Interactions between Integrin α9β1 and VCAM-1 Promote Neutrophil Hyperactivation and Deep Vein Thrombosis in a Murine Model of Lung Cancer

Abstract

Background: Venous thromboembolism (VTE) is highly prevalent in patients with lung cancer and is associated with increased morbidity and mortality. Ectopic secretion of granulocyte colony-stimulating factor (G-CSF), a critical regulator of neutrophil trafficking, is commonly observed in lung cancer. In recent years, compelling evidence has emerged implicating neutrophils and neutrophil extracellular traps (NETosis) in cancer-associated thrombosis. Integrin α9β1 is highly expressed on neutrophils, upregulated upon neutrophil activation, promotes NETosis, and is known to stabilize neutrophil adhesion to activated endothelium via multiple ligands including vascular cell adhesion molecule 1 (VCAM-1). Interactions between integrin α9 and VCAM-1 have been reported to reduce neutrophils apoptosis. Given the relevance of integrin α9 in neutrophil migration and apoptosis, granulopoiesis, and thromboinflammation, here we evaluated the mechanistic role of neutrophil integrin α9β1 in the pathophysiology of cancer-associated thrombosis. Methods: Lung cancer was induced by the intravenous administration of 3X10 5 Lewis lung carcinoma cells. Deep vein thrombosis (DVT) was induced in neutrophil-specific α9 -/- (α9 fl/flMrp8Cre +/-) and littermate controls (α9 fl/flMrp8Cre -/-) by partial ligation of the inferior vena cava (IVC). Plasma levels of the thrombin-antithrombin (TAT) complex, fibrinogen, G-CSF, elastase, and myeloperoxidase (MPO) were assessed by ELISA. To evaluate the potential mechanisms by which neutrophil integrin α9-VCAM-1 interactions promote DVT, we performed unbiased RNA sequencing from 1) control neutrophils stimulated with and without VCAM-1 and, 2) neutrophils of α9 fl/flMrp8Cre +/- and α9 fl/flMrp8Cre -/- mice stimulated with VCAM-1. Computational modeling, docking, virtual screening, and the cell-free assay were used to identify small-molecule inhibitors of integrin α9-VCAM-1 interaction. Results: Mice with lung cancer exhibited significantly increased levels of TAT, G-CSF, blood neutrophil/lymphocyte ratio, cell-free DNA, elastase, and MPO, suggesting a hypercoagulable and inflammatory state. Tumor-bearing and non-tumor-bearing neutrophil-specific α9 -/- mice exhibited reduced DVT severity, as evident by reduced IVC thrombus weight and thrombus length compared to littermate controls, while thrombosis incidence was comparable between the groups. Reduced DVT severity in neutrophil-specific α9 -/- mice was also associated with decreased neutrophil content and citrullinated histone H3 (CitH3) expression in IVC thrombi. Integrin α9 deficient neutrophils incubated with VCAM-1 exhibited reduced release of elastase and MPO, decreased endothelial cell apoptosis, and reduced adhesion to the activated endothelium at venous shear rate compared to control neutrophils incubated with VCAM-1. Bioinformatic analysis of RNA sequencing data from VCAM-1 stimulated neutrophils from α9 fl/flMrp8Cre +/- and control mice showed distinct transcriptional profiles. Hierarchical clustering of gene expression revealed gene clusters that were highly downregulated in stimulated neutrophils from α9 fl/flMrp8Cre +/- mice. Gene ontology enrichment revealed that highly downregulated genes corresponded to biological processes involved in the inflammatory response, cytokine production, NF-κB and ERK signaling pathways, granulocyte migration, and chemotaxis. Virtual screening and cell-free assay identified macitentan as a potent inhibitor of integrin α9-VCAM-1 interactions. In vitro treatment with macitentan reduced neutrophil adhesion to VCAM-1 and also to activated endothelial cells at venous shear rate. In vivo, macitentan treatment at a dose of 5 mg/kg exhibited reduced DVT severity, decreased plasma levels of elastase and MPO, and reduced neutrophil and CitH3 levels in IVC thrombi. Conclusion: Using genetic and pharmacological approaches in combination with transcriptomic and pharmacological studies, we described a previously unknown pathway involving integrin α9 and VCAM-1 in promoting cancer-associated thrombosis. We found that integrin α9-VCAM1 axis is critical for neutrophil hyperactivation, and its pharmacological inhibition significantly reduces DVT severity. The results of this study suggest that neutrophil integrin α9 is a key factor regulating neutrophil hyperactivation and cancer-associated thrombosis.