Fig 2: Human pluripotent stem cell (hPSC)-derived endothelial progenitors as a model for studying Wnt-mediated barriergenesis.(A) Overview of the endothelial differentiation and Wnt treatment protocol. (B) Immunocytochemistry analysis of CD34 and CD31 expression in D5 endothelial progenitor cells (EPCs) prior to magnetic-activated cell sorting (MACS). Hoechst nuclear counterstain is overlaid in the merged image. Scale bars: 200 µm. (C) Flow cytometry analysis of CD34 and CD31 expression in D5 EPCs prior to MACS. (D) Immunocytochemistry analysis of ß-catenin and GLUT-1 expression in Passage 1 ECs treated with Wnt3a or control. Hoechst nuclear counterstain is overlaid. Arrowheads indicate smooth muscle-like cells (SMLCs). Scale bars: 200 µm. (E) Quantification of the percentage of GLUT-1+ ECs in control- and Wnt3a-treated conditions. Points represent replicate wells from two independent differentiations of the IMR90-4 line, each differentiation indicated with a different color. Bars indicate mean values. p-value: two-way ANOVA.

Fig 3: 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-1a 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-?) 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.

Fig 4: SAMHD1 determines CNDAC sensitivity of primary AML cells. (A) Correlation of SAMHD1 protein levels and CNDAC concentrations that reduce cell viability by 50% (IC50s) in bone-marrow-derived leukaemic blasts derived from 24 therapy-naïve AML patients. Cells were co-immunostained for CD33, CD34, CD45 (surface markers) and intracellular SAMHD1 and the mean fluorescence intensity (MFI) was analysed by flow cytometry. ATP assays were performed in three technical replicates to determine the CNDAC IC50 values. Linear regression analyses were performed using GraphPad Prism. (B) CNDAC IC50 values in bone-marrow-derived leukaemic blasts derived from six therapy-naïve AML patients either treated with VPX virus-like particles (VPX-VLPs, cause SAMHD1 depletion), VPR virus-like particles (VPR-VLPs, negative control), or left untreated. Horizontal lines and error bars indicate means ± SD of three technical replicates. p-values were determined by two-tailed Student’s t-tests (*p < 0.05; **p < 0.01; ***p < 0.001). (C) CNDAC dose-response curves in primary AML cells of one exemplary patient (Patient E) treated with VPX-VLPs, VPR-VLPs or left untreated. IC50 values represent means ± SD of three technical replicates. (D) Representative Western blots indicating SAMHD1 levels in primary AML cells derived from Patient E in response to treatment with VPX-VLPs. (E) CNDAC-triphosphate (CNDAC-TP) levels as determined by LC-MS/MS in primary AML cells derived from Patient E in response to treatment with VPX-VLPs