Application of synthetic peptide targeting CLDN12 in reduction of hepatic metastasis of pancreatic cancer

Abstract

The invention relates to an application of a synthetic peptide targeting CLDN12 in reduction of hepatic metastasis of pancreatic cancer. Research finds that the tight junction protein Claudin12 (CLDN12) is expressed on the surface of pancreatic cancer cells and also expressed on fibroblasts, and the CLDN12 on the fibroblasts and the CLDN12 on the pancreatic cancer cells mediate an adhesion effect through homologous interaction and guide migration and invasion of the pancreatic cancer cells. The adhesion effect of CLDN12-mediated fibroblasts and pancreatic cancer cells is blocked, and the invasion of the pancreatic cancer cells dominated by the fibroblasts can be blocked, so that the invasion and metastasis of the pancreatic cancer are reduced.

Description

Technical Field

[0001] The present invention belongs to the technical field of tumor targeting, and specifically relates to the application of a synthetic peptide targeting CLDN12 in reducing liver metastasis of pancreatic cancer. Background Art

[0002] Pancreatic cancer is an insidious disease, and once diagnosed, it is often accompanied by local invasion and distant metastasis. Cancer associated fibroblasts (CAFs) are an important part of the tumor microenvironment, which can directly affect tumor cell behavior through secreted factors or cell-to-cell contact, thereby changing the movement and invasion of tumor cells. Therefore, designing strategies to target fibroblasts and reduce the risk of pancreatic cancer metastasis is an urgent direction to be worked on. Summary of the invention

[0003] The purpose of the present invention is to overcome the deficiencies of the prior art and provide a use of a synthetic peptide targeting CLDN12 in reducing pancreatic cancer liver metastasis.

[0004] The technical solution of the present invention is as follows:

[0005] Application of synthetic peptides targeting CLDN12 in reducing liver metastasis of pancreatic cancer. Synthetic peptides targeting CLDN12 are used to inhibit the invasion and metastasis of pancreatic cancer.

[0006] Furthermore, the synthetic peptide targeting CLDN12 is a specific targeting polypeptide of fibroblast tight junction protein, and the amino acid sequence of the synthetic peptide targeting CLDN12 is YNSHLNRKFEP.

[0007] Compared with the prior art, the present invention has the following beneficial effects:

[0008] 1. The study found that the tight junction protein CLDN12 is expressed on the surface of pancreatic cancer cells and also on fibroblasts. CLDN12 on the surface of fibroblasts and CLDN12 on the surface of pancreatic cancer cells mediate cell adhesion through homotypic interactions, guiding the migration and invasion of pancreatic cancer cells. Blocking the adhesion of fibroblasts and pancreatic cancer cells mediated by CLDN12 can block the invasion of pancreatic cancer cells dominated by fibroblasts, thereby reducing the invasion and metastasis of pancreatic cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] Figure 1 Schematic diagram of the CLDN12 protein structure of the present invention and the CLDN12 targeting polypeptide C12-pm and control Con-p polypeptide sequences;

[0010] Figure 2 Schematic diagram of CLDN12 promoting adhesion of iMEF cells and Pan02 cells in the present invention;

[0011] Figure 3 This is a schematic diagram of the CLDN12 targeting polypeptide C12-pm in the present invention blocking the adhesion of iMEF and Pan02 cells;

[0012] Figure 4 This is a schematic diagram of iMEF cells with low CLDN12 expression in the present invention inhibiting the invasion of Pan02 in Matrige1 matrix gel;

[0013] Figure 5 Schematic diagram of the CLDN12 targeting peptide C12-pm in the present invention inhibiting pancreatic cancer liver metastasis in mice. DETAILED DESCRIPTION

[0014] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0015] like Figure 1-5 As shown, the application of synthetic peptides targeting CLDN12 in reducing liver metastasis of pancreatic cancer;

[0016] In order to verify the experimental effect of the present invention, the following in vitro and in vivo verifications were performed:

[0017] 1. Materials and Methods

[0018] 1. Cell lines and animals

[0019]

[0020] 2. Main Reagents

[0021]

[0022]

[0023] 3. Plasmids used

[0024]

[0025] 4. Experimental methods

[0026] I. Isolation of Mouse Pancreatic Cancer-associated Fibroblasts

[0027] 1) Fresh PDAC tissue from the KPC genetically engineered mouse model was used as the material, where the material was sterile fresh surgical tissue, and mouse CAFs were constructed by enzymatic digestion.

[0028] 2) Wash the material 2-3 times with 1× phosphate buffered saline (PBS, Hyclone) to remove blood contamination.

[0029] 3) Use ophthalmic scissors to fully chop the material, place it on a shaker and digest it with digestion solution (DNase 200 μg / ml, hyaluronidase 100 μg / ml, collagenase IV 2 mg / ml) at 37°C for 1 hour.

[0030] 4) The digestion was stopped with DMEM containing fetal bovine serum (FBS), and then filtered through a 100 μm nylon mesh (BD Biosciences), followed by centrifugation at 2000 rpm, 4°C for 5 min.

[0031] 5) Resuspend the cells and culture them in DMEM containing 10% FBS, 1% penicillin and streptomycin.

[0032] 6) The cells were cultured at 37°C in a 5% CO2-air humidified atmosphere.

[0033] II. Construction of cell lines with low expression of Claudin12 using shRNA method

[0034] 1) Preparation of 293T cells: 293T cells were cultured in a cell culture dish and virus packaging was performed when the cells grew well and the density reached 50-80%.

[0035] 2) Mixture reagent preparation:

[0036] a. Prepare DNA mixture: Take a 1.5 ml EP tube, mark the information, add 200 μl of jetprimebuffer (buffer), 0.75 μg of pSPAX2 plasmid, 0.25 μg of pMD2.G plasmid, and 1 μg of target plasmid (plasmid carrying shRNA) to each tube, and mix gently.

[0037] b. Add 4 μl of jetprime buffer transfection reagent to the DNA mixture and let it stand at room temperature for 10 minutes.

[0038] 3) Cell culture medium replacement: Take out the 293T cell culture dish from the cell culture incubator, discard the culture medium, and add 2 ml of DMEM culture medium containing 10% FBS.

[0039] 4) Add mixture reagent: Gently add the mixed mixture reagent dropwise into the 293T cell culture dish, shake gently to mix, and place in the incubator to continue culturing.

[0040] 5) Change cell medium: After 6-8 hours (or 8-12 hours), replace with fresh complete medium and continue culturing.

[0041] 6) Collecting virus solution:

[0042] a. After 48 hours of culture, pipette the 293T cell culture medium into a 15 ml centrifuge tube and centrifuge at 3000 rpm for 10 min. Collect the supernatant after centrifugation and dispense it into new 1.5 ml EP tubes and store them in a -80 °C refrigerator for subsequent experiments.

[0043] b. Add 2 ml of complete culture medium to the 293T cell culture dish from which the culture medium has been aspirated and continue culturing for 72 hours. Collect the virus solution according to method a, divide it into portions and store it in a -80°C refrigerator or use it directly for subsequent virus infection.

[0044] 7) Virus infection of target cells:

[0045] a. Cell preparation: Culture the target cells to be infected in a cell culture dish, wait for them to adhere to the wall, and change the medium when the density reaches 30-50%;

[0046] b. Mix the collected virus solution with complete culture medium in a certain ratio and add it to the target cell culture dish. Add Polybrene (1:1000) transfection reagent and place it in the incubator for further culture.

[0047] c. The plasmid used for this transfection carries a GFP fluorescent label. After the virus solution infects the target cells for a certain period of time, the fluorescence intensity of the cells can be observed under a fluorescence microscope, or the cells can be collected and the proportion of GFP-positive cells can be detected by FlowCytometry; or after 48 hours of culture, the cells can be cultured with a medium containing puromycin (the concentration to be used should be found in advance), and one well of cells should be reserved as a blank control (not infected with lentivirus). Observe cell death regularly, change the medium in time, and digest and passage normally.

[0048] Screening was carried out until all blank control cells died, and the cells were expanded and cultured with 2 μg / ml of puromycin, and RNA or protein was extracted to verify the knockdown effect.

[0049] III. CFSE fluorescent labeling of cells

[0050] Collect cells and fully disperse them into single suspended cells; wash once with serum-free medium, centrifuge at 1000 rpm for 5 min; discard the supernatant, and wash three times with 1× PBS. Resuspend the cells to a concentration of 1-3×10 7 cells / mL in 1×PBS single cell suspension. Add CFSE solution to the single cell suspension to a final concentration of 1μM. Incubate at 37℃ for 10min, add 9 times the volume of PBS, and centrifuge at 1000rpm for 5min. Discard the supernatant, add 9ml of complete culture medium containing 10% fetal bovine serum, repeat the centrifugation step and set aside.

[0051] IV. Cell Adhesion Assay

[0052] 1) 1×10 5 pancreatic cancer cells.

[0053] 2) After the cells attach and fuse to form a monolayer membrane, wash and replace the medium.

[0054] 3) The constructed iMEF with GFP and knockdown of CLDN12 (iMEF shCldn12 ) cells and control cells (iMEF sh -nc ) with 1×10 5 The above cell layer was placed in each well, centrifuged at 100 g for 5 min, and then cultured in an incubator at 37° C. and 5% CO 2 for 15 min.

[0055] 4) Wash with PBS three times to remove non-adherent cells, and then detect and count the proportion or number of adherent cells by flow cytometry or fluorescence photography.

[0056] V. Cell invasion assay

[0057] 1) Marking: Use a marker pen and a ruler to draw three horizontal lines on the bottom of the 96-well plate as marking lines.

[0058] 2) Plating: The constructed iMEF with GFP and knockdown of CLDN12 (iMEF shCldn12 ) cells and control cells (iMEF sh-nc ) were seeded with mCherry-labeled tumor cells in a 96-well plate at a ratio of 2:1.

[0059] 3) Scratch: When the cells have adhered to the wall overnight and the degree of confluence reaches 90%, use the gun tip to draw a vertical line through the center of the well plate perpendicular to the well plate and the marked horizontal line so that the scratch intersects with the marked line.

[0060] 4) Washing: Discard the culture medium and wash the removed cells with PBS.

[0061] 5) Add 40 μl of Matrigel and place the 96-well plate in a 37° C., 5% CO 2 incubator for 1 hour to allow the Matrigel to solidify.

[0062] 6) Add 100 μl of DMEM complete medium to each well and place in a 37°C, 5% CO2 incubator for further culturing for 24 h.

[0063] 7) Photographing and observing: Take out the cell culture plate and observe the invasion of cells into the scratched area under an inverted fluorescence microscope.

[0064] 8) Data analysis: ImageJ software was used to analyze the number of cell invasion.

[0065] VI. Pancreatic cancer cell and fibroblast co-injection liver metastasis model

[0066] 1) Mouse pancreatic cancer cells Pan02-GFP-Luc, iMEF sh-nc and iMEF shCldn12 Digest with trypsin, centrifuge at 1000 rpm for 5 min, discard the supernatant, wash twice with sterile PBS, add 1 ml sterile PBS to resuspend the cells and count them.

[0067] 2) Pan02-GFP-Luc cells and fibroblasts were mixed at a ratio of 1:3 (5×10 Pan02-GFP-Luc cells were injected into each mouse). 5 , fibroblasts 1.5×10 6 ) in a ratio of 1:1 and then mix evenly with matrix gel in a ratio of 1:1 (matrix gel tends to solidify at room temperature, so a pre-cooled pipette tip is required and the operation must be carried out on ice).

[0068] 3) C57BL / 6 mice were anesthetized using isoflurane gas anesthesia. After the mice were anesthetized, the peritoneum of the mice was cut open using ophthalmic scissors to fully expose the abdominal cavity. The spleen of the mice was lifted with forceps, and 20 μl of the matrix gel cell suspension was extracted with an insulin needle and slowly injected into the spleen of the mice.

[0069] 4) Small animal in vivo imaging: D-luciferin potassium salt solution was prepared with PBS without calcium and magnesium, the working solution concentration was 30 mg / ml, and the storage solution was kept away from light. After 2 weeks of tumor bearing, D-luciferin potassium salt solution was injected intraperitoneally at 150 mg / kg body weight. After about 10 minutes of reaction, the mice were anesthetized with a gas anesthesia machine, and the spleen tumor-bearing site and liver site were imaged by small animal in vivo imaging to detect the fluorescence intensity of Pan02-GFP-Luc. Alternatively, the mice were killed by cervical dislocation, and the liver and spleen tissues were removed. The small animal in vivo imaging was used to take pictures again, and the fluorescence intensity of Pan02-GFP-Luc at the tumor site was counted.

[0070] VII. Peptide Synthesis

[0071] The peptides C12-pm (synthetic peptide) and Con-p used in this study were synthesized by Shanghai Qiangyao Biotechnology Co., Ltd. (purity>95%), and the storage solution was dissolved in 30% DMSO; before use, they were diluted with PBS to the working concentration; in in vitro cell experiments, the final concentration of the peptide was 400uM, and the stimulation time was 24h.

[0072] VIII. In vivo peptide therapy for liver metastasis

[0073] Five days after Pan02 and CAFs were inoculated into the spleen of mice, tumor formation was detected by IVIS, and tumor-bearing mice were randomly divided into different treatment groups (8 mice per group). C12 peptide mimetic (C12-pm: YNSHLNRKFEP) and control peptide (Con-p: LYQY) (China Peptides, Shanghai, China) were dissolved in 30% DMSO water stock solution before use and added to PBS so that the final concentration of DMSO was less than 0.5% of the final volume of the culture medium. The peptide (500μg / 200μL / mouse) was injected intravenously into the mice. The injection was performed once through the tail vein and repeated every other day for a total of six injections. After the treatment period, all mice were euthanized after one week of continuous observation. Liver tissue was taken and liver metastasis was detected by IVIS.

[0074] 2. Results and Analysis

[0075] 1. CLDN12 is a four-transmembrane protein, whose structure from the N-terminus to the C-terminus contains two U-shaped ring structures ECL1 and ECL2. The specific structural diagram is shown in Figure 1 .

[0076] 2. Use iMEF shCldn12 and control group iMEF sh-nc Taking advantage of the expression of GFP in cells, the adhesion of iMEF cells to Pan02 was detected by flow cytometry. shCldn12 Adhesion to Pan02 is less than that to iMEF sh-nc cell( Figure 2 )

[0077] 3. Label iMEF cells with CFSE. Stimulate Pan02 or iMEF cells with C12-pm or control peptide (400uM) for 24h. Add stimulated iMEF (CFSE) cells to the monolayer of pancreatic cancer cells Pan02 to detect adherent cells. Adherent cells were detected by flow cytometry or fluorescence photography. The results showed that C12-pm treatment of iMEF or Pan02 can reduce the adhesion between iMEF and Pan02 ( Figure 3 ).

[0078] 4. Mouse pancreatic cancer cells Pan02-mcherry and iMEF cells were co-cultured for scratch test, and the cell surface was covered with matrix gel. After 24 hours, the invasion of tumor cells into the wound area was evaluated. shCldn12 Compared with iMEF cells sh-nc The number of invasion of Pan02-mcherry cells in the cell co-culture group was higher ( Figure 4 ).

[0079] 5. C12-pm was applied to the liver metastasis model of Pan02-GFP-Luc cells co-injected with CAFs and liver metastasis was detected. After 5 days of tumor bearing, the mice were randomly divided into two groups and injected with C12-pm and Con-p through the tail vein every two days for a total of six injections. Tumor liver metastasis was observed on the 21st day. The results showed that C12-pm treatment significantly reduced tumor liver metastasis ( Figure 5 ).

[0080] Although the present invention has been described in detail with reference to the aforementioned embodiments, it is still possible for those skilled in the art to modify the technical solutions described in the aforementioned embodiments, or to make equivalent substitutions for some of the technical features therein. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. Use of a synthetic peptide targeting CLDN12 in reducing pancreatic cancer liver metastasis, characterized in that: Synthetic peptides targeting CLDN12 are used to inhibit pancreatic cancer invasion and metastasis.

2. The use of a synthetic peptide targeting CLDN12 according to claim 1 in reducing liver metastasis of pancreatic cancer, characterized in that: The synthetic peptide targeting CLDN12 is a specific targeting polypeptide of fibroblast tight junction protein, and the amino acid sequence of the synthetic peptide targeting CLDN12 is YNSHLNRKFEP.

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