[Cell loss in retinal ischemia is associated with increased necroptosis]. / Zellverlust bei retinaler Ischämie ist assoziiert mit vermehrter Nekroptose.

BACKGROUND: Retinal ischemia plays a central pathophysiological role in numerous eye diseases, such as glaucoma. In addition to apoptosis, autophagy, necroptosis and ferroptosis are among the cell death mechanisms of ischemia; however, their role is not clearly understood and controversially discussed. OBJECTIVE: The aim of this study is to gain an improved understanding of the role of alternative cell death mechanisms such as autophagy and necroptosis after retinal ischemia. Based on this, future autophagy-based or necroptosis-based therapeutic approaches could be developed. MATERIAL AND METHODS: Retinal ischemia reperfusion was induced in one eye of 6 to 8­week-old rats by temporarily increasing the intraocular pressure to 140 mm Hg (60 min), followed by reperfusion. The untreated contralateral eye served as a control. Retinas after ischemia and control retinas were examined 7 days after ischemia immunohistochemically with markers for retinal ganglion cells (RGC), astrocytes (GFAP) as well as an autophagy (LAMP1) and a necroptosis marker (RIPK3) (n = 6/group). RESULTS: Ischemia reperfusion resulted in both significant RGC loss (p ≤ 0.001) and a significant increase of astrocyte area (p = 0.026) after 7 days. Interestingly, the number of autophagic LAMP1 positive cells was unchanged 7 days after ischemia (p = 0.272), whereas the number of necroptotic RIPK3 positive cells was significantly increased (p ≤ 0.001). CONCLUSION: Necroptotic processes appear to be activated 7 days after ischemia reperfusion, contributing to retinal cell death and activation of astrocytes. Late autophagic processes are not activated 7 days after ischemia. Necroptosis-associated parameters could therefore be targeted as an early therapeutic approach after ischemia in the future.

Protection against Oxidative Stress by Coenzyme Q10 in a Porcine Retinal Degeneration Model.

Oxidative stress plays an important role in neurodegenerative diseases, including glaucoma. Therefore, we analyzed if the antioxidant coenzyme Q10 (CoQ10), which is also commercially available, can prevent retinal degeneration induced by hydrogen peroxide (H2O2) in a porcine organ culture model. Retinal explants were cultivated for eight days, and H2O2 (500 µM, 3 h) induced the oxidative damage. CoQ10 therapy was applied (700 µM, 48 h). Retinal ganglion cells (RGCs) and microglia were examined immunohistologically in all groups (control, H2O2, H2O2 + CoQ10). Cellular, oxidative, and inflammatory genes were quantified via RT-qPCR. Strong RGC loss was observed with H2O2 (p ≤ 0.001). CoQ10 elicited RGC protection compared to the damaged group at a histological (p ≤ 0.001) and mRNA level. We detected more microglia cells with H2O2, but CoQ10 reduced this effect (p = 0.004). Cellular protection genes (NRF2) against oxidative stress were stimulated by CoQ10 (p ≤ 0.001). Furthermore, mitochondrial oxidative stress (SOD2) increased through H2O2 (p = 0.038), and CoQ10 reduced it to control level. Our novel results indicate neuroprotection via CoQ10 in porcine retina organ cultures. In particular, CoQ10 appears to protect RGCs by potentially inhibiting apoptosis-related pathways, activating intracellular protection and reducing mitochondrial stress.

In a novel autoimmune and high-pressure glaucoma model a complex immune response is induced.

Background: The neurodegenerative processes leading to glaucoma are complex. In addition to elevated intraocular pressure (IOP), an involvement of immunological mechanisms is most likely. In the new multifactorial glaucoma model, a combination of high IOP and optic nerve antigen (ONA) immunization leads to an enhanced loss of retinal ganglion cells accompanied by a higher number of microglia/macrophages in the inner retina. Here, we aimed to evaluate the immune response in this new model, especially the complement activation and the number of T-cells, for the first time. Further, the microglia/macrophage response was examined in more detail. Methods: Six-week-old wildtype (WT+ONA) and ßB1-connective tissue growth factor high-pressure mice (CTGF+ONA) were immunized with 1 mg ONA. A wildtype control (WT) and a CTGF group (CTGF) received NaCl instead. Six weeks after immunization, retinae from all four groups were processed for immunohistology, RT-qPCR, and flow cytometry, while serum was used for microarray analyses. Results: We noticed elevated numbers of C1q+ cells (classical complement pathway) in CTGF and CTGF+ONA retinae as well as an upregulation of C1qa, C1qb, and C1qc mRNA levels in these groups. While the complement C3 was only increased in CTGF and CTGF+ONA retinae, enhanced numbers of the terminal membrane attack complex were noted in all three glaucoma groups. Flow cytometry and RT-qPCR analyses revealed an enhancement of different microglia/macrophages markers, including CD11b, especially in CTGF and CTGF+ONA retinae. Interestingly, increased retinal mRNA as well as serum levels of the tumor necrosis factor α were found throughout the different glaucoma groups. Lastly, more T-cells could be observed in the ganglion cell layer of the new CTGF+ONA model. Conclusion: These results emphasize an involvement of the complement system, microglia/macrophages, and T-cells in glaucomatous disease. Moreover, in the new multifactorial glaucoma model, increased IOP in combination with autoimmune processes seem to enforce an additional T-cell response, leading to a more persistent pathology. Hence, this new model mimics the pathomechanisms occurring in human glaucoma more accurately and could therefore be a helpful tool to find new therapeutic approaches for patients in the future.

Neural extracellular matrix regulates visual sensory motor integration.

Visual processing depends on sensitive and balanced synaptic neurotransmission. Extracellular matrix proteins in the environment of cells are key modulators in synaptogenesis and synaptic plasticity. In the present study, we provide evidence that the combined loss of the four extracellular matrix components, brevican, neurocan, tenascin-C, and tenascin-R, in quadruple knockout mice leads to severe retinal dysfunction and diminished visual motion processing in vivo. Remarkably, impaired visual motion processing was accompanied by a developmental loss of cholinergic direction-selective starburst amacrine cells. Additionally, we noted imbalance of inhibitory and excitatory synaptic signaling in the quadruple knockout retina. Collectively, the study offers insights into the functional importance of four key extracellular matrix proteins for retinal function, visual motion processing, and synaptic signaling.

Glaucoma Animal Models beyond Chronic IOP Increase.

Glaucoma is a complex and multifactorial disease defined as the loss of retinal ganglion cells (RGCs) and their axons. Besides an elevated intraocular pressure (IOP), other mechanisms play a pivotal role in glaucoma onset and progression. For example, it is known that excitotoxicity, immunological alterations, ischemia, and oxidative stress contribute to the neurodegeneration in glaucoma disease. To study these effects and to discover novel therapeutic approaches, appropriate animal models are needed. In this review, we focus on various glaucoma animal models beyond an elevated IOP. We introduce genetically modified mice, e.g., the optineurin E50K knock-in or the glutamate aspartate transporter (GLAST)-deficient mouse. Excitotoxicity can be mimicked by injecting the glutamate analogue N-methyl-D-aspartate intravitreally, which leads to rapid RGC degeneration. To explore the contribution of the immune system, the experimental autoimmune glaucoma model can serve as a useful tool. Here, immunization with antigens led to glaucoma-like damage. The ischemic mechanism can be mimicked by inducing a high IOP for a certain amount of time in rodents, followed by reperfusion. Thereby, damage to the retina and the optic nerve occurs rapidly after ischemia/reperfusion. Lastly, we discuss the importance of optic nerve crush models as model systems for normal-tension glaucoma. In summary, various glaucoma models beyond IOP increase can be utilized.

Investigation of Inter- and Intra-Day Variability of Tear Fluid Regarding Flow Rate, Protein Concentration as well as Protein Composition.

Purpose: The purpose of this study was to present the determination of inter- and intra-day variations in tear flow rate, and tear fluid protein concentration, as well as protein composition regarding their impact for future biomarker studies. Methods: Tear fluid was collected noninvasively from 18 healthy subjects by performing Schirmer tests at 4 different time points repetitive in a period of 2 days. The tear flow rate on the Schirmer test strips was measured. Proteins were extracted from strips and quantified using amino acid analysis. Protein composition was analyzed by the strips data-independent (DIA) based mass spectrometry. To exclude any impairments to health, volunteers underwent a detailed neurological as well as an ophthalmological examination. Results: Whether tear fluid was collected from oculus sinister or oculus dexter did not affect the tear flow rate (P ≈ 0.63) or protein concentration (P ≈ 0.97) of individual subjects. Moreover, protein concentration was independent from the tear volume, so that a change in volume may only influence the total protein amount. When the examination days were compared, investigation of tear flow rate (P ≈ 0.001) and protein concentration (P ≈ 0.0003) indicated significant differences. Further, mass spectrometric analysis of tear fluid revealed 11 differentially regulated proteins when comparing both examination days. Conclusions: Our findings provide evidence of inter-day variation in tear flow rate, tear proteome concentration, and composition in healthy subjects, suggesting that inter-day variation needs to be taken into consideration in biomarker research of tear fluid. Identified proteins were assigned to functions in the immune response, oxidative and reducing processes, as well as mannose metabolism.

Changes of Subjective Symptoms and Tear Film Biomarkers Following Lenticule Extraction.

PURPOSE: To investigate the influence of lenticule extraction on subjective symptoms and objective biomarkers of dry eye and to clarify relationships between markers and find indicators for subjective symptoms after lenticule extraction. METHODS: Right eyes of myopic patients undergoing lenticule extraction surgery (n = 35) were examined preoperatively and 5 and 90 days postoperatively using established clinical dry eye examination methods (tear film break-up time [BUT], Schirmer test, lissamine green and fluorescein staining, and Ocular Surface Disease Index (OSDI) questionnaire). A patient subset was also examined after 1 year (n = 14). Tear samples were eluted from Schirmer strips and then measured with enzyme-linked immunosorbent assays (calcitonin gene-related peptide (CGRP), interleukin-1ß, IL-6, IL-8, matrix metallopeptidase 9 [MMP-9], nerve growth factor, and tumor necrosis factor-α). Postoperatively, unpreserved ofloxacin and dexamethasone eye drops were given (four times a day for 10 days). RESULTS: BUT decreased at days 5 (P = .023) and 90 (P = .025). Lissamine green staining increased at day 90 (P = .036). OSDI values increased at day 5 (total values, vision-related function, ocular symptoms, all P < .001, but not environmental triggers) and at day 90 (vision-related function, P = .017). A downregulation of CGRP (P = .006) and MMP-9 levels (P = .042) was observed on day 5 compared to day 90. CONCLUSIONS: Due to incongruity of patient symptoms, clinical signs, and tear protein changes, no predictive indicator was found, but some patients reported increased discomfort. Changes after lenticule extraction are not exclusively due to dry eye. [J Refract Surg. 2023;39(9):597-604.].

HSP27 induced glaucomatous damage in mice of young and advanced age.

Introduction: Age-related diseases such as glaucoma, a leading cause of blindness, are having an upward trend due to an aging society. In glaucoma, some patients display altered antibody profiles and increased antibody titers, for example against heat shock protein 27 (HSP27). An intravitreal injection of HSP27 leads to glaucoma-like damage in rats. We now aimed to investigate if aged mice are more prone to this damage than younger ones. Methods: We intravitreally injected HSP27 into young (1-2 months) and aged (7-8 months) mice to compare glaucomatous damage. Respective age-matched controls received PBS. Not injected eyes served as naive controls. Results: Optical coherence tomography 4 weeks after injection showed no changes in retinal thickness in all groups at both ages. Cell counts and RT-qPCR revealed a significant reduction in RGC numbers in HSP27 mice at both ages. Comparing aged and young HSP27 mice, no differences in Rbpms and Pou4f1 (RGCs) expression was detected, while the Tubb3 expression (neuronal cells) was significantly upregulated in aged HSP27 animals. Neither microglia/macrophages nor (resident) microglia counts revealed significant differences in HSP27 mice at both ages. Nevertheless, increased relative Iba1 and Tmem119 expression was detected in young and aged HSP27 mice. Aged HSP27 mice displayed a significantly lower Iba1 expression than young ones, whereas Cd68 levels were upregulated. A larger GFAP+ area and an upregulation of GFAP expression in HSP27 animals of both ages indicated a macrogliosis. Also, elevated Il1b and Nos2 expression levels were observed in young and aged HSP27 mice. However, only Il1b levels were upregulated when comparing 7-8 months to 1-2 months old animals. A larger HSP25+ area was seen in aged HSP27 animals, while Hspb2 expression levels were downregulated in both HSP27 groups. The aged HSP27 group displayed an upregulated Hspb2 expression compared to young mice. Furthermore, a higher optic nerve degeneration score was noted in young and aged HSP27 groups. Discussion: These findings indicate that an intravitreal injection of HSP27 led to RGC loss accompanied by inflammation. Age-dependent effects (7-8 months vs. 1-2 months) were not very prominent. The results suggest a potential role of extracellular HSP27 in the development of glaucoma.

Etiology of Idiopathic Macular Holes in the Light of Estrogen Hormone.

The aim of this review was to identify a new potential explanation for the development of macular holes in relation to the female sex and to explain the possible underlying pathways. This approach was based on the evaluation of anatomical, physiological, and morphological analyses currently available in the literature. The findings showed that estrogen exerts a protective effect on the neuroretina and may influence Müller and cone cells. Both cell types are responsible for the building of the fovea structure. However, this protection may be lost due to the sudden decrease in estrogen levels during menopause. In conclusion, the fovea cones, through its sensitivity to estrogen and high energy consumption, may be very vulnerable to damage caused by a sudden changes in the concentration of estrogen in menopausal females. Such changes may result in cone degeneration, and thus a destroyed structure of the fovea, and may lead to the development of a hole in the fovea, as in the case of macular holes. This review revealed that under the decreasing influence of estrogen may cones play a key role with regard to the etiology of the development of macular holes. This aspect may be of strategic importance in prophylactic therapy for the prevention of the development of macular holes in premenopausal females or after ocular trauma.

Establishment of a primary porcine retinal pigment epithelium monolayer to complement retinal ex vivo cultures.

Impaired function of the retinal pigment epithelium (RPE) resembles a hallmark in many retinal diseases; thus, co-cultivation models with RPE and retinal explants are useful to investigate these. Here, we present an easy-to-handle direct co-cultivation protocol, containing a functional, primary RPE monolayer and retinal explants. We describe in detail steps for establishing the monolayer and the direct co-cultivation. Techniques, which allow users to investigate the integrity and functionality of the RPE monolayer, are presented and the co-cultivation was tested under stress conditions.

Increased Angiopoietin-1 and -2 levels in human vitreous are associated with proliferative diabetic retinopathy.

BACKGROUND: Diabetic retinopathy is a frequent complication of diabetes mellitus and a leading cause of blindness in adults. The objective of this study was to elucidate the diabetic retinopathy pathophysiology in more detail by comparing protein alterations in human vitreous of different diabetic retinopathy stages. METHODS: Vitreous samples were obtained from 116 patients undergoing pars plana vitrectomy. Quantitative immunoassays were performed of angiogenic factors (VEGF-A, PIGF, Angiopoietin-1, Angiopoietin-2, Galectin-1) as well as cytokines (IL-1ß, IL-8, IFN-γ, TNF-α, CCL3) in samples from control patients (patients who don't suffer from diabetes; n = 58) as well as diabetes mellitus patients without retinopathy (n = 25), non-proliferative diabetic retinopathy (n = 12), and proliferative diabetic retinopathy patients (n = 21). In addition, correlation analysis of protein levels in vitreous samples and fasting glucose values of these patients as well as correlation analyses of protein levels and VEGF-A were performed. RESULTS: We detected up-regulated levels of VEGF-A (p = 0.001), PIGF (p<0.001), Angiopoietin-1 (p = 0.005), Angiopoietin-2 (p<0.001), IL-1ß (p = 0.012), and IL-8 (p = 0.018) in proliferative diabetic retinopathy samples. Interestingly, we found a strong positive correlation between Angiopoietin-2 and VEGF-A levels as well as a positive correlation between Angiopoietin-1 and VEGF-A. CONCLUSION: This indicated that further angiogenic factors, besides VEGF, but also pro-inflammatory cytokines are involved in disease progression and development of proliferative diabetic retinopathy. In contrast, factors other than angiogenic factors seem to play a crucial role in non-proliferative diabetic retinopathy development. A detailed breakdown of the pathophysiology contributes to future detection and treatment of the disease.

Islet Co-Expression of CD133 and ABCB5 in Human Retinoblastoma Specimens.

BACKGROUND: The role of CD133 und ABCB5 is discussed in treatment resistance in several types of cancer. The objective of this study was to evaluate whether CD133+/ABCB5+ colocalization differs in untreated, in beam radiation treated, and in chemotherapy treated retinoblastoma specimens. Additionally, CD133, ABCB5, sphingosine kinase 1, and sphingosine kinase 2 gene expression was analyzed in WERI-RB1 (WERI RB1) and etoposide-resistant WERI RB1 subclones (WERI ETOR). METHODS: Active human untreated retinoblastoma specimens (n = 12), active human retinoblastoma specimens pretreated with beam radiation before enucleation (n = 8), and active human retinoblastoma specimens pretreated with chemotherapy before enucleation (n = 7) were investigated for localization and expression of CD133 and ABCB5 by immunohistochemistry. Only specimens with IIRC D, but not E, were included in this study. Furthermore, WERI RB1 and WERI ETOR cell lines were analyzed for CD133, ABCB5, sphingosine kinase 1, and sphingosine kinase 2 by the real-time polymerase chain reaction (RT-PCR). RESULTS: Immunohistochemical analysis revealed the same amount of CD133+/ABCB5+ colocalization islets in untreated and treated human retinoblastoma specimens. Quantitative RT-PCR analysis showed a statistically significant upregulation of CD133 in WERI ETOR (p = 0.002). No ABCB5 expression was detected in WERI RB1 and WERI ETOR. On the other hand, SPHK1 (p = 0.0027) and SPHK2 (p = 0.017) showed significant downregulation in WERI ETOR compared to WERI RB1. CONCLUSIONS: CD133+/ABCB5+ co-localization islets were noted in untreated and treated human retinoblastoma specimens. Therefore, we assume that CD133+/ABCB5+ islets might play a role in retinoblastoma genesis, but not in retinoblastoma treatment resistance.

Heat Shock Protein Upregulation Supplemental to Complex mRNA Alterations in Autoimmune Glaucoma.

Glaucomatous optic neuropathy is a common cause for blindness. An elevated intraocular pressure is the main risk factor, but also a contribution of the immune system seems likely. In the experimental autoimmune glaucoma model used here, systemic immunization with an optic nerve homogenate antigen (ONA) leads to retinal ganglion cell (RGC) and optic nerve degeneration. We processed retinae for quantitative real-time PCR and immunohistology 28 days after immunization. Furthermore, we performed mRNA profiling in this model for the first time. We detected a significant RGC loss in the ONA retinae. This was accompanied by an upregulation of mRNA expression of genes belonging to the heat shock protein family. Furthermore, mRNA expression levels of the genes of the immune system, such as C1qa, C1qb, Il18, and Nfkb1, were upregulated in ONA animals. After laser microdissection, inner retinal layers were used for mRNA microarrays. Nine of these probes were significantly upregulated in ONA animals (p < 0.05), including Hba-a1 and Cxcl10, while fifteen probes were significantly downregulated in ONA animals (p < 0.05), such as Gdf15 and Wwox. Taken together, these findings provide further insights into the pivotal role of the immune response in glaucomatous optic neuropathy and could help to identify novel diagnostic or therapeutic strategies.

Changes of Subjective Symptoms and Tear Film Biomarkers following Femto-LASIK.

Femtosecond laser-assisted in situ keratomileusis (Femto-LASIK) represents a common treatment modality in refractive surgery and shows excellent results in terms of safety, efficacy, predictability, and long-term stability. However, patients may be affected by dry eye symptoms. The aim of this study was to identify a potential association between subjective dry eye symptoms, objective dry eye markers, and possible changes in the tear film, which could be a target for future therapy development. Therefore, clinical (dry eye) examinations (OSDI, Schirmer test, lissamine green and fluorescein staining, BUT, visual acuity) were carried out before LASIK as well as 5 and 90 days post-OP. The dry eye marker MMP-9, cytokines (IL-1ß, IL-8), and pain markers (NGF, CGRP) were quantified in tear samples with immunoassays. In addition, correlation analyses were performed. Clinical examinations revealed an upregulated OSDI score 5 days post-OP and an increased lissamine green staining score 90 days post-OP. Downregulated CGRP levels were noted 5 days post-OP, while other protein markers were not significantly altered after Femto-LASIK. Hence, Femto-LASIK surgery induced subjective symptoms like that of dry eye which could objectively rather be classified as Femto-LASIK-related discomfort. In the future, this could possibly be better detected and treated using pain markers such as CGRP.

Towards an Understanding of Retinal Diseases and Novel Treatment.

The Special Issue, "Towards an Understanding of Retinal Diseases and Novel Treatment", provides comprehensive information on retinal diseases such as glaucoma, age-related macular degeneration (AMD), diabetic retinopathy, retinitis pigmentosa (RP), and others [...].

Impact of Primary RPE Cells in a Porcine Organotypic Co-Cultivation Model.

The pathological events of age-related macular degeneration are characterized by degenerative processes involving the photoreceptor cells, retinal pigment epithelium (RPE), and the Bruch's membrane as well as choroidal alterations. To mimic in vivo interactions between photoreceptor cells and RPE cells ex vivo, complex models are required. Hence, the aim of this study was to establish a porcine organotypic co-cultivation model and enlighten the interactions of photoreceptor and RPE cells, with a special emphasis on potential neuroprotective effects. Porcine neuroretina explants were cultured with primary porcine RPE cells (ppRPE) or medium derived from these cells (=conditioned medium). Neuroretina explants cultured alone served as controls. After eight days, RT-qPCR and immunohistology were performed to analyze photoreceptors, synapses, macroglia, microglia, complement factors, and pro-inflammatory cytokines (e.g., IL1B, IL6, TNF) in the neuroretina samples. The presence of ppRPE cells preserved photoreceptors, whereas synaptical density was unaltered. Interestingly, on an immunohistological as well as on an mRNA level, microglia and complement factors were comparable in all groups. Increased IL6 levels were noted in ppRPE and conditioned medium samples, while TNF was only upregulated in the ppRPE group. IL1B was elevated in conditioned medium samples. In conclusion, a co-cultivation of ppRPE cells and neuroretina seem to have beneficial effects on the neuroretina, preserving photoreceptors and maintaining synaptic vesicles in vitro. This organotypic co-cultivation model can be used to investigate the complex interactions between the retina and RPE cells, gain further insight into neurodegenerative pathomechanisms occurring in retinal diseases, and evaluate potential therapeutics.

Protein Profiling of WERI-RB1 and Etoposide-Resistant WERI-ETOR Reveals New Insights into Topoisomerase Inhibitor Resistance in Retinoblastoma.

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.