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2.  Inflammatory Cytokines in Eyes with Uveal Melanoma and Relation with Macrophage Infiltration 
Eyes with uveal melanoma have been shown to have multiple cytokines and chemokines in the aqueous humor. This study was conducted to determine whether a relation exists between the presence of these cytokines and the prognostic factors and survival of uveal melanoma.
Purpose.
The presence of an inflammatory phenotype, characterized by an increased expression of HLA antigens and an immunologic infiltrate, carries a bad prognosis in uveal melanoma. This study was conducted to determine whether the aqueous humor (AqH) from eyes with uveal melanoma contains inflammatory cytokines and whether their presence is associated with inflammation.
Methods.
Immediately after enucleation, AqH was obtained from 37 eyes containing uveal melanoma. Samples were stored at −80°C until use. Fifteen different cytokines were measured with a multiplex bead array. Intratumoral macrophages were analyzed by immunohistochemistry and immunofluorescence staining. The presence of specific cytokines was compared with histopathologic, genetic, and clinical tumor characteristics, as well as patient survival.
Results.
Several cytokines showed significantly higher expression in the AqH of uveal melanoma–containing eyes than in the AqH of eyes undergoing cataract surgery. MCP-3 was associated with the presence of CD68+ macrophages. Correlations were found between some cytokine levels and a few known prognostic factors of uveal melanoma, but cytokine levels were not of predictive value for survival.
Conclusions.
Uveal melanoma–containing eyes often carry increased levels of inflammation-related cytokines in their AqH. However, the presence of most specific cytokines was not related to the presence of macrophages, clinical or histopathologic parameters, or prognosis.
doi:10.1167/iovs.10-5526
PMCID: PMC3261048  PMID: 20538984
3.  Presence and phenotype of dendritic cells in uveal melanoma 
Background
Uveal melanoma arises in an immune‐privileged site and can itself add to the immunosuppressive environment. Previous studies on cutaneous melanoma have shown the presence of tolerogenic dendritic cells (DCs), which could play an important role in the progression of the tumour.
Aim
To examine the presence and functional status of DCs in a small series of uveal melanomas.
Methods
10 cases of uveal melanoma were examined for the expression of FXIIIa, CD68, human leucocyte antigen (HLA)‐DR, CD40, CD83, transforming growth factor βR1 and indolamine 2,3 dioxygenase by immunohistochemical analysis on sections embedded in paraffin wax.
Results
CD68‐positive macrophages were present in all of the tumours and were evenly distributed throughout. DCs expressing FXIIIa‐positive were seen in 7 cases, and were often found concentrated in foci within the tumour mass. These cells were dendritic and expressed high levels of HLA‐DR. The DCs did not express the maturation markers CD83 or CD40. In one case, concentration of DCs around the area of tumour necrosis was observed, and some of these cells expressed CD83.
Conclusion
Numerous tolerising antigen‐presenting cells may play a role in melanoma‐related immunosuppression in the eye, although activation of DCs may be associated with tumour necrosis.
doi:10.1136/bjo.2006.110908
PMCID: PMC1955657  PMID: 17347328
4.  Genetics of Primary Intraocular Tumors 
Primary intraocular neoplasms are tumors that originate within the eye. The most common malignant primary intraocular tumor in adults is uveal melanoma and the second is primary intraocular lymphoma or vitreoretinal (intraocular) lymphoma. The most common malignant intraocular tumor in children is retinoblastoma. Genetics plays a vital role in the diagnosis and detection of ocular tumors. In uveal melanoma, monosomy 3 is the most common genetic alteration and somatic mutations of BAP1, a tumor suppressor gene, have been reported in nearly 50% of primary uveal melanomas. The retinoblastoma gene RB1 is the prototype tumor suppressor gene—mutations in RB1 alleles lead to inactivated RB protein and the development of retinoblastoma. Immunoglobulin heavy chain (IgH) or T-cell receptor (TCR) gene rearrangement is observed in B-cell or T-cell primary vitreoretinal lymphoma, respectively. Other factors related to the genetics of these three common malignancies in the eye are discussed and reviewed.
doi:10.3109/09273948.2012.702843
PMCID: PMC3436423  PMID: 22834783
Choroidal malignant melanoma; primary intraocular lymphoma; primary vitreoretinal lymphoma; retinoblastoma; uveal melanoma
5.  High levels of Hdmx promote cell growth in a subset of uveal melanomas 
The p53 tumor suppressor pathway is inactivated in cancer either via direct mutation or via deregulation of upstream regulators or downstream effectors. P53 mutations are rare in uveal melanoma. Here we investigated the role of the p53 inhibitor Hdmx in uveal melanoma. We found Hdmx over-expression in a subset of uveal melanoma cell lines and fresh-frozen tumor samples. Hdmx depletion resulted in cell-line dependent growth inhibition, apparently correlating with differential Hdm2 levels. Surprisingly, p53 knockdown hardly rescued cell cycle arrest and apoptosis induction upon Hdmx knockdown, whereas it effectively prevented growth suppression induced by the potent p53 activator Nutlin-3. In addition, two compounds inhibiting Hdmx function or expression, SAH-p53-8 and XI-011, also elicited a growth inhibitory effect in a partly p53-independent manner. These findings suggest a novel, growth-promoting function of Hdmx that does not rely on its ability to inhibit p53. We provide evidence for a contribution of p27 protein induction to the observed p53-independent G1 arrest in response to Hdmx knockdown. In conclusion, our study establishes the importance of Hdmx as an oncogene in a subset of uveal melanomas and widens the spectrum of its function beyond p53 inhibition.
PMCID: PMC3433101  PMID: 22957303
Uveal melanoma; Hdmx; p53; Nutlin-3; p27; SAH-p53-8; XI-011; retinoblastoma
6.  Mda-9/Syntenin Is Expressed in Uveal Melanoma and Correlates with Metastatic Progression 
PLoS ONE  2012;7(1):e29989.
Uveal melanoma is an aggressive cancer that metastasizes to the liver in about half of the patients, with a high lethality rate. Identification of patients at high risk of metastases may provide indication for a frequent follow-up for early detection of metastases and treatment. The analysis of the gene expression profiles of primary human uveal melanomas showed high expression of SDCBP gene (encoding for syndecan-binding protein-1 or mda-9/syntenin), which appeared higher in patients with recurrence, whereas expression of syndecans was lower and unrelated to progression. Moreover, we found that high expression of SDCBP gene was related to metastatic progression in two additional independent datasets of uveal melanoma patients. More importantly, immunohistochemistry showed that high expression of mda-9/syntenin protein in primary tumors was significantly related to metastatic recurrence in our cohort of patients. Mda-9/syntenin expression was confirmed by RT-PCR, immunofluorescence and immunohistochemistry in cultured uveal melanoma cells or primary tumors. Interestingly, mda-9/syntenin showed both cytoplasmic and nuclear localization in cell lines and in a fraction of patients, suggesting its possible involvement in nuclear functions. A pseudo-metastatic model of uveal melanoma to the liver was developed in NOD/SCID/IL2Rγ null mice and the study of mda-9/syntenin expression in primary and metastatic lesions revealed higher mda-9/syntenin in metastases. The inhibition of SDCBP expression by siRNA impaired the ability of uveal melanoma cells to migrate in a wound–healing assay. Moreover, silencing of SDCBP in mda-9/syntenin-high uveal melanoma cells inhibited the hepatocyte growth factor (HGF)-triggered invasion of matrigel membranes and inhibited the activation of FAK, AKT and Src. Conversely syntenin overexpression in mda-9/syntenin-low uveal melanoma cells mediated opposite effects. These results suggest that mda-9/syntenin is involved in uveal melanoma progression and that it warrants further investigation as a candidate molecular marker of metastases and a potential therapeutic target.
doi:10.1371/journal.pone.0029989
PMCID: PMC3258266  PMID: 22267972
7.  Bevacizumab and intraocular tumors: an intriguing paradox 
Molecular Vision  2012;18:2454-2467.
Purpose
Bevacizumab, a humanized monoclonal antibody to vascular endothelial growth factor-A (VEGF-A), was originally developed as an anti-tumor treatment. In ocular oncology, it is being used to treat macular edema due to radiation retinopathy, but it may also be useful for the treatment of primary uveal melanoma (UM) or its metastases. We determined the effect of bevacizumab on the growth of B16F10 cells inside the eye and on B16F10 and UM cells cultured in vitro.
Methods
B16F10 melanoma cells were placed into the anterior chamber of the eye of C57Bl/6 mice and tumor growth was monitored after injection of different doses of bevacizumab or mock injection. In addition, the effect of bevacizumab on in vitro growth of B16F10 and human UM cells and on the expression of VEGF-A, GLUT-1, and HIF-1α was evaluated.
Results
Following intraocular injection of bevacizumab into murine B16 tumor-containing eyes, an acceleration of tumor growth was observed, with the occurrence of anterior chamber hemorrhages. Bevacizumab did not affect proliferation of B16F10 cells in vitro, while it inhibited UM cell proliferation. Expression analysis demonstrated that addition of bevacizumab under hypoxic conditions induced VEGF-A, GLUT-1 and HIF-1α in B16F10 cells as well as in UM cell lines and two of four primary UM tumor cultures.
Conclusions
In contrast with expectations, intraocular injection of bevacizumab stimulated B16F10 melanoma growth in murine eyes. In vitro exposure of B16 and human UM cells to bevacizumab led to paradoxical VEGF-A upregulation. The use of VEGF inhibitors for treatment of macular edema (due to radiation retinopathy) after irradiation of UM should be considered carefully, because of the possible adverse effects on residual UM cells.
PMCID: PMC3472924  PMID: 23077404
9.  Bevacizumab Suppression of Establishment of Micrometastases in Experimental Ocular Melanoma 
The administration of the anti-VEGF agent, bevacizumab, decreases tube formation and migration of human ocular melanoma cells, cultured alone or with HUVECs in vitro. The authors found that systemic bevacizumab decreased the size of ocular melanoma and the number of micrometastases in a mouse model of metastatic ocular melanoma. These findings are important with regard to a potential systemic therapy for patients with ocular melanoma with micrometastasis to the liver, as there is currently no effective treatment.
Purpose.
This study was undertaken to determine whether anti-vascular endothelial growth factor (VEGF) therapy inhibits growth of primary uveal melanoma and spread of its hepatic micrometastases.
Methods.
The human uveal melanoma cell lines Mel290 and Mel 270, HUVECs, mouse B16LS9 melanoma cells, and mouse vascular endothelial cells were separately cultured or co-cultured and incubated with bevacizumab or IgG1. The level of VEGF protein in the culture medium was measured by ELISA. In vitro angiogenesis and invasion assays were performed under bevacizumab or IgG1 treatment. Mel290 or B16LS9 cells were inoculated into NU/NU or C57Bl/6 mouse eyes which were enucleated after 7 days. The sizes of the intraocular tumors were determined. Time and dosage experiments were performed by using 50 or 250 μg bevacizumab starting at day 1 or 4 after inoculation. Hepatic micrometastases were enumerated. Proliferation, apoptosis, and angiogenesis markers were detected in the ocular tumor by immunofluorescence staining.
Results.
Bevacizumab significantly reduced the level of VEGF in the culture media from human uveal melanoma cells, mouse melanoma cells, and co-cultured cells. It also inhibited cell tube formation and decreased in vitro invasion of tumor cells. In the mouse model, bevacizumab suppressed primary ocular melanoma growth and the formation of hepatic micrometastases in a dose-dependent manner. Furthermore, immunohistochemical staining showed decreased Ki67 and unchanged caspase 3 expression after treatment with bevacizumab.
Conclusions.
Treatment with bevacizumab suppressed in vitro growth and in vivo hepatic micrometastasis of ocular melanoma cells. Bevacizumab is a potential therapeutic agent for the treatment of uveal melanoma micrometastases.
doi:10.1167/iovs.09-4755
PMCID: PMC2874122  PMID: 20089875
10.  Impact of Tumor-Associated Macrophages in LHBETATAG Mice on Retinal Tumor Progression: Relation to Macrophage Subtype 
In the present study, the impact of tumor-associated macrophages on retinal tumor growth in LHBETATAG mice was evaluated.
Purpose.
To determine the distribution of tumor-associated macrophages (TAMs) during retinoblastoma tumor development, examine the contribution of bone marrow–derived TAMs in retinoblastoma tumors, and evaluate the supportive role of TAMs in tumor growth in a transgenic retinoblastoma mouse model.
Methods.
The time course of macrophage infiltration in transgenic retinoblastoma tumors was assessed by immunohistochemistry at different time points in tumorigenesis. The origin of TAMs in transgenic retinoblastoma tumors was determined by transplanting 107 bone marrow cells from green fluorescent protein (GFP)–positive 16-week-old mice into age-matched, irradiated LHBETATAG mice via tail vein injections. Macrophage depletion was performed by subconjunctival (SC) delivery of liposomal clodronate.
Results.
The density of TAMs increased from 4 to 12 weeks of age in mice with small to medium tumors (P = 0.037) and remained stable in the later stages of disease (i.e., 16 weeks old with large tumors; P = 0.20). In 16-week-old mice, 38% (2.5 ± 3.2 cells per 400× high-power field) of TAMs were GFP-positive, bone marrow–derived macrophages. Total TAM depletion was associated with a significant decrease in the expression levels of MMP-9 (P = 0.014) and mature vessels (P < 0.001) and a nonsignificant decrease in the density of neovessels (P = 0.94). The density of M2-polarized TAMs did not change significantly after TAM depletion (P = 0.68). After M1-polarized TAM depletion, the tumor burden increased (P = 0.056).
Conclusions.
This work extends understanding of the complex role that macrophages play in retinoblastoma. Macrophage modulation in the tumor microenvironment is a critical factor in retinoblastoma tumor progression.
doi:10.1167/iovs.09-4255
PMCID: PMC2868494  PMID: 20053982
11.  Bevacizumab Suppresses Establishment of Micrometastases in Experimental Ocular Melanoma 
Purpose
This study was undertaken to determine whether anti-vascular endothelial growth factor (VEGF) therapy inhibits growth of primary uveal melanoma and spread of its hepatic micrometastasis.
Methods
Human uveal melanoma cell lines Mel290, Mel 270, and HUVECs, mouse B16LS9 melanoma cells and mouse vascular endothelial cells were separately cultured or co-cultured and incubated with bevacizumab or IgG1. The level of VEGF protein in the culture media was measured by ELISA. In vitro angiognesis and invasion assays were performed under bevacizumab or IgG1 treatment. Mel290 or B16LS9 cells were inoculated into NU/NU or C57Bl/6 mice eyes which were enucleated after 7 days. The sizes of the intraocular tumors were determined. Time and dosing experiments were performed using 50μg or 250μg of bevacizumab starting at day 1 or 4 after inoculation. Hepatic micrometastases were enumerated. Proliferation, apoptosis and angiogenesis markers were detected in the ocular tumor by immunofluorescence staining.
Results
Bevacizumab significantly reduced the level of VEGF in the culture media from human uveal melanoma cells, mouse melanoma cells, and co-cultured cells. Bevacizumab also inhibited cell tube formation and decreased in vitro invasion of tumor cells. We found in a mouse model that bevacizumab suppressed primary ocular melanoma growth and the formation of hepatic micrometastases in a dose-dependent manner. Furthermore, immunohistochemical staining showed decreased Ki67 and unchanged caspase 3 expression after treatment with bevacizumab.
Conclusions
Treatment with bevacizumab suppressed in vitro growth and in vivo hepatic micrometastasis of ocular melanoma cells. Bevacizumab is a potential therapeutic agent for the treatment of uveal melanoma micrometastases.
doi:10.1167/iovs.09-4755
PMCID: PMC2874122  PMID: 20089875
13.  Activation of tonsil dendritic cells with immuno-adjuvants 
BMC Immunology  2008;9:10.
Background
Dendritic cells (DC) play the key role in directing antigen-specific immune responses and manipulating their function may be a useful tool for immunotherapy. The balance between immune stimulation and tolerance is particularly important at mucosal interfaces, where discrimination between dangerous pathogens and innocuous antigens takes place. In humans, although much is known about the responses of monocyte derived DC, relatively little is known about effect of immuno-stimulatory adjuvants on DC found in tonsil.
Results
To examine this, tonsil DC were isolated and cultured with potent DC activators; IFNγ, anti-CD40 antibody, LPS and Poly I:C either singly or in combination. To measure maturation and activation, DC were examined for changes in the expression of HLA-DR, HLA- class I, CD83, CD40, CD80 and CD86 and the release of IL12p70.
The DC isolated from tonsil were a mixed population containing both myeloid and plasmacytoid DC, but all showed similar responses. Tonsil DC released IL12p70 upon stimulation with IFNγ , anti-CD40 antibody, and LPS, but unlike monocyte-derived DC, they did not increase the expression of cell surface activation molecules above those induced by culture alone. Poly I:C, a potent stimulator of laboratory generated DC inhibited the activation of tonsil DC by other adjuvants.
Conclusion
As the response of this mixed population of DC does not mirror that of DC generated in vitro, this may have implications for other tissue residing DC and might be an important consideration for immunotherapy.
doi:10.1186/1471-2172-9-10
PMCID: PMC2311271  PMID: 18366670
14.  Triamcinolone acetonide and anecortave acetate do not stimulate uveal melanoma cell growth 
Molecular Vision  2008;14:1752-1759.
Purpose
Radiotherapy-induced radiation retinopathy can develop in over 40% of eyes treated for uveal melanoma. Triamcinolone acetonide (TA) and anecortave acetate (AA) can be used to treat radiation retinopathy. It is not known whether TA or AA has any effect on potentially still viable uveal melanoma cells in the choroid after radiotherapy. We therefore studied the effect of these drugs on the proliferation of uveal melanoma cell lines in vitro. Furthermore, as these drugs are supposed to counteract vascular leakage, we determined their effect on the expression and production of the proangiogenic vascular endothelial growth factor-A (VEGF-A), the antiangiogenic pigment epithelium-derived factor (PEDF), and thrombospondin-1 (TSP-1) in uveal melanoma cells.
Methods
Three uveal melanoma cell lines were treated in vitro with TA or AA. Cell proliferation was measured by counting cells and using the Water-Soluble Tetrazolium Salt-1 (WST-1) assay. VEGF-A and PEDF production was measured by ELISA, and intracellular expression of angiogenic-associated genes including VEGF-A, PEDF, and TSP-1 was determined by real-time quantitative RT–PCR.
Results
We found no effect of TA or AA on tumor cell growth or production of VEGF-A and PEDF in any of the three uveal melanoma cell lines tested. Regarding expression as measured by RT–PCR, TA had an inhibiting effect on TSP-1 in only one cell line, and no effect on VEGF-A or PEDF. AA showed a similar lack of effect.
Conclusions
Since TA and AA do not stimulate uveal melanoma cell growth, it seems to be safe to use these drugs to treat radiation retinopathy after irradiation for uveal melanoma. Additional experiments using more cell lines or primary tumor cell cultures are needed to validate this conclusion. Furthermore, the results of our study suggest that TA does not exert its antileakage effect through downregulation of VEGF-A or upregulation of TSP-1 or PEDF in uveal melanoma cell lines. It is possible that TA and AA influence these pro- and antiangiogenic factors only under hypoxic circumstances. Further investigation is needed.
PMCID: PMC2556975  PMID: 18836566
15.  Macrophages feel their age in macular degeneration 
The Journal of Clinical Investigation  2007;117(11):3182-3184.
Macular degeneration, during which the posterior part of the eye known as the macula suffers from thinning, atrophy, and bleeding caused by abnormal angiogenesis (blood vessel formation), predominantly affects elderly adults and results in the loss of central vision. In this issue of the JCI, Kelly et al. investigate the regulation of innate immune cells, specifically macrophages, in ocular neovascularization following eye injury in mice (see the related article beginning on page 3421). They found that, as the mice aged, increased expression of IL-10 by senescent macrophages and changes in their expression of other cytokines altered the ability of these cells to restrain trauma-induced angiogenesis in the eye. These data provide insight into the effect of senescence on macrophage function and angiogenesis and have important implications for age-related diseases such as macular degeneration.
doi:10.1172/JCI34070
PMCID: PMC2045629  PMID: 17975664

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