<em>Ex vivo</em> expanded ieILC1-like NK cells induce significant tumor cell death and demonstrate metabolic flexibility under hypoxic conditions — ASN Events
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  3. Ex vivo expanded ieILC1-like NK cells induce significant tumor cell death and demonstrate metabolic flexibility under hypoxic conditions

Ex vivo expanded ieILC1-like NK cells induce significant tumor cell death and demonstrate metabolic flexibility under hypoxic conditions (#157)

Alistaire D Ruggiero Sherman 1 , Keene Lee 1 , Mehri Monavarian 1 , Imran Mohammad 1 , Xinyuan Liu 1 , John Sunwoo 1
  1. Stanford University, Stanford, CA, United States

Background: Hypoxia-induced natural killer (NK) cell dysfunction remains a significant obstacle to NK cell therapies against solid tumors. We identified a subset of CD103+CD49a+ NK cells, termed ieILC1-like NK cells, in primary head and neck squamous cell carcinomas that demonstrate enhanced effector functions [1]. A novel ex vivo expansion method for these cells was developed [1], and we report that expanded ieILC1-like NK cells achieve substantial tumor cell clearance under 1% oxygen conditions. To understand the mechanisms behind their enhanced cytotoxicity, we investigated the metabolic adaptations of these cells under hypoxia.
Methods: Expanded ieILC1-like NK cells were generated by co-culturing conventional NK (cNK) cells with PCI-13 tumor cells (1:1) in the presence of IL-15 (10 ng/mL) [1]. Hypoxic exposure (1% oxygen) was performed for up to 96 hours (n=3–6/group). Cytotoxic capacity was assessed using Incucyte Live Cell analysis at an effector-to-target (E:T) ratio of 1:2.5. Flow cytometry evaluated mitochondrial mass, activity, and transporter expression, while quantitative gene expression analyzed metabolic genes. Single-Cell ENergetics by Profiling Translation Inhibition (SCENITH) assessed single-cell metabolic pathway reliance. Statistical significance was determined using unpaired t-tests.
Results: ieILC1-like NK cells exhibited superior killing capacity (p=0.03) and increased proportions of CD25+ (p=0.012) and Ki67+ (p<0.01) cells. Metabolic profiling revealed increased percentages of MitoTracker Red+ (p=0.002), MitoTracker Green+ (p=0.005), 2NBDG+ (p=0.043), and GLUT1+ (p<0.0001) cells, with decreased MitoSox+ (p=0.014), CD98+ (p=0.0003), and CD71+ (p=0.03) cells. Upregulated VEGF (p=0.02) and LDHA (p<0.0001) expression corresponded with heightened glucose dependence (p=0.035) after hypoxia exposure compared to cNK cells.
Conclusions: These findings demonstrate that ieILC1-like NK cells possess enhanced cytotoxic and proliferative capabilities, supported by unique metabolic reprogramming to thrive in hypoxic environments. This work reveals a novel anti-tumor capacity of ieILC1-like NK cells, and supports clinical investigation of these cells for solid tumor treatment.