Implication of Tissue-Factor Bearing Microparticles In Thrombosis Associated with Breast Cancer
Authors: Mullier François , Gheldof Damien , Robert Severine , Bailly Nicolas , Devel Philippe , Doyen Chantal , Chatelain Christian , Michiels Carine , Chatelain Bernard , Dogné Jean-Michel 
Published: October 2019

Abstract
Abstract 3165

Introduction:
Patients with cancer have a 7- to 10-fold overall increased risk of developing venous thromboembolism (VTE). Some tumors shed small membrane vesicles called microparticles (MPs) containing tissue factor (TF) and membrane phospholipids (PL) leading to procoagulant activity (PCA). TF, the most potent initiator of coagulation cascade, plays a critical role in hemostasis. The circulation of active TF associated with MPs has been considered as a risk factor for VTE in cancer.

Aims:
The primary objectives of this study were to characterize structure, size and PCA of tumor-cell derived MPs released by breast cancer cells MDA-MB231 (MDA) using thrombin generation (TGT), flow cytometry (FCM) and transmission electron microscopy (TEM). The secondary objectives were to determine the PCA of MDA and MPs derived from MDA in order to study the effect of stirring on MPs production and PCA. The study of the effects of the size of MP on PCA and the contribution of TF and PL to the PCA will also be performed.

Methods:
In vitro generation of MPs: Cultured MDA-MB-231 breast cancer cells were adjusted to the desired concentrations (600000 cells/ml) in PBS. Cells suspensions were incubated for 45 min at 37°C under stirring condition or without any stirring. Samples were then centrifuged at 4500g for 15 min and the cell supernatants (Sup) were used for EM, FCM and TGT. Alternatively, MP fractions were filtered through 0.1, 0.22, 0.45 or 0.65μm membranes (Ultrafree-MC) and subjected to activity assays.
PCA: TGT was performed with Calibrated Automated Thrombogram (CAT). Cells suspensions or in vitro-generated MP fractions from 500000 cells were used as the source of TF and PL and added to normal pool plasma (NPP).
Counting and expression of TF and MUC-1: Quantification of MPs and expression of TF (CD142) and MUC-1 (CD227) on MDA and MDA Sup were studied by flow FCM. The size of PMPs was defined using a blend of monodisperse fluorescent beads (Megamix).
Tumor MPs sizing: A 5 μL sample of cells or MP fraction derived from 500000 cells diluted 100X was gently put onto a non-BSA precoated formvar grid and allowed to settle undisturbed for 48h before analysis under a TECNAI 10 TEM.

Results:
Effects of MDA-MB-231 and MDA-MB-231 Sup on PCA
MDA and MDA Sup significantly increased the active thrombin in comparison to human NPP alone. Indeed, for example the lagtime was reduced by 11,7-fold and 7,2-fold, respectively. The difference between MDA and its Sup were highly significant (p<0,01) but this result is due a loss of MPs by centrifugation as shown by the differences in MPs concentration measured by FCM. Therefore, TF is mostly present in a non active encrypted configuration on the surface of MDA-MB-231.
Effect of stirring on PCA of MDA-MB-231
Comparison of MPs concentration positive and negative for anti-MUC-1 and anti-TF in MDA before and after stirring showed that the stirring did not lead to a significantly increased number of MPs, as confirmed by TGT.
Contribution of TF and PL to the PCA
The relative importance of TF and PL in the PCA of MPs was assessed by comparing the TGT curves with or without addition of 4 μM PL. By comparing lagtime, TTP and Peak, we concluded that both PL and TF contributed to PCA of MPs, but at different stages of the coagulation cascade. The lagtime reduction with 0,1μm filtrated Sup MP showed that these particles provide FT to initiate the coagulation. This result supports that active TF mainly come from MPs. The difference in peak can be explained by the PL provided by tumor cells.
Effects of MPs sizes on PCA
The lagtimes of Sup and 0,65 μm filtered Sup (0,65μm Filt-Sup) were respectively reduced by 8,6-fold and 6,6-fold in comparison to NPP alone. The differences before and after 0,65 μm filtration of the MDA Sup were highly significant (p<0,01). The same PCA was found with cell Sup filtered at 0.45 and 0.65μm whereas it lowered progressively with filters from 0.45 to 0.1 μm. Indeed, in FCM and TEM, we found very few MPs between 0.45 and 0.65 μm. Conversely to FCM, EM showed that MPs derived from MDA-MB-231 are comprised between 30 nm and 200 nm and that the vast majority were under 100 nm. Such results were in agreement with FCM and TGT.

Conclusions:
TGT, FCM and TEM are very interesting methods that should be combined to adequately determine the phenotype of tumor-cell derived MPs whatever their size. MDA-MB-231 cells release spontaneously MPs of sizes comprised between 30 nm and 200 nm. These MPs have a strong PCA due to the expression of TF and PL.

Disclosures:
No relevant conflicts of interest to declare.