Figure 6.

To test whether NETs produced outside the collagen region induced tumor invasion within a TIME, we separately stimulated the neutrophils in the microchannel with 500 nM PMA. (i) Schematic illustration of the different steps shows that the TIME-on-Chip easily enables on-demand attachment and detachment of the components to initially induce the neutrophils in the microchannel to produce NETs, add the conditioned media to the spheroids, and re-attach the device to enable NETs-spheroid co-culture for analysis. (ii) More than 60% of the neutrophils in the microchannel NETosed upon PMA stimulation for 6 h. But even after 24 h of co-culture, the spheroids do not appear significantly distorted as seen in the brightfield images (scale bars represent 150 µm). (iii) No significant difference was observed in the distortion of the spheroids with the PMA-stimulated NETs present in the channel or with direct stimulation of the spheroids with PMA (data collected for n = 3 spheroids, mean ± SEM, t test, ns = not significant).