Orthotopic xenograft model

For statistical purposes, three nude mice were used in each group. A total of 1×10⁶ luciferase-stable GBM#P3 cells were injected into the brains of 15 4-week-old male nude mice. One week later, tumours were examined using bioluminescence imaging (PerkinElmer IVIS Spectrum; Waltham, MA, USA). We found that in the 15 mice, one had no intracranial tumor formation and two had spinal metastases, so we excluded them. Our inclusion criteria included successful tumor implantation, uniform tumor size, and no spinal metastasis. Exclusion criteria included that there was no intracranial tumor formation, the tumor volume was significantly larger or smaller than that in other mice, and spinal metastasis. Then, we classified the remaining 12 mice as numbers 1-12, used excel software to generate random numbers, arranged the random numbers in reverse order, and then divided the mice into four groups(each group has 3 mice) from top to bottom: control group (n=3), safranal group (n=3), TMZ group (n=3) and TMZ+safranal group (n=3). Tumour volume was monitored using bioluminescence imaging. Mice were euthanized after 2 weeks of treatment.Cages were placed in the same house, keeping the external environment consistent.