Circadian control of circulating tumour-derived extracellular vesicle secretion affects targeted therapy efficacy.

Extracellular vesicles (EVs) are cell-derived nanoparticles that mediate intercellular communication, whereas circulating tumour-derived EVs (ctEVs) promote tumour progression. However, their circadian dynamics remain poorly understood owing to the lack of tools capable of resolving vesicle biogenesis dynamics. Here we introduce ctEV-CLOCK, a strategy enabling 12-h resolution isolation of nascent ctEVs. The method integrates metabolic glycoengineering-based timestamping with tumour-marker-guided selective click tagging, achieving enrichment of ctEV populations within defined time windows. Using this approach, we uncover circadian oscillations in ctEV abundance and functional properties across multiple mouse tumour models. These oscillations are accompanied by time-of-day-dependent alterations in vesicle protein profiles. Importantly, synchronizing targeted therapy with peak ctEV protein abundance markedly enhances therapeutic efficacy. Together, these findings position ctEV circadian dynamics as a regulatory layer in tumour biology and provide a framework for circadian-aligned cancer chronotherapy beyond immune-focused strategies.