Abstract 236: Modeling multi-step retinoblastoma genesis with cone reporter retinal organoids

Abstract

Abstract Purpose: This study aims to develop a human retinoblastoma organoid (RBRO) model that recapitulates the cell-of-origin and multi-step retinoblastoma genesis. Background: Retinoblastomas originate from maturing cone photoreceptor precursors with biallelic RB1 inactivation. Rb1 mutant animal models fail to recapitulate retinoblastomagenesis with a cone precursor cell-of-origin, likely due to human-specific cone development features. In explanted fetal retina, pRB-depleted post-mitotic cone precursors proliferate, followed by a 3-5 month premalignant indolence phase before retinoblastoma-like masses emerge at tissue ages similar to retinoblastomas in vivo. However, tissue availability limits research with this disease model. RB1−/- retinal organoids (ROs) provide a potential alternative, as they demonstrate cone proliferation, but they deteriorate before forming indolent premalignant lesions or malignant retinoblastoma foci. Here, we developed methods with which to examine the effects of cone precursor pRB loss in otherwise healthy retinal tissue. Methods: We generated cone-reporter GNAT2-EGFP iPSC lines through CRISPR knock-in of EGFP-P2A at the N-terminus of GNAT2 in WTC11-mTagRFPT-LMNB1. A second round of CRISPR editing produced homozygous RB1 knockout. Chimeric RB1 WT ROs and RB1-null RBROs were generated from cone reporter iPSCs mixed with unedited parental iPSCs. ROs and RBROs were embedded in hydrogel and live-imaged episodically to track EGFP+ cone proliferation dynamics. scRNA-seq was carried out on FACS isolated EGFP+ RB1-null cones at various ages. Results: In RB1 WT ROs, EGFP+ cone precursors appeared at d34 and adopted cone morphology at ~d120, with rapid inner segment growth between ~d120 and ~d150. Immunohistochemistry confirmed cone-specific EGFP expression. In RB1-null RBROs, bi-weekly live confocal imaging revealed initial EGFP+ RB1−/− cone proliferation followed by a pre-malignant indolence phase starting at ~d150. The majority of the initially proliferating cones were Ki67-negative with some adopting mature cone photoreceptor morphology. Nascent retinoblastoma-like foci co-expressing EGFP, cone markers, and Ki67 formed in five of six RBROs after d281, a tissue age that equates to the first post-natal month when early retinoblastomas typically emerge. scRNA-seq of EGFP+ RB1−/− cones from the initial proliferation, indolence, and retinoblastoma-like stages revealed distinct molecular signatures. Conclusions: We generated a human retinoblastoma organoid model that recapitulates the cell-of-origin and timing of multi-step retinoblastomagenesis, with each tumorigenesis stage harboring distinct molecular signatures. This model may enable the identification of epigenetic and transcriptomic changes underlying malignant progression and screening for compounds that efficiently block this process. Citation Format: Jinlun Bai, David S. Koos, Kayla Stepanian, Kevin Stachelek, Bhavana Bhat, Scott Fraser, Rex Moats, David Cobrinik. Modeling multi-step retinoblastoma genesis with cone reporter retinal organoids [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 236.