Anti-Metastatic Function of Extracellular Vesicles Derived from Nanog-Overexpressing Melanoma

Abstract

A metastatic melanoma cell line B16-F10 (F10) was modified to a more undifferentiated state by Nanog overexpression. The produced cell line Nanog+F10 showed a higher metastatic potential than F10. Instead of whole cells, the extracellular vesicles (EVs) therefrom were investigated about their possible role as an autovaccine against metastasis. EVs from Nanog+F10 cells (Nanog+F10-EVs) could suppress the metastasis, contrasting the EVs from less metastatic F10 cells (F10-EVs) enhanced metastasis. The involvement of TGF-β1 in the role of Nanog+F10-EVs was analyzed, as TGF-β1 was a secretory cytokine being affected most intensively by Nanog overexpression. It was suggested to be crucial that the TGF-β1 concentration in Nanog+F10-EVs should be as low as 1.6 pg/μg for its metastasis-suppressive role. In response to Nanog+F10-EVs, immunoreaction was observed in liver, indicating the specific decrease in the number of tumor-promotive CD163-positive macrophages. These indicate a possibility of Nanog+F10-EVs as a novel autovaccine candidate against melanoma metastasis.

Novel Roles of Nanog in Cancer Cells and Their Extracellular Vesicles

“…Therefore, liver was focused on as a predominant target organ, and the number and volume of metastatic colonies were quantitatively analyzed. As a result, those of Nanog + F10 increased 2.5 and 2.4 times, respectively, compared to B16-F10 [ 20 ]. At the same time, in vitro tests were conducted separately to investigate cell proliferation and migration.…”

“…Livers were separated and metastatic colonies were analyzed 2 weeks later. As a result, F10-EV increased metastasis, but Nanog + F10-EV suppressed metastasis [ 20 ].…”

“…Tgf-β1 gene expression level and protein concentration (pg/μg EVs) in those cell lines—and EVs therefrom—were analyzed. The concentration of Tgf-β1 protein in EVs and the effect on metastasis were summarized as F10-EV (3.9 pg/μg EVs, promotive), Nanog + F10-EV (1.6 pg/μg EVs, suppressive), and Tgf-β1 (−) -F10-EV (0.5 pg /μg EVs, suppressive) [ 20 ]. At high concentrations of Tgf-β1, the role of Tgf-β1 was the promotion of metastasis.…”

“…Regarding (ii), we first examined the involvement of macrophages according to the test schedule. As a result of examining the expressions of six types of macrophage markers (pan-macrophage [ CD68 , F4/80 ], M1-type [ CD80 , CD86 ], and M2-type [ CD163 , CD206 ] macrophage markers), it was revealed that only the expression of the tumor-promotive M2-type marker CD163 was significantly reduced [ 20 ].…”

“…There are many studies on cancer cell-derived EVs. However, there are only a few papers [ 20 , 52 , 53 ] reporting metastasis-suppression effects. Among them, only one paper [ 20 ] addresses the Nanog -dependent phenomenon.…”