Search Results (13)

Recent advancements in aqueous humor (AH) cell-free DNA (cfDNA) genomics have opened new avenues for ex vivo molecular profiling of retinoblastoma (RB), the most common pediatric intraocular malignancy, where biopsy is typically prohibited. While these insights offer a genetic blueprint of the tumor, they lack multi-omic molecular phenotyping, which is essential for understanding the functional state. Extracellular vesicles (EVs), naturally present in AH, are promising by offering time-resolved phenotypic information. We employed multiplex bead-based flow cytometry and Single Extracellular Vesicle Nanoscopy (SEVEN) to analyze EV phenotypes in AH from a cohort of five RB, with three uveal melanoma (UM) and two age-matched glaucoma (GLC) samples serving as controls. The studies identified CD133-enriched EVs uniquely in RB AH, absent in both GLC and UM AH. This was corroborated by further analysis of five RB cell lines, including two commercial (Y79, Weri) and three in-house developed lines, confirming CD133 enrichment and supporting its role as an RB-specific EV marker. Single-vesicle analysis demonstrated a strong association of CD133 with CD81 and CD63, with minimal CD9 presence. These results, validated through complementary techniques, position CD133 as a critical marker in RB-derived EVs, paving the way for enhanced multi-omic RB characterization and potential advancements in clinical diagnostics. Full article

Protein tyrosine phosphatase receptor type E (PTPRE) is a member of the “classical” protein tyrosine phosphatase subfamily and regulates a variety of cellular processes in a tissue-specific manner by antagonizing the function of protein tyrosine kinases. PTPRE plays a tumorigenic role in different human cancer cells, but its role in retinoblastoma (RB), the most common malignant eye cancer in children, remains to be elucidated. Etoposide-resistant RB cell lines and RB patients display significant higher PTPRE expression levels compared to chemosensitive counterparts and the healthy human retina, respectively. PTPRE promotor methylation analyses revealed that PTPRE expression in RB is not regulated via this mechanism. Lentiviral PTPRE knockdown (KD) induced a significant decrease in growth kinetics, cell viability, and anchorage-independent growth of etoposide-resistant Y79 and WERI RB cells. Caspase-dependent apoptosis rates were significantly increased and a re-sensitization for etoposide could be observed after PTPRE depletion. In vivo chicken chorioallantoic membrane (CAM) assays revealed decreased tumor formation capacity as well as reduced tumor size and weight following PTPRE KD. Expression levels of miR631 were significantly downregulated in etoposide-resistant RB cells and patients. Transient miR631 overexpression resulted in significantly decreased PTPRE levels and concomitantly decreased proliferation and increased apoptosis levels in etoposide-resistant RB cells. These impacts mirror PTPRE KD effects, indicating a regulation of PTPRE via this miR. Additionally, PTPRE KD led to altered phosphorylation of protein kinase SGK3 and—dependent on the cell line—AKT and ERK1/2, suggesting potential PTPRE downstream signaling pathways. In summary, these results indicate an oncogenic role of PTPRE in chemoresistant retinoblastoma. Full article

In childhood, retinoblastoma (RB) is the most common primary tumor in the eye. Long term therapeutic management with etoposide of this life-threatening condition may have diminishing effectiveness since RB cells can develop cytostatic resistance to this drug. To determine whether changes in receptor-mediated control of Ca²⁺ signaling are associated with resistance development, fluorescence calcium imaging, semi-quantitative RT-qPCR analyses, and trypan blue dye exclusion staining patterns are compared in WERI-ETOR (etoposide-insensitive) and WERI-Rb1 (etoposide-sensitive) cells. The cannabinoid receptor agonist 1 (CNR1) WIN55,212-2 (40 µM), or the transient receptor potential melastatin 8 (TRPM8) agonist icilin (40 µM) elicit similar large Ca²⁺ transients in both cell line types. On the other hand, NGF (100 ng/mL) induces larger rises in WERI-ETOR cells than in WERI-Rb1 cells, and its lethality is larger in WERI-Rb1 cells than in WERI-ETOR cells. NGF and WIN55,212-2 induced additive Ca²⁺ transients in both cell types. However, following pretreatment with both NGF and WIN55,212-2, TRPM8 gene expression declines and icilin-induced Ca²⁺ transients are completely blocked only in WERI-ETOR cells. Furthermore, CNR1 gene expression levels are larger in WERI-ETOR cells than those in WERI-Rb1 cells. Therefore, the development of etoposide insensitivity may be associated with rises in CNR1 gene expression, which in turn suppress TRPM8 gene expression through crosstalk. Full article

Background: Retinoblastoma (RB) is the most common eye cancer in children that has a high mortality rate when left untreated. Mouse models for retinoblastoma have been established but are time- and cost-intensive. The aim of this work was to evaluate an orthotopic transplantation model of retinoblastoma in zebrafish that also allows for tracking migratory routes and to explore advantages and disadvantages with respect to drug testing. Methods: Three fluorescence-labeled retinoblastoma cell lines (RB355, WERI-RB-1, Y79) were injected into the left eye of two-day-old zebrafish, while the un-injected right eye served as control. The migratory trajectories of injected retinoblastoma cells were observed until 8 days post injection (dpi), both in lateral and dorsal view, and measuring fluorescence intensity of injected cells was done for RB355 cells. Results: Time until the onset of migration and routes for all three retinoblastoma cell lines were comparable and resulted in migration into the brain and ventricles of the forebrain, midbrain and hindbrain. Involvement of the optic nerve was observed in 10% of injections with the RB355 cell line, 15% with Y79 cells and 5% with WERI-RB-1 cells. Fluorescence intensity of injected RB355 cells showed an initial increase until five dpi, but then decreased with high variability until the end of observation. Conclusion: The zebrafish eye is well suited for the analysis of migratory routes in retinoblastoma and closely mirrors patterns of retinoblastoma metastases in humans. Full article

Host defense peptides represent an important component of innate immunity. In this work, we report the anticancer properties of a panel of hyper-charged wholly cationic antimicrobial dodecapeptides (CAPs) containing multiple canonical forms of lysine and arginine residues. These CAPs displayed excellent bactericidal activities against a broad range of pathogenic bacteria by dissipating the cytoplasmic membrane potential. Specifically, we identified two CAPs, named HC3 and HC5, that effectively killed a significant number of retinoblastoma (WERI-Rb1) cells (p ≤ 0.01). These two CAPs caused the shrinkage of WERI-Rb1 tumor spheroids (p ≤ 0.01), induced intrinsic apoptosis in WERI-Rb1 cells via activation of caspase 9 and caspase 3, cleaved the PARP protein, and triggered off the phosphorylation of p53 and γH2A.X. Combining HC3 or HC5 with the standard chemotherapeutic drug topotecan showed synergistic anti-cancer activities. Overall, these results suggest that HC3 and HC5 can be exploited as potential therapeutic agents in retinoblastoma as monotherapy or as adjunctive therapy to enhance the effectiveness of currently used treatment modalities. Full article

Certain combinations of common variants in exon 3 of OPN1LW and OPN1MW, the genes encoding the apo-protein of the long- and middle-wavelength sensitive cone photoreceptor visual pigments in humans, induce splicing defects and have been associated with dyschromatopsia and cone dysfunction syndromes. Here we report the identification of a novel exon 3 haplotype, G-C-G-A-T-T-G-G (referring to nucleotide variants at cDNA positions c.453, c.457, c.465, c.511, c.513, c.521, c.532, and c.538) deduced to encode a pigment with the amino acid residues L-I-V-V-A at positions p.153, p.171, p.174, p.178, and p.180, in OPN1LW or OPN1MW or both in a series of seven patients from four families with cone dysfunction. Applying minigene assays for all observed exon 3 haplotypes in the patients, we demonstrated that the novel exon 3 haplotype L-I-V-V-A induces a strong but incomplete splicing defect with 3–5% of residual correctly spliced transcripts. Minigene splicing outcomes were similar in HEK293 cells and the human retinoblastoma cell line WERI-Rb1, the latter retaining a cone photoreceptor expression profile including endogenous OPN1LW and OPN1MW gene expression. Patients carrying the novel L-I-V-V-A haplotype presented with a mild form of Blue Cone Monochromacy or Bornholm Eye Disease-like phenotype with reduced visual acuity, reduced cone electroretinography responses, red-green color vision defects, and frequently with severe myopia. Full article

Retinoblastoma (Rb) is a pediatric intraocular malignancy that is proposed to originate from maturing cone cell precursors in the developing retina. The molecular mechanisms underlying the biological and clinical behaviors are important to understand in order to improve the management of advanced-stage tumors. While the genetic causes of Rb are known, an integrated understanding of the gene expression and metabolic processes in tumors of human eyes is deficient. By integrating transcriptomic profiling from tumor tissues and metabolomics from tumorous eye vitreous humor samples (with healthy, age-matched pediatric retinae and vitreous samples as controls), we uncover unique functional associations between genes and metabolites. We found distinct gene expression patterns between clinically advanced and non-advanced Rb. Global metabolomic analysis of the vitreous humor of the same Rb eyes revealed distinctly altered metabolites, indicating how tumor metabolism has diverged from healthy pediatric retina. Several key enzymes that are related to cellular energy production, such as hexokinase 1, were found to be reduced in a manner corresponding to altered metabolites; notably, a reduction in pyruvate levels. Similarly, E2F2 was the most significantly elevated E2F family member in our cohort that is part of the cell cycle regulatory circuit. Ectopic expression of the wild-type RB1 gene in the Rb-null Y79 and WERI-Rb1 cells rescued hexokinase 1 expression, while E2F2 levels were repressed. In an additional set of Rb tumor samples and pediatric healthy controls, we further validated differences in the expression of HK1 and E2F2. Through an integrated omics analysis of the transcriptomics and metabolomics of Rb, we uncovered a significantly altered tumor-specific metabolic circuit that reduces its dependence on glycolytic pathways and is governed by Rb1 and HK1. Full article

Chemotherapy resistance is one of the reasons for eye loss in patients with retinoblastoma (RB). RB chemotherapy resistance has been studied in different cell culture models, such as WERI-RB1. In addition, chemotherapy-resistant RB subclones, such as the etoposide-resistant WERI-ETOR cell line have been established to improve the understanding of chemotherapy resistance in RB. The objective of this study was to characterize cell line models of an etoposide-sensitive WERI-RB1 and its etoposide-resistant subclone, WERI-ETOR, by proteomic analysis. Subsequently, quantitative proteomics data served for correlation analysis with known drug perturbation profiles. Methodically, WERI-RB1 and WERI-ETOR were cultured, and prepared for quantitative mass spectrometry (MS). This was carried out in a data-independent acquisition (DIA) mode. The raw SWATH (sequential window acquisition of all theoretical mass spectra) files were processed using neural networks in a library-free mode along with machine-learning algorithms. Pathway-enrichment analysis was performed using the REACTOME-pathway resource, and correlated to the molecular signature database (MSigDB) hallmark gene set collections for functional annotation. Furthermore, a drug-connectivity analysis using the L1000 database was carried out to associate the mechanism of action (MOA) for different anticancer reagents to WERI-RB1/WERI-ETOR signatures. A total of 4756 proteins were identified across all samples, showing a distinct clustering between the groups. Of these proteins, 64 were significantly altered (q < 0.05 & log2FC |>2|, 22 higher in WERI-ETOR). Pathway analysis revealed the “retinoid metabolism and transport” pathway as an enriched metabolic pathway in WERI-ETOR cells, while the “sphingolipid de novo biosynthesis” pathway was identified in the WERI-RB1 cell line. In addition, this study revealed similar protein signatures of topoisomerase inhibitors in WERI-ETOR cells as well as ATPase inhibitors, acetylcholine receptor antagonists, and vascular endothelial growth factor receptor (VEGFR) inhibitors in the WERI-RB1 cell line. In this study, WERI-RB1 and WERI-ETOR were analyzed as a cell line model for chemotherapy resistance in RB using data-independent MS. Analysis of the global proteome identified activation of “sphingolipid de novo biosynthesis” in WERI-RB1, and revealed future potential treatment options for etoposide resistance in RB. Full article

Ursodeoxycholic (UDCA) and tauroursodeoxycholic (TUDCA) acids have shown neuroprotective properties in neurodegenerative diseases, but differential effects of the two bile acids have been poorly explored. The aim of this study was to evaluate the neuroprotective effects of UDCA versus TUDCA in a neuroretinal degeneration model and to compare transcriptionally regulated pathways. The WERI-Rb-1 human cone-like cell line and retinal explants were exposed to albumin and TUDCA or UDCA. Viability, cell death, and microglial activation were quantified. Transcriptionally regulated pathways were analyzed after RNA sequencing using the edgeR bioconductor package. Pre-treatment of cone-like cells with UDCA or TUDCA significantly protected cells from albumin toxicity. On retinal explants, either bile acid reduced apoptosis, necroptosis, and microglia activation at 6 h. TUDCA induced the regulation of 463 genes, whilst 31 genes were regulated by UDCA. Only nineteen common genes were regulated by both bile acids, mainly involved in iron control, cell death, oxidative stress, and cell metabolism. As compared to UDCA, TUDCA up-regulated genes involved in endoplasmic reticulum stress pathways and down-regulated genes involved in axonal and neuronal development. Either bile acid protected against albumin-induced cell loss. However, TUDCA regulated substantially more neuroprotective genes than UDCA. Full article

Retinoblastoma (RB) is a primary intraocular malignancy in childhood. Relapses may develop and cause secondary cancers during later development. This study was set up to identify optimal cell culture conditions for RB cell growth in vitro and to optimize tumor growth in an in vivo model. RB cell lines (Y79 and WERI-Rb1) were cultivated under three different in vitro conditions and apoptosis, proliferation and cell growth, as well as expression profiles of two epithelial-mesenchymal transition (EMT) markers, were analyzed. EMT gene expression profiles were not generally changed, whereas apoptosis levels, tumor cell proliferation, and in vitro growth were significantly influenced by different cell culture conditions. In order to optimize the time-limited chick chorioallantoic membrane (CAM) assay, we investigated two different time points of tumor cell inoculation (embryonic development day EDD8 and EDD10) as well as three different cell concentrations. We showed that inoculation at EDD8 led to decreased tumor formation and chicken viability, whereas different cell concentrations did not change size and weight of developing tumors. Our findings demonstrate that medium conditions in vitro as well as the starting point for CAM inoculation in ovo significantly influence the experimental outcome of investigations using RB cell lines. Full article

Retinoblastoma (RB) represents the most common malignant childhood eye tumor worldwide. Several studies indicate that the extracellular matrix (ECM) plays a crucial role in tumor growth and metastasis. Moreover, recent studies indicate that the ECM composition might influence the development of resistance to chemotherapy drugs. The objective of this study was to evaluate possible expression differences in the ECM compartment of the parental human cell lines WERI-RB1 (retinoblastoma 1) and Y79 and their Etoposide resistant subclones via polymerase chain reaction (PCR). Western blot analyses were performed to analyze protein levels. To explore the influence of ECM molecules on RB cell proliferation, death, and cluster formation, WERI-RB1 and resistant WERI-ETOR cells were cultivated on Fibronectin, Laminin, Tenascin-C, and Collagen IV and analyzed via time-lapse video microscopy as well as immunocytochemistry. We revealed a significantly reduced mRNA expression of the proteoglycans Brevican, Neurocan, and Versican in resistant WERI-ETOR compared to sensitive WERI-RB1 cells. Also, for the glycoproteins α1-Laminin, Fibronectin, Tenascin-C, and Tenascin-R as well as Collagen IV, reduced expression levels were observed in WERI-ETOR. Furthermore, a downregulation was detected for the matrix metalloproteinases MMP2, MMP7, MMP9, the tissue-inhibitor of metalloproteinase TIMP2, the Integrin receptor subunits ITGA4, ITGA5 and ITGB1, and all receptor protein tyrosine phosphatase β/ζ isoforms. Downregulation of Brevican, Collagen IV, Tenascin-R, MMP2, TIMP2, and ITGA5 was also verified in Etoposide resistant Y79 cells compared to sensitive ones. Protein levels of Tenascin-C and MMP-2 were comparable in both WERI cell lines. Interestingly, Fibronectin displayed an apoptosis-inducing effect on WERI-RB1 cells, whereas an anti-apoptotic influence was observed for Tenascin-C. Conversely, proliferation of WERI-ETOR cells was enhanced on Tenascin-C, while an anti-proliferative effect was observed on Fibronectin. In WERI-ETOR, cluster formation was decreased on the substrates Collagen IV, Fibronectin, and Tenascin-C. Collectively, we noted a different ECM mRNA expression and behavior of Etoposide resistant compared to sensitive RB cells. These findings may indicate a key role of ECM components in chemotherapy resistance formation of RB. Full article

The aim of this study was to develop a novel folate receptor-targeted drug delivery system for retinoblastoma cells using a promising anticancer agent, curcumin-difluorinated (CDF), loaded in polymeric micelles. Folic acid was used as a targeting moiety to enhance the targeting and bioavailability of CDF. For this purpose, amphiphilic poly(styrene-co-maleic acid)-conjugated-folic acid (SMA-FA) was synthesized and utilized to improve the aqueous solubility of a highly hydrophobic, but very potent anticancer compound, CDF, and its targeted delivery to folate overexpressing cancers. The SMA-FA conjugate was first synthesized and characterized by ¹H NMR, FTIR and DSC. Furthermore, the chromatographic condition (HPLC) for estimating CDF was determined and validated. The formulation was optimized to achieve maximum entrapment of CDF. The particle size of the micelles was measured and confirmed by dynamic light scattering (DLS) and transmission electron microscopy (TEM). Cytotoxicity studies were conducted on (Y-79 and WERI-RB) retinoblastoma cells. Results showed that the solubility of CDF could be increased with the newly-synthesized polymer, and the entrapment efficiency was >85%. The drug-loaded nanomicelles exhibited an appropriate size of <200 nm and a narrow size distribution. The formulation did not show any adverse cytotoxicity on a human retinal pigment epithelial cell (ARPE-19), indicating its safety. However, it showed significant cell killing activity in both Y-79 and WERI-RB retinoblastoma cell lines, indicating its potency in killing cancer cells. In conclusion, the folic acid-conjugated SMA loaded with CDF showed promising potential with high safety and pronounced anticancer activity on the tested retinoblastoma cell lines. The newly-formulated targeted nanomicelles thus could be a viable option as an alternative approach to current retinoblastoma therapies. Full article

A naturally new cyclopeptide, clavatustide C, was produced as a stress metabolite in response to abiotic stress elicitation by one of the hydrothermal vent fluid components Zn in the cultured mycelia of Aspergillus clavatus C2WU, which were isolated from Xenograpsus testudinatus. X. testudinatus lives at extreme, toxic habitat around the sulphur-rich hydrothermal vents in Taiwan Kueishantao. The known compound clavatustide B was also isolated and purified. This is the first example of a new hydrothermal vent microbial secondary metabolite produced in response to abiotic Zn treatment. The structures were established by spectroscopic means. The regulation of G1-S transition in hepatocellular carcinoma cell lines by clavatustide B was observed in our previous study. The purpose of the present study was to verify these results in other types of cancer cell lines and elucidate the possible molecular mechanism for the anti-cancer activities of clavatustide B. In different human cancer cell lines, including pancreatic cancer (Panc-1), gastric cancer (MGC-803), colorectal cancer (SW-480), retinoblastoma (WERI-Rb-1) and prostate cancer (PC3), clavatustide B efficiently suppressed cell proliferations in a dose-dependent manner. Although different cancer cell lines presented variety in Max effect dose and IC₅₀ dose, all cancer cell lines showed a lower Max effect dose and IC₅₀ dose compared with human fibroblasts (hFB) (p < 0.05). Moreover, significant accumulations in G1 phases and a reduction in S phases (p < 0.05) were observed under clavatustide B treatment. The expression levels of 2622 genes including 39 cell cycle-associated genes in HepG2 cells were significantly altered by the treatment with 15 μg/mL clavatustide B after 48 h. CCNE2 (cyclin E2) was proved to be the key regulator of clavatustide B-induced G1-S transition blocking in several cancer cell lines by using real-time PCR. Full article