An Optimized In Vivo Model for Long-Term Two-Photon Imaging of Glioblastoma Growth at Single-Cell Resolution [dataset]

Background. Glioblastoma multiforme (GBM) is among the most lethal primary brain tumours, with a median survival of ~15 months and a prognosis that has remained essentially unchanged despite decades of clinical research effort. Preclinical models capable of resolving tumour growth, invasion, and microenvironmental interactions at single-cell resolutions are critical for advancing for evaluating therapeutic strategies. Methods. We developed an orthotopic mouse GBM model combining intracranial implantation of GL261 murine glioma cells with a chronic cranial window preparation in C57BL/6J mice. Six implantation conditions were systematically compared, including four capillary scar sealants, over a 32-day longitudinal imaging period using one-photon epifluorescence and high-resolution two-photon microscopy. We introduce a tumor deep-localization index to quantify the spatial distribution of tumour expansion relative to depth below the dura. Results. Tumorsphere implantation produced significantly greater glioma cell infiltration into peri-tumoral tissue than cell suspension injection, independently of inter-animal variability. Complex scar sealing approaches introduced variable tumour outcomes and severe optical artifacts. Careful deep tumorsphere implantation without scar sealing yielded predominantly intraparenchymal growth, confirmed by the lowest tumor deep-localization index values. Conclusions. Deep tumorsphere implantation without additional scar sealing provides a reproducible, optically favourable model well-suited to longitudinal single-cell resolution imaging of GBM progression and invasion.