P14.22.a a Novel Immunocompetent Orthotopic Mouse Model to Study Embryonal Tumors With Multilayered Rosettes (Etmrs): Development and Characterization

Abstract BACKGROUND Embryonal tumors with multilayered rosettes (ETMR) are rare and highly aggressive pediatric brain tumors, characterized by poor prognosis and a median survival below one year. Diagnostic markers include multilayered rosettes, amplification of the C19MC microRNA cluster, and elevated LIN28A expression. To improve understanding of ETMR pathogenesis and support the development of immunotherapies, immunocompetent preclinical models are urgently needed. However, such models remain scarce. This study aims to generate and characterize a novel orthotopic ETMR mouse model in an immunocompetent setting. MATERIAL AND METHODS The murine primitive neuroectodermal tumor (PNET) cell line DF1-MYC, engineered via a MYC-overexpressing RCAS system in a C57BL/6J background, was used for tumor induction. Cells and derived allografts were implanted orthotopically into the supratentorial region of four weeks old NSG (immunodeficient) and NTV-a p53ᶠloxed (NP53) immunocompetent mice. Tumor development and survival were monitored, with survival curves calculated using the Kaplan-Meier method. Formalin-fixed, paraffin-embedded brain tissues were analyzed by hematoxylin and eosin (H&E) staining, as well as immunohistochemistry for Ki67, c-Myc and Lin28a. Transcriptomic profiling was performed by RNA sequencing (RNA-seq). RESULTS Direct implantation of DF1-MYC into NP53 mice resulted in low tumor incidence, with only one small tumor exhibiting multilayered rosettes. To improve model consistency, tumors established in NSG mice were subsequently allografted into NP53 hosts. The resulting model, termed MYC384, showed 70% tumor penetrance and a median survival of 36 days. Histopathological evaluation revealed features characteristic of human ETMR, including poorly differentiated, proliferative cells with multilayered rosettes. Further molecular refinement is underway via overexpression of LIN28A and c-Myc in the MYC384 model to enhance ETMR fidelity. CONCLUSION The MYC384 model constitutes a promising immunocompetent platform for ETMR research, reproducing key histological characteristics observed in human disease. It offers a valuable resource for studying tumor biology and evaluating novel therapeutic approaches.