Application of GCLC in enhancing BNCT drug cellular uptake

Abstract

The application discloses application of GCLC in enhancing cell uptake of BNCT drugs. Boron neutron capture therapy (BNCT) has become a promising method for treating osteosarcoma, and the treatment effect depends to a great extent on 10 Accumulation and distribution of B compounds in tumors. The application provides a gene marker capable of enhancing tumor cell uptake of BNCT drugs and application thereof, and proves that targeting GCLC can overcome the limitation of tumor heterogeneity on BNCT drug uptake by utilizing tumor-specific vulnerability.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biological medicine, and in particular to the application of GCLC in enhancing the cell uptake of BNCT drugs. BACKGROUND

[0002] Osteosarcoma is a highly invasive primary bone tumor that mainly affects children and adolescents. The main treatment for osteosarcoma is chemotherapy combined with surgical resection, and radiotherapy is used for sacrum, pelvis and spine that cannot be operated. However, neither of these two treatment methods can achieve satisfactory therapeutic effect. In order to overcome the recurrence caused by poor response to chemotherapy, insufficient surgical margins and poor penetration of intraosseous radiation, boron neutron capture therapy (BNCT) has become a promising method for the treatment of osteosarcoma due to its strong penetration ability and few side effects. This model requires selective accumulation of non-radioactive boron-10 (10B) compounds in tumor cells, followed by irradiation with thermal neutrons. The subsequent 10 B capture reaction produces high linear energy transfer (LET) alpha particles and recoil lithium-7 (7Li) nuclei. Due to its extremely short range of radiation, the cytotoxic effect of these particles is mainly limited to 10 B-uptake tumor cells, significantly protecting adjacent healthy tissues. However, the therapeutic effect of BNCT depends largely on the accumulation and distribution of 10 B compounds within the tumor. Preclinical and clinical studies have consistently shown that there is significant heterogeneity in the intratumoral 10 B uptake. Specifically, the 10 B levels in some tumor regions are lower than the concentration in the blood stream. This refractory tumor cell subpopulation can survive from BNCT treatment, greatly weakening the therapeutic effect. Therefore, 10 Intratumoral heterogeneity of 10 B uptake is a major limiting factor for the success of BNCT.

[0003] Current strategies to improve 10 B delivery mainly focus on pharmacology and formulation innovation, such as increasing the dose of clinically certified boron phenylalanine (BPA), developing new 10 B carriers with enhanced tumor selectivity and concentration, such as boron sodium (BSH) and new nano-carrier boron drugs. Although these methods show great potential, they still face various challenges such as low delivery efficiency, in vivo stability and immune safety risks. SUMMARY

[0004] In view of the deficiencies of the prior art, the present application 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 application adopts the following technical solutions.

[0006] The present application provides the use of a GCLC inhibitor or a pharmaceutically acceptable salt thereof, which use comprises any one of the following:

[0007] 1) use in the manufacture of a medicament for enhancing BNCT drug uptake;

[0008] 2) use in the manufacture of a medicament for treating cancer.

[0009] In the present application, the pharmaceutically acceptable salt refers to a salt of an active compound and is prepared by reacting the active compound with a suitable organic or inorganic acid or acid derivative. The pharmaceutically acceptable salt includes but is not limited to hydrochloride, sulfate, phosphate, citrate, hydrobromide, acetate, benzoate, benzenesulfonate, tartrate, carbonate, citrate, gluconate, lactate, malate, methanesulfonate, stearate, valerate, nitrate, sodium salt, calcium salt, potassium salt, zinc salt and meglumine salt.

[0010] In the present application, the term "enhancing" refers to the fact that the uptake amount of a tumor tissue to a boron-containing drug is significantly higher than that of a normal tissue and the case where no GCLC inhibitor or pharmaceutically acceptable salt thereof is used, by the technical solutions provided in the present application, 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, while achieving an extension of the drug retention time in tumor cells by at least 67% (relative to the prior art), thereby improving the enrichment efficiency of boron-10 (¹ 0 ) in tumor cells in BNCT treatment, ultimately enhancing the local killing effect of alpha particles and lithium nuclei after neutron irradiation, and reducing the damage to normal tissues.

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

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

[0013] Further, the cancer is osteosarcoma.

[0014] In the present application, osteosarcoma is a primary bone malignancy originating from osteoblasts or bone mesenchymal cells. According to the histological type, it can be divided into conventional type osteosarcoma, capillary dilatation type osteosarcoma, small cell type osteosarcoma, low-grade central type osteosarcoma, secondary osteosarcoma. The tumor prone sites include long bone epiphysis, such as distal femur, proximal tibia, proximal humerus; including axial skeleton, such as skull, spine, pelvis; including extraneous tissues, such as thigh, hip, lung, breast. Its molecular typing includes TP53 deletion type, RB1 deletion type, PARP inhibitor sensitive type.

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

[0016] In the present application, the term "GCLC" is also known as GCL, GCS, GLCL, GLCLC, CNSHA7, and the gene ID is 2729. In the context of the present application, GCLC includes wild type, mutant or fragment thereof. The term covers full-length, unprocessed GCLC, and any form of GCLC derived from processing in cells. The term covers naturally occurring variants of GCLC (e.g. splice variants or allelic variants). The term covers, for example, 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 inhibitor includes naturally occurring and artificially synthesized.

[0018] Further, 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, Cre loxP gene recombination technology.

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

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

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

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

[0024] In the present application, the term "pharmaceutical composition" refers to a composition comprising at least one biologically active compound. The pharmaceutical composition of the present application can 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 composition of the present application can contain any conventional non-toxic pharmaceutically-acceptable carriers, adjuvants or vehicles. In some cases, pharmaceutical acids, bases or buffers can be used to adjust the pH of the formulation to improve the stability of the formulated compound or its administration form. The term parenteral as used in the present application includes subcutaneous, intradermal, intravenous, intramuscular, intraarticular, intraarterial, intrasynovial, intrasternal, intrathecal, intralesional, and intracranial injection or infusion techniques. The pharmaceutical composition of the present application can be administered to a subject by any route as long as the target tissue is reached.

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

[0026] In the present application, the term "therapeutically effective amount" refers to a dosage that can achieve the treatment of cancer in a subject, depending on the patient, the severity of the disease, and the route and medium for administration. The amount of active compound in such a therapeutically effective composition is an appropriate dosage. Those skilled in the art should know or be able to obtain an appropriate dosage through simple experiments.

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

[0028] In some embodiments, the BNCT drug comprises boron phenylalanine, boron safenamide, novel nanocarrier boron drug, antibody conjugated boron drug.

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

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

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

[0032] Further, the GCLC inhibitor is an siRNA targeting GCLC.

[0033] Further, 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 application, the term "pharmaceutically acceptable excipient" refers to any pharmaceutical excipient or pharmaceutical carrier which does not itself induce the production of antibodies harmful to the individual receiving the composition, and which can be administered without undue toxicity, and generally means a nontoxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type, which is not toxic to the host. Suitable carriers can be large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids and amino acid copolymers. Such carriers are well known in the art. The pharmaceutically acceptable carrier in the pharmaceutical composition can include fluids such as water, saline, glycerol and ethanol. There can also be auxiliary substances such as wetting or emulsifying agents, pH buffering substances, and the like.

[0036] Further, the pharmaceutically acceptable excipient comprises a pharmaceutically acceptable carrier, diluent, excipient, glidant, preservative, dye / colorant, flavoring agent, surfactant, wetting agent, dispersing agent, suspending agent, stabilizer, isotonic agent, solvent, surfactant or emulsifying agent.

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

[0038] Further, the cancer is osteosarcoma.

[0039] The fourth aspect of the present application 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 application: The experimental evidence provided by the present application shows that GCLC as 10 B uptake modulator can address the long-standing challenges in BNCT: 10 Heterogeneity of B compounds and often insufficient intra-tumoral accumulation. Targeting GCLC can overcome these limitations by exploiting tumor-specific vulnerabilities. BRIEF DESCRIPTION OF DRAWINGS

[0041] Figure 1 To explore the role of GCLC in BNCT by genome-wide CRISPR screening 10B Damage mechanism results. a, Experimental workflow; b, Venn diagram of differentially expressed genes, BNCT-specific genes (pink) represent genes uniquely identified in the neutron vs. control comparison, while BNCT + BPA-specific genes (green) represent genes uniquely identified in the BNCT vs. control (BPA-treated) comparison, the overlapping region indicates genes common to both comparisons; c-e, GO enrichment analysis for BNCT vs. control (BPA-treated) comparison, c, biological process, d, cellular component, e, molecular function; f, cell survival rate histogram.

[0042] Figure 2 For CRISPR screening 10 B uptake key regulators are GCLC results. a, Experimental workflow schematic; b, flow cytometry analysis results; c, volcano plot depicting FACS-sorted groups and upregulated (red) and downregulated (blue) genes in the U2OS-KO cell pool with a significance threshold of |log2FC| > 2, P < 0.05; d-e, KEGG (d) and GO enrichment analysis (e) of differentially expressed genes between FACS-sorted and U2OS-KO mixed cells.

[0043] Figure 3 For GCLC-KO cells enhance U2OS cells 10 B uptake results. a, GCLC vs. U2OS cells 10 Effect of B uptake, b-c, 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; d-e, representative images (d) and statistical analysis of colocalization index (e) of B drugs and lysosomes. 10 B uptake results. a, GCLC vs. U2OS cells 10 Effect of B uptake, b-c, 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; d-e, representative images (d) and statistical analysis of colocalization index (e) of B drugs and lysosomes.

[0044] Figure 4 For GCLC gene affects U2OS cells 10 B uptake mechanism results. a, volcano plot analysis of transcriptomic profiling; b, GO enrichment analysis of differentially expressed genes between GCLC-KO and wild-type U2OS cells after 2 h of BSH treatment; c, representative fluorescence microscopy images showing the uptake of FITC-dextran (70 kDa) by GCLC-KO and WT U2OS cells after 2 h of incubation at 1 mg / mL, with DAPI blue counterstaining of the nuclei, scale bar: 20 µm; d, quantitative analysis of mean fluorescence intensity of FITC-dextran uptake; e-f, representative images (e) and statistical analysis of relative fluorescence intensity (f) of ROS levels in WT U2OS and GCLC-KO cells validated with DCFH-DA probe.

[0045] Figure 5 GCLC deletion enhances stress-induced macropinocytosis and promotes 10 Results of B uptake, where a is the GCLC-deleted U2OS and HOB-SV40 cells at different time points, 10 B uptake process, b is the fluorescence imaging of the effect of GCLC deletion on U2OS cell macropinocytosis, c is a schematic diagram of stress-induced promotion of 10 B uptake macropinocytosis.

[0046] Figure 6 GCLC gene on 10 Results of in vivo experimental verification of the effect of GCLC on 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 flow 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 using Western blot analysis, e is the fluorescence imaging of BSH-CY5 distribution in the tumor, f is the statistical results of B concentration in tumor tissue. 10 DETAILED DESCRIPTION

[0047] The technical solutions in the embodiments of the present application will be described clearly and completely below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application.

[0048] EMBODIMENT

[0049] I. Materials and methods

[0050] 1、 10 B compounds and animal models: The first generation boron compound L-boron phenylalanine-fructose compound injection (BPA-F) used in this study comes from Steboronine ® . The second generation boron compound is synthesized and provided by the Institute of High Energy Physics, Chinese Academy of Sciences, i.e., a rhodamine B or Cy5-labeled polyamidoamine dendrimer-mercaptoundecahydrodecaborate (PAMAM-BSH) conjugate, prepared according to the method of Liu et al. (Journal of Controlled Release, 2025, DOI:10.1016 / j.jconrel.2025.113863).

[0051] ​All animal housing 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 Vital River Laboratory Animal Technology Co., Ltd.

[0052] 2. Cell culture: U2OS, HEK293FT and MDA-MB-231 cell lines were purchased from China National Cell Resource Center. Normal osteoblast cell line HOB-SV40 (SNL-593) was purchased from ScienCell Bioscience. All cell lines were identified by short tandem repeat (STR) analysis 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 medium (Damas Life, DA298U1) containing 10% FBS and G418. MDA-MB-231 cells were cultured in RPMI-1640 medium (Damas Life, DA298U9) containing 10% FBS. HOB-SV40 cells were cultured in the specialized medium provided by ScienCell (SNLM-593).

[0054] 3. Construction of stable knockout library cell pool: The whole genome human CRISPR GeCKOv2 A knockout library (GenScript, HG789) was used, which contains 3 unique sgRNAs for each gene and 1000 non-targeting control sgRNAs. Each miRNA was 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 proteins of wild type and CRISPR library transduced U20S cells were extracted using RIPA lysis buffer and quantified protein concentration by BCA assay. Proteins were denatured at 100 °C for 10 min with 5x loading buffer (Beyotime, P0015L), separated by SDS-PAGE using 10% HEPES precast gel (YEASEN, LK202) and transferred to PVDF membrane. Membrane was blocked with 5% bovine serum albumin (BSA) in TBST and then incubated with anti-Cas9 (Abeam, ab203933) and anti-GAPDH (Abeam, ab8245) antibodies overnight at 4 °C. After washing, the membrane was incubated with secondary antibodies for 1 h at room temperature. Protein bands were visualized using a chemiluminescence detection system (Tanon 5200). In addition, western blot was also used to: (i) verify the knockdown efficiency of GCLC in U20S cells; (ii) assess the GCLC knockout in HOB-SV40 and MDA-MB-231 cells; and (iii) confirm the silencing efficiency of GCLC siRNA in the in vivo osteosarcoma model.

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

[0058] 5. Survival screening after neutron irradiation: Prior to BNCT at the Spallation Neutron Source Science Center, U20S-KO cells were incubated with 20 pg / mL BPA-F for 2 h in serum-free medium. The control group did not use BPA-F but received irradiation under the same conditions. After incubation overnight and attachment, the surviving cells were collected.

[0059] 6. Fluorescence-activated cell sorting of top 10% high boron-accumulating cells: The U20S-KO cell pool was incubated with 50 pg / mL rhodamine B-labeled PAMAM-BSH for 2 h in serum-free medium. The top 10% cells with the highest fluorescence intensity were sorted by fluorescence-activated cell sorting (FACS) to enrich the high boron-accumulating cell population.

[0060] 7. DNA / RNA extraction: Genomic DNA of BPA-treated and untreated groups and high-uptake and normal-uptake cell populations was extracted using the Genomic DNA Purification Kit (Thermo Fisher, KIT0103) following 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% confluence, cells were treated with 50 pg / mL PAMAM-BSH for 2 hours. When the cell confluence reached 80%, cells were incubated with 50 pg / mL PAMAM-BSH for 2 hours. Total RNA was extracted using TRIzol (Thermo, 15596026CN), snap-frozen in liquid nitrogen, and submitted for RNA sequencing (RNA-seq).

[0062] 8. Genome-wide screening and analysis: Dual-end FASTQ files were generated using the Illumina platform. Reads were assembled using PANDAseq and analyzed using the MAGeCK algorithm against the GeCKOv2 library (Addgene; Sanjana et al., Nat Methods, 2014). Since this is a knockout-based screen, sgRNA abundance is inversely 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: Targeting plasmids for GCLC were purchased from GenScript. Lentivirus packaging and transduction were performed as described in 3 with an MOI of 10. Puromycin selection was performed for 72 hours to generate stable knockout cell lines.

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

[0065] 11. Lysosome co-localization imaging: Logarithmically growing U2OS wild type and knockout cells were seeded in four-quadrant dishes (CSR Biotech, D35-20-1.5-N). When the cell confluence reached approximately 70%, cells were stained with Hoechst 33342 and LysoBrite Red (Beyotime), followed by incubation with 50 pg / mL Cy5-labeled PAMAM-BSH for 10 minutes. Three-channel time-lapse imaging was performed using a structured illumination microscope (HIS-SIM). Co-localization 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 gene expression matrix. Differential expressed genes (DEGs) were subjected to GO and KEGG enrichment analysis and visualized by volcano plot.

[0067] 13. Macropinocytosis pathway validation: U2OS wild type and GCLC-KO cells were seeded in glass-bottomed dishes. When the cells reached 70% confluence, 25 μg / mL fluorescein isothiocyanate-dextran (FITC-Dextran) (MCE, HY-128868A) was added to the culture medium and incubated at 37°C for 20 min. After washing, the 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 of FITC-dextran positive vesicles and total fluorescence intensity in each cell were quantified using ImageJ software.

[0068] 14. Reactive oxygen species (ROS) level detection: Wild type and GCLC-KO cells were seeded in glass-bottomed dishes. When the cells reached the logarithmic growth phase, they were incubated with 10 μM DCFH-DA probe (Beyotime, S0033S) at 37°C for 30 min in the dark. After incubation, the cells were washed with phosphate buffered saline (PBS). Images were acquired using an inverted fluorescence microscope. The mean fluorescence intensity (MFI) was calculated from 5 random fields in each group using ImageJ for statistical analysis.

[0069] 15. Confocal imaging of PAMAM-BSH uptake kinetics: Wild type and GCLC-KO U2OS cells were seeded in four-quadrant dishes and incubated with 50 μg / mL Cy5-labeled PAMAM-BSH. Cells were fixed at 2, 5, 8, 10, and 12 s 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 experiment: HOB-SV40 and U2OS cells and their corresponding GCLC-KO cell lines were seeded in four-well culture dishes and incubated with 50 pg / mL Cy5-labeled PAMAM-BSH for 2 h. After boron compound incubation, 10 pM DCFH-DA was added to assess cellular ROS levels. Then, cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. Non-specific binding sites were blocked with 5% BSA. Immunofluorescence staining was performed with Alexa Fluor ® 594-labeled anti-ARPC3 antibody (1 :200 dilution), and counterstained with DAPI for nuclei. Multichannel fluorescence images were acquired using a confocal microscope. Semi-quantitative analysis of ARPC3 fluorescence intensity and Cy5 signal intensity (representing boron uptake) was performed using ImageJ software. Colocalization analysis was performed to assess the enrichment of ARPC3 at macropinosome sites.

[0071] 17. Boron uptake of GCLC knockout cell lines: GCLC-KO derivatives of HOB-SV40, U2OS, and MDA-MB-231 cell lines were seeded in 6-well plates and incubated with 50 pg / mL PAMAM-BSH for 2 h. Then, cells were subjected to nitric acid digestion, followed by ICP-MS analysis to quantify intracellular boron concentration.

[0072] 18. In vivo validation: Four-week-old male BGCLC / c nude mice (SPF level) were anesthetized and U2OS osteosarcoma cells (5 x 10 6 cells in 10 pL PBS) were implanted in situ into the right tibial plateau. Tumor formation was confirmed by micro-CT at day 14 post-implantation. Mice were randomly assigned to experimental groups and received intratumoral injection of GCLC siRNA (10 nmol / 20 g body weight; Ribobio, R10043.10) every 3 days for 1 week. Subsequently, mice were intravenously injected with 30 pg B / 20 g body weight of Cy5-labeled PAMAM-BSH. At 0.5, 1, 2, 4, 6, 12, and 24 h post-injection, mice were imaged using an IVIS Spectrum imaging system. Fluorescence in the tumor area was quantified using Living Image software. After the last imaging, mice were euthanized, and blood, tumor, and peritumoral tissues were collected and subjected to ICP-MS analysis after digestion to determine boron concentration and calculate tumor-to-non-tumor (T / N) and tumor-to-blood (T / B) ratios.

[0073] II. Experimental Results

[0074] 1. Genome-wide analysis of osteosarcoma cell BNCT cellular response

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

[0076] There are approximately 1203 overlapping genes between the two groups, of which 1070 significant genes are specific to neutron irradiation + BPA (BNCT), and 1411 genes are specific to neutron irradiation. Figure 1 (b) The statistical results suggest specific implications after the introduction of BPA. 10 B. Efficacy. Neutron irradiation therapy and BNCT therapy not only share common response components in their differentially expressed gene profiles but also exhibit unique changes. This difference suggests a significant difference in the induced biological effects of the two treatments.

[0077] Comparative analysis revealed that BPA exposure shifted the BNCT response from a “passive repair mode” to a “metabolic stress mode,” characterized by enhanced DNA damage without immediately killing cells. Instead, tumor cells were forced to activate a broader range of stress defense pathways—including antioxidant systems and DNA repair modules—to survive. Differential enrichment of cell cycle checkpoint genes further supported this shift. These results clearly validated… 10 B neutrons introduce intrinsic cellular stress, thereby enhancing BNCT-induced cytotoxicity through multiple mechanisms. Figure 1 (c, d, e). Furthermore, in our screening, BNCT induced approximately 40% cytotoxicity in U2OS-KO mixed cells ( Figure 1 (f) in this context may be limited not only by genetic resistance but also by... 10 The suboptimal limitation of B accumulation. Therefore, enhancing intracellular... 10 B uptake may be key to maximizing the efficacy of BNCT. Targeted 10 Regulators of B uptake are a promising strategy that could overcome the current limitations of BNCT in osteosarcoma.

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

[0079] In order to improve10 The absorption efficiency of B is something researchers are designing for the next generation. 10 Much work has been done on boron delivery agents. BSH, with 12 boron atoms per molecule and a stable icosahedral configuration, has a higher boron density compared to BPA. Furthermore, polyamidoamine dendritic macromolecular conjugated BSH (PAMAM-BSH) provides a higher boron loading per particle through enhanced permeability and retention, and improves pharmacokinetics. However, its uptake mechanism still needs further elucidation to enhance its potential for clinical application. To investigate this, we utilized PAMAM-BSH covalently labeled with a fluorescent probe (rhodamine B) to achieve visualization and quantification of 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-BSH-rhodamine B uptake by fluorescence-activated cell sorting and performed next-generation sequencing (next-generation sequencing). Figure 2 (a, b in the original text).

[0080] Based on these results from highly uptake cells, sgRNAs targeting GCLC (glutamate cysteine ​​ligase catalytic subunit) were significantly enriched, indicating that GCLC knockout promoted 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. This means that the internalization and intracellular transport of PAMAM-BSH mainly depend on endocytosis and related membrane transport mechanisms. Figure 2 (d) The simultaneous enrichment of phospholipid metabolism, sphingolipid signaling, and ER protein processing pathways suggests that a coordinated cellular adaptation involves membrane remodeling and stress signaling, which frequently occurs under metabolic reprogramming or treatment-induced stress conditions, contributing to cellular adaptation to higher stress levels. 10 B concentration. GO enrichment analysis further confirmed the involvement of these functional modules ( Figure 2 (e in the text).

[0081] To verify GCLC in 10 To understand the role of beta-blocker uptake, we constructed a stable U2OS cell line of GCLC knockout. Wild-type and knockout cell lines were incubated with PAMAM-BSH, and intracellular bioavailability was quantified using inductively coupled plasma mass spectrometry (ICP-MS). 10 B concentration ( Figure 3 (a) In PAMAM-BSH treatment, the GCLC-KO cell line showed significantly better performance compared to wild-type cells. 10B concentration significantly increased, confirming the robustness of the screening strategy. To investigate whether PAMAM-BSH uptake involves the lysosomal pathway, we labeled lysosomes with LysoTracker and analyzed the colocalization of PAMAM-BSH-RhB with lysosomes using confocal microscopy (Fig. 1 Figure 3 b, c). GCLC-KO cells showed higher colocalization index than wild type, and quantitative analysis further confirmed that the fluorescence intensity of GCLC-KO cells was significantly higher than that of wild type, supporting the important role of lysosome-mediated endocytosis in 10 B uptake (Fig. 1 Figure 3 d, e).

[0082] In summary, loss of GCLC function significantly enhanced the uptake of the high boron compound PAMAM-BSH by U2OS cells, establishing its key role in regulating intracellular 10 B accumulation. This uptake-based forward screening strategy using PAMAM-BSH-RhB successfully identified a key functional gene, GCLC, that coordinates oxidative stress response and endocytosis. 10 B delivery.

[0083] 3. Molecular basis of GCLC-enhanced uptake of 10 B by U2OS cells

[0084] To further elucidate the molecular basis of enhanced cellular uptake of boron compounds by GCLC-KO cells, we performed transcriptome (RNA-seq) analysis comparing GCLC-KO U2OS cells with wild type controls. Differential gene expression analysis showed that loss of GCLC induced transcriptome changes, with many genes significantly upregulated (Fig. 2 Figure 4 a).

[0085] GO and KEGG pathway enrichment analysis of the upregulated gene set in GCLC-KO cells highlighted significant enrichment of functional categories related to membrane endocytosis, vesicle transport, lysosomal processes, and endocytosis (Fig. 2 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 loss can lead to rapid depletion of intracellular GSH. Given that glutathione is the major intracellular antioxidant, this depletion impairs 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 was significantly increased in the GCLC-KO group compared to the WT control, with a fluorescence intensity increase of about 10-fold (Fig. 2 Figure 4 c).Figure 4 Overall, these results confirm that the absence of GCLC leads to GSH depletion, triggering a pronounced oxidative stress. This redox imbalance can be a key driver of downstream functional reprogramming, including enhanced endocytic activity.

[0086] In addition, 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 vesicle formation and membrane recycling components also showed upregulated expression, suggesting that GCLC-deficient cells have an intrinsic enhanced endocytic capacity at the transcriptional level. We therefore hypothesized that the enhanced endocytosis is caused by increased vesicle uptake. However, uptake of nanoparticles (PAMAM-BSH) by the endocytic pathway tends to go to 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 d). In addition, we quantified the subcellular colocalization between Cy5-labeled PAMAM-BSH and lysosomes. A significant colocalization was observed in GCLC-KO cells compared to WT cells, indicating that internalized 10 B compounds are more efficiently and rapidly trafficked to the lysosomal compartment. These results support the hypothesis that GCLC loss promotes cellular oxidative adaptation, enhancing vesicle-mediated uptake and trafficking to intracellular organelles, leading to 10 B accumulation. In line with the RNA-seq data, this data identifies endocytosis as a top enriched pathway.

[0087] To functionally confirm that the enhanced macropinocytosis is a potential mechanism for the increased 10 B uptake in GCLC-KO cells, we used FITC-dextran conjugates (70 kDa) as an indicator to validate our hypothesis, as the dextran cannot enter the cell by receptor-mediated endocytosis but can be efficiently internalized by macropinocytosis. Confocal microscopy analysis revealed a significant increase in fluorescence intensity within GCLC-KO cells, demonstrating that more FITC-dextran was internalized compared to WT cells ( Figure 4 e, f). Suggesting that GCLC deficiency promotes rat monocyte activity, providing a mechanistic explanation and strong functional evidence for enhanced 10 B compound uptake.

[0088] 4. GCLC deficiency promotes macropinocytosis to enhance PAMAM-BSH uptake

[0089] The PAMAM-BSH compound carries a total positive charge, which may promote electrostatic interactions with the negatively charged cell membrane, thus facilitating macropinocytosis. To observe whether PAMAM-BSH deficiency in GCLC directly leads to macropinocytosis... 10 For drug uptake, we incubated Cy5-labeled PAMAM-BSH with GCLC-KO and wild-type U2OS cells at multiple time intervals, followed by immediate fixation. Intracellular distribution and uptake efficiency of PAMAM-BSH were assessed using super-resolution confocal laser scanning microscopy. Figure 5 (a) Based on these results, it is clear that KO cells exhibit a significant increase in vesicles associated with macropinocytosis compared to wild-type cells. Simultaneously, PAMAM-BSH retention on the cell membrane is significantly reduced. 10 Intracellular uptake of compound B occurred at an accelerated rate within the same timeframe. These findings are consistent with our initial hypothesis. Furthermore, ICP-MS analysis further confirmed these observations, indicating that in GCLC-KO cells... 10 The accumulation of B increases ( Figure 5 (b) in the middle.

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

[0091] 5. Tumor cells showed higher sensitivity to macropinocytosis induced by GCLC deficiency.

[0092] Although we have already clarified the role of GCLC in promoting 10 The mechanism of drug uptake is unclear, but whether cancer cells exhibit better sensitivity to this process compared to normal cells remains a key determinant of therapeutic potential. To address this issue, we constructed GCLC-KO HOB-SV40 (primary human osteoblasts derived from healthy donors) and MDA-MB-231 (triple-negative breast cancer) cell lines, respectively, and compared them with the U2OS KO model to quantify the effects of PAMAM-BSH treatment under the same conditions using ICP-MS. 10B concentration. Results show that GCLC-KO U2OS and MDA-MB-231 cells have significantly higher uptake of 10 B than GCLC-KO HOB-SV40 cells. In addition, GCLC-KO HOB-V40 cells show increased 10 B uptake compared to WT HOB-SV40 cells, although this increase is significantly lower than that of tumor cells ( Figure 6 a). These results indicate that GCLC knockout broadly enhances tumor cell uptake of drugs containing 10 B, and tumor cells show higher sensitivity to macropinocytosis induced by GCLC deficiency. Targeting GCLC can significantly increase tumor 10 B uptake with minimal impact on normal tissues, consistent with the principle of BNCT and suggesting potential clinical utility.

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

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

[0095] Based on the above results, targeting GCLC as a 10 B uptake modulator can address the long-standing challenges in BNCT: 10 ​The heterogeneity of B compounds and the often insufficient intratumoral accumulation. Targeting GCLC can overcome these limitations by exploiting a tumor-specific vulnerability, the enhanced sensitivity of cancer cells to oxidative stress-induced macropinocytosis. Given the poor clinical outcome of osteosarcoma due to tumor heterogeneity and radioresistance, especially in patients with metastatic or recurrent disease, the technical solution of the present invention is particularly important for BNCT treatment of osteosarcoma. While this study focused on osteosarcoma, the applicability of GCLC modulation to other tumor types can be a direction for future improvements. Our preliminary data in MDA-MB-231 cells suggest that GCLC deficiency also enhances 10 B uptake, indicating its potential broader application value.

[0096] The above detailed the present invention. For those skilled in the art, without departing from the purpose and scope of the present invention, and without unnecessary experiments, the present invention can be implemented in a wider range under the same parameters, concentrations and conditions. Although the present invention gives examples, it should be understood that further improvements can be made to the present invention. In summary, according to the principle of the present invention, this application intends to include any change, use or improvement of the present invention, including changes made by conventional techniques known in the art, which deviates from the scope disclosed in this application.

Claims

1. Use of a GCLC inhibitor or a pharmaceutically acceptable salt thereof in the preparation of a medicament for enhancing the uptake of a BNCT drug; the GCLC inhibitor is an siRNA targeting GCLC, and the sequence of the siRNA is as shown in SEQ ID NO: 1-2; the BNCT drug is selected from PAMAM-BSH, BSH.

2. A pharmaceutical composition for enhancing BNCT drug uptake, characterized by, the pharmaceutical composition comprises a BNCT drug, a GCLC inhibitor or a pharmaceutically acceptable salt thereof; the BNCT drug is selected from PAMAM-BSH, BSH; the GCLC inhibitor is an siRNA targeting GCLC, and the sequence of the siRNA is as shown in SEQ ID NO: 1-2.

3. The pharmaceutical composition of claim 2, wherein, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient.

4. The pharmaceutical composition of claim 3, wherein, the pharmaceutically acceptable excipient comprises a pharmaceutically acceptable carrier, diluent, excipient, glidant, preservative, dye / colorant, flavor enhancer, wetting agent, dispersing agent, suspending agent, stabilizer, isotonic agent, solvent, surfactant or emulsifying agent.

5. Use of a pharmaceutical composition according to any one of claims 2 to 4 for the manufacture of a medicament for the treatment of cancer, characterized in that, the cancer is selected from osteosarcoma, breast cancer.

6. A method of enhancing BNCT drug uptake in vitro, characterized in that, the method is achieved by administering a GCLC inhibitor; the BNCT drug is selected from PAMAM-BSH, BSH; the GCLC inhibitor is an siRNA targeting GCLC, and the sequence of the siRNA is as shown in SEQ ID NO: 1-2.

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