Integrating Ex Vivo Isotope Labeling with Mass Spectrometry Imaging to Spatially Trace Heterogeneous Metabolic...

Spatially resolved isotope tracing provides a powerful means for achieving deeper and more accurate characterization of metabolic activities in biological tissues. However, its application in mammals remains limited by several key technical challenges, including complex surgical procedures, the need for high tracer doses, and poor suitability by clinical tissue samples. In this study, we developed a novel spatially resolved isotope tracing method by integrating ex vivo tissue labeling with airflow-assisted desorption electrospray ionization mass spectrometry imaging (AFADESI-MSI). Using liquid chromatography-mass spectrometry, we identified 263 labeled metabolites in ex vivo U-¹³C glucose-labeled rat kidney tissue, including amino acids, nucleotides, organic acids, and lipids. Moreover, AFADESI-MSI enabled the in situ characterization of 27 labeled metabolites. This method allowed the simultaneous visualization of complex metabolic networks and their spatially dynamic activities without the need for in vivo tracer injection. Our results revealed that the kidney cortex exhibited significantly higher glucose uptake efficiency and gluconeogenic activity compared with the medulla, whereas the medulla demonstrated greater activity in reducing pyruvate to lactate. We anticipate that this method will provide new strategies and analytical tools for investigating metabolic heterogeneity in clinical tumors and other complex tissues.