Graduate Research Assistant Children's Hospital Los Angeles Los Angeles, California, United States
Abstract: Retinoblastoma, the most prevalent childhood intraocular malignancy, originates from maturing cone photoreceptor precursors with biallelic RB1 inactivation. 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 mirroring in vivo disease. Research on this transition is limited by fetal tissue availability, but CRISPR engineered retinal organoids (ROs) provide a promising alternative. This study introduces a robust organoid platform to model and investigate the retinoblastoma indolence-malignancy transition. We generated cone-reporter iPSC lines through CRISPR knock-in of EGFP-P2A at the GNAT2 locus. A second round of CRISPR editing produced homozygous RB1 knockout. Chimeric RB1+/+ ROs and RB1-/- RBROs were generated from edited iPSCs mixed with unedited parental iPSCs. ROs and RBROs were embedded in hydrogel and live-imaged episodically to track EGFP+ cone proliferation dynamics. Deep full-length scRNA-seq was carried out on FACS isolated EGFP+ RB1+/+ and RB1-/- cones at various timepoints. In RB1 WT ROs, EGFP specifically, robustly and innocuously labeled immature and mature cones. In 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 morphology. Nascent retinoblastoma-like foci co-expressing EGFP, cone markers, and Ki67 formed after ~d280, a tissue age that equates to the first post-natal month when early retinoblastomas typically emerge. Single cell transcriptomics of RB1-/- cones from multiple tumorigenesis stages showed distinct molecular signatures of proliferation, differentiation and stress cell states and suggest a role of p53 pathway in indolence entry and escape. In summary, we established a human retinoblastoma organoid model that faithfully recapitulates the cell-of-origin and timing of multi-step retinoblastomagenesis. This model revealed distinct molecular signatures at each tumorigenesis stage and highlighted altered expression of a p53 pathway regulator during the retinoblastoma indolence-malignancy transition.
