Multiplexed Profiling of Individual Extracellular Vesicles Reveals Lung Cancer‐Associated Protein Signatures

Vollständiger Abstract

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ABSTRACT Lung cancer is frequently diagnosed after curative treatment windows have narrowed, creating a need for minimally invasive biomarkers that report tumor development at earlier stages. Extracellular vesicles (EVs) are promising analytical targets because they carry molecular cargo from their originating cells, but tumor‐associated EV signals can be rare in blood and obscured in bulk measurements. Here, we present Cygnus ( Cy clic‐imaging g ateway to n anovesicles u nderlying s ignature), an end‐to‐end workflow that integrates cyclic immunofluorescence imaging with multiscale analysis of individual EVs. Cygnus preserves vesicle‐level measurements, quantifies marker co‐expression, resolves EV subpopulations, and summarizes single‐vesicle phenotypes into sample‐level profiles. In a genetically engineered mouse model of lung adenocarcinoma, Cygnus revealed dynamic changes in EV protein composition and identified an EpCAM‐ and/or CTSH‐positive EV subpopulation before tumors were detectable by radiography. In a pilot clinical cohort ( n = 35), plasma EV profiling showed lung cancer‐associated marker patterns compared with non‐cancer controls and nominated a CTSH/PDL1/MET marker combination for future validation. These findings support multiplexed individual‐EV profiling as a strategy for defining candidate lung cancer‐associated EV signatures and position Cygnus as an integrated workflow for translating vesicle‐level heterogeneity into sample‐level EV profiles.