Primary Uveal Melanoma Cell Lines Research Articles

Calcium Electroporation Reduces Viability and Proliferation Capacity of Four Uveal Melanoma Cell Lines in 2D and 3D Cultures.

Simple SummaryCalcium electroporation (CaEP) is an innovative anti-tumor treatment modality that induces cell death by introducing supraphysiological concentrations of calcium into cells with a limited effect on normal cells. The objective of the present study is to assess the effect of CaEP in uveal melanoma (UM) cell lines in comparison to electrochemotherapy (ECT) with bleomycin using 2D monolayer cell cultures as well as 3D tumor spheroid models in four different UM cell lines. The morphological changes of the spheroids, the cell viability, growth rate as well as the cytotoxic effect of electroporation (EP) with calcium chloride and bleomycin were evaluated with various drug concentrations. The results of CaEP and ECT both suggest a comparable dose-dependent reduction in cell viability and proliferation rate in all tested 2D cell lines and 3D tumor spheroids. These data point out that CaEP is an established anticancer treatment causing cell death by ATP depletion in in vitro and in vivo, representing an efficient alternative therapy with a lower cytotoxic potency for the local UM tumor control.Electrochemotherapy (ECT) is the combination of transient pore formation following electric pulse application with the administration of cytotoxic drugs, which enhances the cytotoxic effect of the applied agent due to membrane changes and permeabilization. Although EP represents an established therapeutic option for solid malignancies, recent advances shift to the investigation of non-cytotoxic agents, such as calcium, which can also induce cell death. The present study aims to evaluate the cytotoxic effect, the morphological changes in tumor spheroids, the effect on the cell viability, and the cell-specific growth rate following calcium electroporation (CaEP) in uveal melanoma (UM) 2D monolayer cell cultures as well as in 3D tumor spheroid models. The experiments were conducted in four cell lines, UM92.1, Mel270, and two primary UM cell lines, UPMD2 and UPMM3 (UPM). The 2D and 3D UM cell cultures were electroporated with eight rectangular pulses (100 µs pulse duration, 5 Hz repetition frequency) of a 1000 V/cm pulse strength alone or in combination with 0.11 mg/mL, 0.28 mg/mL, 0.55 mg/mL or 1.11 mg/mL calcium chloride or 1.0 µg/mL or 2.5 µg/mL bleomycin. The application of calcium chloride alone induced an ATP reduction only in the UM92.1 2D cell cultures. Calcium alone had no significant effect on ATP levels in all four UM spheroids. A significant decrease in the intracellular adenosine triphosphate (ATP) level was documented in all four 2D and 3D cell cultures for both CaEP as well as ECT with bleomycin. The results suggest a dose-dependent ATP depletion with a wide range of sensitivity among the tested UM cell lines, control groups, and the applied settings in both 2D monolayer cell cultures and 3D tumor spheroid models. The colony formation capacity of the cell lines after two weeks reduced significantly after CaEP only with 0.5 mg/mL and 1.1 mg/mL, whereas the same effect could be achieved with both applied bleomycin concentrations, 1.0 µg/mL and 2.5 µg/mL, for the ECT group. The specific growth rate on day 7 following CaEP was significantly reduced in UM92.1 cell lines with 0.5 and 1.1 mg/mL calcium chloride, while Mel270 showed a similar effect only after administration of 1.1 mg/mL. UM92.1 and Mel270 spheroids exhibited lower adhesion and density after CaEP on day three in comparison to UPM spheroids showing detachment after day 7 following treatment. CaEP and bleomycin electroporation significantly reduce cell viability at similar applied voltage settings. CaEP may be a feasible and inexpensive therapeutic option for the local tumor control with fewer side effects, in comparison to other chemotherapeutic agents, for the treatment of uveal melanoma. The limited effect on normal cells and the surrounding tissue has already been investigated, but further research is necessary to clarify the effect on the surrounding tissue and to facilitate its application in a clinical setting for the eye.

Electrochemotherapy with Bleomycin Enhances Radiosensitivity of Uveal Melanomas: First In Vitro Results in 3D Cultures of Primary Uveal Melanoma Cell Lines

Simple SummaryUveal melanoma (UM) is the most common primary intraocular tumor in adults. Treatment options for UM include radiotherapy, thermotherapy and tumor resection. Electrochemotherapy (ECT) is a new therapeutic modality for local tumor control in various cancer entities. The current study assesses the radiosensitizing effect of concomitant ECT with bleomycin and irradiation on 3D tumor spheroids with primary and radioresistant uveal melanoma cell lines. The evaluation of the radiosensitizing effect of ECT as a drug delivery system was based on the changes in the spheroid growth, the cell viability as well as the cytotoxic long-term effect of the combined treatment. The primary cell lines showed a higher radiosensitivity and required lower irradiation and bleomycin doses in comparison to cell lines originating from previously irratiated tumors. ECT should be further assessed for its applicability in clinical settings as a therapeutic radiosensitizing option for radioresistant tumors. Electrochemotherapy (ECT) is emerging as a complementary treatment modality for local tumor control in various cancer entities. Irradiation is an established therapeutic option for oncologic patients, which is commonly combined with chemotherapy due to its insufficient targeting ability. The efficiency of radiotherapy for tumors can be enhanced with different radiosensitizers. ECT can potentiate the radiosensitizing effect of chemotherapeutic agents such as bleomycin. The present study aims to evaluate the radiosensitizing effect of concomitant ECT with bleomycin on 3D tumor spheroids with primary and radioresistant uveal melanoma cell lines (UPMD2, UPMM3, UM92.1, Mel270) and irradiation. The changes in the spheroid growth and the cell viability as well the cytotoxic long-term effect of the combination treatment were evaluated with various combinations of electroporation settings and bleomycin concentrations as well as radiotherapy doses. A broad range of radiosensitivity was documented among the spheroids from different uveal melanoma cell lines. The primary cell lines showed a higher radiosensitivity and required lower irradiation and bleomycin doses. The maximal tumor control with a reduction of cell survival <10% was achieved with a 5 Gy irradiation only in the primary uveal melanoma cell lines and in combination with all tested ECT settings, whereas the same result could be obtained in UM92.1 spheroids only after ECT with 20 Gy irradiation. Based on the spheroid growth and the measurement of the cross-sectional area, the Mel270 spheroids, originating from a previously irradiated recurrent uveal melanoma, required higher doses of bleomycin and ECT settings after irradiation with 5 Gy in order to achieve a significant growth reduction. No significant difference could be demonstrated for the reduction of cell viability in the combination therapy with 20 Gy and 1000 V/cm between 1 and 2.5 µg/mL bleomycin even in Mel270 spheroids, underlying the importance of a drug delivery system to potentiate the radiosensitizing effect of agents in lower doses. ECT should be further assessed for its applicability in clinical settings as a therapeutic radiosensitizing option for radioresistant tumors and a sufficient local tumor control with lower chemotherapy and irradiation doses.

Mitochondrial DNA polymorphisms and biogenesis genes in primary and metastatic uveal melanoma cell lines

Purpose: This study was designed to identify mitochondrial (mt) DNA variations in primary and metastatic uveal melanoma (UM) cell lines and their relation with cell metabolism to gain insight into metastatic progression.Method: The entire mtDNA genomes were sequenced using Sanger sequencing from two primary UM cell lines (92.1 and MEL270) and two cell lines (OMM2.3 and OMM2.5) derived from liver metastases of the MEL270 patient. The mtDNA copy numbers determined by the ratio of nDNA versus mtDNA. qRT-PCR was used to evaluate expression levels of mitochondrial biogenesis genes.Results: Sequencing showed that cell line MEL270 and metastases-derived OMM2.3 and OMM2.5 cell lines had homoplasmic single nucleotide polymorphisms (SNPs) representing J1c7a haplogroup, whereas 92.1 cells had mtDNA H31a haplogroup. mtDNA copy numbers were significantly higher in primary cell lines. The metastatic UM cells showed down-regulation of POLG, TFAM, NRF-1 and SIRT1 compared to their primary MEL270 cells. PGC-1α was downregulated in 92.1 and upregulated in MEL270, OMM2.3 and OMM2.5.Conclusions: Our finding suggests that within metastatic cells, the heteroplasmic SNPs, copy numbers and mitochondrial biogenesis genes are modulated differentially compared to their primary UM cells. Therefore, investigating pathogenic mtDNA variants associated with cancer metabolic susceptibility may provide future therapeutic strategies in metastatic UM.

Expression of Caveolin-1 in Human Cutaneous and Uveal Melanoma Cells

Caveolin-1 can act as a tumour promoter or suppressor depending on the cancer type and stage. In melanoma, information available concerning its expression is ambiguous. In this study, we investigated caveolin-1 mRNA and protein expression levels in human melanoma cell lines of different origin and progression stages. Metastatic cutaneous (WM-266-4, A375), primary cutaneous (WM- 115, IGR-39) and primary uveal (mel-202, 92-1) cells were used for quantitative RT-PCR, Western blotting and confocal microscopy. We observed significantly higher expression of caveolin-I mRNA in cutaneous than in uveal melanoma cells. In accordance, immunostaining of caveolin-I was stronger in cutaneous cell extracts, while protein bands of uveal origin displayed weak signals. Finally, we detected differences in the caveolin-I subcellular pattern of distribution between primary and metastatic cells. Overall, this is the first demonstration of caveolin-1 expression in human primary uveal melanoma cell lines and observation that the origin of cells (uveal/cutaneous) has an impact when considering the utility of caveolin-I as a melanoma cell marker.

Novel mouse model for primary uveal melanoma: a pilot study

To establish a mouse model with the aim of studying the tumour biology and metastasis formation of uveal melanoma. Two human primary uveal melanoma cell lines (UMT2 and UMT42) were injected into the choroid of BALB/c nude mouse eyes. Intraocular tumour growth and metastasis formation in the liver and lungs were assessed after 13 to 22 weeks. Formalin-fixed, paraffin-embedded material was processed via haematoxylin and eosin staining for histological examination and periodic acid Schiff staining to search for extravascular matrix patterns. Immunohistochemistry for Melan A, CD34 and Ki67 was performed to assess the expression of a melanocytic lineage marker, angiogenesis and proliferative activity. All eyes injected with UMT2 cells, but only 25% of eyes treated with UMT42, developed intraocular tumour growth. The morphology of intraocular melanomas resembled that of primary tumours and showed signs of malignancy, including retinal invasion, optic nerve invasion and scleral penetration with extraocular tumour growth. UMT2 tumours formed extravascular matrix patterns exclusively. Most of the tumour cells expressed Melan A. Intratumoural angiogenesis was detected in both tumour entities. Proliferative activity was verified in all but one tumour. However, no metastases appeared in the liver or lungs. The mouse model presented with the UMT2 cell line allows for investigations of tumour biology of the primary UM because of the high degree of similarity between the tumours generated in the mouse eyes and the corresponding primary human UM. Unfortunately, the model is not suitable for investigations of metastasis formation.

Differential Expression of Chemokine Receptors on Uveal Melanoma Cells and Their Metastases

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. 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 CXCR4 and CCR7. The effect of the liver and liver-borne factors on the expression of CXCR4 and CCR7 was determined after intracameral, intrasplenic, and subcutaneous transplantation of uveal melanoma cells in nude mice. Chemotactic responses of melanoma cells to liver-borne factors were determined by in vitro chemotaxis assays using protein extracts of hepatocytes and striated muscle tissue. All the primary uveal melanoma cell lines expressed CXCR4 and CCR7 message and protein, whereas the metastases cell lines expressed little or no chemokine receptor. Extracts of human liver cells stimulated chemotaxis of uveal melanoma cells, which could be inhibited by anti-CXCR4 antibody. Liver-borne factors also induced the downregulation of CXCR4 and CCR7 on uveal melanoma cells. Uveal melanoma cells maintained their high expression of CXCR4 and CCR7 after intracameral transplantation. However, CXCR4 and CCR7 expression was sharply reduced in liver metastases arising from intraocular melanomas. CXCR4 and CCR7 provide directional migration of uveal melanoma cells toward the liver, the most common site for the formation of uveal melanoma metastases. However, soluble factors elaborated by hepatocytes induce the downregulation of CXCR4 and CCR7 on metastatic uveal melanoma cells.

Effect of an anti‐CD54 (ICAM‐1) monoclonal antibody (UV3) on the growth of human uveal melanoma cells transplanted heterotopically and orthotopically in SCID mice

We have shown that administration of a novel anti-CD54 monoclonal antibody (UV3) results in long-term survival of SCID mice bearing human myeloma xenografts. Previous studies have demonstrated a link between the expression of CD54 and the progression of uveal melanoma. Our study assessed the expression of CD54 on 7 human uveal melanoma cell lines and 3 cell lines established from uveal melanoma metastases. In vivo studies examined the efficacy of systemic and local administration of UV3 antibody on the progression of uveal melanoma cells transplanted either heterotopically or orthotopically into SCID mice. Five of the 7 primary uveal melanoma cell lines and all 3 of the metastases cell lines expressed CD54. Intraperitoneal injection of either IgG or F(ab')2 fragments of UV3 significantly inhibited the growth of subcutaneous and intraocular melanomas. Subconjunctival injection of either IgG or F(ab')2 fragments of UV3 produced a significant reduction in the growth of intraocular melanomas, even if the antibody was administered after the appearance of intraocular tumors. The results indicate that both primary and metastatic human uveal melanoma cells express CD54. The marked inhibition of intraocular and subcutaneous uveal melanoma progression suggests that UV3 antibody is a promising therapeutic agent for further evaluation in patients with uveal melanoma. This is especially noteworthy, as no existing therapeutic modality prevents metastasis of uveal melanoma or prolongs the survival of patients with uveal melanoma.

Angiostatin produced by certain primary uveal melanoma cell lines impedes the development of liver metastases.

To evaluate the ability of human uveal melanomas to produce angiostatin in vitro and the effect of angiostatin on the development of liver metastases in vivo. Human uveal melanoma cell lines (OCM1, OCM3, MEL202, MEL285, 92-1, OM431, and OMM1) were assayed for their ability to produce angiostatin in vitro by an angiostatin bioassay and by Western blot analysis. The OCM3 and OMM1 tumor cells were inoculated either in the posterior or the anterior segment of nude mice. One group of mice in each experiment underwent enucleation and hepatic metastases were assayed by histopathologic and liver function analysis. OCM1, OCM3, and 92-1 cell lines significantly inhibited bovine endothelial cell proliferation in vitro and generated 38-Kd angiostatin molecules. Enucleation of eyes containing OCM3 in the posterior segment resulted in a higher number of metastatic foci (26.5) in that group compared with the nonenucleated group of mice (11.17). After enucleation, elevated levels of serum aspartate transaminase and alanine aminotransferase were observed in mice bearing OCM3 in either anterior or posterior segments. The enucleation of eyes containing OMM1 (nonangiostatin-producing cells) had no significant effect on liver metastasis. By removing a source of angiostatin, enucleation of melanoma-containing eyes may unwittingly exacerbate the metastatic potential of uveal melanomas. In certain circumstances, enucleating a melanoma-containing eye may unwittingly exacerbate metastatic disease. The results also suggest that exogenous angiostatin may have potential therapeutic implications in the management of patients with primary intraocular melanomas.

Human uveal melanoma cells produce macrophage migration-inhibitory factor to prevent lysis by NK cells.

Human uveal melanoma arises in an immune privileged ocular environment in which both adaptive and innate immune effector mechanisms are suppressed. Uveal melanoma is the most common intraocular tumor in adults and is derived from tissues in the eye that produce macrophage migration-inhibitory factor (MIF), a cytokine that has recently been demonstrated to produce immediate inhibition of NK cell-mediated lytic activity. Although NK cell-mediated lysis of uveal melanomas is inhibited in the eye, melanoma cells that disseminate from the eye are at risk for surveillance by NK cells. Moreover, uveal melanoma cells demonstrate a propensity to metastasize to the liver, an organ with one of the highest levels of NK activity in the body. Therefore, we speculated that uveal melanomas produced MIF as a means of escaping NK cell-mediated lysis. Accordingly, seven primary uveal melanoma cell lines and two cell lines derived from uveal melanoma metastases were examined for their production of MIF. MIF was detected in melanoma culture supernatants by both ELISA and the classical bioassay of macrophage migration inhibition. Melanoma-derived MIF inhibited NK cell-mediated lysis of YAC-1 and uveal melanoma cells. Cell lines derived from uveal melanoma metastases produced approximately twice as much biologically active MIF as cultures from primary uveal melanomas. Inhibition of NK cell-mediated killing by uveal melanoma-derived MIF was specifically inhibited in a dose-dependent manner by anti-MIF Ab. The results suggest that human uveal melanoma cells maintain a microenvironment of immune privilege by secreting active MIF that protects against NK cell-mediated killing.

Establishment and characterization of primary and metastatic uveal melanoma cell lines

We report on the establishment and characterization of 2 primary (EOM-3, EOM-29) and 3 metastatic uveal melanoma cell lines (OMM-1, OMM-2, OMM-3) and further cytogenetic characterization of a previously described primary uveal melanoma cell line (OCM-1). Only a few long-term growing primary uveal melanoma cell lines have as yet been established, while of metastatic uveal melanoma cell lines we have found no descriptions. The morphology of the in vitro cultured cells varied from spindle to epithelioid. The cell lines were characterized by immunocytochemistry, electron microscopy and cytogenetical analysis. The relative growth rate was determined by bromodeoxyuridine (BUdR) incorporation. The melanocytic origin of the cell lines was determined by positive staining with antibodies identifying melanoma-associated antigens. Melanosomes and pre-melanosomes were indeed observed by electron microscopy in all cell lines. The stem-cell karyotype was found to be normal in 3 cell lines (EOM-29, OMM-2, OMM-3) and abnormal in 3 others (EOM-3, OCM-1, OMM-1) showing a net loss of chromosome 6. The OCM-1 and the OMM-1 cell lines even demonstrated a large amount of structural chromosomal aberrations, the former being near-tetraploid and the latter triploid. The EOM-29 cell line, cultured from a ciliary body melanoma, did not show the previously described chromosome 3 and 8 abnormalities.

Effects of interferon alfa and gamma on human uveal melanoma cells in vitro.

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. The effects of human interferon alfa and gamma on proliferation and expression of immunologically important molecules of human uveal melanoma cells in vitro were studied. A propidium iodide assay was used to determine proliferation and immunostaining with monoclonal antibodies was applied to detect changes in antigen expression on two primary uveal melanoma cell lines, Mel 202 and 92-1. Interferon alfa inhibited proliferation of cell line 92-1 at a concentration of 50 IU/ml, but had no effect on cell line Mel 202, while interferon gamma inhibited growth of both cell lines. Only interferon gamma had a visible effect on cell morphology. With respect to the immunomodulatory effects, interferon alfa increased monomorphic HLA class I expression, but did not affect HLA class II expression. Interferon gamma induced not only HLA class I but also class II expression. The effects on HLA expression were locus-specific with the strongest effects observed for HLA-B and DR products. Small differences were observed with respect to the susceptibility of two different melanoma cell lines to antiproliferative effects and to modulation of antigen expression. The effects of interferon alfa and gamma on human uveal melanoma cells in vitro suggest a potential role of these cytokines in the treatment of patients with uveal melanoma. In particular, the immunomodulatory effects of these cytokines in vitro imply that treatment of patients with these cytokines might stimulate a beneficial antimelanoma immune response in vivo.