title: Novel method for extracting exosomes of hepatocellular carcinoma cells.
abstract: OBJECTIVE
  To develop a novel method for the rapid and efficient extraction of exosomes secreted by tumor cells.
  
  
  METHODS
  Unlike the traditional extraction method, the supernatants of cell cultures were concentrated, and the exosomes were isolated promptly and effectively using a novel nanomaterial called ExoQuick. Coomassie brilliant blue staining was used for protein quantification, and the morphology of the exosomes extracted by both methods was visualized by transmission electron microscopy. Exosome marker proteins were detected by Western blot analysis. Two potential hepatoma-associated proteins, tissue transglutaminase 2 (TGM2) and annexin A2, were analyzed.
  
  
  RESULTS
  The exosomes separated by the new extraction assay based on the nanomaterial were disc-shaped, intact vesicles with lipid bilayer membranes. They were approximately 30-100 nm in diameter, which is similar to the diameter of exosomes isolated by the traditional method. The protein concentration of exosomes extracted by the new method was approximately 780 μg/10(8) cells, and therefore, it was 19 times higher than that of exosomes extracted in the traditional manner. There were differences between the total proteins of Huh-7 cells and the exosomal proteins. Typical exosome proteins, such as the transmembrane protein CD63 and heat shock protein 70, were confirmed. Two potential hepatoma-associated proteins were also identified. TGM2 was first found to exist in the exosomes of human liver cancer cells, but annexin A2 was not secreted into exosomes.
  
  
  CONCLUSIONS
  The new extraction method based on the nanomaterial is quick and efficient. The cancer-associated protein TGM2 can be secreted through an exosome-mediated non-classical secretion pathway, and it may be a valuable tumor marker.
authors.affiliation: Lin Zhu, Third School of Clinical Medicine, Southern Medical University, Guangzhou 510515, Guangdong Province, China.
authors.forename: Lin
authors.initials: L
authors.lastname: Zhu
authors.affiliation: Lin Zhu, Third School of Clinical Medicine, Southern Medical University, Guangzhou 510515, Guangdong Province, China.
authors.forename: Xiu-Hua
authors.initials: XH
authors.lastname: Qu
authors.affiliation: Lin Zhu, Third School of Clinical Medicine, Southern Medical University, Guangzhou 510515, Guangdong Province, China.
authors.forename: Yu-Lin
authors.initials: YL
authors.lastname: Sun
authors.affiliation: Lin Zhu, Third School of Clinical Medicine, Southern Medical University, Guangzhou 510515, Guangdong Province, China.
authors.forename: Yang-Ming
authors.initials: YM
authors.lastname: Qian
authors.affiliation: Lin Zhu, Third School of Clinical Medicine, Southern Medical University, Guangzhou 510515, Guangdong Province, China.
authors.forename: Xiao-Hang
authors.initials: XH
authors.lastname: Zhao
mesh_terms: D017306:Annexin A2 / Q000378:metabolism; D014408:Biomarkers, Tumor / Q000378:metabolism; D006528:Carcinoma, Hepatocellular / Q000378:metabolism*; D045744:Cell Line, Tumor; D003584:Cytological Techniques; D055354:Exosomes / Q000378:metabolism*; D019204:GTP-Binding Proteins / Q000378:metabolism; D006801:Humans; D008051:Lipid Bilayers / Q000737:chemistry; D008113:Liver Neoplasms / Q000378:metabolism*; D046529:Microscopy, Electron, Transmission; D000091345:Protein Glutamine gamma Glutamyltransferase 2; D011503:Transglutaminases / Q000378:metabolism; D014407:Tumor Cells, Cultured; D014461:Ultracentrifugation
chemical_list: C472822:ANXA2 protein, human; D017306:Annexin A2; D014408:Biomarkers, Tumor; D008051:Lipid Bilayers; C000717608:TGM2 protein, human; D000091345:Protein Glutamine gamma Glutamyltransferase 2; D011503:Transglutaminases; D019204:GTP-Binding Proteins
keywords: Annexin A2; Exosome; Membrane vesicles; Nanomaterials; Tissue transglutaminase 2