Abstract
Metabolic analysis of tumor cells is crucial for understanding tumor metabolic reprogramming, and single-cell metabolic accumulation studies provide precise insights into tumor biology. In this work, we established a microfluidic assay for single-cell metabolic accumulation analysis by combining droplet-generated hydrogel microspheres for single-cell encapsulation with a microchamber culture chip for microsphere isolation and 3D cultivation. This system enables efficient accumulation of secreted metabolites from individual cells for downstream analysis. Coupled with mass spectrometry, this system revealed metabolic accumulation heterogeneity among individual cells of the same tumor type. Moreover, metabolic profiling of different tumor types, including A549, HepG2, and HCT116, demonstrated distinct metabolic patterns, enabling cell classification based on accumulation differences. The method supports high-throughput, multiplexed, and dynamic metabolic analysis in a 3D single-cell context, offering a scalable strategy for decoding tumor heterogeneity with potential application in cell-state characterization and advancing oncology research.