Behaviorome profiling of anti-tumor and pro-tumor human neutrophil subtypes in a microphysiological system

Neutrophils exhibit a paradoxical role in cancer, either combating the tumor or facilitating its progression. Studies in mice have shown that polarizing neutrophils from a pro-tumor "N2" phenotype to an anti-tumor "N1" phenotype can serve as a strategy for immunotherapy. Understanding the behavioral differences between N1 and N2 human neutrophil subtypes in the solid tumor tissue is important for developing novel cancer immunotherapy. However, it is challenging to monitor neutrophil-cancer interactions in the human tumor tissue. In this study, we developed a human cell-based microphysiological system to quantify both real-time and end-point behaviors of N1-like and N2-like neutrophils during their interactions with pancreatic cancer in a 3D collagen matrix. We first validated that N1-like neutrophils showed higher levels of pro-inflammatory hallmarks including ROS production, IL-8 secretion, and NET release and 2D cancer cytotoxicity than N2-like neutrophils. We found that N2-like neutrophils showed greater migration to cancer cells and higher motility after migration than N1-like neutrophils. N1-like neutrophils engaged in longer contact with tumor spheroids and infiltrated greater into tumor spheroids than N2-like neutrophils. N1-like neutrophils also attenuated the progression of tumor spheroids in terms of invasion, proliferation (Ki-67 expression), and epithelial-mesenchymal transition (vimentin expression) compared to N2-like neutrophils, although tumor apoptosis was not affected. Our microphysiological system reveals the distinct "behavioromes" of anti-tumor and pro-tumor human neutrophil subtypes in pancreatic cancer, which may inform the design and development of novel cancer immunotherapies aiming to modulate specific neutrophil behaviors.