In vitro studies

BPA

BPA (L-isomer) was kindly supplied by Stella Chemifa Corporation (Osaka, Japan) and was converted to fructose complex before incubation³⁵. Thus, BPA used in the present study was used as a fructose complex.

Cell lines and cell culture

In this study, we used two human GSC lines (HGG13, HGG30) and a mouse GSC line (TS). (Fig. 1a–c). Under the institutional review board-approved protocol according to National Institute of Health (NIH) guidelines, the two human GSC lines (HGG13 and HGG30) had been established from surgical specimens of malignant gliomas after obtaining the written informed consent from the donors, as previously described^{36,37,38,39,40}. In the present study, these human GSC lines were used after obtaining approval from the Research Ethics Committee of Osaka Medical College (Approval No.: E14-2023).

These human GSCs were classified as mesenchymal-type GSCs based on their gene expression profiles³⁷. HGG13 and HGG30 cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM)/F12 (Thermo Fisher Scientific, Indianapolis, IN, USA) supplemented with 20 ng/mL epidermal growth factor (EGF; PeproTech, Rocky Hill, NJ, USA), 20 ng/mL basic fibroblast growth factor (bFGF; PeproTech), B27 supplement without vitamin A (Thermo Fisher Scientific), 200 ng/mL heparan sulfate, GlutaMAX (Thermo Fisher Scientific), 100 U/mL penicillin, and 100 ng/mL streptomycin (Thermo Fisher Scientific) at 37 °C in a 5% CO₂ atmosphere^37,41.

TS cells are murine GSCs established by overexpressing H-RAS V12 in normal neural stem/progenitor cells isolated from the subventricular zone of adult B6 mice harboring a homozygous deletion of the INK4a/Arf locus⁴². TS cells were cultured in DMEM/F12 (Sigma, St. Louis, MO, USA) supplemented with 20 ng/mL EGF (PeproTech), 20 ng/mL bFGF (PeproTech), B27 supplement without vitamin A (Thermo Fisher Scientific), 200 ng/mL heparan sulfate, 100 U/mL penicillin, and 100 ng/mL streptomycin (Thermo Fisher Scientific) at 37 °C in a 5% CO₂ atmosphere⁴².

Quantitative real-time PCR analysis for gene expression

Total RNA was extracted from cells using the RNA extraction kit (miRNeasy Mini #217,004; QIAGEN, Tokyo, Japan) and subjected to cDNA synthesis using the Transcriptor First-Strand cDNA Synthesis Kit (SuperScript VILO cDNA Synthesis Kit #11754-050; Thermo Fisher Scientific). The reaction was run in the LightCycler (Roche Diagnostics, Basel, Switzerland) according to the TaqMan assay protocol. The reaction consisted of a denaturation step (95 °C for 15 min), and the amplification and quantification process was repeated 45 times (95 °C for 15 s and 60 °C for 40 s). Data were analyzed using the LightCycler 3 software. The mRNA levels of SLC3A2, LAT1 (SLC7A5), ATB^0,+ (SLC6A14), Nrf2, HO-1 (HMOX1), and SOD2 were measured. Primer details are provided in Supplementary Table S1. Relative mRNA levels were calculated as the ratio of the target gene to GAPDH and β-actin by referring to each sample’s mRNA levels of GAPDH and β-actin.

In vitro uptake of BPA in GSCs

HGG13, HGG30, and TS cells were incubated with 5-aminolevulinic acid (ALA) (Cosmo Bio, Tokyo, Japan)-containing medium (0, 33, 100, 300, and 900 μΜ) for 24 h. After incubation, the cells were centrifuged and washed with phosphate-buffered saline (PBS). Cells were then exposed to 1 mM BPA-containing medium for 6 h, following which the cells were centrifuged and washed twice with PBS. The cells were then digested for 24 h with 1 N nitric acid solution (Wako Pure Chemical Industries, Osaka, Japan). The intracellular concentration of ¹⁰B was measured using inductively coupled plasma atomic emission spectroscopy (ICP-AES; iCAP6000 emission spectrometer, Hitachi High-Technologies, Tokyo, Japan). All the above experiments were performed under dark conditions to avoid the photodynamic effect on ALA-exposed tumor cells.

Animal experiments

Animal ethics

All of the animal experiments in this study were approved by the Ethical Committee on the Care and Use of Laboratory Animals of Osaka Medical College (Approval No. 30036) and Kyoto University Research Reactor Institute (KURNS; Kumatori, Osaka, Japan) (Approval No. P10-8), respectively. Animal housing and the following animal methods and procedures were performed in accordance with the regulations and guidelines of our institutions (OMC and KURNS), both of which comply with the ARRIVE Guidelines 2.0. (PLoS Bio 8(6), e1000412,2010).

Mouse glioma model using a GSC line (HGG13)

Seven-week-old male BALB/c nude mice [19–23 g body weight (b.w.)] were used for the HGG13 cell transplantation (Japan SLC, Hamamatsu, Japan). All animals were raised in Specific pathogen-free (SPF)-level animal rooms and fed a standard diet. Mice were anesthetized with an intraperitoneal injection of medetomidine (0.3 mg/kg), midazolam (4 mg/kg), and butorphanol (5 mg/kg) and placed in a stereotactic frame (model 51,625; David Kopf Instruments, Tujunga, CA, USA). A midline scalp incision was made, following which a small burr hole was drilled 2 mm right lateral to the bregma using an electric drill. A 25-μL Hamilton microsyringe with a 22-gauge needle (model 1700RN, Hamilton Bonaduz, Bonaduz, Switzerland) was inserted at 4 mm depth from the skull and withdrawn from 1 mm to the target in the brain (3 mm from the skull surface). Since HGG13 cells form spheroid, the cells were dispersed by pipetting and confirmed to be single-celled using a microscope. Ten thousand HGG13 cells suspended in 2 μL of Serum-free DMEM were injected at a rate of 1 μL/min using an automatic infusion pump. The needle was placed for 2 min after infusion and withdrawn slowly. The burr hole of the skull was sealed with bone wax, and the scalp was sutured with a sterilized clip.

Boron biodistribution studies using the HGG13-implanted glioma model

BPA biodistribution was evaluated 14 days after HGG13 cell implantation into the mouse brain. ALA or PBS (80 mg/kg b.w.) was orally administered to mice 24 h before BPA administration. BPA (250 mg/kg b.w., equivalent to 12 mg ¹⁰B/kg b.w.) was administered intraperitoneally. After 2.5 h of BPA administration, the mice were euthanized, and the tumor and blood were collected to measure the tissue ¹⁰B concentration using ICP-AES as we described previously⁴³.

In vivo BNCT studies using the HGG13-implanted glioma model

The in vivo BNCT study was performed using the HGG13-implanted glioma model mice at the Kyoto University Reactor (KUR) seven days after tumor cell implantation. Forty-four glioma-bearing mice were randomly divided into six groups: group 1, untreated control (n = 5); group 2, ALA-only control (n = 5); group 3, neutron irradiation-only control (n = 7); group 4, neutron irradiation following ALA (n = 6); group 5, neutron irradiation following BPA (n = 10); and group 6, neutron irradiation following the combination of ALA and BPA (n = 9).

ALA was administered orally at 80 mg/kg b.w. to animals in groups 2, 4, and 6, 24 h before BPA administration. BPA (250 mg/kg b.w., equivalent to 12 mg/kg ¹⁰B/kg b.w.) was administered intraperitoneally to groups 5 and 6, and PBS was administered to animals in other groups. Mice in groups 3 to 6 were irradiated at a reactor power of 1 MW at the Heavy Water Neutron Irradiation Facility (HWNIF) for 50 min, 2.5 h after BPA administration. Sham irradiation was performed on mice in groups 1 (neither BPA nor ALA) and 2 (no BPA, only ALA pre-loading). After implantation surgery, the mice’s body weight and neurological function were monitored daily. One day before death became imminent (defined by significant weight loss and a lack of activity or severe neurological deficits such as paralysis or ataxia and the onset of seizures), the mice were euthanized in accordance with the ARRIVE guidelines 2.0.

The therapeutic effects of BNCT were evaluated by measuring the survival time of glioma-bearing mice. The median survival time was calculated, and Kaplan–Meier survival curves were plotted for all groups. An overall log-rank test was performed to test the equality of the survival curves among groups.

Estimating of physical and equivalent doses delivered to GB13 glioma-bearing mice brain tumor model in the BNCT experiments

Estimation of the physical dose and equivalent dose delivered to the mice was done as described previously^44,45. The BNCT dose is the sum of the physical dose attributed to the ¹⁰B (n, α) ⁷Li and ¹⁴N (n, p) ¹⁴C capture reactions, the ¹H (n, n) ¹H scattering reaction, and gamma rays, which can be described by the following equation:

where D_B (boron dose) = 7.43 × 10⁻¹⁴ (Gy cm²/μg ¹⁰B/g) × Boron concentration (μg ¹⁰B/g) × thermal neutron fluence (1/cm²), and D_N (nitrogen dose) = 6.78 × 10⁻¹⁴ (Gy cm²/wt.%) × nitrogen concentration (weight%) × thermal neutron fluence (1/cm2). The above numerical values were taken from a previous study⁴⁶. The thermal neutron fluence was measured by the radioactivity of a gold foil (0.05 mm thick, 3 mm diameter) that was set at the surface of the heads of the irradiated mice. D_H is the physical dose due to the elastic scattering between epithermal or fast neutrons and the hydrogen nucleus⁴⁷. D_γ is the measured dose of gamma rays mixed in the neutron beam⁴⁸. To evaluate the therapeutic effect of BNCT, each radiation dose value was multiplied by the relative biological effectiveness (RBE) for each tissue to obtain the equivalent dose, as follows:

In BNCT, the relative biological effectiveness (RBE) is expressed as the compound biological effectiveness (CBE), which corresponds to the biological properties of the boron compounds. Its value for BPA is 3.8 for glioma and 0.9 for the brain⁴⁹. RBE for nitrogen (RBEN) and hydrogen (RBEH) is 3.0⁵⁰.

Expression of SLC3A2, LAT1 (SLC7A5), and ATB^0,+ in HGG13-derived tumors

We examined the effect of ALA on the expression of SLC3A2, SLC7A5, and ATB^0,+ in tumors derived from the HGG13 implanted glioma mice model. Fourteen days after HGG13 cell implantation, mice were administered ALA (80 mg/kg b.w.) or PBS (control). Twenty-four hours later, the mice were euthanized, and tumors were excised from the brains to extract RNA. Gene expression analysis was performed using RT-PCR.

Statistical analysis

In vitro data were generated from at least three independent experiments. All statistical analyses were performed using JMP Pro ver. 13 for Mac (SAS Institute Inc., Cary, NC, USA). Data are presented as mean ± SE. Comparison between two groups was performed using the Student’s t-test. A p-value less than 0.05 was considered statistically significant. Survival of the GSC-implanted mice was calculated for each group using the Kaplan–Meier method, and the log-rank test was used to determine the differences between survival curves. A Cox proportional hazards regression model was used to analyze the survival data, with Bonferroni’s correction for multiple comparisons.

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• Source: https://www.nature.com/articles/s41598-023-37296-6