Background: Glioblastoma (GB), is an incurable brain tumor characterized by extreme malignancy and invasiveness, and the cellular mechanisms underlying such a severe phenotype are not completely understood. Although calcium (Ca2+) plays an important part in tumor proliferation and infiltration, it remains unclear whether Ca2+ signaling in GB cells is related to its location within the tumor and on the infiltrative potential of the cells.
Methods: In this study, we developed a stably transfected GL261 cell line that coexpresses a red fluorescent protein for actin cytoskeleton staining and the intracellular Ca2+ sensor, GCaMP6s. By means of intravital 2-photon imaging, we have characterized the morphological and functional properties of cells at different locations within the tumor.
Results: Our results showed that cells located at the tumor core are densely packed and rounded in shape, contrasting sharply with the polarized morphology observed in the peripheral cells. This anatomical heterogeneity corresponded to notable variations of the physiological phenotype: cells at the tumor core displayed low Ca2+ activity and very limited motility, while peripheral cells displayed intense Ca2+ activity and increased migration rates. Moreover, peripheral cells formed a cellular ensemble characterized by synchronized Ca2+ activity accompanied by a directionally biased collective motility.
Conclusions: These findings suggest that GB cells manifest activity patterns depending upon their spatial location within the tumor and that these correlate with their migration.
Keywords: calcium imaging; glioblastoma; intravital 2-photon imaging; tumor heterogeneity.
© The Author(s) 2025. Published by Oxford University Press, the Society for Neuro-Oncology and the European Association of Neuro-Oncology.