Abstract Body: Introduction: Colorectal cancer associated venous thrombosis (CRC-VT) is a major cause of mortality in CRC patients, yet the mechanisms linking CRC to thrombosis remain unclear. NETosis, a process in which neutrophils release prothrombotic extracellular traps (NETs), is elevated in various cancers. However, the mechanisms of cancer-associated NETosis and its contribution to CRC-VT remain unknown.We hypothesized that CRC associated reactive oxygen species (ROS) reprogram neutrophil toward a pro-NETotic phenotype promoting a prothrombotic state.
Methods:C57BL/6J wild-type (WT) mice were subcutaneously injected with murine CRC cells (MC38). Neutrophil subpopulations were characterized by spectral flow cytometry. Spontaneous and agonist induced NETosis in bone marrow-derived neutrophils was quantified by immunostaining. Pharmacological and genetic approaches were used to assess contribution of ROS to NETosis. Plasma thrombin generation potential was measured with phospholipids ± tissue factor (TF). Venous thrombosis susceptibility was evaluated using inferior vena cava (IVC) stenosis model. DNase I treatment for NET degradation, Ly6G-mediated neutrophil depletion, and Nox2-KO mice were used to assess causality. Nox2-NADPH oxidase is the key superoxide generating enzyme in neutrophils and drives NETosis.
Results: C57BL/6J mice bearing CRC-tumors exhibited elevated circulating neutrophils and H3Cit (a marker of NETosis). Spectral flow cytometry showed reduced maturation marker CD16 but elevated migration/activation markers (CD11b, CD62L), co-localized with H3Cit, indicating a CRC-driven-pro-NETotic reprogramming. Neutrophils displayed altered nuclear morphology with loss of lobulation, suggesting expansion of immature subsets. Bone marrow-derived neutrophils showed increased spontaneous and agonist-induced NETosis, suppressed by Nox2 inhibition or antioxidants.
Plasma from tumor-bearing mice showed enhanced thrombin generation potential ± exogenous TF. Finally, in IVC stenosis model, tumor-bearing mice developed larger thrombi, which were significantly reduced by DNase I treatment or Ly6G-mediated neutrophil depletion. Furthermore, tumor-bearing Nox2-KO mice showed reduced NET formation and venous thrombosis, suggesting that Nox2 mediated NETosis contributes to thrombosis in CRC model.
Conclusions: ROS-driven neutrophil reprogramming links CRC to venous thrombosis. Our data may support ROS/NETs as potential targets to reduce CRC-VT burden.