Extracellular vesicles (EVs) are lipid bilayer-delimited particles, naturally released from a cell. Initially regarded as waste products, nowadays EVs draw interest due to their role in intercellular communications. In breast cancer (BC), EVs play a role in the interplay between cancer cells and tumor microenvironment, and in the preparation of pre-metastatic niche in distant organs. Since they are present in many biofluids, EVs could have an exciting potential as liquid biopsy-derived biomarkers. Aim of this study was to assess EVs plasma levels in patients affected by early and metastatic BC using Single Molecule Array (SiMoA), a digital ELISA able to quantify low concentrations of target molecules.
SiMoA assays were developed based on a bead-immune separation of EVs followed by the direct quantification of the target on top of the magnetic beads. As target we used well know markers of EVs, i.e. CD9 and CD81, while the beads were functionalized to capture CD63, another EV-associated tetraspanin. Patients affected by early (n=56) and metastatic BC (n=44) were prospectively recruited at Istituti Clinici Scientifici Maugeri (Pavia, Italy). A control group of healthy subjects was also enrolled. For each patient a blood sample was collected and plasma was isolated for analysis.
The anti-CD63/anti-CD9 and anti-CD63/anti-CD81 SiMoA assays allowed the analysis of EVs directly in unprocessed plasma with good reproducibility performance. EVs levels were significantly higher in early BC than healthy controls by both assays. Metastatic BC, instead, showed lower levels of EVs as compared to healthy controls and early BC by both assays. By calculating the fraction of CD81-expressing EVs related to the amount of those expressing CD9 we observed that EVs from metastatic BC samples were enriched in CD81 as compared to healthy and early BC.
EVs levels measured by SiMoA increased in early BC and decreased in metastatic BC, suggesting that EVs reflect functional changes occurring during BC progression. As perspective, the identification of more specific markers will help to quantify subpopulation of EVs associated to different BC stages, improving early diagnosis and favoring timely intervention.
Prof. Fabio Corsi.
Has not received any funding.
All authors have declared no conflicts of interest.