Abstract A041: A microphysiological system to quantify the interactions of human neutrophil subpopulations with pancreatic tumor spheroids

Abstract Cancer cells recruit immune cells to the solid tumor tissue, which can either promote or inhibit the progression. Studies in mice suggest that polarizing neutrophils from a pro-tumor (N2) phenotype to an anti-tumor (N1) phenotype may serve as a promising strategy for immunotherapy. Understanding the mechanisms by which N1 and N2 human neutrophil subpopulations interact with cancer cells and influence the tumor microenvironment (TME) is important for solid tumor immunotherapy in the clinic. It is difficult to monitor immune cell-cancer interactions in the human TME. Microphysiological models are microfluidic platforms that consist of cells, explants, spheroids, or organoids to study pathological processes with single-cell resolution. In this study, we engineered a human cell-based microphysiological system to quantify the interactions of N1 and N2 human neutrophils with pancreatic tumor spheroids. LPS, IFN-γ, and IFN-β-polarized N1-like or TGF-β-polarized N2-like HL-60 human neutrophils and PANC-1 tumor spheroids were embedded in 3D collagen hydrogel mimicking the extracellular matrix of the tumor tissue and loaded into the polydimethylsiloxane- fabricated microfluidic system. Time-lapse epifluorescence imaging and end-point confocal imaging were performed to capture neutrophil motility, contact with, and infiltration into tumor spheroids. We first confirmed that N1-like neutrophils were CD11bhigh/CD54high/CD62Llow/CXCR2low and that N2-like neutrophils were CD11bhigh/CD54low/CD62Lhigh/CXCR2high using flow cytometry. We then validated that N1-like neutrophils showed higher levels of ROS production, IL-8 secretion, and NET release than N2-like neutrophils (p<0.0001). When challenged with tumor spheroids in the system, N2-like neutrophils showed a higher motility than N1-like neutrophils in speed (3.23 vs. 1.68 µm/min, p<0.0001), directionality (0.29 vs. 0.26, p<0.01), and displacement (31.60 vs. 18.08 µm, p<0.0001). While N1-like neutrophils showed a similar frequency of contact with tumor spheroids as N2-like neutrophils (1.68 vs. 1.58 times/h, p>0.99), N1-like neutrophils showed a longer duration of contact than N2-like neutrophils (32.7 vs. 20.3 min, p<0.0001). We found a higher number of N1-like neutrophils that infiltrated into tumor spheroids than N2-like neutrophils (4.6 vs. 3.0 cells/spheroid, p=0.01), although the infiltration depth was similar (7.4 vs. 6.0 µm, p=0.36). 2D co-cultures showed that N1-like neutrophils induced apoptosis of PANC-1 cancer cells, while N2-like neutrophils attenuated chemotherapy-induced cancer cell apoptosis (p<0.0001). The system also enabled us to measure the effect of N1-like and N2-like neutrophils on the apoptosis, proliferation (Ki-67), and epithelial-mesenchymal transition (vimentin) of tumor spheroids. Our research sheds light on the “behaviorome” of human neutrophil subpopulations in cancer. Our microphysiological model will serve as a complementary tool to animal models for mechanistic studies, immunotherapy testing, and personalized medicine using patient-derived cells. Citation Format: Shuai Shao, Daniel Duncko, Caroline N Jones. A microphysiological system to quantify the interactions of human neutrophil subpopulations with pancreatic tumor spheroids [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor Immunology and Immunotherapy; 2024 Oct 18-21; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2024;12(10 Suppl):Abstract nr A041.