Mouse Melanoma Cells Exosomes Induced MEF Cells Expression Invade Ability

BackgroundExosomes is a structure of a double layer of phospholipid released from molecules a class of membrane vesicles in eukaryotic cells,, it will transmit to signal from one cell to another cell in the molecular level. Components of exosomes contains the information carried by the main source of the cells, it determine exosomes’ characteristics. Tumor-derived cell exosomes have complete mRNAs, microRNAs, proteins, so tumor cells-dericed exosomes carry tumor information.Studies have shown that tumor can "cross-talk" with the surrounding environment so affect the surrounding normal stromal cells. Mesenchymal cell phenotype changes and release metalloproteinases and chemokines in order to reduce cell adhesion, destroy the basement membrane and pro-angiogenic generation in favor of the invasion and metastasis of tumor cells. In the process that tumor cells change normal cells, is there tumor cell derived exosomes communication?Mouse melanoma B16-F10 cells and their homologous MEF cells were used to study. We study on the effect of B16-F10 cells exosomes on invasion and migration ability of MEF cells and verify that tumor cells transfer invasive exosomes communication to a normal cell. From exosomes point of view, we interpreted the phenomenon of tumor cell invasion and metastasis. It may provide a new way of thinking methods to control tumor invasion and metastasis. Purpose1. Determination and structural characterization Preparation of B16-F10 cells in exosomes.2. Analysis of the invasion of B16-F10 cells exosomes for the MEF to verify tumor cells into normal cells passed invasive exosomes communication. Methods1. 100000 × g ultracentrifugation was adopted to saparete exosomes from the B16-F10 cell culture supernatant, nanoparticle tracking analysis was used to meature exosomes size. Exosomes was glutaraldehyde, embedded in epoxy resin, after lead citrate stained, ultrastructure was observed under transmission exosomes electron microscopy, Western-blot analysis was to determin protein TSG101, TYRP2 expressions of exosomes.2. Mouse embryonic fibroblasts(MEF) were purified from C57 mouse embryos in 13.5 days. The different concentrations exosomes and MEF cells were co-cultured under dynamic real-time label-free cell analysis technology(RTCA) recording dynamic changes of MEF cell invasion and migration ability. Collecting co-culture 48 h MEF cells, MMP-2, MMP-9, E-cadherin mRNA expression was detected by qPCR, MMP-2, MMP-9, E-cadherin protein expression levels was determined by western-blot. Using CFSE fluorescent to dye exosomes, confocal fluorescence microscopy observed fusion of exosomes and MEF cells. Results1. B16-F10 cells exosomes had a diameter of about mainly in the 90 ~ 430 nm, the average diameter of 169.7 ±4.62 nm, and had a structure lipid vesicle bilayer membrane, and TSG101, TYRP2 were expressed.2. B16-F10 cells in exosomes and MEF cells were co-cultured 72 h, MEF cell in exosomes concentration of 0.6g / L group was most invasive. MMP-2, MMP-9 protein and mRNA expression was stronger in MEF cells in co-cultured group than that in the control group(p <0.05), E-cadherin protein and RNA expression was lower(p <0.05). Migration of two groups has no significant change(p> 0.05). After CFSE-exosomes and MEF cells were co-cultured 6h, green fluorescence was observed between the MEF cells CFSE-exosomes, occasionally watching green fluorescence in MEF cells. After co-cultured 12 h, green fluorescence was appeared in the visible part of MEF cells. After co-cultured 18 h, green fluorescence appeared nearly half of the MEF cells and when cultured for 48 h, the green fluorescence were seen in almost all of the MEF cells. Conclusion1. Exosomes that mainly in the 90 ~ 430 nm, the average particle diameter of 169.7 ± 4.62 nm and had a lipid bilayer membranes and protein expression profiles TSG101, TYRP2 can be prepared by using 100000×g ultracentrifugation method.2.B16-F10 cells exosomes induce MEF cells invasive, which suggested that tumor cells transmitted invasive exosomes communicationin to normal cells between cancer cells and normal cells.