Study population

A total of 42 patients with first-onset brain tumors were enrolled. Among those, nine patients were excluded as they had different tumor types (central neurocytoma, hemangiopericytoma, pilocytic astrocytoma, cavernoma, cortical dysplasia or diffuse astrocytoma, malignant lymphoma, and metastases). Consequently, 33 patients with gliomas were enrolled in the study (see Supplementary Tables 1 and 2 [Online Resource 1]). Ten IDH-mutant gliomas and 16 IDH-wild gliomas were included.

Overall, 24 healthy volunteers were also enrolled after a thorough examination by a neurologist to confirm the absence of both neurological abnormalities and cognitive impairments (see Supplementary Table 1 [Online Resource 1]). Healthy volunteers were recruited from the participants who had joined a clinical trial that aimed to assess the effects of polyphenols on cognition in older adults without dementia (Clinical Study Number: jRCTs041180064 and UMIN000021596) [24].

Blood and urine sampling

Blood (plasma) and urine samples were collected from patients with gliomas and healthy volunteers. In patients with gliomas, these samples were collected after surgery. The collected samples were immediately stored on ice, followed by centrifugation at 3,000 rpm for 30 min in an ice-cold environment. Subsequently, the samples were stored at − 80 °C until amino acid analysis. All samples were collected between 2019 and 2022 at Kanazawa University Hospital.

Brain tissues were obtained from all 33 enrolled patients. The number of blood and urine samples differed because some samples could not be collected before surgery. Blood and urine samples were collected from 26 to 18 individuals, respectively.

Removal of brain tissues by craniotomy

A craniotomy was performed to remove brain tumors and obtain brain tissues. Following treatment and an institutional review board-approved protocol, surplus tumor tissues were resected to obtain brain tissues for amino acid measurements. Vivid tumor samples were collected from tumor core areas without necrosis. The extent of brain tumor resection was based on the standard treatment, thereby eliminating the risk of adverse events directly related to the study.

Brain tissues for amino acid measurements consisted of non-tumor and tumor sections. The non-tumor tissues were distant from the excised tumor lesions, which were systematically removed for approach or extended resection of the lesion at the time of standard tumor resection using electrophysiologic functional monitoring and intraoperative awake brain mapping. Pathologists and neurosurgeons distinguished between tumor and non-tumor areas during surgery. The pathologist diagnosed the tumor area using rapid intraoperative pathological examination, while the neurosurgeon used intraoperative findings and postoperative hematoxylin and eosin staining of the excised brain tissues. Resected brain samples were immediately frozen at − 80 °C to preserve the in vivo concentration of amino acids without being influenced by amino acid-degrading enzymes or tissue denaturation. The evaluation focused on the white matter, where gliomas frequently occur, to minimize the impact of the collection site. Although different brain regions exhibit varying amino acid levels [30], this study disregarded the influence of brain regions since the non-tumor and tumor areas were essentially from the same brain region. All samples were collected between 2019 and 2022 at Kanazawa University Hospital.

Determination of chiral amino acids by 2D high-performance liquid chromatography

D- and L-amino acids were evaluated using a 2D high-performance liquid chromatography system (Nanospace SI-2 series, Shiseido, Tokyo, Japan), as previously described [8, 18]. Initially, NBD-amino acids were isolated in the first dimension using a microbore-ODS column prepared in a fused silica capillary (1000 mm × 0.53 mm i.d., 45 °C, Shiseido, Tokyo, Japan). The isolated fractions were subsequently transferred to the second dimension, which consisted of a narrow-bore enantioselective column, KSAACSP-001 S (250 mm × 1.5 mm i.d., 25 °C, prepared in collaboration with Shiseido), for the determination of D- and L-enantiomers. The mobile phases for the second dimension comprised mixed solutions of MeOH and MeCN containing formic acid. NBD-AAs were detected using fluorescence at 530 nm, with excitation at 470 nm.

Immunohistochemistry

We examined cases of central nervous system WHO grade 2 (9 cases), grade 3 (8 cases), and grade 4 (14 cases) using paraffin blocks from glioma cases stored in our laboratory, in addition to brain tissues from the cases in this study (see Supplementary Table 3 [Online Resource 1]). Histological diagnosis was determined according to the revised WHO criteria [34]. A non-tumor area was used as normal (10 cases). This procedure was performed in cases where sufficient tissue could be removed for staining.

In brief, 4-µm-thick tissue sections were stained using the standard hematoxylin and eosin staining technique. The slides were immunostained using the Envison+ System (Dako, Tokyo, Japan). Slides were autoclaved at 120 °C for 10 min in Target Retrieval Solution (pH 6.0; Dako, Glostrup, Denmark) after deparaffinization using Fast Solve. Quenching was performed using 3% hydrogen peroxide (H2O2) in methanol for 20 min, followed by blocking with 5% skim milk in TBS-T. Subsequently, sections were incubated overnight at 4 °C with a 1:200 dilution of DAO rabbit polyclonal antibody (HPA038654, Sigma, St Louis, MO, USA) and 1:200 dilution of serine racemase (SRR) (A-4) mouse monoclonal antibody (SC-365217, Santa Cruz, Dallas, TX, USA). The corresponding secondary antibody was applied at room temperature for 1 h after washing with TBS-T, and color development was achieved using 3,3-diaminobenzidine tetrahydrochloride (DAB Substrate Kit SK-4100, Vector, Akasaka, Japan) for 2–5 min. Hematoxylin was used for counterstaining (Wako Pure Chemical Industries, Ltd., Osaka, Japan). Images were acquired using a BZ-X700 microscope (Keyence, Osaka, Japan).

The positive rate of tumor cells in three high-magnification fields of view under a microscope was counted for each molecule, and the average value was calculated.

Human GBM mouse model

Animal experiments were performed according to our previous study, following the same protocol approved by the institutional review board [32]. We generated a mouse brain tumor model of human GBM by transplanting 10 × 105 KGS01 cells, derived from a human patient-derived GBM cell line established at Kanazawa University, into the brains of nude mice (BALB/cSlc-nu/nu, Charles River Laboratories, Osaka, Japan) (n = 5). Previous reports have confirmed that KGS01 cells are glioma-initiating cells capable of self-renewal in vitro and replicating the original tumor characteristics in a mouse xenograft model [2, 16]. The procedure involved drilling a hole into the skull, 3 mm outside the bregma, using a microdrill; next, stereotactic injection of glioma cells was performed to a depth of 3 mm below the dura mater. After 28 days, all mice were euthanized, and their brains were excised. The excised brains were stained with hematoxylin and eosin and that brain tumors were confirmed in all mice (see Supplementary Fig. 1 [Online Resource 2]).

Cell culture

KGS01 cells were cultured in a neurosphere formation medium containing DMEM/F12 (Gibco, Life Technologies, Carlsbad, CA, USA) supplemented with recombinant human epidermal growth factor at 20 ng/mL (Sigma–Aldrich, St. Louis, MO, USA), recombinant human basic fibroblast growth factor at 20 ng/mL (Sigma–Aldrich), MACS® NeuroBrew-21 supplement without vitamin A (Miltenyi Biotec, North Rhine-Westphalia, Germany), and GlutaMAX (Gibco) and 1% penicillin/streptomycin (Gibco).

Cell viability assay

Cell viability was assessed using an AlamarBlue assay (Bio-Rad Laboratories, Hercules, CA, USA). Neurospheres were dissociated into single cells using StemPro Accutase (Thermo Fisher Scientific), and the cells were seeded at a density of 3.0 × 103 cells/200 µL in a 96-well Costar ultra-low attachment plate (Corning) in asparagine-free medium (amino free DMEM/MEM essential amino acids/L-Alanyl-L-Glutamine/1%FBS/100uM Glycine medium) with 20 µL AlamarBlue. The cells were treated with different concentrations of D-Asn (only DMSO, 10 µM, 100 µM, and 1000 µM) after 4 h of incubation. The relative numbers of viable cells were determined by measuring the absorbance using a microplate reader (Bio-Rad Laboratories) at 0 and 72 h after adding amino acids. The average fluorescence values from the six wells in each group were calculated and plotted. Cells were treated with various concentrations of D-Asn to evaluate the effect of D-amino acids on cell proliferation. In addition, the concentration of amino acids in the medium was measured at 0 and 72 h after adding D-Asn.

Study approval

The Ethics Committee of Kanazawa University Hospital approved this study, which was conducted according to the Declaration of Helsinki. The IRB number for patients with gliomas was No. 2893, and that for heathy controls was No. 2016 − 417. All participants provided written informed consent and were informed of their right to withdraw from the study at any time.

All animal experiments were performed according to the guidelines of Kanazawa University for animal care and approved by the Institute for Experimental Animals, Kanazawa University Advanced Science Research Center (registration number AP-214259).

Statistical analyses

Data are presented as mean ± standard error of the mean, calculated using SPSS Statistics version 23 (IBM Inc, Tokyo, Japan). Statistical analysis was performed using Student’s t-test and the Mann–Whitney U test when comparing two groups. One-way ANOVA with Tukey’s multiple comparison test was used for multiple group comparisons. Statistical significance was set at p < 0.05.