Application of GCLC in enhancement of BNCT drug cellular uptake

Abstract

The invention discloses an application of GCLC in enhancement of BNCT drug cellular uptake. Boron neutron capture therapy (BNCT) has become a promising method for osteosarcoma treatment, and the treatment effect of the BNCT depends on accumulation and distribution of 10B compounds in tumors to a great extent. The invention provides a gene marker capable of enhancing BNCT drug uptake of tumor cells and application of the gene marker, and proves that targeted GCLC can overcome limitation of tumor heterogeneity on BNCT drug uptake by utilizing the vulnerability of tumor specificity.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to the application of GCLC in enhancing the cellular uptake of BNCT drugs. Background Art

[0002] Osteosarcoma is a highly aggressive primary bone tumor that primarily affects children and adolescents. The main treatment for osteosarcoma is chemotherapy combined with surgical resection, and radiotherapy is used for inoperable sacrum, pelvis, and spine. However, neither of these two treatments can achieve satisfactory therapeutic effects. In order to overcome the recurrence caused by poor chemotherapy response, insufficient surgical margins, and poor radiation penetration into the bone, boron neutron capture therapy (BNCT) has strong penetration ability and few side effects and has become a promising method for the treatment of osteosarcoma. This model requires the selective accumulation of non-radioactive boron-10 ( 10 B) compound, which is then irradiated with thermal neutrons. 10 The B capture reaction produces high linear energy transfer (LET) alpha particles and recoil lithium-7 (7Li) nuclei. Due to their extremely short radiation range, the cytotoxic effects of these particles are mainly limited to 10 BNCT uptake of tumor cells significantly protects adjacent healthy tissues. However, the therapeutic effect of BNCT depends largely on 10 Preclinical and clinical studies have consistently shown that the accumulation and distribution of compound B in tumors 10 There was significant heterogeneity in B uptake. Specifically, some tumor regions 10 B levels are lower than blood concentrations. This refractory tumor cell subpopulation may survive BNCT treatment, greatly weakening the therapeutic effect. 10 Intratumoral heterogeneity of B uptake is a major limiting factor for the success of BNCT.

[0003] Current improvement 10 Strategies for B delivery have primarily focused on pharmacological and formulation innovations, such as increasing the dose of clinically approved borylphenylalanine (BPA), developing novel agents with enhanced tumor selectivity and concentration, and 10 B carriers, such as boron sodium (BSH) and novel nanocarriers for boron drugs. Although these approaches show great potential, they still face multiple challenges, such as low delivery efficiency, in vivo stability, and immune safety risks. Summary of the Invention

[0004] In view of the deficiencies of the existing technology, the present invention provides a gene marker capable of enhancing the uptake of BNCT drugs by tumor cells and its application.

[0005] To achieve the above object, the present invention adopts the following technical solutions:

[0006] The first aspect of the present invention provides a use of a GCLC inhibitor or a pharmaceutically acceptable salt thereof, wherein the use comprises any one of the following:

[0007] 1) Application in the preparation of drugs for enhancing BNCT drug uptake;

[0008] 2) Application in the preparation of drugs for treating cancer.

[0009] In the present invention, the pharmaceutically acceptable salt refers to a salt of the active compound and is prepared by reacting the active compound with a suitable organic or inorganic acid or acid derivative. Pharmaceutically acceptable salts include, but are not limited to, hydrochlorides, sulfates, phosphates, citrates, hydrobromides, acetates, benzoates, benzenesulfonates, tartrates, carbonates, citrates, gluconates, lactates, malates, methanesulfonates, stearates, valerates, nitrates, sodium salts, calcium salts, potassium salts, zinc salts, and meglumine salts.

[0010] In the present invention, the term "enhancement" means that the technical solution provided by the present invention enables the uptake of boron-containing drugs by tumor tissue to be significantly higher than that of normal tissue and the case where GCLC inhibitors or pharmaceutically acceptable salts thereof are not used, and meets the following quantitative standards: the tumor-to-blood boron concentration ratio (T / B) ≥ 3.2:1, or the tumor-to-normal tissue boron concentration ratio (T / N) ≥ 3.7:1, and at the same time, the retention time of the drug in the tumor cells is prolonged by at least 67% (relative to the prior art), thereby increasing the boron-10 (¹ 0 B) enrichment efficiency, ultimately enhancing the local killing effect of α particles and lithium nuclei after neutron irradiation and reducing damage to normal tissues.

[0011] In some embodiments, the drug can be used alone or in the form of a pharmaceutical composition.

[0012] In some embodiments, the cancer is selected from osteosarcoma, glioma, head and neck tumors, and melanoma.

[0013] Furthermore, the cancer is osteosarcoma.

[0014] For the purposes of this invention, osteosarcoma is a primary bone malignancy originating from osteoblasts or mesenchymal cells. Based on histological subtype, it can be categorized as conventional osteosarcoma, telangiectatic osteosarcoma, small cell osteosarcoma, low-grade central osteosarcoma, and secondary osteosarcoma. Tumors commonly occur in the metaphysis of long bones, such as the distal femur, proximal tibia, and proximal humerus; in axial bones, such as the skull, spine, and pelvis; and in extraosseous tissues, such as the thigh, buttocks, lung, and breast. Molecular subtypes include TP53 deficiency, RB1 deficiency, and PARP inhibitor sensitivity.

[0015] In the present invention, the term "inhibitor" refers to a substance that targets, reduces or inhibits GCLC protein activity or mRNA expression activity.

[0016] In the present invention, the term "GCLC," also known as GCL, GCS, GLCL, GLCLC, CNSHA7, and with Gene ID 2729, is used. In the context of the present invention, GCLC includes wild-type, mutant, or fragments thereof. The term encompasses full-length, unprocessed GCLC, as well as any form of GCLC derived from processing in cells. The term also encompasses naturally occurring variants of GCLC (e.g., splice variants or allelic variants). For example, the term encompasses the GCLC gene, GCLC protein, human GCLC, and GCLC from any other vertebrate source, including mammals such as primates and rodents (e.g., mice and rats).

[0017] In some embodiments, the GCLC inhibitors include naturally occurring and synthetic ones.

[0018] Furthermore, the GCLC inhibitor is a compound that has a specific inhibitory effect on GCLC.

[0019] In some embodiments, the GCLC inhibitor includes a compound targeting GCLC used in any one of RNA interference technology, CRISPR technology, antisense oligonucleotide technology, TALEN technology, ZFN technology, and Cre loxP gene recombination technology.

[0020] Furthermore, the GCLC inhibitor is a compound targeting GCLC used in RNA interference technology.

[0021] Furthermore, the GCLC inhibitor is siRNA targeting GCLC.

[0022] Furthermore, the sequence of the siRNA is shown in SEQ ID NO: 1-2.

[0023] A second aspect of the present invention provides a pharmaceutical composition for enhancing the uptake of a BNCT drug, the pharmaceutical composition comprising a BNCT drug, a GCLC inhibitor or a pharmaceutically acceptable salt thereof.

[0024] As used herein, the term "pharmaceutical composition" refers to a composition comprising at least one biologically active compound. The pharmaceutical compositions of the present invention may be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally, or via an implanted reservoir. In some embodiments, oral administration is preferred. The pharmaceutical compositions of the present invention may contain any conventional non-toxic pharmaceutically acceptable carrier, adjuvant, or vehicle. In some cases, pharmaceutically acceptable acids, bases, or buffers may be used to adjust the pH of the formulation to improve the stability of the formulated compound or its dosage form. The term parenteral as used herein includes subcutaneous, intradermal, intravenous, intramuscular, intraarticular, intraarterial, intrasynovial, intrasternal, intrathecal, intralesional, and intracranial injection or infusion techniques. The pharmaceutical compositions of the present invention may be administered to the recipient by any route that reaches the target tissue.

[0025] Furthermore, the pharmaceutical composition comprises a therapeutically effective amount of a BNCT drug and a GCLC inhibitor or a pharmaceutically acceptable salt thereof.

[0026] As used herein, the term "therapeutically effective amount" refers to a dose that achieves cancer treatment in a subject, and will depend on the patient, the severity of the condition, and the route and medium used for administration. The amount of active compound in such therapeutic compositions is a suitable dose. Those skilled in the art will be aware of suitable doses or will be able to determine them through simple experimentation.

[0027] Furthermore, the subject refers to any individual of interest, including humans, other mammals, preferably primates, and particularly preferably humans.

[0028] In some embodiments, the BNCT drugs include borophenylalanine, borsafenamic acid, novel nanocarrier boron drugs, and antibody-conjugated boron drugs.

[0029] In some embodiments, the GCLC inhibitor is a compound that has a specific inhibitory effect on GCLC.

[0030] Furthermore, the GCLC inhibitor includes a compound targeting GCLC used in any one of RNA interference technology, CRISPR technology, antisense oligonucleotide technology, TALEN technology, ZFN technology, and Cre loxP gene recombination technology.

[0031] Furthermore, the GCLC inhibitor is a compound targeting GCLC used in RNA interference technology.

[0032] Furthermore, the GCLC inhibitor is siRNA targeting GCLC.

[0033] Furthermore, the sequence of the siRNA is shown in SEQ ID NO: 1-2.

[0034] In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient.

[0035] In the present invention, the term "pharmaceutically acceptable excipient" refers to any pharmaceutical excipient or pharmaceutical carrier that does not induce the production of antibodies harmful to the individual receiving the composition and can be used without excessive toxicity, generally referring to non-toxic solid, semi-solid or liquid fillers, diluents, encapsulating materials or any type of formulation aids. Suitable carriers can be large, slowly metabolized macromolecules, such as proteins, polysaccharides, polylactic acid, polyglycolic acid, polymerized amino acids and amino acid copolymers. Such carriers are well known to those of ordinary skill in the art. The pharmaceutically acceptable carrier in the pharmaceutical composition can include fluids, such as water, saline, glycerol and ethanol. Auxiliary substances, such as wetting agents or emulsifiers, pH buffer substances, etc., can also be present in such vehicles.

[0036] Furthermore, the pharmaceutically acceptable excipients include pharmaceutically acceptable carriers, diluents, excipients, glidants, preservatives, dyes / colorants, flavor enhancers, surfactants, wetting agents, dispersants, suspending agents, stabilizers, isotonic agents, solvents, surfactants or emulsifiers.

[0037] The third aspect of the present invention provides the use of the pharmaceutical composition according to the second aspect of the present invention in the preparation of a product for treating cancer, wherein the cancer is selected from osteosarcoma, glioma, head and neck tumor, and melanoma.

[0038] Furthermore, the cancer is osteosarcoma.

[0039] A fourth aspect of the present invention provides a method for enhancing BNCT drug uptake in vitro, which is achieved by inhibiting the expression of GCLC.

[0040] Advantages and beneficial effects of the present invention: The experimental evidence provided by the present invention shows that GCLC 10 B uptake modulators can address long-standing challenges in BNCT: 10 Compound B is heterogeneous and often exhibits insufficient intratumoral accumulation. Targeting GCLCs could overcome these limitations by exploiting tumor-specific vulnerabilities. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 To explore through genome-wide CRISPR screening 10B. Damage mechanism results diagram, where a is the experimental workflow; b is a Venn diagram of differentially expressed genes, BNCT-specific genes (pink) represent genes uniquely identified in the comparison between neutron and control, while BNCT + BPA-specific genes (green) represent genes uniquely identified in the comparison between BNCT and control (BPA treatment), and the overlapping area represents genes common to the two comparisons; ce is the GO enrichment analysis of the comparison between BNCT and control (BPA treatment), where c is biological process, d is cellular component, and e is molecular function; f is a statistical chart of cell survival rate.

[0042] Figure 2 Screening for CRISPR 10 B shows the GCLC results for key regulatory factors in uptake. (a) Schematic diagram of the experimental workflow; (b) flow cytometry analysis results; (c) a volcano plot depicting upregulated (red) and downregulated (blue) genes in the FACS-sorted group and the U2OS-KO cell pool, with a significance threshold of |log2FC| > 2, P < 0.05; (d) KEGG (d) and GO enrichment analysis (e) of differentially expressed genes between FACS-sorted and U2OS-KO pooled cells.

[0043] Figure 3 Enhancement of U2OS cells for GCLC-KO cells 10 B Uptake results, where a is GCLC uptake of U2OS cells 10 Bc are representative fluorescence images (b) and statistical analysis of mean fluorescence intensity (c) of BSH-Cy5 uptake in GCLC-KO cells and wild-type U2OS cells; de are the uptake of BSH-Cy5 in GCLC-KO cells and U2OS cells. 10 B Representative images of fluorescence colocalization of drugs and lysosomes (d) and colocalization index (e).

[0044] Figure 4 GCLC gene affects U2OS cells 10 B. Mechanistic results of uptake. (a) Volcano plot analysis of transcriptome profiles; (b) GO enrichment analysis of differentially expressed genes between GCLC-KO and wild-type U2OS cells after 2-hour BSH treatment; (c) Representative fluorescence microscopy images showing FITC-dextran (70 kDa) uptake by GCLC-KO and WT U2OS cells after 2-hour incubation at 1 mg / mL. Cell nuclei were counterstained with DAPI blue. Scale bar: 20 µm; (d) Quantitative analysis of mean fluorescence intensity of FITC-dextran uptake; (e) Representative images (e) and relative fluorescence intensity statistics (f) of ROS levels in WT U2OS and GCLC-KO cells validated by the DCFH-DA probe.

[0045] Figure 5 GCLC deficiency enhances stress-induced macropinocytosis in tumor cells and promotes 10 B shows the uptake results of GCLC-deficient U2OS and HOB-SV40 cells at different time points. 10 B is the uptake process, b is the fluorescence imaging of the effect of GCLC deficiency on macropinocytosis in U2OS cells, and c is the stress-induced promotion 10 B Schematic diagram of uptake macropinocytosis.

[0046] Figure 6 GCLC gene pair 10 Figure 1 shows the in vivo experimental verification results of the effect of B uptake, where a is the quantitative analysis of PAMAM-BSH uptake in GCLC-KO cell lines using ICP-MS, b is a schematic diagram of the in vivo experiment, c is a representative CT image of the osteosarcoma animal model, d is the detection of GCLC levels in tumor tissue by Western blot analysis, e is the fluorescence imaging of BSH-CY5 distribution in tumors, and f is the fluorescence imaging of BSH-CY5 in tumor tissues. 10 Statistical results of B concentration. DETAILED DESCRIPTION

[0047] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without making any creative efforts shall fall within the scope of protection of the present invention.

[0048] Example

[0049] 1. Materials and Methods

[0050] 1. 10 B compound and animal model: The first generation boron compound used in this study, L-boron phenylalanine-fructose complex injection (BPA-F), is derived from Steboronine ® The second-generation boron compound, synthesized and provided by the Institute of High Energy Physics, Chinese Academy of Sciences, is a polyamidoamine dendrimer-mercaptodecaborate (PAMAM-BSH) conjugate labeled with rhodamine B or Cy5. The preparation method follows that of Liu et al. (Journal of Controlled Release, 2025, DOI:10.1016 / j.jconrel.2025.113863).

[0051] All animal husbandry and experimental procedures were performed in accordance with the guidelines approved by the Institutional Animal Care and Use Committee (IACUC) of Tsinghua University. Male BGCLC / c nude mice (1–4 weeks old, 18–20 g) were purchased from Beijing Weitonglihua Laboratory Animal Technology Co., Ltd.

[0052] 2. Cell Culture: U2OS, HEK293FT, and MDA-MB-231 cell lines were purchased from the China National Cell Resource Center, and the normal osteoblast cell line HOB-SV40 (SNL-593) was purchased from ScienCell Bioscience. All cell lines were authenticated by short tandem repeat (STR) profiling and confirmed to be free of mycoplasma contamination.

[0053] U2OS cells were cultured in McCoy's 5A medium (FEIMOBIO, M3781) supplemented with 10% fetal bovine serum (FBS). HEK293FT cells were cultured in high-glucose DMEM (Damas Life, DA298U1) supplemented with 10% FBS and G418. MDA-MB-231 cells were cultured in RPMI-1640 medium (Damas Life, DA298U9) supplemented with 10% FBS. HOB-SV40 cells were cultured in a proprietary medium (SNLM-593) provided by ScienCell.

[0054] 3. Construction of stable knockout library cell pool: Use the whole-genome human CRISPR GeCKOv2 A knockout library (GenScript, HG789), which contains 3 unique sgRNAs per gene and 1000 non-targeting control sgRNAs. Each miRNA is targeted by four different sgRNAs to ensure comprehensive knockout coverage.

[0055] Lentiviral particles were produced by co-transfecting HEK293FT packaging cells with the GeCKOv2 plasmid library and a lentiviral packaging kit (GeneChem, YK-LVP-05). U2OS cells were transfected at a multiplicity of infection (MOI) of 0.3 to achieve 300-fold library coverage. After 72 hours of incubation, cells were selected with 1.5 µg / mL puromycin (MCE, HY-K1057-1) for 72 hours to establish a stable U2OS-KO cell pool.

[0056] 4. Western Blot Analysis: Total protein from wild-type and CRISPR library-transduced U2OS cells was extracted using RIPA lysis buffer, and protein concentration was quantified by BCA assay. Proteins were denatured with 5× loading buffer (Beyotime, P0015L) at 100°C for 10 minutes, separated by SDS-PAGE using a 10% HEPES precast gel (YEASEN, LK202), and transferred to a PVDF membrane. The membrane was blocked with 5% bovine serum albumin (BSA) in TBST and incubated overnight at 4°C with anti-Cas9 (Abcam, ab203933) and anti-GAPDH (Abcam, ab8245) antibodies. After washing, the membrane was incubated with secondary antibodies for 1 hour at room temperature. Protein bands were visualized using a chemiluminescence detection system (Tanon 5200). In addition, western blotting was used to: (i) validate the knockdown efficiency of GCLC in U2OS cells; (ii) assess GCLC knockdown in HOB-SV40 and MDA-MB-231 cells; and (iii) confirm the silencing efficiency of GCLC siRNA in an in vivo osteosarcoma model.

[0057] GCLC siRNA sequence: 5′-GAAGGAGGCTACTTCTATA-3′ (SEQ ID NO:1); 5′-GGAACAATGTCCGAGTTCA-3′ (SEQ ID NO:2).

[0058] 5. Survival Screening After Neutron Irradiation: Before BNCT at the Spallation Neutron Source, U2OS-KO cells were incubated with 20 μg / mL BPA-F in serum-free medium for 2 hours. A control group was irradiated under the same conditions without BPA-F. After overnight incubation and adherence, surviving cells were harvested.

[0059] 6. Fluorescence-activated cell sorting (FACS) of high boron-accumulating cells (top 10%): The U2OS-KO cell pool was incubated with 50 μg / mL rhodamine B-labeled PAMAM-BSH in serum-free medium for 2 hours. The top 10% of cells with the highest fluorescence intensity were sorted by fluorescence-activated cell sorting (FACS) to enrich for the high boron-accumulating cell population.

[0060] 7. DNA / RNA Extraction: Genomic DNA was extracted from BPA-treated and untreated groups, as well as from high- and normal-uptake cell populations, using a genomic DNA purification kit (Thermo Fisher, KIT0103) according to the manufacturer's protocol. RNA was removed with RNase A, and DNA was eluted with elution buffer for high-throughput sequencing.

[0061] For transcriptome analysis, wild-type and GCLC knockout U2OS lines were seeded in 6-well plates. At 80% confluency, cells were treated with 50 μg / mL PAMAM-BSH for 2 hours. When cells reached 80% confluency, they were incubated with 50 μg / mL PAMAM-BSH for 2 hours. Total RNA was extracted using TRIzol (Thermo Fisher Scientific, 15596026CN), snap-frozen in liquid nitrogen, and submitted for RNA sequencing (RNA-seq).

[0062] 8. Whole-genome screening and analysis: Paired-end FASTQ files were generated using the Illumina platform. Reads were assembled using PANDAseq and analyzed using the MAGeCK algorithm with reference to the GeCKOv2 library (Addgene; Sanjana et al., Nature Methods, 2014). Because this was a knockout-based screen, sgRNA abundance was negatively correlated with gene function. Volcano plots were generated to identify candidate genes (GCLC), followed by GO and KEGG enrichment analysis.

[0063] 9. Construction of Single-Gene Knockout Cell Lines: GCLC targeting plasmids were purchased from GenScript. Lentiviral packaging and transduction were performed as described in 3, with an MOI of 10. Stable knockout cell lines were generated by puromycin selection for 72 hours.

[0064] 10. Quantification of boron uptake by inductively coupled plasma mass spectrometry (ICP-MS): Wild-type and single-gene knockout U2OS cells were seeded in 6-well plates. The cells were then incubated with 50 μg / mL PAMAM-BSH or 20 μg / mL BPA-F for 2 hours. After nitric acid digestion, boron content in the samples was quantified by ICP-MS.

[0065] 11. Lysosomal Colocalization Imaging: U2OS wild-type and knockout cells in logarithmic growth phase were plated in four-quadrant culture dishes (CSR Biotech, D35-20-1.5-N). When cells reached approximately 70% confluence, they were stained with Hoechst 33342 and LysoBrite Red (Beyotime) and subsequently incubated with 50 µg / mL Cy5-labeled PAMAM-BSH for 10 minutes. Three-channel time-lapse imaging was performed using structured illumination microscopy (HIS-SIM). Colocalization and uptake levels were analyzed using ImageJ and Image-Pro Plus software.

[0066] 12. Transcriptome Sequencing and Analysis: RNA-seq libraries were prepared and sequenced using the Illumina platform. Raw reads were quality-trimmed using Trimmomatic, aligned to the human reference genome (GRCh38.p14) using HISAT2, and quantified using FeatureCounts to generate a gene expression matrix. GO and KEGG enrichment analysis was performed on differentially expressed genes (DEGs) and visualized using volcano plots.

[0067] 13. Verification of the Macropinocytosis Pathway: U2OS wild-type and GCLC-KO cells were seeded in glass-bottom culture dishes. When cell confluency reached 70%, 25 μg / mL fluorescein isothiocyanate-dextran (FITC-Dextran) (MCE, HY-128868A) was added to the culture medium and incubated at 37°C for 20 minutes. After washing, cells were fixed with 4% paraformaldehyde and stained with Hoechst 33342 (for nuclear staining). Confocal images were acquired using a Zeiss LSM880 microscope. The number and total fluorescence intensity of FITC-dextran-positive vesicles per cell were quantified using ImageJ software.

[0068] 14. Reactive oxygen species (ROS) level measurement: Wild-type and GCLC-KO cells were seeded in glass-bottom culture dishes. When cells reached the logarithmic growth phase, they were incubated with 10 μM DCFH-DA probe (Beyotime, S0033S) at 37°C in the dark for 30 minutes. After incubation, cells were washed with phosphate-buffered saline (PBS). Images were acquired using an inverted fluorescence microscope. The mean fluorescence intensity (MFI) was calculated from five random fields of view in each group for statistical analysis using ImageJ.

[0069] Confocal Imaging of PAMAM-BSH Uptake Kinetics: Wild-type and GCLC-KO U2OS cells were seeded in four-quadrant culture dishes and incubated with 50 μg / mL Cy5-labeled PAMAM-BSH. Cells were fixed at 2, 5, 8, 10, and 12 seconds after incubation, washed with PBS, and imaged using a Zeiss LSM880 microscope. Image analysis of uptake kinetics was performed using ImageJ.

[0070] 16. Stress-induced macropinocytosis assay: HOB-SV40 and U2OS cells and their corresponding GCLC-KO cell lines were seeded in four-quadrant culture dishes and incubated with 50 μg / mL Cy5-labeled PAMAM-BSH for 2 hours. After incubation with boron compounds, 10 μM DCFH-DA was added to assess cellular ROS levels. The cells were then fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. Nonspecific binding sites were blocked with 5% BSA. Alexa Fluor ® Immunofluorescence staining was performed using a 594-conjugated anti-ARPC3 antibody (1:200 dilution), and nuclei were counterstained with DAPI. Multichannel fluorescence images were acquired using a confocal microscope. ARPC3 fluorescence intensity and Cy5 signal intensity (representing boron uptake) were semiquantitatively analyzed using ImageJ software. Colocalization analysis was performed to assess ARPC3 enrichment at macropinosomes.

[0071] Boron Uptake in GCLC Knockout Cell Lines: GCLC-KO derivatives of the HOB-SV40, U2OS, and MDA-MB-231 cell lines were seeded in 6-well plates and incubated with 50 μg / mL PAMAM-BSH for 2 hours. The cells were then digested with nitric acid and analyzed by ICP-MS to quantify intracellular boron concentrations.

[0072] 18. In vivo validation: 4-week-old male BGCLC / c nude mice (SPF grade) were anesthetized and U2OS osteosarcoma cells (5×10 6 Cells (10 μL of GCLC siRNA, dissolved in 10 μL of PBS) were orthotopically implanted into the right tibial plateau. Tumor formation was confirmed by micro-CT on day 14 after implantation. Mice were randomly assigned to experimental groups and received intratumoral injections of GCLC siRNA (10 nmol / 20 g body weight; Ribobio, R10043.10) every 3 days for 1 week. Subsequently, mice were intravenously injected with Cy5-labeled PAMAM-BSH at 30 μg B / 20 g body weight. Mice were imaged using an IVIS Spectrum imaging system at 0.5, 1, 2, 4, 6, 12, and 24 hours after injection. Fluorescence in the tumor area was quantified using Living Image software. After the final imaging session, mice were euthanized, and blood, tumor, and peritumoral tissues were collected, digested, and analyzed by ICP-MS to determine boron concentrations and calculate tumor-to-nontumor (T / N) and tumor-to-blood (T / B) ratios.

[0073] 2. Experimental Results

[0074] 1. Genome-wide analysis of BNCT cell responses in osteosarcoma cells

[0075] To investigate the genomic determinants of cellular response to BNCT, we performed a genome-wide CRISPR-Cas9 knockout screen in the U2OS human osteosarcoma cell line. Cells transduced with a genome-wide sgRNA library were assigned to two conditions to decode the mechanisms of BNCT therapy: (1) control versus neutron irradiation, and (2) control versus treatment with a clinically approved 10 Neutron irradiation of B carrier BPA pretreatment, BPA is used to promote 10 B accumulation in tumor cells. After treatment, surviving cells were collected and subjected to next-generation sequencing (NGS) to quantify the enrichment or depletion of sgRNAs, revealing genes essential for survival or death under each condition ( Figure 1 a) in the above example.

[0076] There were approximately 1203 overlapping genes between the two groups, of which 1070 significant genes were unique to neutron irradiation + BPA (BNCT) and 1411 genes were unique to neutron irradiation ( Figure 1 b). The statistical results suggest that the specific 10 B. Effectiveness. Neutron irradiation and BNCT not only shared a common response component in the differential gene profiles but also exhibited unique changes. This difference suggests that the two treatment conditions have significant differences in inducing biological effects.

[0077] Comparative analysis showed that BPA exposure shifted the BNCT response from a "passive repair mode" to a "metabolic stress mode," characterized by enhanced DNA damage without immediate cell killing. Instead, tumor cells were forced to activate a wider range of stress defense pathways—including antioxidant systems and DNA repair modules—to survive. This shift was further supported by differential enrichment of cell cycle checkpoint genes. These results clearly validated 10 B neutrons induce intrinsic cellular stress, thereby enhancing BNCT-induced cytotoxicity through multiple mechanisms ( Figure 1 In addition, in our screening, BNCT induced approximately 40% cytotoxicity in U2OS-KO mixed cells ( Figure 1 f), which may be limited not only by genetic resistance but also by 10 Suboptimal limitation of B accumulation. Therefore, enhanced intracellular 10 B uptake may be the key to maximizing the efficacy of BNCT. 10 Regulators of B uptake are a promising strategy to overcome the current limitations of BNCT in osteosarcoma.

[0078] 2. GCLC knockout enhances the uptake of PAMAM-BSH in U2OS cells

[0079] To improve10 B absorption efficiency, researchers are designing a new generation 10 Much work has been done on boron delivery agents. BSH, with 12 boron atoms per molecule in a stable icosahedral configuration, has a higher boron density compared to BPA. In addition, polyamidoamine dendrimer-conjugated BSH (PAMAM-BSH) provides a higher boron payload per particle and improved pharmacokinetics through enhanced permeability and retention effects. However, its uptake mechanism still needs to be further elucidated to improve its potential for clinical application. To investigate this, we utilized PAMAM-BSH covalently labeled with a fluorescent probe (Rhodamine B) to visualize and quantify intracellular accumulation. After treating the U2OS-KO library with PAMAM-BSH-Rhodamine B for 2 hours, we isolated the top 10% of cells with the highest PAMAM BSSH Rhodamine B uptake by fluorescence-activated cell sorting and performed next-generation sequencing ( Figure 2 a, b in ).

[0080] Based on the results of these high-uptake cells, sgRNAs targeting GCLC (glutamate cysteine ​​ligase catalytic subunit) were significantly enriched, indicating that knockout of GCLC promoted the cellular uptake of PAMAM-BSH ( Figure 2 c). Further analysis showed that the endocytic pathway was significantly enriched, and several related pathways including the PI3K-Akt signaling pathway, actin cytoskeleton regulation, and lysosomal pathway were also upregulated accordingly, implying that the cellular internalization and intracellular trafficking of PAMAM-BSH mainly depend on endocytosis and related membrane transport mechanisms ( Figure 2 The simultaneous enrichment of phospholipid metabolism, sphingolipid signaling, and ER protein processing pathways suggests a coordinated cellular adaptation involving membrane remodeling and stress signaling, which is often seen under metabolic reprogramming or therapeutically induced stress conditions and helps cells adapt to higher 10 B concentration. GO enrichment analysis further verified the involvement of these functional modules ( Figure 2 e in the above example).

[0081] In order to verify the 10 To investigate the role of GCLC in B uptake, we constructed a U2OS cell line with stable GCLC knockout. Wild-type and knockout cell lines were incubated with PAMAM-BSH, and inductively coupled plasma mass spectrometry (ICP-MS) was used to quantify the intracellular 10 B concentration ( Figure 3 (a) Under PAMAM-BSH treatment, the GCLC-KO cell line showed a significantly decreased expression of β-actin in the GCLC-KO cell line compared with the wild-type cell line. 10To investigate whether PAMAM-BSH uptake is involved in the lysosomal pathway, we labeled lysosomes with LysoTracker and analyzed the colocalization of PAMAM-BSH-Rhodamine B with lysosomes using confocal microscopy ( Figure 3 GCLC-KO cells showed a higher colocalization index than wild-type cells. Quantitative analysis further confirmed that the fluorescence intensity of GCLC-KO cells was significantly higher than that of wild-type cells, supporting the role of lysosome-mediated endocytosis in 10 The important role of B uptake ( Figure 3 d, e in ).

[0082] In conclusion, loss of GCLC function significantly enhanced the uptake of the high-boron compound PAMAM-BSH by U2OS cells, establishing its role in regulating intracellular 10 This uptake-based forward screening strategy using PAMAM-BSH-Rhodamine B successfully identified regulators of 10 B delivered key functional genes, GCLC became the central node for coordinating oxidative stress response and endocytosis.

[0083] 3. GCLC enhances the growth of U2OS cells 10 Molecular basis of B uptake

[0084] To further elucidate the 10 To investigate the molecular basis for enhanced cellular uptake of compound B, we performed transcriptome (RNA-seq) analysis comparing GCLC-KO U2OS cells with wild-type controls. Differential gene expression analysis revealed that GCLC depletion induced transcriptome alterations, with many genes significantly upregulated ( Figure 4 a) in the above example.

[0085] GO and KEGG pathway enrichment analysis of the upregulated gene sets in GCLC-KO cells highlighted significant enrichment of functional categories related to membrane endocytosis, vesicle trafficking, lysosomal processing, and endocytosis ( Figure 4 b). Notably, pathways related to redox regulation and oxidative stress response were also enriched. Since GCLC encodes the catalytic subunit of glutamate-cysteine ​​ligase, its deletion may lead to rapid depletion of intracellular GSH. Given that glutathione is the major intracellular antioxidant, such depletion would impair the redox buffering capacity of the cell. To experimentally verify the induction of oxidative stress in GCLC-KO cells, we measured reactive oxygen species (ROS) levels using the fluorescent probe 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA). The results showed that the baseline ROS in the GCLC-KO group was significantly elevated compared with the WT control group, with the fluorescence intensity increasing by approximately 10-fold ( Figure 4 (c) Overall, these results confirm that GCLC depletion leads to GSH depletion, triggering significant oxidative stress. This redox imbalance may be a key driver of downstream functional reprogramming, including enhanced endocytic activity.

[0086] Furthermore, RNA-seq data indicated increased expression of several genes involved in actin cytoskeleton remodeling and membrane ruffling, both key steps in endocytic vesicle formation. Genes encoding components of vesicle formation and membrane recycling also showed upregulated expression, suggesting that GCLC-deficient cells possess intrinsically enhanced endocytic capacity at the transcriptional level. Therefore, we hypothesized that enhanced endocytosis results from increased vesicle uptake. However, nanoparticles (PAMAM-BSH) taken up via the endocytic pathway tend to end up in lysosomes. To test this, we labeled lysosomes in WT and GCLC-KO U2OS cells and measured PAMAM-BSH uptake using rhodamine B and Cy5 fluorescence over a fixed incubation period ( Figure 4 In addition, we quantified the subcellular colocalization between Cy5-labeled PAMAM-BSH and lysosomes. Significant colocalization was observed in GCLC-KO cells compared to WT cells, indicating that internalized 10 Compound B is more efficiently and rapidly transported to the lysosomal compartment. These results support the hypothesis that GCLC depletion promotes cellular oxidative adaptation, thereby enhancing vesicle-mediated uptake and trafficking to intracellular organelles, leading to 10 Increased accumulation of B. This is consistent with RNA-seq data that identified endocytosis as the top enriched pathway.

[0087] To functionally confirm that the enhanced macropinocytosis is the 10 To investigate the potential mechanism of increased B uptake, we used FITC-dextran conjugate (70 kDa) as an indicator to verify our hypothesis. This dextran cannot enter cells through receptor-mediated endocytosis but is effectively internalized through macropinocytosis. Confocal microscopy analysis showed that the fluorescence intensity in GCLC-KO cells was significantly increased, indicating that more FITC-dextran was internalized compared to WT cells. Figure 4 e, f). This indicates that GCLC deficiency promotes the activity of rat monocytes. 10 The enhanced uptake of compound B provides a mechanistic explanation and strong functional evidence.

[0088] GCLC deficiency enhances PAMAM-BSH uptake through oxidative stress-induced macropinocytosis

[0089] The PAMAM-BSH compound carries an overall positive charge, which may promote electrostatic interactions with negatively charged cell membranes and facilitate the initiation of macropinocytosis. 10 B. Drug uptake process. We incubated Cy5-labeled PAMAM-BSH with GCLC-KO and wild-type U2OS cells at multiple time intervals and then immediately fixed them. The intracellular distribution and uptake efficiency of PAMAM-BSH were assessed using super-resolution confocal laser scanning microscopy ( Figure 5 Based on these results, it is clear that KO cells exhibit a significant increase in vesicles associated with macropinocytosis compared to wild-type cells. Concomitantly, the retention of PAMAM-BSH on the cell membrane is significantly reduced. 10 The intracellular uptake of compound B occurred at an accelerated rate within the same time frame. These findings were consistent with our initial hypothesis. In addition, ICP-MS analysis further confirmed these observations, showing that the intracellular uptake of compound B in GCLC-KO cells 10 The accumulation of B increased ( Figure 5 b) in the above example.

[0090] Based on these results, we proposed a GCLC-mediated regulation of 10 B uptake model ( Figure 5 Figure c): GCLC deficiency leads to decreased glutathione, triggering oxidative stress and upregulating macropinocytosis. This enhanced macropinocytosis activity enables the non-selective uptake of extracellular fluid and solutes, thereby increasing 10 This stress-induced macropinocytosis model elucidates a new mechanism for regulating the uptake of therapeutic agents in U2OS cells under GCLC-deficient stress conditions and proposes a method to enhance the internalization of high-drug loading and high-molecular-weight macropinocytosis. 10 Potential strategies for the uptake of B drugs (such as PAMAM-BSH, etc.).

[0091] 5. Tumor cells show increased sensitivity to GCLC-deficiency-induced macropinocytosis

[0092] Although we have demonstrated the role of GCLC in promoting 10 Although the mechanism of action of GCLC in drug uptake is unclear, whether cancer cells exhibit enhanced sensitivity to this process compared to normal cells remains a key determinant of therapeutic potential. To address this question, we generated GCLC-KO HOB-SV40 (primary human osteoblasts derived from healthy donors) and MDA-MB-231 (triple-negative breast cancer) cell lines and compared them to the U2OS KO model to quantify GCLC using ICP-MS under the same conditions by PAMAM-BSH treatment. 10B concentration. The results showed that GCLC-KO U2OS and MDA-MB-231 cells 10 B uptake was significantly higher in GCLC-KO-HOB-SV40 cells. In addition, GCLC-KO HOB-V40 cells showed a 10 B uptake was increased, although this increase was significantly lower than that in tumor cells ( Figure 6 These results indicate that GCLC knockout broadly enhances tumor cell responsiveness to 10 Tumor cells showed increased sensitivity to GCLC-deficiency-induced macropinocytosis. Targeting GCLC significantly increased tumor 10 The uptake of B was minimal, with minimal impact on normal tissues, which is consistent with the principle of BNCT and demonstrates potential clinical practical value.

[0093] In order to further verify the effect of GCLC expression on complex environment in vivo 10 To investigate the effect of B uptake, we established a U2OS xenograft mouse model and silenced GCLC expression in tumor tissues by siRNA injection ( Figure 6 Tumor formation was confirmed by computed tomography (CT) ( Figure 6 Western blot analysis confirmed that GCLC was significantly suppressed in tumors ( Figure 6 Subsequently, fluorescently labeled BSH-Cy5 (10 mg / kg) was injected via the tail vein and monitored using a small animal imaging system. 10 B distribution.

[0094] The results showed that boron accumulation in tumors peaked at 6 hours after injection ( Figure 6 e). ICP-MS analysis showed that GCLC silenced tumors 10 B concentrations were approximately 16% higher than those in wild-type tumors ( Figure 6 In the figure (f), the ratios of tumor to normal tissue (T / N) and tumor to blood (T / B) were 3.2 and 3.7, respectively, compared with 2.1 and 2.5 in the wild-type group. These results indicate that GCLC silencing significantly enhances 10 B accumulates in tumors with limited impact on adjacent tissues and blood. In vivo experiments confirmed that GCLC-targeted interventions improve BNCT 10 B. Feasibility of uptake.

[0095] Based on the above results, it is shown that GCLC can be used as 10 B uptake modulators can address long-standing challenges in BNCT: 10The heterogeneity of B compounds and the lack of intratumoral accumulation that often occurs. Targeting GCLC can overcome these limitations by exploiting a tumor-specific vulnerability, namely the enhanced sensitivity of cancer cells to oxidative stress-induced macropinocytosis. Since tumor heterogeneity and radioresistance in osteosarcoma lead to poor clinical outcomes, especially in patients with metastatic or recurrent disease, the technical solutions of the present invention are particularly important for BNCT treatment of osteosarcoma. Although this study focused on osteosarcoma, the applicability of GCLC modulation to other tumor types may be a future direction of improvement. Our preliminary data in MDA-MB-231 cells suggest that GCLC deficiency also enhances 10 B, indicating its potential for wider application.

[0096] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention can be implemented over a wide range under equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the present invention and without the need for unnecessary experimentation. Although the present invention provides embodiments, it will be understood that further improvements can be made to the present invention. In short, according to the principles of the present invention, this application is intended to include any variations, uses, or improvements to the present invention, including changes made by conventional techniques known in the art that depart from the disclosed scope of this application.

Claims

1. Use of a GCLC inhibitor or a pharmaceutically acceptable salt thereof, characterized in that: The application includes any of the following: 1) Application in the preparation of drugs for enhancing BNCT drug uptake; 2) Application in the preparation of drugs for treating cancer; Preferably, the drug can be used alone or in the form of a pharmaceutical composition.

2. The use according to claim 1, characterized in that The cancer is selected from osteosarcoma, glioma, head and neck tumor, and melanoma; Preferably, the cancer is osteosarcoma.

3. The use according to claim 1, characterized in that The GCLC inhibitors include naturally occurring and artificially synthesized ones; Preferably, the GCLC inhibitor is a compound that has a specific inhibitory effect on GCLC.

4. The use according to claim 1, characterized in that The GCLC inhibitors include compounds targeting GCLC used in any one of RNA interference technology, CRISPR technology, antisense oligonucleotide technology, TALEN technology, ZFN technology, and Cre loxP gene recombination technology; Preferably, the GCLC inhibitor is a GCLC-targeting compound used in RNA interference technology.

5. The use according to claim 1, characterized in that The GCLC inhibitor is a siRNA targeting GCLC; Preferably, the sequence of the siRNA is shown in SEQ ID NO: 1-2.

6. A pharmaceutical composition for enhancing BNCT drug uptake, characterized in that: The pharmaceutical composition includes a BNCT drug, a GCLC inhibitor or a pharmaceutically acceptable salt thereof; Preferably, the pharmaceutical composition comprises a therapeutically effective amount of a BNCT drug and a GCLC inhibitor or a pharmaceutically acceptable salt thereof; Preferably, the BNCT drugs include boranophenylalanine, boroxafenamic acid, novel nanocarrier boron drugs, and antibody-conjugated boron drugs.

7. The pharmaceutical composition according to claim 6, characterized in that The GCLC inhibitor is a compound that has a specific inhibitory effect on GCLC; Preferably, the GCLC inhibitor includes a GCLC-targeting compound used in any one of RNA interference technology, CRISPR technology, antisense oligonucleotide technology, TALEN technology, ZFN technology, and Cre loxP gene recombination technology; Preferably, the GCLC inhibitor is a GCLC-targeting compound used in RNA interference technology; Preferably, the GCLC inhibitor is a siRNA targeting GCLC; Preferably, the sequence of the siRNA is shown in SEQ ID NO: 1-2.

8. The pharmaceutical composition according to claim 6, characterized in that The pharmaceutical composition further comprises a pharmaceutically acceptable excipient; Preferably, the pharmaceutically acceptable excipients include pharmaceutically acceptable carriers, diluents, excipients, glidants, preservatives, dyes / colorants, flavor enhancers, surfactants, wetting agents, dispersants, suspending agents, stabilizers, isotonic agents, solvents, surfactants or emulsifiers.

9. Use of the pharmaceutical composition according to any one of claims 6 to 8 in preparing a product for treating cancer, characterized in that: The cancer is selected from osteosarcoma, glioma, head and neck tumor, and melanoma; Preferably, the cancer is osteosarcoma.

10. A method for enhancing BNCT drug uptake in vitro, characterized in that: The method is achieved by inhibiting the expression of GCLC.

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