Nanobody-based capture of extracellular vesicles as a diagnostic strategy for prostate cancer

Abstract

Extracellular vesicles (EVs) are small membrane-enclosed entities released from different cell types. EVs are promising biomarkers for the diagnosis and monitoring of various malignancies, including

prostate cancer1. From the heavy-chain-only VHH library, we selected three different nanobodies tar- geting EVs from the PC3 cell line. Nanobody genes were cloned into a vector and fused with the GFP

protein. Production was optimized in E. coli cells, where nanobodies mainly accumulated in inclusion bodies. They were purified from these fractions. SDS-PAGE confirmed the purity of the protein samples obtained. The selected nanobodies were immobilized on a polymethylacrylate polymer, which was

used for the isolation of EVs from prostate cancer cell lines (PC3) and epithelial cells of prostate (RWPE- 1) cell lines. Their ability to discriminate between EVs from PC3 and RWPE-1 cells was evaluated using

fluorescence-activated cell sorting (FACS). EV samples were tested for protein and lipid content.

This work highlights the potential of nanobodies as selective tools for the recognition of EVs. The ob- tained candidates may serve as a basis for novel diagnostic strategies targeting tumor-derived vesicles

in prostate cancer. Furthermore, the integration of nanobody-based capture with standardized EVs characterization could provide robust platforms for clinical biomarker discovery. Future studies will focus on validating binding specificity in complex biological samples and exploring their application in liquid biopsy approaches.