Identification Of Glioma Derived Exosome And Its Proteomic Analysis

Gliomas (tumors arising from glial cells, such as astrocytes, oligodendrocytes, ependymal cells, and choroid plexus cells) are the most common primary tumors of the central nervous system (CNS) with a incidence rate of the whole neural tumor about 40%-50%. The prognosis for most patients harboring gliomas remains grim despite advances in surgery, radiation therapy, and chemotherapy, because the feature of this kind of tumor is high-invaded and relapse. For example, a patient with a glioblastoma multiforme still faces a mean survival time of less than 1 year. However, recent years have seen an exponential increase in tumor immunologic and molecular biological knowledge about gliomas that may lead to the promise of cancer immunotherapeutic strategies, including cellular vaccines, molecular vaccines, antitumor immunogenetherapy and so on. But because many disadvantage and defections of these strategies have been found in the process of researches, the application of cancer immunotherapy is largely limited on clinic cure.The term"exosome"was first used in 1987 to describe a kind of microvesicles released during the study process of reticulocyte maturation conducted by Johnstone et al. After that lots of researches related to exosomes have revealed that there are many other kinds of cell can secrete exosome except for reticulocyte, including blood cells, epithelial cells, neurocytes, tumor cells (such as melanoma, lung carcinoma, mesothelioma, breast cancer, ovarian cancer andleukemic cell) and so on. Western blot, flow cytometric analysis and MALDI-TOF-MS have identified many cellular proteins in exosomes derived from various cellular sources, most of which are related to immunological function, such as MHC-I, MHC-II, HSPs, costimulatory molecules (i.e. CD86, ICAM-1) and so on. In addition, tumor cell derived exosomes contain tumor- associated antigens. Except for the biological function of eliminating obsolete proteins during the reticulocyte maturation, exosomes primarily mediate intercellular communication by transferring material among cells, which enables exosomes to play a role in modulating immune responses. Exosome secretion could be a regulated process participating in the transfer of molecules such as MCH-I and MHC-II between immune cells, which makes it possible to active T cells and amplify the immune response. DCs and tumor derived exosome also can elicit efficient antitumor response. Several phase I clinical trials have been successfully finished and no side-effects have been found. Exosomes represent a cell free vaccine which may be a little bit easier to define, store and manipulate compared to a cell-based vaccine. The feasibility and the safety of exosome assessed in phase I clinical trials are encouraging for the future development of exosomes in cancer immunotherapy. However, the research about glioma derived exosomes hasn't been reported. So we conduct this trial to prove that glioma just like most cells can also secrete exosome-like vesicles, and initially analyze the protein composition by SDS-PAGE and Coomassie Blue staining. Finally,we identify some immunological molecule by western-blot in order to offer a theoretical foundation for its utilization in anti-glioma immunotherapy.Method performed as follow:1. Glioma cells had been derived from tow patients bearing astroglioma identified by pathological diagnosis, one of which was grade II and the other grade III. Before the exosome was collected, glioma cells were traditionally cultured in 2 weeks. Then the purification of exosome from culture supernatants was proceeded by differential ultracentri- fugation. The appearance characterization of exosome was observed under electron microscopy.2. The protein composition of exosome was initially analyzed by 10%SDS-PAGE and Coomassie Blue staining, and the data was obtained from ImageQuant TL software.3. For confirming the hypothesis of hsp70, western-blotting was performed following one-dimensional SDS-PAGE.Obtained results:1. Glioma cell just like most cells can also secrete exosome-like vesicles. The morphologic characterization of exosome is: spherical or oval-shaped membrane vesicles, syngeneous in size, 60-150 nm in diameter with the average about 100nm.2. The data obtained from SDS-PAGE and software shows that both cell lines derived exosome contains similar protein quantity and composition. The appearance molecular weights (MW) of all these proteins are among 270kD and 30kD, which is consistent withother cells derived exosomes that have been reported.3. Hsp70 is detected to present in glioma derived exosome by western-blotting.Conclusions:1. It is confirmed that glioma cells also have the ability to secrete exosome-like vesicles, and the extraction purified by our method is exosome, not apoptosis bodies or other subcellular organelles.2. All the data obtained from this experiment and other articles that have reported reveals a common constituent between glioma cell derived exoosme and other cells.3. The presence of hsp70 reveals some properties of immunomodulation for glioma derived exosome.The novel points:1. For the first time the exosome is proved to be secreted by glioma, and our trial facilitates coming researches of glioma derived exosome.2. The proteomic analysis is initially performed, and hsp70 is identified to offer a theoretical foundation for its utilization in anti-glioma immunotherapy.