The role of exosomes in the extracellular signaling of different molecular subgroups of pediatric Medulloblastomas

Abstract

Medulloblastoma (MB) is the most frequent malignant brain neoplasm in children and adolescents. MB is now considered a genetically heterogeneous disease, and it is subdivided into four molecular subgroups (WNT, Shh, Group 3 and Group 4), each with its clinical and genetic characteristics. Exosomes are vesicles secreted by cells, especially under conditions of cellular stress. The exosomes contain in their interior proteins, lipids, DNA fragment, mRNA and miRNA. These vesicles have been implicated in tumorogenesis, particularly in tumor progression and metastatic spread. Yet, little is known, about the role of miRNAs in this intra and extracellular signaling process in MB. Objectives: This study aims to describe and to compare the expression profile of miRNAs contained in intracellular and secreted exosomes in MB cell lines of the four molecular subgroups of the disease. Materials and methods: cells from the commercial strains of the 4 molecular subgroups of MB: MED5R (WNT); DAOY, UW473, UW402 (SHH TP53 mutant) and ONS-76 (wild-type TP53 SHH); MED-114FH (Group 3) and CHLA-01-MED (Group 4) will be cultured in DMEM media, with and without vesicle-free fetal bovine serum supplementation. After the cells reach confluence, the culture medium will be collected and the exosomes isolated by the ultracentrifuge method, following the protocol of the miRCURY Exosome Kit (Quiagen). Electron microscopy and NanoSight will characterize the exosomes. The miRNA will be extracted from the isolated exosomes using the miRNeasy Micro kit (Quiagen). Subsequently, isolated miRNAs will undergo miRNA sequencing and validation of hypo and hyperexpressed miRNAs by quantitative real-time PCR. Statistical analysis: The AgiMicroRNA AFE (Agilent Resource Extraction) package from the Bioconductor library will be used to measure miRNA differential expression and the Mann-Whitney test, supported by IBM® SPSS Statistical software v.20 (SPSS Inc, IL , USA), to gauge differences in miRNA expression levels from the samples from each of the four groups. We hope to clarify if there is a specific profile of exosomes secreted by each molecular subgroup of MB. We also hope to apply this knowledge to the refinement of the MB molecular classification, as well as to support new therapeutic targets for this highly malignant and aggressive neoplasm. (AU)