Novel Strategy for Acquiring Metabolically-Tagged Nascent Extracellular Vesicles: Implications for Identifying Surface Protein Markers of Extracellular Vesicles From Neuroblastoma Cells Cultured With Native Serum.

Tumour-derived extracellular vesicles (tdEVs) have emerged as a promising representative of cancer manifestation that can be accessed non-invasively through liquid biopsy. Selective examination of tdEVs requires their isolation, which relies on tumour-specific surface markers. These markers are often identified using cancer cell lines cultured in EV-depleted serum or serum-free conditions to avoid interference by exogenous EVs in serum. However, these nutrient-deprived media can alter gene expression and the proteomic composition of EVs. This study aims to develop a method to identify potential EV surface markers for paediatric neuroblastoma from tumour cell lines grown in native serum. Our methodology enables distinguishing tumour-specific EVs from the exogenous serum EVs, without prior knowledge of any tumour-specific surface markers. By metabolically incorporating an azide-tagged sugar analogue into nascent glycoproteins, we differentially marked only tumour-derived EVs and captured them using copper-catalysed click chemistry-mediated biotinylation and affinity enrichment. Subsequent analysis through mass spectrometry and western blotting led to the identification of gap junction protein GJC1 (connexin 45) as a potential surface marker for neuroblastoma EVs. This methodology not only aids in EV surface profiling but also has significant implications for time-resolved and spatial EV studies in various biological contexts, including disease development, progression, therapy resistance, and cellular communication.