Comprehensive profiling of lung cancer markers for early screening and detection

Abstract

Lung cancer remains the leading cause of cancer-related mortality worldwide, largely due to late-stage diagnosis and the lack of effective non-invasive screening strategies. The stability of extracellular vesicles in biofluids, combined with their capacity to present cell-derived surface proteins, has led to growing interest in their potential as minimally invasive cancer biomarkers. While EV-based assays are not currently implemented for early cancer diagnosis, methodological challenges related to EV isolation, detection sensitivity and assay reproducibility remain key barriers to their clinical translation. The aim of this study was to comprehensively profile EV-associated surface markers relevant to lung cancer and to evaluate their robustness and detectability across different assay formats, isolation methods, and biological matrices. A multi-step analytical workflow was implemented using four non- small-cell lung cancer (NSCLC) cell lines (A549, NCI-H1975, NCI-H460 and NCI-H520). Cell-surface marker expression was first characterised by flow cytometry to establish a biological reference. EVs were subsequently isolated using three approaches: polyethylene glycol (PEG) precipitation, size-exclusion chromatography (Izon qEV1), and polymer-based precipitation (SubXTM), and further characterised by nanoparticle tracking analysis (NTA). EV surface marker detection was performed using antibody-based bead immunoprecipitation coupled to Luminex FLEXMAP 3D® assays in both single-plex and multiplex formats. Plasma spike-in experiments using tumour-derived EVs were performed to assess assay sensitivity in a clinically relevant matrix, and plasma samples from patients with metastatic lung cancer were analysed to explore translational feasibility. Distinct and biologically meaningful EV surface marker profiles were observed across lung cancer models. CD9 and CD63 were consistently detected across cell line-derived EVs, plasma spike-in experiments and patient plasma samples, with CD63 producing the strongest signal. Multiplex immunoprecipitation improved detection of several lower-abundance markers, including CD24, CD90, and CD274, compared with single-plex capture. Both Izon and SubXTM isolation methods enabled detection of tumour-derived EVs in plasma, however, Izon SEC produced a higher marker signal intensities across spike concentrations. In conclusion, this study establishes a robust technical framework for EV surface marker profiling in lung cancer and demonstrates the feasibility of immunoprecipitation-based bead assays for detecting biologically relevant EV markers. These findings support the potential of EV-associated surface proteins as candidates for future non-invasive lung cancer screening and early detection strategies.