Development of a high-sensitivity assay for direct quantitation of EGFr on extracellular vesicles in plasma

Extracellular vesicles (EVs) play an important role in intercellular communication and hold promise for cancer diagnostics and therapeutic monitoring, particularly in Epidermal Growth Factor receptor (EGFr)-driven malignancies. This study aims to develop a method to quantitate EGFr on EVs directly in plasma samples without using an EV purification step first. Additionally assays for quantitating the total concentrations of EVs were established. The assays were developed using Single molecule array (Simoa) technology with antibodies targeting the EV surface markers EGFr, CD9, CD63 and CD81. Plasma samples from healthy individuals and cancer patients were used for assay development, validation and testing. In this pilot study we observed a lower concentration of EGFr on EVs in cancer patients as compared to healthy individuals, though the difference was not statistically significant. The total EV concentrations were significantly increased in plasma from cancer patients compared to healthy individuals (p < 0.0001). Positive correlations were observed among the total EV assays (r = 0.6, p < 0.0001). The developed EV assays present non-invasive methods for quantitating both total EVs and EGFr on EVs directly in plasma samples. Next step will be a comprehensive validation using large cohorts of cancer samples to explore the clinical relevance.