Connexin 45 is a novel suppressor of melanoma metastasis.

The expression spectra of connexin (Cx) isoforms were investigated in three mouse melanoma cell lines: B16-F1 (F1), B16-F10 (F10), and B16-BL6 (BL6). Metastatic potential intensity was higher in the order of F1, F10, and BL6. A remarkable behavior of Cx45 was found among 20 isoforms. The expression level of Cx45 was highest in F1 and lowest in BL6. It was inductively predicted that Cx45 might be a novel suppressor of metastasis. A Cx45-overexpressing BL6 cell line (Cx45 +BL6) was developed and its properties were compared with those of a wild-type cell line of BL6 (W-BL6). Compared to W-BL6, Cx45 +BL6 showed reduced wound healing, Transwell® permeability, and matrix metalloproteinase 9 expression, suggesting the suppression of cellular migration and invasion. The expression of E-cadherin and integrin ß1 in Cx45 +BL6 was also lower than in W-BL6, suggesting reduced cell adhesion. The decrease in cell adhesion was supported by the cell washing-out assay. In contrast, no difference between W-BL6 and Cx45 +BL6 was observed in cell proliferation, suggesting no effect on cell-cycle regulating factors. Finally, an in vivo assay revealed a significant decrease in the number of metastatic colonies of Cx45 +BL6 (176 ± 25/lung) in comparison with those of W-BL6 (252 ± 23/lung) in a mouse model. In conclusion, Cx45 is a novel suppressor of melanoma metastasis. Supplementary Information: The online version contains supplementary material available at 10.1007/s10616-022-00563-x.

Moderate hyperglycemia suppresses melanoma metastasis to liver.

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 pro-inflammatory 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.

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

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-ß1 in the role of Nanog+F10-EVs was analyzed, as TGF-ß1 was a secretory cytokine being affected most intensively by Nanog overexpression. It was suggested to be crucial that the TGF-ß1 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.

Effect of Nanog overexpression on the metastatic potential of a mouse melanoma cell line B16-BL6.

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-ß1 was down-regulated, because TGF-ß1 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.