TWI485159B - A microvesicle membrane protein and application thereof - Google Patents

Description

A microbubble membrane protein and its application

The present invention relates to a newly discovered phosphorylated CSE1L (chromosome segregation 1-like protein) (GenBank No. U33286) microbubble membrane protein and application thereof.

Cellular microvesicles are small particles derived from cell membranes that are released into the extracellular environment and are about 0.1 μm to 100 μm in diameter. Microbubbles are involved in the transfer of proteins, genes, small RNAs, hormone receptors, cell organelles, etc. between cells and cells, and thus are associated with various diseases, including cancer (Simak 2006; Cocucci 2009; Muralidharan-Chari 2010). Invasion and metastasis of tumor cells are a major factor in the death of cancer patients. Tumor cells undergo cancer invasion and cancer metastasis by secreting proteases and decomposing extracellular matrices. The microvesicles released by tumor cells are rich in proteases that decompose the extracellular matrix, thereby playing an important role in the invasion and metastasis of cancer cells (Cocucci 2009).

CSE1L protein (chromosome segregation 1-like protein) or cellular apoptosis susceptibility protein (GenBank accession number U33286) is highly expressed in tumor tissues of various cancers ( Brinkmann 1995; Tung 2009; Tai 2010). CSE1L was previously discovered by Scherf et al. to be phosphorylated by extracellular signal-regulated kinase (ERK) and is therefore a tyrosine phosphorylated protein, and Scherf et al. found tyrosine phosphate. The function of the CSE1L protein is to promote the transport of cellular proteins from the cytoplasm to the nucleus (Scherf 1998).

The data currently available indicate that the patents associated with the CSE1L protein include: US 6,664,057 for the identification of a novel amplicon on human chromosome 20q13.2 associated with cancer. (amplicon), this amplicon contains the CSE1L gene. US 6,072,031 relates to CSE1L's complementary DNA (cDNA) and amino acid sequences for detecting the expression and amplification of the CSE1L gene in normal cells and cancer cells, and introducing the antisense CSE1L gene sequence into the living Apoptosis can be inhibited in cells. US 6,207,380 is about the performance of polypeptides and polynucleotides derived from keratin/cytokeratin, CSE1L or mat-8 in human urinary tract tissues for detection, diagnosis, monitoring, bio-angiography, prevention, and treatment of urinary tract in individuals. Systemic diseases (such as urinary system cancer). US 6,207,380 also discloses antibodies that specifically bind to urinary tract tissue keratin/cytokeratin, CSE1L or mat-8 encoded polypeptides or proteins that are useful in the treatment of urinary tract diseases. Thus, US 6,207,380 describes the use of antibodies that specifically bind to urinary tract tissue of keratin/cytokeratin, CSE1L or mat-8 to treat urinary tract diseases. US 6,232,086 discloses a complementary deoxyribonucleic acid and amino acid sequence of CSE1L for detecting the expression and amplification of the CSE1L gene in normal cells and cancer cells. US 6,156,564 is a method for detecting human proliferating cells, which comprises measuring the amount of CSE1L protein in a human tissue sample, and detecting that the human protein exhibits at least two higher CSE1L protein expressions than normal non-proliferating cells. More than double. US 6,440,737 is an antisense compound, composition and method for regulating the expression of the CSE1L gene. US20080081339 is directed to measuring dozens of individual fluid antibodies, including CSE1L "autoantibody" (non-CSE1L protein) in body fluids as a diagnostic marker for prostate cancer. US20050260639 relates to the detection of CSE1L in "cancer cells" isolated from body fluids or body tissues for the diagnosis of pancreatic cancer. WO 2009/052573 relates to the detection of hundreds of transcriptional ribonucleic acids, including CSE1L, in "cancer cells" isolated from body fluids or body tissues to diagnose gastrointestinal cancer. US20100120074 relates to the measurement of CSE1L protein in body fluids for the diagnosis of metastatic cancer. From the above description, it is known that the contents of these prior art (patent) are to detect the cell level CSE1L gene expression or the humoral CSE1L protein.

One aspect of the invention relates to a method of isolating, binding or detecting microvesicles in vitro for the diagnosis or treatment of a disease.

In one aspect of the invention, a method of diagnosing a tumor comprises the steps of: 1) obtaining a body fluid of a mammal; 2) detecting CSE1L or phosphorylation of microvesicles in the body fluid of the test subject in vitro; The amount of CSE1L or phosphorylated CSE1L in the body fluid of normal individuals was used as a control group in vitro; 3) the difference in the amount of CSE1L or phosphorylated CSE1L in the microcapsules of the body fluid of the tested individual and the control group, It is discriminated whether the subject has a tumor.

In one aspect of the invention, a method for diagnosing a tumor comprises the steps of: 1) obtaining a body fluid of a mammal; 2) detecting the amount of phosphorylated CSE1L in the body fluid of the test subject in vitro; 2) detecting phosphorylation in a body fluid of a normal individual in vitro; The amount of CSE1L was used as a control group; 3) The presence or absence of a tumor was determined based on the difference in the amount of phosphorylated CSE1L in the body fluid of the test individual and the control group.

In one aspect of the invention, a method for isolating microvesicles from a body fluid comprises the steps of: 1) obtaining a body fluid of a mammal; 2) isolating the antibody-bound microvesicles in vitro with an antibody that binds to CSE1L or phosphorylated CSE1L. .

In one aspect of the invention, a method for detecting microvesicles in a body fluid comprises the steps of: 1) obtaining a body fluid of a mammal; 2) detecting the microvesicles bound to the antibody with an anti-CSE1L antibody or an anti-phosphorylated CSE1L antibody in vitro.

In one aspect of the invention, there is also a method of binding to an antibody or cell microbubble that binds to CSE1L or phosphorylated CSE1L for use in a method of preventing or treating a disease. The priority disease is cancer.

In one aspect of the invention, the invention also relates to the use of a composition comprising an anti-CSE1L antibody or an anti-phosphorylated CSE1L antibody in combination with cellular microvesicles for the diagnosis or treatment of a disease. The priority disease is cancer.

Other uses, features, and advantages of the invention will be apparent from the description and drawings.

Cellular microvesicles are produced by cells, distributed in cell membranes, or released from cells, and found in body fluids and cell culture media (Simak 2006; Cocucci 2009; Muralidharan-Chari 2010). The microvesicles of the present invention include and are suitable for microbubbles that bind to cell membranes, and microvesicles released from cells. The microvesicles of the present invention also include and are suitable for cell microvesicles of all sizes.

The present invention firstly found that CSE1L regulates cell-forming microvesicles, phosphorylated CSE1L is located on the membrane of microvesicles, and phosphorylated CSE1L is ubiquitous in the serum of cancer patients. Microbubbles play an important role in a variety of diseases, especially cancer progression (Simak 2006). Therefore, it can be combined with CSE1L or Phosphorylated CSE1L-bound compositions, such as anti-CSE1L antibodies or anti-phosphorylated CSE1L antibodies and their derivatives, as well as pharmaceutical ingredients, can be used to detect or bind to humoral microvesicles and phosphorylated CSE1L in body fluids to diagnose or control disease. Microbubbles are involved in the transfer of cells, cells, etc. between proteases, genes, small RNAs, hormone receptors, cell organelles, etc., and thus with a variety of diseases, including coagulation diseases, wound healing, atherosclerosis, coronary artery disease, Diabetes, blood diseases, infectious diseases, inflammatory diseases, nervous system diseases, cancer and other deterioration (Simak 2006; Cocucci 2009; Muralidharan-Chari 2010). Therefore, protein molecules of microbubbles, especially protein molecules located on the microbubble membrane, can be used as markers for diagnosing diseases. In addition, antibodies or compositions that bind to microbubble membrane proteins can be utilized in combination with microvesicles for medical imaging or for treating diseases.

Another finding in the present invention is that CSE1L is linked to the Ras and ERK cell signaling pathways, and the activated Ras and ERK signaling pathways phosphorylate CSE1L, which is ubiquitously present in the serum of cancer patients. In addition, in the diagnosis of cancer, the sensitivity of detecting cancer phosphorylated CSE1L is higher than that of detecting serum non-phosphorylated CSE1L. Therefore, phosphorylated CSE1L has clinical applicability for cancer diagnosis.

Another finding of the present invention is that phosphorylated CSE1L is present in body fluids of cancer patients, and although CSE1L is also present in normal (healthy) human body fluids, it is difficult to detect phosphorylated CSE1L in normal human body fluids (Example 5 ). Previous studies have indicated that a protein that can be secreted by a cell, if it has both a phosphorylated form and a dephosphorylated form, is not a phosphorylated form or a dephosphorylated form of the protein. Secretion (Konishi 1994; Fendrick 1997). Therefore, another finding of the present invention is that phosphorylated CSE1L is a secreted protein, and phosphorylated CSE1L can be detected in body fluids of cancer patients.

Another finding of the present invention is that phosphorylated CSE1L is a membrane protein of microvesicles, and antibodies against CSE1L are capable of finding and binding to tumors. The microbubbles released by the tumor cells still remain in the surrounding environment of the tumor. Therefore, the use of a CSE1L binding agent or a phosphorylated CSE1L binding agent in combination with a therapeutic drug or a cytotoxic agent can be applied to cancer treatment. In addition, CSE1L binding agents or phosphorylated CSE1L binding agents can be used in medical imaging of diseases in combination with substances that emit fluorescence, radiation or other detectable messages.

Another aspect of the invention relates to the use of compositions or antibodies that bind CSE1L or phosphorylated CSE1L for the isolation and analysis of microvesicles in biological fluids, including cell conditioned media. The method or kit kit comprising a compound or antibody that binds to CSE1L or phosphorylated CSE1L, suitable for, but not limited to, ultracentrifugation, immunoprecipitation, affinity purification, micro Microfiltration, flow cytometry or fluorescence activated cell sorter, enzyme-linked immunosorbent assay, antibody microarray, biochips, chromatography (chromatography), immunoblotting, microfluidic systems, microfluidic chips, and other immunological techniques.

Another aspect of the invention relates to the isolation, analysis, or incorporation of microvesicles to diagnose or monitor a disease using a method or kit comprising a CSE1L binding agent or a phosphorylated CSE1L binding agent. The analysis of CSE1L or phosphorylated CSE1L can be quantitative or qualitative analysis, and the results of the analysis are combined with CSE1L or in the humoral microvesicles of one or more organisms that have no disease (as a control). The results of the analysis of phosphorylated CSE1L were compared. If the amount of CSE1L or phosphorylated CSE1L in the humoral microbubbles of the tested individuals is different from that of the control group, the status of the tumor, such as the presence or absence of tumor and tumor malignancy, can be indicated.

Since the microbubbles in the body fluid may be dissolved, the phosphorylated CSE1L contained in the microbubbles can be released into the body fluid. Another aspect of the invention relates to the isolation or analysis of phosphorylation of CSE1L in a body fluid to diagnose or monitor a disease by a method or kit comprising a binding agent that binds to phosphorylated CSE1L (eg, an antibody that phosphorylates CSE1L). The analysis of phosphorylated CSE1L can be qualitative or quantitative, and the results of the analysis are compared to the results of simultaneous analysis of one or more humoral CSE1L in humoral organisms (as a control). If the amount of CSE1L in the body fluid of the test subject is different from that of the control group, the status of the tumor, such as the presence or absence of tumor and tumor malignancy, can be indicated.

The invention also provides a kit for use in the above detection and diagnosis. For diagnostic or detection applications, the kit may comprise any or all of the following: a detection reagent, a buffer, an anti-phosphorylated CSE1L antibody or may bind to a phosphorylated CSE1L protein or a phosphorylated CSE1L polypeptide and may display a body fluid or Chemicals for phosphorylation of CSE1L protein or phosphorylated CSE1L polypeptide in body fluid microvesicles Peptides and other molecules.

"Biological fluid" as used in the present invention refers to a fluid sample that can be separated from any part of the organism, including but not limited to: blood, serum, plasma, urine, lymph, cerebrospinal fluid, sputum, pleural fluid, Nipple aspirate, respiratory fluid, intestinal fluid, genitourinary fluid, milk, tears, saliva, lymph, semen, cerebrospinal fluid, ascites, amniotic fluid, tumor cyst fluid. Also included are cell conditioned media. The microvesicles in the body fluid sample also include microbubbles present on the cell membrane of the cells within the biological fluid sample. The cell conditioned medium is a medium that has been cultured for a period of time. "Acquiring a bodily fluid sample" means obtaining a bodily fluid sample for use in the methods described herein.

By "tumor" as used herein, an individual has the presence of a cell that causes cancer. Cells that cause cancer often have uncontrolled proliferation, immortality, metastatic potential, and typical characteristics of certain cell types and cell markers. In some cases, the cancer cells are in the form of a tumor, but they may also be present in the body alone or in a separate cell form (such as leukemia cells) circulating in the bloodstream.

The "subject" as used in the present invention refers to an animal whose cells produce microbubbles. Animals include mammals, especially humans.

The "normal subject" as used in the present invention means that the individual has no tumor or other disease.

The "test subject" as used in the present invention refers to an individual to be detected for the presence or absence of a tumor or other disease.

As used herein, "in vitro" refers to an environment other than a living individual, typically an artificial environment, such as a test tube or culture environment.

"Immunological techniques" as used herein refers to any antibody-based detection technique, including but not limited to, dot blot hybridization, immunoblotting, immunoprecipitation, enzyme-bound immunosorbent assay (ELISA). , and radioimmunoassay (RIA) technology.

The "dot blot assay" as used in the present invention refers to immobilizing a sample on the surface of paper, glass fiber, or plastic sheet, and detecting the protein to be detected by a plurality of methods, including direct or indirect labeling of the antibody. The presence of the protein to be detected forms a distinct detection signal.

"Tumor targeting" as used in the present invention refers to compound preference or priority The ability to bind to tumors. A tumor-targeted pharmaceutical ingredient means that the drug component can preferentially or preferentially bind to tumor tissue.

The "cancer therapy" as used herein refers to a technique, step, or substance that can control tumor growth, invasion, metastasis, or cancer mortality.

"Medical imaging" as used in the present invention refers to a technique for clinically or medically scientifically researching an image of a human or animal tissue.

The "therapeutic agent" as used in the present invention means an agent or a compound having cytotoxicity, growth inhibition, or immunosuppressive effect on cancer cells.

The term "antibody" as used in the present invention means: (i) an immunoglobulin, an immunoglobulin polypeptide immunologically active portion (ie, an immunoglobulin) that specifically binds to a corresponding antigen (such as CSE1L or phosphorylated CSE1L). a polypeptide, or fragment thereof, comprising an antigen binding site, which binds to a specific antigen (such as CSE1L or phosphorylated CSE1L); or (b) a derivative of any polypeptide or fragment of an immunoglobulin that binds to an antigen ( Such as CSE1L or phosphorylated CSE1L).

The antibodies of the invention can be produced by known techniques and methods. For example, antibodies such as monoclonal antibodies, hybridomas, recombinant antibodies, phage displays, and the like are produced.

The anti-CSE1L antibody, or the anti-phosphorylated CSE1L antibody and the microbubble binding reaction of the present invention are detected by immunological principle techniques. In a typical immunoassay, cells, microbubbles, or antibodies can be immobilized on a support (column, membrane, or colloid), and unreacted or unbound material can be separated to detect the relationship between these antibodies and microbubbles. Combine. Methods of immobilizing cells, microvesicles or antibodies are known techniques. For example, they can be directly immobilized to a solid phase material via a chemical linking reaction or a physical adsorption method. Alternatively, the antibody of the present invention may be indirectly immobilized to a solid phase substance adsorbing an antibiotic protein (avidin) or a streptavidin after biotinylated biotinylated. When antibodies are bound to magnetic particles, not only antibodies, including microbubbles, can be detected quickly and conveniently and separated using magnets. In addition, when an antibody recognizing a plurality of antigens, such as a multispecific antibody, is used, the antibody binds to CSE1L or phosphorylates CSE1L on the microvesicles, and then can also bind to other antigenic proteins. Alternatively, the antibody may be immobilized to a solid phase material via Protein A or G (protein G) or the like.

The time for immobilizing the antibody according to the present invention is not particularly limited, and the antibody may be immobilized before, after, or at the same time as the mixed contact with the body fluid sample. The immobilized antibody may be immobilized using any solid phase material including fibrous membranes, particles, fibrous carriers, etc. made of glass, organic polymer, polystyrene, silica gel, alumina, activated carbon, and the like. . For example, the antibody of the present invention can be immobilized to the inner wall of a reaction vessel of a test tube, tray, dish, or bead.

Detection or quantification of microvesicles using the antibodies of the invention, including but not limited to the following immunological methods, for example, fluorescent antibody method, enzyme-bound immunosorbent assay (ELISA), radioimmunoassay (RIA), immunohistochemical staining (See Monoclonal Antibodies: Principle and Practice, 3rd ed. (1996) Academic Press), immunoblotting, immunoprecipitation.

The "average amount" calculation method of the present invention is to first determine the value or concentration of CSE1L or phosphorylated CSE1L in each sample in one or more samples, and then calculate CSE1L or phosphorylated CSE1L in The average or average concentration of these samples. The average value can be added to the total value by the individual values of the multiple samples, and the average value can be divided by the number of values. "Average amount of microvesicles of CSE1L" refers to the average amount of CSE1L protein in microvesicles in one or more control body fluid samples. "Average amount of phosphorylated CSE1L of microvesicles" refers to the average amount of phosphorylated CSE1L protein in microvesicles in one or more control body fluid samples. "Average amount of phosphorylated CSE1L in a body fluid sample" refers to the average amount of phosphorylated CSE1L protein in one or more control body fluid samples. The amount of CSE1L or phosphorylated CSE1L in the microcapsules of the tested body fluid sample, or the amount of phosphorylated CSE1L in the sample of the test body fluid, is higher than the average amount of CSE1L or phosphorylated CSE1L in the body fluid sample of the control group, and refers to the control group. The amount of CSE1L or phosphorylated CSE1L was increased in the body fluid sample compared to the body fluid sample. The "increased amount" is typically at least 10%, or at least 20% or 50%, or 100%, or at least 2 times, or at least 5 times, or can be as high as 10 or even 20 times.

The "cut-off value" or "positive judgment value" as used in the present invention refers to a threshold value for distinguishing and judging whether the subject to be tested has a disease or a disease thereof. The cut-off value is a statistically appropriate value for disease diagnosis, in which the mean and standard deviation (SD) of the test values are obtained from the same type of patient test and calculation. When the patient's test value is less than this threshold, the patient is considered negative (eg, no tumor), when the patient's The test value is greater than or equal to the threshold and the patient is considered positive (eg, with a tumor).

A "pharmaceutical composition" as used in the present invention refers to a combination of one or more drugs and one or more adjuvants, pharmaceutical excipients.

The "pharmaceutical excipient, diluent or carrier" as used in the present invention includes any pharmaceutical excipient, diluent or carrier approved by a government regulatory agency, such as phosphate buffer, water, Water oil emulsion, etc. Also included are the agents used in any pharmacopoeia.

The "kit" or "reagent kit" as used in the present invention refers to a kit equipped with all reagents necessary for analysis or measurement. The kit contains one or more reagent items including, but not limited to, compounds, composition components, instruments or equipment, and the like. The kit of the present invention may or may not be accompanied by instructions for use or operation.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly used technical and scientific terms. The present invention is not limited to the specific detection methods, detection protocols, and detection reagents described in the present invention, as these detection methods and reagents can be appropriately changed to achieve the same result and purpose. The scientific terms used in the present invention are intended to be illustrative, and are not intended to limit the scope or the scope of the invention.

The CSE1L protein and its encoding gene are known techniques. The DNA sequence of the CSE1L gene (GenBank Accession No. U33286) is shown below: Sequence 1 (SEQ ID NO.: 1)

The amino acid sequence of the CSE1L protein (protein ID number AAC50367.1) is shown below: Sequence 2 (SEQ ID NO.: 2)

The amino acid sequence of the phosphorylated peptide used to generate the anti-phosphorylated CSE1L (protein ID number AAC50367.1) antibody is as follows: SEQ ID NO.: 3 LYpEYpLKKTLDPDPAC [Tp represents phosphorylation of phosphothreonine, Yp represents Phosphoryl tyrosine (phosphotyrosine)

Example antibody

The antibodies used in the examples were: anti-p21/ras antibody (EP1125Y) (Epitomics, Burlingame, USA); anti-CSE1L antibody (3D8), anti-MAPK1/MAPK3 antibody (phospho T202/204, G15-B) (Abnova, Taipei) , Taiwan); anti-CSE1L antibody (24), anti-phosphotyrosine antibody (PY20), anti-phospho-serine and phospho-serine/threonine antibody (22A/pSer/Thr) (BD Pharmingen , USA); anti-β-tubulin antibody (D66) (Sigma, USA); anti-β-actin antibody (Ab-5), anti-GFP antibody (Ab-1) (Lab Vision, USA); anti-MMP -2 antibody (H-76), anti-phosphorylated threonine antibody (H2) (Santa Cruz Biotechnology, USA); goat anti-mouse IgG (Jackson ImmunoResearch Laboratories, USA); goat linked to Alexa Fluor 488 (or 568) Anti-mouse (or anti-rabbit) IgG secondary antibody (Molecular Probes, USA).

Gene expression vector

pZIP-v-H-ras is an expression vector that expresses the v-H-Ras protein and contains a neomycin transfection selection marker provided by Dr. Channing J Der. pcDNA-CSE1L is an expression vector that expresses the CSE1L protein and contains a neomycin transfection selection marker, which was previously constructed by itself (Tung 2009). CSE1L short hairpin RNA (shRNA), which reduces the expression of CSE1L gene in cells (SC-29909-SH, Santa Cruz Biotechnology), and its control-controlled shRNA vector (SC-108060, Santa Cruz Biotechnology), contains 嘌呤The puromycin transfection selection marker was purchased from Santa Cruz Biotechnology.

Cell and gene transfection

B16F10 melanoma cells were purchased from the American Type Culture Collection (USA). DMEM medium (Dulbecco's Modified Eagle's Medium) supplemented with 10% fetal bovine serum (FBS), 100 units/mL penicillin, 100 mg/mL streptomycin and 2 mM glutamate at 37 ° C, The cells were cultured under humidified 5% CO ₂ atmosphere. The cells were transfected with a gene expression vector using a lipofectamine reagent Lipofectamine plus reagent (Invitrogen, USA). Transfected cells were screened for 3 weeks with high concentrations of neomycin and puromycin. Multi-drug resistant lines (>100) were collected and propagated into large numbers of cells. The transfected cells are maintained in medium containing neomycin and puromycin. The cells used in the experiments described herein were cultured in medium without neomycin and puromycin.

Immune dot method

The cells were washed with phosphate buffered saline (PBS) and the cells were collected by scraping. RIPA radioimmunoprecipitation buffer [25 mM tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl, pH 7.2), 0.1% sodium dodecyl sulfate (SDS), 0.1% Triton X- 100, 1% sodium deoxycholate, 150 mM sodium chloride (NaCl), 1 mM ethylenediaminetetraacetic acid (EDTA), 5 mM sodium orthovanadate, 1 mM benzylsulfonated fluoride (phenylmethylsulfonyl fluoride), 10 μg/mL aprotinin, 5 μg/mL leupeptin, 25 mM β-glycerophosphate, 5 mM sodium fluoride, dissolved cells . Protein concentration was determined in a BCA protein assay kit (Pierce, USA). 50 μg of the protein sample was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The protein was transferred to nitrocellulose membranes (Amersham Pharmacia, UK). Nitrocellulose membrane at 4 ° C and containing 1% bovine serum albumin (BSA, bovine serum albumin), 50 mM tris (hydroxymethyl) aminomethane hydrochloride (Tris-HCl, pH 7.6), 150 mM sodium chloride, The immunostaining blocking buffer of 0.1% Tween-20 was reacted for 16 hours. The nitrocellulose membrane was then reacted with the primary antibody for 1 hour at room temperature, followed by reaction with a horseradish peroxidase-conjugated secondary antibody for 1 hour. Protein content was determined using a Forte Western blotting system (Forte Western HRP Substrate; Millipore, USA).

Immunoprecipitation

Wash the cells with phosphate buffer, collect the cells, add appropriate amount of radioimmunoprecipitation buffer, cleave on ice or at 4 ° C for 20 minutes, centrifuge at 12,000 rpm for 10 minutes, and take the supernatant; use BCA protein test kit (Pierce) Protein concentration. Quantitative cell lysate (500 μg), corresponding antibody, and 30 μL of protein A/G-beads (protein A/G-beads) were added to the immunoprecipitation buffer [50 mM tris(hydroxymethyl)aminomethane) Hydrochloride (Tris-HCl, pH 7.5), 150 mM sodium chloride in a test tube, incubate slowly at 4 ° C overnight; after immunoprecipitation, centrifuge at 3,000 rpm for 5 minutes at 4 ° C to protein A / G- The beads were centrifuged to the bottom of the tube; the supernatant was carefully aspirated, and the protein A/G-beads were washed 3-4 times with 1 mL of radioimmunoprecipitation buffer; finally, 50 μL of 2 times sodium dodecyl sulfate protein was added. SDS protein loading buffer, boiled in boiling water for 10 minutes; analyzed by immunoblotting method. The control group immunoprecipitation was carried out with an anti-GFP mouse antibody.

In immunoprecipitation with serum samples, serum and agarose-conjugated anti-phosphothreonine antibodies (H2) (Santa Cruz Biotechnology) in phosphate buffer, at 4 Incubate slowly overnight with °C; after immunoprecipitation, centrifuge at 3,000 rpm for 5 minutes at 4 °C. After careful removal of the supernatant, wash the pellet 3-4 times with 1 mL of phosphate buffer, and finally add 50 μL of 2X SDS protein loading buffer in boiling water. Cook for 10 minutes; then analyze by immunoblotting. The control group immunoprecipitation was carried out with agarose-conjugated normal mouse IgG conjugated to agarose.

Immunofluorescence experiment and microbubble counting

The cells grown on a 12x12 mm coverslip were centrifuged at 1000 rpm for 10 minutes. The cells were washed with phosphate buffer and fixed in 4% paraformaldehyde phosphate buffer, then treated with methanol, and then reacted with phosphate buffer containing 0.1% bovine serum albumin. One hour. The cells were allowed to react with the primary antibody for one hour, washed with phosphate buffer, and then reacted with Alexa Fluor 488 (or 568) goat anti-mouse (or anti-rabbit) IgG secondary antibody for one hour and then washed with phosphate buffer. The cells were observed under an inverted fluorescent microscope. Three independent experiments were performed in the experiment. Each experiment contained two coverslips, and each cover slip was randomly observed for 5 fields. Three hundred cells were observed in each experiment and the microvesicles on the surface of these cells were counted. The number of microbubbles counted by three independent experiments was plotted and statistical standard deviation was shown.

Cell proliferation analysis

The same number of cells (1 x 10 ⁴ cell number/plate) were seeded in a 100-mm cell culture dish. After cell seeding, the number of cells was counted every 24 hours using a trypan blue exclusion assay. The culture solution was updated every three days. Three cells are counted at each time point, and each plate is counted only once.

Conditioned medium

The same number of cells were seeded in a 100-mm cell culture dish, and the cells were first grown to sub-confluence and washed with phosphate buffer, and then the cells were cultured in serum-free medium for 48 hours. The number of cells was measured, and the culture solution was collected, and the collected culture solution was centrifuged at 10,000 rpm for 10 minutes, and the supernatant was collected to remove any possible suspended cells or cell debris.

Microbubble collection

Cellular conditioned medium and microvesicles in serum are collected essentially by size exclusion chromatography and ultra-high speed centrifugation techniques. Briefly, cell conditioned medium or serum was injected into a Sepharose 2 column (Amersham Biosciences, USA). Each 1 ml of liquid flowing through the column was collected separately, and the absorbance at 280 nm was measured with a photometric instrument. The collected protein liquid containing more than 500,000 kDa was centrifuged at 105,000 g for one hour at 4 °C. The precipitate containing the microbubbles was centrifuged, uniformly mixed with 50 μL of phosphate buffer and stored at low temperature.

GST-CSE1L fusion protein synthesis and CSE1L protein purification

The GST-CSE1L fusion protein was synthesized using a wheat germ cell-free protein synthesis system (CellFree Sciences, Japan). Briefly, the CSE1L protein synthesis sequence of the pcDNA-CSE1L vector was cleaved by restriction enzymes, and the CSE1L protein synthesis sequence was ligated into the pPEU-E01 wheat germ expression vector (CellFree Sciences). Two micrograms (μg) of CSE1L wheat germ expression vector was added to a test tube containing a transcription premix solution (CellFree Sciences), and transcription reaction was carried out at 37 ° C for 6 hours. Ten microliters (μL) of the transcriptional message ribonucleotide (mRNA) product was mixed with 10 μl of wheat germ extract (WEPRO 3240, Cell Free Sciences) and this mixture was injected into SUB-AMIX (CellFree Sciences) The lower layer of the test tube was subjected to a protein translation reaction at 26 ° C for 16 hours to synthesize a GST-CSE1L fusion protein. Using GST Purification Module (Bulk GST Purification Modules, Amersham Pharmacia, USA) to Glutathione-Sepharose The GST-CSE1L fusion protein was purified by glutathione-Sepharose 4B beads (Amersham Pharmacia, USA). Next, the tube containing the fusion protein was washed with a phosphate buffer, and the fusion protein was isolated with reduced glutathione (Amersham Pharmacia). The GST-CSE1L fusion protein was cleaved with thrombin (thrombin; Sigma Chemicals, USA) at a concentration of 3 units per 100 μg of fusion protein for 16 hours at 22 °C. Thrombin and GST were removed using an Amicon® Ultra-4 centrifugal filter set (Millipore, USA). Purified CSE1L was confirmed by immunoblotting using an anti-CSE1L antibody. CSE1L protein concentration was determined in a BCA protein assay kit (Pierce, USA).

Tissue microarray sections and immunohistochemical reactions

The cancerous tissue and adjacent non-cancerous tissue paraffin blocks were cut to a moderate size, and the paraffin blocks were arranged and made into tissue microarray sections. Immunohistochemical reactions were performed on tissue microarray sections at a thickness of 4 μm. After sectioning the paraxylene in xylene and rehydrating with ethanol, it was immersed in citrate buffer at 95 ° C for 10 minutes. Immunohistochemical reactions were performed using the Histostain kit (Zymed, USA) labeled with the labeled streptavidin-biotin method. The endogenous peroxidase activity of the tissue sections was masked with 3% water-soluble hydrogen peroxide, and then incubated with 5% bovine serum albumin for 1 hour at room temperature to cover non-specific staining. The sections were reacted with 50-fold diluted antibody for 1 hour at room temperature, then reacted with biotinylated secondary antibodies, and finally streptavidin-labeled peroxidase. reaction. The sections were developed with diaminobenzidine and washed with distilled water and finally counterstained with Mayer's hematoxylin.

Cell invasion analysis with Matrigel

Matrigel Matrigel (BD Pharmingen, USA) was diluted 1:10 in DMEM and at 8 °C with polyvinylpyrrolidone-free 8 μm pore size polycarbonate filter (polyvinylpyrroide-free polycarbonate filters; Costar, USA) After standing for 16 hours, after washing the filter paper 4 times with DMEM, it was placed in microchemotaxis chambers. The cells were treated with 0.1% trypsin-EDTA and the cells were resuspended in DMEM medium containing 10% fetal calf serum, after which the cells were washed with serum-free DMEM medium. Finally, cells (3 x 10 ⁵ ) were suspended in DMEM (200 μL) and placed in the upper compartment of the microchemotaxis. A cell culture medium (300 μL) containing 20% fetal bovine serum was placed under the microchemotaxis chamber as a cell invasion attractant. After incubating for 10 hours in a cell culture incubator, the cells on the upper layer of the microchemotaxis compartment filter paper were completely wiped off with a cotton swab. The cells under the filter paper in the upper compartment of the microchemotaxis were fixed with methanol and stained with Liu's A and Liu's B reagents, and then counted under a microscope. The test was repeated three times, and each test included four replicates. For each sample, 10 microscopic field counts were randomly selected to invade cells under the microchemotaxis filter paper, and the counts were averaged.

Immunogold electron microscopy

The cells were washed with phosphate buffer and fixed in hydroxyethyl-piperazine ethanesulafonic acid (HEPES) buffer containing 0.5% glutaraldehyde and 2% paraformaldehyde. The pH was 6.8) for 15 minutes, and then the cells were reacted in hydroxyethylpyridazine ethanesulfonic acid buffer containing 2% paraformaldehyde at 4 ° C for 14 days. The sample was further dehydrated with 80% ethanol and treated with Lowicryl HM20 resin (Polysciences, Japan) and polymerization was carried out for 24 hours. The cell-containing resin blocks were subjected to ultrathin sectioning, and then the sections were placed on nickel grids coated with 2% neoprene (Neoprene, Ohken, Japan). After the sample was treated with 100% ethanol for 3 minutes, it was immersed in 0.01 M ethylenediaminetetraacetic acid (EDTA, pH 7.2) at 65 ° C for 24 hours. The sample was washed three times with phosphate buffer and reacted with phosphate buffer containing 1% bovine serum albumin and 0.1% Tween-20 for 15 minutes. The sample was then reacted with an antibody diluted in phosphate buffer (1:30) for 1 hour, washed three times with phosphate buffer, and then reacted with gold-labeled secondary antibodies. Wash three times with phosphate buffer. The sample was then stained with uranyl acetate and observed under an electron microscope by Hitachi H-7000 (Hitachi, Japan).

Gelatin zymography assay

The volume of the cell conditioned medium was adjusted according to the number of cells, and the microbubbles collected from the cell conditioned medium were taken for gelatin zymography. The microbubbles are dissolved in a 2X sodium dodecyl sulfate protein loading buffer (SDS protein loading buffer) containing no reducing agent dithiothreitol or 2-Mercaptoethanol. 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis containing 1 mg/mL gelatin. The gel was then washed twice with 2.5% Triton X-100 in water for 30 minutes to remove sodium dodecyl sulfate (SDS), and then the gel was placed in gelatin zymography buffer [50 mM tris (hydroxyl) Aminomethane hydrochloride (Tris-HCl, pH 7.6), 200 mM sodium chloride, 10 mM calcium chloride (CaCl ₂ )] was allowed to stand at 37 ° C for 24 hours. The gel was stained with Comassie blue staining solution (0.125% Comassie blue R-250, 50% methanol, 10% acetic acid) for 30 minutes, then decolorized with decolorizing solution (20% methanol, 10% acetic acid, 70% water) until clear The transparent strip appears.

Animal cancer metastasis experiment

C57BL/6 mice (6- to 7 weeks (N=18) and 14 to 15 weeks (N=26)) in a standard environment (22 ° C; 50% humidity; 12-hour light/dark cycle) Laboratory Animal Center, Taiwan) is housed in animal rooms. The experiment included 4 groups [ie injection of B16-dEV cells (N=11), B16-CSE1L cells (N=14), B16-ras cells (N=8), and B16-Ras/anti-CSE1L cells (N=11). Mice], and mice of different ages were evenly distributed in four groups. Each mouse was injected with 100 μL of phosphate buffer containing 3 × 10 ⁴ cells from the tail vein. The mice were sacrificed three weeks after the injection. The number of tumors in the lungs of mice was counted by visual inspection and microscopic examination. The breeding and experimental procedures of the mice were in accordance with the specifications of the Taiwan Laboratory Animal Management Committee. There were 3 mice injected with B16-dEV cells, 10 mice injected with B16-CSE1L cells, 4 mice injected with B16-ras cells, and 1 mouse injected with B16-Ras/anti-CSE1L cells. Lost life after 3 weeks of injection, and lung tumor statistics were excluded. In addition, there were 1 mouse injected with B16-dEV cells, 1 mouse injected with B16-CSE1L cells, 0 mice injected with B16-ras cells, and 3 small B16-Ras/anti-CSE1L cells. Rats, which did not grow tumors, were also excluded from tumor statistics.

Patient and tumor sample

The tumor samples were obtained from 115 colorectal cancer patients from Changhua Christian Hospital in Taiwan, and cancer tissues and serum samples were obtained without cancer treatment. The tumor specimen is obtained at the time of diagnosis and surgery, following the guidelines approved by the Institutional Human Body Testing Committee (IRB) and informed consent of the patient. The classification and classification of tumors is based on the sixth edition of the United States Joint Commission's cancer staging manual. Serum samples from healthy donors were obtained from 60 healthy individuals (mean age 61.0 ± 8.7 years; age range, 22-71 years). The collected blood is allowed to stand at room temperature for at least 30 minutes to form a clot. The sample was then centrifuged at 1300 g for 20 minutes at 4 ° C, and serum was collected. The serum was centrifuged at 10,000 rpm for 10 minutes, and the supernatant was collected to remove any possible suspended cells or cell debris and stored at -80 °C for subsequent testing. All specimens are labeled with specific labels to protect patient privacy. All specimens were not thawed prior to analysis. The clinicopathological features of the patients are summarized in Table 1.

Grade 1 tumor (grade 1): well differentiated, grade 2 tumor (grade 2): moderately differentiated, grade 3 tumor (grade 3): low grade differentiation. N0: no regional lymph node metastasis, N1: 1-3 regional lymph node metastasis, N2: metastasis to more than 4 regional lymph nodes, M0: no distant cancer metastasis, M1: distant cancer metastasis.

Animal living tumor imaging experiment

Anti-CSE1L antibodies or mouse IgG antibodies were bound to Qdot 800 quantum dots using the Qdot 800 Antibody Conjugation Kit (Invitrogen, USA) according to the manufacturer's protocol. Normal mouse IgG antibodies that bind to Qdot 800 quantum dots will be used in control group experiments. Briefly, 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid amber imidate (SMCC, N-Succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate ) Activate Qdot with dithiothreitol Perform antibody reduction. The reduced antibody was mixed with activated Qdot to carry out a coupling reaction, and the reaction was terminated with 2-Mercaptoethanol. The Qdot-conjugated antibody was purified using ultrafiltration and size exclusion chromatography. The antibody concentration of the coupled Qdot was determined using a spectrophotometer.

C57BL/6 mice (National Laboratory Animal Center, Taiwan) from 6 to 7 weeks old were housed in an animal room under standard conditions (22 ° C; 50% humidity; 12 hours light/dark cycle), each mouse was backed The lateral skin was injected with 100 μL of phosphate buffer containing 3 × 10 ⁴ B16-CSE1L cells. Three weeks after the injection, the dorsal tumor-bearing mice were injected with 100 μL of anti-CSE1L antibody containing 500 pmole-conjugated Qdot from the tail vein. The control group was injected with equal amounts of normal IgG from mice conjugated with Qdot 800 quantum dots. Tumor imaging was detected after 0, 1 and 4 hours of injection using a non-invasive Xenogen IVIS 200 in vivo imaging system [excitation: 525/50 nm; emission (emission): 832/65 nm].

Production of antibodies against phosphorylated CSE1L

The phosphorylated peptide was synthesized using a solid phase method: LTpEYpLKKTLDPDPAC (Tp represents phosphorylated threonine; Yp represents phosphorylated tyrosine), and non-phosphorylated peptide: LTEYLKKTLDPDPAC. Keyhole limpet haemocyanin (KLH) is coupled to the phosphorylated peptide via the thiol of the cysteine (cysteine) at the N-terminus of the phosphorylated peptide, and is used as an antigen to inject and immunize New Zealand rabbits. (New Zealand rabbit) five times. One week after the last immunization, immune sera were collected. The IgG antibody was purified using a Protein G column (Amersham Pharmacia, Sweden). The antibody is purified using a phosphorylated peptide affinity column, which is then adsorbed with a non-phosphorylated peptide to remove non-anti-phosphorylated CSE1L antibodies therein. Antibody titers and specificity were analyzed by enzyme-linked immunosorbent assay and immunoblot method.

Dot blotting

Equal amounts (5 μL) of each purified microbubble suspension were individually injected into a 96-well dot-blot manifold apparatus (BRL, USA) nitrocellulose membranes (Amersham Pharmacia). Nitrocellulose membrane at room temperature and containing 1% bovine serum Albumin, 50 mM tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl, pH 7.6, 150 mM sodium chloride, 0.1% Tween-20 in an immunostaining blocking buffer for one hour. Then with primary antibody The reaction was carried out at 4 ° C for 16 hours and then reacted with a secondary antibody conjugated with horseradish peroxidase (HRP) for 1 hour. Protein was determined using a Forte Western blotting system (Forte Western HRP Substrate; Millipore, USA). content.

Enzyme combined immunosorbent assay (ELISA)

Each serum was diluted 3-fold in phosphate buffer, twice repeated and equal (100 μL) placed in 96-well NUNC Immunoplate MaxiSorb well plates (96-well Nunc Immunoplate MaxiSorb plates; Nunc, Denmark), reacted at 4 °C 16 hours. Then, the suspended serum in the well was aspirated, and the phosphate buffer containing 1% of bovine serum albumin was reacted with the serum adsorbed in the well at room temperature for 1 hour. The phosphate buffer containing 1% bovine serum albumin was aspirated, and the wells of the well plate were simply washed with PBST (0.05% Tween-20 in phosphate buffer). The wells were reacted with biotin-conjugated anti-CSE1L antibodies or biotinylated anti-phospho CSE1L antibodies for 1 hour. The biotin-labeled antibody was prepared by biotinylation of an anti-CSE1L antibody or an anti-phosphorylated CSE1L antibody using a Biotin Labeling Kit-NH2 kit (Dojindo Laboratories, Japan). The wells were then washed with PBST and reacted with streptavidin-conjugated horseradish peroxidase (R&D Systems, USA) for 30 minutes, the wells of the wells were washed with PBST, and immunosorbed with enzymes. The reagent (R&D Systems) was reacted for 20 minutes. The wells of the well plate were then washed with PBST. Three wells containing bovine serum albumin were used as background values, and three wells containing bovine serum albumin but with all other enzymes in combination with immunosorbent reagents were used as a control group for correction. The absorbance at 450 nm was measured in a disk spectrometer (Thermo Multiskan EX, Thermo Fisher Scientific, USA) in 30 minutes. Each specimen was analyzed twice.

Statistical Analysis

Data analysis was performed using SPSS 14.0 statistical software. Statistical differences were analyzed using a two-tailed Fisher's exact test. A value of less than 0.01 is statistically significant With.

Example

Example 1: Establishment of B16-dEV cells, B16-CSE1L cells, B16-ras cells, and B16-Ras/anti-CSE1L cells.

B16F10 cells were transfected with pcDNA empty vector, pZIP empty vector, pcDNA-CSE1L vector, pZIP-vH-ras vector, CSE1L shRNA vector, or control shRNA vector to establish B16-dEV cells, B16-CSE1L cells, B16-ras Cells and B16-Ras/anti-CSE1L cells. Figure 1 shows the results of Ras and CSE1L protein expression in B16-dEV, B16-ras, B16-CSE1L, and B16-Ras/anti-CSE1L cells by immunoblotting (Fig. 1).

Figure 1 shows the expression of Ras and CSE1L proteins in B16-dEV cells, B16-CSE1L cells, B16-ras cells, and B16-Ras/anti-CSE1L cells by anti-Ras antibody, anti-CSE1L antibody, and immunoblotting method. the amount. The expression level of β-actin was a control.

Example 2: Transfection of cancer cells with vH-ras oncogene induces microbubble formation.

On the surface of B16F10 cells transfected with the v-H-ras oncogene (ie, B16-ras cells), many vesicular microvesicles were found (Fig. 2A). Microbubbles that are forming or developing can also be found at the cytoplasm of the cell and at the base of the pseudopodia (Fig. 2B). Therefore, the v-H-ras gene regulates the production of cellular microvesicles. Microbubbles contain a large amount of protease (Taraboletti 2002). Immunofluorescence experiments confirmed the presence of matrix metalloproteinase-2 (MMP-2) in the microvesicles of B16-ras cells (Fig. 2C). Analysis by immunoblotting method showed that transfection of VH-ras oncogene with B16F10 cells increased the amount of microbubble MMP-2 protein; while treatment with PD98059 (an inhibitor of ERK activity) decreased the cell microstimulation stimulated by vH-ras. An increase in the amount of vesicular MMP-2 protein (Fig. 2D). Cell chromatin staining with 4',6-biindole-3-phenylindole dihydrochloride (DAPI; 4',6-diamidino-2-phenylindole) showed no chromatin condensation or chromatin fragments in these cells. Apoptosis. Therefore, the Ras-ERK message pathway regulates the generation of cellular microvesicles.

Figure 2 shows that vH-ras oncogene transfection induces microbubble formation in cancer cells. (A) Microscopic images and microbubble counts of B16F10 cells, B16-dEV cells, and B16-ras cells. (B) The microscopic image shows the pseudopod base of B16-ras cells and the microbubbles being formed on the cell membrane. (C) Anti-MMP-2 antibody immunofluorescence showed MMP-2 in microvesicles of B16-ras cells. (D) Immunoblotting method, analysis of microbubbles of B16-dEV and B16-ras cells treated with 50 μM PD98059 or its lysing agent dimethyl sulphoxide (DMSO, dimethy sulphoxide) and cultured for 48 hours under serum-free conditions. The amount of MMP-2.

Example 3: Transfection of vH-ras oncogene increases the secretion of CSE1L by cancer cells.

The results of the immunoblotting method indicated that transfection of the v-H-ras gene into B16F10 cells increased the secretion of CSE1L protein; whereas PD98059 treatment decreased the secretion of CSE1L protein in the cells stimulated by v-H-ras (Fig. 3). Transfection of B16F10 cells with v-H-ras gene or treatment with PD98059 did not significantly affect intracellular CSE1L expression (Fig. 3). Therefore, the Ras-ERK message pathway regulates the secretion of CSE1L by cells.

Figure 3 shows that transfection of the vH-ras oncogene increases the secretion of CSE1L by cancer cells. The CSE1L expression and secretion amount of B16-dEV and B16-ras cells treated with dimethyl sulfoxide (DMSO) or 50 μM PD98059 and cultured for 24 hours under serum-free conditions were analyzed by immunoblotting.

Example 4: CSE1L is a phosphorylated protein.

The phosphorylation of CSE1L was studied using the GST-CSE1L fusion protein synthesized by the wheat germ cell-free protein synthesis system and the purified CSE1L protein. The results of the immunoblotting method showed that the CSE1L protein reacted with antibodies against phosphorylated serine and phospho-serine/threonine, and against phosphotyrosine (Fig. 4). Therefore, CSE1L is a serine/threonine and tyrosine phosphorylated protein.

Figure 4 shows that CSE1L is a phosphorylated protein. Phosphorylation of CSE1L protein was analyzed by anti-phosphorylated serine and phosphorylated threonine antibodies, and anti-phosphotyrosine antibodies and immunoblots. The CSE1L protein is obtained by thrombin-decomposing a GST-CSE1L fusion protein synthesized by the wheat germ cell-free protein synthesis system.

Example 5: Transfection of vH-ras oncogene increased phosphorylation of CSE1L protein in cancer cells, and phosphorylated CSE1L protein was present in cancer serum.

B16-dEV and B16-ras cells were treated with anti-CSE1L antibody and immunoprecipitation technique, immunoprecipitated with dimethyl sulfoxide (DMSO) or 50 μM PD98059, and cultured for 24 hours under serum-free conditions. The immunoprecipitate was then analyzed by immunoblotting using an anti-phosphorylated threonine antibody labeled with horseradish peroxidase. The results indicated that transfection of the v-H-ras oncogene increased the phosphorylation of CSE1L protein in cells; whereas PD98059 treatment reduced the phosphorylation of CSE1L protein in cells increased by transfection of v-H-ras oncogene (Fig. 5A). Thus, Ras-ERK signaling regulates phosphorylation of CSE1L protein. CSE1L is a secreted protein and therefore also tests whether the Ras-ERK signaling pathway regulates the secretion of phosphorylated CSE1L protein. The conditioned medium of B16-dEV and B16-ras cells treated with dimethylarsine (DMSO) or 50 μM PD98059 and cultured for 24 hours under serum was immunoprecipitated with anti-CSE1L antibody and immunoprecipitation technique. The immunoprecipitate was then analyzed by immunoblotting using an anti-phospho-threonine antibody labeled with horseradish peroxidase. The results indicated that transfection of the v-H-ras oncogene increased the secretion of CSE1L protein in the cell; whereas PD98059 treatment reduced the secretion of the phosphorylated CSE1L protein in the cells transfected with the v-H-ras oncogene (Fig. 5B).

Immunoprecipitation of serum from patients with colorectal cancer (N=36) and healthy blood donors (N=36) with agarose-conjugated anti-phosphothreonine antibodies and immunoprecipitation . The immunoprecipitate was further analyzed by anti-CSE1L antibody by immunoblotting. The results showed that the threonine phosphorylated CSE1L protein was present in the immunoprecipitate of the serum of cancer patients, but not in the immunoprecipitates of healthy blood donors (Fig. 5C). On the other hand, the results of immunoblotting analysis showed that although the amount of CSE1L in the serum of these cancer patients was higher than the amount of CSE1L in the serum of healthy blood donors, the difference was not as good as that of cancer patients with serum phosphorylation CSE1L and healthy blood donation. The difference in the amount of phosphorylated CSE1L in serum was significant (Fig. 5D). These results indicate that secreted phosphorylated CSE1L can be a diagnostic marker for cancer.

Figure 5 shows that transfection of the vH-ras oncogene increases the phosphorylation of CSE1L protein in cancer cells, and that phosphorylated CSE1L protein is present in the serum of cancer patients. (A) Analysis of B16-dEV treated with dimethylammonium (DMSO) or 50 μM PD98059 with anti-phosphorylated threonine antibody, anti-CSE1L antibody, and immunoprecipitation, and cultured for 24 hours under serum-free conditions CSE1L phosphorylation status in B16-ras cells. In addition, a control group immunoprecipitation reaction was carried out with a mouse antibody against GFP. (B) Analysis of B16-dEV and B16 cultured in serum-free conditions for 24 hours with anti-phosphorylated threonine antibody, anti-CSE1L antibody, and immunoprecipitation reaction with dimethyl sulfoxide (DMSO) or 50 μM PD98059 Phosphorylation status of CSE1L protein secreted by -ras cells. The control group immunoprecipitation was carried out with an anti-GFP mouse antibody. (C) Analysis of phosphorylation of CSE1L protein in serum of cancer patients by agarose-conjugated anti-phosphorylated threonine antibody, anti-CSE1L antibody, and immunoprecipitation reaction. The serum of cancer patients was obtained from 36 patients with colorectal cancer (10 μL per patient), and the blood of healthy blood donors was obtained from 36 healthy persons (10 μL per person). Controlled immunoprecipitation was performed with agarose-conjugated normal mouse IgG. (D) Immunoblotting method, the amount of CSE1L protein in the serum of the above cancer patients (36 colorectal cancer patients, 10 μL per patient) and healthy blood donor serum (36 healthy persons, 10 μL per person) were analyzed.

Example 6: Phosphorylated ERK and CSE1L were highly expressed in colorectal cancer tumors, and the relative staining intensity was consistent.

Analysis of 115 colorectal cancer specimens by tissue microarray and immunohistochemistry showed that phosphorylated ERK and CSE1L were 100% (115/115) and 99.1%, respectively (114/ The cancer sample of 115) was highly expressed (Fig. 6A). Adjacent normal tissues had only relatively weak phosphorylated ERK and CSE1L expression (Fig. 6A). The sequential staining of colorectal cancer tissues and immunohistochemical techniques were used to analyze the relative staining intensity of phosphorylated ERK and CSE1L in colorectal cancer. It was also found that phosphorylated ERK and CSE1L were mostly (97.4%; 112/115). The relative staining intensity of colorectal cancer tumor samples is consistent. This indicates a close relationship between phosphorylated ERK and CSE1L in the progression of colorectal cancer (Fig. 6B).

Figure 6 shows that phosphorylated ERK and CSE1L are highly expressed in colorectal cancer tumors, and the relative staining intensity is consistent. (A) Analysis of the expression levels of phosphorylated ERK and CSE1L in colorectal cancer by immunohistochemical techniques and tissue microarray sections consisting of 115 cancer specimens. Left panel: non-tumor tissue; right panel: colorectal cancer tumor. Original magnification: 400 times. (B) The relative staining intensity of phosphorylated ERK and CSE1L in colorectal cancer tumors was analyzed by immunohistochemistry and adjacent serial sections of colorectal cancer tissues. Arrows indicate the location of phosphorylated ERK and CSE1L relative to the table staining intensity. Original magnification: 40 times.

Example 7: CSE1L regulates the generation of cancer cell microvesicles induced by the vH-ras oncogene.

B16-Ras/anti-CSE1L cells were established by transfecting B16-ras cells with an expression vector carrying the CSE1L shRNA gene to inhibit CSE1L expression in B16-ras cells. Microscopic examination revealed that reduction of CSE1L expression in B16-ras cells inhibited the generation of cancer cell microvesicles induced by the v-H-ras oncogene (Fig. 7A). In addition, B16F10 cells were transfected with a vector carrying the CSE1L gene to allow cells to highly express CSE1L to establish B16-CSE1L cells. Microscopic examination revealed that high expression of CSE1L in cells also induced microbubble formation (Fig. 7B). Using immunofluorescence studies, it was found that both CSE1L and MMP-2 are located in microvesicles that are being formed in the cytoplasm and pseudopodia base (Fig. 7C). These results indicate that CSE1L regulates the generation of cancer cell microvesicles induced by the v-H-ras oncogene.

Figure 7 shows that CSE1L regulates the generation of cancer cell microvesicles induced by the vH-ras oncogene. (A) Microscopic images and microbubble counts of B16-Ras cells and B16-Ras/anti-CSE1L cells. (B) Microscopic image and microbubble count of B16-dEV cells and B16-CSE1L cells. (C) Immunofluorescence technique studies showing that CSE1L and MMP-2 proteins are located in the microvesicles that are being formed on the base of the pseudopod and on the cell membrane of B16-ras cells.

Example 8: CSE1L regulates cancer cell invasion induced by the vH-ras oncogene.

The conditioned medium of B16-dEV, B16-ras, B16-CSE1L, and B16-Ras/anti-CSE1L cells treated with dimethyl sulfoxide (DMSO) or 50 μM PD98059 and cultured for 24 hours under serum-free conditions was collected. The microvesicles in these conditioned media were then separated. The MMP-2 gelatinase activity of the microvesicles in these conditioned media was analyzed by gelatin zymography. The results showed that transfection of vH-ras oncogene increased MMP-2 gelatinase activity of microvesicles released into conditioned medium; PD98059 inhibited microbubble MMP-2 gelatinase increased by transfection of vH-ras oncogene Activity (Figure 8A). In addition, high expression of CSE1L by cells also increased MMP-2 gelatinase activity of microvesicles released by cells into conditioned medium (Fig. 8A). Furthermore, reducing CSE1L expression in B16-ras cells inhibited the increase in microbubble MMP-2 gelatinase activity of the v-H-ras oncogene (Fig. 8A). Matrigel-based invasion of B16-dEV, B16-Ras, B16-CSE1L, and B16-Ras/anti-CSE1L cells using Matrigel Matrigel Assays), the results indicate that vH-ras oncogene transfection increases cancer cell invasiveness (Fig. 8B), and high expression of CSE1L in cells also increases cancer cell invasiveness (Fig. 8B), using CSE1L shRNA to reduce CSE1L in B16-ras cells. Expression also inhibited the invasiveness of cancer cells increased by transfection of the vH-ras oncogene (Fig. 8B). Therefore, CSE1L regulates cancer cell invasion induced by the v-H-ras oncogene.

Figure 8 shows that CSE1L regulates cancer cell invasion induced by the vH-ras oncogene. (A) B16-dEV, B16-ras, B16-CSE1L, and B16-Ras treated with dimethyl sulfoxide (DMSO) or 50 μM PD98059 and cultured for 24 hours under serum-free conditions by gelatin zymography /anti-CSE1L cells, MMP-2 gelatinase activity of the released microvesicles. (B) Cell invasiveness analysis of B16-dEV, B16-Ras, B16-CSE1L, and B16-Ras/anti-CSE1L cells using Matrigel. The number of invading cells (mean ± standard deviation; cell number / field of view) were B16-dEV cells: 109.4 ± 12.6; B16-CSE1L cells: 406.8 ± 23.9; B16-Ras cells: 311.25 ± 32.4; B16-Ras/anti - CSE1L cells: 46.75 ± 8.7.

Example 9: CSE1L regulates cancer cell metastasis induced by the vH-ras oncogene.

MMP-2 plays a crucial role in tumor invasion (Taraboletti 2002). Immunofluorescence showed that CSE1L and MMP-2 were co-located in the microvesicles of B16-ras cells and B16-CSE1L cells, and CSE1L was preferentially accumulated in the microvesicles (Fig. 9A). Animal experimental studies showed that the expression of CSE1L in B16F10 cancer cells was increased by the expression vector carrying the CSE1L gene, and the ability of cancer cells to metastasize to the lung was increased by 361.5% (P<0.05) (Fig. 9B). The ability of the v-H-ras oncogene to transfect B16F10 cancer cells increased the ability of cancer cells to metastasize to the lung by 246.1% (P < 0.05) (Fig. 9B). Reduction of CSE1L expression in B16-ras cells by CSE1L shRNA inhibits the ability of cancer cells with increased vH-ras oncogenes to metastasize to the lung (100%, P<0.01); although B16-Ras cells and B16-Ras/anti The growth rate of CSE1L cells was similar (Fig. 9B and C). Therefore, CSE1L regulates cancer cell metastasis induced by the v-H-ras oncogene.

Figure 9 shows that CSE1L regulates cancer cell metastasis induced by the vH-ras oncogene. (A) Cellular immunofluorescence analysis demonstrated that CSE1L and MMP-2 are co-located in the microvesicles of B16-ras cells and B16-CSE1L cells, and that CSE1L preferentially accumulates in microvesicles rather than like MMP-2. In the cell. (B) CSE1L regulates cancer cell metastasis induced by the vH-ras oncogene. The above figure is a representative photograph showing the lung tumors of mice induced by injection of B16-dEV, B16-ras, B16-CSE1L, and B16-Ras/anti-CSE1L cells into C57BL/6 mice. The mean number of lung tumors (mean ± standard deviation; number of tumors per mouse) of these cells injected into mice were B16-dEV cells: 13.7 ± 4.8; B16-CSE1L cells: 47 ± 15.8; B16-Ras cells : 32.2 ± 8.5; B16-Ras/anti-CSE1L cells: 7 ± 3.6. (C) Cell count growth curves of B16-dEV, B16-CSE1L, B16-Ras, and B16-Ras/anti-CSE1L cells. The graph represents three independent experimental results.

Example 10: CSE1L is located in the microvesicle membrane, and antibodies against CSE1L are able to find and bind tumors.

Immunofluorescence studies showed that CSE1L was located in the microbubble membrane (Fig. 10A). Immunogold electron microscopy studies further showed that both CSE1L and MMP-2 are located in microvesicles, and CSE1L is mainly located in the microbubble membrane (Fig. 10B). The microbubbles released by the tumor cells still remain in the surrounding environment of the tumor. CSE1L is located in the microbubble membrane, indicating that CSE1L can be a target for cancer therapy. After injecting B16-CSE1L cells into the back of C57BL/6 mice to induce tumors, and then injecting anti-CSE1L antibodies labeled with Qdot 800 quantum dots via tails, the tumors on the back of the mice emit near-infrared fluorescent signals of quantum dots. (NIR fluorescence signals), this result indicates that antibodies against CSE1L can find and bind to tumors. The control mice were injected with normal dot IgG labeled with quantum dots, and under the same conditions, the tumor did not emit near-infrared fluorescence signals of quantum dots (Fig. 10C). Therefore, antibodies against CSE1L are able to find and bind to tumors.

Figure 10 shows that CSE1L is located in the microvesicle membrane and that antibodies against CSE1L are able to find and bind to the tumor. (A) Cellular immunofluorescence analysis demonstrated that CSE1L is located on the membrane of microvesicles produced by B16-CSE1L cells. (B) Immunoelectron microscopy analysis showed that CSE1L (18-nm nanogold, indicated by the arrow) and MMP-2 (12 nm nanogold) were distributed on the membrane of microvesicles formed by B16-CSE1L cells. Original magnification: 250,000 times. (C) Combined images showed that tumor-infected C57BL/6 mice were injected with B16-CSE1L cells, and tumors were emitted 4 minutes after the quantum dot-labeled anti-CSE1L antibody (right mouse, N=3) via the tail. Near-infrared fluorescent signal of quantum dots. The control mice (left mouse, N=3) were normal IgGs that were labeled with quantum dots, and the tumors did not emit near-infrared fluorescence signals of quantum dots under the same conditions.

Example 11: Specific binding of an anti-phosphorylated CSE1L antibody to a phosphorylated CSE1L protein.

New Zealand rabbits were immunized with a synthetic phosphorylated CSE1L peptide to produce an anti-phospho CSE1L antibody. The titer of the affinity-purified antibody was analyzed by enzyme-binding immunosorbent assay and the results are shown in Table 2 (Table 2). The results of the immunoblotting method indicated that the anti-phosphorylated CSE1L antibody specifically binds to phosphorylated CSE1L (Fig. 11).

Figure 11 shows the specific binding of anti-phosphorylated CSE1L antibodies to phosphorylated CSE1L protein. Specific anti-phosphorylation of CSE1L antibody against phosphorylated CSE1L protein by immunoblotting and B16-dEV and B16-ras cell lysates treated with dimethyl hydrazine (DMSO) or 50 μM PD98059 for 12 hours Combine. Anti-CSE1L antibodies and pre-immune sera were also tested as controls.

*O.D. (optical density).

Example 12: Phosphorylated CSE1L protein is located in cellular microvesicles.

Using anti-phospho CSE1L antibody and immunofluorescence techniques, the results showed that the phosphorylated CSE1L protein was located in cellular microvesicles (Fig. 12).

Figure 12 shows that the phosphorylated CSE1L protein is located in cellular microvesicles. Cellular immunofluorescence analysis with anti-phospho-CSE1L antibody and B16-CSE1L cells demonstrated that the phosphorylated CSE1L protein was located in the microvesicles, and the phosphorylated CSE1L was preferentially accumulated in the microvesicles.

Example 13: Microvesicles isolated from serum of cancer patients contain phosphorylated CSE1L protein.

The microvesicles were separated from the serum of colorectal cancer patients and healthy blood donors, and each purified microbubble suspension was equally injected (5 μL) into a 96-well dot-blot manifold. Dot blot analysis was performed using an anti-CSE1L antibody and an anti-phosphorylated CSE1L antibody, respectively. The results showed that the study samples isolated from the serum of cancer patients had a higher prevalence of CSE1L protein than the study samples isolated from the serum of healthy blood donors. CSE1L was detected in 96.5% (111/115) of the study samples isolated from the serum of cancer patients, while only 6.6% (4/60) of the blood samples from healthy blood donors were able to be detected. CSE1L (Fig. 13A). There was a significant difference between the cancer group and the healthy group (P<0.01). Using the synthetic CSE1L protein as a standard, the CSE1L cut-off value of the study sample isolated from the serum of the cancer patient was determined as 21 ng/mL. On the other hand, the results of the analysis also showed that the study samples isolated from the serum of cancer patients had a higher prevalence of phosphorylated CSE1L relative to the study samples isolated from the serum of healthy blood donors. Phosphorylated CSE1L was detected in 98.2% (113/115) of the study samples isolated from the serum of cancer patients, while only 3.3% (2/60) of the blood samples from healthy blood donors were isolated. Phosphorylated CSE1L was detected (P < 0.01) (Fig. 13B). Using the synthetic phosphorylated CSE1L peptide as a standard, the phosphorylated CSE1L cut-off value of the study sample isolated from the serum of cancer patients was determined as 15 ng/mL.

Figure 13 shows that microvesicles isolated from the serum of cancer patients have a higher prevalence of CSE1L and phosphorylated CSE1L relative to microvesicles isolated from the serum of healthy blood donors. The prevalence of microvesicles in the serum of CSE1L (A) and phosphorylated CSE1L (B) in colorectal cancer patients and healthy humans was detected by dot blot hybridization using anti-CSE1L antibody and anti-phosphorylated CSE1L antibody, respectively. * Significant statistical differences.

Example 14: In the diagnosis of cancer, detection of serum phosphorylated CSE1L is superior to detection of serum CSE1L.

The results of the immunoprecipitation showed that the phosphorylated CSE1L protein was present in the cancer serum (Fig. 5C). The prevalence of CSE1L and phosphorylated CSE1L in serum of patients with colorectal cancer was analyzed by enzyme-linked immunosorbent assay (ELISA). The results showed that 92.1% (106/115) of the cancer patients were phosphorylated CSE1L-positive, while only 1.6% (1/60) of the healthy donors were positive for phosphorylated CSE1L (P<0.01). ) (Figure 14). Using the synthetic phosphorylated CSE1L peptide as a standard, the phosphorylation CSE1L cutoff for cancer patients was determined as 3 ng/mL. The results of enzyme-binding immunosorbent assay showed that 65.2% (75/115) of the cancer patients were CSE1L-positive, while 13% (8/60) of the healthy blood donors were CSE1L-positive. Using the synthetic CSE1L protein as a standard, the serum CSE1L cut-off value of cancer patients was determined as 8 ng/mL. Phosphorylation of CSE1L in serum was measured in serum, with sensitivity and specificity of 92.1% and 98.3%, respectively. The CSE1L test for cancer in serum was measured with sensitivity and specificity of 65.2% and 86.6%. Therefore, in the diagnosis of cancer, detection of serum phosphorylated CSE1L is superior to detection of serum CSE1L.

Figure 14 shows that cancer patient sera have a higher prevalence of phosphorylated CSE1L relative to phosphorylated CSE1L in healthy blood donor serum. The prevalence of phosphorylated CSE1L in serum of colorectal cancer patients and healthy blood donors was measured by enzyme-bound immunosorbent assay. * Significant statistical differences.

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BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a diagram showing the expression of Ras and CSE1L proteins in B16-dEV, B16-ras, B16-CSE1L, and B16-Ras/anti-CSE1L cells by immunoblotting using immunoblotting. .

Figure 2 is a diagram showing Example 2 of the present invention, showing that v-H-ras oncogene transfection induces the generation of microvesicles in cancer cells.

Figure 3 is a diagram showing Example 3 of the present invention, showing that transfection of the v-H-ras oncogene increases the secretion of CSE1L by cancer cells.

Figure 4 is a diagram showing Example 4 of the present invention, showing that CSE1L is a phosphorylated protein.

Figure 5 is a diagram showing Example 5 of the present invention, showing that v-H-ras oncogene transfection increases phosphorylation of CSE1L protein in cancer cells, and that phosphorylated CSE1L protein is present in cancer serum.

Figure 6 is a diagram showing Example 6 of the present invention, showing that phosphorylated ERK and CSE1L are highly expressed in colorectal cancer tumors, and the relative staining intensity is consistent.

Figure 7 is a diagram showing Example 7 of the present invention, showing that CSE1L regulates the generation of cancer cell microvesicles induced by the v-H-ras oncogene.

Figure 8 is a diagram showing Example 8 of the present invention, showing that CSE1L regulates cancer cell invasion induced by the v-H-ras oncogene.

Figure 9 is a diagram showing Example 9 of the present invention, showing that CSE1L regulates cancer cell metastasis induced by the v-H-ras oncogene.

Figure 10 is a diagram of Example 10 of the present invention showing that CSE1L is located in the microvesicle membrane and that antibodies against CSE1L are capable of finding and binding to the tumor.

Figure 11 is a diagram of Example 11 of the present invention showing the specific binding of an anti-phosphorylated CSE1L antibody to a phosphorylated CSE1L protein.

Figure 12 is a diagram showing Example 12 of the present invention showing that the phosphorylated CSE1L protein is located in cellular microbubbles.

Figure 13 is a diagram showing Example 13 of the present invention showing that the microvesicles isolated from the serum of cancer patients have a higher prevalence of CSE1L and phosphorylated CSE1L relative to microvesicles isolated from the serum of healthy blood donors.

Figure 14 is a diagram showing Example 14 of the present invention showing phosphorylation of CSE1L relative to serum of healthy blood donors, and serum of patients with cancer has a higher prevalence of phosphorylated CSE1L.

Claims (11)

Use of an anti-CSE1L antibody or an anti-phosphorylated CSE1L antibody in the preparation of a kit for detecting CSE1L or phosphorylated CSE1L on a microbubble membrane in a body fluid sample for in vitro detection of CSE1L of microvesicles in a body fluid sample Or a method of phosphorylating CSE1L, the method comprising the steps of: (1) obtaining a body fluid sample of the subject to be tested; (2) isolating the microvesicles in the sample; (3) in vitro, using an anti-CSE1L antibody or an anti-phosphorylated CSE1L An antibody, mixed and reacted with the sample; and (4) measuring the amount of CSE1L or phosphorylated CSE1L of the microvesicle in the sample of the body fluid being tested. The use according to claim 1, wherein the amount of CSE1L or phosphorylated CSE1L in said sample is detected by dot blot assay. The use according to claim 1, wherein the amount of CSE1L in said sample is detected by binding of an anti-CSE1L antibody and microvesicles; the amount of phosphorylated CSE1L in said sample is anti-phosphorylated CSE1L antibody and microvesicle Combine for testing. The use of claim 1 wherein the individual is an animal. The use of an anti-phosphorylated CSE1L antibody for the preparation of a kit for phosphorylating CSE1L in a body fluid sample for in vitro, the kit for in vitro detection of phosphorylated CSE1L in a body fluid sample, the method comprising the steps of: (1) Obtaining a body fluid sample of the subject to be tested; (2) mixing and reacting with the sample of the body fluid to be tested with an anti-phosphorylated CSE1L antibody; and (3) measuring the amount of phosphorylated CSE1L of the sample of the body fluid to be tested. The use according to claim 5, wherein the amount of phosphorylated CSE1L in said sample is detected by enzyme-bound immunosorbent assay (ELISA). The use according to claim 5, wherein the amount of phosphorylated CSE1L in said sample is detected by binding of an anti-phosphorylated CSE1L antibody to phosphorylated CSE1L. The use according to claim 5, the individual being an animal. A tumor targeting vector conjugated to an anti-CSE1L antibody or an anti-phosphorylated CSE1L antibody. A kit for the in vitro detection of CSE1L or phosphorylated CSE1L on a microvesicle membrane isolated from a body fluid sample, the kit comprising the antibody of claims 1 to 4. A kit for the in vitro detection of phosphorylated CSE1L in a body fluid sample, the kit comprising the antibody of claims 5-8.