Jean-Claude A. Marshall

BACKGROUND: Previous studies identified the human nonmetastatic gene 23 (NME1, hereafter Nm23-H1) as the first metastasis suppressor gene. An inverse relationship between Nm23-H1 and expression of lysophosphatidic acid receptor 1 gene (LPAR1, also known as EDG2 or hereafter LPA1) has also been reported. However, the effects of LPA1 inhibition on primary tumor size, metastasis, and metastatic dormancy have not been investigated. METHODS: The LPA1 inhibitor Debio-0719 or LPA1 short hairpinned RNA (shRNA) was used. Primary tumor size and metastasis were investigated using the 4T1 spontaneous metastasis mouse model and the MDA-MB-231T experimental metastasis mouse model (n = 13 mice per group). Proliferation and p38 intracellular signaling in tumors and cell lines were determined by immunohistochemistry and western blot to investigate the effects of LPA1 inhibition on metastatic dormancy. An analysis of variance-based two-tailed t test was used to determine a statistically significant difference between treatment groups. RESULTS: In the 4T1 spontaneous metastasis mouse model, Debio-0719 inhibited the metastasis of 4T1 cells to the liver (mean = 25.2 liver metastases per histologic section for vehicle-treated mice vs 6.8 for Debio-0719-treated mice, 73.0% reduction, P < .001) and lungs (mean = 6.37 lesions per histologic section for vehicle-treated mice vs 0.73 for Debio-0719-treated mice, 88.5% reduction, P < .001), with no effect on primary tumor size. Similar results were observed using the MDA-MB-231T experimental pulmonary metastasis mouse model. LPA1 shRNA also inhibited metastasis but did not affect primary tumor size. In 4T1 metastases, but not primary tumors, expression of the proliferative markers Ki67 and pErk was reduced by Debio-0719, and phosphorylation of the p38 stress kinase was increased, indicative of metastatic dormancy. CONCLUSION: The data identify Debio-0719 as a drug candidate with metastasis suppressor activity, inducing dormancy at secondary tumor sites.

Little is known about the effect of blue light on inducing melanocytic malignant transformation. We chose to investigate the effect of blue light (475 nm wavelength) on the proliferation rates of uveal melanoma cells. In addition, we tested two different intraocular lenses to determine the possible effects of ultraviolet absorbing and blue light filtering intraocular lenses on the changes in proliferation. Four human uveal melanoma cell lines (92.1, MKT-BR, OCM-1, SP6.5) were exposed to blue light with and without the presence of ultraviolet absorbing and blue light filtering intraocular lenses. Cells covered by aluminum foil were used as a control. The proliferation rate of the cells compared with the control was then assessed using the Sulforhodamine-B proliferation assay. Cells exposed to blue light showed a statistically significant (P<0.05) increase in proliferation. Those exposed to blue light through a standard ultraviolet absorbing intraocular lens showed a smaller increase in proliferation, whereas those exposed with a blue light filtering intraocular lens showed no increase in proliferation than the control in all four cell lines. The exposure of cells to blue light led to an increase in proliferation in all cell lines compared with the control. The use of blue light filtering intraocular lenses abolished these increases in proliferation in the four cell lines. These results indicate that blue light filtering intraocular lenses may have a protective effect on the proliferation rates of uveal melanoma cells exposed to blue light.

PURPOSE: Uveal melanoma (UM) is the most common intra-ocular tumor in adults. Despite advances in diagnosis and treatment, the survival rate of UM has not increased in the last several decades. Approximately 50% of patients will die as a consequence of metastatic disease with the majority of metastases localized to the liver. Due to the lack of lymphatics in the eye, hematogenous dissemination is the predominant means by which UM cells escape the primary site. Our laboratory has recently demonstrated the presence of circulating malignant cells (CMCs) in the blood using both animal models and clinical trails involving UM patients. Current data suggests that all UM patients will be positive for CMCs after diagnosis. Furthermore, some of the phenotypic changes that are necessary for metastatic growth may occur while the cells are circulating in the blood. In this study, we evaluated the efficiency of a panel of antibodies to immunomagnetically isolate CMCs for the purpose of in vitro expansion and genetic, immunological, and phenotypic characterization. METHODS: In this study, five human uveal melanoma cell lines (92.1, MKT-BR, OCM-1, SP6.5, and UW-1) were immunostained with a panel of antibodies against known melanoma cell surface markers. Staining with monoclonal antibodies PAL M2, NKI C3, NKI/Beteb, and 9.2.27 permitted the generation of a cell surface expression profile in these cell lines. The five human UM cell lines and 92.1 transfected with GFP were subsequently spiked into human blood at concentrations ranging from 1x10(6) cells/ml to 10 cells/ml. Cells were immuno-magnetically isolated at concentrations as low as 10 cells/ml. RESULTS: Immunomagnetic isolation of all five human UM cell lines tested at concentrations down to 10 cells/ml human blood was achieved only when antibodies were used in combination. Individually, the antibodies did not permit isolation of cells at physiologically relevant concentrations. CONCLUSIONS: The immunomagnetic isolation method presented in this study can be used to isolate CMCs at physiologically relevant concentrations and at sensitivities comparable to those seen in polymerase chain reactions (PCR). In addition, our data suggests that our method is more efficient and reliable for the isolation of CMCs in UM than the methods currently used.