PMC

Panel A and B. B-CSC and R-CSC grew in spheres and were characterized as CD24⁻/CD44⁺ or CD24⁻/CD105⁺ cells, respectively (A). B-CSC and R-CSC lacked cytokeratin (CK) that was acquired when cultured in epithelial differentiating conditions (EPITH. DIFF.) for 14 days (D14), as compared with basal condition (D0) (B). Panel C. B-CSC and R-CSC cultured for 14 days (D14) in endothelial differentiating conditions under hypoxia (ENDOTH. DIFF.) acquired the endothelial-specific markers CD31, VEGFR2, VE-cadherin (VE-CAD) and vWF and the ability to organize into capillary-like structures. Original magnification: immunofluorescence staining: x400; tubulogenesis: x200. Nuclei were counterstained with Hoechst dye.

Panel A and B. Effect of 1–10 μM Sunitinib (S1-S10) and of 25–250 μg/ml Bevacizumab (B25-B250) on proliferation (A) and apoptosis (B) of B-CSC and R-CSC before (Undiff, black columns) and after the endothelial differentiation (Diff., white columns). Panel C and D. The effect of Bevacizumab and Sunitinib on endothelial differentiated CSC was compared to that on total breast tumor-derived endothelial cells (BTEC) or on normal endothelial cells (HUVEC). Data are mean ± SD of five different experiments (A and B) or three different experiments (C and D). Student's t test was performed: **= p < 0.001, *= p < 0.05 drug treated vs CTL cells.

Tumors were generated by subcutaneous implant of B-CSC carrying an empty vector (Ctl) or a sFlk1 vector (sFLK1) (n = 8 per experimental group). Selected animals carrying Ctl tumors were treated daily with Sunitinib (SUN) from day 4. Panel A. Reduction of tumor volume and vascularization in sFLK1 and SUN tumors in respect to Ctl. Vessels quantification is the mean ± SD erythrocyte containing structures/field in at least 10 fields per tumor. Student's t test was performed: **= p < 0.001, *= p < 0.05 vs Ctl. Panel B. Representative micrographs of tumor sections stained with hematoxylin and eosin (upper panels) or with Masson's trichromic reaction (blu: connective, red: cells, yellow: erythrocytes; lower panels). The star indicated necrotic areas and the arrows vessels. Original magnification: H/H: 100x; Trichromic: 200x.

Panel A. Both sFlk1 releasing tumors (sFLK1) and Sunitinib treated tumors (SUN) showed reduction of murine vessels in respect to control tumors (Ctl), evaluated as murine β2-microglobuline-positive structures by immunohistochemistry (n = 8 per experimental group). Murine vessels only represented a part of total vessels in Ctl and sFLK but not in SUN tumors. Data are the mean ± SD of β2-microglobulin positive structures containing erythrocytes (β2⁺)/field or total erythrocytes containing structures in at least 10 fields per tumor. Student's t test was performed: **= p < 0.001, *= p < 0.05 vs Ctl; $$= p < 0.001, $= p < 0.05 vs β2⁺/field. Panel B. SUN tumors and not sFLK1 tumors showed reduction of human vessels, evaluated as structures co-expressing vWF and human HLA Class I (vWF⁺/HLA⁺) by immunofluorescence. Data are the mean ± SD of vWF⁺/HLA⁺ structures/field in at least 10 fields per tumor. Student's t test was performed: **= p < 0.001 vs Ctl. Panel C. Representative micrographs of Ctl, sFLK1 and SUN tumor sections showing murine β2-microglobulin positive structures (upper panels). Representative micrographs of Ctl, sFLK1 and SUN tumor sections showing positive immunofluorescence staining for vWF (red) /human HLA class-I (green) positive structures, for human CD31 (hCD31, red) or for vWF (red)/αSMA (green). Original magnification: x200 (upper panels) and x400 (lower panels).

Panel A. Quantitative RT-PCR analysis showing the increase of HIF-1 alpha but not of HIF-2 alpha by B-CSC incubated for 3 hours in endothelial-differentiating condition in hypoxia in the presence and absence of 1 μM Sunitinib (S1) or 100 μg/ml Bevacizumab (B100). Data were normalized to GAPDH mRNA and to 1 for time 0, expressed as relative quantification (RQ) and are mean ± SD of three different experiments. Student's t test was performed: **= p < 0.001 vs Basal; $= p < 0.001 vs Hypoxia. Panel B. Western blot micrograph and densitometric analysis of HIF-1 alpha expression. Data, shown as arbitrary units, are representative of three different experiments and were normalized to vinculin expression. Student's t test was performed: **= p < 0.001 vs Basal; $= p < 0.001 vs CTL. Panel C and D. Expression of endothelial differentiation markers by control B-CSC infected with a scramble shRNA (shCTL) and in B-CSC lacking HIF-1 alpha (shHIF-1α), HIF-2 alpha (shHIF-2α) or both (shHIF-1α+shHIF-2α). In the representative FACS analyses, the gray filled area shows binding of the specific antibody and the dark line the isotypic control (C). In the histogram, the percentage of expression is reported (D). Data are mean ± SD of five different experiments. Student's t test was performed: **= p < 0.001 vs shCTL. Panel E. Quantitative RT-PCR analysis showing the reduction of the stem-cell associated markers Oct4-A, Vimentin and Nanog in shCTL cells after endothelial differentiation in respect to basal condition, but not in endothelial differentiated shHIF-1α+shHIF-2α cells. Data were normalized to GAPDH mRNA and to 1 for time 0 and expressed as relative quantification (RQ). Data are mean ± SD of three different experiments. Student's t test was performed: **= p < 0.001 vs Basal.

Panel A. Representative FACS analyses showing the expression of endothelial differentiation markers by B-CSC infected with lentiviruses carrying an empty vector (Ctl) or the soluble VEGF trap sFlk1 (sFLK1 cells) or treated with an anti-VEGFR2 and an anti-VEGFR1 neutralizing Abs (anti-VR2 and anti-VR1). The grey area shows binding of the specific antibody and the dark line the isotypic control. Panel B. Western blot micrograph showing the presence of the soluble VEGFR2 (sVR2) in the supernatant of cells expressing the soluble VEGF trap sFlk1 (sFLK1 cells) but not an empty vector (Ctl). Panel C. Quantitative RT-PCR analyses showing the acquisition of the expression of VR2 and TIE-2 after 14 days of endothelial differentiation by Ctl, sFLK1 B-CSC and by B-CSC incubated with the anti-VR1, but not by B-CSC incubated with the anti-VR2, during differentiation process. Data were normalized to GAPDH mRNA and to 1 for undifferentiated CSC (Basal) and expressed as relative quantification (RQ). Data are mean ± SD of three different experiments. ANOVA with Newmann-Keuls' multicomparison test was performed: *= p < 0.05 and **= p < 0.001 vs CTL; $ = p < 0.05 and $$ = p < 0.001 vs Basal. Panel D. Quantitative RT-PCR analysis showing the expression of the stem cell markers (Oct4-A, Vimentin and Nanog) by CSC (Basal) and by B-CSC differentiated in the presence of anti-VR2 antibody (anti-VR2). The expression of stem cell markers were significantly decreased in B-CSC differentiated into endothelial cells and in B-CSC differentiated into endothelial cells in the presence of anti-VEGFR1 neutralizing Ab (anti-VR1) and in sFLK1 cells compared with basal condition. Data were normalized to GAPDH mRNA and to 1 for undifferentiated CSC (Basal) and expressed as relative quantification (RQ). Data are mean ± SD of three different experiments. ANOVA with Newmann-Keuls' multicomparison test was performed: *= p < 0.05 and **= p < 0.001 vs CTL; $ = p < 0.05 and $$ = p < 0.001 vs Basal. Panel E. The percentage of VEGFR2⁺ cells at different time points during endothelial differentiation was assessed in control B-CSC cells (CTL), or in the sFLK1 cells or in cells treated with 1 μM Sunitinib (S1) or with an anti-VEGFR2 neutralizing Ab (anti-VR2). Data are mean ± SD of three different experiments. Student's t test was performed: **p < 0.001 vs CTL. Panel F. VEGFR2 Tyr951 phosphorylation was detected in B-CSC cells expressing an empty vector (CTL), or the soluble VEGF trap sFlk1 (sFLK1 cells) by Western blot analysis of cell lysates immunoprecipitated with an anti-VEGFR2 Ab. VEGFR2 phosphorylation levels are expressed as the ratio of phosphorylated VEGFR2 to total VEGFR2. Data are representative of three different experiments.

Panel A and B. 1 μM Sunitinib (S1), but not 100 μg/ml Bevacizumab (B100), impaired the hypoxia-mediated endothelial differentiation of B-CSC (A) and R-CSC (B) as shown by the lack of acquisition of endothelial specific markers. In the representative FACS analyses, the grey area shows binding of the specific antibody and the dark line the isotypic control. In the lower histogram, the percentage of expression is reported. Data are mean ± SD of five different experiments. Student's t test was performed: **= p < 0.001, *= p < 0.05 vs CTL. Panel C. Quantitative RT-PCR analysis showing the acquisition of the expression of endothelial markers VEGFR2 (VR2) and TIE-2 by B-CSC after endothelial differentiation (CTL) in respect to undifferentiated B-CSC (Basal). Sunitinib (1 μM, S1) but not Bevacizumab (100 μg/ml, B100) abrogated VEGFR2 and TIE-2 mRNA expression. Total breast tumor-derived endothelial cells (BTEC) were used as positive control of differentiation. Data were normalized to GAPDH mRNA and to 1 for undifferentiated CSC (Basal) and expressed as relative quantification (RQ). Data are mean ± SD of three different experiments. ANOVA with Newmann-Keuls' multicomparison test was performed: *= p < 0.05 and **= p < 0.001 vs CTL; $ = p < 0.05 and $$= p < 0.001 vs Basal. Panel D. Quantitative RT-PCR analysis showing the reduction of Oct4-A and Nanog by CSC differentiated into endothelial cells (CTL) in respect to undifferentiated CSC (Basal). CSC differentiated in the presence of 1 μM Sunitinib (S1), but not of 100 μg/ml Bevacizumab (B100) maintained these markers. Data were normalized to GAPDH mRNA and to 1 for Basal and expressed as relative quantification (RQ). Data are mean ± SD of three different experiments. ANOVA with Newmann-Keuls' multicomparison test was performed: *= p < 0.05 and **= p < 0.001 vs CTL; $ = p < 0.05 and $$ = p < 0.001 vs Basal. Panel E. Effect of Bevacizumab and Sunitinib on endothelial-differentiated CSC organization into capillary-like structures. Quantitative evaluation and representative micrographs show the formation of capillary-like structures by control cells (CTL) and by cells treated with 1 μM Sunitinib (S1) or 100 μg/ml Bevacizumab (B100). Data are expressed as the mean ± SD of the length of capillary-like structures, evaluated by the computer analysis system in arbitrary units (a.u.) in at least 10 different fields. Four different experiments per group were carried out in duplicate. Original magnification x200. Student's t test was performed: **= p < 0.001 vs CTL.