Figure 2

Connexin-43 expression marks the sites of micrometastases. (a)-(d), (h) Immunohistofluorescence fixed sections of mouse lung after 12 hours of tail vein injection of 4T1-GFP cells or connexin-43 (Cx43) mutant variants C61S, G138R, or Cx43OE. Autofluorescence of lung tissue and 4T1-GFP fluorescence (green fluorescent protein (GFP) channel green color) and Cx43 signal (Cy5-channel red color), and nuclear staining (DAPI channel blue color) were overlaid. Panels (a)-(d) show images of lung sections taken at low magnification. Panel (a) shows the sections from a lung after tail vein injection of control 4T1-GFP tumor cells. Cx43 (red) was upregulated in vessels containing tumor cells (yellow arrow), while vessels not containing tumor cells from the same lung had barely detectable Cx43 signal (white arrows). Lung sections with C61S expressing tumor cells exhibited similar pattern of Cx43 expression (b), whereas those with the dominant-negative G138R expressing cells exhibited decreased staining of Cx43 (c). In contrast, in lung sections with Cx43 overexpressing tumor cells, a significant increase in the number of intravascular tumor cells, an increase in the number of vessels containing adherent tumor cells, and induction of Cx43 was seen (d). Most of the vessels with Cx43 staining appeared to contain tumor cells (yellow arrows). Panels (e)-(h) show higher-magnification images of lung sections containing tumor cells overexpressing Cx43. Panel (e) shows a DIC image of a vessel within the lung containing tumor cells; (f) shows the Cx43 signal in the same vessel; and (g) is an overlay showing the induction of Cx43 in both endothelium and tumor cells in red color. Panel (h) shows another overlay of the same image of the green (tumor cells, autofluoresence of lung tissue and red blood cells), blue (nuclei), and red (Cy5 for Cx43) channels. Scale bars in (c), (d) are 100 μm (the same scale is used in (a) and (b)) and the scale bar in (h) is 25 μm (the same scale is used in (e), (f), and (g)).