human juvenile foreskin lymphatic endothelial cells lecs  (PromoCell)

Journal: eLife

Article Title: Lymphatic endothelium stimulates melanoma metastasis and invasion via MMP14-dependent Notch3 and β1-integrin activation

doi: 10.7554/eLife.32490

Figure Lengend Snippet: ( a ) Schematic of the experimental pipeline. ( b ) Confocal images of LEC spheroids (PECAM-1, red) in 3D fibrin matrix (left panel), LEC spheroids co-cultured with WM852 (green, middle panel) or Bowes (green, left panel). The area enclosed in the white square is shown enlarged below each panel. Melanoma cells were stained with GFP (green), and nuclei were counterstained with Hoechst 33342. Maximum intensity Z-projections of confocal stacks are shown. ( c,d ) Growth rates of the 3D LEC primed WM852* ( c ) and Bowes* ( d ) derived tumors (n = 8 for both cell types) compared to control WM852 (n = 7) and Bowes (n = 8) tumors, respectively. ( e, f ) Distant organ metastasis, detected by bioluminescence imaging of luciferase signal, in liver ( e ) and lung ( f ) of SCID mice subcutaneously injected with WM852 alone or co-cultured with LECs (WM852*). Upper panels: representative images of the indicated organs, each box represents an organ from one mouse. Bottom panel: quantification of luciferase signal, each dot represents the luciferase value in one sample. Horizontal line indicates the average, vertical bars represent SEM. *: p<0.05. n.s., non-significant.

Article Snippet: Human primary juvenile foreskin lymphatic endothelial cells (LEC) were obtained from Promocell, and adult dermal LECs from Lonza.

Techniques: Cell Culture, Staining, Derivative Assay, Imaging, Luciferase, Injection

Journal: eLife

Article Title: Lymphatic endothelium stimulates melanoma metastasis and invasion via MMP14-dependent Notch3 and β1-integrin activation

doi: 10.7554/eLife.32490

Figure Lengend Snippet: ( a ) mRNA expression of a panel of LEC markers ( CD34 , PROX1 , FLT4 ) in 3D LEC primed Bowes and WM852 cells (*) before the cells were used for the in vivo xenograft assay. Expression in monotypic-cultured LEC was used as a control and set to one. ( b ) End point analysis of the volume (left panel) and weight (right panel) of the Bowes/Bowes* and WM852/WM852* tumors. A dot represents one mouse. Error bars indicate s.e.m. *: p<0.05. n.s., non-significant ( c ) Immunohistochemistry of xenograft sections from the LEC co-cultured WM852* (left panels) or Bowes* (right panels) stained for mouse Lyve-1 (red) to detect mouse lymphatic vessels and human MMP14 (green) as a marker of the human melanoma cells. Nuclei were counterstained with Hoechst 33342. Scale bar = 100 µm. ( d, e ) Luciferase signal from lymph nodes isolated from mice bearing WM852 (n = 7) and WM852*(n = 8) ( d ), or Bowes and Bowes* (n = 8) ( e ); each line shows lymph nodes from one mouse derived tumors. ( f ) Quantitative-PCR (q-PCR) for human Alu sequences from lung genomic DNA isolated from mice bearing WM852 and WM852* derived tumors. Each dot in the graph represents the luciferase signal intensity obtained from one isolated organ per mouse, and the red line marks the median of the samples. ( g ) Luciferase signal from liver and lungs isolated from mice bearing Bowes or Bowes* derived tumors (n = 8); each box represents an organ from one mouse.

Article Snippet: Human primary juvenile foreskin lymphatic endothelial cells (LEC) were obtained from Promocell, and adult dermal LECs from Lonza.

Techniques: Expressing, In Vivo, Xenograft Assay, Cell Culture, Immunohistochemistry, Staining, Marker, Luciferase, Isolation, Derivative Assay, Real-time Polymerase Chain Reaction

Journal: eLife

Article Title: Lymphatic endothelium stimulates melanoma metastasis and invasion via MMP14-dependent Notch3 and β1-integrin activation

doi: 10.7554/eLife.32490

Figure Lengend Snippet: ( a ) Schematic of the workflow for cell separation from the melanoma-LEC 2D co-cultures. ( b ) WM852-LEC co-culture (left), flow through of the magnetic column (middle) and the separated elute (right) were stained for GFP (green), PECAM (red), and PROX-1 (magenta) and visualized by indirect immunofluorescence using high-content imaging. Panels of 16 (4 × 4) images are shown. ( c ) Relative mRNA fold changes of the indicated targets in LECs and melanoma cells derived from monotypic cultures or co-cultures (*) followed by magnetic separation. Average of three independent experiments, error bars indicate SEM. *: p<0.05. ( d ) Cell count at different time points following magnetic separation of WM852 (left) and Bowes (right) from monotypic culture or after LEC priming in the co-culture. Graphs show an average of two independent experiments, error bars indicate SD.

Article Snippet: Human primary juvenile foreskin lymphatic endothelial cells (LEC) were obtained from Promocell, and adult dermal LECs from Lonza.

Techniques: Co-Culture Assay, Staining, Immunofluorescence, Imaging, Derivative Assay, Cell Counting

Journal: eLife

Article Title: Lymphatic endothelium stimulates melanoma metastasis and invasion via MMP14-dependent Notch3 and β1-integrin activation

doi: 10.7554/eLife.32490

Figure Lengend Snippet: ( a–b ) Generally Applicable Gene-set Enrichment (GAGE) for RNA-seq pathway analysis of LEC primed ( a ) WM852* and ( b ) Bowes* cells. Samples were compared to their respective cells derived from monotypic cultures. Three biological replicates per sample group and four run replicates were used. In the heatmap, red represents upregulated and green downregulated pathways in WM852* and Bowes*. Pathways enriched in both cell lines are marked with red text if they were similarly upregulated or downregulated, and blue text if they were differentially upregulated or downregulated. Pathways enriched only in one cell line are marked with black text. Pathways with underlined text were used for further analysis. ( c ) Heatmap depicting average expression fold change of the differentially expressed ECM-receptor interaction pathway genes in the RNA-seq of LEC primed WM852* and Bowes* cells. The WM852 and Bowes cells from monotypic cultures were used as controls, and set to one. Red represents upregulated and blue downregulated genes in WM852* and Bowes*. Adjusted p-values are less than 0.05 for all genes shown. ( d ) Relative mRNA fold change of the indicated targets in WM852 and WM852*. *:p<0.05; **p<0.001; n.s., non-significant. ( e ) Representative images of Notch3 immunohistochemistry in the WM852 and WM852* derived xenografts. Scale bar = 50 µm. ( f ) Representative confocal images of Notch3 staining (red) in different GFP expressing melanoma cell lines (GFP positive cells shown in the inset) cultured in the presence (*, upper panels) or absence (bottom panels) of LECs. Nuclei are counterstained with Hoechst 33342. The dashed line indicates the LEC-melanoma (below the line) border. Scale bar = 50 µm. Full size confocal images are available as a .

Article Snippet: Human primary juvenile foreskin lymphatic endothelial cells (LEC) were obtained from Promocell, and adult dermal LECs from Lonza.

Techniques: RNA Sequencing Assay, Derivative Assay, Expressing, Immunohistochemistry, Staining, Cell Culture

Journal: eLife

Article Title: Lymphatic endothelium stimulates melanoma metastasis and invasion via MMP14-dependent Notch3 and β1-integrin activation

doi: 10.7554/eLife.32490

Figure Lengend Snippet: ( a ) Right panels: representative confocal images of MMP14 (red) expression in WM852 and Bowes co-cultured with LECs (*, upper panels) and from monotypic culture (bottom panels). Nuclei were counterstained with Hoechst 33342. GFP-expressing melanoma cells are shown white in the inset. Arrowheads indicate MMP14 localization to the cell-cell contacts. The dashed line indicates the LEC-melanoma (below the line) border. Scale bar = 50 µm. Left panel: quantification of MMP14 intensity analysed in four images per condition from two independent experiments. More than 100 cells were always analysed per condition. Average is shown, error bars represent SD; *: p<0.05. n.s., non-significant. ( b ) Quantification of the 3D sprouting index of WM852 and WM852* treated with the indicated siRNAs for 72 hr followed by magnetic separation and the 96 hr fibrin assay. The graph represents the average of three images per condition in each of the two independent experiments, error bars indicate SEM; *: p<0.05. n.s., non-significant. ( c ) Representative confocal images of MMP14 (green) and Notch3 (red) in WM852 and WM852*. Arrowheads indicate the cell-cell junction where MMP14 and Notch3 co-localize. Nuclei were counterstained with Hoechst 33342. The dashed line indicates the LEC–melanoma (below the line; GFP positive cells (white) in the inset) border. Scale bar = 50 µm. ( d ) mRNA fold change of the indicated targets in WM852 and WM852* upon treatment with the indicated siRNA for 72 hr and following magnetic separation. Graphs show the average of three independent experiments, error bars indicate SEM, *: p<0.05; **: p<0.01. n.s., non-significant ( e ) Representative confocal images of Notch3 staining (red) in WM852* treated with the indicated siRNAs for 72 hr. Nuclei were counterstained with Hoechst 33342. The dashed lines indicate the LEC-WM852 (GFP positive cells (white) in the inset) border. Scale bar = 50 µm. ( f ) Quantification of Notch3 signal intensity of WM852* treated as in ( e ) and described in ( a ). Error bars indicate SD; *: p<0.05. Full size confocal images are available as .

Article Snippet: Human primary juvenile foreskin lymphatic endothelial cells (LEC) were obtained from Promocell, and adult dermal LECs from Lonza.

Techniques: Expressing, Cell Culture, Staining

Journal: eLife

Article Title: Lymphatic endothelium stimulates melanoma metastasis and invasion via MMP14-dependent Notch3 and β1-integrin activation

doi: 10.7554/eLife.32490

Figure Lengend Snippet: ( a ) Flow cytometry analysis of MMP14 levels in WM852 and WM852*. Mean fluorescence intensity normalized to WM852 is shown, error bars represent SD across three independent experiments; *: p<0.05. ( b ) Left panels: representative images of MMP14 staining (red) in WM165 and WM793 cells (GFP positive cells (white) in the insets) cultured in the presence (*, upper panels) or absence (bottom panels) of LECs. Nuclei were counterstained with Hoechst 33342. The dashed line indicates the LEC-melanoma (below the line) border. Scale bar = 50 µm. Right panel: quantification of MMP14 signal intensity for the indicated cell lines. Average of the intensity is shown from four images/condition from two independent experiments. More than 100 cells were always analysed per condition. Error bars indicate SD. n.s., non-significant ( c ) Confocal images of Bowes (upper panels) and WM852 (lower panels) stained with TGN46 (green) and MMP14 (red) antibodies, nuclei were counterstained with Hoechst 33342. Scale bar: 25 µm. ( d ) Relative sprouting index of WM852 and WM852* treated with the vehicle (DMSO) or panMMP inhibitor GM6001 for 48 hr during co-culture and after magnetic separation as well as during the 3D fibrin assay. Error bars indicate SEM; **: p<0.01. ( e ) MMP14 mRNA fold change in WM852 and WM852* treated with the indicated siRNAs for 72 hr; *: p<0.05; **: p<0.01. n.s., non-significant. Full size confocal images are available as .

Article Snippet: Human primary juvenile foreskin lymphatic endothelial cells (LEC) were obtained from Promocell, and adult dermal LECs from Lonza.

Techniques: Flow Cytometry, Fluorescence, Staining, Cell Culture, Co-Culture Assay

Journal: eLife

Article Title: Lymphatic endothelium stimulates melanoma metastasis and invasion via MMP14-dependent Notch3 and β1-integrin activation

doi: 10.7554/eLife.32490

Figure Lengend Snippet: ( a ) Representative confocal images of active β1-integrin (12G10) staining (red) in the indicated melanoma cell lines (GFP positive cells (white) in the inset) in the presence (*, upper panels) or absence (bottom panels) of LECs. Nuclei were counterstained with Hoechst 33342. The dashed line indicates the LEC-melanoma border. Scale bar = 50 µm. ( b ) Quantification of 3D sprouting index in WM852 and WM852* mock treated or treated with β1-integrin blocking antibody (AIIB2) during the 96 hr fibrin growth assay. Graph shows the average of at least three images per condition per two independent experiments, error bars indicate the SEM; *: p<0.05. n.s., non-significant. ( c ) Representative confocal image of active β1-integrin (12G10, red) and MMP14 (green) staining of WM852* (white cells in the inset). Nuclei were counterstained with Hoechst 33342. The dashed lines indicate the border between LEC and WM852 (white, GFP positive WM852 cells in the inset). The right and bottom panels show an enlargement of the area enclosed within the white square as a merge, Notch3 (red) and MMP14 (green) in separated channels. Scale bar = 50 µm. ( d,f ) Representative confocal images of WM852* treated with the indicated siRNAs for 72 hr and stained for active β1-integrin with 12G10 (d, red), or total β1-integrin with P5D2 (f, red) antibodies. Nuclei were counterstained with Hoechst 33342. The dashed lines indicate the LEC-WM852 borders (white, GFP positive WM852 cells in the inset) border. Scale bar = 50 µm. Quantification of the average 12G10 ( e ) and total β1-integrin ( g ) signal intensity in WM852* (white) cells. Four images/condition were quantified from two independent experiments. More than 100 cells were always analysed per condition; error bars indicate SD. *: p<0.05. n.s., non-significant. Full size confocal images are available as a .

Article Snippet: Human primary juvenile foreskin lymphatic endothelial cells (LEC) were obtained from Promocell, and adult dermal LECs from Lonza.

Techniques: Staining, Blocking Assay, Growth Assay

Journal: eLife

Article Title: Lymphatic endothelium stimulates melanoma metastasis and invasion via MMP14-dependent Notch3 and β1-integrin activation

doi: 10.7554/eLife.32490

Figure Lengend Snippet: ( a ) Quantification of active β1-integrin (12G10) intensity in the indicated melanoma cell lines from monotypic cultures or after co-culture with LEC (*). Average of the intensity was calculated in four images/condition from two independent experiments. More than 100 cells were always analysed per condition. Error bars indicate SD, *: p<0.05; n.s., non-significant. ( b ) Left and middle panels: representative confocal images of WM852 and WM852* stained with 9EG7 antibody for active β1-integrin (red). Nuclei were counterstained with Hoechst 33342 (blue). Dashed line indicates LEC-melanoma border. Scale bar = 50 µm. Right panel: quantification of 9EG7 intensity in the indicated cell types performed as in ( a ). ( c ) Representative confocal images of β1-integrin staining (red) in the different GFP expressing melanoma cell lines (GFP positive cells white in the inset) cultured in the presence (*, upper panels) or absence (bottom panels) of LECs. Nuclei were counterstained with Hoechst 33342. The dashed line indicates the LEC-melanoma (below the line) border. Scale bar = 50 µm. ( d ) Quantification of total β1-integrin intensity in the indicated melanoma cell lines from monotypic cultures or co-cultured with LEC (*) as described in ( a ). n.s., non-significant ( e ) Left panels: Immunoblot of indicated cell lines derived from monotypic or LEC co-cultures and separated by magnetic separation prior to sample preparation and stained with the indicated antibodies. Right panel: Percentages of β1-integrin intensities normalized to actin from two independent experiments. Error bars indicate SD; *: p<0.05; n.s., non-significant. Full size confocal images are available as a .

Article Snippet: Human primary juvenile foreskin lymphatic endothelial cells (LEC) were obtained from Promocell, and adult dermal LECs from Lonza.

Techniques: Co-Culture Assay, Staining, Expressing, Cell Culture, Western Blot, Derivative Assay, Sample Prep