Quantifying the dual effect of anti-tumor and pro-tumor human neutrophils on natural killer cell behaviors in a microphysiological system

Neutrophils can promote tumor progression by inhibiting the antitumor activity of natural killer (NK) cells known as the first line of defense against cancer. Studies in mice show that neutrophils can be polarized toward either an anti-tumor “N1” or a pro-tumor “N2” state. However, it is unknown how N1 and N2 neutrophil subtypes influence NK cell behaviors differently in human cancer. It is also challenging to monitor neutrophil-NK cell interactions in the human tumor tissue. Here, we engineered a human cell-based microphysiological system to measure the distinct effect of N1 and N2 neutrophil subtypes on NK cell migration, motility, tumor cytotoxicity, and tumor infiltration. We fabricated a three-channel microfluidic chip using standard lithography. To model the preferential migration of NK cells toward different neutrophil subtypes (scenario 1), the two side channels of the microfluidic chip were seeded with LPS, IFN-gamma, and IFN-beta-polarized N1 and TGF-beta-polarized N2 HL-60 neutrophils respectively and the central channel was seeded with NK-92MI cells. To model NK cell cytotoxicity against tumor (scenario 2), the side channels were seeded with a mixture of NK-92MI cells, PANC-1 pancreatic tumor spheroids, and N1 or N2 neutrophils. All cells were embedded in 3D collagen hydrogel to mimic the extracellular matrix of the pancreatic tumor tissue. Time-lapse imaging and end-point confocal imaging were performed to capture NK cell migration and motility in scenario 1 and tumor spheroid apoptosis and NK cell-tumor infiltration in scenario 2. NK-92MI cells showed both a higher percentage of migration (12.07 vs. 5.11% at t=24 h, p<0.001) and a higher maximum rate of migration (1.31 vs. 0.73%/h, p<0.01) to N1 neutrophils than to N2 neutrophils over 24 h. NK-92MI cells showed a higher motility after migration to N2 neutrophils than N1 neutrophils in speed (1.07 vs. 0.80 µm/min at t=12 h), displacement (9.89 vs. 6.89 µm at t=12 h), and directionality (0.43 vs. 0.36 at t=24 h) (p< 0.0001). Hence, NK-92MI cells showed preferential migration to N1 over N2 neutrophils, although they slowed down after migration to N1 compared to N2 neutrophils. Moreover, N1 neutrophils restored NK cell cytotoxicity against tumor spheroids (0.97 vs. 1.01, p>0.99) while N2 neutrophils suppressed it (0.97 vs. 0.54, p<0.0001), although both N1 and N2 neutrophils inhibited NK cell infiltration in tumor spheroids (control vs. N1 vs. N2: 8.04 vs. 5.87 vs. 5.49%, p<0.01). We also found that N1 neutrophils secreted a higher level of NK cell chemokine IP-10 and induced higher expressions of activation markers CD107a and IFN-gamma by NK-92MI cells than N2 neutrophils. This study reveals the dual role of human neutrophils in modulating NK cell behaviors and the complex neutrophil-NK cell crosstalk, suggesting reprogramming neutrophils to reverse the immunosuppression on NK cells as a potential therapeutic strategy for cancer.