中性粒细胞胞外诱捕网参与卵巢癌相关静脉血栓形成

Aims: Ovarian cancer patients have increased risk of venous thrombosis. However, the responsible mechanism of ovarian cancer associated venous thrombosis is not fully understood. This study aimed to investigate the formation of neutrophil extracellular traps (NETs) and its pathologic role in ovarian cancer patients. Methods: Human neutrophils were isolated from peripheral blood by density gradient centrifugation. Neutrophils were incubated in various conditions and tested for NETs formation in vitro. NETs formation was quantified by observing neutrophils stained with DNA-binding dye under fluorescent microscope. Several benign tumor sera and ovarian cancer sera were analyzed by cytokine array and ELISA. Serum NETs levels were measured by sandwich ELISA using anti-neutrophil elastase (NE) and anti-double strand DNA antibody. Clinical records of ovarian cancer patients who underwent surgery at our institution were retrospectively reviewed to evaluate the association between serum NETs levels and clinical. Immunohistochemistry with NETs marker, citrullinated histone H3 (CitH3) and myeloperoxidase (MPO), was performed for the samples of intravascular thrombosis in ovarian cancer patient. Results: Neutrophils could be isolated from peripheral blood with high purity. Conditioned media collected from OVCAR8 and OVKATE induced neutrophils to form NETs strongly. Serum from ovarian cancer patient predisposed neutrophils to form NETs more strongly than that from benign tumor patient (Median of index: 0.55 vs. 1.64; p < 0.05). Serum granulocyte-colony stimulating factor (G-CSF) levels from ovarian cancer patients were significantly higher than those from benign patients (Mean [pg/mL]: 4.5 vs. 9.9; p < 0.05). The stimulation with recombinant G-CSF induces neutrophils to form NETs. By cytokine array, Insulin-like Growth Factor 1 (IGF-1), C-C motif chemokine ligand 5 (CCL5), Angiogenin and Epidermal Growth Factor (EGF) were upregulated in ovarian cancer serum. However, when we verify CCL5 levels using multiple samples, there were no significant differences between benign tumor and ovarian cancer. Neutrophils isolated from ovarian cancer patients form NETs more greatly than neutrophils from benign tumor patients (Mean of index: 1.12 vs. 2.34; p < 0.05). Serum NETs level was significantly higher in ovarian cancer patients than in benign tumor patients (Median index: 0.0318 vs. 0.0667; p < 0.05). High NETs concentration was related to poor prognosis, increased neutrophil count, high platelet count, elevated CA125 level, advanced stage and venous thrombosis. Among them, thrombosis was significantly related to high NETs concentration in multi-variate analysis. Immunohistochemical analysis revealed NETs foci in thrombosis in ovarian cancer patients. Conclusion; It is suggested that ovarian cancer predisposed neutrophils to form NETs, which might have a causal effect on venous thrombosis.