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

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 (Cyclic-imaging gateway to nanovesicles underlying signature), 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.