Figure 3.

Direct contact is required for the growth inhibitory effect of neutrophils. Neutrophil anticancer effect was assayed by indirectly co-incubating A549 cancer cells with human neutrophils from healthy donors at effector to target (E:T) cell ratio of 20:1 using transwell model. Cells were incubated at 37℃ in a humidified 5% CO₂ incubator for 24h. Following washing steps to remove the neutrophils and the remaining viable tumor cells number was assessed by using CCK8 kit (a). Cell cycle and apoptosis of A549 were assayed using flow cytometry (d, e, f, g). In addition, we further seted out to determine tumor cells growth when wiping off the co-cultured neutrophils. After 24 hours further culturing, tumor cells growth was examined using CCK8 kit (b). In parallel, neutrophil lysate was also used in the antitumor assay and the result was measured using CCK8 assay (c). Neutrophils surrounded along tumor cells was observed under SEM (h), scale bar, 20 μm. Data are representative of four (a-g) or three (h) independent experiments. Mean and SD are presented. Statistical analysis was performed by one way ANOVA; ***P<0.001, compared with control group.