Abstract 3364: Combined enrichment of plasma EpCAM-positive extracellular vesicles (EVs) and circulating tumor cells (CTCs) from a single tube of cancer patient blood samples, for subsequent molecular analysis

• Published in: Cancer research (Chicago, Ill.) Ill.), 2023-04, Vol.83 (7_Supplement), p.3364-3364
• Main Authors: Bregola, Giulia, Masotti, Martina, Mattei, Valentina, Moras, Martina, Ratti, Claudio, Picardo, Francesco, Simonelli, Cecilia, Manaresi, Nicolò, Fontana, Francesca
• Format: Article
• Language: English
• ISSN: 1538-7445; 1538-7445
• DOI: 10.1158/1538-7445.AM2023-3364

Background: EVs are secreted by cells in physiological and pathological conditions and carry selected molecules, such as proteins, DNA and RNA; specific EVs released by neoplastic cells are defined as “tumor-derived extracellular vesicles (tdEVs)”. tdEVs have been demonstrated to be involved in promoting cell growth and survival, shaping the tumor microenvironment and increasing invasiveness and metastatic activity. Thanks to their systemic availability in body fluids and their role in tumor pathogenesis, tdEVs have gained attention in the liquid biopsy area, as a possible biomarker for tumor diagnosis, prognosis and monitoring and also as possible therapeutic targets. Beyond being a target for the capture of CTCs, the Epithelial Cell Adhesion Molecule (EpCAM) was also recently employed for the detection of EVs released by tumors. Here we present a method (for research use only) based on the CellMag™ technology (Menarini Silicon Biosystems, MSB) to carry out parallel enrichment of EVs and CTCs from whole blood through an EpCAM-based magnetic enrichment. Method: Whole blood samples from stage III/IV prostate (n=5) or breast cancer patients (n=6) and healthy donors (n=4) were collected in CellRescue™ tubes (MSB). After centrifugation, EpCAM+ EVs and CTCs were enriched from the plasma and cell fraction respectively, using the CellMag™ Epithelial CTC kit (MSB); CTCs were counted at the CellTracks Analyzer II (MSB). EVs were enriched for qualitative evaluation by Transmission Electron Microscopy (TEM) and for miRNA analysis by Real-time PCR, using the TaqMan™ Advanced miRNA Human Serum/Plasma panel. Results: CTCs counts ranged from 0 to 68; TEM analysis showed a higher density of EpCAM+ extracellular microvesicles (0,1-1µm) in breast cancer patients’ samples (n=2) compared to controls (n=2). Endogenous hsa-miR-16-5p control was detected in all analyzed samples (n=9 patients’ samples, n=2 controls). One control and two patients’ samples were tested for a panel of 188 serum/plasma miRNAs. In both the breast and the prostate cancer patients a higher percentage of miRNAs was found with respect to healthy control (31%, 32% vs. 17%). Among these, several oncogenic miRNAs with diagnostic and prognostic value (such as hsa-miR-125b-5p or hsa-miR-26a-5p) were exclusively found in patients’ samples, and further confirmed to be related to ErbB2 pathway in breast cancer using the miRPathDB 2.0 database, despite the CTCs count was 0 for both these patients’ samples. Conclusi