389 Ex vivo expanded ieILC1-like NK cells induce significant tumor cell death and demonstrate metabolic flexibility and macrophage reprogramming capacity under hypoxic conditions

• DOI: 10.1136/jitc-2024-sitc2024.0389
• Publication Date: 2024-11-05T14:58:58Z
• Authors: Alistaire D Ruggi...; Keene Lee; Xinyuan Liu; Imran A Mohammad; John B Sunwoo
• Keywords: Reprogramming; Ex vivo

<h3>Background</h3> Natural killer (NK) cells offer unique therapeutic potential given their antigen-independent cytotoxicity. NK cell dysfunction in hypoxic tumor microenvironments remains a barrier for therapies against solid tumors. Through single-cell RNA sequencing of primary head and neck squamous carcinomas, we identified CD103⁺CD49a⁺ (termed ieILC1-like cells) that demonstrate upregulated expression effector molecules.¹ We developed novel method differentiating expanding these <i>ex vivo.</i>¹ Here, found induce significant clearance under 1% oxygen conditions. To determine attributes associated with enhanced cytotoxicity, investigated the metabolic fitness stemness cells, impact on macrophages <h3>Methods</h3> Expanded were generated by culturing conventional (cNK) irradiated PCI-13 (1:1) IL-15 (10ng/mL).¹ cNK placed hypoxia chambers set to up 96 hours (n=3–6/group). Conditioned medium (CM) was collected after exposure. Incucyte Live Cell analysis determined vivo</i> killing capacity post-exposure (E:T = 1: 2.5). Mitochondrial mass activity, amino acid transporter expression, activation markers measure flow cytometry post-exposure. Metabolism genes NADH/NAD concentrations measured quantitative gene enzymatic assays performed duplicate. Macrophage chemoattraction assessed transwell migration polarized (1x10⁵ cells/M-type) exposed CM. polarization unpolarized CM, measuring M-type cytometry. All data met normality equal variance assumptions. One-way ANOVAs or unpaired t-tests statistical differences. <h3>Results</h3> showed improved (p=0.03), increased percentages CD25⁺ (p=0.012), MitoTracker Red⁺ (p=0.002), Green⁺ (p=0.005), CD43⁺ (p=0.02) CD38⁺ (p=0.13) reduced CD98⁺ (p=0.0003) CD71⁺ (p=0.03) demonstrated LDHA (p&lt;0.0001) an 85% increase NADH:NAD compared CM from attracted more anti-tumorigenic M1 (p=0.018) fewer pro-tumorigenic M2 (p=0.016), CD206⁺ exposure (p=0.04). <h3>Conclusions</h3> uniquely co-occurs mitochondrial stemness, anti-tumor macrophage attraction settings. This work reveals supports clinical investigation treatment. <h3>Reference</h3> Moreno-Nieves UY, Tay JK, Saumyaa S, Horowitz NB, Shin JH, Mohammad IA, Sunwoo JB. Landscape innate lymphoid human cancer divergent states microenvironment. <i>Proceedings National Academy Sciences</i>, 2021;<b>118</b>(28):e2101169118.