Comprehensive serum metabolic snapshots enable diagnosis, prognosis, and monitoring for brainstem gliomas [II]

Brainstem gliomas (BSG) is a highly malignant central nervous system childhood tumors with a poor prognosis of a 5-year survival rate of < 10%. Metabolism during radiotherapy is a dynamic and precisely programmed process, improving clinical outcomes and guiding therapy decisions of BSG. Here, we for the first time construct diagnostic and prognostic assays of BSG via circulating metabolites based on both cross-sectional study and longitudinal cohort study with 106 BSG patients. We employ nanoparticle enhanced laser desorption/ionization mass spectrometry to characterize high-resolution static and dynamic snapshots of metabolites during BSG radiotherapy. We show that this serological tool reaches the area under the curve of 0.933 for BSG diagnosis in an independent blind test and predicts risk of patients with significant differences (p < 0.05) in prognostic outcomes. We further identify eight distinct temporal patterns of metabolite regulation associated with radiotherapy responses and tracked the metabolic trajectory via dynamic metabolic snapshots throughout radiotherapy process. If further validated, this framework could be extended to derive comprehensive metabolic pictures for cancers including but not limited to BSG. Overall design: SPF-grade female BALB/c nude mice, aged 4-6 weeks and weighing 18-20 g, were used as experimental subjects in this study, following refinement of the experimental methods based on previous study. All animal care and experiments were approved (#XHEC-F-2023-031) by the Animal Ethics Committee of Xinhua Hospital, School of Medicine, Shanghai Jiao Tong University. To establish the orthotopic intracranial xenograft model, DIPG-XH-01 cells were transduced with lentiviral vectors encoding enhanced green fluorescent protein (eGFP) and firefly luciferase (ffLuc). Positively transduced cells were subsequently sorted by fluorescence-activated cell sorting (FACS) to achieve a purity greater than 95% prior to implantation. Under deep anesthesia, mice were secured on a stereotactic frame, and a cranial burr hole was drilled at stereotactic coordinates relative to the lambda suture: 0.8 mm posterior, 1 mm lateral to the midline, and 4.5 mm depth. A total of 1×106 cells suspended in 3 µL phosphate-buffered saline (PBS) were infused via Hamilton syringe at a controlled rate of 0.3 µL/min. To minimize reflux, the injection needle was retained for 5 minutes post-infusion. Tumor engraftment was validated 72 hours post-surgery by intraperitoneal administration of D-luciferin potassium salt (75 mg/kg; Promega) followed by bioluminescent signal acquisition using the IVIS Spectrum In Vivo Imaging System (PerkinElmer). At the humane endpoint, mice were euthanized. Brain tumor tissue and normal mice brainstem tissues were collected and subjected to RNA sequencing. To establish the subcutaneous tumor model, 3×106 DIPG-XH-01 cells were resuspended in a 1:1 mixture with chilled Matrigel matrix (Corning), maintained on ice throughout preparation. A 100 µL aliquot of the cell-Matrigel suspension was injected subcutaneously into the left dorsal flank of mice using a sterile 25-gauge needle. Tumor volume was measured using a vernier caliper. Radiotherapy was initiated in subcutaneous tumor-bearing mice when the tumor size exceeded 100 mm3. Mice in the radiotherapy group were administered a daily radiation dose of 2 Gy at the tumor site for five consecutive days. Tumor tissues before and after radiotherapy were collected and subjected to RNA sequencing.