JP2015227292A - Pancreatic cancer cell invasion metastasis inhibition vaccine - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a vaccine which can particularly effectively inhibit invasion and metastasis of pancreatic cancer cells.SOLUTION: A pancreatic cancer cell invasion metastasis inhibition vaccine according to the present invention comprises peptides having part of an amino acid sequence (sequence number 1) of human insulin-like growth factor 2 mRNA binding protein 3.

Description

  The present invention relates to a vaccine capable of effectively suppressing pancreatic cancer cell infiltration and metastasis.

  “Tumor” refers to a cell that has proliferated abnormally, and refers to a state in which cell growth continues even if the cause of the abnormal growth disappears or is removed. Among tumors, benign tumors grow slowly and do not metastasize. Therefore, in general, there is no problem if it is excised, and it can be said that there is no difference in life even if it is left without being excised. On the other hand, unlike benign tumors, malignant tumors, that is, cancers, proliferate rapidly and metastasize to lymph nodes and other organs. Therefore, for example, even if removed by surgical operation, cancer cells that remain even slightly, or cancer cells that have already metastasized to lymph nodes or other organs may start to proliferate again. Therefore, cancer has a poor prognosis after treatment is completed, and the survival rate of each cancer has been investigated after 5 years. Generally, after 5 years since the cancer disappeared by treatment. If there is no recurrence before, it is finally considered a cure.

  Pancreatic cancer is said to have the worst prognosis among cancers. This is because the pancreas is a retroperitoneal organ and is difficult to detect at an early stage, and the pancreatic cancer cells have extremely high motility. Invasion into bile ducts, nerves, etc., metastasis to nearby lymph nodes, and distant metastasis to the liver.

  As described above, the reason that cancer is a malignant tumor is that it infiltrates and metastasizes to other tissues in particular, and it is thought that the prognosis will be good if there is no invasion and metastasis. Therefore, techniques for suppressing cancer cell invasion and metastasis have been developed.

  For example, in Patent Documents 1 and 2, a gene encoding NK4, which is an intramolecular fragment having a terminal hairpin in a hepatocyte growth factor (HGF) molecule followed by four kringle domains, is infiltrated or metastasized into cancer cells. The invention used for the suppression of the above is described.

  By the way, insulin-like growth factor 2 mRNA binding protein 3 (Insulin-like Growth Factor 2 mRNA-Binding Protein 3, hereinafter sometimes abbreviated as “IGF2BP3”) is mainly present in the nucleolus, and insulin-like growth factor It binds to the 5 'UTR of the 5' untranslated region 3 of the II RNA and inhibits translation of insulin-like growth factor II. In the invention described in Patent Document 3, a gene encoding IGF2BP3 is listed as one of genes whose overexpression is used for detection of cancer.

JP 2003-250549 A JP 2008-7514 A Special table 2011-516077 gazette

  As described above, various techniques for suppressing invasion and metastasis of cancer cells have been studied, but there is no particular reason why pancreatic cancer cells tend to infiltrate and metastasize. It was not necessarily effective in suppressing cell invasive metastasis.

  Accordingly, an object of the present invention is to provide a vaccine that can particularly effectively suppress invasion and metastasis of pancreatic cancer cells.

  This inventor repeated earnest research in order to solve the said subject. As a result, human insulin-like growth factor 2 mRNA binding protein 3 found mainly in nucleolus in general cells is found in cell membrane processes, which are essential intracellular structures for invasion and metastasis in pancreatic cancer cells. The present invention was completed by finding that it exists and is greatly involved in invasion and metastasis of pancreatic cancer cells.

  Hereinafter, the present invention will be described.

  [1] A pancreatic cancer cell invasion and metastasis-suppressing vaccine comprising a peptide having a part of the amino acid sequence of human insulin-like growth factor 2 mRNA binding protein 3 (SEQ ID NO: 1).

  [2] The pancreatic cancer cell invasion and metastasis-suppressing vaccine according to [1], wherein the peptide has 8 or more and 12 or less amino acids.

  [3] The pancreatic cancer cell invasion and metastasis-suppressing vaccine according to [1] or [2], wherein the peptide has an amino acid sequence selected from amino acid sequences of SEQ ID NOs: 2 to 49.

  [4] The pancreatic cancer cell invasion and metastasis-suppressing vaccine according to any one of [1] to [3], further including a peptide having a part of the amino acid sequence of KIF20A in addition to the peptide.

  [5] The pancreatic cancer cell invasion and metastasis-suppressing vaccine according to any one of [1] to [4], wherein the peptide is capable of binding to both HLA-A24 and HLA-A2.

  Human insulin-like growth factor 2 mRNA binding protein 3 (IGF2BP3) according to the present invention is present in the plasma membrane processes of pancreatic cancer cells, is involved in improving the motility of pancreatic cancer cells, and plays a major role in invasion and metastasis thereof. It is considered to have played. Further, according to the experimental findings of the present inventors, when the expression of the IGF2BP3 gene is inhibited, the motility of pancreatic cancer cells is impaired, and the invasion and metastasis of such pancreatic cancer cells is significantly suppressed. Therefore, if a peptide having a part of the amino acid sequence of IGF2BP3 is used as an active ingredient of a vaccine, pancreatic cancer cells expressing IGF2BP3 are damaged by cytotoxic T cells (CTL) activated by the peptide vaccine. As a result, highly invasive and metastatic cells can be reduced and invasive metastasis can be suppressed. On the other hand, IGF2BP3 is highly expressed in important organs in the fetal period, but some expression is observed in the testis in adult organs, but there is no expression in important organs. Furthermore, since IGF2BP3 is a protein derived from living cells, a peptide having a partial amino acid sequence is considered to be harmless and safe. Therefore, the peptide vaccine according to the present invention is very useful as it can safely suppress the invasion and metastasis of pancreatic cancer cells.

FIG. 1 is an immunocytochemical fluorescence-labeled photograph of human pancreatic ductal adenocarcinoma cells S2-013 and PANC-1 which have been promoted to form cell membrane protrusions by culturing on fibronectin. The green part shows the part labeled with the anti-IGF2BP3 antibody, and the red part shows the actin filaments labeled with phalloidin. The arrow indicates the part where IGF2BP3 is localized in the cell membrane projection, and the length of the bar is 10 μm. FIG. 2 is a Z-stack (multilayer uptake) photograph of the S2-013 cell line cultured on fibronectin in a confocal immunofluorescence microscope test. The blue part shows nuclei stained with DAPI and the green part shows stained IGF2BP3. The arrow indicates a portion where IGF2BP3 is localized in the cell membrane projection, and it is indicated that IGF2BP3 is accumulated in the cell membrane projection. A portion surrounded by a white frame indicates an enlarged area. The lower and right panels each show a cross section of the yellow line. The length of the bar is 10 μm. FIG. 3 shows IGF2BP3-RNAi S2-013 cell line (siIGF-1 and siIGF-2) into which siRNA targeting IGF2BP3 mRNA is introduced, and control RNAi S2-013 cell line (Scr-1 and Scr-2). It is a photograph which shows the result of having analyzed the protein expressed in <3> by Western blot. FIG. 4 shows the number of cells migrated to the wound area in the motility assay of the IGF2BP3-RNAi S2-013 cell line and the control RNAi S2-013 cell line to the wound area. FIG. 5 is a graph showing the number of cells migrated to the wound area in the motility assay of the IGF2BP3-RNAi S2-013 cell line and the control RNAi S2-013 cell line to the wound area. “*” Indicates that there is a significant difference at p <0.001 with respect to the control in the t-test. FIG. 6 shows that a wound region is formed by removing a part of the S2-013 strain cells grown confluently in a monolayer with a pipette tip, and an advanced site of pancreatic cancer cells that begin to migrate toward the wound region is immunized. It is a dyed photograph. The green part shows the part dye | stained with the anti- IGF2BP3 antibody. The arrow indicates IGF2BP3 present in the cell membrane protrusion formed at the advanced site of the S2-013 cell line that has begun to migrate. The length of the bar is 10 μm. FIG. 7 shows the number of cells infiltrated in a transwell invasive assay of control RNAi S2-013 cell line, IGF2BP3-RNAi S2-013 cell line, and IGF2BP3-RNAi S2-013 cell line that restored IGF2BP3 expression. It is a graph. “*” Indicates that there is a significant difference at p <0.005 relative to the control in the t-test. FIG. 8 is a photograph of a tumor formed in the pancreas stained with hematoxylin-eosin in a nude mouse transplanted with control RNAi S2-013 cell line or IGF2BP3-RNAi S2-013 cell line into the pancreas. “R” indicates luminal peritoneal tissue, “P” indicates muscle tissue, and “N” indicates normal tissue. FIG. 9 shows Z in confocal immunofluorescence microscopy of control RNAi S2-013 cells stimulated with fibronectin, IGF2BP3-RNAi S2-013 cells, and IGF2BP3-RNAi S2-013 cells restored to IGF2BP3 expression. -Stack (multi-layer capture) photograph. The blue part shows nuclei stained with DAPI, and the red part shows actin filaments labeled with phalloidin. Arrows indicate actin filaments present in cell membrane processes. The lower and right panels each show a cross section of the white line portion. The length of the bar is 10 μm. FIG. 10 shows cell membrane protrusions relative to the total number of cells of control RNAi S2-013 cells stimulated with fibronectin, IGF2BP3-RNAi S2-013 cells, and IGF2BP3-RNAi S2-013 cells restored to IGF2BP3 expression. It is a graph which shows the ratio of a cell. “*” Indicates that there is a significant difference at p <0.001 with respect to the control RNAi S2-013 cell line or the IGF2BP3-RNAi S2-013 cell cell that restored IGF2BP3 expression in the t-test. FIG. 11 is a photograph of the morphology of control RNAi S2-013 cell line and IGF2BP3-RNAi S2-013 cell line stimulated with fibronectin using a phase contrast microscope.

  The pancreatic cancer cell invasion and metastasis-suppressing vaccine according to the present invention contains a peptide having a part of the amino acid sequence (SEQ ID NO: 1) of human insulin-like growth factor 2 mRNA binding protein 3 (IGF2BP3) as an active ingredient.

  Insulin-like growth factor (IGF) is a polypeptide whose amino acid sequence is highly similar to that of insulin and causes a reaction such as mitogenesis in the same manner as insulin in cell culture. IGF-2 is believed to be the primary growth factor required for early development, secreted by the brain, kidneys, pancreas and muscle in mammals, acting more specifically than IGF-1, and in adults by insulin It can be seen at a concentration 600 times that of. The function of IGF is regulated by a family of genes known as IGF binding proteins, but IGF gene expression is regulated by proteins that bind to its mRNA.

  IGF2BP3 is one of the regulators of IGF gene expression and is mainly present in the nucleolus, whereas the present inventor considered that the cell membrane is a site essential for motility in pancreatic cancer cells. It has been found that it is accumulated in the processes and is greatly involved in the motility of pancreatic cancer cells. For example, if the IGF2BP3 gene is knocked out in pancreatic cancer cells, it loses motility and does not cause invasion or metastasis. Therefore, the present inventor has conceived the present invention that pancreatic cancer cell invasion and metastasis can be suppressed by using a peptide having a partial amino acid sequence of IGF2BP3 as a vaccine.

  The peptide which is an active ingredient of the pancreatic cancer cell invasion and metastasis-suppressing vaccine according to the present invention has a part of the amino acid sequence of human IGF2BP3. The amino acid sequence of human IGF2BP3 is shown in SEQ ID NO: 1, and the peptide of the present invention has a partial sequence.

  In the present invention, “part of the amino acid sequence” means that it is not the entire amino acid sequence, but the number of amino acids is preferably 5 or more and 15 or less. If the number of amino acids is 5 or more, it may be possible to cause an immune response more reliably. On the other hand, when the number of amino acids is 15 or less, sufficient binding to HLA on cytotoxic T cells can be ensured. The number of amino acids is preferably 6 or more, more preferably 7 or more, particularly preferably 8 or more, more preferably 14 or less, still more preferably 12 or less, and particularly preferably 10 or less.

  Specific examples of the peptide used as an active ingredient in the pancreatic cancer cell invasion and metastasis-suppressing vaccine according to the present invention include those having an amino acid sequence selected from the amino acid sequences of SEQ ID NOs: 2 to 49.

  Although only 1 type may be used for the said peptide, it can also use 2 or more types together, and can select what has a positive immune response. In this case, the number of peptides can be, for example, 2 or more and 10 or less. The number of peptides is more preferably 3 or more, more preferably 6 or less, and still more preferably 4 or less.

  In addition to the IGF2BP3 peptide, the pancreatic cancer cell invasion and metastasis-suppressing vaccine according to the present invention may contain a peptide having a part of the amino acid sequence of a protein other than IGF2BP3. For example, even if it is a peptide having a weak vaccine effect alone, when used in combination with the IGF2BP3 peptide according to the present invention, an anticancer effect such as an excellent invasion metastasis inhibiting effect on pancreatic cancer cells is exhibited. there is a possibility. Examples of such proteins other than IGF2BP3 include KIF20A. Since KIF20A (kinesin family member 20A) is involved in the invasion and metastasis of pancreatic cancer cells, the invasion and metastasis of pancreatic cancer cells can be achieved by using a peptide having a partial amino acid sequence together with the IGF2BP3 peptide according to the present invention. Can be suppressed synergistically.

  Examples of the selection criteria for the peptide to be blended in the pancreatic cancer cell invasion and metastasis-suppressing vaccine according to the present invention include binding power to HLA on cytotoxic T cells of the recipient. If a peptide having a strong binding force is selected, a more effective immune response may be obtained in the recipient. For example, HLA-A24 can be mentioned as an HLA often found in Japanese. In addition, if a peptide having binding ability with other HLA such as HLA-A2 is selected, the application range of the recipient to whom the vaccine of the present invention is effective can be expanded.

  The peptide used in the present invention can be appropriately prepared by methods known to those skilled in the art. For example, DNA encoding a desired peptide is amplified by PCR using the human IGF2BP3 gene as a template, introduced into a microorganism such as Escherichia coli, Bacillus subtilis, or yeast using an appropriate vector, transformed, cultured, What is necessary is just to refine. Also, it can be chemically synthesized by a solid phase method or the like.

  What is necessary is just to select the dosage form of the pancreatic cancer cell invasion metastasis suppression vaccine which concerns on this invention suitably according to a desired administration route. For example, in the case of subcutaneous injection, a solution or an emulsion may be used, or a solution or an emulsion may be prepared as a freeze-dried product. In the case of transmucosal administration, tablets such as sublingual tablets, ointments, creams and the like may be used.

  The pancreatic cancer cell invasion and metastasis-suppressing vaccine according to the present invention may contain additive components according to the dosage form. Examples of additive components include solvents such as water and ethanol; salts such as sodium chloride, potassium chloride, sodium bicarbonate, potassium bicarbonate; immune enhancers such as incomplete Freund's adjuvant, polysaccharides such as pullulan, and complete Freund's adjuvant. A water-soluble polymer such as a relatively low molecular weight polyethylene glycol; a base material such as a relatively high molecular weight polyethylene glycol; and a pH adjuster such as citric acid, lactic acid, and potassium carbonate.

  You may select in advance the peptide mix | blended with the pancreatic cancer cell invasion metastasis suppression vaccine which concerns on this invention. For example, a serum sample can be obtained from a planned administration person, an immune reaction against the test peptide can be tested, and a peptide capable of obtaining a high CTL inducing ability can be selected.

  The dose of the vaccine of the present invention may be appropriately adjusted according to the severity, sex, age, etc. of the patient. For example, the dose of each peptide per administration is 1 mg or more, about 15 mg or less, More preferably, it can be set to about 3 mg or more and 5 mg or less.

  The administration method may be appropriately selected. For example, subcutaneous injection is preferably performed in the armpit or in the vicinity of the groin.

  The vaccine of the present invention is preferably administered twice or more for an efficient immune response. For example, it is preferable to administer 5 times or more at an administration interval of 5 days or more and 10 days or less, preferably once a week. However, when there is a symptom such as redness or fever at the injection site, for example, administration can be resumed after omission of one or more administrations. The upper limit of the number of administrations is not particularly limited, and may be set until a sufficient immune response is obtained unless symptoms such as inflammation and pain appear in the patient.

  When the vaccine of the present invention is administered twice or more, the peptide to be blended as an active ingredient may be appropriately changed. For example, it is possible to appropriately obtain a blood sample from a patient, specify a peptide that activates cytotoxic T cells and a peptide that does not activate, and select a peptide that provides a good immune response.

  Although the subject of administration of the vaccine of the present invention is pancreatic cancer patients, it can also be administered to patients whose pancreatic cancer cells have metastasized to lymph nodes or other organs.

  EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention is not limited by the following examples, but may be appropriately modified within a range that can meet the purpose described above and below. Of course, it is possible to implement them, and they are all included in the technical scope of the present invention.

Example 1: Confirmation of location of IGF2BP3 in metastatic pancreatic cancer cells The location of IGF2BP in pancreatic ductal adenocarcinoma (PDAC) cells was confirmed using an immunocytochemical technique. Two types of PDAC cells were used: moderately differentiated PDAC cells (S2-013 strain) and undifferentiated PDAC cells (PANC-1 strain).

(1) Culture of pancreatic cancer cells S2-013 strain, a sub-system of human PDAC cell SUIT-2 strain, was obtained from Prof. Takeshi Iwamura of Miyazaki University. The PDAC cell PANC-1 strain was purchased from American Type Culture Collection. These cells, in Dulbecco's modified Eagle's medium containing 10% fetal calf serum (FCS) inactivated by heat (DMEM, manufactured by Gibco-BRL), were cultured in a humidified atmosphere containing 5% CO _2.

(2) Confocal immunofluorescence microscopy test A glass coverslip was coated with 10 μg / mL fibronectin (Sigma-Aldrich) for 1 hour at room temperature. Each PDAC cell was seeded on the fibronectin-coated coverslips and incubated for 5 hours. Next, the cells were fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100, coated with a blocking solution (3% BSA / PBS), and then incubated with an anti-IGF2BP3 primary antibody or the like for 1 hour. . Furthermore, fluorescent dyes Alexa488-, Alexa546-, Alexa594- or Alexa647-conjugated secondary antibody (manufactured by Cytoskeleton) were used in the presence or absence of rhodamine-conjugated phalloidin (manufactured by Cytoskeleton). Some primary antibodies were coupled with green or red fluorophores using commercially available antibody labeling technology ("Zenon (R)" from Life technologies). Visualization was performed with a microscope (“Zeiss LSM 510 META microscope” manufactured by Carl Zeiss), and photographs of the obtained microscopic images are shown in FIGS.

(3) Results and discussion When the S2-013 strain or the PANC-1 strain, which is a pancreatic ductal adenocarcinoma cell, was cultured on fibronectin to promote the formation of cell membrane protrusions, as shown by the arrow part shown in the photograph of FIG. IGF2BP3 was accumulated in cell membrane processes. According to FIG. 2, which is a Z-stack (multilayer uptake) photograph in a confocal immunofluorescence microscope test, in the S2-013 strain stimulated with fibronectin, IGF2BP3 was found in cytoplasmic granules localized in cell membrane processes. Expression could be confirmed.

  From these findings and the above results, it was clarified that IGF2BP3 is important for pancreatic cancer cell motility and invasion because IGF2BP3 accumulates in cell membrane processes, which are essential sites for cell motility.

Example 2: IGF2BP3 knockdown test in metastatic pancreatic cancer cells (1) IGF2BP3-RNA interference In order to clarify the influence of IGF2BP3 on the invasion and metastasis of pancreatic cancer cells, an siRNA specific to the IGF2BP3 gene is expressed. Was used to inhibit the expression of the IGF2BP3 gene in the S2-013 cell line.

GP2-293 packaging cells (Clontech) were added to scrambled negative controls (OriGene Technologies "
TR30013 ") or a pGFP-V-RS vector (OriGene Technologies) that generates replication-defective lentiviruses incorporating siRNA targeting IGF2BP3 mRNA (" TG310221 "from OriGene Technologies). Subsequently, the S2-013 cell line was infected with GP-293 packaging cells containing each siRNA. 48 hours after infection, the S2-013 cell line is transferred to a flask and cultured in DMEM medium containing 0.5 μg / mL puromycin for 7 days to stably express siRNA targeting IGF2BP3 mRNA. A large number of clonal cells derived from the S2-013 cell line were obtained. The degree of suppression of IGF2BP3 expression in each clonal cell was analyzed by Western blot. The results are shown in FIG. In FIG. 3, “Scr” is a control clone that constantly expresses scrambled negative control siRNA, and “siIGF” is a clone that constantly expresses IGF2BP3-specific siRNA and suppresses IGF2BP3 expression. “GAPDH” is glyceraldehyde-3-phosphate dehydrogenase as an internal control indicating that an equal amount of each sample is used in Western blot analysis.

  As shown in FIG. 3, knockdown of IGF2BP3 was confirmed by immunoblotting. In the following experiments, Scr-1 and Scr-2, which are clonal cells in which the expression of the IGF2BP3 gene was confirmed to be suppressed by Western blot analysis, were used.

  Separately, it was confirmed by in vitro MTT assay that suppression of IGF2BP3 gene expression did not affect the proliferation of pancreatic ductal adenocarcinoma cells (S2-013 strain or PANC-1 cells).

(2) Wound area motility assay In each wound healing assay, a plastic pipette tip was used to make a cruciform cut as a wound area in a confluent monolayer cell. After marking some wound areas to be observed, photographs were taken using a phase contrast microscope. Cells that migrated to the marked wound area were observed for 1 to 8 hours and photographed continuously. The degree of blockage of the wound area by the migrated cells was quantified by counting the migrated cells. The photograph of a cell is shown in FIG. 4, and a quantitative value is shown in FIG.

  As shown in the results shown in FIG. 4, by suppressing the expression of the IGF2BP3 gene, the cell migration to the wound region of the confluent culture S2-013 strain was inhibited. Moreover, as shown in FIG. 5, the number of cells that migrated to the wound area was significantly reduced by inhibiting the expression of the IGF2BP3 gene by RNA interference.

(3) Immune cell staining of advanced cell movement section Using a plastic pipette chip, a cross-shaped cut was made in confluent S2-013 cell line, and then cell migration to the wound area was promoted. After 4 hours, the cells were immunostained with a primary antibody and incubated with a phosphor-conjugated secondary antibody as in Example 1 above. The cell movement advanced part that began to migrate to the wound region was observed with a confocal laser scanning fluorescence correlation spectroscopy microscope (“Zeiss LSM 510 META microscope” manufactured by Carl Zeiss). The results are shown in FIG. As shown in FIG. 6, IGF2BP3 was accumulated in the cell advanced part where actin of the S2-013 cell line that started migration was polymerized.

(4) Matrigel Invasion Assay 3.0 × 10 ⁴ cells were seeded in the upper chamber of Matrigel Invasion Chamber (24 well plate, pore size: 8 μm, manufactured by Becton Dickinson). Serum-free medium was added to the upper chamber and a solvent containing 5% FCS was added to the lower chamber. Cells were incubated for 22 hours in the upper chamber. Then, three independent areas were observed with a microscope, and cells infiltrated by lysing Matrigel into the lower chamber were counted. The same experiment was repeated three times, and the S2-013 control clone (Scr-1 and Scr-2) constitutively expressing scrambled negative control siRNA and IGF2BP3 specific siRNA constitutively expressed to suppress IGF2BP3 expression. The S2-013 clones (siIGF-1 and siIGF-2) were compared. The results are shown in FIG.

  As a result of the invasiveness assay using Matrigel Invasion Chamber as shown in FIG. 7, the invasiveness of S2-013 strains (siIGF-1 and siIGF-2) in which the expression of IGF2BP3 was inhibited by RNA interference was found to be in the control (Scr -1 and Scr-2) were significantly suppressed.

  Further, in order to amplify cDNA encoding all amino acids of IGF2BP3, RT-PCR was performed using RNA of S2-013 strain cells as a template. The obtained PCR product was introduced into a pCMV6-Entry vector (produced by Origen) that generates a myc-DDK tag at the C-terminus. Hereinafter, the obtained vector is referred to as “IGF2BP3-pCMV6”. In order to restore IGF2BP3 expression in siIGF-1 and siIGF-2 clone cells, IGF2BP3-pCMV6 was transfected into siIGF-1 and siIGF-2 clone cells using X-tremeGENE HP DNA Transfection Reagent (manufactured by Roche). It was. The results are shown in FIG. “*” In FIG. 7 indicates that cells transfected with IGF2BP3-pCMV6 significantly recovered cells infiltrated at p <0.005 in the t-test compared to cells transfected with the mock vector. Show.

  As shown in FIG. 7, transfection of IGF2BP3-pCMV6 into IGF2BP3-RNAi S2-013 cell line has been found to invalidate the suppression of cell invasion caused by IGF2BP3-RNAi by restoring IGF2BP3 expression. It was issued.

(5) Orthotopic transplantation of mice and tumor cells 6-week-old sterile female athymic nude mice (BALB / cSlc-nu / nu) were purchased from Japan SLC Co., Ltd. according to the Kochi University Research Institute Animal Care and Use Guidelines Handled. An S2-013 control clone (Scr-1 and Scr-2) that constitutively expresses scrambled negative control siRNA and an S2-013 clone (siIGF) that constitutively expresses IGF2BP3-specific siRNA and suppresses IGF2BP3 expression −1 and siIGF-2) were surgically and orthotopically implanted into the pancreas of each mouse to form tumors in the mouse pancreas. 42 days after transplantation, the mice were sacrificed, stained with hematoxylin-eosin, and examined for the presence of infiltration into the retroperitoneum from the tumor formed in the pancreas of the mice and the presence of metastasis to the lung and liver. In addition, tumors formed in the mouse pancreas were weighed. Table 1 shows the measurement results, the presence or absence of distant metastasis to the retroperitoneum, and FIG. 8 shows a stained photograph. In FIG. 8, “R” indicates a cavity peritoneum tissue, “P” indicates a muscle tissue, and “N” indicates a normal tissue.

  As shown in Table 1, the invasion from the tumor formed in the mouse pancreas to the retroperitoneum was observed in clone cells (siIGF−) in which IGF2BP3 expression was suppressed compared to control clone cells (Scr-1 and Scr-2). 1 and siIGF-2) were significantly lower. In addition, pancreatic cancer tissue derived from control clonal cells metastasized to the liver and lung, whereas clonal cell derived pancreatic cancer tissue in which IGF2BP3 expression was suppressed did not metastasize.

  In addition, as shown in FIG. 8, pancreatic cancer tissue derived from control clone cells invaded the entire pancreatic tissue of the mouse. In particular, the boundary between tumor tissue and normal tissue was not clear in the control sample. On the other hand, most of the clonal cell-derived pancreatic cancer tissue in which IGF2BP3 expression was suppressed was encased in mouse stromal cells and clearly separated from mouse normal pancreatic tissue. In mice transplanted with control clone cells, the surface of the peritoneum is covered with a relatively thick cancer cell layer seeded from pancreatic cancer tissue derived from control clone cells, and the cancer cells infiltrate to the muscle layer. It was. On the other hand, in the mice transplanted with clonal cells in which IGF2BP3 expression was suppressed, cancer cells were not observed in most of the peritoneum. In addition, mice transplanted with control clone cells showed metastases in the lung and liver, whereas mice transplanted with clone cells with suppressed IGF2BP3 expression had no metastases to the lungs and liver. It was. From the above results, it can be said that IGF2BP3 can be a novel therapeutic target molecule for pancreatic cancer by inhibiting the expression of IGF2BP3 in pancreatic cancer cells and completely preventing metastasis to the lung, liver and the like. .

(6) Discussion From the above results, IGF2BP3 particularly promotes invasion and metastasis of pancreatic cancer cells, and in the formation of pancreatic tumors in vivo, the decrease in the expression level of IGF2BP3 is 1) the increase of tumors in the pancreas And 2) it affects the invasion of adjacent tissues of the pancreas and 3) affects the metastasis to other tissues.

Example 3: Role of IGF2BP3 in the formation of cell membrane projections of cells Using a confocal microscope, the polymerization of actin filaments and the three-dimensional structure of cell membrane projections in S2-013 cell line stimulated with fibronectin were examined. The detailed conditions of the confocal microscope were the same as in Example 1 above. The results are shown in FIGS.

  FIG. 9 is a photograph of an actin filament formed directly under the cell membrane. As shown in FIG. 9, compared to the S2-013 control clone (Scr-1) that constitutively expresses scrambled negative control siRNA, IGF2BP3-specific siRNA was constitutively expressed and S2-013 expression was suppressed. The amount of polymerized actin filaments in the clone (siIGF-1) is small. This result shows that transfection of IGF2BP3-pCMV6 into IGF2BP3-RNAi S2-013 cell line restores IGF2BP3 expression and restores actin filament polymerization directly under the cell membrane.

  FIG. 10 shows the ratio of cells having cell membrane processes in all cells. As shown in FIG. 10, the proportion of cells having cell membrane protrusions in the clones (siIGF-1 and siIGF-2) in which IGF2BP3 expression was suppressed is significantly smaller than that in the control clones (Scr-1 and Scr-2).

  FIG. 11 is a photograph using a phase contrast microscope of control clonal cells (Scr-1) and clonal cells (siIGF-1) in which IGF2BP3 expression was suppressed. As shown in FIG. 11, the control clonal cells were spindle-shaped and exhibited a fibroblast-like morphology, while the clonal cells in which IGF2BP3 expression was suppressed were round and exhibited an epithelial cell-like morphology. Since fibroblast-like morphology is often more prone to invasion and metastasis than epithelial cell-like morphology, this result indicates that clonal cells with suppressed IGF2BP3 expression infiltrated more than control clonal cells with suppressed IGF2BP3 expression. Shows low metastatic potential.

Claims (5)

  A pancreatic cancer cell invasion and metastasis-suppressing vaccine comprising a peptide having a part of the amino acid sequence of human insulin-like growth factor 2 mRNA binding protein 3 (SEQ ID NO: 1).   The vaccine for suppressing pancreatic cancer cell invasion and metastasis according to claim 1, wherein the peptide has 8 or more amino acids.   The pancreatic cancer cell invasion and metastasis-suppressing vaccine according to claim 1 or 2, wherein the peptide has an amino acid sequence selected from amino acid sequences of SEQ ID NOs: 2 to 49.   The pancreatic cancer cell invasion and metastasis-suppressing vaccine according to any one of claims 1 to 3, further comprising a peptide having a part of the amino acid sequence of KIF20A in addition to the peptide.   The pancreatic cancer cell invasion and metastasis-suppressing vaccine according to any one of claims 1 to 4, wherein the peptide is capable of binding to both HLA-A24 and HLA-A2.