Acquision of Resistance to Doxorubicin By Breast Cancer Cells MCF7 Enhances Their Procoagulant Properties and Alters the Efficacy of Antithrombotic Agents to Inhibit Thrombin Generation
Authors: Rousseau Aurélie , Van Dreden Patrick , Khaterchi Amir , Mbemba Elisabeth , Larsen Annette , Elalamy Ismail , Gerotziafas Grigorios T. 
Published: October 2019

Abstract
Introduction: A major problem associated with breast cancer chemotherapy is the subsequent development of resistance to chemotherapeutic agents (multidrug résistance-MDR). Hypercoagulability, increased risk of venous thromboebolism (VTE) and high rate of failure of the antithrombotic treatment with LMWHs have been observed in patients with disease resistant to the chemotherapy.
Aim of the study: In the present study we investigated if acquisition of MDR by breast cancer cells MCF7 is associated with modification of their procoagulant potency and induces alteration of the efficacy of the antithrombotic agents. We evaluated the capacity of wild type MCF7 cells (MCF7/WT) and doxorubicin resistant cells (MCF7/DR) to trigger thrombin generation (TG) and to modify the antithrombotic activity of the LMWH enoxaparin, and the specific direct and indirect FXa inhibitors (apixaban and fondaparinux).
Materials and Methods: Pre-treatment of MCF-7 cells for several weeks with increasing concentrations of doxorubicin, induced the acquisition of chemo-resistance phenotype documented by the expression the MDR1-Pgp. Tissue factor (TF) and MDR1-Pgp expression by MCF7/WT and MCF7/DR cells were assessed by flow cytometry and western blot assays. Reverse transcriptase - polymerase chain reaction (RT-PCR) for TF mRNA expression was also performed. Thrombin generation of normal platelet poor plasma (PPP) in the presence of MCF7 cells was assessed with the Calibrated Automate Thrombogram® assay (Diagnostica Stago). TG in the presence of MCF7 cells was also assessed in PPP spiked with clinically relevant concentrations of enoxaparin, apixaban or fondaparinux. The efficiency of the studied agents in the presence of MCF7 cells was compared to that in the control experiment where TG was triggered by PPP-Reagent® 5 pM TF. The antithrombotic agents were compared on the bases of the concentration which inhibited 50% TG (IC50).
Results: The MCF7/DR cells showed higher expression of TF as compared to MCF7/WT. The TF expression by MCF7/DR was correlated with the expression of the MDR1/P-gp. The MCF7/DR cells significantly enhanced TG as compared to MCF7/WT cells. The three studied agents significantly inhibited TG at plasma concentrations usually achieved at doses for thromboprophylaxis. The presence resistant MCF7 cells did not significantly modify the antithrombotic potency of fondaparinux. The inhibitory effect of enoxaparin and apixaban on thrombin generation was partially reversed when TG was triggered by MCF7/DR as compared to that triggered by MCF7/WT or in the control experiment.
Table 1. Modification of the inhibitory potency of the antithrombotic agents by MCF7 cells on thrombin generation in function of the acquisition of MDR phenotype. IC50 for MRI PPP + TF/PPL PPP + MCF7/WT PPP+ MCF7/DR fondaparinux (anti-Xa IU/mL) 0.21 ± 0.03 0.28 ± 0.06 0.30 ± 0.04 enoxaparin (anti-Xa IU/mL) 0.10 ± 0.02 0.12 ± 0.05 0.23 ± 0.08* apixaban (ng/ml) 14 ± 0.9 19 ± 3.1 26 ± 1.4* *p<0.05 versus MCF7/WT
PPL: procoagulant phospholipids
MRI: mean rate of the propagation phase of thrombin generation
IC50: concentration that inhibits 50% the MRI
Conclusions: The acquisition of the chemo-resistance phenotype by the breast cancer cells MCF7 is associated with enhancement of their procoagulant properties. The procoagulant fingerprint of the chemo-resistant MCF7 cells is characterized by increased expression of TF which is correlated with enhanced thrombin generation. The chemo-resistant MCF7 cells reverse the antithrombotic effect of enoxaparin and apixaban but not that of fondaparinux.

Disclosures
Van Dreden: Diagnostica Stago: Employment.