Abstract: Glioblastoma (GBM) is one of the deadliest brain tumors, with limited therapeutic options due to its extreme heterogeneity, largely driven by glioblastoma stem cells (GSCs). GSCs exhibit self-renewal, plasticity, and therapy resistance, making them key players in tumor progression and recurrence. Targeting GSCs is an unmet clinical challenge requiring a deeper understanding of the mechanisms underpinning their stemness. GBM hijacks neurodevelopmental pathways, and GSCs share transcriptomic similarities with neural stem cells of the developing brain, called basal or outer radial glia (bRG). Those cells were shown to exhibit a morphological heterogeneity which is thought to regulate their proliferation and migration. Similarly, our recent work revealed that also GSC morphology is tightly linked to their function. These findings prompted us to conduct an in-depth examination of the GSC morphology. By employing a cohort of patient-derived 2D and 3D in vitro model system, we identify cell morphology as a new layer of GSC heterogeneity. While bRG cells have been morphologically categorized based mainly on the number of their cellular protrusions, GSCs exhibited further inter- and intra-patient morphological heterogeneity. This is particularly pertinent to the length and thickness of the protrusions, tendency to form cell-cell networks, and cell flatness. The analysis of these features, coupled with the measure of their in vitro clonogenic ability, allowed us to identify different morphoclasses with specific self-renewal potentials. We further performed a transcriptomic profiling and revealed morphoclass-intrinsic molecular traits. The gene expression profile of morphoclasses associated with higher clonogenic potential was enriched in pathways linked to tumor growth and aggressiveness. Moreover, the enriched pathways play fundamental roles in neurodevelopment, including the maintenance of bRG cells, the promotion of neuronal process growth, and their morphogenesis. Hence, this study highlights that GSC morphology and stemness are intricately linked, likely through neurodevelopmental morphoregulatory pathways. This provides rationale for a potential future targeting of morphoregulators to therapeutically target GSCs and disrupt GBM progression.
