Application of NCP-STI combined with chemotherapeutic drugs in the preparation of drugs for treating tumors

Abstract

The application provides application of NCP-STI and a chemotherapeutic drug in combination in preparation of a drug for treating tumors, and belongs to the technical field of biological medicines.The application proposes that NCP-STI combined with gemcitabine (NCP-STI / Gem) can be used for tumor chemotherapy-immune combined treatment, realizes synchronous inhibition of in-situ tumors and remote metastasis, and has good biocompatibility.In addition, the application proposes that NCP-STI can promote Gem to enter cells, significantly enhances tumor cell apoptosis, also enhances immunogenicity of tumor cells, and effectively relieves an immunosuppressive tumor microenvironment (TME). The application further proposes that NCP-STI / Gem can significantly inhibit tumor growth and metastasis, and prolongs the survival period of a tumor-bearing organism.Meanwhile, NCP-STI / Gem does not cause obvious reduction of body weight of the organism, and has no significant influence on main organs and related blood biochemical indexes.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and in particular relates to the application of NCP-STI in combination with chemotherapy drugs in the preparation of drugs for treating tumors. Background Technology

[0002] Tumor metastasis is the process by which tumor cells detach from the primary tumor, migrate to other sites through the bloodstream or lymphatic system, form metastatic cell clones, and maintain the growth of the recurrent tumor. Metastasis is a fundamental biological characteristic of malignant tumors and one of the leading causes of treatment failure and death in the vast majority of cancer patients. Currently, treatments for tumor metastasis mainly include chemotherapy and targeted therapy. Chemotherapy primarily uses anti-tumor drugs to directly kill primary and metastatic tumor cells; however, traditional chemotherapy drugs often cause severe toxic side effects because they lack the ability to distinguish between normal and tumor cells. Targeted therapy mainly targets various stages of tumor metastasis, using specific methods to inhibit specific metastatic pathways at certain stages of metastasis, thereby suppressing tumor growth. For example, gene therapy can regulate the transcription and expression of metastasis-related genes, or small molecule inhibitors can be used to target and prevent angiogenesis at the tumor site, thereby reducing the nutrient supply to the tumor and inhibiting its growth. However, molecular targeted therapy often only inhibits one signaling pathway and cannot achieve satisfactory therapeutic effects. Due to the high heterogeneity of tumors, it may also lead to further deterioration and increased invasiveness, and even distant metastasis. Therefore, the discovery and design of safer and more effective anti-tumor drugs and treatment strategies are of great significance for inhibiting tumor metastasis, improving the treatment effect of metastatic tumors, and reducing toxic side effects.

[0003] Tumor drug sensitivity refers to the degree to which tumor cells respond to drugs. If tumor cells are highly sensitive to drugs, they will be effectively killed during drug treatment, leading to better therapeutic effects. With high drug sensitivity, patients only need to receive lower drug doses and shorter treatment cycles, effectively reducing adverse reactions and side effects. Conversely, decreased tumor cell sensitivity to drugs, also known as tumor resistance, is one of the main causes of drug treatment failure in clinical practice.

[0004] Glycobiology research has confirmed that sialic acid on the cell surface is an important target in clinical research, closely related to tumor invasion, metastasis, drug resistance, immune escape, and viral infection processes. Its content is determined by the balance between neuraminidase and sialyltransferase. However, current methods for intervening in sialic acid expression are still relatively limited, mainly due to: (1) neuraminidase cannot block the intracellular sialic acid biosynthesis pathway, and sialic acid recovers rapidly after clearance; (2) sialyltransferase inhibitors have low activity, or their molecular structure has limitations that hinder their biological application. Currently, there are no studies in this field on the combined use of sialyltransferase inhibitors with chemotherapeutic drugs to treat tumors, inhibit tumor metastasis, and improve tumor drug sensitivity. Summary of the Invention

[0005] In view of this, the object of the present invention is to provide the application of core-shell nanoparticles (NCP-STI) that deliver transition state sialyl transferase inhibitors in combination with chemotherapeutic drugs in the preparation of medicaments for treating tumors.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0007] This invention provides the application of NCP-STI in combination with chemotherapy drugs in the preparation of drugs for treating tumors.

[0008] This invention also provides the application of NCP-STI in combination with chemotherapy drugs in the preparation of drugs that inhibit tumor growth.

[0009] This invention also provides the application of NCP-STI in combination with chemotherapy drugs in the preparation of drugs that inhibit tumor metastasis.

[0010] This invention also provides the application of NCP-STI in combination with chemotherapy drugs in the preparation of drugs that promote tumor cell apoptosis.

[0011] Preferably, the chemotherapeutic agent includes a pyrimidine chemotherapeutic agent, such as gemcitabine, 5-fluorouracil, or cytarabine.

[0012] Preferably, the tumor includes melanoma, breast cancer, lung cancer, stomach cancer, or pancreatic cancer.

[0013] Preferably, the mass ratio of NCP-STI to gemcitabine is (15-100):(1-20).

[0014] Preferably, the NCP-STI and gemcitabine are administered separately.

[0015] Preferably, the NCP-STI is administered by injection, and the gemcitabine is administered by injection or orally.

[0016] The present invention also provides a combination therapy for tumors, the drug comprising NCP-STI and gemcitabine.

[0017] The beneficial effects of this invention are:

[0018] This invention is the first to propose that NCP-STI combined with gemcitabine (NCP-STI / Gem) can be used for combined chemotherapy and immunotherapy in tumors, achieving simultaneous inhibition of both primary tumors and distant metastases, with good biocompatibility. This invention is also the first to propose that NCP-STI can effectively inhibit sialic acid on condensed nucleoside transporter 1 (CNT1), promoting Gem entry into cells, causing strong DNA damage and cell cycle arrest, and significantly enhancing tumor cell apoptosis; it also effectively triggers the exposure of the immunogenic cell death (ICD) signaling molecule calreticulin (CRT) to the cell membrane and increases adenosine triphosphate (ATP) secretion, enhancing the immunogenicity of tumor cells. Furthermore, NCP-STI / Gem can increase the proportion and infiltration of CD8+ T cells, NK cells, and M1 macrophages, while decreasing the proportion of Tregs and MDSCs, effectively alleviating the immunosuppressive tumor microenvironment (TME). This invention is also the first to propose that NCP-STI / Gem can significantly inhibit tumor growth and metastasis, and prolong the survival of tumor-bearing organisms. Meanwhile, NCP-STI / Gem does not cause a significant decrease in body weight and has no significant effect on major organs and related blood biochemical indicators. Attached Figure Description

[0019] Figure 1 This describes the synthetic route for transition state sialyltransferase inhibitors.

[0020] Figure 2 The results of the in vivo distribution experiment of NCP-STI are shown, where a represents the accumulation of NCP-STI in major organs at different time points, H: heart, Li: liver, S: spleen, Lu: lung, K: kidney, T: tumor; b represents the quantitative results of the average fluorescence intensity of tumor tissue.

[0021] Figure 3 A schematic diagram of an in vivo desialylation experiment;

[0022] Figure 4 The results of flow cytometry analysis of NCP-STI inhibition of sialylation in 4T1-Luc tumor tissue are shown. In the figure, a represents the result of NCP-STI inhibition of α-2,3-sialylation in 4T1-Luc tumor tissue, and b represents the result of NCP-STI inhibition of α-2,6-sialylation in 4T1-Luc tumor tissue. n = 3, **P < 0.01, ***P < 0.001;

[0023] Figure 5The results of NCP-STI inhibition of sialylation immunofluorescence staining in 4T1-Luc tumor tissue are shown. In the figure, a represents the result of inhibition of α-2,3-sialic acid in 4T1-Luc tumor tissue, and b represents the result of inhibition of α-2,6-sialic acid in 4T1-Luc tumor tissue. Scale bar: 25 μm.

[0024] Figure 6 Flow cytometry results of apoptosis in 4T1-Luc cells after different treatments, where a is the flow cytometry atlas and b is the quantitative analysis, n=3, ****P<0.0001;

[0025] Figure 7 Flow cytometry results of apoptosis in B16-F10 cells after different treatments, where a is the flow cytometry atlas and b is the quantitative analysis, n=3, ****P<0.0001;

[0026] Figure 8 The results of 4T1-Luc cell colony formation after different treatments are shown in Figure a. Figure a shows the 4T1-Luc cell colony formation, and Figure b shows the quantitative analysis results. n = 3, ***P < 0.001.

[0027] Figure 9 Representative B16-F10 cell colony formation results after different treatments, where a is a diagram of B16-F10 cell colony formation and b is the quantitative analysis result, n=3, ***P<0.001;

[0028] Figure 10 KEGG analysis of differentially expressed genes in 4T1-Luc cells after treatment with PBS and NCP-STI / Gem;

[0029] Figure 11 Flow cytometry results of 4T1-Luc cells after different treatments, where a is the flow cytometry atlas and b is the quantitative analysis result, n=3, ****P<0.0001;

[0030] Figure 12 Immunofluorescence images and quantitative analysis results of γ-H2AX in representative 4T1-Luc cells after different treatments, where a is the immunofluorescence image of γ-H2AX and b is the quantitative analysis result. Scale bar: 25 μm, n=3, ****P<0.0001;

[0031] Figure 13 Representative 4T1-Luc cells after different treatments: comet electrophoresis images and quantitative analysis results. (a) Comet electrophoresis image, (b) Quantitative analysis results. Scale bar: 50 μm, n = 3, ****P < 0.0001.

[0032] Figure 14Immunoblot images of CNT1 and CNT1 sialylation in 4T1-Luc cells treated with PBS or NCP-STI;

[0033] Figure 15 Uptake of 4T1-Luc cells under different treatments after 2 hours of incubation with Gem, n=3, ***P<0.001;

[0034] Figure 16 The uptake of Gem by B16-F10 cells after 2 hours of incubation with different treatments was calculated. n=3, ***P<0.001;

[0035] Figure 17 A schematic diagram illustrating how NCP-STI enhances the sensitivity of tumor cells to Gem chemotherapy;

[0036] Figure 18 Dosing regimen for 4T1-Luc tumor-bearing mice;

[0037] Figure 19 To analyze the expression level of CRT on the surface of 4T1-Luc cells after different treatments by flow cytometry, n=3, ****P<0.0001;

[0038] Figure 20 Representative immunofluorescence images of CRT expression on the surface of 4T1-Luc cells from different groups. Scale bar: 20 μm;

[0039] Figure 21 Flow cytometry analysis and representative immunofluorescence images of CRT expression levels on the surface of B16-F10 cells after different treatments are shown. In the figure, a is the result of flow cytometry analysis and b is a representative immunofluorescence image. Scale bar: 20 μm, n=3, ****P<0.0001;

[0040] Figure 22 Representative immunohistochemical images of CRT expression in mouse tumor tissues after different treatments. Scale bar: 50 μm;

[0041] Figure 23 Extracellular ATP content of 4T1-Luc and B16-F10 cells after different treatments, where a represents the result of 4T1-Luc cells and b represents the result of B16-F10 cells, n=3, ****P<0.0001;

[0042] Figure 24 CD8 in 4T1-Luc tumors after different treatments + The ratio of T cells to NK cells, where a represents CD8+. + The proportion of T cells, b is the proportion of NK cells, n = 3, *P < 0.05;

[0043] Figure 25CD8 in tumor tissue after different treatments + Representative immunofluorescence images of T cell and NK cell infiltration, where a represents CD8+. + T cell results, b represents NK cell results, scale bar: 50μm;

[0044] Figure 26 The proportion of M1 macrophages in 4T1-Luc tumors after different treatments, n=3, *P<0.05;

[0045] Figure 27 The proportions of Tregs and MDSCs in 4T1-Luc tumors after different treatments are shown, where a is the proportion of Tregs and b is the proportion of MDSCs, n=3, *P<0.05, **P<0.01;

[0046] Figure 28 Dosing regimen for B16-F10 tumor-bearing mice;

[0047] Figure 29 Tumor growth curves of different groups of B16-F10 tumor-bearing mice, n=5, **P<0.01, ***P<0.001;

[0048] Figure 30 Tumor growth curves of individual B16-F10 tumor-bearing mice in different groups, n=5;

[0049] Figure 31 Photographs and weights of tumors dissected after the experiment in different groups are shown, where a is a tumor photograph and b is the weight result, n=5, **P<0.01, ***P<0.001;

[0050] Figure 32 Representative H&E and TUNEL stained images of tumors dissected after the experiment, scale bar: 50μm;

[0051] Figure 33 Survival curves of B16-F10 tumor-bearing mice after different treatments, n=5, **P<0.01;

[0052] Figure 34 The curves showing the changes in body weight of B16-F10 tumor-bearing mice after different treatments, n=5;

[0053] Figure 35 H&E staining of major organs in the Control group and NCP-STI / Gem group, scale bar: 50 μm;

[0054] Figure 36 Dosing regimen for 4T1-Luc orthotopic tumor-bearing mice;

[0055] Figure 37Tumor growth curves of different groups of 4T1-Luc tumor-bearing mice, n=5, **P<0.01, ****P<0.0001;

[0056] Figure 38 Tumor growth curves of individual 4T1-Luc tumor-bearing mice in different groups, n=5;

[0057] Figure 39 Photographs and weights of 4T1-Luc tumors dissected after the experiment in different groups are shown. a is a tumor photograph and b is weight. n=5. **P<0.01;

[0058] Figure 40 Representative H&E, Ki67 and TUNEL staining images of tumors, scale bar: 50 μm;

[0059] Figure 41 Bioluminescence images and quantitative analysis results of different groups of isolated lung tissues are shown, where a is the bioluminescence image and b is the quantitative analysis result, n=5, ****P<0.0001;

[0060] Figure 42 Representative images of Bouin's staining of lung tissue, H&E staining of lung and liver tissues after treatment, and the number of lung metastatic nodules are shown. In the image, a is the stained image and b is the number of lung metastatic nodules. n = 5. ***P < 0.001, ****P < 0.0001.

[0061] Figure 43 The curves showing the changes in body weight of 4T1-Luc tumor-bearing mice after different treatments, n=5;

[0062] Figure 44 H&E staining images of major organs in 4T1-Luc tumor-bearing mice after different treatments. Scale bar: 50 μm.

[0063] Figure 45 Blood biochemical analysis of different groups of 4T1-Luc tumor-bearing mice, n=5. Detailed Implementation

[0064] This invention provides the application of NCP-STI in combination with chemotherapy drugs in the preparation of drugs for treating tumors.

[0065] In this invention, the preparation method of NCP-STI and related descriptions are described in the invention patent application number 202211244787.7, and will not be repeated here. In this invention, the chemotherapeutic drugs preferably include pyrimidine chemotherapy drugs. Pyrimidine chemotherapy drugs have the same mechanism of action: after entering the human body, they are activated by deoxycytosine kinase and metabolized by cytosine nucleoside deaminase. The main metabolites are incorporated into DNA within cells, interfering with DNA replication, thereby inhibiting tumor growth. In this invention, the pyrimidine chemotherapy drugs preferably include gemcitabine, 5-fluorouracil, or cytarabine. This invention uses gemcitabine, a pyrimidine antitumor drug, as an example for illustration. The tumors preferably include melanoma, breast cancer, lung cancer, gastric cancer, or pancreatic cancer. The mass ratio of NCP-STI to gemcitabine is preferably (15-100):(1-20), more preferably 15:(5-20). In the combined drug therapy described in this invention, NCP-STI and gemcitabine are preferably administered separately. The preferred administration method for NCP-STI is injection, and the preferred administration method for gemcitabine is injection or oral administration. This invention does not impose any particular limitations on the order or interval of administration of NCP-STI and gemcitabine, as long as they are administered separately. This invention does not impose any particular limitations on the specific source of gemcitabine. This invention first investigated the enhancement of gemcitabine (Gem) chemosensitivity and immunogenic cell death (ICD) effects of NCP-STI in 4T1-Luc cells. Simultaneously, 4T1-Luc tumor-bearing mice were selected to examine the ability of NCP-STI / Gem to reverse the immunosuppressive tumor microenvironment. Finally, the therapeutic efficacy and biosafety of NCP-STI / Gem in B16-F10 ectopic melanoma and 4T1-Luc orthotopic breast cancer were systematically evaluated. The results showed that NCP-STI / Gem could achieve effective chemotherapy-immunotherapy, inhibit tumor growth and metastasis, and had good biocompatibility.

[0066] This invention also provides the application of NCP-STI in combination with chemotherapy drugs in the preparation of drugs that inhibit tumor growth, metastasis, or promote tumor cell apoptosis. In this invention, the NCP-STI, chemotherapy drugs, methods of combination administration, and tumor-related aspects are the same as described above and will not be repeated here.

[0067] This invention also provides a combination therapy for tumors, comprising NCP-STI and gemcitabine. In this invention, the drug uses NCP-STI and gemcitabine as the two sole active ingredients. Preferably, the drug is formulated as an oral or injectable formulation. This invention does not impose any particular limitation on the specific preparation method of the above-mentioned drug formulation; conventional methods in the art can be used.

[0068] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0069] Unless otherwise specified, the following embodiments are all conventional methods.

[0070] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0071] The experimental materials used in the following examples were sourced from the following sources: (1) DOPA, Avanti Polar Lipids, USA; (2) DiR probe and gemcitabine, MedChemExpress, USA; (3) Tetrahydrofuran, chloroform, and anhydrous ethanol, Beijing Tongguang Fine Chemical Co., Ltd.; (4) Ultrapure RNA extraction kit, Jiangsu Kangwei Century Biotechnology Co., Ltd.; (5) DOPC, DSPE-PEG 2000, and cholesterol, Shanghai Avit Pharmaceutical Technology Co., Ltd.; (6) Annexin V-FITC / PI apoptosis detection kit, Beijing Biosen Biotechnology Co., Ltd.; (7) Triton X-100, calcium chloride dihydrate, n-hexanol, and cyclohexane, Sigma-Aldrich, USA; (8) Anti-Calreticulin antibody, Anti-CNT1 antibody, and Anti-GAPDH antibody, Abcam, UK; (9) Biotinylated lectin MAL II, SNA-I and sugar-free blocking buffer CFBS, Shenzhen Xinbosheng Biotechnology Co., Ltd.; (10) 100mm cell culture dish, 6-well cell culture plate, 24-well cell culture plate, confocal glass bottom culture dish, Corning, USA; (11) DAPI staining solution, 0.5% crystal violet solution, PBS buffer solution, 4% paraformaldehyde, various fluorescently labeled secondary antibodies, Bouin's fixative, Beijing Solarbio Science & Technology Co., Ltd.; (12) RPMI-1640 medium, penicillin-streptomycin double antibody, 0.25% trypsin-EDTA, protein marker, PE-Cy7 labeled anti-mouse CD45 antibody, Pierce protein A / G magnetic beads, fetal bovine serum, Thermo Fisher Scientific, USA; (13) D-luciferin potassium salt, primary antibody dilution buffer, secondary antibody dilution buffer, serum-free blocking buffer, 5×SDS loading buffer, RIPA lysis buffer, cell cycle detection kit, comet electrophoresis kit, γ-H2AX DNA damage detection kit and enhanced ATP detection kit, Shanghai Beyotime Biotechnology Co., Ltd.; (14) APC-labeled anti-mouse CD3 antibody, FITC-labeled anti-mouse CD4 antibody, PE-labeled anti-mouse CD8 antibody, FITC-labeled anti-mouse CD49b antibody, PE-labeled anti-mouse CD80 antibody, FITC-labeled anti-mouse CD206 antibody, APC-labeled anti-mouse F4 / 80 antibody, APC-labeled anti-mouse Gr-1 antibody, PE-labeled anti-mouse CD11b antibody, Violet 450-labeled anti-mouse CD25 antibody, PE-labeled anti-mouse FOXP3 antibody, BioLegend, Inc., USA; other reagents are commercial materials.

[0072] The specific culture methods for mouse breast cancer cell line 4T1-Luc and mouse melanoma cell line B16-F10 are as follows:

[0073] The stable transfected mouse breast cancer 4T1 cell line (4T1-Luc) and the mouse melanoma cell line B16-F10 were provided by Professor Wang Yiguang's research team at the School of Pharmaceutical Sciences, Peking University. Both cell lines were cultured in 100mm cell culture dishes containing 10% FBS, 1% penicillin-streptomycin (100 U / mL penicillin, 100 μg / mL streptomycin), and 1% glutamine in RPMI 1640 medium, and incubated at 37°C, 5% CO2, and 95% relative humidity. When cell confluence reached 80%, the medium was discarded, and the cells were washed twice with PBS. The cells were then digested for 1 min with trypsin containing 0.25% EDTA, and the digestion was terminated by adding an equal volume of complete culture medium. The cell suspension was collected, centrifuged at 1000 rpm for 5 min, the supernatant was discarded, and the cells were resuspended in complete culture medium and passaged at a 1:6 ratio.

[0074] Inbred female BALB / c mice (6-8 weeks old) and C57BL / 6 mice (6-8 weeks old) were purchased from the Beijing Vital River Laboratory Animal Center and housed in the SPF laboratory animal facility. All animal experiments were conducted in accordance with international guidelines for animal experiments.

[0075] All quantitative experimental data were obtained at least three times independently and presented as mean ± standard error (Mean ± SEM). Significance analysis was performed using GraphPad Prism (8.0.1) software via unpaired two-tailed t-tests. Unless otherwise stated, P < 0.05 (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001) was considered statistically significant.

[0076] Example 1

[0077] Preparation of core-shell nanoparticles (NCP-STI) for delivering transition-state sialyl transferase inhibitors

[0078] (1) Hexanol and Triton X-100 were dispersed in cyclohexane at a concentration of 1.5 M and a concentration of 0.6 M to obtain an oil phase dispersion system;

[0079] (2) Add 50 μL of 500 mM CaCl2·2H2O to 4 mL of the above oil phase dispersion system to obtain CaCl2 microemulsion;

[0080] (3) Add 50 μL of 25 mM transition-state sialyltransferase inhibitor to 4 mL of the above oil phase dispersion system, then add 160 μL of 20 mM DOPA (dissolved in chloroform) to obtain an inhibitor microemulsion; wherein, the transition-state sialyltransferase inhibitor has the structure shown in Formula I, and the synthetic route of the transition-state sialyltransferase inhibitor is as follows. Figure 1 As shown.

[0081]

[0082] (4) Stir the CaCl2 microemulsion and the inhibitor microemulsion at room temperature for 30 minutes, and add the inhibitor microemulsion dropwise to the CaCl2 microemulsion. Stir the mixture for another 30 minutes to carry out coordination polymerization. Then add 16 mL of ethanol and stir for 20 minutes to break the emulsion and obtain the core nanoparticles.

[0083] (5) The core nanoparticles were washed with ethanol once, washed twice with chloroform / ethanol (V / V=1:1), and redispersed in 80 μL of tetrahydrofuran (THF) containing DOPC, DSPE-PEG 2000 and cholesterol (molar ratio of 2:1:1) to obtain the core-shell nanoparticle precursor.

[0084] (6) The core-shell nanoparticle precursor was added dropwise to a 500 μL ethanol / PBS system (V / V = 3:7) and stirred at 50 °C until the ethanol and THF were completely evaporated, yielding core-shell nanoparticles delivering a transition state sialyltransferase inhibitor, denoted as NCP / STI. The NCP / STI used in subsequent examples were all prepared in this example.

[0085] Example 2

[0086] Establishment of a tumor-bearing mouse model

[0087] 4T1-Luc and B16-F10 tumor cells with 80% confluence were digested with trypsin containing 0.25% EDTA, centrifuged and the supernatant was discarded. The cells were washed once with PBS, resuspended in serum-free RPMI-1640 medium, and the cell density was adjusted to 1×10⁶ cells / year. 7 100 μL of the sample was injected into the fourth pair of mammary pads (4T1-Luc) of BALB / c mice and the axilla (B16-F10) of C57BL / 6 mice.

[0088] In vivo distribution experiment of NCP-STI

[0089] To investigate the distribution of NCP-STI in mice, this invention injected DiR-labeled NCP-STI into the tail vein of 4T1-Luc tumor-bearing mice. In vivo imaging was used to evaluate the accumulation of NCP-STI in major organs and tumor tissues at different time points. Specifically, the preparation method of NCP-STI nanonuclei was the same as steps (1) to (4) in Example 1. When resuspending the nuclei in tetrahydrofuran, DiR probes were added at a dosage of 2 μg per mouse. The organic solvent was removed by solvent evaporation to obtain DiR-labeled NCP-STI.

[0090] 4T1-Luc tumor-bearing mice were randomly divided into 5 groups of 3 mice each, and DiR-labeled NCP-STI was administered via tail vein. Mice were sacrificed at 1 h, 3 h, 8 h, 24 h and 48 h after administration, and tumors and major organs were removed for fluorescence imaging. The fluorescence intensity was quantitatively analyzed using LivingImage software.

[0091] The results are as follows Figure 2 As shown, within 24 hours, the fluorescence signal of NCP-STI in mouse tumor tissue continuously increased over time, indicating that NCP-STI gradually accumulated in the tumor tissue; after 24 hours, the fluorescence signal in mouse tumor tissue decreased, indicating that the NCP-STI accumulated in the tumor tissue was gradually degraded and metabolized.

[0092] Example 3

[0093] Evaluation of NCP-STI's Inhibition of Sialization in Tumor Tissue

[0094] Given that NCP-STI can effectively accumulate at tumor sites, the ability of NCP-STI to inhibit sialylation in tumor tissue was further investigated using flow cytometry and immunofluorescence staining. The specific methods are as follows:

[0095] 4T1-Luc tumor-bearing mice (constructed using the same method as in Example 2) were randomly divided into two groups of three mice each. Figure 3The administration regimens were PBS and NCP-STI (obtained in Example 1) (15 mg / kg), administered every two days for a total of 4 times. On the 8th day after administration, the mice were euthanized by cervical dislocation, the tumor was dissected and divided in half, and one half was used to prepare tumor single cell suspensions using a DSC-400 single cell suspension preparation instrument. The expression of sialic acid on the suspensions was detected by flow cytometry. The method was as follows: (1) The tumor single cell suspensions were washed twice with CFBS, and biotinylated lectin MAL II or SNA-I diluted 1:200 was added. The suspensions were incubated at room temperature for 45 min and washed twice with CFBS. (2) Fluorescent secondary antibody diluted 1:500 was added. The suspensions were incubated at room temperature in the dark for 30 min and washed twice with CFBS. (3) The cells were resuspended in PBS containing 1% FBS, passed through a 300-mesh cell sieve, and the mean fluorescence intensity (MFI) was detected by flow cytometry. The number of cells collected for each sample was 10,000. The other half was fixed in 4% paraformaldehyde for 24 hours, and then prepared into 5μm paraffin sections for lectin immunofluorescence staining. The specific operation steps are as follows:

[0096] (1) Place the sections in xylene I and II for 10 min each, then in anhydrous ethanol for 10 min, then in 95% ethanol for 5 min, then in 90% ethanol for 5 min, then in 80% ethanol for 5 min, then in 70% ethanol for 5 min, and finally in distilled water for 5 min to dewax to water; (2) Immerse the sections in EDTA antigen retrieval solution (pH=8.0), microwave on high for 3 min, then on low for 15 min for antigen retrieval, and wash with PBS 3 times for 5 min each time; (3) Draw circles around the tissue with an immunohistochemical pen, and block with BSA at room temperature for 30 min; (4) Add diluted primary antibody, incubate at 4℃ overnight, and wash with PBS 3 times for 5 min each time; (5) Add diluted fluorescently labeled secondary antibody, incubate at room temperature in the dark for 1 h, and wash with PBS 3 times for 5 min each time; (6) Add DAPI staining solution, incubate at room temperature in the dark for 10 min, and wash with PBS 3 times for 5 min each time; (7) After the sections are dried, mount them with anti-fluorescence quenching mounting medium. Images were acquired using the Vectra Polaris fully automated quantitative pathological imaging system to analyze the expression of sialic acid in tumor tissues.

[0097] Flow cytometry results as follows Figure 4 As shown, NCP-STI can significantly inhibit the expression of α-2,3-sialic acid and α-2,6-sialic acid in 4T1-Luc tumor tissue, with an inhibition effect of more than 50%.

[0098] Immunofluorescence staining can more intuitively determine the differences in sialic acid expression in tumor tissues after different treatments, and the results are as follows: Figure 5 As shown, NCP-STI can effectively inhibit the expression of sialic acid in tumor tissue (green fluorescent signal).

[0099] Example 4

[0100] Cellular chemosensitivity evaluation

[0101] This embodiment utilizes Annexin V-FITC / PI cell apoptosis assay, colony formation assay, RNA-seq, cell cycle assay, DNA damage assay, Western blotting assay, immunoprecipitation (IP) assay, and intracellular Gem content assay to investigate the mechanism by which NCP-STI enhances the sensitivity of Gem chemotherapy.

[0102] 4.1 Apoptosis Analysis

[0103] 4T1-Luc or B16-F10 cells with 80% confluence were digested with trypsin containing 0.25% EDTA, and the supernatant was discarded by centrifugation. Tumor cells were diluted with complete culture medium and seeded into 6-well cell culture plates at 2 × 10⁶ cells per well. 5 Cells. Cell culture plates were incubated overnight at 37°C in a 5% CO2 incubator. The culture medium was discarded, and the cells were washed twice with PBS. The cells in the wells were then divided into four groups of n=3:

[0104] (1) Control group, treated with PBS for 48 hours; (2) NCP-STI group, treated with NCP-STI for 24 hours, then with PBS for 24 hours; (3) Gemcitabine (Gem) group, treated with PBS for 24 hours, then with Gem for 24 hours; (4) NCP-STI / Gem group, treated with NCP-STI for 24 hours, then with Gem for 24 hours.

[0105] In (2) and (4), the dosage of NCP-STI was the same, which was 1 mg / mL; in (3) and (4), the dosage of Gem was the same, which was 30 ng / mL.

[0106] The apoptosis levels of 4T1-Luc and B16-F10 tumor cells after different treatments were detected by flow cytometry using the Annexin V-FITC / PI apoptosis detection kit. Specifically, after treatment with different drugs, the culture medium was discarded, and the cells were washed twice with PBS, digested with trypsin containing 0.25% EDTA, and collected. Each sample was resuspended in 100 μL of 1× Binding Buffer, and 5 μL of Annexin V-FITC and 5 μL of PI staining solution were added. The mixture was gently mixed and incubated at room temperature in the dark for 15 min. Then, 400 μL of 1× Binding Buffer was added, mixed, and the cells were passed through a 300-mesh sieve. The fluorescence intensity of FITC and PI in the cells was detected by flow cytometry. 10,000 cells were collected for each sample.

[0107] The results are as follows Figure 6 and Figure 7 As shown, by Figure 6 It can be seen that over 97% of 4T1-Luc cells in the Control group, NCP-STI group, and low-dose Gem group were Annexin V / PI double-negative, indicating normal cells. However, in the mixed-dose group (NCP-STI / Gem), 19.2% of 4T1-Luc cells were Annexin V / PI double-positive, indicating late-stage apoptosis, and another 18.7% were Annexin V single-positive, indicating early-stage apoptosis. Compared to NCP-STI or Gem alone, the NCP-STI / Gem mixed-dose administration significantly enhanced 4T1-Luc cell apoptosis.

[0108] Depend on Figure 7 It can be seen that over 94% of B16-F10 cells in the Control group, NCP-STI group, and low-dose Gem group were Annexin V / PI double-negative, indicating normal cells; while in the NCP-STI / Gem group, 4.0% of B16-F10 cells were Annexin V / PI double-positive, indicating late-stage apoptosis, and another 39.3% of B16-F10 cells were Annexin V single-positive, indicating early-stage apoptosis. Unlike 4T1-Luc cells, NCP-STI / Gem mainly induced early apoptosis in B16-F10 cells, possibly because different types of tumor cells have different sensitivities to the chemotherapeutic drug Gem. These results collectively indicate that NCP-STI can effectively improve the chemosensitivity of various tumor cells to Gem.

[0109] 4.2 Cloning experiment

[0110] Colony formation assays are an effective method for examining the sensitivity of cells to drugs in terms of proliferative capacity. To further confirm the effect of NCP-STI on the sensitivity of tumor cells to Gem chemotherapy, a lower dose of Gem (10 ng / mL) was selected for colony formation assays. Specifically, 4T1-Luc or B16-F10 cells with a confluence of 80% were digested with trypsin containing 0.25% EDTA, centrifuged, and the supernatant was discarded. The tumor cells were diluted with complete culture medium and seeded into 6-well cell culture plates at 2 × 10⁶ cells per well. 3 Cells. Cell culture plates were incubated overnight at 37°C in a 5% CO2 incubator. The culture medium was discarded, and the cells were washed twice with PBS. The cells in the wells were then divided into four groups of n=3:

[0111] (1) Control group, treated with PBS for 8 days; (2) NCP-STI group, treated with NCP-STI for 1 day, then with PBS for 7 days; (3) Gem group, treated with PBS for 1 day, then with Gem for 7 days; (4) NCP-STI / Gem group, treated with NCP-STI for 1 day, then with Gem for 7 days.

[0112] In (2) and (4), the dosage of NCP-STI was the same, which was 1 mg / mL; in (3) and (4), the dosage of Gem was the same, which was 10 ng / mL.

[0113] After different drug treatments were completed, the culture medium was discarded, the cells were washed twice with PBS, fixed with 4% paraformaldehyde for 10 min, stained with 0.5% crystal violet solution for 10 min, washed thoroughly twice with PBS, photographed with a camera, and the number of cell clones formed in each group was counted using ImageJ.

[0114] The results are as follows Figure 8 and Figure 9 As shown, the results indicate that even with a reduced Gem dosage, NCP-STI can still significantly inhibit the formation of tumor cell colonies, suggesting that it can effectively improve the chemosensitivity of tumor cells to Gem. Unlike apoptosis experiments, in colony formation experiments, low-dose Gem partially inhibits the formation of colonies, mainly for two reasons: (1) fewer tumor cells are initially plated in colony formation experiments; and (2) Gem interacts with tumor cells for a longer period of time.

[0115] 4.3 RNA-seq analysis

[0116] To explore the mechanism by which NCP-STI / Gem enhances the chemosensitivity of tumor cells, RNA-seq analysis was performed on 4T1-Luc cells treated with PBS and NCP-STI / Gem. Specifically, tumor cells were treated according to the cell plating and drug administration regimens described in section 4.1. After treatment with different drugs, total RNA was extracted from the cells. The specific steps are as follows:

[0117] (1) Thoroughly remove the culture medium, add 1 mL of TRIzon reagent, mix thoroughly by pipetting, incubate at room temperature for 5 min, and transfer the sample to an RNase-free centrifuge tube; (2) Add 200 μL of TRIzon. Pal, shake vigorously for 15s, place at room temperature for 2min, centrifuge at 12000rpm for 10min; (3) transfer the upper aqueous phase to a new RNase-free centrifuge tube, add an equal volume of 70% ethanol, vortex to mix, add to the adsorption column already loaded into the collection tube, centrifuge at 12000rpm for 20s, and discard the waste liquid; (4) add 700μLLRW1 solution to the adsorption column, centrifuge at 12000rpm for 20s, and discard the waste liquid; (5) add 500μLLRW2 solution to the adsorption column, centrifuge at 12000rpm for 20s, discard the waste liquid, and repeat this step once; (6) place the adsorption column at room temperature for 2min, dry it thoroughly, place it in a new RNase-free centrifuge tube, add 50μL DEPC water to the adsorption column, place at room temperature for 1min, centrifuge at 12000rpm for 1min, and collect the RNA solution; (7) use a UV spectrophotometer to detect the RNA purity and concentration, and store at -80℃.

[0118] The extracted total RNA samples were sent to Beijing Novogene Technology Co., Ltd. for subsequent library construction and sequencing analysis.

[0119] See results Figure 10 KEGG (Kyoto Encyclopedia of Genes and Genomes) enrichment analysis showed that DNA damage repair pathways related to cell cycle, DNA replication, and homologous recombination were activated in NCP-STI / Gem-treated 4T1-Luc cells, suggesting that NCP-STI / Gem may enhance the chemosensitivity of tumor cells by inducing DNA damage.

[0120] 4.4 Cell cycle detection

[0121] To confirm the regulatory effect of NCP-STI / Gem on tumor cell cycle, the cell cycle distribution of 4T1-Luc cells after different treatments was analyzed by flow cytometry using a cell cycle assay kit. Specifically:

[0122] (1) Treat 4T1-Luc tumor cells according to the cell plating and drug administration protocol in 4.1. After drug treatment, discard the culture medium, wash twice with pre-cooled PBS, digest with trypsin containing 0.25% EDTA and collect the cells; (2) Add 1 mL of pre-cooled 70% ethanol to each sample, gently pipette to mix, and fix at 4℃ for 2 h; (3) Centrifuge to remove ethanol and wash twice with pre-cooled PBS; (4) Add 0.5 mL of PI staining solution to resuspend the cells and incubate at room temperature in the dark for 30 min; (5) Pass through a 300-mesh cell sieve and detect the fluorescence intensity of PI by flow cytometry. The number of cells collected for each sample is 10,000.

[0123] The results are as follows Figure 11 As shown, compared with the Control group, 79.74±5.54% of cells in the NCP-STI / Gem group were in the G0 / G1 phase (mitotic quiescence phase), while only 8.51±2.92% of cells were in the S phase (DNA synthesis phase). This indicates that NCP-STI / Gem can effectively arrest 4T1-Luc cells from the G0 / G1 phase to the S phase, thereby inhibiting tumor cell proliferation.

[0124] 4.5 DNA Damage Experiment

[0125] The level of cellular DNA damage after different treatments was investigated using γ-H2AX immunofluorescence staining and comet electrophoresis.

[0126] The specific steps for γ-H2AX immunofluorescence staining are as follows:

[0127] (1) Digest 4T1-Luc or B16-F10 cells with a confluence of 80% using trypsin containing 0.25% EDTA, centrifuge and discard the supernatant, then dilute the tumor cells with complete culture medium and seed them into confocal glass-bottomed culture dishes at 2 × 10⁶ cells per well. 4 (1) Treat tumor cells according to the drug administration protocol in 4.1; (2) After different drug treatments, discard the culture medium, wash twice with PBS, and fix with 4% paraformaldehyde for 10 min; (3) Treat with immunostaining blocking solution for 10 min, add diluted γ-H2AX antibody, incubate overnight at 4℃, and wash twice with PBS; (4) Add diluted fluorescent secondary antibody, incubate at room temperature in the dark for 1 h, and wash twice with PBS; (5) Add DAPI staining solution, incubate at room temperature for 10 min, wash twice with PBS, observe with laser confocal microscope and quantitatively analyze fluorescence intensity with Image J.

[0128] The specific operating procedures for comet electrophoresis experiments are as follows:

[0129] (1) Tumor cells were treated according to the cell plating and drug administration protocol in 4.1. After different drug treatments, the culture medium was discarded, the cells were washed twice with PBS, digested with trypsin containing 0.25% EDTA, and the cells were collected. The cell density was adjusted to 1×10⁻⁶. 6(1) Spread 300 μL of preheated 1% normal melting point agarose gel on a frosted glass slide, cover with a coverslip, place at 4°C for 10 min to solidify, and gently remove the coverslip; (2) Mix 100 μL of cells from step 1 with an equal volume of 0.7% low melting point agarose, take 180 μL and spread evenly on the first layer of gel, place at 4°C for 10 min to solidify; (3) Place the glass slide in a petri dish, pour in pre-cooled lysis buffer, and submerge the gel surface by at least 0.3 cm. (5) Dissolve the slide overnight at 4℃ in the dark, remove the slide and rinse it three times with ddH2O; (6) Place the slide in a horizontal electrophoresis tank, pour in enough pre-cooled electrophoresis buffer, place at room temperature for 20 min, and then electrophores at 20V / 200mA for 30 min; (7) Place the slide in a petri dish, add neutral buffer, neutralize twice at 4℃, 10 min each time; (8) Add 100μL of PI staining solution to the slide, stain at room temperature in the dark for 10 min, then rinse it three times with ddH2O, cover with a coverslip, take a picture under a fluorescence microscope and perform quantitative analysis using CASP software.

[0130] The results are as follows Figure 12 and 13 As shown, by Figure 12 It can be seen that the 4T1-Luc cells in the Control group showed almost no green fluorescence signal in the nuclei and very low γ-H2AX content; while after NCP-STI / Gem treatment, the green fluorescence signal in the nuclei was significantly enhanced, the γ-H2AX content was greatly increased, and almost all nuclei showed obvious DNA damage. Similarly, comet electrophoresis experiments also showed that, compared with the Control group, the cells in the NCP-STI / Gem group showed obvious tailing, indicating a severe level of DNA damage. Figure 13 ).

[0131] 4.6 Western Blotting Experiment

[0132] (1) Digest 4T1-Luc cells with 80% confluence using trypsin containing 0.25% EDTA, centrifuge and discard the supernatant. Dilute the tumor cells with complete culture medium and seed them into 6-well cell culture plates at 2 × 10⁶ cells per well. 5Cells. The cell culture plate was placed in a 37℃, 5% CO2 constant temperature incubator overnight, the culture medium was discarded, and the cells were washed twice with PBS. The cells in the well plate were divided into two groups according to n=3, and PBS or 1mg / mL NCP-STI were added to treat them for 2 days respectively; (2) After the drug treatment, the culture medium was discarded, the cells were washed twice with PBS, digested with trypsin containing 0.25% EDTA and the cells were collected. RIPA lysis buffer containing a mixture of protease and phosphatase inhibitors was added, and the cells were thoroughly mixed and lysed at 4℃ for 20min; (3) Centrifuged at 15000rpm for 15min at 4℃, the supernatant was collected, the protein concentration was detected by BCA method, 5×SDS loading buffer was added according to the ratio, and the cells were thoroughly mixed and boiled at 100℃ for 10min; (4) The pre-made gel was placed in the electrophoresis tank, the protein sample and protein marker were added to the sample well, and electrophoresis was performed at 80V for 30min, and then electrophoresis was performed at 110V. (5) Activate the PVDF membrane in anhydrous methanol, remove the gel and cut off the stacking gel. Place the sponge, filter paper, gel, PVDF membrane, filter paper and sponge on the black side of the transfer box in sequence. Insert the transfer box into the transfer tank in the correct polarity direction and transfer the membrane at a constant current of 200mA for 2h. (6) Take out the PVDF membrane, wash it three times with 0.1% TBST for 5min each time, place it in serum-free blocking solution and block it at room temperature for 20min. (7) Wash it three times with 0.1% TBST for 5min each time, add the diluted primary antibody solution, and incubate overnight at 4℃. (8) Wash it three times with 0.1% TBST for 5min each time, add the diluted secondary antibody solution, incubate at room temperature for 1h, and then wash it three times again with 0.1% TBST for 5min each time. Mix equal volumes of chemiluminescent solutions A and B, drop them onto the PVDF membrane, and expose and photograph it using a gel imaging system.

[0133] 4.7 IP Experiment

[0134] (1) Cells treated with different methods were lysed and centrifuged according to method 4.6 to obtain protein lysis buffer; (2) Take an appropriate amount of protein lysis buffer, add 5×SDS loading buffer, mix thoroughly, boil at 100℃ for 10 min, and keep as Input component; (3) Add the corresponding antibody to the remaining protein lysis buffer, incubate overnight at 4℃ by rotation, add an appropriate amount of protein A / G magnetic beads, and rotate at room temperature for 2 h; (4) Separate the magnetic bead complex using a magnetic separator and wash thoroughly three times with RIPA lysis buffer; (5) Add an appropriate amount of 2×SDS loading buffer, mix thoroughly, boil at 100℃ for 10 min, separate the magnetic beads using a magnetic separator, and keep the supernatant as IP component; (6) Perform Western blotting detection on the Input component and IP component together.

[0135] Intracellular transport of Gem cells is primarily mediated by condensed nucleoside transporter 1 (CNT1). Results are shown below. Figure 14 NCP-STI does not affect the expression of CNT1 protein, but it significantly inhibits its α-2,3-sialylation and α-2,6-sialylation.

[0136] 4.8 Analysis of intracellular Gem content

[0137] Numerous studies have demonstrated that alterations in glycosylation, particularly sialylation, can affect protein structure and function. To investigate whether decreased CNT1 protein sialylation affects intracellular Gem accumulation, tumor cells treated with different methods were incubated with Gem for 2 hours, and intracellular Gem content was detected using LC-MS / MS. The mechanism by which NCP-STI enhances Gem chemosensitivity was explored. The specific procedures are as follows:

[0138] (1) Accurately weigh an appropriate amount of Gem, add 0.1M HCl to prepare a stock solution of 100 μg / mL, and then serially dilute with 0.1M HCl to prepare working solutions of 1, 5, 10, 20, 50 and 100 ng / mL; (2) Establish a standard curve with Gem concentration as the abscissa (x) and peak area as the ordinate (y) using LC-MS / MS; (3) Digest 4T1-Luc and B16-F10 cells with a confluence of 80% using trypsin containing 0.25% EDTA, centrifuge and discard the supernatant. Dilute the tumor cells with complete culture medium and seed them into 6-well cell culture plates, 2 × 10⁶ cells per well. 5 (4) The cell culture plate was placed in a constant temperature incubator of 37℃ and 5% CO2 overnight. The culture medium was discarded and the cells were washed twice with PBS. The cells in the well plate were divided into two groups according to n=3. PBS or 1mg / mL NCP-STI was added to each group for 1 day, and then 30ng / mL Gem was added for 2 hours. (5) The culture medium was discarded and the cells were washed twice with PBS. The cells were digested with trypsin containing 0.25% EDTA and collected. The cells were counted and treated overnight with 0.1M HCl. Protein was precipitated with an equal volume of methanol. The intracellular Gem content was determined by LC-MS / MS and standard curve.

[0139] The results are as follows Figure 15 and Figure 16 As shown, the intracellular Gem content in both 4T1-Luc cells and B16-F10 cells was significantly higher in the NCP-STI / Gem group than in the Gem group (5.71 times and 4.69 times, respectively).

[0140] The results above indicate that NCP-STI promotes Gem entry into cells by inhibiting sialic acid on the surface of tumor cells, especially sialic acid on the CNT1 protein, causing severe DNA damage and cell cycle arrest, thereby inducing tumor cell apoptosis and enhancing the chemosensitivity of Gem (see [link to relevant documentation]). Figure 17 ).

[0141] Example 5

[0142] ICD effect analysis

[0143] Existing evidence suggests that gem cells (GEM) induce immunogenic cell death (ICD) during chemotherapy, triggering an anti-tumor immune response. This study aims to investigate whether NCP-STI, while improving gem chemosensitivity, enhances the triggering of the ICD effect.

[0144] This embodiment mainly uses flow cytometry, immunofluorescence staining, and immunohistochemistry to examine the changes in ICD effector markers after different treatments, including calreticulin (CRT) and adenosine triphosphate (ATP).

[0145] 5.1 Detection of CRT expression

[0146] CRT proteins are originally located in the endoplasmic reticulum, where they participate in the regulation of calcium ion concentration and protein folding. When tumor cells undergo ICD (intracytoplasmic Depression) effects, CRT proteins are exposed to the cell membrane, sending a "eat me" signal and triggering the body's anti-tumor immune response.

[0147] First, the expression of CRT in 4T1-Luc cells after different treatments was quantitatively analyzed by flow cytometry. The specific operation steps are as follows:

[0148] (1) Treat tumor cells according to the cell plating and drug administration regimen in Example 4.1. After different drug treatments, discard the culture medium, wash twice with PBS, digest with trypsin containing 0.25% EDTA and collect the cells; (2) Add 1:200 diluted CRT antibody, incubate at room temperature for 1 h, wash twice with PBS; (3) Add 1:500 diluted fluorescent secondary antibody, incubate at room temperature in the dark for 30 min, wash twice with PBS; (4) Add PI staining solution, pass through a 300-mesh cell sieve, and use flow cytometry to detect the fluorescence intensity of FITC in the PI negative cell population. The number of cells collected for each sample is 10,000.

[0149] The results are as follows Figure 19 As shown, NCP-STI and low-dose Gem did not increase CRT expression, but after NCP-STI / Gem treatment, CRT expression increased significantly, nearly 14 times higher than the Gem group, effectively promoting the release of "eat me" signals from tumor cells.

[0150] Subsequently, laser confocal microscopy was used to visualize the CRT exposure of 4T1-Luc cells. Compared with flow cytometry, immunofluorescence staining can more intuitively determine the changes in CRT distribution on the cell membrane after different treatments. The specific operation steps are as follows:

[0151] (1) Tumor cells with a confluence of 80% were digested with trypsin containing 0.25% EDTA, centrifuged and the supernatant was discarded. The tumor cells were then diluted with complete culture medium and seeded into confocal glass-bottomed culture dishes, 2 × 10⁶ cells per well. 4 (1) Treat tumor cells according to the drug administration regimen in Example 4.1; (2) After different drug treatments, discard the culture medium, wash twice with PBS, and fix with 4% paraformaldehyde for 10 min; (3) Treat with immunostaining blocking solution for 10 min, add diluted CRT antibody, incubate at room temperature for 1 h, and wash twice with PBS; (4) Add diluted fluorescent secondary antibody, incubate at room temperature in the dark for 30 min, and wash twice with PBS; (5) Add DAPI staining solution, wash twice again with PBS, and observe the distribution of CRT on the cell membrane (green fluorescent signal) using a laser confocal microscope.

[0152] Experimental results are as follows Figure 20 As shown in the figure, similar to the quantitative results of flow cytometry, compared with the control group, the green fluorescence signal of CRT on the cell membrane of 4T1-Luc cells was significantly increased after NCP-STI / Gem treatment, indicating that NCP-STI / Gem can effectively induce CRT to migrate from the endoplasmic reticulum to the cell membrane and trigger the ICD effect.

[0153] Similarly, the above flow cytometry and immunofluorescence staining experiments were performed on B16-F10 cells, and the results were as follows: Figure 21 As shown, flow cytometry and immunofluorescence staining also fully confirmed that NCP-STI / Gem can promote the exposure of CRT on the cell membrane.

[0154] To further investigate the differences in CRT expression in tumor tissues of tumor-bearing mice after different treatments, a 4T1-Luc tumor-bearing mouse model was established according to the method in Example 2. BALB / c mice were randomly divided into 4 groups, with 3 mice in each group. Figure 18 The mice were administered PBS, Gem, NCP-STI, and NCP-STI / Gem, respectively, with Gem at a dose of 5 mg / kg and NCP-STI at a dose of 15 mg / kg. On day 9 post-administration, mice were euthanized by cervical dislocation, tumors were dissected, fixed in 4% paraformaldehyde for 24 hours, and 5 μm paraffin sections were prepared for CRT immunohistochemical staining as follows:

[0155] (1) The sections were treated with xylene I and II for 10 min each, anhydrous ethanol for 10 min, 95% ethanol for 5 min, 90% ethanol for 5 min, 80% ethanol for 5 min, and 70% ethanol for 5 min, and washed with distilled water for 5 min to dewax to water; (2) The sections were immersed in Tris-EDTA antigen retrieval solution (pH=9.0), and heated in a microwave oven on high for 3 min and on low for 15 min for antigen retrieval. The sections were washed with PBS 3 times for 5 min each time; (3) The sections were immersed in 3% H2O2 solution and incubated at room temperature in the dark for 30 min to inactivate endogenous peroxidase. The sections were washed with PBS 3 times for 5 min each time; (4) The sections were circled around the tissue with an immunohistochemical pen, diluted primary antibody was added, and the sections were incubated at 4℃ overnight. The sections were washed with PBS 3 times. , 5 min each time; (5) Add diluted HRP-labeled secondary antibody, incubate at room temperature in the dark for 1 h, wash 3 times with PBS, 5 min each time; (6) Add DAB chromogenic solution, rinse the slide with tap water in time to stop the chromogenic process; (7) Stain with hematoxylin for 5 min, rinse with tap water, differentiate with 1% hydrochloric acid alcohol for 3 s, rinse with tap water, hematoxylin blue for 10 s, rinse with tap water; (8) Place the slide in 70% ethanol for 5 min, 80% ethanol for 5 min, 90% ethanol for 5 min, 95% ethanol for 5 min, anhydrous ethanol for 10 min, and xylene I and II for 10 min each to dehydrate and clear; (9) After the slide is dried, mount it with neutral resin; use a digital pathological slide scanner to collect images and analyze the expression of CRT in different organs and tissues.

[0156] Experimental results are as follows Figure 22 As shown, CRT distribution was low in the Control and NCP-STI groups, while CRT was highly expressed on the cell membrane in the NCP-STI / Gem group. This indicates that NCP-STI / Gem can effectively trigger ICD effects in vivo and enhance the immunogenicity of tumor cells. Based on this result, the consistency of NCP-STI / Gem triggering ICD effects in vivo and in vitro is also verified.

[0157] 5.2 ATP detection

[0158] ATP plays an indispensable role in maintaining normal cellular energy supply. When tumor cells undergo ICD (Intracellular Discharge) effects, intracellular ATP is released into the extracellular environment, sending a strong "detect me" immune signal and triggering the body's anti-tumor immunity. An enhanced ATP detection kit was used to determine the ATP content in the supernatant of tumor cells after different treatments. Tumor cells were treated according to the cell plating and drug administration regimen in Example 4.1. After different drug treatments, the cell supernatant was collected. The supernatant and an equal volume of ATP detection working solution were added to a black-background 96-well plate, and the chemiluminescence value was measured using a multifunctional microplate reader.

[0159] Experimental results are as follows Figure 23 As shown, after NCP-STI / Gem treatment, 4T1-Luc and B16-F10 cells secreted significantly more extracellular ATP than the Control group, NCP-STI group, and low-dose Gem group, indicating that NCP-STI / Gem can effectively promote the release of ATP from tumor cells, trigger the ICD effect, and thus enhance the immunogenicity of tumor cells.

[0160] Example 6

[0161] Tumor tissue immune cell subset analysis

[0162] Given that the inhibition of sialic acid on tumor cells can disrupt its interaction with Siglecs on immune cells, thereby breaking the immunosuppressive tumor microenvironment (TME), and that NCP-STI / Gem can effectively induce tumor ICD effects, we used flow cytometry combined with immunofluorescence to analyze the immune cell subsets and infiltration in 4T1-Luc tumor tissue.

[0163] 4T1-Luc tumor-bearing mice were randomly divided into 4 groups of 3 mice each. Figure 18 The mice were administered PBS, Gem, NCP-STI, and NCP-STI / Gem, respectively, with Gem at a dose of 5 mg / kg and NCP-STI at a dose of 15 mg / kg. On day 9 post-administration, the mice were euthanized by cervical dislocation, and the tumors were dissected and bisected.

[0164] Half of the tumor cells were prepared into single-cell suspensions using a DSC-400 single-cell suspension preparation instrument and divided into 5 groups: (1) APC-labeled anti-mouse CD3 antibody, PE-labeled anti-mouse CD8 antibody and PE-Cy7-labeled anti-mouse CD45 antibody were added at a ratio of 1:200 to detect CD8 in tumor tissue. +(2) NK cells were detected in tumor tissue by adding APC-labeled anti-mouse CD3 antibody and FITC-labeled anti-mouse CD49b antibody at a dilution of 1:200; (3) Treg cells were detected in tumor tissue by adding FITC-labeled anti-mouse CD4 antibody, Violet 450-labeled anti-mouse CD25 antibody and PE-labeled anti-mouse FOXP3 antibody at a dilution of 1:200; (4) MDSC cells were detected in tumor tissue by adding APC-labeled anti-mouse Gr-1 antibody and PE-labeled anti-mouse CD11b antibody at a dilution of 1:200; (5) M1 macrophages were detected in tumor tissue by adding PE-labeled anti-mouse CD80 antibody, FITC-labeled anti-mouse CD206 antibody and APC-labeled anti-mouse F4 / 80 antibody at a dilution of 1:200, and blank tubes and single-labeled sample tubes were set up at the same time. Incubate on ice in the dark for 30 minutes, wash twice with PBS, and finally resuspend the cells in PBS containing 1% FBS. After passing through a 300-mesh cell sieve, perform flow cytometry analysis.

[0165] The other half of the tumor was fixed in 4% paraformaldehyde for 24 hours, and 5μm paraffin sections were prepared. The tumor tissue sections were then subjected to CD8 and CD49b immunofluorescence staining according to the method in Example 3. Subsequently, CD8 in the tumor tissue was observed using the Vectra Polaris fully automated quantitative pathological imaging system. + The infiltration of T cells and NK cells.

[0166] In TME, CD8 + T cells and NK cells are the main effector cells that kill tumor cells, therefore CD8 + The expansion of T cells and NK cells is an important indicator of the body's ability to produce an effective anti-tumor immune response. For example... Figure 24 As shown, flow cytometry revealed that, compared to the Control group, the NCP-STI / Gem group had significantly higher levels of CD8+. + The proportions of T cells and NK cells increased by 3.96-fold and 2.24-fold, respectively, which to some extent reflects that NCP-STI / Gem can promote CD8 cell growth. + Infiltration of T cells and NK cells in tumor tissue.

[0167] Immunofluorescence staining was performed on tumor tissues from each group of mice, and CD8+ was visualized and analyzed after different treatments. + Changes in the ratio of T cells to NK cells. For example... Figure 25 As shown, CD8 in the NCP-STI / Gem group + The number and infiltration of T cells and NK cells were significantly higher in the control group, NCP-STI group, and Gem group than in the control group, further demonstrating that NCP-STI / Gem can promote CD8. +Infiltration of T cells and NK cells in tumor tissue.

[0168] TAM (tumor endothelial cells) is an important component of TME (tumor endothelial cells), playing an indispensable role in tumor cell proliferation, metastasis, drug resistance, and angiogenesis. Based on different macrophage polarization types, they can be divided into M1 and M2 types, with M1 macrophages generally considered to be tumor-killing macrophages, playing an anti-tumor and immune-boosting role. Figure 26 As shown, compared with the Control group, the proportion of M1 macrophages in the tumor tissue of the NCP-STI / Gem group was significantly increased, indicating that NCP-STI / Gem can effectively stimulate TAM to differentiate into M1 macrophages and exert an adaptive immune response against tumors.

[0169] In immunosuppressive tumor microenvironments (TMEs), regulatory T cells (Tregs) and myeloid suppressor cells (MDSCs) are key cellular subsets involved in tumor immune escape. Flow cytometry was used to detect the proportions of Tregs and MDSCs in tumor tissues; the experimental results are as follows: Figure 27 As shown in the figure, compared with the Control group, the proportions of Tregs and MDSCs in the NCP-STI / Gem group decreased by 3.16 times and 2.09 times, respectively, effectively alleviating the immunosuppressive TME. In summary, NCP-STI / Gem can create an immune-active TME, shifting the homeostatic balance of immunosuppression and immunopromotion towards a direction favorable to tumor immunotherapy.

[0170] Example 7

[0171] Efficacy evaluation of drugs for B16-F10 ectopic melanoma

[0172] 7.1 Pharmacodynamic evaluation of ectopic tumor growth

[0173] A B16-F10 ectopic melanoma model was established according to the method in Example 2. C57BL / 6 mice were randomly divided into 4 groups, with 5 mice in each group. Figure 28 The mice were administered PBS, Gem, NCP-STI, and NCP-STI / Gem, respectively, with Gem at a dose of 20 mg / kg and NCP-STI at a dose of 15 mg / kg. Starting from the first administration, the long axis (L) and short axis (W) of the mouse tumors were measured every other day using electronic calipers. The tumor volume (V) was calculated using the following formula, and a tumor growth curve was plotted.

[0174] V = 0.5 × L × W 2

[0175] The results are as follows Figure 29 and Figure 30As shown in the figure, compared to the Control group, both the NCP-STI and Gem administration groups alone showed certain tumor-suppressing effects. This is because they each have certain regulatory effects on the tumor microenvironment and tumor tissue. NCP-STI can inhibit sialic acid on the surface of tumor cells, disrupt the interaction between sialic acid and Siglecs, promote the activation of TME, and delay tumor growth. Gem has a certain cytotoxic effect on tumor cells and can trigger the ICD effect, resulting in a slight inhibition of tumor volume. Combining the two to form the NCP-STI / Gem group significantly inhibits tumor growth by disrupting the Siglecs-sialic acid immune axis, enhancing the sensitivity of Gem chemotherapy, and effectively triggering the ICD effect, achieving a "1+1>2" effect.

[0176] On day 12 after drug administration, mice in each group were sacrificed by cervical dislocation, and tumors and major organs (heart, liver, spleen, lung, and kidney) were removed. The tumor tissue was photographed and weighed, and the results are as follows: Figure 31 As shown in the figure, compared with other groups, the NCP-STI / Gem group showed a significant reduction in tumor weight, consistent with the tumor growth curve results, demonstrating a good efficacy of chemotherapy-immunotherapy combination therapy.

[0177] After photographing and weighing the tumor tissue, it was fixed in 4% paraformaldehyde for 24 hours to prepare 5μm paraffin sections. The paraffin sections were then stained with H&E as follows: (1) The sections were treated with xylene I and II for 10 min each, anhydrous ethanol for 10 min, 95% ethanol for 5 min, 90% ethanol for 5 min, 80% ethanol for 5 min, 70% ethanol for 5 min, and then washed with distilled water for 5 min to dewax to water; (2) The sections were stained with hematoxylin for 10 min and then rinsed with distilled water; (3) The sections were stained with eosin for 3 min and then rinsed with distilled water; (4) The sections were treated with 70% ethanol for 5 min, 80% ethanol for 5 min, 90% ethanol for 5 min, 95% ethanol for 5 min, anhydrous ethanol for 10 min, and xylene I and II for 10 min each to dehydrate and clear; (5) After the sections were dried, they were mounted with neutral resin. Images were acquired using a digital pathological slide scanner, and the pathological condition was analyzed.

[0178] Simultaneously, TUNEL immunohistochemical staining was performed on the slides. The specific steps are as follows:

[0179] (1) The sections were sequentially treated with xylene I and II for 10 min each, anhydrous ethanol for 10 min, 95% ethanol for 5 min, 90% ethanol for 5 min, 80% ethanol for 5 min, and 70% ethanol for 5 min, and washed with distilled water for 5 min to dewax to water; (2) The sections were circled around the tissue with an immunohistochemical pen, proteinase K was added, and the sections were incubated at 37°C for 20 min for antigen retrieval. The sections were washed with PBS 3 times, 5 min each time; (3) 0.5% Triton was added. X-100 solution, incubate at room temperature for 5 min to break the membrane, wash with PBS 3 times, 5 min each time; (4) Immerse the slice in a solution containing 3% H2O2, incubate at room temperature in the dark for 30 min to inactivate endogenous peroxidase, wash with PBS 3 times, 5 min each time; (5) Add TUNEL detection solution, incubate at 37℃ for 1 h, wash with PBS 3 times, 5 min each time; (6) Add Streptavidin-HRP reaction solution, incubate at 37℃ for 30 min, wash with PBS 3 times, 5 min each time; (7) Add (8) Add DAB staining solution and rinse the slides with tap water to stop the staining process; (9) Stain with hematoxylin for 5 min, rinse with tap water, differentiate with 1% hydrochloric acid alcohol for 3 s, rinse with tap water, hematoxylin blue for 10 s, rinse with tap water; (10) Soak the slides in 70% ethanol for 5 min, 80% ethanol for 5 min, 90% ethanol for 5 min, 95% ethanol for 5 min, anhydrous ethanol for 10 min, and xylene I and II for 10 min each to dehydrate and clear the slides; (11) After the slides are dried, mount them with neutral resin. Use a digital pathological slide scanner to acquire images and analyze tumor cell apoptosis.

[0180] The results are as follows Figure 32 As shown in the figure, compared with the control group, the NCP-STI / Gem group showed extensive apoptosis, significant DNA damage, enlarged intercellular spaces, nuclear pyknosis, and strongly eosinophilic cytoplasm in tumor tissue. Pharmacodynamic experiments collectively demonstrate that NCP-STI / Gem can effectively induce apoptosis and necrosis in tumor tissue, thereby significantly inhibiting tumor growth.

[0181] 7.2 Mouse survival analysis

[0182] The B16-F10 ectopic melanoma model was grouped and administered the same as in 7.1. The major and minor diameters of the mouse tumors were measured using electronic calipers, and the tumor volume was calculated. Once the tumor volume exceeded 1500 mm... 3 This means that the mouse is considered dead.

[0183] Survival results of different groups of B16-F10 tumor-bearing mice are as follows: Figure 33As shown, all mice in the Control group died on day 21 after tumor implantation; mice in the Gem and NCP-STI groups began to die on day 20 after tumor implantation, and all mice in both groups died by day 25; all mice in the NCP-STI / Gem group survived on day 20 after tumor implantation, and 80% of the mice were still alive by day 25, indicating that NCP-STI / Gem can significantly prolong the survival of B16-F10 tumor-bearing mice.

[0184] 7.3 In vivo safety analysis

[0185] Starting with the first administration on July 1, the body weight of mice in each group was measured using an electronic balance the following day, and a curve of body weight change was plotted.

[0186] Changes in body weight of tumor-bearing mice during the experiment are as follows Figure 34 As shown, no significant decrease in body weight was observed in any of the mouse groups, indicating that the combined administration of NCP-STI / Gem has good biocompatibility.

[0187] Major organs fixed in 4% paraformaldehyde for 24 hours according to 7.1 were prepared into 5μm paraffin sections. The paraffin sections were stained with H&E according to the method in 7.1, and the fine morphological structure of the tissue was observed by digital tissue scanner.

[0188] H&E staining results of major organs in mice of the Control group and NCP-STI / Gem group are as follows: Figure 35 As shown, after treatment with NCP-STI / Gem, no obvious inflammation or microstructural damage was observed in the heart, liver, spleen, lungs, and kidneys of tumor-bearing mice, further demonstrating that NCP-STI / Gem has good biocompatibility.

[0189] Example 8

[0190] 4T1-Luc in situ breast cancer efficacy evaluation

[0191] The high metastasis rate of malignant tumors exacerbates the difficulty of clinical cancer treatment and is one of the main reasons for poor patient prognosis and even death. Triple-negative breast cancer in mice is highly prone to metastasis in its late stages, accelerating the mortality process. Based on this, a 4T1-Luc orthotopic breast cancer model was constructed to investigate the inhibitory effect of NCP-STI / Gem on orthotopic tumor growth and metastasis.

[0192] 8.1 Pharmacodynamic evaluation of in situ tumor growth

[0193] A 4T1-Luc orthotopic breast cancer model was established according to the method in Example 2. BALB / c mice were randomly divided into 4 groups, with 5 mice in each group. Figure 36The dosing regimens included PBS, Gem, NCP-STI, and NCP-STI / Gem, with Gem administered at a dose of 5 mg / kg and NCP-STI at a dose of 15 mg / kg. Starting with the first dose, tumor volume was measured every other day using electronic calipers, and tumor growth curves were plotted.

[0194] The results are as follows Figure 37 As shown. Unlike B16-F10 tumors, NCP-STI showed negligible inhibitory effects on the growth of 4T1-Luc in situ tumors. This may be because 4T1-Luc tumors exhibit a higher degree of immunosuppression, and the activation of the immune microenvironment caused by simple sialic acid blockade is insufficient to inhibit tumor growth. The Gem group showed moderate tumor inhibition, with an average tumor volume of 1000 mm² at 18 days. 3 Approximately 18 days after tumor implantation, the tumor volume in the NCP-STI / Gem group mice was 480 mmHg. 3 The results were significantly smaller than those in the Control and Gem groups, indicating that NCP-STI / Gem effectively inhibited the growth of breast cancer in mice, which is attributed to the potent combination of chemotherapy and immunotherapy.

[0195] according to Figure 38 It can be seen that the trends reflected by the tumor growth curves of individual mice in different groups are basically consistent with the average tumor growth trends of each group. On day 18 after tumor implantation, the maximum tumor volume in the Control group mice reached 2000 mmHg. 3 The smallest is approximately 1350mm. 3 The NCP-STI / Gem group showed good tumor suppression, with tumor volume in mice within the group all being less than 650 mm². 3 .

[0196] On day 18 after drug administration, mice in each group were sacrificed by cervical dislocation, and tumors and major organs (heart, liver, spleen, lung, and kidney) were removed. The tumor tissue was photographed and weighed, and the results are as follows: Figure 39 As shown, the tumor size and weight were significantly reduced in the NCP-STI / Gem group, consistent with the results of the tumor growth curve.

[0197] After photographing and weighing the tumor tissue, it was fixed in 4% paraformaldehyde for 24 hours to prepare 5μm paraffin sections. The paraffin sections were then stained with H&E according to method 3.2.4, and Ki67 immunohistochemically stained according to method 5.1 in Example 5. Simultaneously, the sections were stained with TUNEL immunofluorescence. The specific operating steps are as follows:

[0198] (1) The sections were sequentially treated with xylene I and II for 10 min each, anhydrous ethanol for 10 min, 95% ethanol for 5 min, 90% ethanol for 5 min, 80% ethanol for 5 min, and 70% ethanol for 5 min, and washed with distilled water for 5 min to dewax to water; (2) The sections were circled around the tissue with an immunohistochemical pen, proteinase K was added, and the sections were incubated at 37°C for 20 min for antigen retrieval. The sections were washed with PBS 3 times, 5 min each time; (3) 0.5% Triton was added. (3) Incubate X-100 solution at room temperature for 5 min to break the membrane, wash with PBS 3 times, 5 min each time; (4) Immerse the sections in a solution containing 3% H2O2 at room temperature in the dark for 30 min to inactivate endogenous peroxidase, wash with PBS 3 times, 5 min each time; (5) Add TUNEL detection solution, incubate at 37℃ for 1 h, wash with PBS 3 times, 5 min each time; (6) Add DAPI staining solution, incubate at room temperature in the dark for 10 min, wash with PBS 3 times, 5 min each time; (7) After the sections are dried, mount them with anti-fluorescence quenching mounting medium. Images were acquired using the VectraPolaris fully automated quantitative pathological imaging system to analyze tumor cell apoptosis.

[0199] The results are as follows Figure 40 As shown, NCP-STI / Gem can effectively induce apoptosis and necrosis in tumor tissue and inhibit the proliferation of tumor cells.

[0200] 8.2 Pharmacodynamic evaluation of tumor metastasis

[0201] Lung tissues extracted in step 8.1 were immersed in a 15 mg / mL D-luciferin potassium solution. After 10 minutes, the bioluminescence of the lung tissues in each group of mice was detected using the IVISSpectrum small animal in vivo optical three-dimensional imaging system, and the bioluminescence intensity was quantitatively analyzed using Living Image software. The results are as follows: Figure 41 As shown. It is worth noting that although NCP-STI cannot inhibit the growth of tumors in situ, it can effectively inhibit the occurrence of lung metastasis. Gem can moderately inhibit the growth of tumors in situ through cytotoxic effects, but it is difficult to interfere with the colonization of circulating tumor cells in lung tissue, thus its ability to inhibit lung metastasis is weak. When the two are used together to form the NCP-STI / Gem group, the bioluminescent signal in the lungs is very low, and there is almost no obvious occurrence of lung metastasis.

[0202] Lung tissue was stained with Bouin's fixative, and the number of metastatic nodules was counted. At the same time, 5 μm paraffin sections were prepared, and H&E staining was performed on the paraffin sections according to the method in 7.1 of Example 7 to analyze the lung metastasis of tumors in different groups.

[0203] The results are as follows Figure 42As shown, isolated lung tissue was fixed and stained using Bouin's fixative, and the number of metastatic nodules in the upper lung was counted. It was found that the Control group had approximately 40 metastatic nodules, while the NCP-STI / Gem group had only about 3. Given that triple-negative breast cancer can also metastasize to the liver in advanced stages, further H&E staining of lung and liver tissue sections revealed obvious metastatic lesions in the Control and Gem groups; however, the NCP-STI / Gem group showed almost no metastatic lesions, and the lung and liver tissues exhibited normal morphology. These results collectively indicate that NCP-STI / Gem has a good tumor metastasis inhibitory effect.

[0204] 8.3 In vivo safety analysis

[0205] Starting with the first dose in 8.1, the body weight of mice in each group was measured using an electronic balance the following day, and a curve of body weight change was plotted. For example... Figure 43 As shown, during multiple administrations, the body weight of mice in each group did not decrease significantly, indicating that NCP-STI / Gem has good biocompatibility.

[0206] Major organs fixed in 4% paraformaldehyde for 24 hours were prepared into 5 μm paraffin sections. These sections were then stained with H&E according to the method described in 7.1 of Example 7, and the fine morphological structure of the tissues was observed using a digital tissue scanner. The results are as follows: Figure 44 As shown, after treatment with NCP-STI / Gem, no inflammation or microstructural damage was observed in the H&E staining results of major organs in tumor-bearing mice, indicating that NCP-STI / Gem is non-toxic to major organs in vivo.

[0207] In the pharmacodynamic evaluation experiment of in situ tumor growth (8.1), 50 μL of blood was collected from the orbital cavity of mice before sacrifice, and various blood biochemical indicators were detected using a fully automated biochemical analyzer. The blood biochemical results are as follows: Figure 45 As shown, there were no significant differences in relevant indicators such as alanine aminotransferase (ALT), albumin (ALB), total protein (TP), creatinine (CREA), uric acid (UA), creatine kinase (CK), and lactate dehydrogenase (LDH) among mice in different drug administration groups, indicating that NCP-STI / Gem has good biocompatibility.

[0208] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. The application of NCP-STI in combination with chemotherapy drugs in the preparation of drugs for treating tumors, characterized in that, The chemotherapy drug includes a pyrimidine chemotherapy drug, wherein the pyrimidine chemotherapy drug is gemcitabine; the tumor is melanoma or breast cancer; the mass ratio of NCP-STI to gemcitabine is (15-100):(1-20).

2. The application of NCP-STI in combination with chemotherapy drugs in the preparation of drugs to inhibit tumor growth, characterized in that, The chemotherapy drug includes a pyrimidine chemotherapy drug, wherein the pyrimidine chemotherapy drug is gemcitabine; the tumor is melanoma or breast cancer; the mass ratio of NCP-STI to gemcitabine is (15-100):(1-20).

3. The application of NCP-STI in combination with chemotherapy drugs in the preparation of drugs to inhibit tumor metastasis, characterized in that... The chemotherapy drug includes a pyrimidine chemotherapy drug, wherein the pyrimidine chemotherapy drug is gemcitabine; the tumor is melanoma or breast cancer; the mass ratio of NCP-STI to gemcitabine is (15-100):(1-20).

4. The application of NCP-STI in combination with chemotherapy drugs in the preparation of drugs that promote tumor cell apoptosis, characterized in that, The chemotherapy drug includes a pyrimidine chemotherapy drug, wherein the pyrimidine chemotherapy drug is gemcitabine; the tumor is melanoma or breast cancer; the mass ratio of NCP-STI to gemcitabine is (15-100):(1-20).

5. The application according to any one of claims 1 to 4, characterized in that, The NCP-STI and gemcitabine were administered separately.

6. The application according to claim 5, characterized in that, The NCP-STI is administered by injection, and the gemcitabine is administered by injection or orally.

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