Membrane Sensing Peptides -Enhanced SiMoA Platform for the Detection of HER2 on Extracellular Vesicles in Metastatic Breast Cancer Patients

Abstract

Extracellular vesicles (EVs) offer a promising avenue for non-invasive, real-time monitoring of metastatic breast cancer (mBC), but clinical application as a liquid biopsy is hindered by their heterogeneity and low abundance. Here we present a Single Molecule Array (SiMoA) platform enhanced by membrane sensing peptides (MSP) for the highly sensitive detection of HER2 on EV membranes (EVs-HER2) and general EVs population (CD9+) directly from plasma samples of mBC patients. The MSP-based SiMoA assay demonstrated superior sensitivity and specificity compared to conventional antibody-based assays, allowing the detection of lower amounts of EVs and discriminating EVs derived from breast cancer patient-derived organoids (BC-PDO) from healthy control-derived organoids (HC-PDO). Concerning the analysis of EVs in plasma samples (n=49 mBC patients, n=30 healthy controls), we observed significantly lower CD9+ EVs levels in mBC patients relative to healthy controls, a trend consistently confirmed across assays. Notably, EVs-HER2 levels were significantly enriched in HER2-positive patients and correlated with clinical HER2 status assessed by immunohistochemistry. Besides, lower CD9+ EVs levels were associated with poorer clinical outcomes, highlighting the potential prognostic utility of EV quantification. Our findings underscore the potential of MSP-enhanced SiMoA platforms for accurate, minimally invasive monitoring of EVs-HER2 in mBC and for monitoring CD9+ EVs levels to assess disease progression.