Evidence of in vivo NET formation in a murine cancer model. (A) The injection of tumor cells in mice increases the immunofluorescence of NETosis biomarkers DNA (blue) and citrullinated Histone 3 (H3) in murine neutrophils after stimulation with PAF; neutrophils from tumor-free mice show only minimal immunofluorescence for DNA and H3, whereas immunofluorescence increases after injection of the tumor (days 7 and 14). Scale bars indicate 20 μm in the top row and 5 μm in the bottom row. VWF- (V) and fibrin-rich thrombi form spontaneously in the lungs of tumor-bearing mice 28 days after tumor injection (n = 4); lung sections are immunostained for DNA (blue), VWF (green), and fibrinogen/fibrin (red). Scale bar indicates 50 μm. White star indicates a VWF- and fibrin-rich thrombus in the pulmonary vasculature. (C) Left, Tumor injection in mice increases the percentage of NET-generating neutrophils (as indicated by H3 hypercitrullination; plotted on the y-axis) from initially ∼1% in tumor-free mice to >80% at day 21 after tumor injection; the steep decline in the percentage of NET-generating neutrophils after day 21 was associated with spontaneous intravascular thrombus formation and may indicate a consumption of NETing neutrophils at the site of thrombosis. Right, Western blot analysis reveals the presence of H3 in the plasma of tumor-bearing mice 28 days after tumor injection. (Figure adapted with permission from Demers et al.¹³ )