Studying the Dynamics of Chromosomal Instability in Glioma within the Neural Microenvironment - ABSTRACT Glioblastoma (GBM) is the most aggressive primary brain tumor, with a median survival of just 14 months. Despite treatments including surgical resection, radiotherapy, and chemotherapy, patient outcomes remain poor, highlighting the urgent need for new therapeutic approaches. GBM’s aggressiveness is driven by extensive inter- and intra-tumoral heterogeneity resulting from diverse genetic and molecular alterations, including amplification of chromosome 7 and mutations in receptor tyrosine kinases (RTKs), such as Epidermal Growth Factor Receptor (EGFR) and Mesenchymal-Epithelial Transition factor receptor (MET). These alterations activate downstream signaling pathways that support tumor progression. Chromosomal instability (CIN), characterized by gains, losses, or rearrangements of chromosomal segments during cell division, is a major contributor to GBM's genetic complexity. CIN facilitates rapid adaptation to selective pressures, including treatment, fostering resistance to therapies. It also promotes the formation and maintenance of extrachromosomal DNA (ecDNA), including extrachromosomal circular DNA (eccDNA), which amplify oncogenic signaling pathways. Tumor cells harbor a mix of ecDNA amplicons, including wild-type EGFR and constitutively active mutant variants of EGFR, such as EGFRvIII, which contribute to genomic instability through disrupted cellular mechanisms such as cytokinesis. Current models, such as patient-derived neurosphere cultures, often fail to maintain key features of GBM, including ecDNA, limiting their reliability for long-term studies. Human-induced pluripotent stem cell (iPSC)- derived 3D brain organoid models, specifically human cortical organoids (hCOs), present a promising alternative for studying GBM biology. These organoids are enriched with synaptically active neurons and astrocytes, mimicking the brain’s neural microenvironment. However, hCOs lack vasculature and immune cells. To complement this, organotypic brain slice cultures (OBSCs), prepared from fresh surgical specimens, preserve native 3D architecture and include vascular, immune, and extracellular matrix components, and can serve as a crucial ex vivo experimental platform for investigating aspects of GBM. This proposal aims to evaluate the maintenance of CIN in GBM cells, with a particular focus on ecDNA, and will address three key questions: (1) How does the neural microenvironment contribute to CIN and maintenance of ecDNA within GBM cells over time? (2) Can neurodevelopmental mechanisms that govern cytokinesis and asymmetric division heterogeneously maintain CIN in GBM cells? (3) Can eccDNA be induced to convert normal neural progenitor cells in hCOs to GBM-like tumor cells to model tumor initiation and progression? By investigating these questions, this research seeks to uncover new therapeutic strategies targeting the mechanisms that sustain CIN in GBM.
