Isolation and identification of exosomes from hypopharyngeal cancer
Exosomes from Fadu cell line was collected, then diluted 1000 times. Concentration and diameter of these exosomes were analyzed, and shoot by electron microscope as shown in Fig. 2a,b. The particle size distribution curve and Table 1 showed that the diameter of 97.4% of diluted exosomes was 117.1nm, and the corresponded concentration was 2.8E+06 particles/ml. The overall particle concentration of exosomes from Fadu cells was 4.2E+10 particles/ml.
Exosomes from FaDu cells mediated induction of CAF-like features in hBMSCs.
To investigate the role of exosomes from FaDu cells in the transition of hBMSCs to CAFs, we collected the exosomes from FaDu cell culture system. Normal BMSCs were co-cultured with collected exosomes and the expression of the CAF-related markers was evaluated on day 7, 14, 21, 28, 35, 42 respectively. Microscopic observation results showed that the number of CAF-like cells was significantly increased in FaDu-exo+BMSC group compared to control (Fig. 3a). The morphology of hBMSCs tended to be spindle or star shaped flat structure, and differentiated into fibroblasts with obvious protein synthesis and secretion activities. As shown in Fig. 3b, western blot results showed that both the CAF-related proteins of hBMSCs, including α-SMA and FAP, were significantly increased in FaDu-exo+BMSC group compared to control. qRT-PCR results confirmed up-regulation of miR-21 level in hBMSCs in the FaDu-exo+BMSC group compared to control (Fig. 3c,d). Flow cytometry results showed that the cell proportion of G1 phase in FaDu-exo+BMSC group significantly decreased, while the cell proportion of G2 phase significantly increased compared to the control (Fig. 3e-g).