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1.
Figure 1

Figure 1. From: Targeting the Transforming Growth Factor-? pathway inhibits human basal-like breast cancer metastasis.

Morphology of MDA-MB-231-derived subclones in 3D organotypic culture. Cells were plated onto a layer of growth factor-reduced Matrigel® matrix as described in Materials and Methods. Confocal microscopy images of 9 day-old 3D cultures labeled with Alexa-488-phalloidin were obtained. Colonies of early (SCP2TR and SCP25TR) and post-dormant (2860TR and 3847TR) bone-tropic subclones displayed a mass-like and grape-like morphology, respectively [46]. In contrast, the two lung-tropic subclones (4175TR and 4173) exhibited a stellate invasive phenotype [46].

Vidya Ganapathy, et al. Mol Cancer. 2010;9:122-122.
2.
Figure 2

Figure 2. From: Targeting the Transforming Growth Factor-? pathway inhibits human basal-like breast cancer metastasis.

Effects of TGF-β antagonists on Smad activation in MDA-MB-231-derived metastatic subclones. A. Cells were starved in serum free DMEM medium overnight and incubated with vehicle or TGF-β antagonist for 15 minutes. Subsequently, TGF-β (100 pM) was added, and cells were incubated for an additional hour. The levels of phosphorylated Smad-2,-3 and -1/5/8 were determined by Western blotting. Induction of Smad-2 and -3 phosphorylation by exogenous TGF-β was effectively inhibited by either LY2109761 (2 μM) or 1D11 (10 μg/ml) in all six subclones. TGF-β induced Smad1 and -5 phosphorylation most strongly in the most metastatic bone-tropic (SCP2TR) and lung-tropic (4175TR and 4173) subclones, and to a lesser extent in the 2860TR and 3847TR cells. Activation of these BMP Smads was also inhibited by both antagonists. B. To assess the ability of TGF-β antagonists to induce dephosphorylation of R-Smads, bone-tropic SCP2TR cells were treated with TGF-β for 1.5 h, followed by treatment with the antagonists for the indicated time-periods. The receptor kinase inhibitor, LY2109761, induced dephosphorylation of activated Smad-2 and -3 more rapidly than the pan- TGF-β neutralizing antibody, 1D11.

Vidya Ganapathy, et al. Mol Cancer. 2010;9:122-122.
3.
Figure 5

Figure 5. From: Targeting the Transforming Growth Factor-? pathway inhibits human basal-like breast cancer metastasis.

Pharmacodynamic effects of TGF-β antagonists in vivo. A. Protein extracts from snap-frozen uninvolved lung tissue of mice treated with vehicle and LY2109761 and of mice treated with controls and 1D11 were prepared and subject to Western blotting using rabbit phosphoSmad2 antibody (1:1000 dilution). Whole cell lysate from SCP2TR was used as a control (Co). Treatment with both antagonists resulted in a reduction of phosphorylated Smad2 levels. Values represent the means and SD of three mice per group. Unpaired 2-sided t-test was used for comparisons. B. RNA was extracted from snap-frozen uninvolved kidneys of mice treated with vehicle or LY2109761 using Trizol reagent (Invitrogen) and purified using RNeasy mini columns (Qiagen) according to the manufacturer's instructions. Transcript levels of CTGF and PAI-1 were assayed using the QuantiTect™ Probe RT-PCR Kit on a Mx4000® Multiplex Quantitative PCR System (Stratagene). Treatment with LY2109761 significantly reduced mRNA levels of both CTGF and PAI-1 relative to GAPDH mRNA. Values represent the means and SD of three mice per group. Unpaired 2-sided t-test was used for comparisons. C. RNA was extracted from snap-frozen lungs of mice treated with vehicle, isotype control antibody or 1D11 and transcript levels determined as described above. No significant reduction in CTGF or PAI-1 mRNA levels could be detected in the 1D11-treated group. Values represent the means and SD of two independent experiments, 3 mice per group. Unpaired 2-sided t-test was used for comparisons.

Vidya Ganapathy, et al. Mol Cancer. 2010;9:122-122.
4.
Figure 4

Figure 4. From: Targeting the Transforming Growth Factor-? pathway inhibits human basal-like breast cancer metastasis.

Effects of TGF-β antagonists on experimental MDA-MB-231 human breast cancer cell metastases in vivo. A. Mice were inoculated with bone-tropic SCP2TR cells via intracardiac injection and treated with vehicle, 13C4 isotype control antibody or 1D11 anti-TGF-β antibody (Left panel) or with vehicle or LY2109761 (Right panel). Bone metastases were monitored by BLI once weekly. Median values per group are shown. Treatment with 1D11 antibody reduced the burden of bone metastases by approximately 70-80% (p = 0.001) compared to treatment with either vehicle or isotype control antibody. Similarly, LY2109761 treatment inhibited bone metastases compared to vehicle controls by approximately 55% (p = 0.039). B. Faxitron analysis of fore-and hind limbs of tumor-bearing animals. Both 1D11 and LY2109761 treatment resulted in significant reductions in the total extent of SCP2TR-induced osteolytic bone lesions. Unpaired 2-sided t-test was used for comparisons. C. Treatment of bone-tropic SCP2TR inoculated mice with the 1D11 antibody was associated with prolongation of survival of the test animals (n = 10) compared to control animals (n = 12) (p = 0.06, Log-Rank test). D. Mice were inoculated with lung-tropic 4175TR cells via tailvein injection and treated with vehicle, 13C4 or 1D11 (Left panel) or with vehicle or LY2109761 (Right panel). Treatment with 1D11 antibody reduced the metastatic burden to lungs by approximately 25-40% (p = 0.001, Kruskall-Wallis test) compared to treatment with either vehicle or isotype control antibody. Similarly, LY2109761 treatment reduced the burden of lung metastases compared to vehicle by approximately 40% (p = 0.079). E. Treatment of mice inoculated with post dormancy bone tropic 2860TR cells with 1D11 antibody reduced the metastatic burden to bones by between 55-80% compared to treatment with vehicle or isotype control antibody (p = 0.019).

Vidya Ganapathy, et al. Mol Cancer. 2010;9:122-122.
5.
Figure 6

Figure 6. From: Targeting the Transforming Growth Factor-? pathway inhibits human basal-like breast cancer metastasis.

Mechanism of action of TGF-β antagonists in vivo. Lung metastases from mice treated with LY2109761 and 1D11 were stained for markers of cell proliferation (Ki-67), apoptosis (TUNEL), and microvascular endothelial cells (CD34). At least 600 nuclei were counted in 5 randomly selected high-power (400 ×) fields in areas of viable tumor to determine the proportion of Ki-67-or TUNEL-positive cells. The total number of CD34+ microvessels were counted in 5 randomly selected high-power (400 ×) fields in areas of viable tumor. A. Neither antagonist affected tumor cell proliferation (Ki-67; p = 0.49 for the LY2109761 group and p = 0.12 for the 1D11 group) or B. apoptosis (TUNEL assay; p = 0.12 for the LY2109761 group and p = 0.06 for the 1D11 group). C. However, microvessel density was significantly reduced in tumors of mice treated with LY2109761 and 1D11 compared with their respective controls (p = 0.018 for the LY2109761 group and p = 0.024 for the 1D11 group). D. Histological staining for tartrate resistant acid phosphatase (TRAP) activity (red color) of bone metastases from representative vehicle-(left images) and 1D11-treated (right images) mice. The numbers of TRAP positive cells per mm2 of tumor adjacent to bone in bone metastases from 1D11-treated mice (n = 9 lesions) relative to vehicle-treated mice (n = 17 lesions) are shown. Treatment with 1D11 was associated with a significant reduction in the number of TRAP-positive osteoclasts (p = 0020). Unpaired 2-sided t-test (± Welch correction) was used for comparisons.

Vidya Ganapathy, et al. Mol Cancer. 2010;9:122-122.
6.
Figure 3

Figure 3. From: Targeting the Transforming Growth Factor-? pathway inhibits human basal-like breast cancer metastasis.

Effects of TGF-β and TGF-β antagonists on cell growth and cell-motility and -invasion of MDA-MB-231 subclones. A. Cells were plated at 2 × 104/well in 24-well plates and incubated in the presence of vehicle (Blue bars), TGF-β (100 pM) (Green bars), 1D11 (10 μg/ml) or LY2109761 (2 μM) (Red bars) or a combination of TGF-β and an inhibitor (Yellow bars) for 72 h and cell numbers determined. Treatment with exogenous TGF-β failed to significantly inhibit growth of any of the subclones, with the exception of SCP2TR cells. Neither 1D11 nor LY2109761 stimulated tumor cell growth of any of the MDA-MB-231 subclones. Means ± SD of at least three independent experiments. Unpaired 2-sided t-test ± Welch correction was used to compare treatment with or without TGF-β antagonist. For cell-motility (B) and -invasion (C) assays, MDA-MB-231 sublines were cultured in uncoated and Matrigel®-coated PET inserts, respectively. Cells were treated with TGF-β (100 pM) and either LY2109761 (2 μM) or 1D11 (10 μg/ml) for 24 h. MDA-MB-231 subclones SCP2TR and 4175TR displayed the greatest motility and invasion, which were further stimulated by exogenous TGF-β. Moreover, both TGF-β pathway antagonists significantly inhibited TGF-β-induced motility and invasion in these two cell lines. Means ± SD of at least three independent experiments. Unpaired 2-sided t-test ± Welch correction was used to compare treatment with TGF-β alone with TGF-β plus inhibitor. D. Lung-tropic 4173 cells were plated onto a layer of growth factor-reduced Matrigel® matrix, followed by treatment with either vehicle or varying concentrations of LY2109761 for 9 days. Phase contrast microscopy images of 9 day-old 3D cultures labeled with Alexa-488-phalloidin were obtained. Numbers refer to the four representative colonies from each culture shown. As can be seen, treatment with LY2109761 inhibited invasion into surrounding Matrigel® in a dose-dependent manner, resulting in a reversal from a stellate to mass-like phenotype.

Vidya Ganapathy, et al. Mol Cancer. 2010;9:122-122.

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