Application of PARP inhibitor in preparation of medicine for improving osteosarcoma carbon ion radioimmune effect

Abstract

The invention belongs to the technical field of radiosensitization, and particularly relates to application of a PARP inhibitor in preparation of a medicine for improving osteosarcoma carbon ion radioimmune effect. Researches find that the PARP inhibitor is combined with C-ions to up-regulate the expression of IFN-beta and PD-L1 by activating a cGAS / STING pathway so as to enhance the radiation immune effect of osteosarcoma. The treatment strategy of combining the PARP inhibitor with the C-ions radiotherapy has a synergistic effect in the aspects of improving the osteosarcoma anti-tumor immune curative effect and improving the survival. Therefore, the PARP inhibitor is taken as a radioimmunity synergist, and the synergistic effect of osteosarcoma radioimmunity can be improved by combining the PARP inhibitor with C-ions radiotherapy.

Description

Technical Field

[0001] This invention belongs to the field of radioimmunoassay enhancement technology, specifically relating to the application of PARP inhibitors in the preparation of drugs that enhance the radioimmunoassay effect of carbon ions in osteosarcoma. Background Technology

[0002] Osteosarcoma, originating from bone-forming mesenchymal cells, is the most common malignant bone tumor, especially prevalent in children and adolescents. Osteosarcoma is considered a radiation-resistant tumor, requiring high-dose radiation therapy (≥70 Gy) to achieve good local control. Carbon ion (C-ions) radiotherapy offers superior physics by depositing dose in the "Bragg Peak" region, providing a better dose distribution and delivering a higher dose to the tumor area while protecting surrounding tissues and organs. Furthermore, C-ions exhibit superior relative biological effectiveness (RBE), demonstrating greater killing effect on tumor cells.

[0003] Tumor immunotherapy is considered the third revolution in clinical oncology treatment and is gradually becoming one of the important strategies for systemic treatment of malignant tumors. ICIs, adoptive immunotherapy, and tumor vaccines have been used in clinical practice, among which ICIs are currently the most widely used. With the development of tumor immunology, many immune checkpoints have been discovered in tumors, including but not limited to PD-1 / PD-L1, lymphocyte activation gene-3, and cytotoxic T lymphocyte antigen-4 (CTLA-4). PD-1 / PD-L1 inhibitors are among the most widely used ICIs.

[0004] Currently, the shortcomings of ICIs are gradually being exposed in clinical practice, mainly their low response rate in most malignant tumors, only 10% to 30%. Radiotherapy can increase the release and expression of tumor-associated antigens, improve the tumor immunosuppressive microenvironment, and activate specific T-cell immune responses, thereby promoting the body's anti-tumor immune response, but the radioimmunotherapy effect in osteosarcoma is not ideal. Summary of the Invention

[0005] In order to develop a new strategy to enhance the radioimmunoassay effect of osteosarcoma and overcome the shortcomings of the existing technology in the unsatisfactory carbon ion radioimmunoassay effect of osteosarcoma, the present invention provides the application of PARP inhibitors in the preparation of drugs to enhance the carbon ion radioimmunoassay effect of osteosarcoma. To achieve the above objective, the present invention adopts the following technical solution.

[0006] One of the objectives of this invention is to provide the application of PARP inhibitors in the preparation of drugs that enhance the carbon ion radioimmunoassay effect in osteosarcoma.

[0007] This invention systematically overcomes the bottleneck of unsatisfactory radioimmunotherapy in osteosarcoma through the synergistic mechanism of PARP inhibitors and carbon ion radiotherapy. In existing technologies, although carbon ion radiotherapy can precisely induce DNA double-strand breaks, the inherently efficient DNA damage repair capacity of osteosarcoma cells (especially the active homologous recombination repair pathway) leads to high tumor cell survival rates. Simultaneously, the regulatory effect of carbon ions on the tumor immune microenvironment is limited, making it difficult to effectively activate the anti-tumor immune response. PARP inhibitors combined with C-ions radiotherapy enhance the radioimmunotherapy effect in osteosarcoma. This dual-pathway enhancement strategy not only solves the deficiency of osteosarcoma's insensitivity to carbon ion radiotherapy but also compensates for its insufficient immune activation, achieving a breakthrough from local radiotherapy to systemic immune enhancement.

[0008] Furthermore, the drug uses the PARP inhibitor as the sole active ingredient or one of the active ingredients.

[0009] Furthermore, when the drug uses the PARP inhibitor as one of its active ingredients, the active ingredient also includes a PD-L1 inhibitor.

[0010] Furthermore, the PD-L1 inhibitor is selected from any one or more of atezolizumab, durvalumab, avelumab, and envorimab.

[0011] Furthermore, the combined administration of the drug with carbon ion radiotherapy produces a synergistic effect, enhancing the radioimmunoassay effect against osteosarcoma.

[0012] Furthermore, the PARP inhibitor includes olaparib.

[0013] The second objective of this invention is to provide a method for enhancing the radioimmunoassay effect of carbon ions in osteosarcoma cells in vitro, comprising the following steps: The osteosarcoma cells were treated in vitro using a PARP inhibitor, wherein the PARP inhibitor included olaparib.

[0014] Carbon ion radiotherapy was administered to osteosarcoma cells that had been treated in vitro.

[0015] Furthermore, when treating the osteosarcoma cells in vitro, the duration of action of olaparib is 12h to 48h.

[0016] Furthermore, when treating the osteosarcoma cells in vitro, the concentration of olaparib is 9.8 μM to 10.2 μM.

[0017] Furthermore, when treating the osteosarcoma cells in vitro, the concentration of olaparib is 10 μM.

[0018] Furthermore, the relative bioequivalent dose of the carbon ion radiotherapy is 10 Gy (RBE).

[0019] Furthermore, the osteosarcoma cells are the human osteosarcoma cell line U2OS and / or the osteosarcoma cells are the mouse osteosarcoma cell line K7M2.

[0020] Compared with the prior art, the present invention has the following beneficial effects: To develop a novel strategy for enhancing the radioimmunotherapy effect in osteosarcoma, this invention provides the application of PARP inhibitors in the preparation of drugs that enhance the radioimmunotherapy effect of carbon ions in osteosarcoma. This invention systematically overcomes the bottleneck of unsatisfactory radioimmunotherapy in osteosarcoma through the synergistic mechanism of PARP inhibitors and carbon ion radiotherapy: In existing technologies, although carbon ion radiotherapy can precisely induce DNA double-strand breaks, the inherently efficient DNA damage repair capacity of osteosarcoma cells (especially the active homologous recombination repair pathway) leads to high tumor cell survival rates. Simultaneously, the regulatory effect of carbon ions on the tumor immune microenvironment is limited, making it difficult to effectively activate the anti-tumor immune response. PARP inhibitors combined with C-ions radiotherapy enhance the radioimmunotherapy effect in osteosarcoma. This dual-pathway synergistic strategy not only addresses the insensitivity of osteosarcoma to carbon ion radiotherapy but also compensates for its insufficient immune activation, achieving a breakthrough from local radiotherapy to systemic immune enhancement.

[0021] The main mechanism by which the PARP inhibitor olaparib acts as a radioimmunoassay enhancer for osteosarcoma includes: inducing more severe DNA damage (DSBs). Damaged DNA, in the form of dsDNA, gradually migrates from the nucleus to the nuclear membrane and is expelled into the cytoplasm, leading to the accumulation of large amounts of dsDNA in the cytoplasm. This dsDNA then binds to and activates the cytoplasmic DNA sensor cGAS. Activated cGAS catalyzes the synthesis of cGAMP from ATP and GTP, which in turn binds to the STING protein, causing it to dimerize. The activated STING protein recruits TBK1, causing it to phosphorylate. pTBK1 then phosphorylates IRF3, thereby upregulating the expression of type I interferon-related genes, upregulating IFN-β and PD-L1 expression, and increasing the expression of chemokines CXCL10 and CCL5. In summary, this invention provides a potential theoretical basis for the clinical application of the PARP inhibitor olaparib in enhancing the radioimmunoassay effect of carbon ion radioimmunoassay in osteosarcoma. The application method provided by this invention can effectively enhance the radioimmunoassay effect in osteosarcoma, overcoming the shortcomings of the existing technology in achieving the desired carbon ion radioimmunoassay effect in osteosarcoma. Attached Figure Description

[0022] Figure 1 This is a schematic diagram of the irradiation, olaparib, and αPD-L1 treatment process for tumor-bearing mice in this invention.

[0023] Figure 2This invention provides a statistical analysis of the effects of olaparib combined with irradiation (C-ions) on the formation of γH2AX / 53BP1 foci in U2OS osteosarcoma cells and the number of foci formed; wherein, Figure 2 Figure A shows the effect of olaparib combined with C-ions irradiation on the formation of γH2AX / 53BP1 foci in U2OS osteosarcoma cells 2 hours later. Figure 2 Figure B in the figure shows the effect of olaparib combined with C-ions irradiation on the formation of γH2AX / 53BP1 foci in U2OS osteosarcoma cells 12 hours later. Figure 2 Figure C in the figure shows the number of γH2AX / 53BP1 foci U2OS osteosarcoma cells 2 hours after irradiation with olaparib combined with C-ions; Figure 2 Figure D in the figure shows the number of γH2AX / 53BP1 foci U2OS osteosarcoma cells 12 hours after irradiation with olaparib combined with C-ions; scale bar: 10 μm; *: p <0.05.

[0024] Figure 3 This invention presents the effects of olaparib combined with irradiation (C-ions or) on the formation of γH2AX / 53BP1 foci in K7M2 osteosarcoma cells (A, B) and the statistical analysis of the number of foci formed (C, D); wherein, Figure 3 Figure A shows the effect of olaparib combined with C-ions irradiation on the formation of γH2AX / 53BP1 foci in K7M2 osteosarcoma cells 2 hours later. Figure 3 Figure B in the figure shows the effect of olaparib combined with C-ions irradiation on the formation of γH2AX / 53BP1 foci in K7M2 osteosarcoma cells 12 hours later. Figure 3 Figure C in the figure shows the number of γH2AX / 53BP1 foci K7M2 osteosarcoma cells 2 hours after irradiation with olaparib combined with C-ions; Figure 3 Figure D in the figure shows the number of γH2AX / 53BP1 foci K7M2 osteosarcoma cells 12 hours after olaparib combined with C-ions irradiation; scale bar: 10 μm; *: p <0.05.

[0025] Figure 4 This study focuses on the enrichment analysis of PARP3 and osteosarcoma-related immune signaling pathways in this invention.

[0026] Figure 5 This invention illustrates the relationship between PD-L1 expression in osteosarcoma and different X-ray doses; wherein, Figure 5 Figure A in the figure shows the relationship between PD-L1 expression in U2OS osteosarcoma and different X-ray doses; Figure 5Figure B in the figure shows the relationship between PD-L1 expression in K7M2 osteosarcoma and different X-ray doses; Figure 5 Figure C in the figure represents the statistical analysis of the relative expression of PD-L1 in U2OS osteosarcoma. Figure 5 Figure D in the figure represents the statistical analysis of relative PD-L1 expression in K7M2 osteosarcoma; ns: p >0.05, *: p <0.05.

[0027] Figure 6 This invention illustrates the relationship between PD-L1 expression in osteosarcoma and different time points following C-ions irradiation; wherein, Figure 6 Figure A in the figure shows the relationship between PD-L1 expression in U2OS osteosarcoma and different time points; Figure 6 Figure B in the figure shows the relationship between PD-L1 expression in K7M2 osteosarcoma and different time points; Figure 6 Figure C in the figure represents the statistical analysis of the relative expression of PD-L1 in U2OS osteosarcoma. Figure 6 Figure D in the figure represents the statistical analysis of relative PD-L1 expression in K7M2 osteosarcoma; ns: p >0.05, *: p <0.05.

[0028] Figure 7 In this invention, the PARP inhibitor olaparib is combined with X-rays or C-ions to enhance radiation to induce PD-L1 expression in osteosarcoma cells; wherein, Figure 7 Figure A shows the PD-L1 expression in U2OS osteosarcoma cells induced by the PARP inhibitor olaparib combined with X-rays or C-ions enhanced radiation. Figure 7 Figure B shows the PD-L1 expression in K7M2 osteosarcoma cells induced by the PARP inhibitor olaparib combined with X-rays or C-ions enhanced radiation. Figure 7 Figure C in the figure represents the statistical analysis of relative PD-L1 expression in U2OS osteosarcoma. Figure 7 Figure D in the figure represents the statistical analysis of the relative expression of PD-L1 in K7M2 osteosarcoma; IR: ionizing radiation; ns: p >0.05, *: p <0.05.

[0029] Figure 8 This study provides an enrichment analysis of PD-L1 and osteosarcoma-related immune signaling pathways in this invention.

[0030] Figure 9 This invention illustrates the accumulation of dsDNA breaks induced by olaparib combined with C-ions in the cytoplasm of osteosarcoma cells; scale bar: 10 μm.

[0031] Figure 10The effect of olaparib combined with C-ions on the co-localization of dsDNA and cGAS in this invention; scale bar: 10 μm.

[0032] Figure 11 This invention relates to the effects of olaparib combined with C-ions on the cGAS / STING signaling pathway and PD-L1 expression in the U2OS osteosarcoma cell line; wherein, Figure 11 Figure A in the figure shows the Western bolting analysis of cGAS, STING, TBK1, p-TBK1, IRF3, p-IRF3, IFN-β, PD-L1, with β-Actin as an internal reference; Figure 11 Image B in the middle~ Figure 11 The G-plot in the figure represents the statistical analysis of the relative expression levels of Western bolting proteins. Figure 11 H diagram in the middle~ Figure 11 The K-plot represents the statistical analysis of relative mRNA expression levels in qRT-PCR; IR: ionizing radiation; ns: p >0.05, *: p <0.05.

[0033] Figure 12 This invention uses ELISA to analyze the expression levels of chemokines CCL5 and CXCL10 in the U2OS osteosarcoma cell line induced by olaparib combined with irradiation (C-ions); wherein, Figure 12 Figure A in the figure shows the expression level of the chemokine CCL5 in the U2OS osteosarcoma cell line; Figure 12 Figure B in the figure shows the expression level of the chemokine CXCL10 in the U2OS osteosarcoma cell line; ns: p >0.05, *: p <0.05.

[0034] Figure 13 This invention relates to the effects of olaparib combined with C-ions on the cGAS / STING signaling pathway and PD-L1 expression in the K7M2 osteosarcoma cell line; wherein, Figure 13 Figure A in the figure shows the Western bolting analysis of cGAS, STING, TBK1, p-TBK1, IRF3, p-IRF3, IFN-β, PD-L1, with β-Actin as an internal reference; Figure 13 Image B in the middle~ Figure 13 The G-plot in the figure represents the statistical analysis of the relative expression levels of Western bolting proteins. Figure 13 H diagram in the middle~ Figure 13 The K-plot represents the statistical analysis of relative mRNA expression levels in qRT-PCR; IR: ionizing radiation; ns: p >0.05, *: p <0.05.

[0035] Figure 14 This invention uses ELISA to analyze the expression levels of chemokines CCL5 and CXCL10 in the K7M2 osteosarcoma cell line induced by olaparib combined with irradiation (C-ions); wherein, Figure 14 Figure A in the figure shows the expression level of the chemokine CCL5 in the K7M2 osteosarcoma cell line; Figure 14 Figure B in the figure shows the expression level of the chemokine CXCL10 in the K7M2 osteosarcoma cell line; ns: p >0.05, *: p <0.05.

[0036] Figure 15 This invention evaluates the siSTING silencing efficiency; wherein, Figure 15 Figure A in the middle and Figure 15 Figure B in the figure shows the efficiency of siSTING at the protein level using Western bolting. Figure 15 The D diagram and Figure 15 Figure F in the figure shows the efficiency of siSTING at the mRNA level using qRT-PCR. Figure 15 Figure C in the middle and Figure 15 Figure E in the figure represents the statistical analysis of the relative expression levels of Western bolting proteins; *: p <0.05.

[0037] Figure 16 This invention relates to the effects of olaparib combined with C-ions on the cGAS / STING signaling pathway and PD-L1 expression in the U2OS (siSTING) cell line; wherein, Figure 16 Figure A in the figure shows the Western bolting analysis of cGAS, STING, TBK1, p-TBK1, IRF3, p-IRF3, IFN-β, PD-L1, with β-Actin as an internal reference; Figure 16 Image B in the middle~ Figure 16 The G-plot in the figure represents the statistical analysis of the relative expression levels of Western bolting proteins. Figure 16 H diagram in the middle~ Figure 16 The K-plot represents the statistical analysis of relative mRNA expression levels in qRT-PCR; IR: ionizing radiation; ns: p >0.05, *: p <0.05.

[0038] Figure 17 This invention uses ELISA to analyze the expression levels of chemokines CCL5 and CXCL10 in the U2OS (siSTING) cell line induced by olaparib combined with irradiation (C-ions); wherein, Figure 17Figure A in the figure shows the expression level of the chemokine CCL5 in the U2OS (siSTING) osteosarcoma cell line; Figure 17 Figure B in the figure shows the expression level of the chemokine CXCL10 in the U2OS (siSTING) osteosarcoma cell line; ns: p >0.05.

[0039] Figure 18 This invention relates to the effects of olaparib combined with C-ions on the cGAS / STING signaling pathway and PD-L1 expression in the K7M2 (siSTING) cell line; wherein, Figure 18 Figure A in the figure shows the Western bolting analysis of cGAS, STING, TBK1, p-TBK1, IRF3, p-IRF3, IFN-β, PD-L1, with β-Actin as an internal reference; Figure 18 Image B in the middle~ Figure 18 The G-plot in the figure represents the statistical analysis of the relative expression levels of Western bolting proteins. Figure 18 H diagram in the middle~ Figure 18 The K-plot represents the statistical analysis of relative mRNA expression levels in qRT-PCR; IR: ionizing radiation; ns: p >0.05, *: p <0.05.

[0040] Figure 19 This invention uses ELISA to analyze the expression levels of chemokines CCL5 and CXCL10 in the K7M2 (siSTING) cell line induced by olaparib combined with irradiation (C-ions); wherein, Figure 19 Figure A in the figure shows the expression level of the chemokine CCL5 in the K7M2 (siSTING) osteosarcoma cell line; Figure 19 Figure B shows the expression level of the chemokine CXCL10 in the K7M2 (siSTING) osteosarcoma cell line; ns: p >0.05.

[0041] Figure 20 This is a schematic diagram illustrating the upregulation effect and mechanism of olaparib combined with irradiation to activate the cGAS / STING / TBK1 / IRF3 signaling pathway in this invention.

[0042] Figure 21 The figures (A) and (B) show the changes in body weight (C-ions) and tumor volume (B) of mice in each treatment group after inoculation in this invention; wherein, Figure 21 Figure A in the figure shows the changes in body weight of mice in each treatment group after inoculation; Figure 21 Figure B in the figure shows the changes in tumor volume in mice of each treatment group after inoculation; *: p <0.05.

[0043] Figure 22 This invention illustrates the changes in tumor weight in mice of different treatment groups after C-ions irradiation; wherein, Figure 22 Figure A shows the changes in tumor weight in mice in each treatment group after C-ions irradiation. The eight samples from left to right are Control group, αPD-L1 group, Olaparib group, αPD-L1+Olaparib group, C-ions group, C-ions+αPD-L1 group, C-ions+Olaparib group and C-ions+αPD-L1+Olaparib group. Figure 22 Figure B in the figure shows the statistical analysis of tumor weight changes in mice in each treatment group after C-ions irradiation; ns: p >0.05, *: p <0.05.

[0044] Figure 23 This is the survival curve of mice in each treatment group after C-ions irradiation in this invention; ns: p >0.05, *: p <0.05.

[0046] Figure 24 CD4+ in tumor tissues of mice in each treatment group after C-ions irradiation in this invention. + and CD8 + T cell infiltration; among which... Figure 24 Figure A shows the CD4+ nucleotides in tumor tissues of mice in each treatment group. + and CD8 + T cell infiltration status; Figure 24 Figure B in the figure shows the CD8+ in tumor tissues of mice in each treatment group. + Statistical analysis of T cell infiltration; Figure 24 Figure C in the figure shows CD4 in tumor tissues of mice in each treatment group. + Statistical analysis of cell infiltration; ns: p >0.05, *: p <0.05.

[0047] Figure 25 CD4+ in the spleens of mice in each treatment group after C-ions irradiation in this invention. + and CD8 + T cell infiltration; among which... Figure 25 Figure A shows the CD4 count of the spleen in mice from each treatment group. + and CD8 + T cell infiltration status; Figure 25 Figure B in the figure shows the CD8+ of the spleen of mice in each treatment group. + Statistical analysis of T cell infiltration; Figure 25 Figure C in the diagram shows the CD4 count of the spleen in mice from each treatment group.+ Statistical analysis of cell infiltration; ns: p >0.05, *: p <0.05. Detailed Implementation

[0048] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments, but this should not be construed as limiting the invention. Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following embodiments are commercially available unless otherwise specified.

[0049] The following is information on the materials and reagents used in the examples: 1. Experimental cells and experimental animals Human osteosarcoma cells U2OS (HTB-96) and mouse osteosarcoma cells K7M2 (CRL-2836) were purchased from ATCC (American Type Culture Collection) and cryopreserved. BALB / c mice (male, 6-8 weeks old).

[0050] 2. Reagents and Materials DMEM medium; Fetal bovine serum (FBS); Penicillin-streptomycin mixture (double antibody); Trypsin; Olaparib: AbMole; Trypsin (EDTA-free); High-efficiency RIPA tissue / cell rapid lysis buffer; PMSF; BCA protein concentration assay kit; BSA standard; Marker (10-250 kDa); Skim milk powder; Antibody diluent; ECL chemiluminescence ultrasensitive colorimetric kit; 5× protein loading buffer; DAPI solution (ready-to-use); Goat serum; Triton X-100; TBST; CCL5 ELISA kit; CXCL10 ELISA kit; Trizol; cDNA kit; Quantitative PCR kit; Enzyme-free 8-tube PCR kit; Isopropanol; DEPC water; GP-transfect-Mate transfection reagent; Human STING siNRA Kit; Mouse STING siNRA Kit; 0.5M EDTA (pH 8.0); DPBS (calcium and magnesium ion-free); DNAse I. RNase-free, HC (50 U / µL); 10×PBS buffer; erythrocyte lysis buffer; Percoll; hyaluronidase; collagenase IV; mouse serum; corn oil; sodium pentobarbital.

[0051] 3. Antibodies γH2AX; 53BP1; Rad51; β-Actin; HRP-conjugated Affinipure Goat Anti-RabbitlgG(H+L); 488; PD-L1 / CD274; β-Actin; cGAS; cGAS; STING; TBK1; p-TBK1; IRF3; p-IRF3; IFN-β; dsDNA; PerCP / Cyanine5.5 anti-mouse CD3; FITC anti-mouse CD4; PE anti-mouse CD8a; APC anti-mouse CD45; anti-CD16 / 32 FC blocker; anti-mouse PD-L1 (αPD-L1).

[0052] Example 1 I. Experimental Methods 1. Cell Culture (1) Cell thawing: Preheat the water bath to 37°C and place the cryopreserved U2OS osteosarcoma cells and K7M2 osteosarcoma cells in the 37°C water bath for rapid thawing. Then add 2 mL of DMEM complete medium (containing 10% (v / v) FBS and 1% (v / v) penicillin-streptomycin), and centrifuge the tubes at 1200 rpm for 3 min. After centrifugation, discard the supernatant, add 5 mL of DMEM complete medium to resuspend the cells, mix the cell suspension evenly, and transfer it to a 60 mm cell culture dish for culture. Then place the cell culture dish in a 37°C cell incubator for culture.

[0053] (2) Cell passage: When the growth density of U2OS osteosarcoma cells and K7M2 osteosarcoma cells reaches 85% of the culture dish area, passage can be performed. After trypsin digestion, the single-cell suspension is gently pipetted, centrifuged and resuspended, and then seeded into new cell culture dishes according to the passage ratio. The cell culture dishes are then placed in a 37°C constant temperature cell incubator and cultured at 90% humidity and 5% CO2.

[0054] 2. Cell irradiation treatment Carbon ions were supplied by the HIMM carbon ion unit at Lanzhou Heavy Ion Hospital. Cell irradiation energy was 120 MeV / u, LET was 80 keV / μm, dose rate was 2 Gy / min, and irradiation was performed vertically.

[0055] 3. PARP inhibitors Olaparib powder (M1664) was purchased from AbMole (USA). The olaparib powder was dissolved in 10 mM DMSO, diluted to 10 μM with DMEM, and added 2 hours before cell irradiation. Negative control samples were treated with the same DMSO concentration as the experimental samples.

[0056] 4. Immunofluorescence (1) Osteosarcoma cells were digested and isolated into single cells using trypsin. After counting, the cell suspension was evenly seeded into laser confocal glass culture dishes and placed in a 37°C, 5% CO2 incubator overnight. After the cells adhered to the culture dish, appropriate intervention treatment was given. Overnight refers to a time ≥12h.

[0057] (2) After cell irradiation, 2 mL of pre-cooled 4% (v / v) paraformaldehyde was added to fix the cells at a specific time point.

[0058] (3) Add 2 mL of PBS to the fixed cells and incubate for 10 min to hydrate the cells, then discard the PBS.

[0059] (4) After rehydration, add 2 mL of 0.5% (v / v) Triton-X100 solution (diluted with PBS) to permeate and perforate the cell membrane. After soaking for 10 min, discard the 0.5% (v / v) Triton-X100 solution.

[0060] (5) Rinse the cells after punching with PBS and block them with 2 mL of 5% (v / v) goat serum for 1 h.

[0061] (6) Remove the blocking solution, wash the cells three times with TBST for 5 min each time, aspirate the TBST dry, and add the primary antibody diluted with antibody dilution buffer: γH2AX (1:200), 53BP1 (1:200), Rad51 (1:200). Place in a humidified chamber and incubate overnight on a shaker at 4°C. Overnight means ≥12 h.

[0062] (7) The next day, aspirate the primary antibody, wash the cells three times with TBST for 5 min each time, aspirate the TBST dry, and add the fluorescent secondary antibody diluted with antibody dilution buffer: Goat Anti Rabbit IgG (H&L)-Alexa Fluor 594 (1:200) and Goat Anti Mouse IgG (H&L)-Alexa Fluor 594 (1:200). Place in a humidified chamber and incubate on a shaker at room temperature for 2 h. After that, aspirate the secondary antibody, wash the cells three times with TBST for 5 min each time, aspirate the TBST dry, add mounting buffer containing DAPI, stain the nuclei and mount the cells, and incubate at room temperature for 20 min.

[0063] (8) Adjust the parameters of the laser confocal microscope, randomly select the field of view to take pictures, and collect and statistically analyze the fluorescence information.

[0064] 5. Gene enrichment analysis Gene enrichment analysis (GSEA) is based on the expression levels of all genes. Genes are ranked in descending order using Signal2Noise as the criterion, and then the pathway to which the gene set belongs is scored; the score is called the ES (Enrichment Score). Permutation tests are performed based on the gene set to calculate significance. p Finally, the standardized ES values ​​(NES values) were adjusted using various tests to obtain the FDR values. GSEA data for osteosarcoma were obtained from the TARGET database. The enrichment results were considered statistically significant if the following criteria were met: ⓵ |NES|>1.0; ⓶ Nominal p<0.05; ⓷ FDR q<0.05.

[0065] 6. Western blotting to detect changes in the expression of related proteins Cellular proteins were extracted and their concentrations were quantified using the BCA method. Stacking and separating gels of appropriate concentrations were prepared according to the molecular size of the target protein. 20 μL of the quantified protein sample and 5 μL of pre-stained protein marker were added to the loading tank, followed by electrophoresis buffer. Electrophoresis, membrane transfer, and blocking with 5% (v / v) skim milk were then performed. The blocked PVDF membrane was placed in diluted primary antibody and incubated overnight on a shaker at 4°C, followed by washing three times with TBST for 10 min each time. The PVDF membrane was then placed in the appropriate secondary antibody and incubated on a shaker at room temperature for 1 h, followed by washing three times with TBST for 10 min each time. Finally, the PVDF membrane was bound to ECL chromogenic solution and exposed in a fully automated chemiluminescence imaging system.

[0066] 7. qRT-PCR (real-time quantitative PCR) detection of changes in the expression levels of related genes. RNA was extracted from U2OS osteosarcoma cells and K7M2 osteosarcoma cells using Trizol, and the RNA was purified by chloroform and isopropanol before subsequent detection experiments. The specific steps are as follows:

[0067] (1) Aspirate the culture medium from the cell culture dish, add 1 mL of Trizol reagent, shake and gently pipette to fully lyse the cells, transfer them to a new EP tube (let stand for 5 min), then add 0.2 mL of chloroform, shake vigorously for 15 s to mix them evenly, and centrifuge at low temperature (10000 g, 15 min).

[0068] (2) Transfer the supernatant in the centrifuged EP tube to a new EP tube, add 0.5 mL of isopropanol, let stand at room temperature for 10 min, and centrifuge at low temperature (10000 g, 10 min) to obtain RNA precipitate. Add 1 mL of 75% (v / v) ethanol to wash the RNA precipitate, centrifuge at low temperature (7000 g, 5 min), discard the supernatant, air dry at room temperature, dissolve the RNA precipitate with 20 μL of DEPC water, mix well, and incubate the RNA mixture at 58 °C for 10 min.

[0069] (3) Take 1 μL of the diluted mixture to detect OD. 260 / OD 280 Value, select OD 260 / OD 280 RNA samples with a concentration of 1.9 were subjected to electrophoresis to check their integrity. A 1% agarose gel was prepared using 1×TAE solution, and 5 μL of sample and marker were added. Electrophoresis was performed at a voltage of 5 V / cm, and the electrophoretic bands were observed under ultraviolet light.

[0070] (4) Take 3 μL of 5×gDNA digester mix and 1 μg RNA into a centrifuge tube and add DEPC water to 15 μL. Mix well and incubate at 42℃ for 2 min in a PCR instrument. Then add 5 μL of 4×Hifair® Ⅲ SuperMix plus and mix well. Incubate at 25℃ for 5 min; 56℃ for 15 min; 85℃ for 5 min in a PCR instrument to obtain 20 μL of cDNA.

[0071] Real-time quantitative qPCR (qRT-PCR) was performed using a 20 μL system according to the instructions of the qPCR SYBR Green Master Mix kit.

[0072] (1) Dilute the above cDNA tenfold with DEPC water, add 10 μL of Hieff® qPCR SYBR Green Master Mix (Low Rox Plus), 0.4 μL of forward and reverse primers (10 μM) and 1 μL of cDNA template to a 0.2 mL PCR eight-tube, add RNase-free H2O to 20 μL, mix and centrifuge, and place on a PCR instrument for amplification; the amplification program is as follows: pre-denaturation 95℃ 1 min, one cycle; denaturation 95℃ 10 s; annealing 58℃ 20 s, extension 72℃ 20 s; denaturation-annealing-extension for 40 cycles; set up three replicates for each sample.

[0073] (2) After amplification, the changes in the amplification curve and melting curve are detected, and the Ct data obtained are used to calculate the changes using 2... -ΔΔCt The calculation method is as follows: △Ct = △Ct of experimental group - △Ct of control group, △Ct = Ct of target gene in experimental group - Ct of internal reference in experimental group. Primer sequences are shown in Table 1.

[0074] Table 1. qRT-PCR primer sequence list Note: (H) is a human sequence, and (M) is a mouse sequence.

[0075] 8. Cell transfection experiment (1) Cell preparation: Logarithmically growing U2OS osteosarcoma cells and K7M2 osteosarcoma cells were used for transfection experiments. Cells were separated by trypsin digestion, and U2OS osteosarcoma cells and K7M2 osteosarcoma cells were counted separately using an automated cell counter. U2OS osteosarcoma cells and K7M2 osteosarcoma cells were seeded into six-well plates (2×10⁻⁶ cells per well). 5 (Cells / well), incubate the seeded cells in six-well plates overnight until cell adhesion occurs, under constant temperature: 37℃, CO2: 5%. Perform subsequent transfection experiments when the cell density reaches approximately 70%. "Overnight" refers to a time ≥12 hours.

[0076] (2) Preparation of transfection complex: ① Place the GP-transfect-mate transfection reagent at room temperature and mix gently before use. ② Add 200 μL of FBS-free medium and the transfection reagent to a 1.5 mL sterile enzyme-free centrifuge tube, mix gently with a pipette, and let stand at room temperature for 5 min. ③ Add 200 μL of FBS-free medium and RNA to another 1.5 mL sterile enzyme-free centrifuge tube, mix gently with a pipette, and let stand at room temperature for 5 min. ④ Add the GP-transfect-mate transfection reagent-medium mixture dropwise to the RNA-medium mixture, mix gently with a pipette, and let stand at room temperature for 17 min before use.

[0077] (3) Transfection process: ① While the transfection complex is settling, change the medium in the six-well plate, adding 1600 μL of FBS-free medium to each well. ② Add 400 μL of the transfection mixture to each well, bringing the final volume to 2000 μL. After adding the mixture, gently shake the plate to ensure even distribution of the complex. ③ Place the six-well plate in a 37°C cell incubator and replace the medium with complete medium after 5 hours. Detect mRNA expression after 36 hours and protein expression after 72 hours.

[0078] 9. ELISA testing (1) Sample collection and storage: Transfer the cell culture medium to a sterile centrifuge tube, centrifuge at 1000g for 10 min at 4℃, and then aliquot the supernatant into small EP tubes and store at -20℃ (for testing within 24 hours, it can be stored at 4℃) to avoid repeated freeze-thaw cycles.

[0079] (2) Reagent preparation: ① Reagent warming: First, place the reagent kit and the sample to be tested at room temperature 30 minutes before the experiment. If crystals appear in the concentrated washing solution, place it in a 37°C warm bath until all crystals are dissolved. ② Gradual dilution of standards: Dilute the standards according to the gradient concentration according to the instructions.

[0080] (3) Detection steps: Wash the plate 3 times and spin dry before adding the standard / sample; set up blank wells, standard wells and sample wells, add 100 μL of standard and test sample to the reaction wells, seal the plate and incubate at 37℃ for 90 min; tap and wash the plate 4 times, add 100 μL of biotinylated antibody working solution to the reaction wells, seal the plate and incubate at 37℃ for 60 min; tap and wash the plate 4 times, add 100 μL of enzyme conjugate working solution to the reaction wells, seal the plate and incubate at 37℃ for 30 min; tap and wash the plate 5 times, add 100 μL of chromogenic substrate to the reaction wells, seal the plate and incubate at 37℃ for 15 min; add 50 μL of stop solution, measure the OD value of each well at 450 nm using an ELISA reader, select dual wavelength detection, and the reference wavelength is 630 nm (within 5 min).

[0081] (4) Calculate the OD value of the standard / sample: Subtract the OD value of the blank well from the OD value of each standard / sample.

[0082] (5) Plot a standard curve using Origin software with the concentration of the standard as the x-axis and the OD value as the y-axis, and calculate the corresponding concentration using the OD value of the sample through the standard curve.

[0083] 10. Establishment and observation of mouse tumor-bearing model (1) Preparation of cell suspension: When the growth density of mouse osteosarcoma cells K7M2 reaches 85% of the culture dish area, the original culture medium is aspirated, digested with trypsin, and a single cell suspension is prepared. The number of K7M2 osteosarcoma cells is counted using a fully automated cell counter, and the cell density of the suspension is calculated.

[0084] (2) Animal husbandry: BABL / c mice were housed in an SPF-grade animal laboratory under the following conditions: temperature (24℃±2℃), humidity (60%), light (12h / 12h day and night alternation), feed (SPF grade, added every 2 days), drinking water (sterile water, changed once a day), and bedding (SPF grade, changed every 3 days). The mice had free access to food and water throughout the process, ensuring animal welfare.

[0085] (3) Establishment of a unilateral tumor-bearing mouse model: The ultraviolet disinfection bench was used, and all necessary items were prepared. The mice were immobilized, and the injection site was disinfected with a sterile cotton ball containing 75% (v / v) alcohol. After thoroughly mixing the cell suspension, 100 μL of the cell suspension (4 × 10⁻⁶ cells / mL) was drawn up using a 1 mL sterile syringe. 6 (1 cell / mouse) Insert the needle subcutaneously at a 45° angle into the right leg, slowly inject, hold for 5 seconds, then withdraw the needle and disinfect the injection site again to ensure that all mice are inoculated at the same site as much as possible.

[0086] (4) After inoculation, mice were continued to be fed, and the long diameter (L) and short diameter (W) of the tumor were measured regularly. The tumor volume was calculated according to the following formula: V = 1 / 2 (L × W) 2 ); In the above formula, the unit of V is mm. 3 .

[0087] 11. Grouping and Treatment Procedures for Tumor-Bearing Mice On day 10 post-vaccination in BABL / c mice, the tumor on the right leg was approximately 100 mm in size. 3Mice bearing tumors were randomly assigned to groups (n=5 per group) using a random number table: control group, αPD-L1 monotherapy group, olaparib monotherapy group, αPD-L1 combined with olaparib group, C-ions 10 Gy (RBE) irradiation group, C-ions 10 Gy (RBE) irradiation combined with αPD-L1 group, C-ions 10 Gy (RBE) irradiation combined with olaparib group, and C-ions 10 Gy (RBE) irradiation combined with αPD-L1 and olaparib group. Survival analysis was performed in separate groups under the same conditions.

[0088] Olaparib powder (M1664) was purchased from AbMole (USA). The olaparib powder was first dissolved in DMSO and then diluted with corn oil to the desired concentration. Negative control samples were treated with the same DMSO concentration as the experimental samples. Three days prior to irradiation, tumor-bearing mice were administered the drug via gavage (ig) daily at a dose of 50 mg / kg / time / day, following the specific procedure described below. Figure 1 As shown.

[0089] The murine PD-L1 inhibitor (αPD-L1, anti-PD-L1 antibody) was purchased from InVioMAb (USA). Following the αPD-L1 manufacturer's label and previous studies, the antibody was diluted with 0.9% sterile saline and administered intraperitoneally (ip) to tumor-bearing mice at a dose of 12.5 mg / kg / time. Negative control samples were treated with the same volume of 0.9% sterile saline as the experimental samples. The specific procedure is as follows: Figure 1 As shown.

[0090] 12. Irradiation treatment of mice Carbon ions were supplied by the HIMM carbon ionization unit at Lanzhou Heavy Ion Hospital. The irradiation energy was 120 MeV / u, the LET was 80 keV / μm, the dose rate was 2 Gy / min, and the irradiation was performed vertically.

[0091] Mice were anesthetized and fixed before irradiation. Lead blocks were used during irradiation to expose the tumor-bearing area to the irradiation field, ensuring that areas outside the tumor were not irradiated.

[0092] 13. Tumor observation and measurement After BABL / c mice were inoculated, tumor growth was observed regularly. The major and minor diameters of the tumors were measured using digital calipers, and the mice were weighed and their weights recorded. Tumor volume was calculated using the following formula:

[0093] V = 1 / 2 (L × W) 2 ); In the above formula, the tumor volume is V, and the unit is mm. 3 L is the major axis and W is the minor axis.

[0094] According to relevant ethical requirements for animal experiments, the tumor volume in mice should not exceed 2000 mmHg throughout the entire observation process. 3 Based on preliminary experiments, this invention selected D21 (7 days after irradiation) as the time for euthanizing mice. Figure 1 Before sacrifice, the size and volume of the tumor were measured. After sacrifice, intact subcutaneous tumor tissue was dissected and removed, and its size and weight were measured and recorded. The survival analysis group's observation and recording endpoint was mouse death or the tumor volume reaching 2000 mmHg. 3 Survival curves were plotted using GraphPad Prism 9.0.

[0095] 14. Flow cytometry detection of tumor-infiltrating lymphocytes (1) Preparation of single-cell suspension: ① Cut the tumor into small pieces with scissors and add 5 mL of digestion solution to the centrifuge tube; ② Digest with digestion solution for 1 h; ③ Grind with a 200 mesh grinding basket; ④ Add DPBS2 to stop digestion and transfer the ground tumor tissue and digestion solution to a 15 mL centrifuge tube; ⑤ Centrifuge at 1500 rpm for 5 min at 4℃; ⑥ Discard the supernatant and resuspend and rinse with pre-cooled DPBS2; ⑦ Centrifuge at 1500 rpm for 5 min at 4℃; ⑧ Resuspend the tumor cells in 4 mL of 40% (v / v) Percoll solution.

[0096] (2) Density gradient centrifugation to obtain TILs: ① Add 4 mL of 80% (v / v) Percoll solution to a new centrifuge tube. Carefully drop 4 mL of 40% (v / v) Percoll containing tumor cells onto the 80% (v / v) Percoll solution along the tube wall using a Pasteur tube, being careful to separate the two into layers; ② Centrifuge at 200g for 30 min, with an ascent rate of 1 and a deceleration rate of 0 (keeping the ascent and deceleration rates as low as possible); ③ Collect immune cells at the junction; ④ Add DPBS2 to wash, centrifuge at 1500 rpm for 5 min at 4℃; ⑤ Count the immune cells at this time; ⑥ Remove the supernatant, add DPBS, and add 1 × 10⁻⁶ DPBS per sample. 6 For each cell sample, add the live / dead dye to each sample, mix well, and let stand for 15 min; ⑦ Add DPBS2 to stop staining, centrifuge at 1500 rpm for 5 min at 4℃; ⑧ Discard the supernatant and resuspend each cell sample in 100 μL of FACSbuffer.

[0097] (3) FC receptor blocking: each cell sample was blocked by adding anti-CD16 / 32 FC blocker and placed in a 4-degree refrigerator for 20 minutes.

[0098] (4) Staining and loading: ① Add CD3, CD4, CD45 and CD8 antibodies to each sample for staining (single positive and mixed positive). Prepare a blank sample for voltage adjustment. After adding the antibodies, incubate at 4°C for 30 min. ② Add 1 mL of DPBS2 to each tube, centrifuge at 1500 rpm for 5 min, and incubate at 4°C. ③ Remove the supernatant and resuspend each tube in 200 μL of DPBS2. ④ Detect using a flow cytometer. Filter the cells through a 200-mesh sieve before loading.

[0099] (5) Flow cytometry analysis: The sample is used for flow cytometry analysis, and statistical analysis is performed based on the percentage of immune cell subset infiltration.

[0100] 15. Flow cytometry detection of infiltrating lymphocytes in the spleen (1) Preparation of single-cell suspension: After euthanizing the mice, the spleen was carefully bluntly separated, the spleen was cut into pieces and placed in a 200-mesh grinding frame, PBS was added while grinding, and finally the sieve was rinsed with PBS. All cells were collected and the number of K7M2 osteosarcoma cells was counted using an automated cell counter. The cell density of the single-cell suspension was calculated.

[0101] (2) Centrifuge the single cell suspension at 1500 rpm for 5 min at 4℃; discard the supernatant, add red blood cell lysis buffer, and place on a shaker for 15 min (4℃).

[0102] (3) Add PBS to stop the red blood cell splitting, then centrifuge at 1500 rpm for 5 min at 4℃; discard the supernatant and add 100 μL of FACSbuffer to resuspend the cells.

[0103] (4) Add anti-CD16 / 32 FC blocker for sealing and place in a 4-degree refrigerator for 25 minutes.

[0104] (5) Add appropriate amounts of CD3, CD4, CD45 and CD8 antibodies to stain the cells (single positive tube and mixed tube), and prepare a blank sample for voltage adjustment. After adding the antibodies, incubate at 4 degrees for 30 min.

[0105] (6) Add 1 mL of DPBS2, then centrifuge at 1500 rpm for 5 min at 4℃; discard the supernatant.

[0106] (7) After resuspending the cells in 200 μL of DPBS, the cells were tested on the instrument; the cells were filtered through a 200-mesh filter before testing.

[0107] (8) Flow cytometry analysis: The sample is used for flow cytometry analysis, and statistical analysis is performed based on the percentage of immune cell subset infiltration.

[0108] 16. Data Statistical Analysis Data analysis was performed using SPSS 23.0 software and GraphPad Prism 9. Unpaired t-tests were used for comparisons between two groups, and one-way ANOVA was used to assess statistical significance for comparisons among multiple groups. Data are expressed as mean ± SD. p <0.05 (*) indicates that the difference is statistically significant. p >0.05 (ns) indicates that the difference is not statistically significant.

[0109] II. Results 1. Olaparib-induced DNA damage in osteosarcoma cells after C-ions irradiation This invention detects the formation of γH2AX and 53BP1foci in U2OS osteosarcoma cells using immunofluorescence assays at 2 h and 12 h after irradiation. Figure 2 Immunofluorescence results showed that the formation of γH2AX and 53BP1 foci reached its peak 2 hours after osteosarcoma cell irradiation. Figure 2 ): After C-ions irradiation, the foci formed by γH2AX and 53BP1 were larger and irregular in shape. Compared with the irradiated group, olaparib combined irradiation induced more, larger, and more irregular γH2AX / 53BP1 foci ( p <0.05). Furthermore, 12 hours after irradiation, the γH2AX / 53BP1 foci in the irradiated group was significantly reduced, while the combined group still had a relatively high γH2AX / 53BP1 foci, suggesting that olaparib combined with irradiation can maintain more persistent DNA damage in U2OS osteosarcoma cells. Figure 2 (Figure B in the diagram).

[0110] This invention detects the formation of γH2AX and 53BP1 foci in K7M2 osteosarcoma cells 2 h and 12 h after irradiation using immunofluorescence experiments. Figure 3 Similar to U2OS osteosarcoma cells, immunofluorescence results showed that the formation of γH2AX and 53BP1 foci in K7M2 osteosarcoma cells reached its peak 2 hours after irradiation. Figure 3 ): After C-ions irradiation, the foci formed by γH2AX and 53BP1 were larger and irregular in shape. Compared with the irradiated group, olaparib combined with irradiation induced more, larger, and more irregular γH2AX / 53BP1 foci ( p <0.05). Furthermore, 12 hours after irradiation, the γH2AX / 53BP1 foci in the irradiated group was significantly reduced, while the combined group still had a relatively high γH2AX / 53BP1 foci, suggesting that olaparib combined with irradiation can also maintain more durable DNA damage in K7M2 osteosarcoma cells. Figure 3 (Figure B in the diagram).

[0111] 2. PARP3 enrichment analysis with osteosarcoma GSEA Olaparib belongs to the pan-PARP family of protein inhibitors and can inhibit multiple members of the family. This invention uses GSEA analysis to explore PARP-related signaling pathways. Based on the median expression level of PARP3, osteosarcoma was divided into high-expression and low-expression groups. GSEA gene enrichment analysis of these data showed that the genes in the PARP3 high-expression group were mainly enriched in immune-related signaling pathways, including: cytoplasmic DNA sensing signaling pathway, chemokine signaling pathway, T cell receptor signaling pathway, B cell receptor signaling pathway, NK cell-mediated cytotoxicity, Toll-like receptor signaling pathway, NOD-like receptor signaling pathway, antigen processing and presentation, FCγR-mediated phagocytosis, cytokine receptor signaling pathway, FcεRI signaling pathway, and primary immunodeficiency diseases (…). Figure 4 ).

[0112] 3. Olaparib enhances C-ions-induced PD-L1 expression in osteosarcoma cells. Previous studies have shown that DNA damage signals induced by ionizing radiation can upregulate the expression of PD-L1 on the surface of tumor cells, and PD-1 / PD-L1 immune checkpoint inhibitors are among the most widely used ICIs in clinical practice. Therefore, this invention uses GSEA enrichment analysis to study PARP-related signaling pathways in osteosarcoma. The results suggest that genes in the PARP3 high-expression group are mainly enriched in multiple immune-related signaling pathways (…). Figure 4 Based on the above research results, the following hypothesis is proposed: Will the PARP inhibitor olaparib enhance X-ray-induced PD-L1 expression in osteosarcoma cells?

[0113] This invention first determined the dose-dependent relationship of PD-L1 expression in U2OS and K7M2 osteosarcoma cells induced by different irradiation dose gradients using Western blotting experiments (protein extraction time was 48 h after irradiation). (See...) Figure 5 The results showed that, compared with the control group, there was no significant change in PD-L1 expression in either osteosarcoma cell line at low dose (2 Gy) (P>0.05), but PD-L1 protein expression gradually increased with increasing irradiation dose. p <0.05); At 12 Gy, although PD-L1 expression still increased, there was no significant difference compared with the 10 Gy group ( P >0.05). Therefore, this invention considers 10 Gy of X-rays to be the optimal irradiation dose for inducing PD-L1 expression in two osteosarcoma cell lines, and selected 10 Gy of X-rays for subsequent experiments.

[0114] Given that C-ions are prescribed in clinical practice using relative biological effect dose (RBE dose) as the unit of dosage, this invention selected 10 Gy (RBE) C-ions for subsequent experiments to meet actual clinical needs. Based on previous colony formation experiments with two cell lines and two irradiations, this invention plotted dose-survival curves after X-ray and C-ions irradiation. Then, using the D10 values ​​corresponding to the dose-survival curves of U2OS osteosarcoma cells and K7M2 osteosarcoma cells, the RBE value for U2OS osteosarcoma cells was calculated to be 2.9; the RBE value for K7M2 osteosarcoma cells was 1.8. 10 Gy (RBE) of C-ions applied to U2OS osteosarcoma cells is equivalent to a physical dose of 3.45 Gy, and 10 Gy (RBE) of C-ions applied to K7M2 osteosarcoma cells is equivalent to a physical dose of 5.56 Gy.

[0115] In osteosarcoma cells, PD-L1 expression after irradiation exhibits both dose-dependent and time-dependent characteristics. Figure 6 In this invention, proteins were extracted at 12h, 24h, and 48h after irradiation for Western blotting experiments. The results showed that, compared to 0h, there was no significant difference in PD-L1 expression in U2OS osteosarcoma cells at 12h after irradiation. P >0.05), while PD-L1 protein expression was increased in K7M2 osteosarcoma cells ( p <0.05); PD-L1 protein expression was significantly increased in both osteosarcoma cell lines at 24h and 48h after irradiation. p <0.05). Furthermore, PD-L1 expression levels were significantly higher after 48 hours of irradiation than at 12 hours and 24 hours (…). p <0.05). Therefore, this invention selected 48 hours after irradiation as the time point for subsequent experiments.

[0116] Based on the above experiments, the optimal irradiation dose and irradiation time for PD-L1 expression in osteosarcoma cells were determined. Figure 5 and Figure 6 So, can olaparib enhance PD-L1 protein expression in osteosarcoma cells induced by ionizing radiation? In this invention, two cell lines were divided into: control group, olaparib group, X-rays group, X-rays + olaparib group, C-ions group, and C-ions + olaparib group. After irradiation for 48 hours, protein was extracted for Western blotting experiments. Figure 7 The results showed that, compared with the control group, the olaparib group failed to induce PD-L1 expression in osteosarcoma cells. P >0.05); at the same RBE dose, the C-ions group showed higher PD-L1 expression ( p<0.05), C-ions, as high-LET rays, have a greater radiobiological advantage in improving the immunogenicity of osteosarcoma cells. Furthermore, compared with the irradiation-only group, the olaparib combined with irradiation group showed higher PD-L1 expression ( p <0.05)( Figure 7 ).

[0117] 4. Enrichment analysis of PD-L1 and osteosarcoma GSEA This invention has experimentally demonstrated that olaparib combined with X-rays can induce a stronger radiomodulatory effect by upregulating PD-L1 expression on the surface of osteosarcoma cells. So, what is the specific mechanism by which olaparib combined with irradiation enhances its immunomodulatory effect? ​​Therefore, this invention divides osteosarcoma into high-expression and low-expression groups based on the median PD-L1 expression level. Gene enrichment analysis (GSEA) of these data showed that the genes in the PD-L1 high-expression group were mainly enriched in immune-related signaling pathways, including: cytoplasmic DNA sensing pathway, antigen processing and presentation, cytokine receptor signaling pathway, NK cell-mediated cytotoxicity, chemokines, Toll-like receptor signaling pathway, T cell receptor signaling pathway, JAK-STAT signaling pathway, B cell receptor signaling pathway, NOD-like receptor signaling pathway, FCγR-mediated phagocytosis, and primary immunodeficiency diseases (such as...). Figure 8 (As shown).

[0118] 5. Olaparib combined with C-ions induces the aggregation of dsDNA in the cytoplasm of osteosarcoma cells. Based on the above research results, olaparib combined with C-ions irradiation induces more severe and complex DNA damage in osteosarcoma cells. Figure 2 and Figure 3 Immunofluorescence indicated that olaparib combined with irradiation could induce the formation of more γH2AX foci in the nuclei of osteosarcoma cells. Figure 2 and Figure 3 γH2AX is an important marker of DNA double-strand breaks (DSBs). So, where do these DNA double-strand breaks go? Therefore, this invention uses confocal immunofluorescence assays to track the destination and location of dsDNA in osteosarcoma cells after C-ions irradiation. Figure 9 ).

[0119] like Figure 9As shown, the fluorescence results 24 hours after C-ions irradiation indicated that, compared with the control group, the fluorescence intensity of dsDNA in the irradiation group and the olaparib combined irradiation group was significantly increased; compared with the irradiation group alone, the fluorescence intensity of dsDNA in the C-ions combined olaparib group was significantly increased. Furthermore, this invention observed that dsDNA in the cell nucleus gradually migrated towards the vicinity of the nuclear membrane and crossed the nuclear membrane to reach the cytoplasm, forming dsDNA foci in the cytoplasm. This indicates that the DNA of DSBs in the cell nucleus entered the cytoplasm, leading to the aggregation of dsDNA in the cytoplasm. Figure 9 ).

[0120] 6. Olaparib combined with C-ions-induced dsDNA binds to cGAS in the cytoplasm of osteosarcoma cytoplasm. Previous GSEA gene enrichment analysis indicated that genes in the PARP3-overexpressing group were mainly enriched in signaling pathways related to osteosarcoma immunity, including: cytoplasmic DNA sensing signaling pathways, chemokine signaling pathways, etc. Figure 4 It is noteworthy that genes in the PD-L1 high-expression group are also primarily enriched in immune-related signaling pathways associated with osteosarcoma, including: cytoplasmic DNA sensing pathway, antigen processing and presentation, cytokine receptor signaling pathway, chemokine signaling pathway, and other signaling pathways (such as...). Figure 8 It is not difficult to see that the immune signaling pathway simultaneously associated with DNA damage is the cytoplasmic DNA sensing signaling pathway, and this invention has demonstrated that PARP inhibitors combined with C-ions can induce more dsDNA accumulation in the osteosarcoma cytoplasm. Figure 9 ).

[0121] cGAS is one of the most important cytoplasmic DNA sensors. It can detect cytoplasmic DNA and trigger the expression of inflammatory genes, activating defense mechanisms. Tumor-derived DNA can activate the cGAS / STING signaling pathway, inducing cellular senescence and anti-tumor immunity. Based on previous results and literature reports, this invention proposes the following hypothesis: Does olaparib combined with irradiation induce upregulation of PD-L1 expression in osteosarcoma cells by activating the cGAS / STING signaling pathway?

[0122] Previous studies have shown that cytoplasmic dsDNA can be sensed by cGAS. To verify the above hypothesis, this invention again used confocal immunofluorescence colocalization to track and locate the sensing process of cytoplasmic dsDNA and cGAS. Figure 11 ).like Figure 10 As shown, the fluorescence results 24 hours after C-ions irradiation indicated that, compared with the control group, the fluorescence of dsDNA and cGAS in the irradiation group and the olaparib combined irradiation group was enhanced and they co-localized and bound in the cytoplasm; compared with the irradiation group alone, the fluorescence of dsDNA and cGAS in the olaparib combined irradiation group was enhanced and they co-localized and bound in the cytoplasm.

[0123] 7. Olaparib combined with C-ions activates the cGAS / STING signaling pathway, upregulating the expression of PD-L1 and chemokines CCL5 and CXCL10. The aforementioned immunofluorescence experiments confirmed that olaparib combined with irradiation induces more significant cytoplasmic dsDNA aggregation, and subsequently, cytoplasmic dsDNA binds to cGAS and activates cGAS activity. Therefore, what are the regulatory effects of olaparib combined with irradiation on the cGAS / STING signaling pathway and its downstream molecules? Based on this, this invention used Western bolting, qRT-PCR, and ELISA experiments to confirm the changes in the expression of the cGAS / STING signaling pathway and its downstream signaling molecules in two osteosarcoma cell lines 48 hours after olaparib combined with irradiation.

[0124] The results of changes in the cGAS / STING signaling pathway and its downstream signaling molecules in U2OS osteosarcoma cells are as follows: Figure 11 As shown, we first explored the activation of the cGAS / STING signaling pathway and upregulation of PD-L1 expression in U2OS cells by olaparib combined with C-ions using Western blotting and qRT-PCR. Figure 11 As shown: Compared with the control group, the C-ions group and the olaparib combined with C-ions group significantly upregulated the expression of cGAS, STING, and PD-L1 at the transcriptional and translational levels. p <0.05), while no significant difference was observed in the olaparib group ( P >0.05); compared to the C-ions group, olaparib combined with the C-ions group significantly upregulated the expression of cGAS, STING, and PD-L1 at both transcriptional and translational levels ( p <0.05). Furthermore, compared to the C-ions group, the combination of olaparib and the C-ions group not only upregulated STING expression in U2OS cells, but also activated phosphorylation of downstream STING molecules TBK1 and IRF3, as well as the type I interferon pathway, leading to upregulation of IFN-β at both transcriptional and translational levels. p <0.05)( Figure 11 As is well known, the type I interferon pathway is a major component of innate immunity and a key factor in radiation-induced and activated tumor-specific T-cell immune responses.

[0125] Furthermore, the results of the ELISA experiment showed that ( Figure 12 Compared to the C-ions group alone, the release levels of CCL5 and CXCL10 in the olaparib plus C-ions group were also significantly increased. P>0.05). These two chemokines are recognized as one of the markers of cGAS-STING pathway activation, which can chemotactically recruit T lymphocytes in tumors to enhance anti-tumor immune responses.

[0126] Does the murine osteosarcoma cell line K7M2 exhibit similar results to the human osteosarcoma cell line U2OS? To this end, this invention further investigates the effects of olaparib combined with C-ions on the activation of the cGAS / STING signaling pathway and upregulation of PD-L1 expression in K7M2 cells using Western blotting and qRT-PCR. Figure 13 As shown: Compared with the control group, the C-ions group and the olaparib combined with C-ions group significantly upregulated the expression of cGAS, STING, and PD-L1 at the transcriptional and translational levels. p <0.05), while no significant difference was observed in the olaparib group ( P >0.05); compared to the C-ions group, olaparib combined with the C-ions group significantly upregulated the expression of cGAS, STING, and PD-L1 at both transcriptional and translational levels ( p <0.05). Furthermore, compared to the C-ions group, the combination of olaparib and the C-ions group not only upregulated STING expression in U2OS cells, but also activated phosphorylation of downstream STING molecules TBK1 and IRF3, as well as the type I interferon pathway, leading to upregulation of IFN-β at both transcriptional and translational levels. p <0.05)( Figure 13 The activation of the cGAS / STING signaling pathway and the upregulation of PD-L1 expression in K7M2 cells were consistent with those in U2OS cells.

[0127] The results of the ELISA experiment on K7M2 cells showed that ( Figure 14 Compared to the control group, the C-ions group and the olaparib combined with C-ions group induced increased release levels of K7M2 cell chemokines CCL5 and CXCL10. p <0.05), while no significant difference was observed in the olaparib group ( P >0.05); compared to the C-ions group alone, the release levels of CCL5 and CXCL10 in the olaparib plus C-ions group were also significantly increased ( P >0.05).

[0128] 8. Investigation into the mechanism by which olaparib, in combination with C-ions, activates the cGAS / STING signaling pathway (verification of STING gene silencing efficiency). Based on the above results, this invention draws the preliminary conclusion that olaparib combined with C-ions can induce the aggregation of cytoplasmic dsDNA, and the binding of cytoplasmic dsDNA and cGAS activates the cGAS / STING signaling pathway and upregulates the expression of downstream signaling molecules. To investigate the reliability of this conclusion, this invention selects to silence the STING gene and uses a siRNA strategy to inhibit STING transcription.

[0129] First, this invention designed three different siRNA primers for the STING gene in human osteosarcoma cells U2OS and murine osteosarcoma cells K7M2. After 48 hours of transfection, the silencing efficiency was evaluated by Western bolting and qRT-PCR. Figure 15 As shown, in U2OS cells, all three human siRNAs inhibited STING expression at both transcriptional and protein levels, but siSTING-H-1 showed the best inhibitory effect. In K7M2 cells, all three murine siRNAs inhibited STING expression at both transcriptional and protein levels, but siSTING-M-2 showed the best inhibitory effect. Therefore, in subsequent experiments, this invention selected siSTING-H-1 and siSTING-M-2 for use (Table 2). Human osteosarcoma cells U2OS are abbreviated as U2OS cells, and murine osteosarcoma cells K7M2 are abbreviated as K7M2 cells.

[0130] The siRNA primers (each with a TT end dangling) are as follows: The nucleotide sequence of siSTING-H-1 is shown in SEQ ID NO.1: 5'-GUGCCUGAUAACCUGAGUATTUACUCAGGUUAUCAGGCAC-3'.

[0131] The nucleotide sequence of siSTING-H-2 is shown in SEQ ID NO.2: 5'-CUGGCAUGGUCAUAUUACATTUGUAAUAUGACCAUGCCAG-3'.

[0132] The nucleotide sequence of siSTING-H-3 is shown in SEQ ID NO.3: 5'-GCAUCAAGGAUCGGGUUUATTUAAACCCGAUCCUUGAUGC-3'.

[0133] The nucleotide sequence of siSTING-M-1 is shown in SEQ ID NO.4: 5'-CCGGAUCCGAAUGUUCAAUTTAUUGAACAUUCGGAUCCGG-3'.

[0134] The nucleotide sequence of siSTING-M-2 is shown in SEQ ID NO.5: 5'-GCACAUUCGUCAGGAAGAATTUUCUUCCUGACGAAUGUGC-3'.

[0135] The nucleotide sequence of siSTING-M-3 is shown in SEQ ID NO.6: 5'-GGCAAAGGAUCCACCAAAUTTAUUUGGUGGAUCCUUUGCC-3'.

[0136] The nucleotide sequence of NC is shown in SEQ ID NO.7: 5'-UUCUCCGAACGUGUCACGUTTACGUGACACGUUCGGAGAA-3'.

[0137] Table 2 Final STING siRNA Sequence Note: (H) is a human sequence, and (M) is a mouse sequence.

[0138] 9. STING gene silencing inhibits the activation of the cGAS / STING signaling pathway and the upregulation of PD-L1, CCL5, and CXCL10 expression induced by olaparib combined with C-ions. Based on the above silencing efficiency verification results, this invention selected siSTING-H-1 and siSTING-M-2 to transfect U2OS and K7M2 cells, respectively, to obtain U2OS (siSTING) cells and K7M2 (siSTING) cells with silenced STING. The two cell lines with silenced STING were divided into: a control group, an olaparib group, an irradiation group, and an olaparib combined with irradiation group. After 48 hours of irradiation, the expression of the cGAS / STING signaling pathway and its downstream related molecules in osteosarcoma cells (siSTING) was verified.

[0139] First, Western blotting and qRT-PCR were used to verify the activation of the cGAS / STING signaling pathway and upregulation of PD-L1 expression in U2OS (siSTING) cells by olaparib combined with C-ions. Figure 16 As shown: Compared with the control group, the C-ions group and the olaparib combined with C-ions group significantly upregulated cGAS expression at both transcriptional and translational levels. p <0.05), while no significant difference was observed in the olaparib group ( p>0.05); compared to the C-ions group, olaparib combined with the C-ions group significantly upregulated cGAS expression at both transcriptional and translational levels ( p <0.05)( p <0.05), cGAS upward trend is the same. Figure 11 The results were consistent. However, silencing STING expression not only significantly inhibited the activation of the classical type I interferon regulatory pathway of STING-TBK1-IRF3 by the C-ions group and the C-ions plus olaparib group, but also significantly inhibited the upregulation of PD-L1 at the transcriptional and translational levels induced by the C-ions group and the C-ions plus olaparib group in U2OS (siSTING) osteosarcoma cells. p >0.05)( Figure 16 ).

[0140] Furthermore, when silencing STING expression inhibits the activation of the classical type I interferon regulatory pathway STING-TBK1-IRF3, ELISA results show that ( Figure 17 Neither the C-ions group alone nor the olaparib combined with C-ions group significantly induced the release of CCL5 and CXCL10 from U2OS (siSTING) osteosarcoma cells. p >0.05).

[0141] For murine K7M2 osteosarcoma cells with silenced STING, this invention also verified the activation of the cGAS / STING signaling pathway and upregulation of PD-L1 expression in K7M2 (siSTING) cells by olaparib combined with C-ions using Western blotting and qRT-PCR. Figure 18 As shown: Compared with the control group, the C-ions group and the olaparib combined with C-ions group significantly upregulated cGAS expression at both transcriptional and translational levels. p <0.05), while no significant difference was observed in the olaparib group ( p >0.05); compared to the C-ions group, olaparib combined with the C-ions group significantly upregulated cGAS expression at both transcriptional and translational levels ( p <0.05)( p <0.05), cGAS upward trend is the same. Figure 13 The results were consistent. Furthermore, silencing STING expression not only significantly inhibited the activation of the classical type I interferon regulatory pathway of STING-TBK1-IRF3 by the C-ions group and the C-ions combined with olaparib group, but also significantly inhibited the upregulation of PD-L1 at both transcriptional and translational levels induced by the C-ions group and the C-ions combined with olaparib group in K7M2 (siSTING) osteosarcoma cells. p >0.05)( Figure 18 ).

[0142] Regarding the expression of chemokines CCL5 and CXCL10 in K7M2 (siSTING) osteosarcoma cells, when STING expression was silenced, the release of chemokines CCL5 and CXCL10 was also inhibited because the classical type I interferon regulatory pathway of STING-TBK1-IRF3 could not be activated. ELISA results showed that ( Figure 19 Neither the C-ions group alone nor the olaparib combined with C-ions group significantly induced the release of CCL5 and CXCL10 from K7M2 (siSTING) osteosarcoma cells. p >0.05).

[0143] Based on the above experimental results, the following conclusions can be drawn from this invention ( Figure 20 Ionizing radiation (C-ions) can cause DNA damage (SSBs and DSBs) in osteosarcoma cells, while PARP inhibitors can induce more severe DNA damage (DSBs) in irradiated osteosarcoma cells. Damaged DNA, in the form of dsDNA, gradually migrates from the nucleus to the nuclear membrane and is expelled into the cytoplasm, leading to a large accumulation of dsDNA in the cytoplasm. Subsequently, the cytoplasmic dsDNA binds to and activates the cytoplasmic DNA sensor cGAS. Activated cGAS catalyzes the synthesis of cGAMP from ATP and GTP, which then binds to the STING protein, causing it to dimerize. The activated STING protein recruits TBK1 and phosphorylates it. pTBK1 then phosphorylates IRF3, thereby upregulating the expression of type I interferon-related genes, such as IFN-β and PD-L1, and increasing the expression of chemokines CXCL10 and CCL5. Figure 20 ).

[0144] 10. C-ions combined with PARP and PD-L1 inhibitors have a synergistic anti-tumor effect. All BABL / c tumor-bearing mice irradiated with C-ions showed good growth before the end of the experiment, with no deaths. The mice's body weight increased steadily over time, and data analysis indicated no statistically significant differences in body weight within or between groups. p >0.05)( Figure 21 (See Figure A in the original text). Analysis of the tumor volume measurements in mice during treatment showed that, compared to the Control group, the αPD-L1 group did not show significant tumor growth remission (see Figure A in the original text). p >0.05)( Figure 21 Figure B in the diagram); while tumor growth was significantly slowed in the Olaparib group, αPD-L1+Olaparib group, C-ions group, X-rays+αPD-L1 group, C-ions+Olaparib group, and C-ions+αPD-L1+Olaparib group (Figure B in the diagram).p <0.05)( Figure 21 (See Figure B in the figure); Furthermore, compared to the C-ions group, tumor growth was significantly slowed in the C-ions+αPD-L1 group, the C-ions+Olaparib group, and the C-ions+αPD-L1+Olaparib group. p <0.05)( Figure 21 (Figure B in the figure), in which the tumor growth remission was most significant in the C-ions+αPD-L1+Olaparib group.

[0145] In this invention, mice in each of the C-ions groups were sacrificed on day 21 to obtain tumor tissue, and the tumor weight was statistically analyzed. Figure 22 The results indicated that there was no significant difference in tumor weight between the αPD-L1 group and the Control group. p >0.05), while the tumor weight of the Olaparib group, αPD-L1+Olaparib group, C-ions group, C-ions+αPD-L1 group, C-ions+Olaparib group, and C-ions+αPD-L1+Olaparib group all decreased to varying degrees. p <0.05)( Figure 22 (See Figure B in the diagram). Similarly, compared to the C-ions group alone, the tumor weight of the C-ions+αPD-L1 group, the C-ions+Olaparib group, and the C-ions+αPD-L1+Olaparib group all decreased to varying degrees. p <0.05, among which the tumor weight reduction was most significant in the C-ions+αPD-L1+Olaparib group, while no significant difference was observed between the C-ions+αPD-L1 group and the C-ions+Olaparib group ( p >0.05)( Figure 22 (Figure B in the diagram).

[0146] The survival outcomes of the mice in each group were as follows: Figure 23 As shown, the survival outcome analysis of the survival group mice suggests that, compared with the control group, the αPD-L1 monotherapy group did not significantly prolong the survival of the mice. p >0.05), while the survival time of mice in the Olaparib group, αPD-L1+Olaparib group, C-ions group, C-ions+αPD-L1 group, C-ions+Olaparib group, and C-ions+αPD-L1+Olaparib group was prolonged to varying degrees. p <0.05)( Figure 23Similarly, compared to the C-ions group alone, the survival of mice in the C-ions+αPD-L1 group, C-ions+Olaparib group, and C-ions+αPD-L1+Olaparib group was significantly prolonged. p <0.05%, with no significant difference in survival between the C-ions+αPD-L1 group and the C-ions+Olaparib group ( p <0.05), while the C-ions+αPD-L1+Olaparib group showed the most significant survival extension ( Figure 23 ).

[0147] 11. C-ions combined with PARP and PD-L1 inhibitors promote tumor CD8. + T cell infiltration This invention uses flow cytometry to analyze tumor-infiltrating lymphocytes (TILs) in mice from each treatment group, and then statistically analyzes the proportions of immune cells after grouping them. The results suggest ( Figure 24 Compared to the control group, the αPD-L1-only group, the Olaparib-only group, and the αPD-L1+Olaparib group showed significantly higher levels of CD4+ in tumor tissues of mice. + and CD8 + T cell infiltration did not increase significantly ( p >0.05), while CD4 in tumor tissues of mice in the C-ions group, C-ions+αPD-L1 group, C-ions+Olaparib group, and C-ions+αPD-L1+Olaparib group was higher than 0.05. + and CD8 + T cell infiltration increased significantly ( p <0.05).

[0148] C-ions combined with PARP inhibitors and PD-L1 inhibitors inhibited CD4+ in mouse tumor tissues. + and CD8 + The infiltration of T cells is slightly different ( Figure 24 Compared to the C-ions group alone, the C-ions+αPD-L1 group, C-ions+Olaparib group, and C-ions+αPD-L1+Olaparib group showed higher levels of CD8+ in tumor tissues of mice. + T cell infiltration increased significantly ( p <0.05), of which C-ions+αPD-L1+Olaparib group CD8 +T cell infiltration was most pronounced in the C-ions+αPD-L1 group and the C-ions+Olaparib group mice, while CD8+αPD-L1 was most prominent in the tumor tissue. + No significant difference was observed in T cell infiltration. p >0.05). However, although C-ions irradiation groups were able to induce CD4 in mouse tumor tissue compared to the non-irradiation group, + T cell infiltration increased significantly ( p <0.05), but there were differences in CD4 levels in mouse tumor tissue among the C-ions group, C-ions+αPD-L1 group, C-ions+Olaparib group, and C-ions+αPD-L1+Olaparib group. + No significant difference was observed in T cell infiltration. p >0.05).

[0149] 12. C-ions combined with PARP and PD-L1 inhibitors promote spleen CD8 + T cell infiltration This invention uses flow cytometry to analyze spleen-infiltrating lymphocytes (SILs) from mice in each treatment group, and then statistically analyzes the proportions of immune cells after grouping them. The results suggest ( Figure 25 Compared to the control group, the αPD-L1-only group, the Olaparib-only group, and the αPD-L1+Olaparib group showed higher levels of CD4+ in the spleen tissue of mice. + and CD8 + T cell infiltration did not increase significantly ( p >0.05), while CD4+ in the spleen tissue of mice in the C-ions group, C-ions+αPD-L1 group, C-ions+Olaparib group, and C-ions+αPD-L1+Olaparib group was higher than 0.05. + and CD8 + T cell infiltration increased significantly ( p <0.05).

[0150] C-ions combined with PARP inhibitors and PD-L1 inhibitors inhibited CD4+ in mouse spleen tissue. + and CD8 + The infiltration of T cells is slightly different ( Figure 25 Compared to the C-ions group alone, the C-ions+αPD-L1 group, C-ions+Olaparib group, and C-ions+αPD-L1+Olaparib group showed higher levels of CD8+ in the spleen tissue of mice. + T cell infiltration increased significantly ( p<0.05), of which C-ions+αPD-L1+Olaparib group CD8 + T cell infiltration was most pronounced, while CD8+ in the spleen tissue of mice in the C-ions+αPD-L1 group and the C-ions+Olaparib group was most significant. + No significant difference was observed in T cell infiltration. p >0.05). However, although C-ions irradiation could induce CD4 in mouse spleen tissue compared to the non-irradiated group, + T cell infiltration increased significantly ( p <0.05) but CD4 levels in the spleen tissue of mice were different among the C-ions group, C-ions+αPD-L1 group, C-ions+Olaparib group, and C-ions+αPD-L1+Olaparib group. + No significant difference was observed in T cell infiltration. p >0.05).

[0151] In summary, the PARP inhibitor olaparib combined with C-ions enhances the radioimmunoassay effect in osteosarcoma by activating the cGAS / STING pathway and upregulating the expression of IFN-β and PD-L1. The treatment strategy of combining the PARP inhibitor olaparib with C-ions and PD-L1 inhibitors synergistically improves the anti-tumor immunotherapy efficacy and survival in osteosarcoma.

[0152] It should be noted that when numerical ranges are mentioned in the claims of this invention, it should be understood that the two endpoints of each numerical range and any value between the two endpoints can be selected. To avoid redundancy, the present invention describes preferred embodiments.

[0153] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including both the preferred embodiments and all changes and modifications falling within the scope of the invention.

[0154] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. Application of PARP inhibitors in the preparation of drugs that enhance the radioimmunoassay effect of carbon ions in osteosarcoma.

2. The application according to claim 1, characterized in that, The drug uses the PARP inhibitor as the sole active ingredient or one of the active ingredients.

3. The application according to claim 2, characterized in that, When the drug uses the PARP inhibitor as one of its active ingredients, the active ingredients also include a PD-L1 inhibitor.

4. The application according to claim 3, characterized in that, The PARP inhibitors include olaparib.

5. A method for enhancing the radioimmunoassay effect of carbon ions in osteosarcoma cells in vitro, characterized in that, Includes the following steps: The osteosarcoma cells were treated in vitro using a PARP inhibitor, wherein the PARP inhibitor included olaparib. Carbon ion radiotherapy was administered to osteosarcoma cells that had been treated in vitro.

6. The method according to claim 5, characterized in that, When treating osteosarcoma cells in vitro, the duration of action of olaparib is 12h to 48h.

7. The method according to claim 5, characterized in that, When treating the osteosarcoma cells in vitro, the concentration of olaparib is 9.8 μM to 10.2 μM.

8. The method according to claim 5, characterized in that, The relative bioequivalent dose of the carbon ion radiotherapy is 10 Gy.

9. The method according to claim 5, characterized in that, The osteosarcoma cells are the human osteosarcoma cell line U2OS and / or the osteosarcoma cells are the mouse osteosarcoma cell line K7M2.

Images