Inhibition of CD146 lessens uveal melanoma progression through reducing angiogenesis and vasculogenic mimicry.

PURPOSE: Anti-angiogenesis drug therapy is ineffective in treating uveal melanoma since it only targets angiogenesis leaving vasculogenic mimicry aside. There is no effective clinical strategy targeting vasculogenic mimicry, yet. We show here that CD146 is a novel target to inhibit uveal melanoma progression since it regulates both uveal melanoma angiogenesis and vasculogenic mimicry activity. METHODS: CD146 inhibition was achieved with its specific siRNAs or antibody AA98. Tube formation and migration of primary human retinal microvascular endothelial cells and tube-like structure formation, migration, invasion of uveal melanoma cells were evaluated after CD146 inhibition. The underlying mechanisms were investigated by Western blot and immunofluorescence. Finally, uveal melanoma cells were injected subretinally into the eyes of nude mice and AA98 was administrated. Tumor size was revealed by H&E staining, and angiogenesis and vasculogenic mimicry were evaluated with CD31-PAS staining. RESULTS: CD146 inhibition induced declines in tube formation and migration of primary human retinal microvascular endothelial cells and tube-like structure formation of uveal melanoma cells. CD146 mediated VEGFR/AKT/p38/NF-κB and FAK/VE-cadherin signal cascades were partially responsible for these biological effects. CD146 blockade by siRNA or AA98 also resulted in inhibition of migration and invasion as well as EMT process of uveal melanoma cells. The physiological relevance of such declines was confirmed by showing that AA98 treatment markedly suppressed the tumor growth, angiogenesis and vasculogenic mimicry induced by implantation of uveal melanoma cells into the eyes of nude mice. CONCLUSIONS: CD146 is a novel mediator of both angiogenesis and vasculogenic mimicry in uveal melanoma. Its antibody AA98 has the potency to be developed as a new antibody drug for treating uveal melanoma. Our results warrant further assessment of CD146 as a potential target to improve therapeutic management of uveal melanoma in a clinical setting.

Additive Role of Immune System Infiltration and Angiogenesis in Uveal Melanoma Progression.

Uveal melanoma (UM) is a malignant tumor that arises in the melanocytes of the uveal tract. It is the most frequent eye cancer, and despite new therapeutic approaches, prognosis is still poor, with up to 50% of patients developing metastasis with no efficient treatment options available. In contrast to cutaneous melanoma, UM is considered an "immune-cold" tumor due to the low mutational burden and the unique immunosuppressive microenvironment. To gain insight into the role of the UM microenvironment in regard to prognosis and metastatic progression, we have performed a pool analysis characterizing the UM microenvironment by using a bioinformatic approach. A variety of scores based on gene expression measuring stromal infiltration were calculated and used to assess association with prognosis. As a result, the highest immune and stromal scores were associated with poor prognosis. Specifically, stromal cells (fibroblasts and endothelial cells), T cells CD8+, natural killer (NK) cells, and macrophages M1 and M2 infiltration were associated with poor prognosis. Contrary to other tumors, lymphocytic infiltration is related to poor prognosis. Only B cells were associated with more favorable prognosis. UM samples scoring high in both angiogenesis (Angio) and antigen presentation (AP) pathways showed a poor prognosis suggesting an additive role of both functions. Almost all these tumors exhibited a chromosome 3 monosomy. Finally, an enrichment analysis showed that tumors classified as high Angio-high AP also activated metabolic pathways such as glycolysis or PI3K-AKT-MTOR. In summary, our pool analysis identified a cluster of samples with angiogenic and inflammatory phenotypes exhibiting poor prognosis and metabolic activation. Our analysis showed robust results replicated in a pool analysis merging different datasets from different analytic platforms.

Density of PAS positive patterns in uveal melanoma: Correlation with vasculogenic mimicry, gene expression class, BAP-1 expression, macrophage infiltration, and risk for metastasis.

Purpose: Periodic acid-Schiff (PAS) positive patterns of vasculogenic mimicry (VM) have been associated with poor prognosis in uveal melanoma (UM). We examined these patterns with digital image analysis and transmission electron microscopy, and correlated them with BAP-1 expression, gene expression class, macrophage infiltration, and metastatic disease in full tumor cross-sections and intratumor regions. Methods: Thirty-two enucleated eyes with UM were stained immunohistochemically (BAP-1, laminin, CD31, and CD68) and with PAS without hematoxylin counterstain. Retrospective data on gene expression class and patient survival were retrieved. Tumor sections were digitally scanned and analyzed with the QuPath Bioimage analysis software, and imaged with transmission electron microscopy. Results: The mean area proportion covered by CD31, laminin, and PAS positive patterns in tumor cross-sections was 0.9% (SD 0.6), 3.0% (SD 1.9), and 8.4% (SD 5.9), respectively. PAS density was statistically significantly greater in tumors with gene expression class 2 (p=0.02). The cumulative 5-year metastasis-free survival decreased for each quartile of increased PAS density (1.0, 0.75, 0.40, and 0.17, p=0.004). Forty percent of the tumors had heterogeneous BAP-1 expression. Intratumor regions with low BAP-1 expression were more likely to harbor VM (p<0.0001), and had statistically significantly greater PAS density (p<0.0001) and number of CD68 positive cells (p=0.01). Conclusions: PAS positive patterns in UM are composed of a mixture of blood vessels and extracellular matrix (ECM), including VM. Increased density of PAS positive patterns correlated with gene expression class and metastasis, and colocated to tumor regions with macrophage infiltration and low BAP-1 expression.

Quantitative Evaluation of Vascular Microcirculation Using Contrast-Enhanced Ultrasound Imaging In Rabbit Models of Choroidal Melanoma.

Purpose: We investigated the application of contrast-enhanced ultrasound (CEUS) to quantify vascular perfusion in two animal models of choroidal melanoma. Methods: B16F10 and choroidal melanoma cell line (OCM1) melanoma xenografts were implanted into the cyclodialysis cleft of rabbits' right eyes. Fundus photography, indocyanine green angiography (ICGA), B-mode ultrasound, and CEUS were used to examine the tumors. Standard vascular parameters of CEUS imaging were evaluated, including the area under the receiver operating characteristic curve (AUC), peak intensity (IMAX), mean transit time (mTT), rise time, and time to peak. Kruskal-Wallis tests followed by Nemenyi tests and Bonferroni corrections were used to analyze the findings. Results: OCM1 and B16F10 animal models of choroidal melanoma were established. Tumor size was larger in the animals receiving B16F10 xenografts compared with the OCM1. Choroidal melanoma received a more abundant blood supply and had higher blood flow velocities than normal eye tissue. The animals receiving B16F10 xenografts had larger blood volume but smaller blood flow velocity compared with the OCM1 models. Vascular mimicry was present in seven of eight OCM1 tumors (87.5%). Growth and blood circulation patterns differed between OCM1 and B16F10 melanomas. Conclusions: CEUS could be used to noninvasively dynamically, and safely detect the microcirculation patterns, quantify blood circulation volume, and velocity in ocular melanoma in animal models. The blood circulation patterns of two models were different in both qualitatively and quantitative analysis under CEUS and ICGA.

Evaluation of nicotinamide as an anti-inflammatory and anti-angiogenic agent in uveal melanoma cell lines.

PURPOSE:: To investigate the effect of nicotinamide on the secretion of pro-an giogenic and pro-inflammatory cytokines in uveal melanoma cell lines. METHODS:: Two human uveal melanoma cell lines (92.1 and OCM-1) were treated with nicotinamide (10 mmol/L) or control media for 48 hours in culture. The su perna tant from each culture was used in sandwich enzyme-linked immuno sorbent assay-based angiogenesis and inflammation arrays to evaluate the effects of exogenously administered nicotinamide on the secretion of a total of 20 pro-an gio genic and pro-inflammatory proteins. RESULTS:: Seven pro-angiogenic cytokines were detected under control conditions for both uveal melanoma cell lines. Treatment with nicotinamide resulted in a significant decrease in secretion of the following pro-angiogenic cytokines: angiogenin, angiopoietin-2, epidermal growth factor, and vascular epithelial growth factor-A in the 92.1 cells; basic fibroblast growth factor in the OCM-1 cells; and placenta growth factor in both cell lines. Among the pro-inflammatory proteins, monocyte chemotactic protein-1 and interleukin-8 were expressed in both untreated cell lines and both were significantly reduced when treated with nicotinamide. CONCLUSIONS:: Results from this in vitro model suggest that nicotinamide may have anti-inflammatory and anti-angiogenic properties, which may open the possibility of using it as a chemopreventive agent for uveal melanoma; however, further studies including animal models are warranted.

Evaluation of nicotinamide as an anti-inflammatory and anti-angiogenic agent in uveal melanoma cell lines / Avaliação da nicotinamida como agente anti-inflamatório e anti-angiogênico em linhas celulares de melanoma uveal

ABSTRACT Purpose: To investigate the effect of nicotinamide on the secretion of pro-an giogenic and pro-inflammatory cytokines in uveal melanoma cell lines. Methods: Two human uveal melanoma cell lines (92.1 and OCM-1) were treated with nicotinamide (10 mmol/L) or control media for 48 hours in culture. The su perna tant from each culture was used in sandwich enzyme-linked immuno sorbent assay-based angiogenesis and inflammation arrays to evaluate the effects of exogenously administered nicotinamide on the secretion of a total of 20 pro-an gio genic and pro-inflammatory proteins. Results: Seven pro-angiogenic cytokines were detected under control conditions for both uveal melanoma cell lines. Treatment with nicotinamide resulted in a significant decrease in secretion of the following pro-angiogenic cytokines: angiogenin, angiopoietin-2, epidermal growth factor, and vascular epithelial growth factor-A in the 92.1 cells; basic fibroblast growth factor in the OCM-1 cells; and placenta growth factor in both cell lines. Among the pro-inflammatory proteins, monocyte chemotactic protein-1 and interleukin-8 were expressed in both untreated cell lines and both were significantly reduced when treated with nicotinamide. Conclusions: Results from this in vitro model suggest that nicotinamide may have anti-inflammatory and anti-angiogenic properties, which may open the possibility of using it as a chemopreventive agent for uveal melanoma; however, further studies including animal models are warranted.

RESUMO Objetivo: Acredita-se que a nicotinamida (NIC) seja capaz de diminuir a angiogênese induzida pelo fator de crescimento endotelial vascular (VEGF). Investigar os efeitos da nicotinamida sobre a secreção de citocinas pró-angiogênicas e pró-inflamatórias em linhagens de células de melanoma uveal humano (UM). Métodos: Duas linhagens de células humanas de UM (92,1 e OCM-1) foram tratadas com NIC (10 mmol/L) ou apenas com meio de cultura por 48 horas. O sobrenadante das culturas obtido após a administração de nicotinamida foi comparado com o sobrenadante das culturas controle quanto à expressão de 20 fatores pró-angiogênicos e pró-inflamatórios, pela técnica de enzyme-linked immunosorbent assay (ELISA). Resultados: Sete citocinas pró-angiogênicas foram detectadas nas condições de controle em ambas as linhagens de células de UM. O tratamento com nicotinamida promoveu uma redução significativa da secreção das seguintes citocinas angiogênicas: Angiogenina, ANG2, EGF e VEGF-A em células 92.1; bFGF em células OCM-1; PIGF em ambas as linhagens celulares. Quanto às proteínas pró-inflamatórias, a expressão de MCP-1 e IL-8 foi significativamente reduzida com a administração de nicotinamida em relação às culturas de células que não receberam o tratamento. Conclusões: Nicotinamida apresenta propriedades anti-inflamatórias e anti-angiogênicas em modelo experimental in vitro. Tais efeitos sugerem a possibilidade de utilizar esta substância na quimioprevenção do UM. Entretanto, estudos com modelos experimentais in vivo são necessários para melhor avaliar o benefício do tratamento do UM com nicotinamida.

The Long Non-Coding RNA RHPN1-AS1 Promotes Uveal Melanoma Progression.

Increasing evidence suggests that aberrant long non-coding RNAs (lncRNAs) are significantly correlated with the pathogenesis, development and metastasis of cancers. RHPN1 antisense RNA 1 (RHPN1-AS1) is a 2030-bp transcript originating from human chromosome 8q24. However, the role of RHPN1-AS1 in uveal melanoma (UM) remains to be clarified. In this study, we aimed to elucidate the molecular function of RHPN1-AS1 in UM. The RNA levels of RHPN1-AS1 in UM cell lines were examined using the quantitative real-time polymerase chain reaction (qRT-PCR). Short interfering RNAs (siRNAs) were designed to quench RHPN1-AS1 expression, and UM cells stably expressing short hairpin (sh) RHPN1-AS1 were established. Next, the cell proliferation and migration abilities were determined using a colony formation assay and a transwell migration/invasion assay. A tumor xenograft model in nude mice was established to confirm the function of RHPN1-AS1 in vivo. RHPN1-AS1 was significantly upregulated in a number of UM cell lines compared with the normal human retinal pigment epithelium (RPE) cell line. RHPN1-AS1 knockdown significantly inhibited UM cell proliferation and migration in vitro and in vivo. Our data suggest that RHPN1-AS1 could be an oncoRNA in UM, which may serve as a candidate prognostic biomarker and target for new therapies in malignant UM.

Novel mouse model for primary uveal melanoma: a pilot study.

BACKGROUND: To establish a mouse model with the aim of studying the tumour biology and metastasis formation of uveal melanoma. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Hypoxia-inducible factor 1 upregulation of both VEGF and ANGPTL4 is required to promote the angiogenic phenotype in uveal melanoma.

PURPOSE: Expression of the hypoxia-inducible factor (HIF)-1-regulated gene product, vascular endothelial growth factor (VEGF), correlates with tumor vascularity in patients with uveal melanoma (UM). While the relationship between HIF-1 and VEGF in cancer is well-studied, their relative contribution to the angiogenic phenotype in UM has not previously been interrogated. Here we evaluate the contribution of HIF-1, VEGF, and a second HIF-1-regulated gene product, angiopoietin-like 4 (ANGPTL4), to angiogenesis in UM. EXPERIMENTAL DESIGN: UM cells were examined for expression of HIF-1α, VEGF, and ANGPTL4. Their contribution to the angiogenic potential of UM cells was assessed using the endothelial cell tubule formation and directed in vivo angiogenesis assays. These results were corroborated in tissue from UM animal models and in tissue from patients with UM. RESULTS: Inhibition of VEGF partially reduced tubule formation promoted by conditioned medium from UM cells. Inhibition of ANGPTL4, which was highly expressed in hypoxic UM cells, a UM orthotopic transplant model, a UM tumor array, and vitreous samples from UM patients, inhibited the angiogenic potential of UM cells in vitro and in vivo; this effect was additive to VEGF inhibition. CONCLUSIONS: Targeting both ANGPTL4 and VEGF may be required for the effective inhibition of angiogenesis in UM.

Analysis of caveolin-1 and phosphoinositol-3 kinase expression in primary uveal melanomas.

BACKGROUND: To evaluate the regulation of blood supply in primary uveal melanomas through caveolin-1 (Cav-1)/phosphoinositol-3 kinase (PI3K). METHODS: The expression of Cav-1 and PI3K was analysed in 51 paraffin sections of metastatic (n = 30) and non-metastastic uveal melanomas (n = 21). Two trained observers quantified Cav-1 and PI3K immunofluorescensce expression by determining intensity of staining and percentage of positive cells. The expression was correlated with known prognostic factors. Besides angiogenesis by means of endoglin expression, the normal vasculature (von Willebrand Factor expression) was evaluated semi-quantitatively. Vasculogenic mimicry (VM) was analysed by CD31/PAS staining. RESULTS: All examined specimens expressed Cav-1 with a mean of 90.34% Cav-1 positive cells (range, 3.23-100%). Metastatic disease was associated with a higher Cav-1 expression. The correlation of Cav-1 with well-established prognostic factors showed a significant association between Cav-1 expression and largest tumour diameter (P = 0.022), tumour node metastasis classification (P = 0.008) and invasion of optic nerve head (P = 0.048). PI3K was expressed by all uveal melanomas with a mean of 87.28% cells showing PI3K expression. A higher level of PI3K was significantly associated with larger height (P = 0.042) and progressed tumour node metastasis stage (P = 0.016). The percentage of PI3K and Cav-1 positive cells were significantly associated (P = 0.034). For PI3K and Cav-1 expression a non-significant association with VM was shown (P = 0.064 and P = 0.072, respectively). No correlation of PI3K or Cav-1 with angiogenesis or mature vasculature was seen (P > 0.05). CONCLUSIONS: Cav-1 expression may be especially up-regulated in larger uveal melanomas. As it was correlated with PI3K expression and VM in this series of uveal melanoma, Cav-1 might induce the formation of VM via the PI3K-signalling cascade.

Intravitreally Injected HCmel12 Melanoma Cells Serve as a Mouse Model of Tumor Biology of Intraocular Melanoma.

PURPOSE: To establish a mouse model with histologic characteristics of uveal melanoma for investigation of intraocular tumor biology of melanoma. METHODS: After injection of 1 × 10(5) of HCmel12 melanoma cells, a cutaneous melanoma cell line, into the vitreous of CX3CR1(+/GFP) or C57Bl/6 mice (n = 12), tumor growth patterns, clinicopathological features, angiogenesis and metastatic behavior were analyzed by histology (hematoxylin and eosin, periodic acid-Schiff without hematoxylin) and immunohistochemistry (HMB45/MART-1-Ab, F4/80-Ab, green fluorescent protein (GFP)-Ab and VE-cadherin-Ab). RESULTS: HCmel12 cells formed intraocularly growing tumor masses, which showed histologic features of intraocular melanoma such as angiotropism, intratumoral endothelial-lined vasculature, vasculogenic mimicry including prognostic significant extravascular matrix patterns, and invasion by inflammatory cells, in particular macrophages. There was no difference in tumor growth characteristics between CX3CR1(+/GFP) and C57Bl/6 mice. Five of 10 mice proceeded to extrascleral tumor growth and three of these developed metastases. CONCLUSIONS: Intraocularly injected HCmel12 cells developed tumor masses with histologic characteristics of aggressive melanoma similar to human uveal melanoma. Since hematogenous dissemination to the liver was not observed, intravitreally injected HCmel12 cells do not qualify as a model for metastasizing intraocular melanoma. However, since the eye represents a semi-closed compartment with access to constant blood supply, these intraocular tumors represent a model for studies of isolated parameters in general tumor biology of intraocular melanoma.

The association of blood angioregulatory microRNA levels with circulating endothelial cells and angiogenic proteins in patients receiving dacarbazine and interferon.

BACKGROUND: Blood biomarkers are needed to monitor anti-angiogenic treatments for cancer. The association of blood levels of microRNAs (miRs) implicated in angiogenesis with circulating endothelial cells (CEC) and with angiogenic proteins was examined in patients administered drugs with anti-angiogenic activity. METHODS: Blood was collected from patients with uveal melanoma enrolled on an adjuvant therapy trial in which they were treated sequentially with dacarbazine and interferon-alfa-2b. Plasma levels of nine angioregulatory miRs, miR-16, 20a, 106a, 125b, 126, 146a, 155, 199a, and 221, were determined by quantitative real time polymerase chain reaction; CEC, by semi-automated immunomagnetic; and plasma angiogenic proteins, by enzyme linked immunosorbent assays. RESULTS: Levels of miR-199a were positively correlated and miR-106a negatively correlated with CEC pre-therapy. Decreases in miR-126 and miR-199a and increases in miR-16 and miR-106a were observed after interferon-alfa-2b, but not after dacarbazine. CEC also increased after treatment with interferon but not after treatment with dacarbazine. Levels of miRs did not correlate with levels of vascular endothelial growth factor, basic fibroblast growth factor, and interleukin-8. Angiogenic proteins also did not change significantly with treatment. CONCLUSIONS: Blood levels of specific angioregulatory miRs are associated with CEC, and changes in specific angioregulatory miRs parallel increases in CEC after treatment with interferon-alfa-2b. Blood levels of specific angioregulatory miRs are not associated with levels of angiogenic proteins. miRs warrant further evaluation as blood biomarkers of angiogenesis.

Transscleral optical spectroscopy of uveal melanoma in enucleated human eyes.

PURPOSE: The aims of this study were to use transscleral optical spectroscopy to analyze normal and tumor-infiltrated areas of enucleated human eyes, and to characterize the spectral properties of uveal melanomas in relation to various morphological features. METHODS: Nine consecutive eyes enucleated for uveal melanoma were examined by transscleral spectroscopy, using a fiber-optic probe that exerted a fixed pressure on the scleral surface. Spectroscopic measurements, covering the wavelength range of 400-1100 nm, were sequentially performed over the uveal melanoma and on the opposite (normal) side of each eye. The eyes were then processed for histological and immunohistochemical analyses. Comparisons between spectral and morphological parameters were performed by Spearman's rank correlation coefficient and unpaired t-test. RESULTS: The average reflection intensity obtained from the normal side of the eyes was higher than that from the tumors. The spectral imprint of hemoglobin was lower and that of water was considerably stronger when compared with the tumor side. The diffuse reflection spectra from the melanomas showed a strong correlation with the degree of tumor pigmentation (Spearman's rho = -0.87, P < 0.0001). A weaker correlation was observed between the amount of hemoglobin-related absorption and the density of intratumoral blood vessels (Spearman's rho = -0.25, P = 0.023). The mean diffuse reflection intensity obtained from the spindle cell melanomas was significantly higher than that from the mixed and epithelioid cell melanomas (P < 0.0001). CONCLUSIONS: Although future in vivo studies are required, these data suggest that transscleral optical spectroscopy is a feasible method for identification and morphological assessment of choroidal tumors.

Suppression of thrombospondin-1 expression during uveal melanoma progression and its potential therapeutic utility.

OBJECTIVES: To determine whether expression of thrombospondin-1 (TSP1), an endogenous inhibitor of angiogenesis, is downregulated during progression of uveal melanoma and whether administration of TSP1 and/or its antiangiogenic peptides attenuate tumor growth. METHODS: Tyrosinase-SV40 T-antigens (Tyr Tag) transgenic mice were used for evaluation of TSP1 expression during tumor progression using immunohistological methods. The therapeutic potential of TSP1 on tumor progression was evaluated either by crossing Tyr Tag mice with a line of transgenic mice overexpressing TSP1 in the eye or by administration of TSP1-mimetic peptide with known antiangiogenic, antitumor activity. Tumor areas were measured in histological sections using Optima software (Media Cybernetics, Inc). RESULTS: The Tyr Tag tumors from 3-week-old mice showed significant TSP1 expression, which was dramatically downregulated in tumors from 12-week-old mice. Furthermore, the development and progression of tumor was significantly delayed in Tyr Tag TSP1 transgenic mice or Tyr Tag mice receiving TSP1-mimetic peptide (100 mg/kg/d). CONCLUSIONS: Expression of TSP1 was decreased with the angiogenic switch during progression of uveal melanoma, and TSP1 and/or its antiangiogenic peptides were effective in attenuation of tumor growth. CLINICAL RELEVANCE: Modulation of TSP1 expression and/or activity may be beneficial in treating uveal melanoma.

In vivo high-frequency, contrast-enhanced ultrasonography of uveal melanoma in mice: imaging features and histopathologic correlations.

PURPOSE: To evaluate the usefulness of in vivo imaging of uveal melanoma in mice using high-frequency contrast-enhanced ultrasound (HF-CE-US) with 2D or 3D modes and to correlate the sonographic findings with histopathologic characteristics. METHODS: Fourteen 12-week-old C57BL6 mice were inoculated into their right eyes with aliquots of 5 × 10(5)/2.5 µL B16LS9 melanoma cells and were randomly assigned to either of two groups. At 7 days after inoculation, tumor-bearing eyes in group 1 (n = 8) were imaged using HF-CE-US to determine the 2D tumor size and relative blood volume; eyes in group 2 (n = 6) were imaged by 3D microbubble contrast-enhanced ultrasound, and the tumor volume was determined. Histologic tumor burden was quantified in enucleated eyes by image processing software, and microvascular density was determined by counting von Willebrand factor-positive vascular channels. Ultrasound images were evaluated and compared with histopathologic findings. RESULTS: Using HF-CE-US, melanomas were visualized as relatively hyperechoic regions. The intraobserver variability of sonographic measurements was 9.65% ± 7.89%, and the coefficient of variation for multiple measurements was 7.33% ± 5.71%. The correlation coefficient of sonographic volume or size and histologic area was 0.71 (P = 0.11) and 0.79 (P = 0.32). The relative blood volume within the tumor demonstrated sonographically correlated significantly with histologic tumor vascularity (r = 0.83; P < 0.001). CONCLUSIONS: There was a positive linear correlation between sonographic tumor measurements and histologic tumor burden in the mouse ocular melanoma model. Contrast-enhanced intensity corresponded with microvascular density and blood volume. HF-CE-US is a real-time, noninvasive, reliable method for in vivo evaluation of experimental intraocular melanoma tumor area and relative blood volume.

Macrophages in uveal melanoma and in experimental ocular tumor models: Friends or foes?

Macrophages belong to the innate immune system and as such constitute one of the first barriers against infection. They play an important role in wound healing, in inflammation and in angiogenesis, but are also essential in the first stage of a "danger response". After scavenging debris, they can digest cellular proteins into smaller pieces, and protein-derived peptides can subsequently be presented to the immune system. Depending on the activation state of the macrophage, this antigen presentation may trigger a full-blown active immune response, or may suppress a potential immune reaction. Macrophages constitute a heterogeneous cell population described by many names, with varying phenotypic characteristics, depending on their tissue location and state of activation. They play important roles in different ocular tissues, including the cornea and the choroid, and have been found to be involved in anti-tumor immune responses in mouse ocular tumor models. One would thus expect macrophages to belong to the "good guys" that help to protect our body against dangers such as cancer. In human uveal melanoma however, a high density of macrophages is associated with a poor prognosis for the patient. Macrophages play a role in promoting angiogenesis, and thus may stimulate tumor growth; in addition, macrophages have also been found to suppress anti-melanoma immune responses. These functions may shift during aging. Taken together, these new observations extend our understanding of the diverse functions of macrophages and show us their different faces, making them either "friends or foes" in human uveal melanoma. A better understanding of these multifaceted cells will help in developing new treatments to prevent the growth of metastases in uveal melanoma patients.

Bevacizumab suppression of 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 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 microg 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.

Constitutive overexpression of pigment epithelium-derived factor inhibition of ocular melanoma growth and metastasis.

PURPOSE: Pigment epithelium-derived factor (PEDF) is known to be an angiogenesis suppressor and to have antitumor effects. This study investigates whether constitutive overexpression of PEDF inhibits the growth and hepatic micrometastasis of ocular melanoma. METHODS: Real-time RT-PCR was used to detect endogenous PEDF expression in human uveal melanoma cell lines and mouse melanoma cells. A lentiviral vector containing a mouse PEDF expression sequence was constructed and transduced into mouse melanoma cells in vitro. Transgene expression was assessed by Western blot analysis. Angiogenesis and transendothelial migration assays were performed in constitutively stable PEDF-overexpressing cells and transduced lentiviral vector control cells. The size and microvessel density of the ocular tumor and the number of hepatic micrometastasis were compared between the mice inoculated with PEDF-overexpressing tumor cells and those mice with the control cell line. RESULTS: Four human uveal melanoma and three mouse melanoma cell lines were found to express PEDF mRNA. Endogenous overexpressing PEDF melanoma cells lost the ability to migrate and form tubes in vitro. In the animal experiment, the size of the ocular melanoma and the number of hepatic micrometastasis were decreased and microvessel density was also reduced in mice inoculated with constitutively overexpressing PEDF melanoma cells. CONCLUSIONS: Lentivirus-mediated gene transfer of PEDF decreased the growth of ocular melanoma and its hepatic micrometastasis in a mouse ocular melanoma model. Dual antitumor/antiangiogenic activities of PEDF suggest that PEDF gene therapy may be considered an approach for the treatment of ocular melanoma.