From Cell Lines to Patients: Dissecting the Proteomic Landscape of Exosomes at Breast Cancer

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Abstract
Breast cancer (BC) is the most common cancer among women worldwide, therefore, the efforts of many scientists are aimed at finding effective biomarkers of this disease. It is known that exosomes are nanosized extracellular vesicles (EVs), which are released from various cell types, including cancer cells. Exosomes are directly involved in governing physiological and pathological processes of an organism by horizontal transfer of functional molecules (proteins, microRNA, etc.) from producing to receiving cells. Since the diagnosis and treatment of BC were improved substantially with exosomes, in this study, we isolated breast carcinoma cells‐derived exosomes, primary endoteliocytes-derived exosomes and blood exosomes from BC patients at 1st stage of disease and investigated their proteomic profile. Exosomes were isolated from the samples by ultrafiltration and ultracentrifugation, followed by mass spectrometric and bioinformatics analysis of the data. Exosomal nature of vesicles has been verified using transmission electron microscopy and flow cytometry. Exosome proteins secreted by MCF-7 and BT-474 cells were found to form 4 clusters, three of which enhanced the malignant potential of cancer cells, and one cluster coincided with a cluster of HUVEC-derived exosome proteins. Despite the different ensembles of proteins in exosomes from the MCF-7 and BT-474 lines, these portions of these proteins are involved in similar biological pathways. Comparison analysis reveals more BC-associated proteins were found in the exosomal content from the blood of BCPs than in the content of conditioned medium of cells mimicking the corresponding cancer subtype (89% and 82% for luminal A BC and MCF-7 cells and 86% and 75% for triple-positive BC and BT-474 cells, respectively). Thus, tumor-associated proteins should be sought not in exosomes secreted by cell lines, but in the composition of blood exosomes of cancer patients, while the contribution of endotheliocyte exosomes to the total pool of blood exosomes can be neglected.

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