Lynn M.G. Gardner

Vascular channel formation by human melanoma cells in vivo and in vitro: vasculogenic mimicry.

Tissue sections from aggressive human intraocular (uveal) and metastatic cutaneous melanomas generally lack evidence of significant necrosis and contain patterned networks of interconnected loops of extracellular matrix. The matrix that forms these loops or networks may be solid or hollow. Red blood cells have been detected within the hollow channel components of this patterned matrix histologically, and these vascular channel networks have been detected in human tumors angiographically. Endothelial cells were not identified within these matrix-embedded channels by light microscopy, by transmission electron microscopy, or by using an immunohistochemical panel of endothelial cell markers (Factor VIII-related antigen, Ulex, CD31, CD34, and KDR[Flk-1]). Highly invasive primary and metastatic human melanoma cells formed patterned solid and hollow matrix channels (seen in tissue sections of aggressive primary and metastatic human melanomas) in three-dimensional cultures containing Matrigel or dilute Type I collagen, without endothelial cells or fibroblasts. These tumor cell-generated patterned channels conducted dye, highlighting looping patterns visualized angiographically in human tumors. Neither normal melanocytes nor poorly invasive melanoma cells generated these patterned channels in vitro under identical culture conditions, even after the addition of conditioned medium from metastatic pattern-forming melanoma cells, soluble growth factors, or regimes of hypoxia. Highly invasive and metastatic human melanoma cells, but not poorly invasive melanoma cells, contracted and remodeled floating hydrated gels, providing a biomechanical explanation for the generation of microvessels in vitro. cDNA microarray analysis of highly invasive versus poorly invasive melanoma tumor cells confirmed a genetic reversion to a pluripotent embryonic-like genotype in the highly aggressive melanoma cells. These observations strongly suggest that aggressive melanoma cells may generate vascular channels that facilitate tumor perfusion independent of tumor angiogenesis.

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

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.

The microcirculation of choroidal and ciliary body melanomas

The microcirculation of ciliary body and choroidal melanomas is remodelled into patterns. The presence of microvascular networks, composed of back-to-back loops that encircle microdomains of tumour, and parallel vessels with cross-linking, are associated with death from metastatic melanoma. The formation of these complex vascular patterns may result from reciprocal interactions between the tumour cell and the extracellular matrix, and pattern formation may reflect an invasive tumour cell phenotype. Ciliary body and choroidal melanomas are among the few forms of cancer treated before a pathologist assigns a grade to indicate whether tumour is likely to follow a benign or aggressive course. There is evidence to suggest that prognostically significant microcirculatory patterns may be detectable by non-invasive imaging techniques that may provide a substitute for biopsy to guide the clinical management of patients with these sight- and life-threatening tumours.

Regulation of Human Cytotrophoblast Morphogenesis by Hepatocyte Growth Factor/Scatter Factor

Abstract In vitro morphogenesis of epithelial cells to form tube-like structures is regulated by hepatocyte growth factor-scatter factor (HGF/SF). The placenta is a rich source of HGF/SF, and its absence in mice has been shown to lead to impaired placental growth and embryonic death. There is no information in the literature regarding in vitro morphogenesis of human cytotrophoblasts or the effect of HGF/SF on this process. In this study, cytotrophoblasts were isolated from human placentae obtained from all three trimesters of gestation and cultured on the recombinant basement membrane matrix (Matrigel). Under these conditions, cytotrophoblasts participated in morphogenetic events including formation of spheroid-like structures, radial linear processes with branching, and invaded Matrigel and formed large, tube-like structures. The presence of a developing lumen was documented in the linear projections arising from spheroids and in the tube-like structures by both confocal and transmission electron microscopy. Immunohistochemistry was used to characterize the phenotype of the cells, and staining with anti-cytokeratin and anti-E-cadherin antibodies confirmed the presence of cytotrophoblasts in both the spheroids and tube-like structures. Recombinant HGF (rHGF) significantly increased the invasive activity of cytotrophoblasts isolated from the first and second (P < 0.001) and third trimesters (P < 0.01). In addition, rHGF significantly increased the percentage of spheroids with branching processes in the first and second trimesters (P < 0.05). Anti-HGF antibody inhibited both these effects in a dose-dependent manner, indicating the specificity of the above findings. This study provides new evidence indicating that HGF/SF regulates invasion and branching morphogenesis of cytotrophoblasts throughout gestation, with maximum effects in the first and second trimester. These findings may help to elucidate the importance of the reduced expression of HGF/SF identified in placentae from women with preeclampsia or intrauterine growth restriction and suggest that HGF/SF may serve as an important candidate in therapeutic intervention strategies.

Distribution of prognostically important vascular patterns across multiple levels in ciliary body and choroidal melanomas

PURPOSE To investigate the validity of assigning patients whose eyes have been removed for ciliary body or choroidal melanoma to risk groups for metastasis based on the identification of microcirculatory patterns in one cross-section taken from the center of the tumor. METHODS Multiple levels were cut through the blocks of 15 ciliary body or choroidal melanomas until the tumor was exhausted. Each level was examined for the presence of microvascular networks and parallel vessels with cross-linking histologic features strongly associated with death from metastatic melanoma. RESULTS The central histologic section did not contain either microvascular networks or parallel vessels with cross-linking in eight tumors, nor were these patterns encountered in any of the more peripheral levels of the tumor. Seven tumors contained at least one focus of either microvascular networks or parallel vessels with cross-linking in the central histologic section. In two tumors, at least one of these patterns appeared in all histologic levels; in five tumors, at least one of these patterns appeared through multiple levels until just before the tumor was exhausted from the block (0.24 to 0.85 mm from the edge of the tumor). CONCLUSIONS This study suggests that the prognostic classification of uveal melanoma based on the histologic profile of the microcirculation may be consistent throughout the tumor depth.

New technique for the cytologic identification of presumed acanthamoeba from corneal epithelial scrapings

PURPOSE To describe a cytologic technique for the rapid identification of presumed Acanthamoeba organisms from corneal epithelial scrapings. METHODS After administering topical anesthesia, we removed the affected corneal epithelium with a scalpel blade. The tip of the blade, containing the scrapings, was washed off into a cuvette with a solution of an alcohol-based fixative for cytology specimens. The blade was immersed in the cuvette and agitated to ensure that the sample was collected. The specimen was fixed for at least 10 minutes and processed by cytospin centrifugation. RESULT Seventy-five patient samples have been studied with this technique, with excellent preservation of the organism. CONCLUSIONS The organism preservation with this technique is superior to that of conventional smears and permits confirmatory organism identification by immunohistochemistry.

An altered gene expression profile is associated with tumour vasculogenesis by human melanoma cells

We have recently observed that tissue sections from uveal melanoma and metastatic cutaneous melanoma generally lack evidence of significant necrosis and contain patterned networks of interconnected loops of extracellular matrix. Similar networks are formed in tissue cultures of highly invasive primary and metastatic melanoma. These networks, which are not formed by melanocytes or poorly invasive melanomas, have the properties of vascular channels and suggest that certain tumours may have an intrinsic ability to undergo vasculogenesis without a contribution from host endothelial cells. Previous studies have identified abnormal co-expression in aggressive melanoma cells of intermediate filaments (an interconverted phenotype), suggesting reversion to a pluripotent cell. We sought to extend the definition of this phenotype at the gene expression level by cDNA microarray analysis. Comparison of aggressive melanomas that form vascular channels with less aggressive cells that do not exhibit this phenotype reveal differences in their gene expression profile. The specific genes expressed in the highly invasive melanoma cultures include those encoding extracellular matrix proteins, signalling and regulatory molecules which are highly consistent with the vasculogenic phenotype exhibited by these cells. The results of this study support the concept that some tumours generate vascular channels independently of conventional angiogenesis and have important implications for the development of therapeutic interventions directed at this novel property of aggressive melanomas.