Phosphatidylserine-Exposing, Tumor-Derived Microparticles Enhance Procoagulant Activity of Platelets and Endothelial Cells after Neo-Adjuvant Chemotherapy in Invasive Breast Cancer
Authors: Zhang Cong , Kou Yan , Zou Lili , Liang Hui , Hou Li , Li Tao , Zhang Yan , Li Baorong , Liu Yingmiao , Zhang Juan , Shi Jialan , WU Changjun 
Published: October 2019

Abstract
Introduction:Breast cancer is the most common cancer in Chinese women, with invasive breast cancer (IBC) accounting for more than 80% of these diagnoses. Recent studies have shown that increased arterial and venous thromboses occur in IBC and may predict poor prognosis. Uncontrolled activation of coagulation cascade is epidemic in cancer treated with neo-adjuvant chemotherapy (NAC), contributing to hypercoagulability resulting in venous thromboembolism and leading to significant morbidity and mortality. Microparticles (MPs) contribute to coagulation and thrombogenesis. However, the role of tumor-derived microparticles (TMPs) in coagulation during NAC is not fully understood, including clearance of TMPs and their effect on activated platelets and endothelial cells (ECs). Previous studies have shown that elevated circulating MPs has clinical significance in patient prognosis. However, the changes in TMP count, morphology and procoagulant activity (PCA) after NAC in IBC are unclear. This study aimed to explore the properties of MPs derived from breast cancer cells (BCMPs) following exposure to high- or low- dose chemotherapeutic agents and evaluate thrombogenic effects of these BCMPs on platelets and ECs.
Methods:BCMPs and MPs were isolated from the blood samples of 25 breast cancer patients treated by NAC and from 20 healthy women (control). Blood samples were taken before and after NAC. MPs were characterized by cell origin and thrombogenicity. In vitro, BCMPs were isolated from BC cell line (high-metastatic MDA-MB-231), pre-exposed to serum-free medium (control), with or without increasing doses of doxorubicin. BCMPs' structure and size were studied using scanning electron microscope (SEM) and Transmission electron microscope. Antigen levels were measured by fluorescence-activated cell sorting. For inhibition assays, isolated BCMPs were pre-treated with lactadherin or anti-TF. Platelets isolated from healthy subjects were treated with BCMPs of different concentrations for 6 h in vitro. Coagulation time, fibrin formation, as well as intrinsic/extrinsic FXa and thrombin were evaluated. BCMPs effects on ECs thrombogenicity were assessed using confocal microscopy, SEM, intrinsic/extrinsic FXa and prothrombinase assays.
Results: We observed the PCA of patients 1, 2 and 6 days after NAC. Changes in MPs expression of BC marker, MUC1, were found in patient subgroups. BCMPs were significantly higher at day 1 and 2 compared to before NAC, and reduced at day 6. Similarly, we found that clotting time of whole blood was significantly shortened at day 1, more so on day 2, and was nearly restored on day 6. In vitro, culture of MDA-MB-231 cells in serum-free medium resulted in BCMPs shedding that further increased with the addition of low- or high- dose doxorubicin (Figure A). Phosphatidylserine (PS) levels were similarly higher in BCMPs compared with their parental MDA-MB-231 cells. BCMPs derived from MDA-MB-231 cells stimulated with high-dose doxorubicin demonstrated markedly elevated levels of PS. Lactadherin can reduce the PCA of BCMPs by 72%. These BCMPs enhance EC thrombogenicity and induce platelet activation or even apoptosis, leading to the high PCA (Figure B-D). Data showed that compared with those treated by PBS-derived BCMPs, ECs incubated with doxorubicin-derived BCMPs increased production of intrinsic and extrinsic FXa complexes and thrombin generation. Using confocal microscopy, a significant co-localized fraction of bound FVa and FXa was observed, indicating that ECs treated with BCMPs were able to offer a biological surface for binding coagulation factors, most likely through externalized PS.
Conclusions: We made four significant observations. (1) The PCA of patients paralleled the release of BCMPs induced by NAC. Moreover, on the second day of NAC treatment, lactadherin significantly inhibited PCA and fibrin generation, while the effect of anti-TF was minimal. (2) BCMPs induce platelet activation or even apoptosis, leading to the high PCA. (3) SEM showed that BCMPs induce the generation of fiber, impact the densities of fiber deposition and promote clot generation in a dose-dependent manner. (4) Isolated BCMPs exerted a strong cytotoxic effect on ECs, converting them to an increased procoagulant phenotype. Circulating PS+BCMPs can reflect and affect thrombogenicity and, therefore, may be used as a biomarker for hypercoagulability states.

Disclosures
No relevant conflicts of interest to declare.