A role for heterologous gap junctions between melanoma and endothelial cells in metastasis
Akihiko Ito, … , Hiroshi Yamasaki, Hiroshi Nojima
Akihiko Ito, … , Hiroshi Yamasaki, Hiroshi Nojima
Published May 1, 2000
Citation Information: J Clin Invest. 2000;105(9):1189-1197. https://doi.org/10.1172/JCI8257.
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A role for heterologous gap junctions between melanoma and endothelial cells in metastasis

Abstract

F10 and BL6 sublines of B16 mouse melanoma cells are metastatic after intravenous injection, but only BL6 cells are metastatic after subcutaneous injection. We found that connexin (Cx) 26 is upregulated in BL6 cells. To examine gap junction formation, we devised a coculture system, in which an opened vein segment was placed at the bottom of a culture dish and then dye-labeled melanoma cells were seeded onto it. Immunohistochemistry indicated that the vein segment preserved the integrity of the endothelial monolayer. In this system, BL6 cells could transfer dye into endothelial cells but F10 cells could not. Transfection with wild-type Cx26 rendered F10 cells competent for coupling with endothelial cells and as spontaneously metastatic as BL6 cells. Conversely, transfection with a dominant-negative form of Cx26 rendered BL6 cells deficient in coupling and less metastatic. In human melanoma lesions, the level of Cx26 expression was low in melanoma cells residing in the basal layer, but significantly upregulated in melanoma cells invading the dermis. The results suggested that Cx26 plays a role in intravasation and extravasation of tumor cells through heterologous gap junction formation with endothelial cells.

Authors

Akihiko Ito, Fumitaka Katoh, Tatsuki R. Kataoka, Morihito Okada, Noriaki Tsubota, Hideo Asada, Kunihiko Yoshikawa, Sakan Maeda, Yukihiko Kitamura, Hiroshi Yamasaki, Hiroshi Nojima

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Figure 3

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Dye-transfer assay using the coculture composed of melanoma cells and IV...
Dye-transfer assay using the coculture composed of melanoma cells and IVC segments. (a and b) Cx26 expression in melanoma cells after culturing on IVC for 4 hours. Transverse sections of the cocultured tissue containing F10 (A in panel a) or BL6 (B in panel b) cells were stained with anti-Cx26 antibody. Arrows indicate the localization of Cx26 protein on the cell membrane. Also, note that endothelial cells of IVC contain much less Cx26 than Cx43. Bar: 20 μm. (c–f) BCECF-labeled F10 (c and e) or BL6 (d and f) cells were seeded onto an IVC segment and observed from directly above the coculture at 2 (c and d) and 4 (e and f) hours using a microscope equipped for epifluorescence. The relative intensity of fluorescence is displayed by the 256-color spectrum; red indicates a greater intensity than blue. Arrowheads indicate BCECF-labeled F10 and BL6 cells. Bar: 20 μm. (g) The number of cells showing dye coupling with endothelial cells was counted in a sample of 500 BCECF-labeled cells observed after 4 hours of coculture. Data are expressed as the mean of three independent experiments. Bars = SE; SE was sometimes too small to be represented. AP < 0.01 by t test when compared with the value obtained from F10 cells. BP < 0.01 by t test when compared with the value obtained from BL6 cells.