Real-time confocal imaging for evaluation of dose-dependent effects of gamma knife radiosurgery on U87 glioblastoma cell line.

purposeTo characterize, in real time, how single-fraction low-dose Gamma Knife stereotactic radiosurgery (GKRS) affects proliferation and cell-cycle dynamics in glioblastoma (GBM) cells across a range of doses. We hypothesized that a 2.5 Gy dose would induce mitotic failure and cell death, whereas low doses (<1 Gy) might elicit adaptive or hormetic responses.

methodsHuman U-87 MG glioblastoma cells were cultured on coated glass-bottom dishes, stained with Hoechst 33,342, and maintained at 37°C/5% CO₂. A custom agarose phantom with an embedded dish and metal grid enabled precise delivery of graded SRS doses. CT-based planning localized the dish in the Leksell Gamma Knife Perfexion®. A single 50% isodose shot delivered 2.5 Gy at the central grid cell (C9), generating adjacent compartments with 2.0, 1.5, 1.0, 0.8, and 0.5 Gy. Live confocal imaging was performed at baseline and at 0, 3, 6, and 24 h post-irradiation. Total cell number and mitotic cells (condensed chromosomes) were quantified per field. Three independent experiments were analyzed using two-way ANOVA with Tukey's post hoc test (α = 0.05).

resultsAt 2.5 Gy, cell numbers declined to near zero by 6 h (Mean normalized adherent nuclear count decreased to approximately 5% of baseline.), associated with marked nuclear abnormalities, including multinucleation and nuclear fragmentation. Mitotic figures were absent at all post-SRS time points. Intermediate doses (1.0-1.5 Gy) caused a transient delay: cell counts increased (up to ~150% at 3-6 h, p < 0.05) before declining by 24 h. In contrast, low doses (0.5-0.8 Gy) resulted in net proliferation. The 0.8 Gy group showed a 182% increase in cell count at 24 h (p = 0.01), with a peak mitotic fraction (~12%) at 6 h versus ~2% in controls (p < 0.01). These findings are consistent with radiation hormesis. Two-way ANOVA confirmed significant effects of dose and time (p < 0.001).

conclusionsTo our knowledge, this is the first study integrating live-cell imaging with GKRS isodose mapping to assess temporal cellular responses to stereotactic radiosurgery. Gamma Knife SRS induces a dose-dependent response in GBM cells: high doses abolish proliferation, whereas sub-therapeutic doses paradoxically stimulate cell-cycle progression. These findings highlight a potential clinical concern, as low-dose regions may transiently promote tumor growth, but may also be exploited to enhance radiosensitivity.