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Abstract

Medulloblastomas (MBs) are the most common form of pediatric brain tumors and are a leading cause of cancer-related deaths in this age group. They are a set of clinically heterogeneous tumors that have been classified into four principal molecular subgroups, namely WNT, SHH, Group 3 (G3), and Group 4 (G4). In particular, the underlying pathology of G4 MBs, such as their cellular and genetic origins, is poorly understood. Recent genomic studies have pointed to the cells arising from the rhombic lip (RL), such as the cerebellar nuclear neurons (CNs) and unipolar brush cells (UBCs), as the putative cells-of-origin of the tumor. Other studies have also identified the putative cancer drivers of G4 MBs, such as Prdm6 and Mycn. To experimentally verify the genetic and cellular origins of G4 MBs, we generated cerebellar organoids, which model the RL of the developing cerebellum that gives rise to CNs and UBCs, as a platform to test these hypotheses. To generate the cerebellar organoids, we optimized our protocol by testing different patterning factors, such as FGF2 and FGFBb, at different concentrations. We demonstrate that the organoids generated mimic the developing neural tube and express the key molecular markers that characterize the RL of the cerebellum, such as Atoh1, Pax6, Olig2, and Tbr2/Calb2. By overexpressing the putative cancer drivers of G4 MBs in these cerebellar organoids, such as Prdm6 and Mycn, we observed a significant increase in the G4 MB marker, Kcna1, at approximately one month after the transfection. These results add evidence to our hypotheses regarding the genetic drivers and cellular origins of G4 MBs and validate the use of cerebellar organoids as a model for studying both normal and aberrant development.