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Results: 6

1.
Figure 1

Figure 1. From: ?1-integrin via NF-?B signaling is essential for acquisition of invasiveness in a model of radiation treated in situ breast cancer.

Phosphorylated-Akt is up-regulated in clinical DCIS specimens. (A) IHC of p-Akt in human DCIS specimens. Formalin-fixed, paraffin-embedded DCIS sections from 24 patients were stained with p-Akt. Phosphorylated-Akt intensity score: 0 = none, 1 = light, 2 = moderate, 3 = heavy. Bar = 100 μm. (B) Intensity and percentage expression pattern for p-Akt in human DCIS specimens. Phosho-Akt percentage score: 0 = <10%, 1 = 10% to 25%, 2 = 25% to 50%, 3 = >50%. (C) Up-regulation of p-Akt is associated with recurrent disease in human DCIS. DCIS, ductal carcinoma in situ; IHC, immunohistochemistry.

Jin-Min Nam, et al. Breast Cancer Res. 2013;15(4):R60-R60.
2.
Figure 3

Figure 3. From: ?1-integrin via NF-?B signaling is essential for acquisition of invasiveness in a model of radiation treated in situ breast cancer.

IR induces apoptosis in an active Akt-overexpressing model of human DCIS in three-dimensional lrECM. (A) Experimental schema. (B) IR-induced apoptosis was specifically observed in the luminal compartment of MCF10A-Akt structures. (Green = α6-integrin; red = cleaved caspase-3; blue = nuclei) Bar = 50 μm. (C) High content image analysis confirmed an increasing percentage of cells positive for cleaved caspase-3 with increasing IR doses. (n = 200 acini, **, P < 1E-7) (D) Concentric measurements of mean intensity of cleaved caspase-3 showed significantly higher signal in the lumina of irradiated acini, compared to unirradiated controls. Dashed lines indicated edge of the acini. DCIS, ductal carcinoma in situ; IR, ionizing radiation; lrECM, laminin-rich extracellular matrix.

Jin-Min Nam, et al. Breast Cancer Res. 2013;15(4):R60-R60.
3.
Figure 2

Figure 2. From: ?1-integrin via NF-?B signaling is essential for acquisition of invasiveness in a model of radiation treated in situ breast cancer.

Phosphorylated-Akt up-regulated MCF10A cells form DCIS-like structures in three-dimensional lrECM cultures and in vivo. (A) MCF10A cells form acinar-like structures with hollow lumina when propagated in three-dimensional lrECM. When p-Akt is overexpressed (MCF10A-Akt), the colonies are significantly larger with cells filling the lumina. Phase-contrast micrographs and IF images stained with α6-integrin or p-Akt are shown. Bar = 10 μm. (B) The average colony size is increased in MCF10A-Akt compared to MCF10A. (C) Experimental schema of in vivo study. The MCF10A-Akt cells were injected intraductally into the mouse mammary duct and subsequently generated DCIS-like lesions. (D) H & E, IHC (β1-integrin, p-Akt and cleaved caspase-3) and IF (Ki-67) staining of intraductal xenografts. H & E stained image from the xenograft is almost identical to clinical human DCIS. Bar = 100 μm. DCIS, ductal carcinoma in situ; IF, immunofluorescence; IHC, immunohistochemistry; lrECM, laminin-rich extracellular matrix.

Jin-Min Nam, et al. Breast Cancer Res. 2013;15(4):R60-R60.
4.
Figure 6

Figure 6. From: ?1-integrin via NF-?B signaling is essential for acquisition of invasiveness in a model of radiation treated in situ breast cancer.

Invasive phenotype emerged in a sub-population of irradiated MCF10A-Akt cells are associated with nuclear translocation of NF-κB. (A) On Day 30 cultures, immunoblotting of nuclear fraction shows up-regulated nuclear translocation of NF-κB p65 in the 8 Gy IR cultures. The intensities of NF-κB were normalized with nuclear protein, Histone H1 (**, P < 0.01, n = 4). (B) IF images of confocal microscopy show β1-integrin (green), NF-κB (red) and nuclei (blue). Bar = 50 μm. (C) The binding of NF-κB to the β1-integrin promoter region is up-regulated in MCF10A-Akt cells that survived after exposure to radiation (WT, wild-type, Mt, mutated oligonucleotide; n = 3, *, P < 0.05). (D-E) The NF-κB inhibitor, JSH-23, was added from day 0 of the second three-dimensional cultures. IR-induced chemoinvasion activity, which was inhibited by NF-κB inhibitor, JSH-23. Bar = 50 μm. IF, immunofluorescence; IR, ionizing radiation; NF-κB, nuclear factor-kappaB; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling.

Jin-Min Nam, et al. Breast Cancer Res. 2013;15(4):R60-R60.
5.
Figure 5

Figure 5. From: ?1-integrin via NF-?B signaling is essential for acquisition of invasiveness in a model of radiation treated in situ breast cancer.

β1-integrin signaling is targeted to suppress invasive recurrence post-IR in MCF10A-Akt cells in three-dimensional lrECM. (A) On Day 30 cultures, up-regulated α5β1-integrin and down-regulated E-cadherin expression observed on the post-IR cultures. Columns, mean intensity (n = 3; **, P < 0.01). (B) Up-regulation of FN and EDA+FN was observed in culture medium of cells post-IR. Columns, mean intensity (n = 5; *, P < 0.05; ***, P < 0.001). (C) Phase-contrast images. IR-induced invasive phenotype was abrogated by AIIB2 compared to IgG. The antibodies were added from day 0 of the second three-dimensional cultures. Bar = 50 μm. (D) Apoptosis was measured by TUNEL-positive cells in AIIB2-treated cultures -/+ IR (mean = 18.1% ± 3.9, P < 0.01). (E-F) Matrigel chemoinvasion was significantly increased in the surviving cells post-IR, and inhibited by β1-integrin (AIIB2, five-fold, P < 0.01) or α5-integrin (P1D6, two-fold, P < 0.05) inhibitory antibodies. DCIS, ductal carcinoma in situ; FN, fibronectin; IgG, immunoglobulin G; IR, ionizing radiation; lrECM, laminin-rich extracellular matrix; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling.

Jin-Min Nam, et al. Breast Cancer Res. 2013;15(4):R60-R60.
6.
Figure 4

Figure 4. From: ?1-integrin via NF-?B signaling is essential for acquisition of invasiveness in a model of radiation treated in situ breast cancer.

An invasive phenotype emerged from a sub-population of cells surviving post-IR in three-dimensional lrECM. (A) Experimental schema of the recurrence model. At Day 12, cultures were exposed to Sham or 8 Gy IR. On Day 15, the colonies were taken out of three-dimensional lrECM, dissociated to make single cells, and expanded on two dimensional. Single cells were re-plated on three-dimensional lrECM and propagated until Day 30 (12 additional days). (B) Phase-contrast micrographs show that a distinct phenotype emerged by Day 30 of culture. Bar = 50 μm. IF images show α6-integrin or β1-integrin (green). Bar = 50 μm. (C) Invasive activity of MCF10A-Akt cells post-IR was quantified using invasion chambers. Graphical representation of the invasive cell numbers were normalized with control, non-irradiated cultures (n = 3; **, P < 0.01). (D) Gelatin zymography shows that MMP-9 secretion was increased in culture medium of IR-treated MCF10A-Akt. (E) Matrix degradation activity was confirmed by fluorescently labeled DQ-gelatin matrix. Degraded gelatin is shown in green (22% ± 7 invasive cells versus 3% ± 1; n = 3; **, P < 0.01). DCIS, ductal carcinoma in situ; IF, immunofluorescence; IR, ionizing radiation; lrECM, laminin-rich extracellular matrix; MMP-9, matrix metalloproteinase-9.

Jin-Min Nam, et al. Breast Cancer Res. 2013;15(4):R60-R60.

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