KR20140147985A - Composition for detecting metastatic tumor - Google Patents

Abstract

The present invention relates to a composition for diagnosing metastatic cancer including preparations capable of detecting CD31 and Tie2 in cancer-associated macrophages, a kit including the composition for diagnosing metastatic cancer, and a method for providing information for diagnosing metastatic cancer by using the diagnosing composition and the kit. The kit for diagnosing metastatic cancer of the present invention is used to not only check development of metastatic cancer by using lymphatic gland of a patient, but also distinguish hyperplastic lymphatic gland and metastatic lymphatic gland having similar symptoms in the lymphatic gland to increase success rate of diagnosing metastatic cancer to be widely used in prevention and treatment of metastatic cancer through early diagnosis.

Description

[Composition for detecting metastatic tumor]

More particularly, the present invention relates to a composition for the diagnosis of metastatic cancer diseases, which comprises a preparation capable of detecting CD31 and Tie2 of tumor-associated macrophages in a lymph node, a metastatic cancer A kit for diagnosing disease, and a method for providing information for diagnosis of metastatic cancer disease using the diagnostic composition or kit.

Macrophages are highly specialized proliferating cells that play an important role in adaptive immunity and innate immunity. Macrophages are classified as M1, or "classically activated" phenotype and M2, or "alternatively activated" phenotype in the in vitro response of the originally induced macrophages. Macrophages acquire the M1 phenotype after LPS (lipopolysaccharide) injection and / or acquire the challenge of IFN-γ (Interferon gamma), secrete high levels of inflammatory-promoting cytokines, and have antimicrobial activity The branch produces reactive oxygen species. In contrast, the M2 phenotype secreting an anti-inflammatory cytokine (e.g., IL-10, TGF-beta, IL-4) that is more associated with tissue repair and inflammation is associated with IL-4 and / or IL- I will. Recent studies suggest that macrophages in tumors have alternatively activated the M2 phenotype rather than the M1 phenotype. In addition, tumor-associated macrophages (TAMs) share a common feature of both types and consist of several distinct cell populations with similar overall similarities to macrophages involved in development.

Tumor-associated macrophages (TAMs) are the most abundant cancer stromal cells in the tumor microenvironment. Recent clinical and experimental studies have shown that these TAMs play a central role in tumor immunosuppression, And provides a suitable microenvironment for the progression. The results of previous studies indicate that the TAMs mediate tumor angiogenesis, increase extracellular matrix degradation, promote tumor cell invasion and migration, inhibit the anti-tumor immune response, It is expected to promote proliferation. The invasion of TAMs promotes the deterioration of most malignant tumors such as breast, cervical, Hodgkin's lymphoma, hepatocellular epithelial carcinoma, lung epithelial carcinoma, ovary, prostate, thyroid, skin melanoma, but CD68, CD163 and the like are also detected in inflammatory macrophages. Therefore, there is a need to develop markers specifically detected in TAMs, but studies on the markers have not yet been reported.

Under these circumstances, the present inventors have made extensive efforts to develop markers specifically detected in TAMs, and as a result, it has been confirmed that TIE2 and CD31 can be used as markers capable of distinguishing inflammatory macrophages from TAMs, Respectively.

It is an object of the present invention to provide a composition for diagnosing metastatic cancer diseases, which comprises an agent capable of detecting CD31 and Tie2 of tumor-associated macrophages in a lymph node.

Another object of the present invention is to provide a kit for the diagnosis of metastatic cancer diseases comprising the above composition.

It is still another object of the present invention to provide a method for providing information for diagnosis of metastatic cancer diseases using the diagnostic composition or kit.

In one embodiment for achieving the above object, the present invention provides a method for detecting the level of a protein selected from the group consisting of TIE2, CD31, or a combination thereof present in tumor-associated macrophages (TAMs) A mRNA level of the gene for the metastatic cancer disease.

The present inventors have focused on tumor-associated macrophages (TAMs) while carrying out various studies to develop a method capable of specifically detecting metastatic cancer diseases. Since the TAMs are located inside or near the tumor and are known to be involved in the process of promoting or inhibiting the tumor, if the TAMs can be detected in the lymph node known as the pathway of the metastatic cancer disease, And predicted that it would be possible to diagnose it effectively. However, the TAMs are a type of macrophages, and the macrophages are known to be distributed in large numbers in the lymph nodes. When diseases such as inflammatory diseases are developed, the macrophages present in the lymph nodes are mutated in a form similar to that of TAMs Therefore, confirming whether the macrophages present in the lymph nodes are TAMs from the patients suspected of having metastatic cancer disease has a disadvantage that the success rate is very low.

As a result of various studies to develop a method for confirming whether macrophages present in the lymph nodes are TAMs, the CD31 or Tie2 protein is specifically expressed only in TAMs in the lymph nodes, and thus the macrophages in the lymph nodes CD31 or Tie2 protein to confirm whether or not the macrophage is TAMs. By confirming whether or not the macrophage is TAMs, it is possible to diagnose metastatic cancer disease in a subject derived from a lymph node sample containing the TAMs Respectively.

The term " CD31 (cluster of differentiation 31) " of the present invention is also referred to as a platelet endothelial cell adhesion molecule (PECAM-1) and plays an essential role in removing neutrophils generated in the body as the age increases. Quot; refers to a protein expressed by a polynucleotide comprising a sequence and 2426 nucleotide sequences coding the same. The amino acid sequence or base sequence of CD31 can be obtained from a known database such as NCBI GenBank, and may be, for example, an amino acid sequence or a base sequence represented by GenBank: M37780.1.

The term "Tie2" of the present invention is a type of tyrosine-protein kinase receptor that is predominantly expressed in epithelial cells and hepatocytes. Tie2 derived from human is composed of 1124 amino acids, An extracellular domain consisting of two Ig-like domains, three EGF-like domains, one Ig-like domain, three fibronectin type-III; Membrane passage domain; And the intracellular tyrosine kinase domain (UniProtKB / Swiss-Prot entry Q02763). The amino acid sequence or base sequence of Tie2 can be obtained from a known database such as NCBI's GenBank. It may be an amino acid sequence or a base sequence represented by NG_011828.1. The Tie2 is known to play an essential role in vasculogenesis, angiogenesis and hematopoiesis, and the ligands binding to Tie2 identified so far include angiopoietin -1, Ang1), angiopoietin-2, Ang2, angiopoietin-3, Ang3 and angiopoietin-4, Ang4 , And these ligands are known to bind to the two Ig-like domains.

The term "diagnosing" of the present invention means identifying the presence or characteristic of a pathological condition. In the present invention, the diagnosis can be interpreted as confirming whether or not the metastatic cancer disease occurs.

The term " metastatic tumor "of the present invention refers to a metastatic cancer, which is a type of cancer caused by the proliferation of metastatic cancer cells, which have been separated from the site where the cancer was first developed, through the blood, . The metastatic cancer disease may be metastatic liver cancer, metastatic bone cancer, metastatic brain cancer, metastatic lung cancer, metastatic peritoneal cancer, metastatic melanoma, metastatic gastric cancer, metastatic eye cancer, metastatic cancer, Tumor, and the like.

The term "agent for measuring mRNA level of a gene " of the present invention means a preparation used for measuring the level of mRNA transcribed from the target gene in order to confirm the expression of the target gene contained in the sample But are not limited to, RT-PCR, competitive RT-PCR, real-time RT-PCR, RNase protection assay (RPA) And may be a probe or primer capable of specifically binding to a target gene used in methods such as Northern blotting and DNA chip analysis.

The term "primer" of the present invention refers to a nucleic acid sequence having a short free 3 'hydroxyl group, capable of forming base pairs with a complementary template and having a starting point for template strand copying ≪ / RTI > The primer can initiate DNA synthesis in the presence of reagents and four different nucleoside triphosphates for polymerization reactions (i.e., DNA polymerase or reverse transcriptase) at appropriate buffer solutions and temperatures. The PCR conditions, the lengths of the sense and antisense primers can be modified based on what is known in the art.

In the present invention, the primer can be used as a means for amplifying and detecting CD31 or Tie2 gene contained in the cDNA after synthesizing cDNA from mRNA obtained from a macrophage contained in a patient's lymph node sample. Herein, the polynucleotide sequence of the primer is not particularly limited as long as it can amplify the CD31 and Tie2 genes contained in the cDNA. Preferably, the polynucleotide sequence of SEQ ID NOS: 7 and 8, which is capable of amplifying the CD31 gene, Or a primer having a polynucleotide sequence of SEQ ID NOs: 29 and 30 capable of amplifying the Tie2 gene can be used.

The term "probe" of the present invention means a nucleic acid fragment such as RNA or DNA corresponding to a short period of a few nucleotides or several hundreds of nucleotides capable of specifically binding to a gene or an mRNA. The oligonucleotide probe, For example, in the form of a single stranded DNA probe, a double stranded DNA probe, an RNA probe, or the like, and may be labeled for easier detection.

In the present invention, the probe may be used as a means for detecting CD31 or Tie2 gene contained in the cDNA after synthesizing cDNA from mRNA obtained from macrophages contained in a patient's lymph node sample. At this time, the probe is not particularly limited in its polynucleotide sequence as long as it can amplify CD31 and Tie2 genes contained in the cDNA.

The term "agent for measuring the level of protein " of the present invention means a preparation used for measuring the level of a target protein contained in a sample, and is not particularly limited to, but preferably western blotting, , Enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), radioimmunodiffusion, Ouchterlony immunodiffusion, rocket immunoelectrophoresis, tissue immuno staining, immunoprecipitation assay (Immunoprecipitation Assay), Complement Fixation Assay (FACS), FACS and protein chip assay.

The term "antibody" of the present invention means a proteinaceous molecule capable of specifically binding to an antigenic site of a protein or peptide molecule. Such an antibody can be prepared by cloning each gene into an expression vector according to a conventional method, A protein encoded by the marker gene can be obtained and can be produced from the obtained protein by a conventional method.

In the present invention, the antibody is used as a means for detecting CD31 or Tie2 protein contained in macrophages present in a patient's lymph node sample, preferably a polyclonal antibody capable of specifically binding to CD31 or Tie2 protein Antibody, monoclonal antibody, or antigen-binding antibody, any part thereof may be included in the antibody of the present invention and may include all immunoglobulin antibodies as well as specific antibodies such as humanized antibodies. In addition, the antibody comprises a functional fragment of an antibody molecule as well as a complete form having two full-length light chains and two full-length heavy chains. A functional fragment of an antibody molecule means a fragment having at least an antigen-binding function, and can be Fab, F (ab ') 2, F (ab') 2, Fv and the like.

According to one embodiment of the present invention, macrophages are present in metastatic lymph nodes (MLNs) induced by inoculation of GFP + B16F1 melanoma cells and are present in the invasion area of the melanoma cells (Fig. 1) As a result of comparing the gene expression patterns of TAMs and other normal or abnormally induced lymph nodes, TIAs present in MLNs specifically detected high levels of TIE2 protein and CD31 protein (FIG. 2), and the production of MLNs in mice (TIE2 + / CD31 + TAMs) in which the TIE2 protein and the CD31 protein are produced at the same time, and the onset of the metastatic cancer disease that produces the MLNs can be diagnosed through the detection of TIE2 + / CD31 + TAMs (FIG. 3), TIE2 + / CD31 + TAMs were specifically detected in MLNs derived from human breast cancer patients, but TIE2 + / CD31 + TAMs were not detected in hyperplastic lymph nodes (Fig. 4) was confirmed.

These results indicate that the TIE2 + / CD31 + TAMs are specifically detected only in MLNs and are not detected in hyperplastic lymph nodes showing similar clinical symptoms. Therefore, diagnostic markers that can significantly improve the diagnostic success rate of metastatic cancer diseases using lymph nodes Can be used.

Accordingly, the composition for diagnosing metastatic cancer disease of the present invention includes an agent for measuring the mRNA level of a gene encoding CD31 or Tie2 or the level of a protein expressed therefrom. In this case, the agent may be a primer or a probe capable of measuring mRNA level of CD31 or Tie2-encoding gene, or may be an antibody capable of measuring CD31 or Tie2 protein level. The antibody may be a polyclonal antibody, a monoclonal antibody or a fragment (Fab, F (ab '), F (ab') 2 and Fv etc.) of the antibody capable of specifically binding to CD31 or Tie2 protein. .

As another embodiment for achieving the above object, the present invention provides a kit for diagnosing metastatic cancer diseases comprising the above composition.

The kit of the present invention diagnoses the onset of metastatic cancer disease by measuring the mRNA level of the gene encoding CD31 or Tie2 or the level of the protein expressed therefrom from the macrophage present in the lymph node of the patient suspected of having metastatic cancer disease But are not limited to, primers, probes or antibodies for measuring the mRNA level of the gene or the level of the protein expressed therefrom, as well as one or more other component compositions suitable for the assay, solutions Or device.

As a specific example, the kit for measuring the mRNA expression level of the CD31 or Tie2 gene of the present invention may be a kit containing essential elements necessary for performing RT-PCR. The RT-PCR kit can be used in combination with a test tube or other appropriate container, a reaction buffer (pH and magnesium concentration varies), deoxynucleotides (dNTPs), Taq polymerase and reverse transcriptase , DNase, RNAse inhibitor, DEPC-water, sterile water, and the like. In addition, it may contain a primer pair specific to a gene used as a quantitative control.

As another example, the kit of the present invention may contain the necessary elements necessary for performing DNA chip analysis. The kit for DNA chip analysis may include a substrate on which a cDNA corresponding to a gene or a fragment thereof is attached as a probe, and a reagent, a preparation, and an enzyme for producing a fluorescent-labeled probe. In addition, the substrate may contain a cDNA corresponding to a quantitative control gene or a fragment thereof.

As another example, the kit of the present invention may be a kit for analyzing a protein chip for measuring the level of a protein expressed from a CD31 or Tie2 gene. The kit may be used for immunological detection of an antibody, A substrate, a suitable buffer solution, a secondary antibody labeled with a chromogenic enzyme or a fluorescent substance, a chromogenic substrate, and the like. The substrate may be a nitrocellulose membrane, a 96-well plate synthesized with a polyvinyl resin, a 96-well plate synthesized from a polystyrene resin, a glass slide glass or the like may be used, But not limited to, peroxidase and alkaline phosphatase. Fluorescent materials include, but are not limited to, FITC, RITC and the like. The coloring substrate is not limited to ABTS Azo-bis (3-ethylbenzothiazoline-6-sulfonic acid) or OPD (o-phenylenediamine), TMB (tetramethylbenzidine).

In another aspect of the present invention, the present invention provides a method for providing information for diagnosing metastatic cancer disease using the diagnostic composition or kit, Measuring the level of a protein selected from the group consisting of TIE2, CD31 and combinations thereof from the macrophages present in isolated lymph nodes of the suspected individual or the mRNA level of the gene encoding the same; And (b) comparing the measured level of the protein or the mRNA level of the gene encoding the same to the level measured from macrophages present in separate lymph nodes of a normal control and judging that the metastatic cancer disease is likely to occur when the level is significantly increased . At this time, the method of measuring the mRNA level of the gene or the level of the protein expressed therefrom is the same as described above.

The term "individual" of the present invention means a living organism in which a metastatic cancer disease can develop. The subject may be any organism of the "mammal" class of higher vertebrate animals that nourishes their offspring with milk secreted by the mammary gland, preferably mice, rats, rabbits, dogs, cats, .

The use of the kit for diagnosing metastatic cancer disease of the present invention can confirm whether or not the metastatic cancer disease has occurred using the lymph node of the patient and also can distinguish the metastatic lymph node from the hyperplastic lymph node showing similar symptoms in the lymph node, It can be widely used for prevention and treatment through early diagnosis of metastatic cancer disease.

Brief Description of the Drawings Fig. 1 is a photograph showing the results of histopathological analysis of metastatic lymph nodes (MLNs) derived from B16F1-GFP melanoma mice, in which (a) represents a representative B16F1-GFP melanoma mouse with a primary tumor, (C) is a photograph showing a state in which hematoxylin and eosin staining (H & E) are performed, (d) is a photograph showing a result of performing GFP fluorescence staining, and (F) is an enlarged photograph of a fluorescent image of a rectangular region in FIG. 1 (d), and FIG. 1 (g) is a photograph showing the result of immunostaining using anti-F4 / This is an enlarged photograph of the immunohistochemical staining of the rectangle.
Figure 2 is a diagram showing the results of the isolation of CD11b + TAMs from MLNs via FACS and the expression of 18 macrophage phenotypes associated with peritoneal macrophages (PMs), peritoneal macrophages (PM-LPS) treated with LPS, and CD11b + TAMs (A) shows the results obtained by staining TAMs isolated from metastatic lymph nodes of B16F1 melanoma mice with anti-CD11b and anti-CD206 antibodies and classifying the cells by FACS (B) shows the results of comparing the expression levels of TGF-β expressed in PM, PM-LPS and CD11b + TAMs, (c) shows the relative expression levels of 18 genes expressed in PM, (E) represents the relative expression level of 18 genes expressed in CD11b + TAMs, (f) represents the relative expression level of 18 genes expressed in PM-LPS, , IL-4 and IL-10 Comparative Results of the.
FIG. 3 is a photograph showing the results of immunostaining of levels of F4 / 80 protein, TIE2 protein and CD31 protein present in normal lymph nodes or metastatic lymph nodes (MLNs) derived from B16F1-GFP melanoma mice, (B) shows the results of immunohistochemical staining of F4 / 80 protein and TIE2 protein in MLNs, and (c) MLNs immunoreactivity in MLNs (D) immunohistochemically stained with F4 / 80 protein and TIE2 protein, (d) immunostained with F4 / 80 protein and CD31 protein, and (e) MLNs The results of immunohistochemical staining of TIE2 protein and CD31 protein in duplicate are shown.
FIG. 4 is a photograph showing the results of immunostaining for the levels of CD163 protein, TIE2 protein and CD31 protein in human breast cancer hyperplastic lymph nodes or MLNs, wherein (a) (B) shows immunostaining of CD163 protein, TIE2 protein and CD31 protein in MLNs, and (c) shows the results of immunohistochemistry of CD163 protein and TIE2 (D) shows immunostaining of CD163 protein and CD31 protein in MLNs, and (e) MLNs in which the TIE2 protein and CD31 protein are double-stranded Immunostaining results are shown.

Hereinafter, the present invention will be described in more detail with reference to examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

Example  1: Confirmation of macrophage infiltration in metastatic lymph nodes of B16F1 melanoma model

To investigate the role of macrophages in metastatic lymph nodes, a stereotactic B16F1 melanoma model was used.

Specifically, 4 × 10 ⁵ B16F1 melanoma cells expressing GFP (B16F1-GFP) were inoculated through the left ankle of an 8-week-old C57BL / 6 male mouse through the left frontal sole, and cultivated for 14 days , Allowing tumor cells having a diameter of 1.0 cm to grow (Fig. 1 (a)). The mice were then sacrificed and metastatic lymph nodes (MLNs) (LN of b in Figure 1) were obtained by collecting primary tumors, local lymph nodes (left underarm and bronchial lymph nodes), and non-local lymph nodes.

To confirm the presence of lymph node metastasis, GFP images from 2.5-mu m-thick sections of the MLNs were obtained using a Zeiss Axio Imager Z1 microscope (Carl Zeiss). Hematoxylin & eosin (H & E) staining was performed under standard conditions and the stained sections were photographed with a Zeiss Axio Imager Z1 microscope. As a result, lymph node metastasis was observed through hematoxylin and eosin staining (H &E; FIG. 1C) and green fluorescence (GFP, FIG. 1D).

In order to confirm the detection of macrophages in the MLNs obtained, immunostaining was performed using an anti-F4 / 80 antibody capable of detecting macrophage specific marker F4 / 80. Specifically, MLNs were fixed in 10% neutral-buffered formalin and embedded in paraffin. Immunohistochemical staining was then performed with rat anti-mouse anti-F4 / 80 monoclonal (clone BM8; eBioscience, San Diego, Calif., USA) antibody. As a result, cancer cells were present in the MLNs (d and f in Fig. 1), and most of the macrophages detected by the antibody were located along the invasion area of the MLNs (e and g in Fig. 1).

From the above results, it was found that macrophages expressing F4 / 80 were present in the invasion area rather than the necrotic areas of GFP + B16F1 melanoma cells.

Example  2: tumor-associated macrophage ( Tumor - associated macrophages , TAMs ) Analysis of gene expression pattern

The CD11b + macrophage population was isolated by performing a fluorescence-activated cell sorting (FACS) method using the MLNs obtained in Example 1 (Fig. 2 (a)). Specifically, the MLNs were filtered through cell strainers (pore size, 70-μm diameter, BD Falcon, Franklin Lakes, NJ, USA) and then washed with FACS buffer (PBS, 0.2% FBS, 2 mM EDTA) And washed. Live cells were then counted using a haemocytometer and trypan blue exclusion. In addition, single cell suspensions were stained with PE-conjugated anti-CD11b antibody (clone M1 / 70, BD Biosciences) and APC-conjugated anti-CD206 antibody (eBiosciences) for 30 min on ice. The dyed samples were cell classified with FACSAria II (Bioscience) and the data were collected and analyzed using software package FACSDiva (BD Bioscience) and FlowJo (Tree star, Ashland, OR, USA).

Tumor-associated macrophages (TAMs) present in the isolated macrophage population and the thyoglycollate-elicited peritoneal macrophages (PM) used as controls Quantitative real-time PCR (qRT-PCR) was performed. At this time, the PM used as the control group was obtained from C57BL / 6 mice. Specifically, 2 mL of 3% brewer thioglycollate medium (Difco, Detroit, MI, USA) was intraperitoneally injected into C57BL / 6 mice, and after 3 days, abdominal cavity was washed with cold PBS, ≪ / RTI > The resulting macrophages were centrifuged at 1000 rpm for 5 minutes, washed three times with PBS, and resuspended in DMEM medium supplemented with 10% FBS (Gibco). The resuspended macrophages were inoculated into a cover glass in a chamber at a cell density of 1 x 10 ⁵ cells / ml, allowed to adhere for 3 hours, and unattached cells were washed. Finally, PM was obtained by allowing the adhered macrophages to react for 3 hours with or without 10 ng / ml of LPS.

Total RNA was extracted from the separated macrophages and qRT-PCR was applied to a cDNA synthesis kit (Takara, Tokyo, Japan) to synthesize cDNA. Then, using the synthesized cDNA, ARG1, CCL2, CD11b , TGF-?, And VEGF genes were amplified by PCR using the method described in Example 1, . At this time, as a control group, a gene encoding cyclophilin A (cycl A) was used, and the primers used were as follows:

ARG1 F: 5'-GAACTCAACGGGAGGGTAACC-3 '(SEQ ID NO: 1)

ARG1 R: 5'-TTTTCATGTGGCGCATTCAC-3 '(SEQ ID NO: 2)

CCL2 F: 5'-TTAAAAACCTGGATCGGAACCAA-3 '(SEQ ID NO: 3)

CCL2 R: 5'-GCATTAGCTTCAGATTTACGGG-3 '(SEQ ID NO: 4)

CD11b F: 5'-ATGCTTACCTGGGTTATGCTTCTG-3 '(SEQ ID NO: 5)

CD11b R: 5'-GCTGATACCGAGGTGCTCCTA-3 '(SEQ ID NO: 6)

CD31 F: 5'-TACTGCAGGCATCGGCAAA-3 '(SEQ ID NO: 7)

CD31 R: 5'-ATACTGGGCTTCGAGAGCATTT-3 '(SEQ ID NO: 8)

CD36 F: 5'-ATACAGAGTTCGTTATCTAGCCAAGGA-3 '(SEQ ID NO: 9)

CD36 R: 5'-CCATTGGGCTGTACAAAAGACA-3 '(SEQ ID NO: 10)

CD68 F: 5'-ATGCGGCTCCCTGTGTGT-3 '(SEQ ID NO: 11)

CD68 R: 5'-GGCCTTTTTGTGAGGACAGTCT-3 '(SEQ ID NO: 12)

CD163 F: 5'-CGCCCCCGCTAGACAGA-3 '(SEQ ID NO: 13)

CD163 R: 5'-CGAATATCTATGTATCGTGAGCAGACT-3 '(SEQ ID NO: 14)

CD206 F: 5'-AACCCAAGGGCTCTTCTAAAGC-3 '(SEQ ID NO: 15)

CD206 R: 5'-ACGCCGGCACCTATCACA-3 '(SEQ ID NO: 16)

F4 / 80 F: 5'-GCCTATTATCTATACCCTCCAGCACATC-3 '(SEQ ID NO: 17)

F4 / 80 R: 5'-TCCATCTCCCATCCTCCACATCAG-3 '(SEQ ID NO: 18)

IL-4 F: 5'-acaggagaagggacgccat-3 '(SEQ ID NO: 19)

IL-4 R: 5'-gaagccctacagacgagctca-3 '(SEQ ID NO: 20)

IL-6 F: 5'-CTGCAAGAGACTTCCATCCAGTT-3 '(SEQ ID NO: 21)

IL-6 R: 5'-AAGTAGGGAAGGCCGTGGTT-3 '(SEQ ID NO: 22)

IL-10 F: 5'-GGCGCTGTCATCGATTTCTC-3 '(SEQ ID NO: 23)

IL-10 R: 5'-TCTTGGAGCTTATTAAAATCACTCTTCA-3 '(SEQ ID NO: 24)

NF-κB F: 5'-TGGCAGCTCTTCTCAAAGCA-3 '(SEQ ID NO: 25)

NF-kB R: 5'-CCCAAGAGTCGTCCAGGTCAT-3 '(SEQ ID NO: 26)

PPAR-y F: 5'-CAGGCCGAGAAGGAGAAGCT-3 '(SEQ ID NO: 27)

PPAR-y R: 5'-GGCTCGCAGATCAGCAGACT-3 '(SEQ ID NO: 28)

TIE2 F: 5'-GAATGGCTCAGGCATTCCA-3 '(SEQ ID NO: 29)

TIE2 R: 5'-AAGGGCCAGAGTTCCTGCGTT-3 '(SEQ ID NO: 30)

TNF-α F: 5'-AGACCCTCACACTCAGATCATCTTC-3 '(SEQ ID NO: 31)

TNF-α R: 5'-CCTCCACTTGGTGGTTTGCT-3 '(SEQ ID NO: 32)

TGF-? F: 5'-CTGAACCAAGGAGACGGAATACA-3 '(SEQ ID NO: 33)

TGF-? R: 5'-CACGTGGAGTTTGTTATCTTTGCT-3 '(SEQ ID NO: 34)

VEGF F: 5'-GAGGAAAGGGAAAGGGTCAAA-3 '(SEQ ID NO: 35)

VEGF R: 5'-TCTGAACAAGGCTCACAGTGAAC-3 '(SEQ ID NO: 36)

cycle F: 5'-TGGAGAGCACCAAGACAGAC-3 '(SEQ ID NO: 37)

cycle R: 5'-TGCCGGAGTCGACAATGAT-3 '(SEQ ID NO: 38)

As a result, TGF-beta was expressed at a high level in PM and LPS-treated PM and TAMs, but it was not significant (Fig. 2b).

The expression level of TGF-beta was set at 100 and relative expression level was calculated. As a result, in the case of PM, CD68, CD36, TGF-beta and CCL2 genes were expressed at a high level ) And LPS-treated PM showed high levels of CD68, CD36, TGF-beta, CCL2, TNF-alpha, CD11b, F4 / 80 and IL-6 genes , The expression levels of NF-κB, TNF-α and IL-6 genes were markedly increased, indicating that the expression of cytokines was increased by LPS treatment.

On the other hand, the expression levels of F4 / 80, ARG1, TIE2 and CD31 genes are increased in TAMs and the expression levels of CCL2, CD36, and CD68 genes (Fig. 2e). In addition, the expression levels of VEGF, IL-4 and IL-10 genes were significantly higher in TAMs than those of PM and LPS-treated PM (Fig. 2f). In addition, overall, the TAMs were found to express TIE2 and CD31 genes at a significantly higher level than TGF-β.

Thus, TAMs could potently over-express angiogenesis promoting factors such as TIE2, CD31 and VEGF as well as cytokines such as IL-4 and IL-10.

Example  3: MLNs Present in TAMs Characterization of

To determine whether the gene expression pattern of TAMs was directly related to protein levels, rabbit-derived polyclonal anti-TEE2 (clone H-176, Santa Cruz Biotechnology) antibody or rat anti-mouse anti-F4 / 80 single Normal lymph nodes and MLNs were immunostained using a clone (clone BM8; eBioscience, San Diego, Calif., USA) antibody. As a result, macrophages that produce F4 / 80 protein in the normal lymph node, but not TIE2 protein were detected (FIG. 3 a), and MLNs detected macrophages that produce both F4 / 80 protein and TIE2 protein (Fig. 3 (b)). In addition, it was confirmed that the macrophages that produce both the F4 / 80 protein and the TIE2 protein were located along the invasive region of the metastatic lymph node rather than the necrotic region of the tumor (Fig. 3b).

In this regard, immunological staining of macrophages in the kidney, liver and spleen of normal mice was performed using anti-TTE2 antibody. As a result, macrophages producing TIE2 protein were not detected, (TAMs) were specifically detected only in MLNs.

On the other hand, whether TIE2 or CD31 protein was produced in TAMs present in MLNs was confirmed by co-immunostaining with F4 / 80 protein. The antibody against the TIE2 protein and the F4 / 80 protein used was the same as described above. As the antibody against the CD31 protein, an anti-mouse polyclonal anti-CD31 antibody (clone M-20; Santa Cruz Biotechnology) Respectively. As a result, TIE2 protein and F4 / 80 protein are simultaneously produced in the TAMs present in MLNs (FIG. 3C), and CD31 protein and F4 / 80 protein are produced simultaneously (FIG. (Fig. 3E).

Therefore, the generation of MLNs can be confirmed by the detection of TAMs (TIE2 + / CD31 + TAMs) in which TIE2 protein and CD31 protein are produced at the same time, and the onset of metastatic cancer disease that produces MLNs is also detected by detection of TIE2 + / CD31 + TAMs It can be diagnosed through

Example  4: TIE2 + / CD31 + TAMs Of breast cancer-related metastatic cancer

The results of Example 3 above suggest that TIE2 + / CD31 + TAMs can be used as markers for the diagnosis of metastatic cancer diseases. In order to confirm whether TIE2 + / CD31 + TAMs can be used as markers for metastatic cancer disease diagnosis in vivo .

Specifically, hyperplastic lymph nodes and MLNs were obtained from normal human and human breast cancer, respectively, and these lymph nodes were immunostained with an antibody against TIE2 protein and an antibody against CD31 protein. At this time, the control group was immuno-stained with an antibody against the CD163 protein, which is known to be predominantly expressed in macrophages.

As a result, the hyperplastic lymph nodes derived from normal persons were immunostained with only CD163 protein, and TIE2 protein and CD31 protein were not immunostained (Fig. 4 (a)). In contrast, MLNs derived from breast cancer patients were immunostained for both CD163 protein, TIE2 protein and CD31 protein (FIG. 4 b). The results were analyzed to demonstrate that TIE2 protein and CD31 protein can be used as markers specific for MLNs.

In order to confirm whether the TIE2 protein and the CD31 protein were partially or wholly present in macrophages present in MLNs, macrophages present in MLNs were immunized with antibodies against TIE2 protein or antibodies against CD31 protein and CD163 Immunostaining was performed jointly using an antibody against the protein. As a result, CD163 protein and TIE2 protein were immunostained at the same level in most macrophages present in MLNs (FIG. 4C), and CD163 protein and CD31 protein were immunostained at the same level (FIG.

These results indicate that TIE2 protein and CD31 protein are commonly produced in all macrophages present in MLNs. Thus, TIE2 protein and CD31 protein are detected in all macrophages present in MLNs. Therefore, all macrophages present in MLNs are called TAMs .

Finally, in order to confirm that the TIE2 protein and the CD31 protein can be produced at the same level in human breast cancer-derived MLNs as in the case of the mouse-derived MLNs, human breast cancer-derived MLNs were assayed for antibody to TIE2 protein and CD31 protein As a result, it was confirmed that TIE2 protein and CD31 protein were immunostained in the same level in human breast cancer-derived MLNs (FIG. 4E).

When diagnosing metastatic cancer disease using human lymph nodes, it is very likely that the hyperplasia lymph nodes and MLNs produced by the inflammatory reaction are not distinguished clinically and diagnostic errors of metastatic cancer diseases are likely to occur . Using the TIE2 + / CD31 + TAMs of the present invention, however, the diagnostic success rate of metastatic cancer diseases using lymph nodes was remarkably determined by using the characteristics of TIE2 + / CD31 + TAMs that were not detected in the hyperplastic lymph nodes and specifically detected only in MLNs It is analyzed that it can be improved.

<110> Gachon University of Industry-Academic cooperation Foundation <120> Composition for detecting metastatic tumor <130> PA130558 / KR <160> 38 <170> Kopatentin 2.0 <210> 1 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 1 gaactcaacg ggagggtaac c 21 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 2 ttttcatgtg gcgcattcac 20 <210> 3 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 3 ttaaaaacct ggatcggaac caa 23 <210> 4 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 4 gcattagctt cagatttacg gg 22 <210> 5 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 5 atgcttacct gggttatgct tctg 24 <210> 6 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 6 gctgataccg aggtgctcct a 21 <210> 7 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 7 tactgcaggc atcggcaaa 19 <210> 8 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 8 atactgggct tcgagagcat tt 22 <210> 9 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 9 atacagagtt cgttatctag ccaagga 27 <210> 10 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 10 ccattgggct gtacaaaaga ca 22 <210> 11 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 11 atgcggctcc ctgtgtgt 18 <210> 12 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 12 ggcctttttg tgaggacagt ct 22 <210> 13 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 13 cgcccccgct agacaga 17 <210> 14 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 14 cgaatatcta tgtatcgtga gcagact 27 <210> 15 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 15 aacccaaggg ctcttctaaa gc 22 <210> 16 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 16 acgccggcac ctatcaca 18 <210> 17 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 17 gcctattatc tataccctcc agcacatc 28 <210> 18 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 18 tccatctccc atcctccaca tcag 24 <210> 19 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 19 acaggagaag ggacgccat 19 <210> 20 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 20 gaagccctac agacgagctc a 21 <210> 21 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 21 ctgcaagaga cttccatcca gtt 23 <210> 22 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 22 aagtagggaa ggccgtggtt 20 <210> 23 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 23 ggcgctgtca tcgatttctc 20 <210> 24 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 24 tcttggagct tattaaaatc actcttca 28 <210> 25 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 25 tggcagctct tctcaaagca 20 <210> 26 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 26 cccaagagtc gtccaggtca t 21 <210> 27 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 27 caggccgaga aggagaagct 20 <210> 28 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 28 ggctcgcaga tcagcagact 20 <210> 29 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 29 gaatggctca ggcattcca 19 <210> 30 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 30 aagggccaga gttcctgcgt t 21 <210> 31 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 31 agaccctcac actcagatca tcttc 25 <210> 32 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 32 cctccacttg gtggtttgct 20 <210> 33 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 33 ctgaaccaag gagacggaat aca 23 <210> 34 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 34 cacgtggagt ttgttatctt tgct 24 <210> 35 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 35 gaggaaaggg aaagggtcaa a 21 <210> 36 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 36 tctgaacaag gctcacagtg aac 23 <210> 37 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 37 tggagagcac caagacagac 20 <210> 38 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 38 tgccggagtc gacaatgat 19

Claims (13)

Comprising an agent that measures the level of a protein selected from the group consisting of TIE2, CD31, and combinations thereof present in tumor-associated macrophages (TAMs) in the lymph nodes, or the mRNA level of a gene encoding the protein. A composition for diagnosing cancer diseases.
The method according to claim 1,
Wherein the agent that measures the level of the gene mRNA comprises a primer or a probe that specifically binds to the gene.
3. The method of claim 2,
Wherein the primer is a primer having a polynucleotide sequence of SEQ ID NOs: 7 and 8 capable of amplifying the CD31 gene or a primer having a polynucleotide sequence of SEQ ID NOs: 29 and 30 capable of amplifying a Tie2 gene.
The method according to claim 1,
Wherein the agent that measures the level of said protein comprises an antibody specific for said protein.
5. The method of claim 4,
Wherein the antibody is selected from the group consisting of a polyclonal antibody, a monoclonal antibody, Fab, F (ab ') 2, F (ab') 2, Fv and combinations thereof.
The method according to claim 1,
Wherein said metastatic cancer disease is a disease selected from the group consisting of metastatic liver cancer, metastatic bone cancer, metastatic brain tumor, metastatic lung cancer, metastatic peritoneal cancer, metastatic melanoma, metastatic gastric cancer, metastatic ocular tumor and combinations thereof.
The method according to claim 1,
Wherein the agent is an agent that measures levels of TIE2 and CD31 proteins or mRNA levels of genes encoding the same.
A kit for the diagnosis of metastatic cancer diseases, comprising the composition of any one of claims 1 to 7.
9. The method of claim 8,
Wherein the kit is an RT-PCR kit, a DNA chip kit or a protein chip kit.
(a) measuring the level of a protein selected from the group consisting of TIE2, CD31, and combinations thereof from macrophages present in isolated lymph nodes of individuals suspected of having metastatic cancer disease or the mRNA level of a gene encoding the same ; And
(b) comparing the measured protein level or the mRNA level of the gene encoding the same to the level measured from macrophages present in separate lymph nodes of a normal control, and determining that the metastatic cancer disease will occur if the level is significantly increased / RTI &gt; a method for providing information for diagnosing metastatic cancer disease.
11. The method of claim 10,
The mRNA level of the gene may be determined by RT-PCR, competitive RT-PCR, real-time quantitative RT-PCR, RNase protection assay (RNase protection method, Northern blotting or DNA chip technology assay.
11. The method of claim 10,
The level of the protein may be determined by Western blotting, enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), radial immunodiffusion, Ouchterlony immunodiffusion, Immunohistochemical staining, immunoprecipitation assays, complement fixation assays, immunofluorescence, immunochromatography, FACS (Immunohistochemical staining), immunofluorescence (immunohistochemical staining), immunoprecipitation assays a fluorescence activated cell sorter analysis and a protein chip technology assay.
11. The method of claim 10,
Wherein said metastatic cancer disease is a disease selected from the group consisting of metastatic liver cancer, metastatic bone tumor, metastatic brain tumor, metastatic lung cancer, metastatic peritoneal cancer, metastatic melanoma, metastatic gastric cancer, metastatic ocular tumor, and combinations thereof.

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