13 research outputs found

    ADAM17 promotes motility, invasion, and sprouting of lymphatic endothelial cells

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    Tumor-associated lymphatic vessels actively participate in tumor progression and dissemination. ADAM17, a sheddase for numerous growth factors, cytokines, receptors, and cell adhesion molecules, is believed to promote tumor development, facilitating both tumor cell proliferation and migration, as well as tumor angiogenesis. In this work we addressed the issue of whether ADAM17 may also promote tumor lymphangiogenesis. First, we found that ADAM17 is important for the migratory potential of immortalized human dermal lymphatic endothelial cells (LEC). When ADAM17 was stably silenced in LEC, their proliferation was not affected, but: (i) single-cell motility, (ii) cell migration through a 3D Matrigel/collagen type I matrix, and (iii) their ability to form sprouts in a 3D matrix were significantly diminished. The differences in the cell motility between ADAM17-proficient and ADAM17-silenced cells were eliminated by inhibitors of EGFR and HER2, indicating that ADAM17-mediated shedding of growth factors accounts for LEC migratory potential. Interestingly, ADAM17 depletion affected the integrin surface expression/functionality in LEC. ADAM17-silenced cells adhered to plastic, type I collagen, and fibronectin faster than their ADAM17-proficient counterparts. The difference in adhesion to fibronectin was abolished by a cyclic RGD peptide, emphasizing the involvement of integrins in the process. Using a soluble receptor array, we identified BIG-H3 among several candidate proteins involved in the phenotypic and behavioral changes of LEC upon ADAM17 silencing. In additional assays, we confirmed the increased expression of BIG-H3, as well as TGFβ2 in ADAM17-silenced LEC. The antilymphangiogenic effects of ADAM17 silencing in lymphatic endothelial cells suggest further relevance of ADAM17 as a potential target in cancer therapy

    Analysis of the effect of ADAM17 silencing on lymphatic endothelial cells (LEC) proliferation.

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    <p>(<b>A</b>) RT-PCR analysis of the expression of members of the EGF receptor family in wild type (WT), M and S1. Positive control (ctrl+)–cDNA from cells that express particular receptors. Reaction mixtures after 40 cycles of quantitative RT-PCR were subjected to electrophoresis in the presence of ethidium bromide (EtBr). The result (shown in photographic negative) is representative of 3 performed experiments. (<b>B</b>) Western blotting analysis of EGFR and HER2 in LEC lysates. (<b>C</b>) Western blotting analysis of HB-EGF in cell lysates and media of LEC sublines M, S1, S2 and of M exposed for 48 h to 25 μM GM6001 (GM). mHB-EGF, membrane HB-EGF; sHB-EGF, soluble HB-EGF. (<b>B, C</b>) β-actin was used as a loading control of lysate proteins; a fragment of blot stained with Coomassie Brilliant Blue after antigen detection procedure was used as a loading control of media proteins. Representative pictures of three independent experiments are shown. (<b>D</b>) Changes in the number of WT, M and S1 cultured in basal medium acquired by cell counting. Bars represent mean ± SD of three independent experiments performed in triplicates.</p

    ADAM17 silencing decreases lymphatic endothelial cells (LEC) migration and sprouting.

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    <p>(<b>A</b>) Fluorescent images of DAPI-labeled M and S1 that transmigrated through a 3D Matrigel/collagen I matrix. (<b>B</b>) Quantification of M and S1 that transmigrated through a Matrigel/collagen I matrix during 16-h incubation in the absence or presence of 100 ng/ml VEGF-C, normalized to untreated M. Bars represent mean ± SD from at least three independent experiments, each analyzed from four microscopic fields per group. *<i>P<</i>0.05 <i>vs</i> M, untreated- or treated with VEGF-C, respectively. (<b>C</b>) Representative bright field images of sprouting spheroids formed by M and S1 embedded in collagen gel. (<b>D, E</b>) Effect of ADAM17 silencing on sprouting of LEC spheroids reflected by the changes in: (<b>D</b>) Number of sprouts and (<b>E</b>) Total length of sprouts. Bars represent mean ± SD from three independent experiments in which at least 10 spheroids per group were analyzed *<i>P<</i>0.01 <i>vs</i> appropriate cell line not treated with VEGF-C; #<i>P<</i>0.05 <i>vs</i> M, untreated- or treated with VEGF-C, respectively.</p

    Analysis of ADAM17 silencing in lymphatic endothelial cells (LEC).

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    <p>(<b>A</b>) Western blotting analysis comparing the levels of ADAM17 in primary LEC vs. hTERT-immortalized LEC. (<b>B</b>) Quantitative RT-PCR analysis of ADAM17 mRNA levels in the wild type LEC (WT), LEC stably transduced with lentiviral vector encoding mock (control) shRNA (M), and LEC stably transduced with lentiviral vectors encoding shRNA sequences targeting ADAM17 –no. 2 alone (S1) or nos. 3 and 5 (S2). Bars represent mean ± SD of three independent experiments performed in duplicates. *<i>P</i><0.01 <i>vs</i> M. (<b>C</b>) Western blotting analysis of ADAM17 protein levels in lysates from the LEC sublines. NS–nonspecific band (<b>D</b>) Flow cytometry analysis of the LEC markers, CD31 and podoplanin, in M and S1. (<b>A, B, C</b>) Shown are representative results of two (<b>A</b>) or three (<b>B, C</b>) independent analyses performed.</p

    Effect of ADAM17 silencing on surface availability of integrins and their involvement in lymphatic endothelial cell (LEC) adhesion.

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    <p>(<b>A</b>) Flow cytometric analysis of α1 and α4 integrin surface expression on M and S1 cultured at the same density. The percentages of α4 integrin-positive cells, as well as the mean fluorescence intensities (MFI, geomean) are presented. A representative result of five independent analyses is shown. (<b>B</b>) Availability of particular integrin chains on the cell surface assessed by the level of cell binding to surfaces coated with specific antibodies. Bars represent mean fluorescence signals of CyQuant GR-stained DNA of the cells that interacted with a given antibody ± SD from two independent experiments performed in duplicates. *<i>P<</i>0.05 <i>vs</i> M. (<b>C</b>) Inhibition of cell adhesion to fibronectin by cyclic RGD peptide analyzed by alamarBlue assay. Bars represent the mean fluorescence signals ± SD from three independent experiments performed in duplicates. *<i>P<</i>0.05 <i>vs</i> control M or control S1.</p

    Differences in protein profiles in lysates and media of M and S1 assessed by the human soluble receptor array.

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    <p>Heat map representation of the proteins whose amounts in lysates and/or media differ by at least 30% between M and S1. The fold differences ([S1]/[M]) were calculated as ratios of the chemiluminescent signal volumes. For any given protein, the readout was considered valid when the chemiluminescent signals in technical duplicates did not differ by more than 10%. To facilitate the calculations for limiting cases, <i>i</i>.<i>e</i>. when a protein was only detected in either M or S1, the mean background chemiluminescence value was taken as a proxy for the lack of signal, and the lower detection limit was set to the lowest value that allowed to distinguish between the background and the signal.</p

    Analysis of the effect of ADAM17 silencing on lymphatic endothelial cells (LEC) proliferation.

    No full text
    <p>(<b>A</b>) RT-PCR analysis of the expression of members of the EGF receptor family in wild type (WT), M and S1. Positive control (ctrl+)–cDNA from cells that express particular receptors. Reaction mixtures after 40 cycles of quantitative RT-PCR were subjected to electrophoresis in the presence of ethidium bromide (EtBr). The result (shown in photographic negative) is representative of 3 performed experiments. (<b>B</b>) Western blotting analysis of EGFR and HER2 in LEC lysates. (<b>C</b>) Western blotting analysis of HB-EGF in cell lysates and media of LEC sublines M, S1, S2 and of M exposed for 48 h to 25 μM GM6001 (GM). mHB-EGF, membrane HB-EGF; sHB-EGF, soluble HB-EGF. (<b>B, C</b>) β-actin was used as a loading control of lysate proteins; a fragment of blot stained with Coomassie Brilliant Blue after antigen detection procedure was used as a loading control of media proteins. Representative pictures of three independent experiments are shown. (<b>D</b>) Changes in the number of WT, M and S1 cultured in basal medium acquired by cell counting. Bars represent mean ± SD of three independent experiments performed in triplicates.</p

    Effect of inhibitors of EGFR family receptors on lymphatic endothelial cells (LEC) motility and viability.

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    <p>(<b>A</b>) Analysis of single cell motility. Bars represent average speed ± SD from two independent experiments of migrating M and S1 incubated in the basal medium (control) or in the basal medium supplemented with AG-1478 (inhibitor of EGFR, 1.5 μM), or with AG-825 (inhibitor of HER2, 1 nM), or with the combination of AG-1478 + AG-825. In each experiment, movements of 30 cells per group were analyzed. *<i>P<</i>0.05 <i>vs</i> control M or control S1 respectively; #<i>P<</i>0.01 <i>vs</i> control M. (<b>B</b>) Viability of LEC incubated for 24 h in the basal medium alone (control) or in the basal medium supplemented with 1.5 μM AG-1478, or with 1 nM AG-825, or with the combination of AG-1478 + AG-825, assessed by alamarBlue assay. Fluorescence intensity of control M or control S1, respectively, was taken as 100%. Bars represent average values ± SD from six independent experiments performed in sextuplicates. *<i>P<</i>0.05 <i>vs</i> control M; #<i>P<</i>0.05 <i>vs</i> M exposed to both inhibitors.</p
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