Naive primary neutrophils play a dual role in the tumor microenvironment.

The tumor microenvironment (TME) is characterized by a network of cancer cells, recruited immune cells and extracellular matrix (ECM) in a hypoxic microenvironment. However, the specific role of neutrophils during tumor development, and their interactions with other immune cells is still not well understood. Thus, there is a need to investigate the interaction between primary neutrophils and natural killer cells and the resulting effects on tumor development. Here we use both standard well plate culture and an under oil microfluidic (UOM) assay with an integrated extracellular cell matrix (ECM) bridge to elucidate how naive primary neutrophils respond to both patient derived tumor cells and tumor cell lines. Our data demonstrated that both patient derived head and neck squamous cell carcinoma (HNSCC) tumor cells and MDA-MB-231 breast cancer cells trigger cluster formation in neutrophils, and the swarm of neutrophils restricts tumor invasion through the generation of reactive oxygen species (ROS) and neutrophil extracellular trap (NETs) release within the neutrophil cluster. However, we also observed that the presence of neutrophils downregulates granzyme B in NK-92 cells and the resulting NETs can obstruct NK cells from penetrating the tumor mass suggesting a dual role for neutrophils in the TME. Further, using label-free optical metabolic imaging (OMI) we observed changes in the metabolic activities of primary neutrophils during the different swarming phases when challenged with tumor cells. Finally, our data demonstrates that neutrophils in direct contact, or in close proximity, with tumor cells exhibit greater metabolic activities (lower nicotinamide adenine dinucleotide phosphate (NAD(P)H) mean lifetime) compared to non-contact neutrophils.