Figure 3

Intratumor vessels and metastatic foci demonstrate increased connexin-43. Immunohistofluorescence for connexin-43 (Cx43) of mouse lung sections 12 days after tail vein injection of 4T1-GFP control cells or of cells expressing Cx43 variants C61S, G138R, or Cx43OE. Lung tissue autofluorescence and tumor cell green fluorescent protein (GFP) fluorescence is pseudocolored green color and the Cx43 signal (Cy5-channel) is shown in red. Nuclear staining with DAPI is shown in blue. Each picture is an overlay of these three channels. (a), (b) Lower-magnification images of lung sections containing control 4T1-GFP tumor cells. Cx43 (red) was upregulated in the vessels that contained tumor cells (yellow arrow) while vessels without tumor cells in them had minimal Cx43 signal (white arrows). (c) A metastatic tumor in the lung with enriched Cx43 signal associated with the tumor vasculature. (d) A higher-magnification image of the same tumor highlighting the region with the high Cx43 signal. (e) Another metastatic tumor with (with a higher-magnification image in (f)) demonstrating the Cx43 upregulation within tumor vessels. (g) One vessel containing tumor cells and exhibiting high Cx43 signal (yellow arrow), while a neighboring vessel with a stack of red blood cells (white arrow) but no tumor cells shows no Cx43 reactivity. (h) A vessel from a control lung with non-immune mouse IgG used in place of the primary antibody showing no staining of a vessel (arrow) in which red blood cells can be seen due to their autofluorescence in the GFP channel. The scale bar in (a), (b), (c), and (e) is 100 μm and the scale bar in (d) and (f) (which are to the same scale as (g) and (h)) is 25 μm.