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Dissecting the transcriptional and chromatin accessibility heterogeneity of proliferating cone precursors in human retinoblastoma tumours by single cell sequencing- opening pathways to new therapeutic strategies?

Lookup NU author(s): Dr Joseph Collin, Dr Rachel Queen, Dr Darin Zerti, Professor David SteelORCiD, Professor Majlinda LakoORCiD



This work is licensed under a Creative Commons Attribution 4.0 International License (CC BY 4.0).


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 RB1 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 human 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 with Seurat graph based-clustering. Integrated scATAC-Seq analysis of Rb tumours and human embryonic/fetal retina samples was performed to identify Rb cone enriched subclusters. 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 subclusters, each characterised by activation of different upstream regulators and signalling pathways, enabling proliferating cone precursors to escape cell cycle arrest and/or apoptosis. Conclusions: Our study provides evidence of Rb tumour heterogeneity and defines molecular pathways that can be targeted to define new treatment strategies.Retinoblastoma, tumour, scRNA-Seq, scATAC-Seq, proliferating cones, cell of origin

Publication metadata

Author(s): Collin J, Queen R, Zerti D, Steel D, Bowen C, Parulekar M, Lako M

Publication type: Article

Publication status: Published

Journal: Investigative Ophthalmology & Visual Science (IOVS)

Year: 2021

Volume: 62

Issue: 6

Print publication date: 01/05/2021

Acceptance date: 19/04/2021

Date deposited: 07/06/2022

ISSN (print): 0146-0404

ISSN (electronic): 1552-5783

Publisher: Association for Research in Vision and Ophthalmology


DOI: 10.1167/iovs.62.6.18


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Funder referenceFunder name
5095/5096Fight for Sight
MR/S035826/1Medical Research Council (MRC)