Orthotopic xenograft model

The animal experiments were approved by The Second Hospital of Hebei Medical University, China and performed in accordance with standard policies. All efforts were made to minimize the number and the suffering of the animals. All procedures performed in studies involving human participants were in accordance with the ethics standards of the institutional and national research committee and with the 1964 Helsinki Declaration. Written consent was collected from all the participants. The study was approved by Ethics Committee of Second Hospital of Hebei Medical University. GBM tumor tissues were collected following previously established procedures.¹⁶ Briefly, the GBM tumor tissues were obtained from patients diagnosed with GBM and undergoing surgery. Excised patient’s tumor tissues were washed with Dulbecco’s modified Eagle’s medium (DMEM) and minced to small pieces. Then the Matrigel (BD Biosciences, Franklin Lakes, NJ, USA) was added to the tumor tissue and planted subcutaneously to the nude mice. The primary tumors were allowed to grow up to 1 cm in diameter. The patient-derived orthotopic xenograft model was established as adapted from previously established protocols.¹⁷ Briefly, nude mice aged 6–8 weeks were anesthetized with sodium pentobarbital (50 mg/kg, intraperitoneal injection). Tumor cells (1 × 10⁴) from patients suspended in 2 μl of culture medium (DMEM with 10% fetal bovine serum) were injected into the right cerebral hemisphere (1 mm to right of midline, 1.5 mm anterior to lambdoidal suture, 3 mm deep) via a 10 μl 26-gauge Gastight 1701 syringe needle (Hamilton Company, Allston, MA, USA). After the injection, mice were observed till they reached a moribund state.

To compare the proliferation rate between the primary and recurrent tumor, the mice were sacrificed 2 months after the injection. The tumor tissues were taken and the volumes were measured. A total of 4 patient-derived samples and 10 mice in each experimental group were used for each assay.