Supplementary Methods, Figures 1 - 8, Tables 1 - 2 from VEGF-Mediated Angiogenesis Links EMT-Induced Cancer Stemness to Tumor Initiation

PDF file - 7604K, Supplemental Figure S1 depicts the increased sensitivity of mesenchymal MTdeltaECad cells to salinomycin as compared to epithelial MTflECad cells. Supplemental Figure S2 shows comparable numbers of mesenchymal MTdeltaECad cells and epithelial MTflECad cells injected i.v. trapped in in lungs and no difference in cell proliferation but higher apoptosis of epithelial MTflECad cells in lung outgrowing lung metastasis. Supplemental Figure S3 presents the assessment of VEGF receptor expression, blood vessel maturation and functionality, and tumor cell proliferation and apoptosis in primary tumors formed by mesenchymal MTdeltaECad cells as compared to tumors of epithelial MTflECad cells. Supplemental Figure S4 shows the upregulated expression of pro-angiogenic genes during EMT by gene ontology and direct gene expression analysis. Supplemental Figure S5 shows an efficient siRNA-mediated ablation of VEGF-A expression in mesenchymal MTdeltaECad cells and that the ablation of VEGF-A expression, treatment with the VEGF receptor inhibitor PTK787 or the addition of recombinant VEGF-A do not affect cell proliferation and mammosphere growth of MTdeltaECad cells or MTflECad cells. Supplemental Figure S6 shows that the forced expression of VEGF-A in epithelial MTflECad cells modestly increases the cells' tumorigenicity and significantly induces tumor microvessel density and vessel functionality, it reduces apoptosis but does not affect tumor hypoxia and cell proliferation. Supplemental Figure S7 shows that other VEGF family members are upregulated in their expression during an EMT in vitro and in vivo, yet that they do not affect tumorigenicity of mesenchymal cancer cells. Supplemental Figure S8 demonstrates that VEGF-A in the supernatant of cultured mesenchymal MTdeltaECad cells induces proliferation of human umbilical vein endothelial cells (HUVEC). Supplemental Table I summarizes the flow cytometry analysis of cell surface markers in NMuMG normal murine mammary gland cell line and in the Py2T and MTflECad/MTdeltaECad murine breast cancer cell lines before and after EMT and cultured on plastic or as spheroids. Supplemental Table II presents the differences in cytokine expression between epithelial MTflECad cells and mesenchymal MTdeltaECad cells as determined by cytokine protein array analysis. Supplemental Methods provide experimental details of the general experimental procedures and reagents used in the main text and of the specific methods used to generate the supplemental data.