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

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. SPF-grade female BALB/c nude mice, aged 5-6 weeks and weighing 18-20 g, were used as experimental subjects in this study, following refinement of the experimental methods based on previous study. In the case of the orthotopic tumor model, the inoculation site was the right caudate nucleus of the mouse brain. When anesthetizing mice with isoflurane, a concentration of 1-2% was maintained once the mice are confirmed to have entered the anesthetized state, in order to sustain the anesthetic effect throughout the experiment. The trypsinized U87MG-Luc cells were resuspended in cold phosphate-buffered saline at 1.0 × 105 cells/μL. A 10 μL microsyringe was used to inject 5 μL of the cell suspension into the right hemisphere of the mouse brain (0.5 mm anterior to the bregma, 2 mm lateral to the midline, and 3.5 mm depth from the skull) at a rate of 1 μL/min. Following the injection, the bone hole was sealed with bone wax, and the incision was sutured. The mice were placed on thermostatic electric blankets and closely monitored until fully resuscitated. Three days post tumor cell injection, tumor implantation and growth were evaluated using the IVIS Lumina Series III in vivo imaging system (PerkinElmer, USA). At the humane endpoint, mice were euthanized. Five brain tumor tissue and four adjacent non-cancerous tissue were collected and subjected to RNA sequencing. To establish the subcutaneous tumor model, mice were inoculated with U87MG-Luc cells (3.0 × 106 cells/site) suspended in Matrigel, and were injected into the axillary region of the right forelimb. Tumor volume was measured using a vernier caliper, and radiotherapy was initiated once the average 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. Four brain tumor samples were collected before and five days after radiotherapy, and then subjected to RNA sequencing, respectively.