Interplay of mechanical and binding properties of Fibronectin type I.

Fibronectins (FNs) are a major component of the extracellular matrix (ECM), and provide important binding sites for a variety of ligands outside and on the surface of the cell. Similar to other ECM proteins, FNs are consistently subject to mechanical stress in the ECM. Therefore, it is important to study their structure and binding properties under mechanical stress and understand how their binding and mechanical properties might affect each other. Although certain FN modules have been extensively investigated, no simulation studies have been reported for the FN type I (Fn1) domains, despite their prominent role in binding of various protein modules to FN polymers in the ECM. Using equilibrium and steered molecular dynamics simulations, we have studied mechanical properties of Fn1 modules in the presence or the absence of a specific FN-binding peptide (FnBP). We have also investigated how the binding of the FnBP peptide to Fn1 might be affected by tensile force. Despite the presence of disulfide bonds within individual Fn1 modules that are presumed to prevent their extension, it is found that significant internal structural changes within individual modules are induced by the forces applied in our simulations. These internal structural changes result in significant variations in the accessibility of different residues of the Fn1 modules, which affect their exposure, and, thus, the binding properties of the Fn1 modules. Binding of the FnBP appears to reduce the flexibility of the linker region connecting individual Fn1 modules (exhibited in the form of reduced fluctuation and motion of the linker region), both with regard to bending and stretching motions, and hence stabilizes the inter-domain configuration under force. Under large tensile forces, the FnBP peptide unbinds from Fn1. The results suggest that Fn1 modules in FN polymers do contribute to the overall extension caused by force-induced stretching of the polymer in the ECM, and that binding properties of Fn1 modules can be affected by mechanically induced internal protein conformational changes in spite of the presence of disulfide bonds which were presumed to completely abolish the capacity of Fn1 modules to undergo extension in response to external forces.

A new alternative mechanism in glioblastoma vascularization: tubular vasculogenic mimicry.

Glioblastoma is one of the most angiogenic human tumours and endothelial proliferation is a hallmark of the disease. A better understanding of glioblastoma vasculature is needed to optimize anti-angiogenic therapy that has shown a high but transient efficacy. We analysed human glioblastoma tissues and found non-endothelial cell-lined blood vessels that were formed by tumour cells (vasculogenic mimicry of the tubular type). We hypothesized that CD133+ glioblastoma cells presenting stem-cell properties may express pro-vascular molecules allowing them to form blood vessels de novo. We demonstrated in vitro that glioblastoma stem-like cells were capable of vasculogenesis and endothelium-associated genes expression. Moreover, a fraction of these glioblastoma stem-like cells could transdifferentiate into vascular smooth muscle-like cells. We describe here a new mechanism of alternative glioblastoma vascularization and open a new perspective for the antivascular treatment strategy.

A pilot study of vasculogenic mimicry immunohistochemical expression in hepatocellular carcinoma.

CONTEXT: The "de novo" formation of fluid-conducting patterns by tumor cells, termed vasculogenic mimicry (VM), is associated with increased mortality in many different solid tumors. OBJECTIVE: To identify VM patterns in hepatocellular carcinoma (HCC) and to determine whether these patterns were associated with more rapid tumor recurrence after orthotopic liver transplantation. DESIGN: Subjects included 20 patients who underwent orthotopic liver transplantation and were found to have HCC in the liver explant. Samples from 5 normal postmortem livers and 5 explanted livers with hepatitis C virus cirrhosis without HCC served as control tissues. Patterned matrix VM expression in HCC was identified by the presence of laminin-positive loops surrounding packets of tumor cells. Time to HCC recurrence after orthotopic liver transplantation was compared between patients with and without patterned VM expression. The relationships among VM in HCC, cause of chronic liver disease, serum alpha-fetoprotein level at the time of diagnosis, tissue expression by epidermal growth factor receptor, and endothelial markers including vascular endothelial growth factor and CD31 were assessed. RESULTS: Patterned matrix VM was identified in 11 of 20 primary HCC tissue samples. Vasculogenic mimicry was absent in all 10 control cases and was not identified in any area of dysplasia. The expression of VM in HCC lesions in liver explants was associated with more rapid posttransplant recurrence (P = .01). Vasculogenic mimicry was not associated with the cause of liver disease, serum alpha-fetoprotein level at time of diagnosis, or expression of epidermal growth factor receptor, vascular endothelial growth factor, or CD31. CONCLUSIONS: Vasculogenic mimicry of the patterned matrix type is present in hepatocellular carcinoma and is associated with tumor recurrence after orthotopic liver transplantation. Vasculogenic mimicry lesions are not associated with endothelial markers in HCC.

Using the direct-injection model of early uveal melanoma hepatic metastasis to identify TPS as a potentially useful serum biomarker.

PURPOSE: To develop a method to screen for serum biomarkers of early hepatic metastasis from uveal melanoma. METHODS: Cytokeratin 18 (TPS) was identified from gene expression profiles as protein generated by highly invasive uveal melanoma cells. Sera were collected from two groups of 15 SCID mice 2 weeks after injection of either tissue culture medium or MUM2B human metastatic uveal melanoma cells into the mouse liver. Serum TPS levels were assayed in 53 healthy human controls, 64 uveal melanoma patients who were disease free for at least 10 years, and 37 patients with metastatic uveal melanoma. RESULTS: After 2 weeks, small hepatic nodules (0.1-2.8 mm; mean, 0.80 mm) developed in 11 of 15 mice injected with MUM2B cells. Serum TPS levels in media-injected mice (84.7 U/L) were substantially lower than levels in MUM2B-injected mice (601 mug/L). TPS levels were significantly higher (P < 0.0001) in patients with metastatic uveal melanoma (139.63 +/- 22.20) than in healthy controls (54.23 +/- 0.01) or in patients free of disease (69.29 +/- 9.76). Significant differences were found between TPS levels before and after the development of hepatic metastases (P < 0.01), and serum TPS levels became elevated in four patients at least 6 months before the detection of hepatic metastases by abdominal ultrasonography. CONCLUSIONS: The direct-injection model of uveal melanoma in the mouse liver may be used to screen for potential serum biomarkers of metastatic uveal melanoma.

Serum markers to detect metastatic uveal melanoma.

BACKGROUND: Osteopontin (OPN) is overexpressed in metastatic uveal melanoma (UM). S-100beta and melanoma-inhibitory activity (MIA) serum levels are elevated in metastatic cutaneous melanoma. The ability of OPN, S-100beta and MIA serum levels to be used as non-invasive markers for detecting metastatic UM was tested. PATIENTS AND METHODS: OPN, S-100beta and MIA levels were measured by ELISA assays in 18 patients with metastatic UM and in 38 patients who were disease-free (DF) for at least 10 years after treatment of the primary tumor. Paired serum samples from 8 patients before and after development of metastasis were analyzed. Forty-four healthy controls (C) were compared to the other two groups. RESULTS: Serum OPN, MIA, and S-100beta levels were significantly higher in patients with metastatic UM as compared to patients who were DF for at least 10 years after treatment (p = 0.0001) or with age-matched controls. Serum OPN, MIA and S-100beta levels were significantly higher (p < 0.005) after metastasis formation than before the clinical detection of metastasis in the 8 patients. Receiver operator characteristic analysis was performed for metastatic patients vs. DF and vs. C, and the area under the curve was calculated for each marker and for the combination of the 3 markers, which was 91%. CONCLUSION: Elevated serum OPN, MIA and S-100beta levels correlate with metastatic UM to the liver. When used in combination, these markers provide a highly sensitive and specific method to detect hepatic metastases and therefore provide for earlier therapeutic intervention that can prolong survival.

Modeling the behavior of uveal melanoma in the liver.

PURPOSE: To model the behavior of uveal melanoma in the liver. METHODS: A 15-muL suspension of metastatic MUM2B or either primary OCM1 or M619 uveal melanoma cells was injected into the liver parenchyma of 105 CB17 SCID mice through a 1-cm abdominal incision. Animals were killed at 2, 4, 6, or 8 weeks after injection. Before euthanatization, 3% FITC-BSA buffer was injected into the retro-orbital plexus of one eye of three mice. Liver tissues were examined by light and fluorescence microscopy, and were stained with human anti-laminin. Vasculogenic mimicry patterns were reconstructed from serial laser scanning confocal microscopic stacks. RESULTS: OCM1a cells formed microscopic nodules in the mouse liver within 2 weeks after injection and metastasized to the lung 6 weeks later. By contrast, M619 and MUM2B cells formed expansile nodules in the liver within 2 weeks and gave rise to pulmonary metastases within 4 weeks after injection. Vasculogenic mimicry patterns, composed of human laminin and identical with those in human primary and metastatic uveal melanomas, were detected in the animal model. The detection of human rather than mouse laminin in the vasculogenic mimicry patterns in this model demonstrates that these patterns were of tumor cell origin and were not co-opted from the mouse liver microenvironment. CONCLUSIONS: There are currently no effective treatments for metastatic uveal melanoma. This direct-injection model focuses on critical interactions between the tumor cell and the liver. It provides for translationally relevant approaches to the development of new modalities to detect small tumor burdens in patients, to study the biology of clinical dormancy of metastatic disease in uveal melanoma, to design and test novel treatments to prevent the emergence of clinically manifest liver metastases after dormancy, and to treat established uveal melanoma metastases.

Comparing vasculogenic mimicry with endothelial cell-lined vessels: techniques for 3D reconstruction and quantitative analysis of tissue components from archival paraffin blocks.

We previously described techniques to generate 3-dimensional reconstructions of the tumor microcirculation using immunofluorescence histochemistry and laser scanning confocal microscopy on serial sections from archival formalin-fixed, paraffin-embedded tissues. By aligning sequential z-stacks in an immersive visualization environment (ImmersaDesk), the need to insert fiduciary markers into tissue was eliminated. In this study, we developed methods to stitch overlapping confocal z-series together to extend the surface area of interest well beyond that captured by the confocal microscope objective and developed methods to quantify the distribution of markers of interest in 3 dimensions. These techniques were applied to the problem of comparing the surface area of nonendothelial cell-lined, laminin-rich looping vasculogenic mimicry (VM) patterns that are known to transmit fluid, with the surface area of endothelial cell-lined vessels in metastatic uveal melanoma to the liver in 3 dimensions. After labeling sections with antibodies to CD34 and laminin, the surface area of VM patterns to vessels was calculated by segmenting out structures that labeled with laminin but not with CD34 from those structures labeling with CD34, or CD34 and laminin. In metastatic uveal melanoma tissues featuring colocalization of high microvascular density [66.4 microvessels adjusted for 0.313 mm2 area (range 56.7 to 72.7)] and VM patterning, the surface area of VM patterns was 11.6-fold greater (range 10.8 to 14.1) than the surface provided by CD34-positive vessels. These methods may be extended to visualize and quantify molecular markers in 3 dimensions in a variety of pathologic entities from archival paraffin-embedded tissues.

Epigenetic reversion of breast carcinoma phenotype is accompanied by changes in DNA sequestration as measured by AluI restriction enzyme.

The importance of microenvironment and context in regulation of tissue-specific genes is well established. DNA exposure to or the sequestration from nucleases detects differences in higher order chromatin structure in intact cells without disturbing cellular or tissue architecture. To investigate the relationship between chromatin organization and tumor phenotype, we used an established three-dimensional assay in which normal and malignant human breast cells can be easily distinguished by the morphology of the structures they make (acinus-like versus tumor-like, respectively). We show that these phenotypes can be distinguished also by sensitivity to AluI digestion in which the malignant cells resist digestion relative to nonmalignant cells. Treatment of T4-2 breast cancer cells in three-dimensional culture with cAMP analogs or a phosphatidylinositol 3-kinase inhibitor not only reverted their phenotype from nonpolar to polar acinar-like structures but also enhanced chromatin sensitivity to AluI. By using different cAMP analogs, we show that cAMP-induced phenotypic reversion, polarization, and shift in DNA organization act through a cAMP-dependent protein-kinase A-coupled signaling pathway. Importantly, inhibitory antibody to fibronectin produced the same effect. These experiments underscore the concept that modifying the tumor microenvironment can alter the organization of tumor cells and demonstrate that architecture and global chromatin organization are coupled and highly plastic.

Role of tumor invasiveness, the extracellular matrix, and chromatin sequestration in the susceptibility of uveal melanoma to herpes simplex virus type 1.

To better understand determinants of susceptibility/resistance of uveal melanomas to herpes simplex virus type 1 (HSV-1) oncolytic therapy, uveal melanoma cell lines of low (OCM1a) and of high (M619, MUM2B) invasive potential were infected with HSV-1 either in the presence or absence of a laminin-rich extracellular matrix (Matrigel). OCM1a cultures were destroyed faster by HSV-1 than M619 and MUM2B cultures. In the presence of Matrigel, all melanoma cultures demonstrated delayed destruction by HSV-1 relative to Matrigel-free cultures. As sequestration of chromatin is a characteristic feature of highly invasive uveal melanomas that is further increased by exposure to laminin, we explored whether chromatin sequestration could be reversed by HSV-1 infection. HSV-1 infection induced a global reversal of chromatin sequestration in highly invasive uveal melanoma cells. However, this viral effect was first observed only 2h following virus infection and required novel protein synthesis from input viral DNA. These findings suggest that tumor invasiveness, the spatial relationship of tumor cells to laminin and chromatin sequestration are determinants of susceptibility/resistance of melanomas to HSV-1 oncolytic therapy. Furthermore, these findings indicate for the first time that HSV-1 infection is associated with global exposure of normally highly sequestered cellular DNA in malignant cells.

Tumor cell plasticity in uveal melanoma: microenvironment directed dampening of the invasive and metastatic genotype and phenotype accompanies the generation of vasculogenic mimicry patterns.

The histological detection of laminin-rich vasculogenic mimicry patterns in human primary uveal melanomas is associated with death from metastases. We therefore hypothesized that highly invasive uveal melanoma cells forming vasculogenic mimicry patterns after exposure to a laminin-rich three-dimensional microenvironment would differentially express genes associated with invasive and metastatic behavior. However, we discovered that genes associated with differentiation (GDF15 and ATF3) and suppression of proliferation (CDKNa1/p21) were up-regulated in highly invasive uveal melanoma cells forming vasculogenic mimicry patterns, and genes associated with promotion of invasive and metastatic behavior such as CD44, CCNE2 (cyclin E2), THBS1 (thrombospondin 1), and CSPG2 (chondroitin sulfate proteoglycan; versican) were down-regulated. After forming vasculogenic mimicry patterns, uveal melanoma cells invaded only short distances, failed to replicate, and changed morphologically from the invasive epithelioid to the indolent spindle A phenotype. In human tissue samples, uveal melanoma cells within vasculogenic mimicry patterns assumed the spindle A morphology, and the expression of Ki67 was significantly reduced in adjacent melanoma cells. Thus, the generation of vasculogenic mimicry patterns is accompanied by dampening of the invasive and metastatic uveal melanoma genotype and phenotype and underscores the plasticity of these cells in response to cues from the microenvironment.

Osteopontin expression and serum levels in metastatic uveal melanoma: a pilot study.

PURPOSE: This was a pilot study conducted to examine the expression of osteopontin in uveal melanoma and to determine whether serum osteopontin can be used in detecting metastatic uveal melanoma. METHODS: Osteopontin mRNA was measured in three uveal melanoma cell lines of various invasive potential by real-time PCR. Tissue sections of primary and metastatic uveal melanomas were stained for osteopontin. Serum osteopontin levels were measured by ELISA assays in 15 patients with metastatic uveal melanoma and in 37 patients who were disease-free for at least 10 years after treatment of the primary tumor. Paired serum samples drawn from eight patients before and after development of metastasis were analyzed. RESULTS: By real-time PCR, highly invasive primary and metastatic uveal melanoma cells expressed 6- and 250-fold excess osteopontin mRNA, respectively, compared with poorly invasive primary uveal melanoma cells. Tissue sections of primary uveal melanomas lacking looping vasculogenic mimicry patterns either did not stain for osteopontin or exhibited weak, diffuse staining. In primary melanomas containing looping vasculogenic mimicry patterns, strong osteopontin staining was detected in the tumor periphery where patterns were located. Diffuse strong expression of osteopontin was detected in eight samples of uveal melanomas metastatic to the liver. Serum osteopontin levels were significantly higher in patients with metastatic uveal melanoma than in patients who had been disease free for at least 10 years after treatment (P = 0.0001) or in age-matched control subjects. Serum osteopontin levels were significantly higher (P = 0.008) after metastasis than before the detection of metastasis in eight patients. When a cutoff of 10 ng/mL was used, the sensitivity and specificity of serum osteopontin in detecting metastatic melanoma was 87.5%, and the area under the receiver operator characteristic curve was 96%. CONCLUSIONS: Osteopontin is expressed diffusely in tissue sections of hepatic metastases from uveal melanoma, and increased serum osteopontin levels correlate with melanoma metastasis to the liver with high specificity and sensitivity.

Distinguishing fibrovascular septa from vasculogenic mimicry patterns.

CONTEXT: Molecular analyses indicate that periodic acid-Schiff (PAS)-positive (laminin-rich) patterns in melanomas are generated by invasive tumor cells by vasculogenic mimicry. Some observers, however, consider these patterns to be fibrovascular septa, generated by a stromal host response. OBJECTIVE: To delineate differences between vasculogenic mimicry patterns and fibrovascular septa in primary uveal melanomas. DESIGN: Frequency distributions, associations with outcome, and thicknesses of trichrome-positive and PAS-positive looping patterns were determined in 234 primary uveal melanomas. Sequential sections of 13 additional primary uveal melanomas that contained PAS-positive/trichrome-negative looping patterns were stained for type I and type IV collagens, laminin, and fibronectin. Real-time quantitative polymerase chain reaction was performed on RNA from cultured uveal melanoma cells for the expression of COL1A1, COL4A2, and fibronectin. RESULTS: Trichrome-positive loops were encountered less frequently than PAS-positive loops (10% vs 56%, respectively). Death from metastatic melanoma was strongly associated with PAS-positive (P < .001) but not with trichrome-positive (P = .57) loops. Trichrome-positive loops were significantly thicker than PAS-positive loops (P < .001). The PAS-positive patterns stained positive for laminin, type I and type IV collagens, and fibronectin. Type I collagen was detected within melanoma cells and focally within some PAS-positive patterns. Real-time quantitative polymerase chain reaction revealed 3-fold, 25-fold, and 97-fold increases, respectively, in expression of COL4A2, fibronectin, and COL1A1 by invasive pattern-forming primary melanoma cells compared with poorly invasive non-pattern-forming cells. CONCLUSIONS: Fibrovascular septa are rare and prognostically insignificant in uveal melanomas, whereas vasculogenic mimicry patterns are associated with increased mortality. Type I collagen, seen focally in some vasculogenic mimicry patterns, may be synthesized by tumor cells, independent of a host stromal response.

Chromatin organization measured by AluI restriction enzyme changes with malignancy and is regulated by the extracellular matrix and the cytoskeleton.

Given that expression of many genes changes when cells become malignant or are placed in different microenvironments, we asked whether these changes were accompanied by global reorganization of chromatin. We reasoned that sequestration or exposure of chromatin-sensitive sites to restriction enzymes could be used to detect this reorganization. We found that AluI-sensitive sites of nonmalignant cells were relatively more exposed compared to their malignant counterparts in cultured cells and human tumor samples. Changes in exposure and sequestration of AluI-sensitive sites in normal fibroblasts versus fibrosarcoma or those transfected with oncogenes, nonmalignant breast cells versus carcinomas and poorly metastatic versus highly invasive melanoma were shown to be independent of the cell cycle and may be influenced by proteins rich in disulfide bonds. Remarkably, regardless of degree of malignancy, AluI-sensitive sites became profoundly sequestered when cells were incubated with laminin, Matrigel, or a circular RGD peptide (RGD-C), but became exposed when cells were placed on collagen I or in serum-containing medium. Disruption of the actin cytoskeleton led to exposure, whereas disruption of microtubules or intermediate filaments exerted a sequestering effect. Thus, AluI-sensitive sites are more sequestered with increasing malignant behavior, but the sequestration and exposure of these sites is exquisitely sensitive to information conferred to the cell by molecules and biomechanical forces that regulate cellular and tissue architecture.

Vasculogenic mimicry.

The term vasculogenic mimicry describes the formation of fluid-conducting channels by highly invasive and genetically dysregulated tumor cells. Two distinctive types of vasculogenic mimicry have been described. Vasculogenic mimicry of the tubular type may be confused morphologically with endothelial cell-lined blood vessels. Vasculogenic mimicry of the patterned matrix type in no way resembles blood vessels morphologically or topologically. Matrix proteins such as laminin, heparan sulfate proteoglycan, and collagens IV and VI have been identified in these patterns. The patterned matrix anastomoses with blood vessels, and systemically injected tracers co-localize to these patterns. Vasculogenic mimicry of the patterned matrix type has been identified in uveal, cutaneous and mucous membrane melanomas, inflammatory and ductal breast carcinoma, ovarian and prostatic carcinoma, and soft tissue sarcomas, including synovial sarcoma rhabdomyosarcoma, osteosarcoma, and pheochromocytoma. Because the microcirculation of many tumors may be heterogeneous -- including incorporated or co-opted vessels, angiogenic vessels, mosaic vessels, and vasculogenic mimicry of the tubular and patterned matrix types -- therapeutic regimens that target angiogenesis alone may be ineffective against highly invasive tumors that contain patterned matrices. Vasculogenic mimicry provides an opportunity to investigate the interrelationships between the genetically dysregulated invasive tumor cell, the microenvironment, and the malignant switch.

Three-dimensional reconstruction of extravascular matrix patterns and blood vessels in human uveal melanoma tissue: techniques and preliminary findings.

PURPOSE: Looping patterns rich in laminin are present in tissue samples of primary aggressive human uveal melanomas and their metastases. Because these extravascular patterns connect to blood vessels and transmit fluid in vitro and in vivo, the three-dimensional configuration of these patterns has been the subject of considerable speculation. In the current study, methods were devised to describe the three-dimensional configuration of looping extravascular matrix patterns in archival human uveal melanoma tissue. METHODS: Twenty-five serial 4-microm-thick sections from primary uveal melanoma tissue were labeled with fluorescence-tagged laminin and examined by confocal microscopy to generate a Z-series within each 4-microm-thick section. The z-series from each section was stacked using an immersive three-dimensional environment (ImmersaDesk; Fakespace, Kitchener, Ontario, Canada) to allow for precise alignment and compensation for distortion artifact. RESULTS: Extravascular matrix patterns that appeared to form loops in two dimensions were shown to represent thin wrappings around branching and twisting cylindrical groupings of melanoma cells. Blood vessels joined with some of these laminin-positive cylindrical wrappings. CONCLUSIONS: In this preliminary study, periodic acid-Schiff (PAS)-positive laminin-rich looping patterns in two-dimensional tissue sections appear to outline cylindrical branching packets of melanoma cells rather than spheroidal nests. The conduction of fluid through this extravascular system may provide a novel delivery system for contrast and diagnostic agents.

An orthotopic model for human uveal melanoma in SCID mice.

The microcirculation of primary uveal melanomas, their precursors, and their metastases is distinctive. Medium-sized and even large primary uveal melanomas typically lack significant zones of necrosis, suggesting that either these tumors are relatively well perfused or they are capable of growth in a severely blood-deprived microenvironment. In addition to normal choroidal vessels that are incorporated into nevi and most primary uveal melanomas, aggressive primary and metastatic uveal melanomas tend to contain patterns of extracellular matrix that surround spheroidal or cylindrical packets of tumor cells. Some components of this branching, looping, and interconnected system of matrix may be perfused. It is now known that the generation of this patterning is a characteristic of genetically dysregulated melanoma cells (nonaggressive tumor cells do not form these patterns and melanomas lacking branching, looping, or interconnected matrix patterns tend to follow a relatively indolent course). We developed an orthotopic model of an aggressive human uveal melanoma by injecting suspensions of the primary human choroidal melanoma cell line (OCM1) into the subretinal space of one eye of 20 SCID mice. All mice were examined daily for tumor growth and tumors developed in every eye within 3 weeks of injection. The tumors were characterized by extraocular extension and the development of looping matrix patterns characteristic of those seen in aggressive human uveal melanoma. As in human uveal melanomas, these patterns were perfused by blood in areas. The orthotopic injection of human uveal melanoma cells into the SCID mouse eye generates a model reproducing the matrix-associated microcirculatory patterns of aggressive primary human uveal melanomas. This model can be used to explore the molecular pathogenesis and modulation of this novel circulation in vivo, to facilitate our understanding of the blood flow to these tumors providing insight into perfusion and drug delivery, to enable testing of pharmacologic modulation of pattern formation and intratumoral blood flow, and to refine noninvasive methods such as confocal scanning laser ophthalmoscopy to detect the presence of these patterns by which ophthalmologists might assess the biological behavior of tumors as noninvasive substitute for biopsy.

Control of melanoma morphogenesis, endothelial survival, and perfusion by extracellular matrix.

The morphogenetic properties of endothelial cells and melanoma cells were tested under varying matrix quantities and distributions and under constant and saturating levels of growth factors. Aggressive melanoma cells self-assembled into cords vasculogenically only when seeded on thin matrices: nonaggressive melanoma cells did not mimic endothelial cell behavior under any matrix thickness. When buried in matrix, however, aggressive melanoma cells generated looping patterns that contained tumor cells and matrix. These patterns were different topologically and compositionally from cord-like structures or blood vessels but were nevertheless capable of conducting dye by microinjection or passive diffusion. When seeded on three-dimensional cultures of nonaggressive nonpattern-forming melanoma cells, prelabeled endothelial cells attached to, penetrated through, and survived for 2 weeks but failed to form vasculogenic cords. In cocultures containing aggressive melanoma cells, endothelial cells survived briefly but formed short cords only in contact with looping patterns formed by the aggressive tumor cells. Time-lapse recording showed that endothelial cells were lysed upon direct contact with aggressive melanoma cells. Looping patterns identified in human tissue samples were composed ultrastructurally of electron-dense material on either side of a layer of tumor cells; scattered red blood cells were seen in this central cellular layer. By immunohistochemistry, patterns labeled with laminin and fibrinogen colocalized to these looping laminin-positive patterns, suggesting the presence of plasma within these patterns from contiguous leaky tumor vessels. These observations are consistent with the perfusion of these patterns in vitro and with repeated demonstrations of the colocalization of intravenous tracers to looping laminin patterns in animal xenograft models by independent groups. Thus, the distribution and localized quantity of extracellular matrix in aggressive melanomas contributes to the regulation of tumor cell morphogenesis, modulates interactions between tumor cells and endothelial cells, and may contribute to an extravascular matrix-directed circulation.

Uveal melanoma cell staining for CD34 and assessment of tumor vascularity.

PURPOSE: Aggressive melanoma cells may express endothelial markers that can be used to calculate microvascular density (MVD). High MVD has been associated with adverse outcome in uveal melanoma. If tumor cells label with endothelial cell markers, then MVD may not accurately reflect a tumor's vascularity. This study was designed to study the influence of melanoma cell labeling by endothelial cell markers on MVD. METHODS: Tissue sections of 200 ciliary body or choroidal melanomas were stained with CD34 alone, and the MVD was calculated by counting discrete foci of CD34 labeling in hot spots, as described previously. From adjacent sections double labeled by fluorescent immunohistochemical stains for S100 protein and CD34, tumor cells labeling with both markers were identified. The relationship between marker coexpression and MVD was tested. Tissue sections were also double labeled for Melan-A and CD34. RESULTS: MVD was found to be associated with death from metastatic melanoma as reported previously. However, colocalization of both Melan-A and S100 protein with CD34 was demonstrated. The labeling of tumor cells by CD34 was associated with an elevated calculation of MVD (P < 0.0001) but not with cell type, mitotic figures, tumor-infiltrating lymphocytes, or PAS-positive patterns. CONCLUSIONS: CD34 may label uveal melanoma cells and may contribute to computation of MVD. Although MVD is prognostically significant in uveal melanoma, this feature is not an exclusive measure of tumor vascularity.

Molecular determinants of human uveal melanoma invasion and metastasis.

The molecular analysis of cancer has benefited tremendously from the sequencing of the human genome integrated with the science of bioinformatics. Microarray analysis technology has the potential to classify tumors based on the differential expression of genes. In the current study, a collaborative, multidisciplinary approach was utilized to study the molecular determinants of human uveal melanoma invasion and metastasis. Uveal melanoma is considered the most common primary intraocular cancer in adults, resulting in the death of approximately 50% of patients affected. Unfortunately, at the time of diagnosis, many patients already harbor microscopic metastases, thus underscoring a critical need to identify prognostic markers indicative of metastatic potential. The investigative strategy consisted of isolating highly invasive vs. poorly invasive uveal melanoma cells from a heterogeneous tumor derived from cells that had metastasized from the eye to the liver. The heterogeneous tissue explant MUM-2 led to the derivation of two clonal cell lines: MUM-2B and MUM-2C. Further morphological and functional analyses revealed that the MUM-2B cells were epithelioid, interconverted (expressing mesenchymal and epithelial phenotypes) highly invasive, and demonstrated vasculogenic mimicry. The MUM-2C cells were spindle-like, expressed only a vimentin mesenchymal phenotype, poorly invasive, and were incapable of vasculogenic mimicry. The molecular analysis of the MUM-2B vs. the MUM-2C clones resulted in the differential expression of 210 known genes. Overall, the molecular signature of the MUM-2B cells resembled that of multiple phenotypes--similar to a pluripotent, embryonic-like genotype. Validation of select genes that were upregulated and down-regulated was conducted by semiquantitative RT-PCR measurement. This study provides a molecular profile that will hopefully lead to the development of new molecular targets for therapeutic intervention and possible diagnostic markers to predict the clinical outcome of patients with uveal melanoma.