Abstract
Since the discovery of the senescence-associated secretory phenotype, the role of senescent hepatic stellate cells (HSCs) in hepatocellular carcinoma (HCC) development has gained increasing attention. Similar to cytokines, extracellular vesicles (EVs) are essential for intercellular communication. However, the function of EVs derived from senescent HSCs in HCC progression has not been extensively studied. The aims of the present study were to characterize the EVs derived from senescent HSCs and determine their role in the tumor microenvironment. Cellular senescence was induced in human hepatic stellate cells (HHSteCs) with various concentrations of etoposide. Induction was confirmed using EdU staining and 53BP1 and p21 immunostaining. EVs were isolated by ultracentrifugation and analyzed by nanoparticle tracking analysis. Multiplex immunoassays were used to compare the levels of growth factors secreted from hepatoma cell lines and macrophage cells pretreated with EVs derived from senescent HHSteCs (senescent EVs) with those pretreated with EVs derived from normal cultured HHSteCs (normal EVs). Treatment with 25 µM etoposide for 3 days was the most effective at inducing senescence in HHSteCs. This finding was confirmed by induction of irreversible cell-cycle arrest, upregulation of 53BP1 and p21 expression, and increased SA-β-gal staining. Senescent HHSteCs released increased quantities of EV particles compared with normally cultured HHSteCs. Multiplex analysis revealed that there was no difference between hepatoma cell lines treated with normal EVs and those treated with senescent EVs in growth factor secretion. In contrast, the secretion of epidermal growth factor (EGF) was increased by macrophage cells treated with senescent EVs compared with those treated with normal EVs. Furthermore, senescent EVs did not affect the viability of hepatoma cells but increased the viability of hepatoma cells co-cultured with macrophage cells. In conclusion, the release of EVs from senescent HSCs was higher compared with normal HSCs. Furthermore, senescent EVs promoted HCC development by upregulating EGF secretion from macrophages.