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Y. Hisada et al.
B16 tumors, which do not produce G-CSF, increased H3Cit in tumors in a PAD4-dependent manner.46,47 We observed a large variation in the H3Cit/H3 ratio in throm- bi from tumor-bearing mice which appears to be due to different levels of H3Cit in the thrombi. At present, we do not know the reason for this range of H3Cit in thrombi from tumor-bearing mice but it may reflect differences in G-CSF levels and the degree of neutrophil priming in the different tumor-bearing mice.
Plasma cfDNA and the NET biomarker, H3Cit, were increased in tumor-bearing mice compared with controls. Tumors are known to release cfDNA into the blood.48 In addition, we observed that BxPc-3 cells express PAD4 and therefore tumors may also contribute to the plasma levels of H3Cit. Thrombi from tumor-bearing mice also had increased levels cfDNA and H3Cit compared with throm- bi from controls. Importantly, administration of DNase I reduced thrombus size in tumor-bearing mice but not in control mice. Similarly, a previous study showed that DNase I did not affect jugular vein occlusion times in con- trol mice but prolonged the time to occlusion in 4T1 tumor-bearing mice.38 We observed that DNase I reduced thrombus size more effectively than depletion of neu- trophils. It is possible that DNase I is more effective than neutrophil depletion because it digests not only NET but also cfDNA which may also be released by cancer cells and may enhance thrombosis by activating factor XII.40,49
In the present study, we observed decreased numbers of red blood cells in thrombi from tumor-bearing mice com- pared with controls. This is consistent with our recent study that showed a decrease in red blood cell-rich areas and an increase in inflammatory cell-rich areas in thrombi from tumor-bearing mice compared with controls.10 In the current study, we did not observe an increase in neu- trophils in thrombi of tumor-bearing mice because these cells were not preserved during the preparation of the
samples for scanning electron microscopy. In addition, thrombi from tumor-bearing mice had a denser fibrin net- work with thinner fibrin fibers compared with thrombi from control mice. In vitro experiments showed that higher thrombin produces thrombi with a denser fibrin network with thinner fibrin fibers.44 In our model of cancer-associ- ated thrombosis, it is likely that tumor-derived, tissue fac- tor-positive extracellular vesicles increase the thrombin concentration in thrombi and this results in the formation of a denser fibrin network with thinner fibers. Indeed, in our previous study,10 we observed that thrombi from BxPc-3 tumor-bearing mice had increased levels of human tissue factor activity derived from extracellular vesicles released from BxPc-3 tumors. In addition, a previous study showed that extracellular vesicles bind to NET via a phos- phatidylserine-histone interaction.50
In summary, we have demonstrated a contribution of neutrophils to venous thrombosis in a mouse model of pancreatic cancer-associated venous thrombosis. Our data, taken together with a clinical study showing an association between circulating H3Cit and VTE in pan- creatic cancer, support the notion that neutrophils and NET formation enhance venous thrombosis in pancreatic cancer.
Acknowledgments
The Animal Surgery Core Laboratory of the McAllister Heart Institute at UNC performed the thrombosis experiments. This work was supported by grants from the National Institutes of Health (to YH T32 HL007149), the John C. Parker Professorship (to NM), the Jochnick Foundation (to CT and HW), the Hungarian National Research, Development and Innovation Office (NKFIH) (129528, to KK) and the Higher Education Institutional Excellence Programme of the Ministry of Human Capacities in Hungary for the Molecular Biology the- matic programme of Semmelweis University (to KK).
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