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. 2020 Aug 1;12(8):2138. doi: 10.3390/cancers12082138

Figure 1.

Figure 1

Cancer–endothelial cell interaction induces expression of endothelial cell (EC) markers in breast cancer cells. (A) Immunofluorescent staining for vascular endothelial cadherin (VE-cadherin) (red) in MCF7-green fluorescent protein (GFP) cells (green), which were added onto a human umbilical vein endothelial cell (HUVEC) monolayer for 24, 48, and 72 h. The protein (B) (full western blot figures (Figure S8) and mRNA (C) levels of VE-cadherin were analyzed in GFP-labeled cancer cells, MCF7-GFP and MDA-MB-231-GFP, which were isolated by fluorescence-activated cell sorting (FACS) after co-culturing with HUVECs for different time points (24, 48, and 72 h). Values are presented as means ± SD (n = 3) (* p ≤ 0.05; ** p ≤ 0.01). (D) The mRNA levels of vascular endothelial growth factor receptor I (VEGFRI), and VEGFRII were determined by qPCR in the isolated MCF7-GFP and MDA-MB-231-GFP cells. Values are presented as means ± SD of the fold changes as compared to the monocultured tumor cells (TCs) (n = 3) (* p ≤ 0.05; ** p ≤ 0.01) (E) The soluble VE-cadherin ectodomains, soluble VE (sVE)-cadherin, shedded by HUVECs into the cell supernatant, were detected in cancer cell lysates with the BV9 antibody by Western blot. sVE-cadherin was not stable in cancer cells and was lost within 24 h (lane R) after the removal of the HUVEC-conditioned medium (full western blot figure. As a positive control, lysates of cancer cells co-cultured with HUVECs were used (two left lanes). (F) Immunofluorescence labeling of VE-cadherin in MCF7 cells treated with HUVEC medium for 48 h showed increased VE-cadherin-positive signal in the nucleus. (G) The positive VE-cadherin staining in the nucleus was biometrically quantified by ImageJ. For the calculation of VE-cadherin-positive signal in the nucleus, we evaluated n = 141 Ctrl cells (gray bar), and n = 130 MCF7-cells treated with HUVEC supernatant (black bar) for 24 h. Means values ± SD are shown (** p ≤ 0.01). (H) MCF7 cells transfected with the VE-cadherin-tdTomato reporter gene were treated with HUVEC culture supernatant, co-cultured with HUVECs (positive control), or monocultured (negative control). The activity of VE-cadherin promoter was quantified by staining the cells with a primary antibody against tdTomato and secondary antibody against tdTomato conjugated with Alexa Fluor 488 (green). Western blots of (B,E) are shown Figure S8, (G) is shown in Figure S9, (C) is shown in Figure S10.