Figure 5. NETs trap both human and murine tumor cells in vitro.
(A) Under static conditions, H59 and A549 cells demonstrate increased adhesion to neutrophil monolayers stimulated with PMA (800 nM) compared with unstimulated neutrophils. Addition of DNAse 1 (1,000 U) or pretreatment of neutrophils with NEi (5 μM) results in levels of adhesion comparable to control. (B) Tumor cells were perfused over neutrophil monolayers at shear rates of 1 dyne/cm/s–1. H59 and A549 cells demonstrate increased adhesion to neutrophils after stimulation with PMA (800 nM) compared with controls. This was abrogated by addition of DNAse 1 (1,000 U) or pretreatment of neutrophils with NEi (5 μM). Data are presented as mean ± SEM from n = 2–4 separate experiments. ***P < 0.01 versus control, DNAse, and NEi. #P < 0.05 versus control. Significance was determined using 1-way ANOVA with Tukey’s HSD post-hoc analysis. (C) Confocal imaging reveals that after PMA stimulation, A549 cells (red) become trapped within webs of extracellular DNA (green) in proximity to neutrophils (blue). (D) Scanning electron microscopy of A549- and PMA-stimulated neutrophils (800 nM) (original magnification, ×3,000). After PMA stimulation, neutrophils flatten (white arrow) and extrude strands consistent with NETs. Strands encompass a cluster of adherent tumor cells (black arrow). (E) Scanning electron microscopy demonstrates that NETs are in direct contact with adherent A549 tumor cells (original magnification, ×3,500). Scale bars: 40 μm (confocal microscopy); 5 μm (electron microscopy). See also Supplemental Figures 2 and 3.