Immunomagnetic isolation and in vitro expansion of human uveal melanoma cell lines
نویسندگان
چکیده
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.
منابع مشابه
Relationship between natural killer cell susceptibility and metastasis of human uveal melanoma cells in a murine model.
PURPOSE The purpose of this study was to determine the susceptibility of human uveal melanoma cells to in vitro and in vivo natural killer (NK) cell-mediated cytolysis and to determine if NK cells influence metastasis from the eye. METHODS Four human uveal melanoma cell lines and one melanoma cell line derived from a metastatic lesion from a patient with uveal melanoma were tested for in vitr...
متن کاملCYTOKINE AND GROWTH FACTOR MODULATION OF CELL CYCLE EVENTS IN HUMAN MELANOMA CELL LINES
Cytokines influence cell cycle events, which in some but not all instances can be associated with melanoma progression. Analysis of the G0/G 1 and S phase fractions of the cell cycle was used to assay the proliferative or inhibitory activity of cytokines against ten human melanoma cell lines, including pairs of cell lines derived from primary and metastatic tissue of individual patients. Cy...
متن کاملDifferential expression of chemokine receptors on uveal melanoma cells and their metastases.
PURPOSE To determine the expression of the chemokine receptors CXCR4 and CCR7 on human uveal melanoma cells and their metastases and the effect of liver-borne factors on the chemotactic responses of uveal melanoma cells. METHODS Four human uveal melanoma cell lines and three cell lines of uveal melanoma metastases were examined by RT-PCR and flow cytometry for their constitutive expression of...
متن کاملRole of epidermal growth factor receptor in the metastasis of intraocular melanomas.
PURPOSE To explore the expression and function of epidermal growth factor receptor (EGFR) expression on human uveal melanoma cells. METHODS Five human uveal melanoma cell lines were examined by flow cytometry for the expression of EGFR. The correlation between EGFR expression and metastasis of uveal melanoma cells was tested in a nude mouse model of intraocular melanoma. The effect of EGFR on...
متن کاملEffects of interferon alfa and gamma on human uveal melanoma cells in vitro.
BACKGROUND Uveal melanoma is a tumour with a high incidence of metastasis and a high mortality rate. Additional therapies to obtain a better local control or an effective treatment of metastases are necessary. Interferons may be applied. METHODS The effects of human interferon alfa and gamma on proliferation and expression of immunologically important molecules of human uveal melanoma cells i...
متن کاملذخیره در منابع من
با ذخیره ی این منبع در منابع من، دسترسی به آن را برای استفاده های بعدی آسان تر کنید
برای دانلود متن کامل این مقاله و بیش از 32 میلیون مقاله دیگر ابتدا ثبت نام کنید
ثبت ناماگر عضو سایت هستید لطفا وارد حساب کاربری خود شوید
ورودعنوان ژورنال:
- Molecular Vision
دوره 14 شماره
صفحات -
تاریخ انتشار 2008