Single-Vesicle Molecular Profiling by dSTORM Imaging in a Liquid Biopsy Assay Predicts Early Relapse in Colorectal Cancer.

BACKGROUND AND OBJECTIVES: Colorectal cancer (CRC) is the third most diagnosed tumor type and the second leading cause of cancer-related mortality. Despite recent improvements in the clinical management of CRC patients both before and after surgery, disease recurrence remains common, with an incidence of about 20-30% within 5 years. Current tissue biopsy techniques are invasive and inadequate for assessing tumor heterogeneity or capturing real-time disease dynamics. In contrast, liquid biopsy offers a promising, minimally invasive alternative. This study aimed to evaluate extracellular vesicle (EV)-associated protein markers, detected through super-resolution microscopy, as potential indicators of recurrence in CRC patients. METHODS: We employed a novel liquid biopsy approach based on the super-resolution imaging (dSTORM) of specific protein markers carried by EVs isolated from the plasma of CRC patients. We analyzed combinations of both surface and intravesicular proteins, including EpCAM, PD-L1, CD81, IL-6, and Cyclin D1. RESULTS: Specific combinations of EV-associated markers were able to distinguish patients with recurrence from those without residual disease. Additionally, we observed correlations between some marker profiles and tumor stage or lymph node involvement. No association was found with mismatch repair system status. CONCLUSIONS: To our knowledge, this is the first study to propose the use of EV-bound proteins for recurrence detection in CRC using super-resolution microscopy within a liquid biopsy framework. These findings support the potential of this approach as a non-invasive tool for CRC monitoring.