In Cellulo Observation of De Novo -Synthesized Nucleotide by Spectral Tracing of Glutamine-Derived Nitrogen Metabolism (QN-STIM) in Cancer Cells

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Abstract De novo nucleotide biosynthesis is directly linked to glutamine-derived nitrogen metabolism in mammalian cells. It is a fundamental process tightly tied to cellular growth, proliferation, and metabolic reprogramming, particularly in cancer cells. Direct detection and imaging of glutamine-derived nitrogen metabolism at single-cell resolution remains a challenge. The conventional liquid chromatography-mass spectrometry-based approach for detecting de novo-synthesized nucleotide studies is mostly performed on metabolites extracted from a group of cells, which makes it inherently destructive. Herein, we introduce a vibrational spectroscopy-based single-cell metabolic detection and imaging framework, “glutamine (Q)-derived nitrogen spectral tracing and imaging (QN-STIM)”. Using this strategy, we detected and mapped nitrogen flow from glutamine to de novo-synthesized nascent nucleotides at the single-cell level. First, a proof-of-concept analysis was conducted in human embryonic kidney HEK293T cells and demonstrated the feasibility of this approach in cancer cells, namely human lung carcinoma A549, human glioblastoma U87 MG, and mouse hepatoma Hepa1-6. QN-STIM technique holds immense potential to be an adjunct tool to advance our understanding of nucleotide dynamics during differentiation, stress adaptation, and immune activation, and to evaluate responses to metabolic drugs in cellulo.

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