Fig 2: Human adipose tissue-derived microvascular endothelial cells (HAMVECs) exhibit morphological, molecular, and functional hallmarks of endothelium.HAMVECs were compared with endothelial cell (EC) controls representative of the predominant endothelial specializations, namely human umbilical vein ECs (HUVECs; macrovascular, venous endothelium), human coronary artery ECs (HCAECs; macrovascular, arterial endothelium), and human dermal microvascular ECs (HDMVECs; microvascular endothelium). a Cobblestone-like morphology of endothelium. Scale bars represent 200 µm. b Abundance of transcripts encoding CD31 (gene: PECAM1), vascular endothelial (VE)-cadherin (gene: CDH5), and von Willebrand Factor (vWF; gene: VWF). Glyceraldehyde-3-phosphate dehydrogenase (gene: GAPDH) was used as a loading control. Dashed line depicts a mean difference of zero; and dotted lines is the equivalence margin (?) used for the two one-sided test for equivalence. Values represent the mean ± 90% confidence interval; mRNA, messenger ribonucleic acid; and Cq, the quantification cycle. c Expression and localization of the corresponding endothelial proteins. Scale bars represent 25 µm. d Internalization of acetylated low-density lipoprotein (AcLDL). Solid and dashed lines represent ECs cultured in the presence and absence of Alexa Fluor 488-conjugated AcLDL, respectively. Values represent mean ± standard deviation. e Capillary-like tubulogenesis by ECs. Scale bars represent 200 µm. All experiments were performed in biological triplicate, using cells derived from three different donors (n = 3 biologically independent samples).

Fig 3: Snail induces stemness properties and endothelium generation of breast cancer cells through Sox2.a qRT-PCR analysis of relative mRNA expression of Sox2 in MCF-7 and ZR75-1 cells stably infected with lentivirus carrying EV or Snail. b qRT-PCR analysis of relative mRNA expression of Sox2 in MCF-7 and ZR75-1 cells transfected with control siRNA or Snail siRNA. c Representative FACS of MCF-7 cells infected with the indicated plasmids CD44-FITC and CD24-PE. Statistical analysis of CD24-/CD44+ rates is shown in the right panel. Results shown are mean ± SD of three independent experiments. d Mammosphere assay of MCF-7 cells infected with indicated plasmids. Graphs show the relative number of mammospheres. Results shown are mean ± SD of three independent experiments. Scale bar, 20 µm. e qRT-PCR analysis of relative mRNA expression of endothelium markers VEGFR2, CD31, and CD105 in MCF-7 and ZR75-1 cells stably infected with the indicated plasmids. f FACS analysis of MCF-7 cells infected with indicated plasmids using CD31-FITC antibodies. Statistical analysis of CD31+ rates is shown in the right panel. Results shown are mean ± SD of three independent experiments. g Representative confocal images of MCF-7 cells infected with the indicated plasmids labelled with endothelium markers CD31 and VWF. The nuclei were stained with DAPI (blue). Scale bar, 20 µm. Graphs show the percentage of endothelium marker-positive cells. Results shown are mean ± SD of three independent experiments. h Lineage tracing of MCF-7 cells stably infected with EV or Snail using CD31pro-GFP, CD105pro-GFP, or CD144pro-GFP. Scale bar, 10 µm. Graphs show the percentage of GFP-positive cells. Results shown are mean ± SD of three independent experiments. i Tube-forming assays and DiI-AcLDL uptake assays with Snail-copGFP-expressing or copGFP-expressing MCF-7 cells. Statistical analysis of tube length is shown in the right panel. Results shown are mean ± SD of three independent experiments. Scale bar, 100 µm. Red: DiI-AcLDL staining. *P < 0.05, **P < 0.01.

Fig 4: Snail induces endothelium generation of breast cancer cells in vitro.a Relative mRNA expression of endothelium markers in MCF-7 cells stably infected with lentivirus carrying EV or Snail was determined by qRT-PCR assay. b Representative confocal images of MCF-7 cells stably infected with lentivirus carrying EV or Snail labelled with endothelium markers CD31 or vWF. The nuclei were stained with DAPI (blue). Scale bar, 20 µm. Graphs show the percentage of endothelium marker-positive cells. Results shown are mean ± SD of three independent experiments. c Representative FACS with the indicated antibodies in MCF-7 and ZR75-1 cells stably infected with EV or Snail. Statistical analyses of CD31-positive or CD144-positive rates are shown in the right panel. Results shown are mean ± SD of three independent experiments. d Endothelium lineage tracing of MCF-7 and ZR75-1 cells stably infected with EV or Snail using CD31 promoter-driven GFP (CD31pro-GFP), CD105 promoter-driven GFP (CD105pro-GFP), or CD144 promoter-driven GFP (CD144pro-GFP). Scale bar, 5 µm. Statistical analysis of GFP-positive rates is shown in the right panel. Results shown are mean ± SD of three independent experiments. e Tube-forming assays and DiI-AcLDL uptake assays with Snail-copGFP-expressing or copGFP-expressing MCF-7 and ZR75-1 cells. Statistical analysis of tube length is shown in the right panel. Results shown are mean ± SD of three independent experiments. Scale bar, 100 µm. Red: DiI-AcLDL staining. *P < 0.05, **P < 0.01.

Fig 5: Sox2 alone fails to induce endothelium generation in breast cancer cells.a qRT-PCR analysis of endothelium markers CD144, CD31, and CD105 in EV-stably or Sox2-stably expressing MCF-7 and ZR75-1 cells cultured with or without VEGF. b FACS analysis with CD31-FITC in EV-stably or Sox2-stably expressing MCF-7 and ZR75-1 cells cultured with or without VEGF. Statistical analysis of CD31+ rates is shown in the right panel. Results shown are mean ± SD of three independent experiments. c IF assays of MCF-7 cells infected with lentivirus carrying Sox2 or EV and cultured with or without VEGF using endothelium markers CD31 and VWF. The nuclei were stained with DAPI (blue). Scale bar, 20 µm. The graphs show the percentage of endothelium marker-positive cells. Results shown are mean ± SD of three independent experiments. d Lineage tracing of EV-stably or Sox2-stably expressing MCF-7 cells with CD31pro-GFP, CD105pro-GFP, or CD144pro-GFP cultured with or without VEGF. Scale bar, 10 µm. Graphs show percentage of GFP-positive cells. Results shown are mean ± SD of three independent experiments. e Tube-forming assays and DiI-AcLDL uptake assays with copGFP-overexpressing or Sox2-copGFP-overexpressimg MCF-7 cells cultured with or without VEGF. Statistical analysis of tube length is shown in the right panel. Results shown are mean ± SD of three independent experiments. Scale bar, 100 µm. Red: DiI-AcLDL staining. *P < 0.05, **P < 0.01.