Investigation of the cell of origin and evolutionary trajectories of human glioblastoma

Abstract Tumor cell of origin is a major determinant of cancer evolution. While the cell of origin of several tumor types has been successfully identified, glioblastoma, a malignant primary brain tumor, remains deprived of such biological information. Although rigorous studies have identified potential candidates among central nervous system cytotypes, a clear consensus of the early tumorigenic steps in glioblastoma has yet to be achieved. Unveiling glioblastoma cell of origin will shed a light on its evolutionary trajectories and, ultimately, better explain disease heterogeneity and poor clinical outcomes. To investigate cell of origin of glioblastoma, I generated multiple technological tools leveraging i) CRISPR/Cas9 genetic engineering, ii) human induced pluripotent stem cells derived cerebral organoids, iii) adeno-associated- and lentiviral gene delivery, iv) genome-tagging dynamic-barcoding. This work laid the foundation of the generation of human pre-clinical model of spontaneous gliomagenesis, thus allowing to trace and characterize early steps of human gliomagenesis. The overarching hypothesis of this research plan is that cell-specific molecular programs are permissive to malignant transformation under the selective pressure of known genetic drivers. To test this hypothesis, the Aim 1 of this work will be focused on characterizing histopathological and transcriptomic alterations following key driver events to provide detailed annotations of early glioblastoma tumorigenesis. To complement Aim 1 and improve our understanding of glioblastoma tumorigenesis, Aim 2 will be focused on developing a reliable platform that will permit to lineage trace glioblastoma cell of origin thanks to a sophisticated CRISPR/Cas9 barcoding technology. Single cell RNA sequencing of these models will generate a computational framework able to identify normal cells susceptible to gliomagenesis and transcriptional programs that lead malignant transformation. Comparative analysis with publicly available single cell RNA sequencing dataset of patient derived glioblastoma, will further advance our knowledge regarding tumor progression. This study will define a comprehensive panoramic of glioblastoma tumorigenesis and clarify its cell of origin, opening new avenues for future clinical therapeutic strategies.