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Neutrophils and cancer-associated thrombosis
counts, such as G-CSF, or are biomarkers of activated neu- trophils and NET, such as NE and H3Cit, in tumor-bearing mice compared with controls. We also observed increased levels of the neutrophil biomarker Ly6G and the NET bio- marker H3Cit in thrombi from tumor-bearing mice. Finally, depletion of neutrophils reduced thrombus size in tumor-bearing mice but not in control mice. The lack of effect on thrombus size in neutrophil-depleted control mice is consistent with a previous study showing that neutrophil depletion did not reduce thrombus size in mice in an IVC stasis model.28 Taken together, these data sug- gest that neutrophils contribute to increased venous thrombosis observed in tumor-bearing mice.
We found that tumor-bearing mice have increased levels of mouse G-CSF and there was a significant positive cor- relation between levels of mouse G-CSF and neutrophil numbers. This suggests that increased levels of G-CSF drive the increase in neutrophil numbers. At present, we
do not know the cellular source of G-CSF in tumor-bear- ing mice but this cytokine is normally expressed by endothelial cells, macrophages and immune cells.45 In sup- port of this, mice with murine breast 4T1 tumors exhibit increased plasma levels of G-CSF and administration of an anti-G-CSF antibody reduces neutrophil counts.37 G-CSF level and neutrophil count may, therefore, represent novel biomarkers of VTE risk in cancer patients.
It has been hypothesized that G-CSF primes neutrophils to undergo NET formation in tumor-bearing mice.37,46 In one study it was found that the percentage of H3Cit-pos- itive neutrophils was increased in tumor-bearing mice compared with controls.37 Furthermore, neutrophils isolat- ed from mice bearing murine 4T1 tumors treated with an anti-G-CSF antibody had significantly less NET formation compared with those from tumor-bearing mice treated with an isotype control.37 In addition, administration of recombinant G-CSF to mice bearing murine melanoma
AB
Figure 5. Effect of neutrophil depletion on venous thrombosis in control and tumor-bearing mice. (A, B) Control and tumor-bearing mice received an anti-mouse Ly6G antibody (clone: 1A8) or a control IgG (5 mg/kg) 24 h and 1 h before inferior vena cava stasis. Thrombi from control (A) and tumor-bearing mice (B) were col- lected at 48 h and weighed. Four to five mice were used for each group. Data were analyzed with either the unpaired t-test or the Mann-Whitney U-test depending on the type of data distribution. *P<0.05.
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Figure 6. Effect of DNase I treatment on venous thrombosis in control and tumor-bearing mice. Mice received vehicle or DNase I (50 U/mouse) 1 h before and 24 h after inferior vena cava stasis. Thrombi from control (A) and tumor-bearing mice (B) were collected at 48 h and weighed. Five to seven mice were used for each group. Data were analyzed with the unpaired t-test. *P<0.05.
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