Expression and functional significance of VE-cadherin in aggressive human melanoma cells: role in vasculogenic mimicry - PubMed (original) (raw)

Expression and functional significance of VE-cadherin in aggressive human melanoma cells: role in vasculogenic mimicry

M J Hendrix et al. Proc Natl Acad Sci U S A. 2001.

Abstract

We recently have introduced the term vasculogenic mimicry to describe the unique ability of aggressive melanoma tumor cells to form tubular structures and patterned networks in three-dimensional culture, which "mimics" embryonic vasculogenic networks formed by differentiating endothelial cells. In the current study, we address the biological significance of several endothelial-associated molecules (revealed by microarray analysis) with respect to expression and function in highly aggressive and poorly aggressive human cutaneous melanoma cell lines (established from the same patient). In a comparative analysis, CD31 was not expressed by any of the melanoma cell lines, whereas TIE-1 (tyrosine kinase with Ig and epidermal growth factor homology domains-1) was strongly expressed in the highly aggressive tumor cells with a low level of expression in one of the poorly aggressive cell lines. Vascular endothelial (VE)-cadherin was exclusively expressed by highly aggressive melanoma cells and was undetectable in the poorly aggressive tumor cells, suggesting the possibility of a vasculogenic switch. Down-regulation of VE-cadherin expression in the aggressive melanoma cells abrogated their ability to form vasculogenic networks and directly tested the hypothesis that VE-cadherin is critical in melanoma vasculogenic mimicry. These results highlight the plasticity of aggressive melanoma cells and call into question their possible genetic reversion to an embryonic phenotype. This finding could pose a significant clinical challenge in targeting tumor cells that may masquerade as circulating endothelial cells or other embryonic-like stem cells.

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Figures

Figure 1

Western blot (A) and semiquantitative RT-PCR (B) analyses of endothelial-associated markers by human melanoma tumor cells. Western blot analysis of VE-cadherin (VE-cad), CD31, and TIE-1 in human cutaneous and uveal melanoma highly aggressive (*) and poorly aggressive cell lines (A). RT-PCR analysis of VE-cadherin (VE-cad) expression in similar melanoma cell samples as shown in A. Equal loading is demonstrated by glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression (B). The aggressive melanoma cells form primitive vasculogenic networks in three-dimensional collagen gels in vitro. (HUVECs were used as positive controls for VE-cadherin expression.)

Figure 2

Morphological and immunohistochemical analyses of highly aggressive and poorly aggressive cutaneous melanoma cells in three-dimensional collagen gels (A–D and F) and in monolayer culture (E). Phase-contrast microscopy (A_–_C) demonstrates the ability of highly aggressive cutaneous C8161 melanoma cells to form a vasculogenic pattern of cordlike networks (A), similar to those formed de novo by human angioblasts (B). Poorly aggressive cutaneous C81–61 melanoma cells were unable to form networks under similar culture conditions (C). Immunohistochemical staining for VE-cadherin (shown as red chromogenic stain) in a cryostat section of C8161 cells highlights developing vasculogenic networks; the negative control (Inset) receiving no primary antibody showed no nonspecific staining by the secondary antibody (D). Immunostaining for VE-cadherin in C8161 monolayer culture demonstrates the intercellular junctions (E). Transmission electron microscopy of developing vasculogenic networks by C8161 cells reveals the endothelial-like nature of the outermost perimeter cells (F). Alignment of melanosomes within the perimeter network cells is shown in the Inset.

Figure 3

Morphological and biochemical analyses of VE-cadherin knockout in highly aggressive human cutaneous C8161 melanoma cells. Cells were treated with sense (A) or antisense (B) oligonucleotides to VE-cadherin over 1 week in three-dimensional collagen gels, followed by periodic acid-Schiff staining to highlight extracellular matrix-rich vasculogenic networks. (This method renders tumor cells not easily detectable.) The tumor cell cultures receiving the control sense (A) or scrambled oligonucleotides (not shown) formed tubular networks that were perfusable with microinjected fluorescent dye (Upper Left Inset; from the tubular region in the box) and appeared as hollow tubes with lumens in longitudinal section (yellow arrowhead; Lower Right Inset). The Lower Right Inset also demonstrates the invasive ability of the C8161 cells (*) to migrate through the collagen gel and the 10-μm size pores (arrows) in the polycarbonate filter to the undersurface of the filter where they form a monolayer in the absence of a matrix. The cultures receiving the antisense oligonucleotide treatment did not form networks (B). Western blot analysis (C) of VE-cadherin expression shows the diminution of VE-cadherin in the antisense-treated cells, but not in the controls (no treatment), sense-treated, or scrambled oligonucleotide-treated cells.

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Expression and functional significance of VE-cadherin in aggressive human melanoma cells: role in vasculogenic mimicry - PubMed (original) (raw)