Functional Analysis of Macrophages and Mirnas in the Metastasis Suppression By iPS Cell-Derived Extracellular Vesicles
Authors: Katayama Noa , Nakano Misato , Saito Mikako 
Published: December 2024

Extracellular vesicles (EVs) carry cell-derived information and play an important role in intercellular communication. In general, cancer cell-derived EVs have the ability to transmit cancer cell-inducing information to tissue cells to which the cancer cells metastasize, thereby promoting metastasis. However, it has been found that cancer cell-derived EVs may also have an inhibitory effect on metastasis. We also found such an effect in mouse melanoma. EVs derived from metastatic B16F10 cells (F10 cells) showed a metastasis-promoting effect, but contrarily, EVs derived from Nanog⁺F10 cells, a higher metastatic cell line than F10, showed a metastasis inhibition effect in a similar manner as anti-metastasis vaccines. Therefore, we hypothesized that EVs derived from poorly differentiated cells have metastasis- inhibition properties, and focused on iPS-EVs, which are even less differentiated than Nanog⁺F10 cells, and examined their ability to suppress melanoma metastasis. The results showed that iPS-EVs had greater inhibitory effect on melanoma metastasis. This result suggests the involvement of immune cells. Therefore, we analyzed the metastasis inhibitory effect of iPS-EVs in vitro using J774.1 macrophage cultured cells. Fluorescently labeled EVs were added to macrophages, and macrophages incorporating EVs were separated by a cell sorter. Isolated macrophages were co-cultured with Nanog⁺F10 on the top of Transwell inserts; after 22 hours, the number of invasive Nanog⁺F10 cells was counted. The results showed that when cultured with macrophages incorporating iPS-EVs, the number of invasive cells decreased compared to controls, indicating that iPS-EVs induced macrophage activation. In this study, miRNAs in EVs were analyzed to elucidate the molecular mechanisms by which iPS-EVs affected immune cells. Differential analysis revealed that 37 miRNAs were up-regulated and 43 miRNAs were down-regulated, respectively significantly in iPS-EVs compared to Nanog⁺F10-EVs. Target genes were determined using TargetScanMouse_8.0 for each of the four miRNAs with most enhanced expression and the four miRNAs with most suppressed expression. Functional association analysis of these genes and immune function-specific keyword analysis were performed to predict the hub genes that should be important for the metastasis suppression mechanism. The top 30 genes with highest relevance scores were selected using Cytoscape software. Of the 30 genes, 5 genes with high relevance scores and 2 miRNAs targeting those genes were predicted. In the immune function-specific keyword analysis, 17 keywords related to suppression of metastasis were pre-defined for each of the three categories of immune response, immune cells, and inflammatory signals. Sixty one genes were found to contain one or more keywords among the functional information of 669 target genes. The five genes involved in the "hub gene" were selected as candidate hub genes, and the miRNAs targeting them were narrowed down to two. As a result, we predicted nine candidate hub genes and two miRNAs (miR-466f-3p and miR-342-5p) that might markedly regulate the expression of these genes. EVs were isolated from each cell line of iPS, F10, and Nanog⁺F10. RNA was extracted from EVs by ultracentrifugation using CD81 as a marker, and miR-466f-3p was quantified. The results confirmed that miR-466f-3p was internalized more in iPS-EVs than in F10-EVs or Nanog⁺F10-EVs.