Doxorubicin Enhances Procoagulant Activity of Endothelial Cells after Exposure to Tumour Microparticles on Microfluidic Devices

Abdulrahman Algarni,John Greenman,Leigh Madden

doi:10.3390/bloods1010006

Abstract

The majority of cancer patients undergoing chemotherapy have a significantly increased risk of venous thromboembolism via a mechanism not yet fully elucidated but which most probably involves tumour microparticles (MP) combined with damaged/activated endothelium. Tumour cell lines (ES-2 and U87) were cultured as 3D spheroids and transferred to biochips connected through to a second chip precultured with an endothelial cell layer (human umbilical vein endothelial cells [HUVECs]). Media were introduced with and without doxorubicin (DOX) to the spheroids in parallel chips under constant flow conditions. Media samples collected pre- and post-flow through the biochip were analysed for tissue factor microparticles (TFMP) and procoagulant activity (PCA). HUVECs were also harvested and tested for PCA at a constant cell number. TFMP levels in media decreased after passing over HUVECs in both conditions over time and this was accompanied by a reduction in PCA (indicated by a slower coagulation time) of the media. The relationship between PCA and TFMP was correlated (r = −0.85) and consistent across experiments. Harvested HUVECs displayed increased PCA when exposed to tumour spheroid media containing TFMP, which was increased further after the addition of DOX, suggesting that the TFMP in the media had bound to HUVEC cell surfaces. The enhanced PCA of HUVECs associated with the DOX treatment was attributed to a loss of viability of these cells rather than additional MP binding. The data suggest that tumour MP interact with HUVECs through ligand-receptor binding. The model described is a robust and reproducible method to investigate cytotoxic agents on tumour spheroids and subsequent downstream interaction with endothelial cells.

Highlights

Cancer is considered a prothrombotic or hypercoagulable state commonly attributed to the ability of malignancy to activate the hemostatic system and interfere with blood clotting [1]
Human blood contains circulating tissue factor which is mainly in the form of microparticles (TFMP) [10], and we have previously shown that the procoagulant activity of in vitro-released tumour
tissue factor microparticles (TFMP), released from 3D tumour spheroids under flow, decreased in concentration after perfusion through biochips coated with human umbilical vein or endothelial cellcells (HUVEC) and this effect is associated with a concurrent, proportional through biochips coated with HUVECs and this effect is associated with a concurrent, proportional increase of procoagulant activity (PCA) of the endothelial cells

Summary

Introduction

Cancer is considered a prothrombotic or hypercoagulable state commonly attributed to the ability of malignancy to activate the hemostatic system and interfere with blood clotting [1]. The haematological balance between pro- and anti-coagulation factors is tipped in favour of a more procoagulant phenotype, in patients with pancreatic or ovarian tumours. This haematological procoagulant state is thought to be driven, or compounded, by tumour-derived microparticles (MP) that are released into the blood; surgical removal of the tumour has been shown to significantly reduce circulating. MP levels in pancreatic cancer patients [2]. Chemotherapy is an independent risk factor for venous thromboembolism (VTE) in patients and a current hypothesis is that the increased risk of VTE is due to an increased release of tumour MP into the blood via tumour apoptosis. The reality is that MP play many roles depending on their parental cells

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