A novel tumor-immune microenvironment (TIME)-on-Chip mimics three dimensional neutrophil-tumor dynamics and neutrophil extracellular traps (NETs)-mediated collective tumor invasion

Neutrophils are the most abundant type of leukocytes in the blood, traditionally regarded as the first immune responders to infections and inflammations. In the context of tumors, neutrophils have been shown to possess both tumor-promoting and tumor-limiting properties. A better understanding of the inter-cellular dynamics between the neutrophils and aggregated tumors could possibly shed light on the different modalities of neutrophil involvement in tumor progression. To study in-vitro the interactional dynamics of neutrophils and growing tumor aggregates, in this work, we engineered a novel, microfluidics-integrated, three-dimensional (3D) tumor-immune microenvironment (TIME)-on-Chip device, and we investigated the effect of neutrophils on the inception of collective 3D invasion of ovarian tumor cells. Herein, tumor spheroids generated and cultured on hydrogel based multi-microwell plates, and embedded within collagen matrix of defined thickness, were magnetically hybrid-integrated with a 3D bioprinting enabled microfluidic system fabricated on a porous membrane and carrying neutrophils. This setting recreated a typical TIME in-vitro to model dynamic neutrophil migration and 3D tumor invasion. Using this device, we observed that neutrophils respond to the growing tumor spheroids through both chemotaxis and generation of neutrophil extracellular traps (NETs). The formation of NETs stimulated the reciprocation of tumor cells from their aggregated state to collectively invade into the surrounding collagen matrix, in a manner more significant compared to their response to known tumor-derived stimulants such as transforming growth factor and Interleukin-8. This effect was reversed by drug-induced inhibition of NETs formation, suggesting that induction of NETs by cancer cells could be a pro-migratory tumor behavior. Further, we additionally report a previously unidentified, location-dictated mechanism of NETosis, in which NETs formation within the stromal extracellular collagen matrix around the spheroids, and not tumor-contacted NETs, is important for the induction of collective invasion of the ovarian tumor cells, thus providing a rationale for new anti-tumor therapeutics research.