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

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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.

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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).
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f4: 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).

Mentions: Melanoma-induced gaps between endothelial cells do not always form beneath the heterocellular contact; therefore, we hypothesized that multiple types of signalling contribute to gap formation, including receptor-ligand interactions and also paracrine signalling. Several studies have shown that metastatic melanoma cells secrete a number of cytokines that affect the behavior of the endothelium under physiological and pathological conditions. For instance, Gutova et al. analysed the media of metastatic A2058 melanoma cells using a cytokine array and found that IL-8 was the most abundant cytokine secreted, followed by Tissue Inhibitor of Metalloproteinase (TIMP)-2, Monocyte chemoattractant protein (MCP)-1 and Interleukin (IL)-650. In our lab, a previous study showed a marked difference between IL-8 concentration in the media of A2058 metastatic melanoma cells (15 ng/ml) and WM35 non-metastatic melanoma cells (0.084 ng/ml)3. Here, to test if A2058 metastatic melanoma cells use signalling pathways through IL-8 receptors C-X-C chemokine receptor type (CXCR)-1 and C-X-C chemokine receptor type (CXCR)-2 to induce gap formation in endothelial monolayers, we used neutralizing antibodies against these receptors. HPMEC monolayers were pre-treated with neutralizing antibodies, gently washed and co-cultured with A2058 metastatic melanoma cells. Results show that endothelial gap formation is significantly reduced when the IL-8 signalling pathway is blocked; however, gap formation is not completely abolished (Fig. 4A and Fig. S2). This suggests that IL-8 is one of the major players in endothelial barrier disruption induced by metastatic melanoma cells, but it is not the only one.


VE-Cadherin Disassembly and Cell Contractility in the Endothelium are Necessary for Barrier Disruption Induced by Tumor Cells
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).
© Copyright Policy - open-access
Related In: Results  -  Collection

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f4: 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).
Mentions: Melanoma-induced gaps between endothelial cells do not always form beneath the heterocellular contact; therefore, we hypothesized that multiple types of signalling contribute to gap formation, including receptor-ligand interactions and also paracrine signalling. Several studies have shown that metastatic melanoma cells secrete a number of cytokines that affect the behavior of the endothelium under physiological and pathological conditions. For instance, Gutova et al. analysed the media of metastatic A2058 melanoma cells using a cytokine array and found that IL-8 was the most abundant cytokine secreted, followed by Tissue Inhibitor of Metalloproteinase (TIMP)-2, Monocyte chemoattractant protein (MCP)-1 and Interleukin (IL)-650. In our lab, a previous study showed a marked difference between IL-8 concentration in the media of A2058 metastatic melanoma cells (15 ng/ml) and WM35 non-metastatic melanoma cells (0.084 ng/ml)3. Here, to test if A2058 metastatic melanoma cells use signalling pathways through IL-8 receptors C-X-C chemokine receptor type (CXCR)-1 and C-X-C chemokine receptor type (CXCR)-2 to induce gap formation in endothelial monolayers, we used neutralizing antibodies against these receptors. HPMEC monolayers were pre-treated with neutralizing antibodies, gently washed and co-cultured with A2058 metastatic melanoma cells. Results show that endothelial gap formation is significantly reduced when the IL-8 signalling pathway is blocked; however, gap formation is not completely abolished (Fig. 4A and Fig. S2). This suggests that IL-8 is one of the major players in endothelial barrier disruption induced by metastatic melanoma cells, but it is not the only one.

View Article: PubMed Central - PubMed

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.

No MeSH data available.


Related in: MedlinePlus