Single-cell RNA-Seq Profiling of the Transcriptomic Response to RB Loss in the Retinoblastoma Cell of Origin
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE223634 | GEO | 2023/01/28
REPOSITORIES: GEO
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Project description:Retinoblastoma (RB) is the most common primary intraocular malignancy of childhood. The diverse malignant and immune cells in tumors determine tumor progression, but the intratumoral heterogeneity, tumor microenvironment and their impact in RB remain poorly characterized. Here, we conducted transcriptomic profiles of around 70,000 cells from tumor samples of seven RB patients with pathological and clinical measurements. Fifteen cell types were identified where the majority were cone precursor-like (CP-like) and MKI67+ cone precursor (MKI67+ CP) cells. These cells expressed markers of cone precursor, suggesting that RB originated from cone precursor cells. The conversion of CP-like to MKI67+ CP led to the loss of photoreceptor function and increased cell proliferation ability in RB. By integrative copy number variation analysis, we found that two subtypes of MKI67+ CP cells were more malignant related to cell proliferation. The tumor microenvironment in RB was composed of three cell types, of which glial cells, including tumor-associated macrophages and astrocytes-like cells, created an immunosuppressive environment and promoted tumor growth and invasion by inhibiting the GALECTIN signaling pathway. Together, our study presents the first transcriptomic map of RB at a single-cell resolution and provides novel insights into the occurrence and progression of RB which will be important to the development of the future targeted therapy and immunotherapy.
2022-05-11 | GSE168434 | GEO
Project description:Retinoblastoma is a childhood retinal tumor that initiates in response to biallelic RB1 inactivation. We show that post-mitotic human cone precursors are uniquely sensitive to the oncogenic effects of Rb depletion. Rb knockdown induced cone precursor proliferation in prospectively isolated populations. SNP-array analysis of two Rb/p130-depleted cone precursor cell lines, revealing no megabase-size loss of heterozygosity (LOH) or copy number alterations (CNAs). SNP-array analysis of one Rb/p130-depleted (tumor 1) or one Rb-depleted (tumor 2) cone precursor-derived tumors, revealing no megabase-size LOH or CNAs, consistent with the lack of DNA copy number alterations in some retinoblastomas. Thus, the cone precursor tumors resembled human retinoblastomas at the molecular cytogenetic level. High resolution SNP-array DNA copy number analyses were performed using CytoScan® HD (Affymetrix, 901835) according to the manufacturer's directions. Data were analyzed using Chromosome Analysis Suite 2.0 (Affymetrix). DNA was extracted from two Rb/p130 depleted cone precursor cell lines and two cryopreserved mouse retinoblastoma samples from eyes xenograted with Rb/p130 or Rb depleted cone precursors. Affymetrix SNP analysis on retinoblastoma cell lines Y79, RB176, and WERI were used as control. Normal human genome 19 was used as reference.
2014-08-25 | E-GEOD-60720 | biostudies-arrayexpress
Project description:Retinoblastoma (Rb) is the most prevalent intraocular malignant tumor in children with less than 30% survival rate globally. Rb genetic predisposition has been well defined for decades, whereas its developmental origin and drug agents remain largely unexplored. Here we developed the first organoidal retinoblastoma model derived from human embryonic stem cells (hESCs) with a biallelic knockout (RB1-/-) or mutagenesis (RB1Mut/Mut) of the RB1 gene, which exhibit extreme consistent properties of Rb tumorigenesis, transcriptome, and genome-wide methylation. Using single-cell RNA-seq and immunostaining of this model, we found that developmentally Rb originated from ARR3+ maturing cone precursors and a new cell type of unfolded protein response occurred in the organoidal retinoblastoma. A key PI3K-Akt pathway was aberrantly regulated, and its strong activator SYK (encoding proto-oncogenic spleen tyrosine kinase) was significantly upregulated in the cancerous organoids. Furthermore, we demonstrated that the SYK inhibitors, A502 and B502, have more significant therapeutic response on the early-stage organoidal retinoblastoma compared to four well-established chemotherapeutic drugs in clinic. Collectively, we established a brand-new organoidal retinoblastoma model derived from human ESCs in-a-dish and discovered its cell-of-origin and potential drug agents, shedding light on development and therapeutics of human cancers.
2020-09-05 | GSE136929 | GEO
Project description:Retinoblastoma is a childhood retinal tumor that initiates in response to biallelic RB1 inactivation. We show that post-mitotic human cone precursors are uniquely sensitive to the oncogenic effects of Rb depletion. Rb knockdown induced cone precursor proliferation in prospectively isolated populations. SNP-array analysis of two Rb/p130-depleted cone precursor cell lines, revealing no megabase-size loss of heterozygosity (LOH) or copy number alterations (CNAs). SNP-array analysis of one Rb/p130-depleted (tumor 1) or one Rb-depleted (tumor 2) cone precursor-derived tumors, revealing no megabase-size LOH or CNAs, consistent with the lack of DNA copy number alterations in some retinoblastomas. Thus, the cone precursor tumors resembled human retinoblastomas at the molecular cytogenetic level.
2014-08-25 | GSE60720 | GEO
Project description:Retinoblastoma (Rb) is a childhood cancer of the developing retina that begins in utero in response to bi-allelic inactivation of RB1 or MYCN amplification, accounting for up to 1% of all tumours in infancy. To gain insights into the transcriptional events of each state cell transition during Rb development, we developed two Rb models via retinal organoid differentiation of RB1 depleted human embryonic stem cell line (RB1-null hESCs) and RB1patient-specific induced pluripotent (iPSC) line harbouring RB1 biallelic mutation (c.2082delC). Both models were characterised by RB1 depletion and a significant increase in the fraction of proliferating cone precursors (RXRγ+Ki67+), which were defined as cell of origin for Rb by the single cell RNA-Seq analysis. The RB1 depleted retinal organoids displayed similar features to Rb tumours including mitochondrial cristae aberration and rosette-like structures and were able to undergo cell growth in an anchorage-independent manner, indicative of cell transformation in vitro. The patient-specific iPSC model displayed an enhanced reduction of amacrine, horizontal and retinal ganglion cells and an accelerated loss of cone photoreceptor markers during transition towards retinoma and Rb, compared to RB1-null hESC. In both models, the Rb cells of origin, intermediate retinoma and/or Rb cells expressed retinal ganglion and horizontal cell in addition to cone markers, a novel finding, which should help to better identify and eradicate these cells within the tumour mass. Application of Melphalan, Topotecan and TW-37 led to a significant reduction in the fraction of proliferating cone precursors, validating the suitability of these in vitro models for testing novel therapeutics for Rb.
2023-03-03 | GSE173447 | GEO
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
2021-07-26 | GSE166174 | GEO
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
2021-07-26 | GSE166173 | GEO
Project description:Gene expression profiling study of Sertoli cells (Sc)-specific Retinoblastoma (Rb) knockout (KO) mice. We generated a Sc-specific Rb knockout (Sc-RbKO) line and characterised its testicular phenotype. Sc-RbKO showed a progressive germ cell depletion leading to infertility and Sc dysfunction. We observed that up to 10 days old, Sc-RbKO mice present a phenotype similar to that of their WT siblings. This age was choosen to study the gene expression changes that would eventually lead to the described phenotypical changes. Sc-RbKO testis gene expression was compared to WT and to Sc-Rb+/- samples. Our Microarray results show changes of many genes involved in cell cycle control, DNA repair, apoptosis and misregulation of genes that may be important for proper Sertoli cell function such as tight junction formation, cell membrane and tissue remodeling. Total RNA extracted from 12 testicular samples, 5 controls, 3 hetorozygotes and 4 homozygotes.
2011-01-01 | E-GEOD-22234 | biostudies-arrayexpress
Project description:Gene expression profiling study of Sertoli cells (Sc)-specific Retinoblastoma (Rb) knockout (KO) mice. We generated a Sc-specific Rb knockout (Sc-RbKO) line and characterised its testicular phenotype. Sc-RbKO showed a progressive germ cell depletion leading to infertility and Sc dysfunction. We observed that up to 10 days old, Sc-RbKO mice present a phenotype similar to that of their WT siblings. This age was choosen to study the gene expression changes that would eventually lead to the described phenotypical changes. Sc-RbKO testis gene expression was compared to WT and to Sc-Rb+/- samples. Our Microarray results show changes of many genes involved in cell cycle control, DNA repair, apoptosis and misregulation of genes that may be important for proper Sertoli cell function such as tight junction formation, cell membrane and tissue remodeling.
2011-01-01 | GSE22234 | GEO
Project description:Although Dicer1 functions as a haploinsufficient tumour suppressor its complete loss of function is selected against during tumourigenesis. Here we show that homozygous loss of Dicer1 prevents retinoblastoma formation in mice by synthetic lethality with combined inactivation of the p53 and Rb tumor suppressor pathways. We show that Dicer1 deficiency is tolerated in Rb-deficient retinal progenitor cells harbouring an intact p53 pathway, but not in absence of p53. The synthetic lethal interaction is mediated by the oncogenic miR-17?92 cluster since its deletion phenocopies Dicer1 loss in this context. Accordingly, miR-17?92- inactivation suppresses retinoblastoma formation in mice and co-silencing of miR17/20a and p53 cooperatively decrease the viability of pre-formed human retinoblastoma cells. These data provide important insights into what underlies selective pressure against loss of Dicer1 during tumorigenesis and indicate that targeting Dicer or miR-17-20a family members should be explored as a potential selective therapeutic approach for retinoblastoma prevention and/or treatment. 23 murine samples
2012-07-05 | E-GEOD-39106 | biostudies-arrayexpress