Fig 1: VM formed by TNBC cells expresses vascular endothelial- and stem cell marker-related genes. Vascular channels of MDA-MB-231 cells in Matrigel were fixed with paraformaldehyde and stained with (A) anti-CD44-FITC antibody, (B) anti-VEGFc antibody, (C) anti-HIF-1α antibody, and (D) anti-CD34 antibody. Alexa Fluor 594 conjugated secondary antibody (B and C) and FITC secondary antibody (D) were used. Fluorescence images (x20 magnification) were captured and corresponding phase contrast images are shown. The experiments were repeated 3 times and representative images are shown. (E-H) Human Angiogenesis Antibody Array: VM cell supernatant analysis of angiogenesis proteins revealed increased levels compared to non-VM cell culture supernatant. (F) VM supernatant on day 7 isolated from MDA-MB-231 cells along with the matching cell culture supernatant of (E) MDA-MB-231 cells were tested on a human angiogenesis protein array. Higher amount of proteins (VEGF, ANG, IL-8, TIMP1, TIMP2, MCP-1 and IFN-γ) based on gray levels or brightness values in VM supernatants (red boxes) as compared with control in (E). The array template is shown in the right panel (G). (H) Quantification of protein expression data, shown as fold change. TNBC, triple-negative breast cancer; VM, vasculogenic mimicry; GRO, growth regulated oncogene; ANG, Angiopoietin; bFGF, basic fibroblast growth factor; EGF, epidermal growth factor; PLGF, placenta growth factor; IFN, interferon; IGF, insulin-like growth factor; IL, interleukin; MCP, monocyte chemoattractant protein; PDGF, platelet-derived growth factor; TGF, transforming growth factor; ENA-78 (CXCL5), epithelial-neutrophil activating peptide; RANTES, regulated on activation, normal T cell expressed and secreted; TIMP, tissue inhibitor of matrix metalloproteinase; VEGF, vascular endothelial growth factor. Cytokines array signal intensities (fold) were plotted (means ± SD), *P<0.05 vs. control.