Single-cell mass spectrometry reveals metabolic divergence mediated by contact-dependent cell-cell interactions

Membrane contact-dependent cell-cell interactions play a critical role in tumor progression. However, elucidating their underlying molecular mechanisms remains challenging due to the lack of in situ analytical approaches at single-cell resolution. Here, we performed a single-cell metabolic analysis in a linearly membrane-connected three-cell system (A-B-C) using nanocapillary-based electrospray ionization mass spectrometry. Molecular profiling revealed that when cell C was induced into apoptosis, its interaction with the directly membrane-contacted cell B drove metabolic divergence between cell B and the distal cell A. This reprogramming was characterized by the upregulation of the pentose phosphate pathway and the Warburg effect, concurrent with the downregulation of the citric acid cycle and associated amino acid metabolism. This reflects adaptive responses in cell B that promote survival and enhance anti-apoptotic capacity. Further analysis demonstrated that these metabolic shifts were associated with reactive oxygen species (ROS), and ROS scavenging significantly attenuated the metabolic differences between cell A and B. Furthermore, the positive correlation between the extent of metabolic changes in cell A and B indicated a stepwise interaction, forming a metabolic gradient that expands single-cell heterogeneity. Collectively, our study establishes an in situ single-cell analysis platform for investigating membrane contact-dependent cell-cell interactions without disrupting physical connections, and provides new insights into metabolic adaptations.