Figure 1

Upregulation of connexin-43 in tumor cell-endothelial cell contact areas in vitro and in vivo. (a)-(d) Immunocytofluorescence detecting connexin-43 (Cx43; red) in co-culture of 4T1-GFP cells (green cytoplasmic staining with blue nuclei) and pulmonary microvascular endothelial cells (PMVECs; blue nuclei without cytoplasmic staining). 4T1-GFP and PMVECs were plated together in co-culture and examined under the green fluorescent protein (GFP) channel (4T1 cells), UV-channel (nuclei), and Cy5-channels (Cx43), and shown as an overlay of all channels indicating the location of 4T1-GFP cells (green), nuclei of both cell types (blue), and Cx43 (red). In (a), (b), and (c), upregulation of Cx43 expression (red) is seen at the contact areas between the tumor cells and PMVECs (white arrows). Cells not in a heterologous contact show little expression of Cx43 (yellow arrows). Panel (d) shows an additional overlay of the DIC image to further emphasize the Cx43 intense expression at the areas of contacts (white arrow) between an endothelial cell on the left and a 4T1-GFP cell on the right. (e) Immunohistofluorescence of a lung metastatic tumor section stained for both Cx43 (red) and CD31 (green) with nuclear DAPI staining in blue. Most endothelial cells within the tumor mass (green) showed an increase in Cx43 staining (arrows). (f) Immunohistofluorescence of a fixed mouse lung section after 12 hours of tail vein injection of tumor cells. Autofluorescence of lung tissue in the GFP-channel and the GFP fluorescence of 4T1-GFP cells were assigned the blue pseudocolor for better contrast with the Cy5-channel Cx43 signal (red). Tumor cells can be seen lodged inside pulmonary microvessels. The image clearly indicates the upregulation of Cx43 in both the endothelial cells (white arrow) and the tumor cells within the vessels adhering to the vessel walls (yellow arrow). The scale bar in (a)-(d) is 10 μm and (e), (f) is 25 μm.