Search for breast cancer proteomic markers in total blood exosomes

Abstract

To improve early detection of cancer, search for tumor markers in biological fluids is of great importance. A significant portion of exosomes is associated with the surface of blood cells, however, the protein spectrum of such exosomes has not been previously studied. The use of total blood exosomes (plasma exosomes and exosomes associated with the surface of blood cells) can not only significantly increase the specificity and sensitivity of existing methods, but also suggest new tumor markers for liquid biopsy. Objective. Search for candidate protein tumor markers of breast cancer by comparing 2D-proteomic maps of total blood exosomes of healthy females (HFs) and breast cancer patients (BCPs). Methods. Exosomes were isolated from plasma and total blood of HFs and BCPs by ultrafiltration followed by ultracentrifugation and were characterized using transmission electron microscopy (TEM) and immunocytochemistry. Protein concentration in exosomes was determined using the NanoOrange Protein Quantitation kit (Invitrogen) commercial kit. Proteomes of exosomes were studied using 2d electrophoresis followed by protein identification by mass spectrometry. Results. Highly purified samples of vesicles from plasma and total blood of no more than 100 nm in size were obtained, on the surface of which markers specific for exosomes were detected by monoclonal antibodies CD9. A comparative analysis of the proteomic maps of exosomal proteins of the HFs and BCPs obtained by 2D-electrophoresis allowed us to establish significant differences in the expression level and protein set in normal and pathological conditions. The 11 proteomic markers of breast cancer were identified by the peptide fingerprint method, of which LRG и у -chain FGB were detected in the composition of exosomes for the first time (according to the Exocarta database). Using MALDI-TOF mass spectrometry, 99 proteins were identified in the exosome preparations of the blood of HFs and BCPs, of which 35% were detected in the composition of the exosomes for the first time (according to the Exocarta database). 17 (53%) proteins associated with breast cancer were detected in total blood exosomes of cancer patients. Conclusion. The results obtained indicate that total blood exosomes are a promising source of diagnostic material for the search for proteomic markers of breast cancer. The identified proteomic tumor markers require further validation.