Profiling Nascent TumorExtracellular Vesicles viaMetabolic Timestamping and Aptamer-Driven Specific Click Chemistry

Tumor-derived extracellular vesicles (tEVs) are essential mediators of tumor progression and therapeutic resistance, yet their secretion dynamics and cargo composition in response to therapies remain poorly understood. Here, we present STAMP, <u>s</u>pecific click-<u>t</u>agging driven by <u>a</u>ptamer for tEV labeled with a <u>m</u>etabolic timestam<u>p</u>, which exploits the unique kinetics and thermodynamics of aptamer to significantly enhance the local concentration of clickable probes on tEVs for their covalent attachment to the timestamp, resulting in the selective microfluidic isolation of nascent tEVs following stimulation. In a PD-L1 antibody-treated model, we demonstrated the feasibility of STAMP and revealed a robust positive correlation between the nascent EpCAM⁺ EV levels and tumor volume. Proteome profiling of isolated nascent tEVs identified previously unknown upregulated vesicle proteins following immunotherapy, including key regulators of immune activation and suppression, suggesting that tumors orchestrate an intricate dual adaptive response through tEV secretion modulation to simultaneously elicit therapeutic sensitivity and resistance. Notably, among the upregulated proteins, we identified HSP70, whose enhanced presentation on tEVs promotes antitumor immunity and inhibits tumor growth. Thus, STAMP provides an effective gateway for studying EV dynamics with cell-origin accuracy and for identifying potential therapeutic targets based on EV transitions.