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.

Nanog, a marker and regulator of the undifferentiated state in embryonic stem cells were anticipated to be an effective enhancer of cancer metastasis. We have developed a Nanog overexpressing mouse melanoma cell line B16-BL6 (BL6). BL6 was well recognized as a cell line with a high metastatic potential. In vitro tests revealed the enhancement of cell proliferation, wound healing activity, and matrix metalloproteinase 9 (MMP9) activity. Nanog-induced up- or down-regulated genes were comprehensively analyzed by transcriptome sequencing using Nanog+BL6 and wild-type BL6. Principally, up-regulated genes were involved in vesicle-aided glucose transport and oxidative phosphorylation, while down-regulated genes were associated with immunosuppression and apoptosis. A marked finding was that TGF-β1 was down-regulated, because TGF-β1 has been well discussed about its suppressive/progressive dual role in cancer. In vivo test showed that the number and volume of metastatic colonies of BL6 to lung were as high as 115 colonies/lung and 5.6 mm3/lung. Under this condition, Nanog overexpression caused a progressive effect (150 colonies/lung, p = 0.25; 9.2 mm3/lung, p = 0.13) rather than a suppressive effect on the metastasis. In this study, the effectiveness of Nanog overexpression in enhancing the metastatic potential of melanoma cell lines has been demonstrated for the first time.

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.

The metastasis of various cancers is promoted by hyperglycemia. In contrast, melanoma and colorectal cancer seemed to be exceptional. We confirmed that the metastasis of melanoma B16-F10 could be suppressed by hyperglycemia. It was attractive from the prognostic point of view of the prevention of metastasis, though the problem of the risk of diabetes remained. Then, the effect of moderate hyperglycemic condition was investigated using a pre-diabetic model mouse (GKKO mouse). The metastasis of B16-F10 cells to liver was focused and the number and volume of metastatic colonies in liver were analyzed. The medians of the number of metastatic colonies in GKKO mice were 0.57-fold (p=0.06) compared to control mice. Analysis of macrophage markers revealed upregulation of CD86, a tumor-suppressive M1-type marker, and downregulation of CD206, a tumor-promotive M2-type marker. A tendency of upregulation of Cxcl10, a proinflammatory cytokine was also observed. Regarding cellular activities of B16-F10, migration activity and invasion activity were reduced by moderate hyperglycemia. In conclusion, metastasis of B16-F10 cells to liver could be suppressed by moderate hyperglycemia without the risk of diabetes. This information should contribute to dietary planning during prognosis.

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