Human glioblastomas harbour a subpopulation of glioblastoma stem cells that drive tumorigenesis. However, the origin of intratumoural functional heterogeneity between glioblastoma cells remains poorly understood. Here we study the clonal evolution of barcoded glioblastoma cells in an unbiased way following serial xenotransplantation to define their individual fate behaviours. Independent of an evolving mutational signature, we show that the growth of glioblastoma clones in vivo is consistent with a remarkably neutral process involving a conserved proliferative hierarchy rooted in glioblastoma stem cells. In this model, slow-cycling stem-like cells give rise to a more rapidly cycling progenitor population with extensive self-maintenance capacity, which in turn generates non-proliferative cells. We also identify rare ‘outlier’ clones that deviate from these dynamics, and further show that chemotherapy facilitates the expansion of pre-existing drug-resistant glioblastoma stem cells. Finally, we show that functionally distinct glioblastoma stem cells can be separately targeted using epigenetic compounds, suggesting new avenues for glioblastoma-targeted therapy.

Significance: Our study reveals lineage-specific mechanisms of ROS control and associated sensitivity to oxidative DNA damage in the basal and luminal progenitor-enriched subsets of normal human mammary cells. We show that the primitive luminal cells contain more mitochondria, show greater uptake of O ₂ , sustain and withstand higher levels of ROS, and have mechanisms that allow them to accrue mutagenic levels of oxidative DNA damage. These findings support a growing body of data suggesting the involvement of primitive luminal cells in the generation of human breast cancers.