All subjects gave their written informed consent for inclusion before they participated in the study. The study was conducted in accordance with the Declaration of Helsinki, and the protocol was approved by the Ethics Committee of the University of California, Davis (Tissue collection project identification code 218204, original approval date 06/01/2005; plasma collection project identification code 1412052-2, approval date 4/16/2019).

The UC Davis Pathology Biorepository at the Comprehensive Cancer Center provides high quality and well-characterized human brain tumor tissue specimens. In this centralized biorepository, all samples were collected after patients’ informed consents and underwent quality control by a clinical neuropathologist. From January 2010 to July 2022, a total of 12 fresh frozen GBM specimens were identified and obtained from the biorepository for metabolomic analysis. Limited deidentified clinical information was abstracted from the medical records within the scope of the approved IRB protocol of the biorepository. All patients underwent the standard Stupp protocol ² for glioblastoma treatment.

The UC Davis Department of Neurosurgery (Dr. Orin Bloch Laboratory) has an IRB approved protocol to collect blood samples from patients with GBM at diagnosis and throughout treatment, along with access to clinical information of these patients. All procedures performed in this study were in accordance with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards. From August 2019 to July 2022, a total of 12 plasma specimens were selected for metabolomic analysis.

All patients underwent the standard Stupp protocol ² for glioblastoma treatment. Blood specimens were collected before surgery during the hospitalization. No anesthesia was administered at the time of the blood collection, but all patients were on high dose oral steroids in the peri-operative periods. Untargeted metabolomic, biogenic amine, and lipidomic analyses for GBM tissue and patient plasma were performed.

Sample preparation and extraction

Metabolites and biogenic amines were extracted as previously described. ¹⁶ Blood plasma or serum was extracted following the protocols first published by V. Matyash et al. ¹⁷ In detail, methanol (1.5 ml) was added to a 200 ml sample aliquot, which was placed into a glass tube with a Teflon-lined cap, and the tube was vortexed. Then, 5 ml of MTBE was added, and the mixture was incubated for 1 h at room temperature in a shaker. Phase separation was induced by adding 1.25 ml of MS-grade water. Upon 10 min of incubation at room temperature, the sample was centrifuged at 1,000 g for 10 min. The upper (organic) phase was collected, and the lower phase was reextracted with 2 ml of the solvent mixture, whose composition was equivalent to the expected composition of the upper phase [obtained by mixing MTBE/methanol/water (10:3:2.5, v/v/v) and collecting the upper phase]. Combined organic phases were dried in a vacuum centrifuge. To speed up sample drying, 200 ml of MS-grade methanol was added to the organic phase after 25 min of centrifugation. Extracted lipids were dissolved in 200 ml of CHCl3/methanol/water (60:30:4.5, v/v/v) for storage.

Using this protocol, lipid extracts in methyl tert-butyl ether phase (MTBA) were separated from proteins and polar hydrophilic small molecules (in the methanol/water phase) in a way that the lipids were found in the top layer of liquid-liquid separations, rather than in the bottom layer.

Decanting the top layer therefore ensured that the extracts were not contaminated by proteins or polar compounds. The top layer was used for lipidomics while the bottom layer (methanol/water phase) was very suitable for the hydrophilic interaction liquid chromatography-mass spectrometry (HILIC-MS) investigations.

Data acquisition

Metabolite profiling using HILIC-MS was performed on the Agilent 1290 UHPLC/Sciex TripleTOF 6600 mass spectrometer. Metabolites (5 μL) were separated using Waters AcquityUPLC BEH amide column (1.7 μm, 2.1 × 50 mm) and a binary mobile phase. Mobile phase A: 100% H ₂O +10 mM Ammonium Formate + 0.125% Formic acid; B: 95:5 ACN/H ₂O + 10 mM Ammonium Formate + 0.125% Formic acid. Chromatographic data acquisition was 15 min long. Data were acquired in data-dependent acquisition mode with a mass range 50-1,500 m/z for MS1 and 40-1,000 m/z for MS2.

Lipidomic data were acquired using the Agilent 1290 UHPLC/Agilent 6530 QTOF (for positive mode) and 6550 QTOF (for negative mode) mass spectrometer. Waters Acquity Premier BEH C18 column (1.7 μm, 2.1 × 50 mm) was used for chromatographic separation applying binary mobile phase system (Positive mode: mobile phase A: 60:40 v/v acetonitrile:water + 10 mM ammonium formate + 0.1% formic acid; mobile phase B: 90:10 v/v isopropanol:acetonitrile + 10 mM ammonium formate + 0.1% formic acid. Negative mode: mobile phase A: 60:40 v/v acetonitrile:water + 10 mM ammonium acetate; mobile phase B: 90:10 v/v isopropanol:acetonitrile + 10 mM ammonium acetate). MS scan range and mass resolution for positive mode were 120-1,200 m/z and 10,000, respectively, and 60-1,200 m/z and 20,000 for negative mode.

Primary metabolism data were acquired by gas chromatography (GC)-MS using an Agilent 7890A GC coupled to a Leco Pegasus HT TOF mass spectrometer. Extracts were dried down, derivatized by methoxyamination and trimethylsilylation, and injected in spitless mode with a temperature gradient from 50-330°C. Mass spectra were acquired at 17 Hz from 85-500 Da. ¹⁶

Raw data processing and metabolite annotation

Acquired raw LC-MS and LC-MS//MS data were processed using the following steps: Raw CSH-C18-TOF (for lipidomics) and HILIC-TTOF (for polar metabolites profiling) MS data were processed using MS-Dial (version 4.9). Data-independent MS/MS deconvolution was performed for comprehensive metabolome analysis. ¹⁶ Raw GC-TOF MS data files were processed using ChromaTOF and metabolomics BinBase database. ¹⁶ Peak heights were used for analysis given the strong correlation between extracted ion peak area and peak height across a wide range of concentrations. For very abundant peaks, ion saturation may occur, and in such cases, peak area may be a slightly better measure than peak height. However, in metabolomics, most compounds are low in abundance. Therefore, peak height is a more robust way to measure metabolite levels because the nose baseline has less impact ( e.g., for peak start/end) on peak height compared to peak area.

Descriptive statistics were used to characterize baseline patient and treatment characteristics. Individual metabolite abundance comparisons at diagnosis and at relapse were performed using GraphPad Prism 9 (version 9.5, San Diego, CA). MetaboAnalyst 5.0 (version5.0, McGill University, Montreal, QC, Canada) ¹⁸ was used to generate principal component analysis (PCA), score plots, heat maps and volcano plots. The processed peak heights with their annotation were imported to MetaboAnalyst, normalized to the total sample median and auto scaled. Paired or unpaired Student’s t-Test was used to identify significantly altered metabolites between the compared groups ( p-value of less than 0.05 was considered significant). ChemRICH (version 2023), a statistical enrichment approach based on chemical similarity rather than sparse biochemical knowledge annotations was used to group the metabolites. ChemRICH sets have a self-contained size where p-values do not rely on the size of a background database.

The protocols are deposited at Protocol IO: https://dx.doi.org/10.17504/protocols.io.n92ldmjkol5b/v1.

Patient demographics are described in Table 1. ³¹ In the tissue cohort, a total of 12 specimens from nine patients were analyzed (nine specimens at diagnosis, three of which had paired specimens at recurrence). The mean age at diagnosis was 49 years old. There was a male predominance of 67%. Most of this cohort was non-Hispanic White in race/ethnicity.

In the plasma cohort, a total of 12 paired specimens (diagnosis and at recurrence) from six patients with GBM were analyzed. The mean age was 54 years old. There was a male predominance of 67%. The original GBM tissue pathology all showed Isocitrate Dehydrogenase ( IDH) wild type. Additional pathology features including epidermal growth factor receptor ( EGFR), O ⁶-methylguanine-DNA methyl-transferase ( MGMT), and Alpha thalassemia/mental retardation syndrome X-linked ( ATRX) status are shown in Table 1. The mean progression free survival of this cohort was 14 months. The mean overall survival was 17 months.

The GBM tissue cohort included nine tumor tissue specimens at diagnosis, three of which had paired tissue specimens at recurrence. A complete list of significantly altered metabolites is available in Table 2. A summary of these changes is shown in Figure 1. Principal component analysis (PCA) showed two grouped clustering trends with significant overlap ( Figure 1A). A heat map of the top 50 significantly altered metabolites, lipids, and biogenic amines, showed differences in cluster trends between tissue samples at diagnosis and at recurrence ( Figure 1B). Dendrogram and heat map were produced from MetaboAnalyst version 5.0 using the default Euclidean distance and Ward linkage. Top 50 altered denotes the top 50 metabolites with significantly altered abundance in each comparison, exhibiting fold changes greater than 1.5. This includes both metabolites with increased and decreased fold changes. There were several significantly upregulated compounds ( Figure 1C) in the lipidomic and biogenic amine analysis. Also included in Figure 1 are scattered column plots for compounds with significant change in abundance at diagnosis and at recurrence ( Figure 1D), which included N-alpha-methylhistamine (P=0.037), 2,3-dihydroxypropyl dihydrogen phosphate with (P=0.029), CE22:6 (P=0.021), LPE 20:4 (P=0.004), LPE 22:6 (P=0.011), LPE 22:4 (P=0.041), CE 20:3 (P=0.031), CE 16:1 (P=0.003), phosphocholine with a (P=0.045), and succinic acid (P=0.025).

When analyzing the three paired tissue specimens at diagnosis and at recurrence, again the PCA plot demonstrated two overlapping clusters ( Figure 2A). The heat map showed more visible separation between tissue samples at diagnosis and at relapse ( Figure 2B). We found 19 compounds that were significantly altered. Volcano plot (using p-value <0.05 and Fold Change cutoff 1.5) revealed that three metabolites were upregulated, and six metabolites were down regulated ( Figure 2C). A complete list of significantly altered metabolites is available in Table 3. Among these metabolites, we found that the levels of the 2-methylbutyryl-L-carnitine (P=0.02) and an unknown compound eluting at 1.84 minutes with an accurate mass of 186.1075 Da (P=0.04) were significantly higher in primary tumors than recurrent GBMs ( Figure 2D).

All six patients enrolled in this cohort had paired plasma specimens at diagnosis and at recurrence. However, these were not the same patients whose GBM tissue were studied in the cohort above. PCA analysis showed evident separation between the metabolomic profiles at diagnosis and at recurrence, except for patient one, whose metabolomic profiles were similar at diagnosis and at recurrence ( Figure 3A). The heatmap of the top 50 altered metabolites showed visible differences between plasma at diagnosis and at recurrence ( Figure 3B). Again, the metabolite profile of patient one at recurrence was similar to its status at diagnosis, while the other patients’ profiles demonstrated differences.

There were 61 compounds that were altered significantly between diagnosis and recurrence in the patient plasma. The representatives were shown in Figure 3C. A complete list of significantly altered metabolites is available in Table 4. The progression free survival of these six patients was shown in Figure 3D. Using ChemRich, we were able to identify that based on chemical structural similarity, amino acids and unsaturated phosphatidylcholines were significantly up regulated and unsaturated fatty acids and phosphatidylethanolamines were downregulated ( Figure 4E). The compounds with significantly increased abundance at recurrence included 2,4-difluorotoluene (P=0.031), diatrizoic acid (P=0.032), indole-3-acetate with (P=0.029), urea (P=0.025), pseudo uridine (P=0.042), and maltose (P=0.035). The compounds with significantly decreased abundance included FA 20:1 (eicosenoic acid) (P=0.017), glucose-1-phosphate (P=0.017), FA 18:2 (linoleic acid) (P=0.017), arginine (P=0.036), FA 20:3 (homo-gamma-linolenic acid) (P=0.036), galactosamine (P=0.007), and FA 18:3 (linolenic acid) (P=0.012) ( Figure 3F).

Both the PCA and heatmap analyses showed separate metabolic profiles from tissues at diagnosis and plasma at diagnosis ( Figure 4A and B). Similarly, the metabolomic profile from tissue at recurrence separated from plasma at recurrence ( Figure 4C and D). When placing all four groups of data in one plot ( Figure 4C and D), we again saw inter-specimen differences between the metabolomic profiles in tissue and plasma, but there were no intra-specimen differences at diagnosis and at recurrence.