Establishment and characterization of primary retinoblastoma cell lines: a comprehensive molecular profiling approach

Retinoblastoma (RB) research is significantly constrained by the limited availability of preclinical models that accurately recapitulate the disease complexity, particularly regarding the context of vitreous seeding, a major driver of therapeutic resistance and treatment failure. Here, we report the establishment and integrative characterization of patient-derived, three-dimensional (3D) spheroid models generated from primary retinal tumors (RBT) and vitreous seeds (RBVS). Across a cohort of 10 cell lines generated from 8 eyes and 8 patients, both RBT and RBVS models demonstrated sustained 3D growth and preserved key phenotypic features of the original tumors. Genome-wide DNA methylation and copy number profiling revealed strong concordance between primary tumors and derived models, supporting their molecular fidelity. RB1 inactivation was identified as the founding driver event, while additional alterations, including BCOR loss-of-function mutations and MYCN, ALK, and IKBKE amplifications, defined clinically aggressive subsets and were retained in corresponding models. Comparative analyses of matched samples showed that 3D spheroid generated models maintain core oncogenic drivers. Collectively, our data establish patient-derived 3D RB models as robust and translationally relevant platforms that recapitulate tumor heterogeneity and evolution. These systems provide a valuable resource for biomarker discovery, mechanistic investigation of resistance pathways, and development of precision therapeutic strategies in RB.