Anti-IL-16 polypeptide and application thereof in prevention of postoperative tumor metastasis of gastric cancer

Abstract

The invention discloses an anti-interleukin-16 (IL-16) polypeptide and application thereof in prevention of postoperative tumor metastasis of gastric cancer, and belongs to the field of biomedicine.The anti-IL-16 polypeptide has the effect of inhibiting IL-16 and can inhibit neutrophil extracellular traps (NETs) generated by polymorphic cells MDSCs (PMN-MDSCs), and then tumor metastasis of patients with gastric cancer is effectively prevented.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and specifically relates to an anti-IL-16 polypeptide and an application thereof in preventing postoperative tumor metastasis of gastric cancer. Background Art

[0002] my country is one of the countries with a high incidence of gastric cancer (GC). Although great progress has been made in GC surgical techniques, perioperative care, and postoperative chemoradiotherapy in recent years, the postoperative recurrence and metastasis rates of GC remain high, seriously shortening the expected survival time of patients after surgery. Finding new key factors and mechanisms that affect GC recurrence and metastasis, thereby providing targeted targets for the clinical treatment of GC, is of great research value.

[0003] In recent years, with the continuous deepening of the study of tumor microenvironment, myeloid-derived suppressor cells (MDSCs) have gradually entered the field of vision of scholars due to their key role in immunosuppression and have become an important research hotspot. MDSCs are heterogeneous myeloid-derived cells composed of myeloid progenitor cells and immature macrophages, immature granulocytes and immature dendritic cells, which play a double-edged sword role in different diseases. Studies have reported that with the clearance of tumors, the body's own immune function has recovered, and MDSCs have shown a downward trend 21 days after rectal cancer surgery; when patients have recurrence and metastasis, tumor cells in the body proliferate again, which may secrete a large number of tumor-derived cytokines, so that immature MDSCs precursor cells are recruited from the bone marrow to the peripheral blood, tumor lesions and lymphoid organs, and then after being activated in the periphery, they proliferate, differentiate and mature in large quantities.

[0004] Interleukin-16 (IL-16) is a multifunctional cytokine that is mainly expressed by CD8 + Produced by T cells, natural killer cells (NK cells) and monocytes. IL-16 plays an important role in the pathological process of diseases such as immune response and inflammation. Studies have reported that the serum IL-16 level in GC samples is significantly higher than that in the healthy control group, and the increased serum IL-16 level is significantly associated with tumor recurrence and poor prognosis. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide an anti-IL-16 polypeptide and its application in preventing postoperative tumor metastasis of gastric cancer.

[0006] The technical solution of the present invention is: an anti-IL-16 polypeptide, whose amino acid sequence is RRKSLQ (shown in SEQ ID No. 1). R is Arg (arginine), K is Lys (lysine), S is Ser (serine), L is Leu (leucine), and Q is Gln (glutamine)

[0007] Use of the anti-IL-16 polypeptide described above in the preparation of a drug for preventing or inhibiting postoperative tumor metastasis of gastric cancer.

[0008] Furthermore, the drug dosage form is an injection.

[0009] A drug for preventing or inhibiting postoperative metastasis of gastric cancer, comprising the anti-IL-16 polypeptide described above.

[0010] Furthermore, the drug also includes a pharmaceutically acceptable carrier.

[0011] Furthermore, the drug dosage form is an injection.

[0012] 1. For the first time, clinical samples and animal models were combined to clarify the expression level of IL-16 before gastric cancer surgery.

[0013] 2. Through in vitro experiments, the mechanism of IL-16 on the production of NETs by PMN-MDSCs after surgery was elucidated in detail.

[0014] 3. For the first time, the mouse surgical tumor removal experiment was conducted, and the IL-16 inhibitory peptide was used to treat the mice, to clarify the effect of IL-16 on postoperative tumor metastasis at the individual level. When the active ingredient of the anti-tumor metastasis drug is the anti-IL-16 peptide, the dose of the anti-tumor metastasis peptide is 100 mg / kg.

[0015] 4. Combining clinical sample verification, animal model experiments and in vitro cell-level mechanism research, the mechanism of postoperative IL-16 regulation on PMN-MDSCs is explained in detail from various levels, providing new targets and ideas for inhibiting the production of NETs and preventing postoperative tumor metastasis.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] The present invention provides a polypeptide for preventing tumor metastasis in gastric cancer patients, which has the function of inhibiting IL-16 and can inhibit polymorphonuclear MDSCs (PMN-MDSCs) from producing neutrophil extracellular traps (NETs), thereby effectively preventing tumor metastasis in gastric cancer patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The cytokine array of the mouse surgical tumor removal model in Example 1 of the present application;

[0019] Figure 2 The content of IL-16 in the serum of the mouse surgical tumor removal model in Example 1 of the present application;

[0020] Figure 3 This is the fluorescent staining of MDSCs after IL-16 stimulates bone marrow cells in Example 1 of the present application;

[0021] Figure 4 The ratio of PMN-MDSCs after IL-16 stimulation of bone marrow cells was detected by flow cytometry in Example 1 of the present application;

[0022] Figure 5 Immunofluorescence staining of NETs marker (MPO) of mouse PMN-MDSCs after conditional treatment in Example 1 of the present application;

[0023] Figure 6 The content of IL-16 in the culture supernatant of PMN-MDSCs of mice after the conditional treatment in Example 1 of the present application;

[0024] Figure 7 Immunofluorescence staining of the NETs marker (MPO) of PMN-MDSCs of gastric cancer patients in Example 1 of the present application;

[0025] Figure 8 The ratio of PMN-MDSCs in the peripheral blood of the postoperative metastasis model of mice detected by flow cytometry in Example 2 of the present application;

[0026] Fig. 9 The solid image of liver nodules, liver tissue HE staining, and Vimentin histochemical staining of the mouse postoperative liver metastasis model in Example 2 of the present application;

[0027] Fig.10 This is a small animal imaging bioluminescence image of the mouse postoperative abdominal metastasis model in Example 2 of the present application. DETAILED DESCRIPTION

[0028] The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples are purchased from commercial channels unless otherwise specified.

[0029] Example 1

[0030] In order to understand the key role of IL-16 in surgically stimulated tumor metastasis, clarify its molecular mechanism of promoting PMN-MDSCs to form NETs and preventing postoperative tumor metastasis. The specific steps are as follows:

[0031] 1. Construction of mouse surgical model

[0032] In vivo experiments were conducted using 6-8 week old female 615 mice, and all animal care and experimental protocols were approved by the Research Ethics Committee of Nankai University (License No.: 2022-SYDWLL-000194). A subcutaneous tumor-bearing mouse model was established by inoculating MFC cells, and tumor removal surgery was performed under a sterile environment. The time point of blood collection in gastric cancer patients was simulated, and the patients were divided into a sham operation group (Sham) and a three-day postoperative group (POD 3). Peripheral blood and serum were collected from the sham operation group and the three-day postoperative group.

[0033] 2. Detection of IL-16 content in serum

[0034] Cytokine array experiments were performed on the serum of the above animal models to search for cytokines that change during gastric cancer surgery. The results showed that compared with the Sham group, the IL-16 level in the POD 3 group was significantly upregulated, such as Figure 1 As shown in (* indicates p<0.05; ** indicates p<0.01; ns indicates no significant difference). The mouse interleukin 16 (IL16) enzyme-linked immunosorbent assay kit (ELK5028) was used to detect the level of IL-16 in the serum of the animal model. Compared with the Sham group, the IL-16 level in the serum of the POD 3 group was significantly increased. The results are shown in Figure 2 As shown (* indicates p < 0.05; ** indicates p < 0.01; ns indicates no significant difference).

[0035] 3. How does IL-16 affect the number of PMN-MDSCs

[0036] Preparation of mouse bone marrow cell suspension: Isolation of mouse tibia: remove the mouse tibia with scissors, and carefully peel off the surface muscle with forceps; soak the tibia in PBS (if sterile treatment is required, soak it in PBS containing 1% PS for 3 minutes and then transfer it to sterile PBS), cut the end of the tibia with surgical scissors, then use a 1mL syringe to absorb an appropriate amount of PBS and gently insert the syringe needle into the bone marrow cavity to flush out the cells, and repeatedly flush several times until all the cells in the bone marrow cavity are flushed out; the flushed cells are filtered with a 70μm nylon cell filter to obtain a cell suspension, and centrifuged at 3000rmp for 5min; the supernatant is discarded, the bottom of the tube is flicked, 1mL of red blood cell lysis buffer is added, and it is placed at room temperature for 1min, and then 5mL of RPMI 1640 culture medium containing 10% FBS is immediately added to the test tube to stop the lysis, centrifuged at 3000rmp for 5min, the supernatant is discarded, and the mouse bone marrow cell suspension is obtained after resuspending with PBS or culture medium. The isolated bone marrow cells were stimulated with IL-16 (40 ng / mL, IL 16Mouse, GLPBIO, GP20578) for 90 min and the number of MDCSs was observed by immunofluorescence staining. Compared with the Con group, the number of MDSCs in the IL-16 group increased significantly, as shown in Figure 2. Figure 3 As shown. Mix well with three fluorescent-labeled specific antibodies [anti-CD11b, Ly6G, Ly6C] and place in 4°C away from light for 1 hour. After the reaction is completed, flow cytometry is used for data acquisition to record the PMN-MDSCs cell ratio. Compared with the Con group, the PMN-MDSCs percentage in the IL-16 group was significantly increased. The results are shown in Figure 4 shown.

[0037] 4. How does IL-16 affect the production of NETs by PMN-MDSCs?

[0038] Peripheral blood was collected from mice three days after surgery into heparin tubes, and peripheral blood mononuclear cells (PBMCs) were separated using Ficoll-Hypaque gradient. PMN-MDSCs (CD 11b + Ly6G + Ly6C low ), and then conditional treatment was performed. The groups were: Con group (PMN-MDSCs), IL-16 group (PMN-MDSCs + 40ng / mL IL-16Mouse), IL-16i group (PMN-MDSCs + 10ng / mL IL-16 neutralizing antibody), Peptide group (PMN-MDSCs + 10ug / mL anti-IL-16 peptide), and immunofluorescence staining of NETs markers (MPO) was performed 90 minutes after treatment. The results showed that compared with the Con group, IL-16 increased the level of MPO; compared with the IL-16 group, the MPO expression levels of the IL-16i group and the Peptide group decreased, and the decrease in the Peptide group was significant, such as Figure 5 The mouse myeloperoxidase (MPO) enzyme-linked immunosorbent assay kit (ELK1662) was used to detect the level of MPO in the supernatant of the above experiment. The results are shown in Figure 6 As shown, compared with the Con group, IL-16 promoted the production of MPO by PMN-MDSCs, and compared with the IL-16 group, the MPO levels in the IL-16i group and the Peptide group were decreased (* indicates p < 0.05; ** indicates p < 0.01; ns indicates no significant difference).

[0039] Whole blood specimens were collected from 50 patients with gastric cancer admitted to the general surgery department of a cancer hospital 3 days after surgery. All patients had histologically confirmed gastric cancer, and all underwent tumor resection surgery by the same group of surgeons. None of the subjects had a history of autoimmune diseases, and the healthy controls had no history of cancer. This study was approved by the Medical Ethics Committee of a cancer hospital, and specimen collection was approved by the patients themselves and they signed informed consent forms. All patients were followed up throughout the process. The blood was drawn into heparin tubes and centrifuged, and peripheral blood mononuclear cells (PBMCs) were separated using a Ficoll-Hypaque gradient. PMN-MDSCs (CD11b + CD33 + HLA - DR - / low CD14 - CD15 + ), and then conditional treatment was performed. The groups were divided into: Con group (PMN-MDSCs), IL-16 group (PMN-MDSCs + 40ng / mL IL-16Human), IL-16i group (PMN-MDSCs + 10ng / mL IL-16 neutralizing antibody), Peptide group (PMN-MDSCs + 10ug / mL anti-IL-16 peptide), and immunofluorescence staining of NETs markers (MPO) was performed 90 minutes after treatment. The results showed that compared with the Con group, IL-16 increased the level of MPO; compared with the IL-16 group, the MPO expression levels of the IL-16i group and the Peptide group decreased, as shown in Figure 2. Figure 7 shown.

[0040] Example 2

[0041] Anti-IL-16 peptide prevents postoperative tumor metastasis in gastric cancer

[0042] 1. Construction of mouse postoperative metastasis model

[0043] The 615 mice with subcutaneous tumors were subjected to tumor resection surgery, and the anti-IL-16 polypeptide of the present invention was administered to observe whether it can reduce the tumor metastasis in the liver metastasis model (LM) and the peritoneal metastasis model (PM). The groups were divided into: Sham group (sham operation + LM / PM group), POD3 group (LM / PM was performed three days after tumor resection surgery), and Peptide group (100 mg / kg anti-IL-16 polypeptide was intraperitoneally injected every three days after surgery, and LM / PM was performed three days after surgery).

[0044] LM, that is, the tumor cell group was inoculated by spleen injection and spleen removal. A sufficient number of MFC cells were cultured and collected; 1×10 6MFC cells were suspended in PBS. Randomly selected mice were weighed and anesthetized by intraperitoneal injection of 0.5% sodium pentobarbital (50 mg / kg). After 3 to 5 minutes of successful anesthesia, the mice were fixed on the operating table with adhesive tape. The skin was disinfected with iodine, and a longitudinal incision of 0.5 to 1 cm long was made under the costal margin of the left posterior axillary line. After entering the abdomen, the willow-shaped spleen was found on the lateral side of the abdomen and pulled out of the abdominal cavity. A 1 mL syringe was used to insert the needle from the lower pole of the spleen, and 0.1 mL of tumor cell fluid was injected into the spleen. After the needle was removed, the needle eye was pressed with an alcohol cotton ball, and the spleen was gently massaged for 5 minutes to allow the tumor cells to enter the liver through the splenic vein. After the pale bulge in the injection area disappeared, the splenic pedicle was ligated, the spleen was removed, and no bleeding was found. The muscles and skin were intermittently sutured in turn to close the abdomen. After surgery, the animals were kept in a Class II animal laboratory.

[0045] PM, that is, tumor cells were inoculated by intraperitoneal injection. 6 luciferase-MFC cells were suspended in PBS and injected intraperitoneally, and tumor growth was recorded by in vivo animal imaging.

[0046] After 14 days of LM / PM, the metastasis was observed, peripheral blood was collected in heparin tubes, and peripheral blood mononuclear cells (PBMCs) were separated using Ficoll-Hypaque gradient. Each group was mixed evenly with fluorescently labeled specific antibodies [anti-CD11b, Ly6G, Ly6C] and placed in a dark tube at 4°C for 1 hour. After the reaction was completed, flow cytometry was used for data acquisition to record the proportion of PMN-MDSCs cells. Compared with the Sham group, the percentage of PMN-MDSCs in the POD 3 group increased; compared with the POD 3 group, the percentage of PMN-MDSCs in the anti-IL-16 peptide group decreased. The results are shown in Figure 2. Figure 8 The tumor nodules in each lobe of the liver were recorded, and the liver tissue was stained with HE and histochemical staining to evaluate liver metastasis. The results are shown in Fig. 9 Compared with the POD 3 group, the percentage of PMN-MDSCs in the anti-IL-16 peptide group decreased, the number of metastatic foci in the liver decreased, and the protein level of Vimentin decreased. The small animal imaging bioluminescence was used to observe the abdominal metastasis. The results showed that compared with the POD 3 group, the anti-IL-16 peptide group could reduce the abdominal metastasis, as shown in Figure 2. Fig.10 shown.

Claims

1. An anti-IL-16 polypeptide, the amino acid sequence of which is shown in SEQ ID No.

1.

2. Use of the anti-IL-16 polypeptide according to claim 1 in the preparation of a drug for preventing or inhibiting postoperative tumor metastasis of gastric cancer.

3. The use according to claim 2, characterized in that: The drug dosage form is an injection.

4. The anti-IL-16 polypeptide of claim 1 may be chemically modified, and the chemically modified polypeptide of any one of claim 1 is defined as a corresponding functional analog of the polypeptide.

5. A drug for preventing or inhibiting postoperative metastasis of gastric cancer, characterized in that: Comprising the anti-IL-16 polypeptide or polypeptide functional analogue described in claims 1 and 4.

6. The drug according to claim 4, characterized in that The polypeptide or its functional analogue also includes a pharmaceutically acceptable carrier.

7. The drug according to claim 4, characterized in that The dosage form of the polypeptide or its functional analogue is injection.

8. The method according to claim 4, wherein the polypeptide or its functional analogue can be used as an active ingredient in a pharmaceutical composition.

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