An intratumorally discovered extracellular vesicle-derived biomarker enables ultrasensitive digital quantification in plasma for early detection of non-small cell lung cancer

BackgroundExtracellular vesicle (EV)-based liquid biopsy remains challenging due to the limited specificity and low abundance of EV cargoes. This study aimed to identify cancer-specific molecules from tissue-derived EVs and evaluate their potential as circulating biomarkers for non-small cell lung cancer (NSCLC). MethodsTissue-derived EVs were isolated by density gradient flotation and subjected to proteomic profiling to identify differentially expressed proteins. Public transcriptomic data, functional enrichment, and variable importance scores were integrated to select candidate biomarkers. Immunogold labeling transmission electron microscopy (TEM) was used to visualize protein expression on the EV membrane using, and an ultrasensitive modified digital EV screening technique (DEST) was employed to quantify protein signals in circulating EVs. The diagnostic performance was evaluated by the area under the curves (AUC). ResultsTissue-derived EVs were isolated from cancer tissues and paired non-cancer lung tissues from patients with NSCLC. Proteomic profiling identified 1,568 tissue-derived EV proteins, of which 904 were differentially expressed. Integrated transcriptomic and proteomic analyses yielded eight cancer-specific, membrane-localized candidate biomarkers, with GLUT1 showing the highest variable importance score. Immunogold labeling TEM confirmed the localization of GLUT1 on the EV membrane. Modified DEST analysis of 47 patients revealed significantly elevated levels of CD63+ EV-derived GLUT1 in the cancer group than in the non-cancer group (P < 0.01). Diagnostic performance reached an AUC of 0.817 in the whole cohort and 0.898 in the stage-I cohort, exceeding that of conventional serum biomarkers. A three-marker panel (CD63+ EV-derived GLUT1, CYFRA 21-1, and CEA) further improved discrimination, with AUCs of 0.853 in the whole cohort and 0.944 in the stage-I cohort. ConclusionCirculating EV-derived GLUT1 was preliminarily identified as a novel biomarker for NSCLC, and its combination with conventional serum biomarkers enhanced diagnostic performance, particularly in early-stage cases.