Stem cell morphology defines functional heterogeneity and therapeutic vulnerabilities in glioblastoma

Glioblastoma (GBM) is an aggressive brain tumor and an unmet clinical need due to its invasiveness and therapy resistance. These features are driven by glioblastoma stem-like cells (GSLCs), which exhibit remarkable functional heterogeneity. While transcriptional profiling has helped define this heterogeneity, it alone does not fully predict cellular behaviors. Because cell morphology is closely linked to function during neurodevelopment, we asked whether integrating morphological and transcriptomic identities could reveal clinically relevant roles of GSLCs.

We employed CellShape-seq, a customised spatial transcriptomics platform integrating cell morphology with transcriptome and applied it to patient-derived GBM organoids. To link GSLC transcriptome to function, we combined assembloid invasion assays, calcium imaging, pharmacological perturbations and time-lapse microscopy.

We identified three GSLC morphological classes corresponding to distinct transcriptomic states and functional behaviors: nonpolar cells, which show differentiation and therapy sensitivity; elongated cells, which are blood-vessel associated, invasive and chemoresistant; and multipolar cells, which form intercellular networks conveying therapy resistance. Importantly, morphological state remains stable throughout interphase and is readily inherited from mother to daughter cells. However, under pharmacological stress GSLCs show striking morphological plasticity. Namely, Temozolomide treatment is associated with GSLC elongation and branching, while YAP inhibition and gap-junction blockage result in a reduction of elongated and multipolar GSLCs, respectively. Finally, combined targeting of these morphoclass-specific vulnerabilities reduces viability in patient-derived organoids.

Our findings demonstrate that cell morphology provides critical insights into GSLC behaviors and establish a rationale for putative morphology-informed therapies to overcome resistance and improve outcomes in GBM.