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

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.

Mouse models of cancer-associated thrombosis

“…However, the study did not analyze the effect of tumors on thrombosis models. A second study also observed increased levels of neutrophils as well as plasma myeloperoxidase and DNA in mice with orthotopic 4T1 tumors [63]. We found increased plasma levels of G-CSF and neutrophils in nude mice with orthotopic BxPc-3 tumors [14] (unpublished data).…”

“…Leal and colleagues examined the effect of orthotopic 4T1 tumors on both arterial and venous thrombosis [63]. The presence of 4T1 tumors shortened the occlusion time in the FeCl 3 carotid artery model and a Rose Bengal/laser-induced jugular vein injury model [63].…”

“…The presence of 4T1 tumors shortened the occlusion time in the FeCl 3 carotid artery model and a Rose Bengal/laser-induced jugular vein injury model [63]. Arterial thrombi from tumor-bearing mice contained Ly6G+ neutrophils, and extracellular fiber-like DNA consistent with the presence of NETs.…”

“…Arterial thrombi from tumor-bearing mice contained Ly6G+ neutrophils, and extracellular fiber-like DNA consistent with the presence of NETs. Interestingly, administration of DNAse I abolished the increase in venous thrombosis in tumor-bearing mice without affecting venous thrombosis in control mice [63]. In contrast, DNAse I treatment prolonged the occlusion time in both control and tumor-bearing mice in FeCl 3 carotid artery model [63].…”

“…Interestingly, administration of DNAse I abolished the increase in venous thrombosis in tumor-bearing mice without affecting venous thrombosis in control mice [63]. In contrast, DNAse I treatment prolonged the occlusion time in both control and tumor-bearing mice in FeCl 3 carotid artery model [63]. These studies suggest that NETs contribute to arterial and venous thrombosis in mice with orthotopic 4T1 tumors.…”