Application of QSOX1 as a target in the elimination of dormant tumor cells

Abstract

The present invention relates to the field of pharmaceutical preparations, and in particular to the use of QSOX1 as a target in eliminating dormant tumor cells. The present invention finds that the gene of resting sulfhydryl oxidase (QSOX1) is highly expressed in dormant tumors, and QSOX1 can inhibit CD8 + The migration of T lymphocytes helps dormant tumors to achieve immune escape, which indicates that QSOX1 can be used as a new target for eliminating dormant tumors. At the same time, the present invention has confirmed in mouse models that compared with the treatment of QSOX1 inhibitor alone or the commonly used PD-1 antibody and chemotherapy drug combination, the present invention combines QSOX1 inhibitors, PD-1 antibodies and chemotherapy drugs to significantly promote CD8 + The invention provides a novel medication scheme for treating tumor dormancy, which has important clinical significance.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical preparations, and in particular to the application of QSOX1 as a target in clearing dormant tumor cells. Background Art

[0002] Dormant tumor cells are characterized by drug resistance and immune escape, and are an important source of tumor recurrence and metastasis. Due to the lack of effective means to monitor and eliminate dormant tumor cells, tumor dormancy is one of the challenges faced in clinical treatment. Therefore, there is an urgent need to find key targets for eliminating dormant tumor cells and develop medication regimens, which is crucial to solving the problems of tumor dormancy and recurrence. The combination of PD-1 antibody drugs and chemotherapy drugs has been used in the first-line treatment of cancers such as esophageal cancer, but some patients still relapse many years after being cured. This shows that the above-mentioned existing combination drug regimens cannot eliminate dormant tumor cells, and there is an urgent need to screen new drug targets for eliminating dormant tumors. Summary of the invention

[0003] The purpose of the present invention is to overcome the shortcomings of the prior art and provide the application of QSOX1 as a target for tumor dormancy treatment. QSOX1 can inhibit the migration of CD8+T lymphocytes, thereby helping dormant tumors to achieve immune escape.

[0004] To achieve the above object, the technical solution adopted by the present invention is:

[0005] In a first aspect, the present invention provides use of a substance for reducing the activity or expression of QSOX1 protein in the preparation of a drug for treating tumors.

[0006] The present invention found that the quiescent sulfhydryl oxidase (QSOX1) gene is highly expressed in dormant tumors, and QSOX1 can inhibit CD8 + The migration of T lymphocytes helps dormant tumors achieve immune escape, which suggests that QSOX1 can be used as a novel target for eliminating dormant tumors.

[0007] Preferably, the medicament comprises treating a tumor by eliminating dormant tumor cells.

[0008] Preferably, the drug promotes CD8 + T cells migrate to eliminate dormant tumor cells.

[0009] In a second aspect, the present invention provides the use of a QSOX1 inhibitor in combination with a PD-1 antibody and a chemotherapy drug in the preparation of a drug for treating tumors.

[0010] The present invention has demonstrated in a mouse model that, compared with the treatment of a single QSOX1 inhibitor or a commonly used PD-1 antibody and chemotherapeutic drug combination, the present invention combines QSOX1 inhibitors, PD-1 antibodies and chemotherapeutic drugs to significantly promote CD8 + Infiltration of T cells clears dormant tumor cells.

[0011] Preferably, the chemotherapy drug includes at least one of paclitaxel and cisplatin.

[0012] In a third aspect, the present invention provides a pharmaceutical composition comprising a QSOX1 inhibitor, a PD-1 antibody and a chemotherapeutic drug.

[0013] Preferably, the QSOX1 inhibitor is Ebselen.

[0014] Preferably, the chemotherapy drug includes at least one of paclitaxel and cisplatin.

[0015] More preferably, the chemotherapy drugs include paclitaxel and cisplatin.

[0016] Preferably, the dosage form of the pharmaceutical composition includes injection.

[0017] In a fourth aspect, the present invention provides the use of a reagent for detecting the level of QSOX1 gene or protein in the preparation of a reagent for diagnosing dormant tumors.

[0018] The present invention analyzes the expression of quiescent sulfhydryl oxidase (QSOX1) in tumor cells and finds that the expression level of QSOX1 protein is significantly higher in dormant tumors than in progressive tumors.

[0019] The beneficial effects of the present invention are:

[0020] The present invention found that the quiescent sulfhydryl oxidase (QSOX1) gene is highly expressed in dormant tumors, and QSOX1 can inhibit CD8 + The migration of T lymphocytes helps dormant tumors to achieve immune escape, which indicates that QSOX1 can be used as a new target for eliminating dormant tumors. At the same time, the present invention has confirmed in mouse models that compared with the treatment of QSOX1 inhibitor alone or the commonly used PD-1 antibody and chemotherapy drug combination, the present invention combines QSOX1 inhibitors, PD-1 antibodies and chemotherapy drugs to significantly promote CD8 + The infiltration of T cells can be used to eliminate dormant tumor cells and prevent tumor recurrence, which is expected to improve the prognosis of patients. The present invention provides a new medication regimen for treating tumor dormancy, which has important clinical significance. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1Figure 1 is a model diagram of mice with progressive tumors and dormant tumors; Figure A is a diagram of the model construction plan for progressive tumors and dormant tumors; Figure B is a photo of mice with progressive tumors or dormant tumors; Figure C is a comparison of progressive tumors and dormant tumors.

[0022] Figure 2 Figure 2 is the expression level of QSOX1 protein in progressive tumors and dormant tumors; Figure A is the fluorescence staining analysis of the expression level of QSOX1 protein in dormant and progressive tumors; Figure B is the statistical graph of the fluorescence values ​​of QSOX1 protein expressed in each group of tumor cells.

[0023] Figure 3 Recombinant protein for QSOX1 inhibits CD8 + T cell migration results; Figure A is an in vitro analysis of QSOX1 protein on CD8 + Figure 2 shows the migration ability of T cells in each treatment group. + T cell count statistics.

[0024] Figure 4 Diagram of the treatment scheme for tumor dormancy in each treatment group.

[0025] Figure 5 The graph shows the results of tumor dormancy in each treatment group.

[0026] Figure 6 Figure 1 is the fluorescence staining result of tumor dormancy in each treatment group; Figure A is the fluorescence staining analysis of tumor cells and CD8 in dormant tumors after drug treatment. + Figure 2 shows the distribution of T cells; Figure B shows the distribution of surviving tumor cells (pan-CK + ) percentage statistics; Figure C is CD8 + T cells (CD8 + ) and tumor cells (pan-CK + ) ratio statistics.

[0027] Figure 7 Figure 1 is the fluorescence staining diagram of the efficacy of each treatment group; Figure A is the fluorescence staining analysis of the proportion of proliferating and apoptotic tumor cells in dormant tumors after drug treatment; Figure B is the proliferating tumor cells (Ki67 + ) percentage statistics; Figure C shows apoptotic tumor cells (Cleaved Caspase-3 + )Percentage statistics chart. DETAILED DESCRIPTION

[0028] In order to better illustrate the purpose, technical solutions and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments.

[0029] Sources of reagents used in the present invention:

[0030] Antifade (Abcam, #ab104139); Ficoll-Paque PLUS density gradient separation solution (#17-1440-02, GE Healthcare); FITC-labeled monoclonal anti-mouse CD8a (eBioscience, #E-AB-F1104C); Carboxyfluorescein Succinimidyl Ester (CFSE, GeneCopoeia, #A001); QSOX1 recombinant protein (R&D Systems, #9257-QS-020); QSOX1 inhibitor Ebselen (TargetMol, #T0825); anti-PD-1 (BioX Cell, #BE0146); Paclitaxel (Selleck, #S1150); Cisplatin (Selleck, #S1166).

[0031] Example 1: Expression of quiescent sulfhydryl oxidase (QSOX1) in tumor cells

[0032] Model construction: The mice used in this example were normal 4-week-old male C57BL / 6 mice (purchased from Guangdong Medical Experimental Animal Center), and the cell lines used were murine esophageal cancer cells mEC2 or mEC25. The mice were subcutaneously injected with gradient numbers (0.2 × 10 6 pcs, 1×10 6 pcs, 5×10 6 The mouse was injected with a 1 mL syringe into the subcutaneous tissue on the right side of the back and the cell suspension (100 μL / mouse) was inoculated near the armpit on the upper right side. The long diameter and short diameter of the tumor were measured with a vernier caliper every other week, and the tumor volume was calculated. Calculation formula: Tumor volume = short diameter 2 × long diameter × 0.5. If the transplanted tumor volume remains unchanged for a long time (>6 months, tumor volume <50mm 3 ), it is defined as a dormant tumor. If the transplanted tumor grows in a short period of time (<1 month, tumor volume>50mm 3 ), it is defined as progressive tumor ( Figure 1 ). This tumor-level dormant animal model has been recognized by international peer experts and published in the top international academic journal PNAS (with the first inventor as the first author).

[0033] After the model was successfully constructed, the mice were killed with an overdose of CO2, the subcutaneous tumor tissue was peeled off with surgical scissors, the tumor tissue was immersed in OCT embedding agent, and slowly placed in liquid nitrogen for 20 seconds to quickly freeze the tissue into blocks, and then sliced ​​using a constant temperature box freezing slicer. After tissue sectioning, multiple immunofluorescence staining was performed to analyze the expression level of QSOX1 protein in progressive tumors and dormant tumors. Among them, green fluorescence represents tumor cells labeled with pan-CK (broad-spectrum keratin); red fluorescence represents QSOX1 protein; blue fluorescence represents cell nuclei labeled with DAPI ( Figure 2 A).

[0034] The specific staining steps are as follows: First, place the frozen sections at room temperature for half an hour, fix them in cold acetone for 10 minutes, and then dry them in a fume hood for 20 minutes. Wash the sections with PBS three times for 5 minutes each time before staining, and then microwave them with EDTA repair solution for 5 minutes. After washing with PBS, add 5% goat serum blocking solution and incubate at room temperature for 1 hour. Add the primary antibody working solution from different species, then place it in a wet box and incubate it at 37°C for half an hour. Wash with PBS three times for 5 minutes each time, add the secondary antibody working solution connected to fluorescent dyes of different wavelengths, and incubate it at 37°C in the dark for 1 hour. After washing with PBS three times, carefully remove the water droplets on the slide with a paper towel, add the antifade agent containing DAPI, gently cover it with a coverslip, and incubate it at room temperature in the dark for 10 minutes. Use a high-resolution laser confocal microscope (ZEISS, #LSM880) for observation and photography. Finally, use ImageJ software to count the fluorescence value of QSOX1 protein expression in tumor cells.

[0035] The results showed that the expression level of QSOX1 protein was significantly higher in dormant tumors compared with progressive tumors ( Figure 2 B).

[0036] Example 2: QSOX1 recombinant protein inhibits CD8 + T cell migration

[0037] Normal C57BL / 6 mice (male, 4 weeks old) were anesthetized with 2% isoflurane, and then blood was collected from the heart. Peripheral blood lymphocytes were separated using Ficoll-Paque PLUS density gradient separation medium according to the manufacturer's instructions. CD8 + T cells were sorted by CytoFLEX flow cytometer (Backman) using FITC-labeled monoclonal anti-mouse CD8a and incubated with Carboxyfluorescein Succinimidyl Ester (5 μM) in a 37°C incubator for 10 min.

[0038] On the other hand, the conditioned medium of mouse esophageal cancer cells mEC2 or mEC25 treated with different methods was used as CD8+ In vitro chemotactic inducers of T cells are:

[0039] (1) PBS control group;

[0040] (2) Treatment group 1: QSOX1 recombinant protein (10 ng / mL);

[0041] (3) Treatment group 2: QSOX1 recombinant protein (10 ng / mL) + QSOX1 inhibitor Ebselen (10 μM).

[0042] The same number (1×10 4 cells) of CD8 + T cells were added to the left well of μ-Slide Chemotaxis (ibidi, #80326), and the same volume (50 μL) of conditioned medium from each group was added to the right well. After culturing at 37°C in an incubator containing 5% CO2 for 12 hours, the migrated T cells were imaged under a fluorescence microscope (Olympus), and the migrated CD8 + T cell count ( Figure 3 A).

[0043] The results showed that compared with the PBS control group, the CD8 + T cells decreased, while the treatment group 2 with the addition of inhibitors could inhibit the activity of QSOX1 and restore CD8 + T cell migration ability Figure 3 B); This indicates that QSOX1 recombinant protein can inhibit CD8 + T cell migration.

[0044] Example 3: Combination therapy for eliminating dormant tumor cells

[0045] In this example, mEC2 cells were injected subcutaneously (1×10 6 60 days after cell injection, mice with tumors less than 3 mm in diameter were divided into groups for treatment:

[0046] (1) Control group: PBS treatment;

[0047] (2) QSOX1 inhibitor group: 10 mg / kg QSOX1 inhibitor Ebselen alone (intraperitoneal injection)

[0048] (3) PD-1 antibody + chemotherapy group: 5 mg / kg anti-PD-1 (tail vein injection), chemotherapy drugs (6 mg / kg paclitaxel and 6 mg / kg cisplatin, intraperitoneal injection)

[0049] (4) PD-1 antibody + chemotherapy + QSOX1 inhibitor group: 10 mg / kg QSOX1 inhibitor Ebselen (intraperitoneal injection), 5 mg / kg anti-PD-1 (tail vein injection), and chemotherapy drugs (6 mg / kg paclitaxel and 6 mg / kg cisplatin, intraperitoneal injection).

[0050] Each group had 4 mice; the drug was administered once every three days for a total of four times. At the same time, the control group was given the same volume of PBS. The tumor volume was measured at the same time as the drug was administered ( Figure 4 ).

[0051] The results showed that compared with the PBS control group, the tumor volume of the PD-1 antibody + chemotherapy group was smaller, and the tumor volume of the combined drug group (PD-1 antibody + chemotherapy + QSOX1 inhibitor) was even smaller. This suggests that inhibiting QSOX1 can improve the efficacy of the combination of PD-1 antibodies and chemotherapy drugs ( Figure 5 ).

[0052] Example 4: Multiple fluorescent staining confirmed that combined drug therapy can eliminate dormant tumor cells

[0053] The drug treatment was the same as in Example 3. After the drug treatment, the excess CO 2 The mice were killed, and the subcutaneous tumor tissue was removed with surgical scissors. The tumor tissue was immersed in OCT embedding agent and slowly placed in liquid nitrogen for 20 seconds to quickly freeze the tissue into blocks, and then sliced ​​using a constant temperature box freezing slicer. After tissue slicing, multiple immunofluorescence staining was performed (see Example 1 for specific staining steps). Among them, green fluorescence represents pan-CK (broad-spectrum keratin) labeled tumor cells; red fluorescence represents CD8 labeled CD8 + T cells; blue fluorescence represents the cell nucleus labeled with DAPI ( Figure 6 A) Statistical analysis of the proportion of surviving tumor cells and CD8 + The proportion of T cells.

[0054] The results showed that a large number of tumor cells survived in both the PD-1 antibody + chemotherapy group (mean survival rate = 24.0%) and the QSOX1 inhibitor group (mean survival rate = 41.0%) (pan-CK + ), while the tumor cells in the combined drug group (PD-1 antibody + chemotherapy + QSOX1 inhibitor (mean survival rate = 3.50%) were basically eliminated ( Figure 6 B). In addition, in the PBS-treated group (mean = 10.0%), the PD-1 antibody + chemotherapy group (mean cell ratio = 25.5%), and the QSOX1 inhibitor group (mean cell ratio = 44.3.0%), the CD8 + T cells (CD8 +) were relatively small, while the combination of drugs (mean cell ratio = 95.0%) could significantly increase the number of CD8 + The proportion of T cells ( Figure 6 C).

[0055] Example 5: Efficacy of combined drug composition for tumor cell proliferation and apoptosis analysis

[0056] Drug treatment was the same as in Example 3. Tumor tissue sections were subjected to multiple immunofluorescence staining (see Example 1 for specific staining steps). Among them, green fluorescence represents tumor cells labeled with pan-CK (broad-spectrum keratin); red fluorescence represents proliferating cells labeled with Ki67; purple fluorescence represents apoptotic cells labeled with Cleaved Caspase-3; blue fluorescence represents cell nuclei labeled with DAPI ( Figure 7 A). Statistical analysis of the proportion of Ki67 tumor cells and Cleaved Caspase-3 + The proportion of tumor cells.

[0057] The results showed that compared with the PD-1 antibody + chemotherapy group (mean Ki67 tumor cell percentage = 8.250%) and the QSOX1 inhibitor group (mean Ki67 tumor cell percentage = 7.750%), the combined drug group (PD-1 antibody + chemotherapy + QSOX1 inhibitor, mean Ki67 tumor cell percentage = 0.875%) had a significantly higher rate of proliferating tumor cells (Ki67 + ) ratio decreased significantly ( Figure 7 B). At the same time, the apoptotic tumor cells (Cleaved Caspase-3 + ) ratio (CleavedCaspase-3 + The mean percentage of tumor cells was 85.25%), which was significantly higher than that of the PD-1 antibody + chemotherapy group (Cleaved Caspase-3 + The mean percentage of tumor cells was 36.25%) or the QSOX1 inhibitor group (Cleaved Caspase-3 + Mean tumor cell percentage = 20.00%) ( Figure 7 C).

[0058] In summary, the present invention provides a novel therapeutic target QSOX1 for tumor dormancy and a very potential combined pharmaceutical composition (QSOX1 inhibitor + PD-1 antibody + chemotherapy) for treating tumor dormancy, which can be used to eliminate dormant tumor cells and prevent tumor recurrence, and is expected to improve patient prognosis.

[0059] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the essence and scope of the technical solution of the present invention.

Claims

1. Use of a substance that reduces the activity or expression of QSOX1 protein in the preparation of a drug for preventing tumor recurrence and metastasis, characterized in that: The substance is Ebselen; the tumor is esophageal cancer.

2. The use according to claim 1, characterized in that The drug treats tumors by eliminating dormant tumor cells.

3. The use according to claim 2, characterized in that The drug promotes CD8 + T cells migrate to eliminate dormant tumor cells.

4. Use of a QSOX1 inhibitor in combination with a PD-1 antibody and a chemotherapy drug in the preparation of a drug for preventing tumor recurrence and metastasis, characterized in that: The QSOX1 inhibitor is Ebselen; and the tumor is esophageal cancer.

5. The use according to claim 4, characterized in that The chemotherapy drug includes at least one of paclitaxel and cisplatin.

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