Single-Cell Analysis of Cell-Cell Communication of Exosome Components Derived from Melanoma Cells

Abstract

When cancer cell-derived exosomes are transported to distant tissue cells and are incorporated in the cells, they may affect the metastasis of the cancer. They may form a niche that promotes cancer metastasis or, on the contrary, contributes to the formation of a niche that suppresses cancer metastasis. However, it is unclear what should determine the property of the niche. We suspect that the concentration of active components in the exosome should be important. Then we intended to investigate the role of a candidate molecule in the exosome quantitatively. The candidate molecule will be injected directly into a target single-cell in tissue and then a cancer cell will be placed on the target cell. The effects of the candidate molecule may be evaluated from the engraftability and the subsequent proliferation of the placed cancer cell. If the effects are promotive, the engraftability and proliferation properties should be enhanced. Another point is the cell-to-cell communication between the exosome introduced-cells and their neighbor cells. It is still unclear whether the exosome information could be transferred to the cell adjacent to the exosome introduced-cell via intercellular molecular passage such as gap junction. Therefore, the transfer of the injected candidate molecule needs to be analyzed quantitatively. We developed a Nanog overexpressing cell line, Nanog+BL6 to obtain a cancer cell line with enhanced metastatic potential. We also developed a Connexin (Cx) 45 overexpressing cell line, Cx45+BL6 to obtain a cancer cell line with reduced metastatic potential. The metastatic potentials of these cell lines as well as of wild BL6, W-BL6 were tested using mice and their metastatic potentials were confirmed to be in the order of Cx45+BL6, W-BL6, Nanog+BL6, with Nanog+BL6 being highest. Among large number of factors that are potentially regulating metastasis of melanoma, Tgf-β1 was focused in this study. The dual role of TGF-β1 in cancer growth and metastasis, whether suppressive or progressive, was well reviewed and its intensity-dependent role was suspected. In fact, we observed that TGF-β1 was reduced both in Nanog+BL6 and in Cx45+BL6. Such apparently contradictory results might reflect the intensity-dependent mechanisms of TGF-β1. Then we aimed at the intensity-dependent role of TGF-β1 in cell-to-cell communication between the target tissue single-cell and neighbor cells. We isolated exosomes according to the conventional method from the cell lines described above. Briefly, melanoma cells were incubated in an exosome-depleted medium at 37 °C for 48 h in the presence of 5% CO2 and then the supernatant was separated by ultracentrifugation. The exosome fraction was confirmed by a marker Hsc70. The protein concentration was quantified by the BCA method and the presence of the Tgf-β1 protein was confirmed. Based on this result, we will adjust the proper concentration range of Tgf-β1 to be injected in the target single-cell. Then we will clarify the concentration-dependent effects of Tgf-β1 on the engraftability and proliferation properties of melanoma cells. Those results will be presented.