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1.
Figure 4

Figure 4. Jagged1-expressing tumor cells have a growth advantage in the bone microenvironment via Notch-dependent crosstalk with osteoblasts. From: Tumor-derived Jagged1 Promotes Osteolytic Bone Metastasis of Breast Cancer by Engaging Notch Signaling in Bone Cells.

(A) Coculture between control or JAG1 OE SCP28 tumor cells and MC3T3-E1 osteoblasts transfected with a Notch reporter and treated with DMSO or MRK-003.
(B) qRT-PCR mRNA expression levels of indicated Notch target genes and TGFβ1 in MC3T3-E1 osteoblasts that were FACS-separated from cocultures in each experimental group. *p < 0.05, **p < 0.01, ***p < 0.001.
(C) Representative images of cocultures from each experimental group. White boxes indicate areas shown at higher magnification in the middle row. Tumor cells cultured alone are shown in the bottom row. Scale bar = 200 μM.
(D) Quantification of tumor cells from cocultures with MC3T3-E1 from each experimental group by luficerase assay. *p = 0.01, **p = 0.007.
(E) Diameter of tumor colonies from cocultures of each experimental group. ***p < 10−7.
(F) Quantification of tumor cells cultured alone from each experimental group.
Data in the figure represent average ± SD. p-values were based on Student’s t-test. See also Figure S4 and Table S1.

Nilay Sethi, et al. Cancer Cell. ;19(2):192-205.
2.
Figure 5

Figure 5. IL-6 is secreted by osteoblasts in a Notch- and Hey1-dependent fashion and stimulates the growth of Jagged1-expressing tumor cells. From: Tumor-derived Jagged1 Promotes Osteolytic Bone Metastasis of Breast Cancer by Engaging Notch Signaling in Bone Cells.

(A) Quantification of control or JAG1 OE tumor cells cocultured with MC3T3-E1 cells and treated with DMSO, 1μM, or 5μM MRK-003 by luficerase assay. *p < 0.05
(B) Quantification of indicated tumor cells cocultured with MC3T3-E1 cells that were treated with Rbpj siRNAs by luficerase assay. *p < 0.05.
(C) Heatmap depicting microarray gene expression profiling of MC3T3-E1 osteoblasts that were FACS-separated from cocultures of each experimental group.
(D) A list of genes with expression levels greater than 3-fold in osteoblasts cocultured with JAG1 OE tumor cells relative to controls.
(E) Quantification of indicated tumor cells cocultured with MC3T3-E1 cells that were treated with Hey1 siRNAs by luficerase assay. **p < 0.005
(F) Quantification of IL-6 levels in conditioned media of control or JAG1 OE tumor cells cultured alone or cocultured with MC3T3-E1 cells in the presence of DMSO, 1μM, or 5μM MRK-003 using ELISA. *** p < 1×10−5.
(G) ELISA quantification of IL-6 levels in conditioned media of indicated tumor cells cocultured with MC3T3-E1 cells treated with Rbpj siRNAs. **p < 0.0005, *** p < 1×10−4.
(H) Quantification of IL-6 levels in conditioned media of indicated tumor cells cocultured with MC3T3-E1 cells treated with Hey1 siRNAs using ELISA. ***p < 0.0005.
(I) Quantification of indicated tumor cells cocultured with MC3T3-E1 cells and treated with IgG, 0.5 μg/ml, or 1.0μg/ml anti-mouse IL-6 by luficerase assay. *p < 0.05, **p = 0.007.
(J) Quantification of indicated tumor cells cocultured with MC3T3-E1 cells and treated with PBS, 10 ng/ml, or 100 ng/ml hIL-6 by luficerase assay. *p < 0.05, ***p < 1×10−5.
Data in the figure represent average ± SD. p-values were based on Student’s t-test.

Nilay Sethi, et al. Cancer Cell. ;19(2):192-205.
3.
Figure 8

Figure 8. Disrupting the Notch pathway with MRK-003 reverses Jagged1-mediated bone metastasis. From: Tumor-derived Jagged1 Promotes Osteolytic Bone Metastasis of Breast Cancer by Engaging Notch Signaling in Bone Cells.

(A) BLI, X-ray, and histological images of bone lesions from representative mice in each experimental group on day 42. Scale bar indicates 200 μM.
(B) (Left) Kaplan-Meier bone metastasis-free survival curve of mice from each experimental group over time. Log rank p = 0.032. (Right) Normalized BLI signals of bone metastasis in mice inoculated with control or JAG1 OE tumor cells and treated with vehicle or MRK-003. *p < 0.05, **p < 0.01 based on repeated measures ANOVA.
(C) Quantification of radiographic osteolytic lesion area of mice hind limbs from each experimental group. *p < 0.05 by Student’s t-test.
(D) Quantification of TRAP+ osteoclasts along the bone-tumor interface of metastases from each experimental group. **p < 0.005, ***p < 1×10−4 by Student’s t-test.
(E) Schematic depicting tumor-stroma interactions of JAGGED1-expressing tumor cells with the bone microenvironment. See text for details.
Data in the figure represent average ± SEM.

Nilay Sethi, et al. Cancer Cell. ;19(2):192-205.
4.
Figure 2

Figure 2. Enforced expression of Jagged1 in breast cancer promotes osteolytic bone metastasis. From: Tumor-derived Jagged1 Promotes Osteolytic Bone Metastasis of Breast Cancer by Engaging Notch Signaling in Bone Cells.

(A) Kaplan-Meier bone metastasis-free survival curve of mice inoculated with control or JAG1 OE SCP28 cells (n = 10).
(B) Normalized BLI signals of bone metastases of mice from each experimental group. ** p < 0.01, * p < 0.05.
(C) Quantification of X-ray osteolytic lesion area of hind limbs from each experimental group.
(D) Quantification of TRAP+ osteoclasts along the bone-tumor interface of metastases from each experimental group.
(E) BLI, X-ray, and histological (H&E and TRAP) images of bone lesions from two representative mice in each experimental group on day 42. Red arrows indicate osteolytic bone lesions in the X-ray images and tumor burden in the histological images. JAGGED1 images have been stained using IHC against JAGGED1. Scale bar = 200 μM.
(F) Top: Ki67 immunohistochemical images of bone metastases from each experimental group. Scale bar = 200 μM. Bottom: quantification of percent Ki67+ tumor cells along bone tumor interface of 5 distinct 20x images from each group (n=3).
Data in the figure represent average ± SEM. p-values were based on Student’s t-test unless otherwise indicated. See also Figure S2.

Nilay Sethi, et al. Cancer Cell. ;19(2):192-205.
5.
Figure 6

Figure 6. Jagged1-expressing tumor cells stimulate osteoclastogenesis and osteolytic bone lesions via Notch-dependent signaling. From: Tumor-derived Jagged1 Promotes Osteolytic Bone Metastasis of Breast Cancer by Engaging Notch Signaling in Bone Cells.

(A) TRAP staining of cocultures of control or JAG1 OE tumor cells with pre-osteoclast Raw 264.7 cells treated with DMSO or 1μM MRK-003 immediately after seeding in the displayed 12-well plate (Early).
(B) TRAP staining of similar experiment as (A) except DMSO or 1μM MRK-003 treatment was initiated 2 days after seeding (Late).
(C) qRT-PCR mRNA expression levels of mouse Apc5 (encoding mouse TRAP) from the experimental groups described in (A) and (B). **p = 0.001, ***p < 10−4, #p > 0.4.
(D) Quantification of TRAP+ osteoclasts from experiment (A). **p < 1×10−4.
(E) Diameter of TRAP+ osteoclasts from experiment (A). ***p < 1×10−7.
(F) Quantification of TRAP+ osteoclasts with the indicated number of nuclei from experiment (A). Number of osteoclasts quantified per group = 86.
(G) Representative images of TRAP+ osteoclasts from experiment (A). Scale bar = 400 μM.
(H) Heatmap depicting qRT-PCR mRNA expression levels of osteoclast differentiation markers in Raw 264.7 cells that have been cocultured under the indicated conditions using mouse-specific primers.
Data in the figure represent average ± SD. p-values were based Student’s t-test. See also Figure S5.

Nilay Sethi, et al. Cancer Cell. ;19(2):192-205.
6.
Figure 7

Figure 7. Disrupting the Notch pathway with MRK-003 reduces osteolytic bone metastasis. From: Tumor-derived Jagged1 Promotes Osteolytic Bone Metastasis of Breast Cancer by Engaging Notch Signaling in Bone Cells.

(A) Normalized BLI signals of bone metastasis in mice (n=10) that have been injected with SCP2 cells and treated with vehicle or MRK-003. *p < 0.05, ** p < 0.005.
(B) BLI and X-ray images of bone lesions from four representative mice in each experimental group.
(C) Kaplan-Meier bone metastasis-free survival curve of mice from each experimental group.
(D) Quantification of total and hind limb bone lesions in vehicle or MRK003-treated mice. *p < 0.05.
(E) Quantification of radiographic osteolytic lesion area of hind limbs of mice from each experimental group.
(F) Quantification of TRAP+ osteoclasts along the bone-tumor interface of metastases of mice from each experimental group.
(G) Histological (H&E and TRAP staining) analysis of bone metastases from each experimental group. Arrowheads indicate bone destruction in histological sections of vehicle mice. Scale bar = 200 μM.
(H) qRT-PCR mRNA expression of Notch target genes and mouse IL-6 in the stromal compartment of bone metastasis from vehicle or MRK-003-treated mice using mouse-specific primers. *p < 0.005, **p < 0.001.
Data in the figure represent average ± SEM; p-values were based on Student’s t-test unless otherwise indicated.

Nilay Sethi, et al. Cancer Cell. ;19(2):192-205.
7.
Figure 1

Figure 1. Tumor-derived Jagged1 is associated with high risk of bone metastasis and functionally important for osteolytic bone metastasis in vivo. From: Tumor-derived Jagged1 Promotes Osteolytic Bone Metastasis of Breast Cancer by Engaging Notch Signaling in Bone Cells.

(A) Heatmap depicting microarray gene-expression profiling of Notch pathway ligands across the 4T1 series.
(B) mRNA expression of JAG1 in the MDA231 cell line and its derivative sublines with distinct bone metastasis properties using qRT-PCR.
(C-D) Kaplan-Meier relapse-free survival curve of patients from the Wang data set (Wang et al., 2005) (C) and bone metastasis-free survival curve of the Minn data set (Minn et al., 2005) (D) with either low or high expression of JAG1.
(E) Western blot analysis showing JAGGED1 protein levels in the control and JAGGED1 KD SCP2 and 1833 sublines.
(F) Kaplan-Meier survival curve of mice (n=10) inoculated with control or JAGGED1 KD SCP2 cells.
(G) Normalized BLI signals of bone metastases of mice (n = 10) from each experimental group. ** p < 0.01, * p < 0.05 by Mann-Whitney test.
(H) Histomorphometric quantification of tumor area of bone lesions from each experimental group.
(I) BLI, X-ray, and histological images of bone lesions from representative mice in each experimental group. Red arrows indicate osteolytic bone lesions. Scale bar = 6mm.
(J) Quantification of hind limb osteolysis from mice in each experimental group using X-ray analysis.
(K) Quantification of TRAP+ osteoclasts along the bone-tumor interface of metastases of mice from each experimental group.
(L) H&E and TRAP staining of bone metastasis from each experimental group. Arrows indicate TRAP+ osteoclasts. Arrowheads indicate areas of overt bone destruction. Scale bar = 200 μM.
Data in the figure represent average ± SEM. p-values were based on Student’s t-test unless otherwise indicated. See also Figure S1.

Nilay Sethi, et al. Cancer Cell. ;19(2):192-205.
8.
Figure 3

Figure 3. Jagged1 is a functional target of the TGFβ-SMAD signaling pathway in breast cancer bone metastasis. From: Tumor-derived Jagged1 Promotes Osteolytic Bone Metastasis of Breast Cancer by Engaging Notch Signaling in Bone Cells.

(A) Gene-set enrichment analysis of the TGFβ-response gene set in a ranked list of differentially expressed genes in strongly versus weakly bone-metastatic MDA231 sublines. p= 0.0212. Right panel: corresponding heatmap of core TGFβ gene set with elevated expression in strongly (red) versus weakly (green) bone-metastatic MDA231 sublines.
(B) mRNA expression of JAG1 in response to TGFβ treatment in the weakly (green) and strongly (red) bone-metastatic MDA231 sublines using previously reported microarray expression profiling data (Kang et al., 2003).
(C) qRT-PCR mRNA expression levels of JAG1 in the SCP28 cell line with inducible (Tet-off) SMAD4 expression (Korpal et al., 2009) under the indicated TGFβ and doxycycline treatment conditions. Data represent average ± SD.
(D) Western blot analysis showing JAGGED1 and SMAD4 protein levels in the indicated cell lines with altered expression of JAGGED1 and SMAD4.
(E) BLI and X-ray images of bone lesions of three representative mice from each experimental group on day 42. White arrows indicate osteolytic lesions in the X-ray images.
(F) Normalized BLI signals of bone metastases in the hind limbs of mice (n = 10) from each experimental group. **p < 0.01, *p < 0.05 by Student’s t-test.
(G) Kaplan-Meier bone metastasis-free survival curve of mice (n=10) from each experimental group.
(H) Quantification of radiographic osteolytic lesion area of hind limbs from each experimental group. Student’s t-test p < 0.001.
Data in the figure represent average ± SEM unless otherwise indicated. See also Figure S3.

Nilay Sethi, et al. Cancer Cell. ;19(2):192-205.

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