The identification of chromosome abnormalities associated with the invasive phenotype of uveal melanoma in vitro.

Tumour cell cultures are often highly heterogeneous, containing sub-populations of cells with differing characteristics. To identify chromosome abnormalities that are associated with the invasive phenotype, we isolated highly invasive uveal melanoma cell populations using the Transwell assay. Using this invasion assay, invasive sub-populations of primary uveal melanoma short-term cultures, and an established cell line, were specifically isolated. A series of sequential assays were undertaken to enrich the invasive population, and the enhanced invasive ability was confirmed by Transwell invasion assay. Chromosome abnormalities in invasive and parental cells were identified by karyotyping and confirmed by comparative genome hybridisation. Invasive sub-populations of uveal melanoma cells were isolated from 3 uveal melanoma short term cultures and a uveal melanoma cell line. In all cases, invasive sub-populations had either acquired additional chromosome abnormalities to those present in the parental cell line, or other abnormalities present in the parental lines were lost. In the established cell line (SOM 157), invasive cells were characterised by widespread chromosomal instability, frequent telomere associations and additional copies of chromosome 20. The invasive phenotype of SOM 196 associated with the presence of a derivative chromosome 5, der(5)t(5;11)(q35;q12) whilst a translocation t(17;20)(q12;q13) was predominant amongst non-invasive cells. In two additional cultures, deletions on chromosome 6q were associated with reduced invasive ability. In conclusion, highly invasive populations of uveal melanoma cells demonstrate chromosomal abnormalities that differ from non-invasive cells. These include chromosome instability and abnormalities of chromosome 20, observations echoing those seen in metastatic uveal melanoma.

Instability of microsatellites is an infrequent event in uveal melanoma.

Microsatellite instability (MSI) is a distinct tumour phenotype that is associated with alterations of DNA mismatch repair and is being increasingly reported in a number of hereditary and sporadic tumours. Numerous reports have suggested that melanocytic neoplasms, including cutaneous melanomas, frequently demonstrate low frequency MSI, whilst a small number of tumours exhibit high frequency MSI. Furthermore, loss of expression of DNA mismatch repair proteins has been associated with progression from benign to malignant disease in melanocytic neoplasms, but the presence or absence of mismatch repair defects in uveal melanomas has yet to be determined. This study was designed to establish whether MSI is a feature of these ocular melanomas. To investigate the prevalence of MSI in uveal melanomas, 52 tumours were analysed by polymerase chain reaction amplification of a panel of microsatellite markers selected for their ability to detect tumours exhibiting defects in DNA mismatch repair mechanisms. MSI was rarely detected in the 52 uveal melanomas analysed. All tumours demonstrated stable microsatellites at five of the six microsatellite markers tested (BAT26, BAT40, APC, D2S123 and Mfd15CA). Only one tumour showed the presence of a single unstable allele at a tetranucleotide marker (MYCL1). These data suggest that high frequency MSI does not occur in these tumours, and that low frequency MSI, in contrast to cutaneous melanoma, is a rare event in malignant melanomas of the uveal tract.

Apoptotic cell death in conjunction with CD80 costimulation confers uveal melanoma cells with the ability to induce immune responses.

Uveal melanoma is a rare malignancy with a poor prognosis despite current therapeutic intervention. The current investigation focuses on the immunogenicity of uveal melanoma cells genetically modified with recombinant adenovirus encoding CD80 (AdCD80) in contrast to their parental counterpart. We demonstrate that costimulation provided by uveal melanoma cells improved immune responses in vitro as determined by mixed lymphocyte tumour cell cultures and cytotoxic T-cell assays using lymphocytes from healthy donors and uveal melanoma patients. Flow cytometry revealed T-cell stimulation by activated CD4+ and CD8+ T cells. Additionally, autologous lymphocytes proliferated in response to CD80-expressing primary uveal melanomas, indicating that this patient group is suitable for immunotherapy. Moreover, this study utilized AdCD80 modified and parental apoptotic tumour cells, loaded onto immature dendritic cells, as a source of tumour antigen. The ability of live or apoptotic tumour cells to stimulate lymphocyte proliferation and activation was determined. Apoptotic uveal melanoma cells expressing CD80 were efficient at inducing an immune response and served as a potent immunogen. The use of apoptotic uveal melanoma cells in combination with expression of costimulatory molecules could prove a novel adjuvant therapy for the treatment of this disease.

Stimulation and inhibition of uveal melanoma invasion by HGF, GRO, IL-1alpha and TGF-beta.

PURPOSE: To investigate potential factors involved in uveal melanoma migration and invasion in vitro. METHODS: Using a microchemotaxis chamber, the effects were studied of a range of stimulators and inhibitors on a series of 10 primary uveal melanomas and 2 uveal melanoma cell lines, by assessing invasion through an 8- micro m pore membrane, precoated with an extracellular matrix solution. In addition, invasion in response to the effect of cells and conditioned media derived from the liver and other tissues was studied for one uveal melanoma culture, by using double-chambered wells, and invasion was assessed through an 8- micro m pore membrane, precoated with synthetic extracellular matrix. In all instances, invading cells were counted under x400 magnification on the lower surface of the membrane. Levels of invasion were correlated with histopathologic markers of prognosis. RESULTS: Conditioned media and cells derived from other tissues, including the liver, increased cellular invasion of the uveal melanoma cell line studied. For specific regulators, maximum stimulation of invasion was induced by hepatic growth factor (HGF), growth-related oncogene (GRO), and macrophage inflammatory protein (MIP)-1beta, whereas significant inhibition was induced by IL-1alpha, TGF-beta1, and TGF-beta2. CONCLUSIONS: The primary site of metastasis in patients with uveal melanoma is the liver. For the degree of site specificity commonly seen, regulators involved in the process may be expressed at the secondary sites, promoting adhesion, migration, invasion, and proliferation of tumor cells. HGF, GRO, MIP-1beta, IL-1alpha, TGF-beta1, and TGF-beta2 may play a significant role in regulating invasion of uveal melanoma cells.

An in vitro assay to assess uveal melanoma invasion across endothelial and basement membrane barriers.

PURPOSE: To develop a modified in vitro invasion assay to assess uveal melanoma invasion across endothelial and basement membrane barriers. METHODS: Using permeable cell culture supports, endothelial cells were grown to confluence on an 8-microM pore polycarbonate membrane precoated with an artificial basement membrane. Primary uveal melanomas were grown as short-term cultures at 37 degrees C and 5% CO2 and invaded through the endothelial cell layer and basement membrane. Invading cells were counted under x400 magnification on the lower surface of the membrane. Levels of invasion were correlated with histopathologic markers of prognosis. The relative invasion of individual tumors was established by comparison of invasion through both endothelial and basement membrane barriers with invasion through basement membrane components alone. RESULTS: A series of 13 primary tumors were studied using the modified invasion assay. Tumors varied in their propensity to permeate both barriers. In all cases the endothelial cell layer reduced invasion, but the effect varied between tumors. CONCLUSIONS: Some tumors were more adept at overcoming the additional endothelial cell layer, whereas invasion of others was severely inhibited. Tumor invasion through the transendothelial model was found to correlate more closely with clinical characteristics associated with invasion, than was invasion through basement membrane components alone. The transendothelial model may represent a more realistic model for the in vitro study of invasion of uveal melanoma cells, providing a useful in vitro system for the investigation of cellular interactions during the invasion process.