Purpose: Uveal melanoma (UM) typically spreads to the liver, where it is incurable, as there are limited therapeutic interventions available. This study aimed to standardize laboratory methods for generating three-dimensional (3D) spheroids using UM cell lines and primary UM (PUM) samples for use in drug screening. Methods: Six UM cell lines and nine PUM, of differing genetic characteristics were cultured in two dimensions (2D) and three dimensions. 3D spheroid formation and growth were time monitored, and ImageJ software was used to calculate cross-sectional areas. PUM spheroids underwent immunohistochemistry for melanoma markers, nuclear BAP1, and cell proliferation. Chromosomal alterations in patient UM biopsies were compared with the corresponding 3D spheroid. In vitro drug assays testing doxorubicin and selumetinib assessed drug penetration and toxicity after 48 hours using imaging and the CellTiter-Glo 3D Cell Viability Assay. Results: All six UM cell lines formed spheroids of varying sizes and compactness; six of the nine PUM samples (67%) also formed spheroids, composed of MelanA+ proliferating melanocytes and admixed macrophages. PUM spheroids were genetically identical to the original sampled tumor. In vitro drug assays showed varying penetrations into UM cell line spheroids, with doxorubicin passing into the spheroid core and selumetinib having an effect largely on peripheral cells. Both drugs caused a dose-dependent reduction in viability of 3D spheroid cells. Conclusions: UM cell lines and PUM samples can successfully generate uniform 3D spheroids. PUM spheroids retain histological and genetic characteristics of the primary tumor. 3D spheroids are an important system for use in future high-throughput drug testing. Translational Relevance: The use of 3D spheroids allows early-phase drug screening and is an important first step toward treatment personalization for UM patients.

Uveal melanoma (UM) is a rare tumour of the eye, characterised by a high propensity to metastasise in half of all patients, most frequently to the liver. Although there are effective treatment options for the primary tumour, once metastasis has occurred prognosis is poor, with overall survival limited to months. Currently, there are no effective treatments for metastatic UM, despite the tumour having a well-defined signalling pathway to which many therapies have been directed. In an effort to develop novel treatment approaches, understanding the role of other signalling molecules, such as microRNAs, is fundamental. MicroRNAs (miRNAs) are small non-coding RNA molecules involved in posttranscriptional gene regulation, resulting in reduced target gene expression and subsequent protein translation. In UM, several dysregulated miRNAs have been proposed to play a functional role in disease progression, whereas others have been put forward as clinical biomarkers of high-risk disease following isolation from blood, plasma and exosomes. Most recently, analyses of large datasets have identified promising prognostic miRNA signatures and panels. This review navigates the plethora of aberrant miRNAs disclosed so far in UM, and maps these to signalling pathways, which could be targeted in future therapies for the disseminated disease.

Uveal melanoma (UM) is a rare aggressive intraocular tumour that spreads most commonly to the liver in tumours with loss of one copy of chromosome 3 (HR-M3); current treatments for metastatic disease remain largely ineffective. Pre-clinical research is increasingly using three-dimensional models that better recapitulate the tumour microenvironment (TME). One aspect of the TME is the acellular extracellular matrix (ECM) that influences cell proliferation, migration and response to therapy. Although commercial matrices are used in culture, the composition and biochemical properties may not be representative of the tumour ECM in vivo. This study identifies UM metastatic risk specific ECM proteins by developing methodology for decellularisation of low- and high- metastatic risk tissue samples (LR-D3 vs. HR-M3). Proteomic analysis revealed a matrisome signature of 34 core ECM and ECM-associated proteins upregulated in HR-M3 UM. Combining additional UM secretome and whole cell iTRAQ proteomic datasets revealed enriched GO and KEGG pathways including ‘regulating ECM binding’ and ‘PI3K/Akt signalling’. Structural analyses of decellularised matrices revealed microarchitecture of differing fibre density and expression differences in collagen 4, collagen 6A1 and nidogen 1, between metastatic risk groups. This approach is a powerful tool for the generation of ECM matrices relevant to high metastatic risk UM.

Purpose: Dual-specificity phosphatase 4 (DUSP4) inactivates factors in the mitogen-activated protein kinase (MAPK) signaling cascade, activated in uveal melanoma (UM) by mutations in upstream G-protein α subunits GNAQ/11 in >90% cases. This study examined whether DUSP4 (1) protein expression in primary UM (pUM) was a biomarker of metastatic risk and (2) knockdown sensitized UM cells to therapeutic agents, selumetinib or doxorubicin. Methods: DUSP4 mRNA data from The Cancer Genome Atlas and DUSP4 protein expression examined using immunohistochemistry in 28 cases of pUM were evaluated for association with clinical, genetic, and histological features. In vitro cytotoxic drug assays tested the efficacy of selumetinib and doxorubicin in UM cell lines with/without small interfering RNA DUSP4 gene silencing. Results: DUSP4 protein expression was observed in 93% of cases, with strong nuclear positivity in 79%. Despite higher DUSP4 messenger RNA levels in disomy 3/wild-type BAP1 UM, there was no significant association of nDUSP4 protein with these metastatic risk predictors or outcome. DUSP4 expression in UM cell lines varied. DUSP4 silencing in Mel202, MP46, and MP41 cells did not affect ERK1/2 or phospho-ERK levels. Despite increased phospho-ERK levels in Mel285, no cell line showed enhanced sensitivity to selumetinib/doxorubicin. Conclusions: DUSP4 protein expression is not a biomarker of UM metastatic risk. DUSP4 plays a complex role in oncogenesis, as reported in other cancers, and further work is required to fully understand its functional role in the MAPK pathway. Translational Relevance: Understanding the role of phosphatases, such as DUSP4, in the control of intracellular signaling cascades will facilitate our ability to identify successful treatment options.

Uveal melanoma (UM) is the most common primary intraocular malignancy in adults, associated with a high tendency for metastasis to the liver. Proton beam therapy (PBT) is the preferred external radiotherapy treatment for primary UM of certain sizes and locations in the eye, due to its efficacy and good local tumour control, as well as its precision to spare surrounding ocular structures. PBT is an effective alternative to surgical enucleation and other non-precision-targeted radiotherapies. Despite this, the radiobiology of UM in response to PBT is still not fully understood. This enhanced knowledge would help to further optimise UM treatment and improve patient outcomes through reducing radiation dosage to ocular structures, treating larger tumours that would otherwise require enucleation, or even offering a treatment strategy for the otherwise fatal liver metastases. In this review, we explore current knowledge of the treatment of UM with PBT, evaluating the biological responses to the therapy. Molecular factors, such as tumour size, oxygen tension levels, DNA damage proficiency, and autophagy, are known to influence the cellular response to radiotherapy, and these will be discussed. Furthermore, we examine innovative strategies to enhance radiotherapy outcomes, such as combination therapies with DNA damage repair and autophagy modulators, as well as advancements in PBT planning and delivery. By integrating current research and emerging technologies, we aim to provide opportunities to improve the therapeutic effectiveness of PBT in UM management.