Molecular and Cellular Insights Into Retinoblastoma: Pathways, Immune Landscape, and Therapeutic Opportunities

Background: Retinoblastoma (RB) is the most common primary intraocular malignancy in children, primarily caused by inactivation of the RB1 tumor suppressor gene. Despite advancements in multimodal therapies, the molecular mechanisms underlying RB progression and its tumor microenvironment (TME) remain poorly understood, limiting the development of effective targeted treatments.

Methods: This study integrates bulk and single-cell RNA sequencing data to characterize the molecular landscape of RB. Differential gene expression analysis, pathway enrichment analysis, and single-sample gene set enrichment analysis (ssGSEA) were performed to uncover key pathways and immune cell populations. Immune checkpoint molecules, m6A RNA modification-related genes, and ferroptosis-associated genes were analyzed to identify potential therapeutic targets. Protein-protein interaction (PPI) networks and cell-cell communication analyses were conducted to explore intercellular signaling within the TME. Additionally, functional validation was performed for CDKN1A, a candidate gene identified from transcriptomic analysis, using shRNA-mediated knockdown and in vitro assays.

Results: Transcriptomic profiling revealed distinct gene expression signatures between RB and normal retinal tissues, including upregulation of oncogenic pathways (e.g., MYC targets and G2/M checkpoint regulation) and downregulation of tumor suppressor pathways (e.g., p53 signaling). Chemotherapy-induced gene expression changes were observed, notably the activation of immune-related pathways such as antigen presentation and NK cell-mediated cytotoxicity. Immune checkpoint molecules (PDCD1, CD274, HAVCR2) exhibited cell-type-specific expression, indicating potential for immunotherapy. Elevated expression of m6A regulators (METTL3, WTAP) and ferroptosis-associated genes (ACSL4, SLC7A11) pointed to novel therapeutic vulnerabilities. Among key regulatory genes, CDKN1A was identified as significantly downregulated in RB. Functional experiments demonstrated that knockdown of CDKN1A inhibited cell proliferation and induced G1 phase arrest in RB cell lines, supporting its potential tumor-promoting role in this context.

Conclusion: This study provides a comprehensive molecular and cellular overview of RB progression and reveals novel therapeutic targets, including immune checkpoints, m6A modification enzymes, ferroptosis regulators, and CDKN1A. Our findings emphasize the need to address tumor heterogeneity and cell-type-specific gene expression in designing effective personalized therapies for RB patients.