Linking Metastatic Behavior and Metabolic Heterogeneity of Circulating Tumor Cells at Single‐Cell Level Using an Integrative Microfluidic System

Abstract Circulating tumor cells (CTCs) are pivotal biomarkers in tumor metastasis, however, the underlying molecular mechanism of CTCs behavioral heterogeneity during metastasis remains unexplored. Here, an integrative workflow is developed to link behavior characteristics to metabolic profiling within individual CTCs, which simulates the metastatic process on a microfluidic system and combined with single‐cell mass spectrometry (MS) detection. Spheroid‐derived HCT116 cells are tracked and extracted via a temporary vascular system, revealing various arrest patterns under biomimetic vascular shear flow. Downstream MS analysis characterizes 17 cellular metabolites and associates metabolic profiles with de‐adhesion behaviors of the same CTCs, identifying a potential high‐metastatic subpopulation with enhanced arrest ability and evaluating critical metabolites involved in metastasis pathways. Additionally, the metastasis‐inhibiting effect of anti‐tumor drug 5‐fluorouracil by reducing high‐metastatic cells in spheroids is elucidated. This approach offers a valuable opportunity to dissect the interplay of the metastatic behavior and metabolic profiles of CTCs and foster insights into the molecular mechanisms underlying behavioral phenotypes in the tumor metastasis process.