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.