Human Neutrophil Isolation and Ex Vivo Neutrophil Migration assay

All neutrophils were isolated from 10 mL of whole human blood with EDTA as an anti-coagulant (Research Blood Components) by negative selection using the EasySep^TM human neutrophil isolation kit (StemCell Technologies Cat #19666). PC3 cells (ATCC) were cultured in RPMI 1640 (Life Technologies) supplemented with 10% FBS (Life Technologies), 10% PenStrep (Life Technologies). PC3 cells were then plated on 24 well plates (Greiner Bio One, Cat. #89131-690) at a density of 60,000 cells/well. A working volume of 500 μL of media was used throughout the experiments. After plating, cells were allowed to adhere for 24h. Following attachment, PC3 cells were treated with 5 μM cabozantinib or DMSO (vehicle control) in unsupplemented serum-free RPMI 1640 for 24h. 200,000 freshly isolated human neutrophils were then placed in the top of the 3 μm polycarbonate transwell insert (Corning, Cat. # 29442-110) in 100 μL of unsupplemented serum-free RPMI 1640. Cells were incubated at 37°C for 4 hours. 100 μL of media was then collected from the bottom of the transwell. 30 μL of sample was then analyzed using flow cytometry to count the number of migrated neutrophils. For blocking experiments, anti-HMGB1 clone 3E8 was added to cabozantinib-treated PC3 cells to a final concentration of 50 μg/mL and incubated for 1hr at 37°C.