Published online Jun 7, 2014. doi: 10.3748/wjg.v20.i21.6651
Revised: February 8, 2014
Accepted: March 4, 2014
Published online: June 7, 2014
AIM: 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/108 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.
CONCLUSION: 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.
Core tip: The traditional extraction assay of exosomes is usually complicated and time consuming. In this manuscript, we investigate a novel assay based on a nanomaterial to extract exosomes more quickly and efficiently compared with the traditional method. A hepatoma-associated protein, tissue transglutaminase 2, was first found to exist in the exosome of the human liver cancer cell line Huh-7. This protein can be secreted through an exosome-mediated non-classical secretion pathway and is a potential tumor marker.