Single-cell metabolic profiling reveals time-associated metabolic trajectories linked to functional changes in anti-tumor NK cells.

Abstract

Cellular metabolism shapes immune-cell function, yet single-cell metabolic heterogeneity remains analytically challenging to resolve. Here, we applied label-free cytometry by electrospray ionization mass spectrometry (CyESI-MS) to NK-92MI, a human natural killer (NK) cell line. Cells were collected from a NK-92MI/HeLa co-culture model at 0 h (resting condition), 6 h (early activation condition), and 72 h (prolonged stimulation condition). The 0 h condition was characterized by baseline cytotoxicity, low interferon gamma (IFN-γ) production, and minimal programmed cell death protein 1 (PD-1) expression. The 6 h condition was characterized by peak cytotoxicity and increased IFN-γ production. The 72 h condition was characterized by reduced cytotoxicity and IFN-γ production relative to their 6 h peaks, together with increased PD-1 expression. Single-cell metabolic profiling revealed time-associated metabolic trajectories linked to these functional changes. Across the dataset, 158 metabolite features were detected. The early activation condition showed changes consistent with antioxidant, central carbon/amino-acid, and lipid remodeling. In contrast, the prolonged stimulation condition showed broader metabolic imbalance. Together, these findings establish a proof-of-concept framework for linking single-cell metabolic trajectories with independently measured changes in NK-cell function.

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