(341c) Profiling of Pan-Cancer Extracellular Vesicle Biomarkers Using Surface Plasmon Resonance Biosensors for Early-Stage Cancer Screening

Cancer is the second leading cause of death in the United States contributing to more than 8.8 million deaths annually. However, over 90% of cancer-related casualties are due to late diagnoses when metastasis has taken place to secondary sites and immunotherapeutic treatment towards the primary tumor is ineffective. Therefore, the development of diagnostic technologies applicable to early stages of cancer is key to improve patient survival. Identifying highly selective, specific, and non-invasive diagnostic markers is urgently needed for cancer screening at initial stages. One promising approach to screening for early-stage, curable cancer types use biomarkers in circulating extracellular vesicles (EVs). EVs are cell-derived sub-micron vesicles comprising surface proteins. With recent improved methods for EV characterization and isolation, new opportunities have emerged to study EVs as biomarkers and mediators of human diseases, including cancer.

Here, we discuss the development of a pan-cancer high-throughput surface plasmon resonance (SPR)-based platform to detect and quantify EV protein biomarkers for early-stage cancer screening with high sensitivity and specificity. For our proof-of-concept model, we use a cancerous mouse cell line (RAW 264.7) to detect and quantify EVs via common EV markers. RAW 264.7 EVs are then transfected and prompted to express disease- and immunotherapeutic-specific biomarkers HER-2 and PD-L1. EVs were isolated via ultrafiltration, with size and concentration determined via nanoparticle tracking analysis (NTA) and atomic force microscopy (AFM). Using a custom-built SPR instrument, successful detection, and quantification of EVs was performed targeting tetraspanin biomarkers (CD63 and CD81) and specific transmembrane disease biomarkers (HER-2 and PD-L1) without the need for a secondary antibody. The limit of detection of our bioassay was determined as 5.1×10⁶ EVs/mL which is several orders of magnitude below the concentration of EVs found in blood (10⁹ - 10¹⁰ EVs/mL), demonstrating its suitability for clinical analysis. Our approach also exhibits high specificity, differentiating between transfected HER-2(+) and PD-L1(+) EVs from RAW 264.7 EVs. We further show our preliminary work developing a functional ultra-low fouling zwitterionic surface with poly-carboxybetaine acrylamide (p-CBAA) brushes for EV detection with clinical undiluted serum samples. In the context of EV detection using SPR biosensors, the use of zwitterionic polymer chemistries has not yet been reported, and their application with EVs is an innovative branch in the field of biosensors and surface chemistry. Our optical biosensor has the potential to be applied as a platform to develop pan-cancer profiling panels for patient selection and early management of cancer.