Immune Cell Analysis of Pancreatic Tumors

Samples were processed and analyzed as previously described (57 (link)). Pancreatic tumors were processed 24 hours after completion of the first cycle of therapy: 5 doses of TH-302 administered once daily and 2 doses of αVEGFR administered every 4 days. The pellet from the Histopaque-1119 (MilliporeSigma, 11191) separation was processed for endothelial cell analysis. CD8⁺ T cells were isolated using a magnetic-activated cell sorting CD8a⁺ T Cell Isolation Kit (Miltenyi Biotec, 130-104-075), activated for 6 hours using the eBioscience Cell Stimulation Cocktail plus protein transport inhibitors (Thermo Fisher Scientific, 00-4975-93), and analyzed by flow cytometry as previously described (56 (link)). Flow data were collected on a 5-laser, 18-color BD LSR II and analyzed using FlowJo version v10.7. A representative illustration of the immune cell gating scheme is provided in Supplemental Figure 5.