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

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

Macrophages enhance Vegfa-driven angiogenesis in an embryonic zebrafish tumour xenograft model

Tumour angiogenesis has long been a focus of anti-cancer therapy; however, anti-angiogenic cancer treatment strategies have had limited clinical success. Tumour-associated myeloid cells are believed to play a role in the resistance of cancer towards anti-angiogenesis therapy, but the mechanisms by which they do this are unclear. An embryonic zebrafish xenograft model has been developed to investigate the mechanisms of tumour angiogenesis and as an assay to screen anti-angiogenic compounds. In this study, we used cell ablation techniques to remove either macrophages or neutrophils and assessed their contribution towards zebrafish xenograft angiogenesis by quantitating levels of graft vascularisation. The ablation of macrophages, but not neutrophils, caused a strong reduction in tumour xenograft vascularisation and time-lapse imaging demonstrated that tumour xenograft macrophages directly associated with the migrating tip of developing tumour blood vessels. Finally, we found that, although macrophages are required for vascularisation in xenografts that either secrete VEGFA or overexpress zebrafish vegfaa, they are not required for the vascularisation of grafts with low levels of VEGFA, suggesting that zebrafish macrophages can enhance Vegfa-driven tumour angiogenesis. The importance of macrophages to this angiogenic response suggests that this model could be used to further investigate the interplay between myeloid cells and tumour vascularisation

ADAM17 Silencing in Mouse Colon Carcinoma Cells: The Effect on Tumoricidal Cytokines and Angiogenesis

ADAM17 (a disintegrin and metalloprotease 17) is a major sheddase for numerous growth factors, cytokines, receptors, and cell adhesion molecules and is often overexpressed in malignant cells. It is generally accepted that ADAM17 promotes tumor development via activating growth factors from the EGF family, thus facilitating autocrine stimulation of tumor cell proliferation and migration. Here we show, using MC38CEA murine colon carcinoma model, that ADAM17 also regulates tumor angiogenesis and cytokine profile. When ADAM17 was silenced in MC38CEA cells, in vivo tumor growth and in vitro cell motility were significantly diminished, but no effect was seen on in vitro cell proliferation. ADAM17-silencing was accompanied by decreased in vitro expression of vascular endothelial growth factor-A and matrix metalloprotease-9, which was consistent with the limited angiogenesis and slower growth seen in ADAM17-silenced tumors. Among the growth factors susceptible to shedding by ADAM17, neuregulin-1 was the only candidate to mediate the effects of ADAM17 on MC38CEA motility and tumor angiogenesis. Concentrations of TNF and IFN gamma, cytokines that synergistically induced proapoptotic effects on MC38CEA cells, were significantly elevated in the lysates of ADAM17-silenced tumors compared to mock transfected controls, suggesting a possible role for ADAM17 in host immune suppression. These results introduce new, complex roles of ADAM17 in tumor progression, including its impact on the anti-tumor immune response

Generation of ADAM17-silenced tumor cell lines and optimization of in vivo 4T1 tumor model

Jednym z celów niniejszej pracy było przygotowanie komórkowych linii nowotworowych: 4T1luc, B16F10, CT26 ze stabilnym wyciszeniem białka ADAM17 metodą transdukcji lentiwirusowej. W tym celu wykorzystano lentiwiusowy system drugiej generacji, w którym komórki HEK293T kotransfekowano trzema plazmidami z genami wirusa, w tym jednym z odpowiednio zaprojektowaną i wklonowaną sekwencją shRNA. Powstałymi wektorami wirusowymi transdukowano komórki docelowe. Wydajność transdukcji oceniano dzięki pomiarom cytometrycznym oraz obserwacjom mikroskopowym, a jej efektywność dzięki metodom Real time PCR oraz Western blotting. Z powodu niskiej wydajności transdukcji komórek 4T1luc, metodę optymalizowano, a kiedy nie przyniosło to oczekiwanych rezultatów, komórki sortowano na sorterze komórkowym, dzięki obecności w stransdukowanych komórkach białka ZsGreen1. W komórkach CT26 oraz B16F10 udało uzyskać się stabilne wyciszenie białka ADAM17, co nie powiodło się w przypadku komórek 4T1luc. Niepowodzenie to może być związane z odpowiedzią interferonową, która została wywołana w komórkach poprzez mechanizm zależny od sekwencji wprowadzanego shRNA. Drugim celem niniejszej pracy była optymalizacja i charakterystyka modelu wzrostu guzów i przerzutowania komórek 4T1luc in vivo. W tym celu myszy Balb/c szczepiono komórkami 4T1luc modyfikując ilość podawanych komórek oraz miejsce szczepienia. Dzięki wysokiej ekspresji lucyferazy, możliwe było obserwowanie wzrostu i metastazy guzów przy użyciu systemu IVIS. Pomiary luminescencji w guzach dokonywane in vivo a także in vitro w komórkach 4T1luc wyizolowanych z guzów oraz z organów do których nastąpiło przerzutowanie, wykazały spadek aktywności lucyferazy w komórkach 4T1luc. Zjawisko to może stanowić problem przy tej metodzie monitorowania wzrostu i przerzutowania nowotworów.One of the aims of this study was to obtain tumor cell lines: 4T1luc, B16F10, CT26 with stable silencing of ADAM17 by a lentiviral transduction. To achieve this goal, a second generation system involving cotransfection of HEK293T cells with three plasmids coding for viral genes including one coding for a specially designed shRNA sequence, was used. The efficiency of transduction of three tumor cell lines was evaluated by flow cytometry and fluorescence microscopy and the levels of ADAM17 silencing were analysed by Real time PCR and Western blotting. The efficiency of 4T1luc transduction remained low, despite strenuous attempts to optimize the process. The cell sorting achievable thanks to the expression of fluorescent ZsGreen1 in transduced cells brought only temporary improvement. It is possible that the failure of ADAM17 silencing in 4T1luc cells using lentiviral system results from a so called interferon response which may be cell-specific and may depend the shRNA sequenceThe second aim of this study was to optimize and characterize the in vivo 4T1 tumor model, especially to analyse its growth rate and metastasic potential. Three experiments were performed, in which different amounts of 4T1luc cells were injected into different mammary glands of Balb/c mice. The cell line 4T1luc expresses luciferase, and thus a tumor growth and an appearance of metastases can be monitored in real time using IVIS bioluminescence imaging. The results of experiments showed decreasing levels of bioluminescence measured in vivo and in vitro, both in cells isolated from tumors and from metastatic organs. This phenomenon may create a serious problem in monitoring of tumor growth and metastasis

Overcoming inefficient secretion of recombinant VEGF-C in baculovirus expression vector system by simple purification of the protein from cell lysate

The first reports about successfully expressed recombinant proteins with the use of a baculovirus vector were published over 30 years ago. Despite the long time of refining this expression system, early problems with the production of baculovirus-derived secretory proteins are still not satisfactorily solved. The high expression level driven by baculoviral promoters often does not result in the desired yield of secreted recombinant proteins, which frequently accumulate inside insect cells and are only partially processed. During our attempts to produce vascular endothelial growth factor C (VEGF-C) with the use of a baculovirus vector we also faced an inefficient secretion of the recombinant protein to culture medium. We were not able to improve the outcome and obtain an acceptable concentration of VEGF-C in the medium by changing the culture conditions or utilizing different signal peptides. However, as a significant amount of native VEGF-C was detected inside the baculovirus-infected cells, we developed a simple method to purify recombinant, glycosylated VEGF-C from a lysate of the cells. The presented results indicate that the lack of a secretory protein in the insect cell culture medium after baculovirus infection does not necessarily signify failure in the production of the protein. As demonstrated by us and contrary to generally accepted views, the lysate of baculovirus-infected cells may constitute a valuable source of the biologically active, secretory protein. (C) 2015 Elsevier Inc. All rights reserved

ADAM17 Promotes Motility, Invasion, and Sprouting of Lymphatic Endothelial Cells

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.

<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.

<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

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

<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