The integrated stress response promotes immune evasion through Lipocalin 2

ExCITE-Seq immune analysis of sorted CD45+ immune cell clusters from orthotopically transplanted KP LUAD with doxycycline-inducible shLcn2 and shCtr KP tumors 8 days after adding doxycycline Overall design: Mice were sedated with ketamine and xylazine and then were injected with 2 ug of APC anti-CD45 (2 ug per mouse diluted in 100 uL PBS, Biolegend 30-F11) retro-orbitally 3 minutes prior to lung collection. The lung lobes were minced on a glass slide and then digested (collagenase IV (Sigma Aldrich C5138), DNAse I (Sigma Aldrich, DN25) in RPMI with 10% FBS) for 35 minutes at 37 degrees celsius. Digestion was stopped with EDTA (1 mM). Digested tissue were filtered into a single cell suspension through a 100 micron filter followed by red blood cell lysis (BD Pharm Lyse 555899). Cells were then washed and suspended in a staining buffer. Cells were then stained with live dead staining (Zombie UV fixabable viability dye, Biolegend #423107) and PeCy7 anti-CD45 (see flow cytometry methods section for staining protocol). Approximately 500,000 lung immune cells from each condition (2 mice per condition) were sorted as live+ IV-CD45 - CD45+ and 50,000 tumor cells were sorted as live+ CD45- GFP+. Sorted samples were multiplexed using cell hashing antibodies (Biolegend) and stained with ADTs (see antibody table). Cells from each sample were pooled and loaded into 10X Chromium.