(A) Phase-contrast microscopy of day 10 primary T-MS generated from samples listed in Table 1. Scale bars: 100 μm. (B) RT-PCR analysis of IL-6, Bmi-1, CK-5, CD44, Oct-4, and β₂μ mRNA in T-MS and in tumor tissues from which T-MS had been obtained. (C) Day 10 primary N-MS and T-MS were obtained from the same patient (see Table 1). RT-PCR analysis of IL-6, Bmi-1, CK-5, BCRP-1, and CD133 and quantitation of IL-6, Bmi-1, and CK-5 mRNA, first normalized onto β₂μ mRNA and then expressed as a ratio of N-MS to T-MS. *P = 0.031, Mann-Whitney test. NA, not available. (D) Breast carcinoma tissues from patients affected by basal-like or ductal breast carcinoma (see Table 2) were subjected to RT-PCR analysis. Shown is the ratio of IL-6 to β₂μ mRNA. ^#P = 0.001, Mann-Whitney test.

(A) RT-PCR analysis of Notch-3 mRNA in day 10 primary N-MS in the presence or absence of IL-6 (10 ng/ml) and in T-MS in the presence or absence of anti–IL-6 (1.5 μg/ml) for 24 hours. (B) RT-PCR analysis of Notch-3 mRNA in MCF-7 cells cultured in the presence or absence of IL-6 (10 ng/ml) and in MCF-7(S) in the presence or absence of anti–IL-6 (1.5 μg/ml) or IL-6 (10 ng/ml) for 24 hours. (C) Day 7 MCF-7(S) generated from MCF-7 cells infected with a pSuper­Puro retroviral vector encoding a Notch-3-specific (N3) or control (CT) shRNA (sh) in the presence or absence of IL-6 (10 ng/ml). Phase-contrast microscopy analysis, MCF-7(S) size distribution (n denotes number of spheroids counted per sample), and Western blot analysis of Notch-3 and β-actin protein levels. *P = 0.034, Monte Carlo χ²test. β₂μ was assessed as quantitative control for RT-PCR analysis. Scale bars: 100 μm.

(A) RT-PCR analysis of CA-IX mRNA in day 10 primary N-MS cultured in the presence or absence of IL-6 (10 ng/ml) for 24 hours. (B) RT-PCR analysis of CA-IX mRNA and Western blot analysis of CA-IX (phosphorylated ERK, total ERK, and β-actin protein levels shown in Figure 4B) in MCF-7 cells exposed to IL-6 (10 ng/ml for 24 hours) in the presence or absence of UO-126 (20 μM) or DMSO, in shNotch-3 and control MCF-7 cells exposed to IL-6 (10 ng/ml for 24 hours), in MCF-7 cells transiently transfected with pNICD3/pEMPTY vector (1 μg), and in MCF-7 cells transfected with pNICD3 and coadministered with UO-126 (20 μM) or DMSO for 24 hours. (C) RT-PCR analysis of IL-6, Notch-3, and CA-IX mRNA in MCF-7 cells exposed to low oxygen (<0.1% O₂) or 100 μM DFX and in N-MS and T-MS exposed to 50 μM DFX for 48 hours. (D) RT-PCR analysis of Notch-3 and CA-IX mRNA in MCF-7 cells in the presence or absence of anti–IL-6 (1.5 μg/ml) and in shNotch-3 and control-infected MCF-7 cells exposed to DFX (100 μM for 24 hours), Western blot analysis of Notch-3 and β-actin protein. β₂μ was assessed as quantitative control for RT-PCR analysis.

(A) Boyden invasion chamber assay in MCF-7 cells, in shNotch-3 and control MCF-7 cells, and in MCF-7 cells transiently transfected with scrambled or CA-IX siRNA (1 μg, 72 hours’ pre-exposure), in the presence or absence of IL-6 (10 ng/ml for 24 hours). n = 5 per group. *P = 0.0001, **P = 0.0001, ^#P = 0.0001, ANOVA. Inset: RT-PCR analysis of CA-IX mRNA in cells administered scrambled or CA-IX siRNA. (B) Boyden chamber invasion assay of day 7 secondary T-MS in the presence or absence of anti-IL6 (1.5 μg/ml for 24 h) or transfected with IL-6 or CA-IX or scrambled siRNA (1 μg, 72 hours’ pre-exposure). n = 3 per group. *P = 0.003, **P = 0.042, ^ΧP = 0.0001, ANOVA. RT-PCR analysis of IL-6 and CA-IX mRNA is shown. (C) Boyden chamber invasion assay of day 7 secondary N-MS exposed to IL-6 (10 ng/ml for 24 hours) in the presence or absence of anti-N3 (1.5 μg/ml for 24 hours) or scrambled or CA-IX siRNA (1 μg, 72 hours’ pre-exposure). n = 3 per group. ^#P = 0.036, ^##P = 0.037, ^ΧP = 0.0001, ANOVA. RT-PCR analysis of IL-6, CA-IX, and β₂μ mRNA is shown. (D) Zymographic analysis of MMP-2 activity in shNotch-3 and control MCF-7 cells in the presence or absence of IL-6 (10 ng/ml for 24 hours) and in MCF-7 cells exposed to IL-6 (10 ng/ml for 24 hours) transfected with CA-IX or scrambled siRNA (1 μg, 72 hours’ pre-exposure). n = 3 per group. *P = 0.032, ^#P = 0.025, ^##P = 0.027, ANOVA.

(A) RT-PCR analysis of IL-6 mRNA in MCF-7 cells and N-MS exposed to IL-6 (10 ng/ml) for 24 hours. (B) MCF-7 cells exposed to IL-6 (10 ng/ml for 24 hours) and assessed at various times (1 or 2 weeks) after the withdrawal of the cytokine. RT-PCR analysis of IL-6, Notch-3, and CA-IX mRNA and Boyden chamber invasion assay (n = 5) and zymographic analysis (n = 3) of MMP-2 activity (24 hours). *P = 0.010, ^#P = 0.012, ^##P = 0.002, ANOVA with post-hoc test for multiple comparisons. (C) MCF-7 cells exposed to IL-6 (10 ng/ml) for 24 hours and assessed 2 weeks after cytokine withdrawal in the presence or absence of anti–IL-6 (1.5 μg/ml) for 24 hours. RT-PCR analysis of IL-6, Notch-3, and CA-IX mRNA and Boyden chamber invasion assay (24 hours). n = 5 per group. **P = 0.004, ANOVA with post-hoc test for multiple comparisons. (D) RT-PCR analysis of IL-6 and CA-IX mRNA, Western blot analysis of Notch-3 and β-actin protein level, and Boyden chamber invasion assay (24 hours) in shNotch-3 and control MCF-7 cells either untreated or exposed to IL-6 for 24 hours and assessed 2 weeks after cytokine withdrawal (n = 5). ^ΧP = 0.001, ANOVA with post-hoc test for multiple comparisons. (E) Boyden chamber invasion assay (24 hours) and RT-PCR analysis of CA-IX mRNA in cells as in C and D transfected with CA-IX or scrambled siRNA (1 μg, 48 hours’ pre-exposure). n = 5 per group. ^ΧΧP = 0.002, ANOVA. β₂μ was assessed as quantitative control for RT-PCR analysis.

(A) Day 7 secondary T-MS, generated from primary T-MS in the presence or absence of the mAb anti–IL-6, which blocks the IL-6 receptor/ligand interaction (1.5 μg/ml). Phase-contrast microscopy analysis and number of MS per well (n = 3). *P = 0.029, **P = 0.042, ANOVA. (B) Phase-contrast microscopy analysis and number of MS per well in day 7 secondary T-MS and N-MS generated from primary MS in the presence or absence of IL-6 (10 ng/ml) and anti–IL-6 (1.5 μg/ml), respectively (n = 3). ^ΧP = 0.027, ^ΧΧP = 0.020, ^#P = 0.048; ^##P = 0.035, ANOVA plus post-hoc tests adjusted for multiple comparisons. (C) RT-PCR analysis of IL-6 mRNA in MCF-7 and day 2 MCF-7–derived spheroids and MCF-7(S) generated in the presence or absence of anti–IL-6 (1.5 μg/ml). Also shown are phase-contrast microscopy analysis and MCF-7(S) size distribution. n denotes the number of spheroids counted for each sample. °P = 0.02, Monte Carlo χ² test. β₂μ was assessed as quantitative control for RT-PCR analysis. Scale bars: 100 μm.

(A) Day 10 primary N-MS and T-MS cultured in the presence or absence of IL-6 (10 ng/ml) or anti–IL-6 (1.5 μg/ml) for 24 hours. RT-PCR analysis of Jagged-1 mRNA. (B) RT-PCR analysis of Jagged-1 mRNA and Western blot analysis of phosphorylated ERK and total ERK protein in MCF-7 cells exposed to IL-6 (10 ng/ml) in the presence or absence of the MEK1 inhibitor UO-126 (20 μM) or DMSO for 24 hours, in shNotch-3 and control MCF-7 cells exposed to IL-6 (10 ng/ml for 24 hours), in MCF-7 cells transfected with 1 μg pNICD3 or empty control vector (pEMPTY) for 24 hours, and in MCF-7 cells transfected with pNICD3 in the presence or absence of UO-126 (20 μM) or DMSO. (C) Day 7 MCF-7(S) generated from MCF-7 cells transfected with Jagged-1–specific or scrambled (JAG1 and SCR, respectively) siRNA (1 μg, 72 hours’ pre-exposure). RT-PCR analysis of Jagged-1 mRNA, phase-contrast microscopy analysis, and MCF-7(S) size distribution. ^#P = 0.001, Monte Carlo χ² test. (D) Day 7 secondary N-MS generated in the presence of IL-6 (10 ng/ml) and in the presence or absence of anti-N3 mAb, which blocks Notch-3 activity (1.5 μg/ml). Shown are phase-contrast microscopy and N-MS size distribution (n denotes number of spheroids counted per sample). ^ΧP = 0.039, **P = 0.009, Monte Carlo χ² plus post-hoc tests adjusted for multiple comparisons. (E) RT-PCR analysis of Jagged-1 and Notch-3 mRNA (ratio to β₂μ) in basal-like or ductal carcinoma tissues. ^##P = 0.005, ^ΧΧP = 0.042, Mann-Whitney test. β₂μ was assessed as quantitative control for RT-PCR; β-actin was assessed as quantitative control for Western blot. Scale bars: 100 μm.

(A) MCF-7 cells in the presence or absence of DFX (100 μM for 48 hours) and in the presence or absence of anti–IL-6 (1.5 μg/ml for 24 hours), with transient transfection with the CA-IX–specific or scrambled siRNA (1 μg for 72 hours), and shNotch-3 and control MCF-7 cells. Shown are Western blot analysis of Notch-3 and β-actin protein and cell death analysis and RT-PCR analysis of Notch-3 and CA-IX mRNA (n = 3). *P = 0.017, **P = 0.008, ***P = 0.002, ANOVA. (B) Cell death analysis and RT-PCR analysis of Notch-3 and CA-IX mRNA in day 7 secondary T-MS exposed to 50 μM DFX for 48 hours in the presence or absence of anti–IL-6 (1.5 μg/ml for 48 hours) or anti-N3 (1.5 μg/ml for 48 hours) or transfected with CA-IX or scrambled siRNA (1 μg for 72 hours). n = 3 per group. ^#P = 0.022, ^##P = 0.025, ^###P = 0.044, ANOVA. (C) RT-PCR analysis and representative IHC analysis of CA-IX protein expression of breast carcinoma tissues from patients affected by basal-like or ductal breast carcinoma (see Table 2). Data are shown as CA-IX/β₂μ mRNA ratio. ^ΧP = 0.002, Mann-Whitney test. β₂μ was assessed as quantitative control for RT-PCR analysis. Scale bar: 100 μm.

(A) HYPO-7, a MCF-7–derived cell population, in the presence of IL-6 or scrambled siRNA (1 μg, 48 hours’ pre-exposure). RT-PCR analysis of IL-6, Notch-3, and CA-IX mRNA; cell death analysis in the presence of DFX (600 μM for 48 hours); and Boyden chamber invasion assay (n = 5) and zymographic analysis (n = 3) of MMP-2 activity (24 hours). *P = 0.042, **P = 0.0001, ***P = 0.015, ANOVA. (B) HYPO-7 cells in the presence of CA-IX or scrambled siRNA (1 μg, 48 hours’ pre-exposure). RT-PCR analysis of IL-6, Notch-3, and CA-IX mRNA; cell death analysis in the presence of DFX (600 μM for 48 hours); and Boyden chamber invasion assay (n = 5) and zymographic analysis (n = 3) of MMP-2 activity (24 hours). ^#P = 0.034, ^##P = 0.0001, ^###P = 0.018, ANOVA. (C) HYPO-7 cells, MCF-7(S), and T-MS exposed to anti–IL-6 (1.5 μg/ml) for 24 hours. RT-PCR analysis of IL-6 mRNA level. β₂μ was assessed as quantitative control for RT-PCR analysis.