with a specialised interval gating strategy to determine the index of platelet activation (IPA). We further assessed cellular ER isoform expression using digital droplet PCR (ddPCR) to determine whether platelet induction capacity is associated with ER isoform expression.
Results
Breast cancer cells induced a significantly (p<0.05) higher IPA compared to the untreated, negative, controls; with T47D cells inducing greater levels than MCF7 cells. Tamoxifen treatment enhanced the ability of MCF7 cells to induce overall platelet activation, but further investigation revealed that T47D cells induced a substantial increase in a small population of CD62p+++ platelets. We are currently analyzing the ddPCR data to determine whether alterations in ER isoform expression are related to the ability of the cells to modulate platelet activation under Tamoxifen treatment.
Conclusions
Hormone-dependent breast cancer cells are able to induce platelet activation the severity of which being potentially linked to the aggressiveness of the tumour, and thus the phenotype. The addition of hormone-therapy treatment differentially affected induction of platelet activation, rivalling that induced by a procoagulant, thrombin. These results highlight the procoagulant nature of breast cancer cells, the effects of hormone-therapy and the need to assess graded levels of platelet activation in investigating the role of platelet-tumour cell interaction in thrombosis and tumour progression.