VE-Cadherin Disassembly and Cell Contractility in the Endothelium are Necessary for Barrier Disruption Induced by Tumor Cells

Abstract

During metastasis, breakdown of the endothelial barrier is critical for tumor cell extravasation through blood vessel walls and is mediated by a combination of tumor secreted soluble factors and receptor-ligand interactions. However, a complete mechanism governing tumor cell transendothelial migration remains unclear. Here, we investigate the roles of tumor-associated signals in regulating endothelial cell contractility and adherens junction disassembly leading to endothelial barrier breakdown. We show that Src mediates VE-cadherin disassembly in response to metastatic melanoma cells. Through the use of pharmacological inhibitors of cytoskeletal contractility we find that endothelial cell contractility is responsive to interactions with metastatic cancer cells and that reducing endothelial cell contractility abrogates migration of melanoma cells across endothelial monolayers. Furthermore, we find that a combination of tumor secreted soluble factors and receptor-ligand interactions mediate activation of Src within endothelial cells that is necessary for phosphorylation of VE-cadherin and for breakdown of the endothelial barrier. Together, these results provide insight into how tumor cell signals act in concert to modulate cytoskeletal contractility and adherens junctions disassembly during extravasation and may aid in identification of therapeutic targets to block metastasis.

Figure 1. Metastatic melanoma cells induce gap formation between endothelial cells.

(A) HPMEC monolayers cultured in direct contact with either A2058 (metastatic) or WM35 (non-metastatic) melanoma cells for 0, 10, 45 and 90 min. Endothelial cell junctions were immunostained with anti-VE-cadherin (green). Yellow outlines show intercellular gaps, arrows show adherens junctions and arrowheads show either A2058 or WM35 melanoma cells. Scale bars: 50 μm. (B) The percentage of endothelial gaps was quantified as the number of pixels within the gap regions divided by the number of pixels in the entire image. Results represent the mean +/−SEM (***p < 0.001 in comparison to monolayers treated with WM35 at equal times, n = 3).

Figure 2. Endothelial cell contractility is necessary for melanoma-induced gap formation and subsequent endothelial barrier breakdown.

(A) HPMEC monolayers stimulated with thrombin or cultured in direct contact with either A2058 (metastatic) or WM35 (non-metastatic) melanoma cells. Endothelial cell contractility was assessed via immunostaining of F-Actin (red) and ppMLC (green). Anisotropy numbers on F-actin images represent a measurement of fiber alignment (mean +/−SEM, n = 3). Scale bars: 10 μm. (B) Phosphorylation levels of MLC were determined by measuring mean fluorescence intensity of ppMLC images. Results represent the mean +/−SEM (n = 3). (C) Co-localization of ppMLC and F-actin filaments was measured from immunofluorescence images and is reported using Pearson’s correlation coefficient. Results represent the mean +/−SEM (n = 3). (D) HPMEC monolayers were pre-treated with inhibitors of contractility for 30 min and were then co-cultured in direct contact with A2058 melanoma cells for 45 min. Endothelial cell junctions were immunostained with anti-VE-cadherin and gap formation was quantified as the number of pixels within the gap regions over the number of pixels in the entire image. Results represent the mean ± SEM. A significant difference was found for the A2058 group between no treatment or DMSO and all the inhibitors. Also, significant differences were found between control and A2058 for no treatment and DMSO groups. No significant differences were observed between control and A2058 treated monolayers for any of the inhibitor treatments. (E) HPMEC monolayers cultured on 8 μm polycarbonate membranes were pre-treated with blebbistatin for 30 min and WM35 or A2058 melanoma cell migration across the monolayer was assessed. Results represent mean +/− SEM. (***p < 0.001, **p < 0.01, *p < 0.05, n = 3).

Figure 3. Stabilization of endothelial VE-cadherin junctions blocks melanoma-induced gap formation and subsequent endothelial barrier breakdown.

HPMEC monolayers were treated with FGF1 for 1 hour and immediately cultured in direct contact with A2058 melanoma cells for 0, 10, 45 and 90 min. Endothelial cell junctions were immunostained with anti-VE-cadherin and gap formation was quantified as the number of pixels within gap regions over the number of pixels within the entire image. Results represent the mean +/−SEM, (***p < 0.001 in comparison to monolayers treated with FGF-1 at equal times, n = 3).

Figure 4. Metastatic melanoma cells use IL-8 signalling and VLA-4/VCAM-1 interactions to induce gap formation and subsequent endothelial barrier breakdown.

(A) HPMEC monolayers were pre-treated with neutralizing antibodies against CXCR1 and CXCR2 receptors for 1 hour and immediately cultured in direct contact with A2058 melanoma cells for 90 min. Endothelial cell junctions were immunostained with anti-VE-cadherin and gap formation was quantified as the number of pixels within gap regions over the number of pixels within the entire image. (B) HPMEC monolayers were cultured in direct contact with either A2058 melanoma cells alone or A2058 melanoma cells pre-treated with VLA-4 neutralizing antibodies. Results represent the mean +/−SEM, (***p < 0.001, **p < 0.01, *p < 0.05, n = 3).

Figure 5. Isolated soluble factors and receptor-ligand interactions are sufficient to disrupt the endothelium and to form intercellular gaps.

(A) Quantification of the percentage of gaps formed in HPMEC monolayers stimulated with increasing concentrations of IL-8 for 45 min. (B) Quantification of the percentage of gaps formed in HPMEC monolayers stimulated with 20 ng/ml IL-8 over a period of 0, 10, 45 and 90 min. (C) Quantification of the percentage of gaps formed in HUVEC monolayers stimulated with either K562-WT cells or K562-VLA-4 positive cells for 0, 10, 45 and 90 min. Statistical analysis was performed between K562-WT and K562-VLA-4 for equal incubation times. Plots represent the mean +/−SEM (*p < 0.05, **p < 0.01, n = 3).

Figure 6. Metastatic melanoma cells induce VE-cadherin phosphorylation via Src activation.

(A) Western blot and densitometric analysis of total and phosphorylated VE-cadherin in endothelial cells in co-culture with either A2058 or WM35 melanoma cells. Endothelial cells were either left untreated or pre-treated with the Src inhibitor PP1 prior to co-culture with melanoma cells. (B) Time-lapse images of endothelial cells expressing the Src FRET biosensor stimulated with either A2058 metastatic melanoma cells WM35 non-metastatic melanoma cells, or media only. Images are pseudocolored showing CFP/FRET ratio. Scale bars represent 5 μm. (C) Quantification of CFP/FRET ratio normalized to background signal (before stimulation) for each time-lapse image set and then normalized to negative control. Plot represent mean +/−SEM (n = 6).

Figure 7. Soluble and receptor-ligand interactions activate Src in endothelial cells resulting in VE-cadherin phosphorylation.

(A) Western blot and densitometric analysis of total and phosphorylated VE-cadherin induced by IL-8 [100, 20 or 10 ng/ml] after 0, 10, 45 and 90 min. (B) Western blot and densitometric analysis of total and phosphorylated VE-cadherin induced by K562-WT or K562-VLA-4 cells after 0, 10, 45 and 90 min. (C) Time-lapse images of endothelial cells expressing the Src FRET biosensor stimulated with different concentrations of IL-8 [100, 20 or 10 ng/ml] or with anti-VCAM-1 antibody [30 μg/ml]. Images are pseudocolored showing CFP/FRET ratio (Bars represent 5 μm). (D,E) Quantification of CFP/FRET ratio normalized to background signal (before stimulation) for each time-lapse image set and then normalized to negative control. Plots represent mean +/−SEM (n = 6).