Isolation and characterization of circulating exosomes from blood in brain tumors

Exosomes (EXs) are crucial in mediating intercellular communication in physiological and pathological conditions. Accordingly, tumor-secreted EXs play an important role in regulating tumor malignancy. Tumor-derived exosomes contain proteins, mRNAs, and miRNAs, which can be delivered between different types of cells and even transferred to distant locations to influence the biological activities of tumors, such as proliferation, invasion and metastasis (1). Clinically, tumor-derived EXs may represent a target for therapeutic intervention and for the development of early diagnostic biomarkers (2). In our laboratory, we have focused on the isolation, morphological analyses, determination of particle concentration, stability and preparations’ purity of EXs isolated by blood samples obtained from patients with cancer before and after ablative surgery. Besides, mass spectrometry (MS) has been used to study their proteomic profile. Blood samples were collected after one week, after one month, and after three months of surgery. The size-exclusion chromatography (SEC) was used to isolate EXs from a small amount of human plasma (1 mL), with most particles being present in fractions 7–10, while the bulk of the plasma proteins were present in fractions 15–20. We show that this method is fast and reliable, as the population of EXs isolated is homogeneous in terms of size, morphology and concentration by Nanoparticle Tracking Analysis (NTA), and Transmission Electron Microscopy (TEM). The presence of exosomal and nonexosomal populations within small EXs was confirmed using standard method (WB) for CD63, CD81, or CD9 markers. Western blot and mass spectrometry analysis was also performed to study the presence and the levels of expression of molecular chaperones Hsp60 and Hsp27. We demonstrated that Hsp60 levels no significant changes in tumor-derived EXs before and one week from brain surgery, but a small decrease was observed one month after surgery. The purpose of this study is provided evidences on the roles of EXs in brain cancer, focusing on the importance of exosomal Hsp as therapeutic targets and biomarkers for early cancer detection.