Page 230 - 2020_01-Haematologica-web

Y. Hisada et al.
performed with GraphPad PRISM version 7.03 (GraphPad Software, La Jolla, CA, USA). For immunofluorescence data and scanning electron microscopy data, bootstrap simulations of the chosen quantiles of the distribution of the measured values were performed42 followed by a Bootstrap Kuiper test for identity of the quantile distributions.43 These statistical analyses are described in detail in the Online Supplementary Material. P values <0.05 were considered statistically significant for all experiments.
Results
Measurement of circulating neutrophil counts and dif- ferent biomarkers in mice bearing human pancreatic tumors
We measured the numbers of neutrophils in whole blood and various circulating biomarkers in the plasma of mice bearing human BxPc-3 pancreatic tumors (1.5-3.9 grams) and in control mice. We observed significant increases in neutrophil numbers and mouse G-CSF levels in tumor-bearing mice compared with those in controls (Figure 1A, B). We did not observe an increase in human G-CSF (data not shown). We found a significant correla- tion between circulating neutrophil count and mouse G- CSF level (r = 0.83, P=0.02, Spearman test). In addition, we found significant increases in the neutrophil bio- marker NE and the NET biomarker H3Cit in tumor-bear- ing mice compared with controls (Figure 1C, D). Finally, the amount of cfDNA was significantly greater in tumor- bearing mice than in controls (Figure 1E).
The human breast cancer cell line MCF7 expresses PAD4.31 We therefore determined whether BxPc-3 cells also express PAD4. Western blotting showed that the BxPc-3 cells did express PAD4 protein (data not shown).
AB
Tumor cells may, therefore, also contribute to plasma H3Cit.
Measurement of neutrophils, histone H3 and citrullinated histone H3 in thrombi from mice bearing human pancreatic tumors
We generated thrombi in control and tumor-bearing mice using the IVC stasis model. Using western blotting, we measured levels of Ly6G (a neutrophil marker), β- actin (a housekeeping gene), histone H3 (a marker of cel- lular content), and H3Cit (a NET biomarker) in thrombi from mice bearing human pancreatic tumors and control mice (Figure 2A). Thrombi from tumor-bearing mice had higher levels of Ly6G, β-actin, histone H3, and H3Cit compared with the levels in thrombi from control mice (Figure 2 B-E). The H3Cit/H3 ratio for thrombi from con- trol mice was ~1 (Figure 2F). Interestingly, there was sig- nificant variation in the H3Cit/H3 ratio in thrombi from tumor-bearing mice (Figure 2F).
Next, we measured the cfDNA and H3Cit content in thrombi from mice bearing human pancreatic tumors and in thrombi from controls using immunofluorescence. Consistent with the data from the western blot analysis, thrombi from tumor-bearing mice had increased levels of cfDNA and H3Cit compared with levels of these bio- markers in thrombi from control mice (Figure 3).
Finally, we analyzed the composition of thrombi from control and tumor-bearing mice by scanning electron microscopy. Red blood cells were decreased in thrombi from tumor-bearing mice compared with thrombi from control mice (Figure 4A). In addition, we observed an increase in fibrin density and a decrease in fibrin fiber thickness in thrombi from tumor-bearing mice compared with thrombi from controls (Figure 4B). Previous studies
CDE
Figure 1. Levels of neutrophils and circulating biomarkers in blood samples. (A-E) Neutrophil counts in whole blood (A) and circulating mouse granulocyte-colony stimulating factor (G-CSF) (B), neutrophil elastase (C), citrullinated histone H3 (H3Cit) (D), and cell-free (cf) DNA (E) in plasma of control and tumor-bearing mice. Nine to 20 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, **P<0.01, ***P<0.001.
220
haematologica | 2020; 105(1)