Project description:Background: Retinoblastoma (Rb) is a malignant neoplasm arising during retinal development from mutations in the RB1 gene. Loss or inactivation of both copies of RB1 results in initiation of retinoblastoma tumours, however additional genetic changes are needed for the continued growth and spread of the tumour. Ex vivo research has shown that in humans, retinoblastoma may initiate from Rb depleted cone precursors. Notwithstanding, it has not been possible to assess the full spectrum of clonal types within the tumour itself in vivo and the molecular changes occurring at the cells of origin, enabling their malignant conversion. To overcome these challenges, we have performed the first single cell (sc) RNA- and ATAC-Seq analyses of primary tumour tissues, enabling us to dissect the transcriptional and chromatin accessibility heterogeneity of proliferating cone precursors in human Rb tumours. Methods: Two Rb tumours each characterised by two pathogenic RB1 mutations were dissociated to single cells and subjected to scRNA-Seq & scATAC-Seq using the 10x Genomics platform. In addition, nine embryonic and fetal retina samples were dissociated to single cells and subjected to scRNA- and ATAC-Seq analyses. The scRNA- and ATAC-Seq data were embedded using Uniform Manifold Approximation and Projection (UMAP) and clustered using Seurat graph based-clustering. Integrated scATAC-Seq analysis of Rb tumours and human embryonic/fetal retina samples was performed to identify Rb cone enriched subsets. Pseudotime analysis of proliferating cones in the Rb samples was performed with Monocle. Ingenuity Pathway Analysis (IPA) was used to identify the signalling pathway and upstream regulators in the Rb cone enriched subclusters. Results: Our single cell analyses revealed the predominant presence of cone precursors at different stages of the cell cycle in the Rb tumours and amongst those identified the G2/M subset as the cell type of origin. scATAC-Seq analysis identified two Rb enriched cone subsets, each characterised by activation of different upstream regulators and signalling pathways, leading proliferating cone precursors to escape cell cycle arrest and/or apoptosis. Conclusions: Our study provides evidence of Rb tumor heterogeneity and defines molecular pathways that can be targeted to define new treatment strategies

Project description:Chemotherapy resistance is one of the reasons for the loss of the eye in retinoblastoma (RB) pa-tients. RB chemotherapy resistance has been investigated in different cell culture models like WERI RB1. Furthermore, chemotherapy resistant RB subclones like the etoposide resistant WERI ETOR cell line have been established to improve understanding of chemotherapy resistance in RB. Ob-jective of this study was to characterize the cell line models of an etoposide sensitive WERI RB1 and its etoposide resistant subclone WERI ETOR by proteomics analysis. Subsequently, quantitative proteomic data served for correlation analysis with known drug perturbation profiles. . WERI RB1 and WERI ETOR were cultured and prepared for quantitative mass spectrometry. Comparative proteomic profiling was performed with electrospray ionization tandem mass spectrometry in data-independent acquisition mode (SWATH). The raw SWATH files were processed using neural networks in library free mode along with machine learning algorithms. Pathway enrichment was performed using the REACTOME pathway resource and correlated to the Molecular Signature Database (MSigDB) hallmark gene set collections for functional annotation. Also, a drug connec-tivity analysis using the L1000 database was used to correlate the mechanism-of-action (MOA) for different anticancer reagents to WERI RB1 / WERI ETOR signatures. A total 4,756 proteins were identified across all samples which revealed a distinct clustering between groups. Of these pro-teins, 64 proteins were significantly altered (q < 0.05 & log2FC |>2|, 22% higher in WERI ETOR). Pathway analysis showed an enriched metabolic pathway for retinoid metabolism and transport pathway in WERI ETOR and for sphingolipid de novo biosynthesis in WERI RB1. In addition, this study showed similar protein signatures of topoisomerase inhibitors and WERI ETOR as well as of ATPase inhibitors, acetylcholine receptor antagonists and VEGFR inhibitors and WERI RB1. In this study, WERI RB1 and WERI ETOR were characterized as a cell line model for chemotherapy re-sistance in RB by using data-independent mass spectrometry. The global proteome revealed the activation of sphingolipid de novo biosynthesis in WERI RB1 and potential treatment options for etoposide resistant RB.

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RB1 depleted pluripotent stem cell retinal organoids recapitulate state cell transitions of retinoblastoma development

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