Tumor-Derived Exosomes Induce the Formation of Neutrophil Extracellular Traps: Implications For The Establishment of Cancer-Associated Thrombosis

Ana C Leal,Tainá Gomes,Elvira M Saraiva,Marcos S Dias,Robson Q Monteiro,Micheli S Sielski,Cristina P Vicente,Natalia C Rochael,Daniella M Mizurini,Claudio C Werneck

doi:10.1038/s41598-017-06893-7

Abstract

Cancer patients are at an increased risk of developing thromboembolic complications. Several mechanisms have been proposed to explain cancer-associated thrombosis including the release of tumor-derived extracellular vesicles and the activation of host vascular cells. It was proposed that neutrophil extracellular traps (NETs) contribute to the prothrombotic phenotype in cancer. In this study, we evaluated the possible cooperation between tumor-derived exosomes and NETs in cancer-associated thrombosis. Female BALB/c mice were orthotopically injected with 4T1 breast cancer cells. The tumor-bearing animals exhibited increased levels of plasma DNA and myeloperoxidase in addition to significantly increased numbers of circulating neutrophils. Mice were subjected to either Rose Bengal/laser-induced venous thrombosis or ferric chloride-induced arterial thrombosis models. The tumor-bearing mice exhibited accelerated thrombus formation in both models compared to tumor-free animals. Treatment with recombinant human DNase 1 reversed the prothrombotic phenotype of tumor-bearing mice in both models. Remarkably, 4T1-derived exosomes induced NET formation in neutrophils from mice treated with granulocyte colony-stimulating factor (G-CSF). In addition, tumor-derived exosomes interacted with NETs under static conditions. Accordingly, the intravenous administration of 4T1-derived exosomes into G-CSF-treated mice significantly accelerated venous thrombosis in vivo. Taken together, our observations suggest that tumor-derived exosomes and neutrophils may act cooperatively in the establishment of cancer-associated thrombosis.

Highlights

The correlation between malignancy and procoagulant states has long been established[1, 2]
The systemic signs of neutrophil activation in 4T1-bearing mice were previously associated with microthrombosis, which coincides with the appearance of NETs23
Mice were subjected to a model of arterial thrombosis in which thrombus formation was triggered in the carotid artery by injury with ferric chloride

Summary

Introduction

The correlation between malignancy and procoagulant states has long been established[1, 2]. Several studies have correlated the increased plasma levels of tumor-derived tissue factor (TF)-positive microvesicles with a prothrombotic state in both animal models[7,8,9] and patients[10]. This correlation has not been observed in a number of cancer types including gastric, colorectal, brain and breast cancer[11,12,13]. Tumor-associated neutrophilia and propensity to NET formation seems to be directed correlated with increased levels of granulocyte colony-stimulating factor (G-CSF)[23,24,25] These tumor models exhibit spontaneous thrombus formation. The prothrombotic state could be reproduced by treating tumor-free mice with G-CSF combined with low doses of LPS23

Methods

Results

Conclusion