CN102803968B - Esophageal cancer marker - Google Patents

Abstract

As a result of immunohistochemical staining of human cancer pathological tissue sections and normal tissue sections with various marker candidates, the antibody against the SLC38A4 protein, out of 80 spontaneously developed types of antigen-specific antibodies against a transporter, was found to react specifically to esophageal cancer tissue and in particular to show high reactivity in esophageal cancer tissue in early well-differentiated stages.

Description

Esophageal Cancer Markers

technical field

The present invention relates to a method for examining esophageal cancer using expression of SLC38A4 (alias: SANT4) protein as an index, a composition for detecting esophageal cancer comprising an anti-SLC38A4 protein antibody, and a method for producing the composition for detecting esophagus comprising anti-SLC38A4 A protein antibody detection kit for esophageal cancer, and a method for screening a drug for treating esophageal cancer using the SLC38A4 protein.

Background technique

Esophageal cancer is a tumor of epithelial origin that occurs in the esophagus. Histologically, it is mainly classified into squamous cell carcinoma of esophageal mucosal epithelium and adenocarcinoma of esophageal adenocarcinoma. In Japan, squamous cell carcinoma accounts for 90% of all esophageal cancers, and the remaining 5% is adenocarcinoma. It can be seen from this that Japanese esophageal cancer is basically squamous cell carcinoma. Moreover, it mostly occurs in men in their 60s, with a male to female ratio of 3 to 5:1. Since there is no outer membrane (serosa) as a morphological feature of the esophagus, the proliferating cancer cells easily infiltrate to the surrounding area and easily metastasize to the lymph nodes, so the progression of canceration is rapid. Coupled with the lack of subjective symptoms, early detection is easily delayed. The prognosis of esophageal cancer is extremely poor among digestive system cancers including gastric cancer and colorectal cancer. The overall 5-year survival rate of esophageal cancer is about 14%. Although the carcinogens are unknown, smoking cessation can reduce the incidence of esophageal squamous cell carcinoma, which suggests that there is an association between smoking and esophageal squamous cell carcinoma.

The diagnosis of esophageal cancer utilizes physical examination findings, imaging diagnosis, and tumor markers. Physical examination findings are almost absent in early stage cancers. Swollen lymph nodes on the right or left upper clavicle are sometimes found in advanced cancer. Among patients diagnosed with esophageal cancer, 74% had dysphagia including esophageal discomfort at the time of diagnosis, and 14% had dysphagia.

In diagnostic imaging, barium sulfate x-rays can be used to easily observe narrowing and/or deformation of the esophagus. However, this test method is difficult to detect early cancer. The most useful in early cancer detection is endoscopy. However, it is often difficult to find superficial cancers that stay on the mucosal surface in ordinary endoscopic observation. For this, staining (chromoendoscopy) is performed with Lugol solution. Lugol's solution stains glycogen-rich normal mucosal squamous epithelium. If the amount of glycogen is significantly reduced due to mucosal canceration and atypical epithelialization, the lesion will not be stained by Lugol's solution and a white non-staining zone will be formed. However, the non-staining band of Lugol's solution is not specific to cancer, and it is also positive in esophagitis and atrophy. Iodine blue staining may also be used, in which case normal mucosa forms unstained bands and cancerous lesions are stained blue. In this staining, it is an indispensable condition for cancerous lesions to be exposed on the surface, but intradermal cancers are not positive. In either case, pathological diagnosis by biopsy combined with endoscopy becomes the definitive diagnosis of esophageal cancer. When esophageal cancer is confirmed, endoscopic ultrasonography and/or CT (computed tomography) are performed in order to determine the depth of invasion (advanced stage). Therefore, it is possible to detect whether there is invasion to the surrounding tissues of esophageal cancer and/or metastasis to lymph nodes and distant organs, thereby diagnosing the advanced stage of esophageal cancer. PET examination is considered to be useful for the evaluation of metastatic lesions that are difficult to judge by CT.

For the diagnosis using tumor markers, as mentioned above, since more than 90% of esophageal cancers in Japan are squamous cell carcinomas, SCC (squamous cell carcinoma), which is an indicator of squamous cell carcinomas of the cervix and esophagus, Related antigens) are more often used. The preoperative diagnosis rate of SCC alone is about 30%. As tumor markers other than SCC, CEA, CYFRA21-1 and the like are used. However, when these markers are used, the positive rate increases with the progression rate of cancer, but the diagnostic rate is low in the case of early cancer. With regard to autoantibodies, p53 antibody has the characteristics of a relatively high positive rate in relatively early cases, but the positive rate is still only about 20-30%.

On the other hand, it has been pointed out that abnormally enhanced expression of transporters can be one of the indicators of carcinogenesis (Patent Documents 1 to 3, Non-Patent Document 1). Significant proliferation and/or metastasis can be cited as a characteristic of cancerous cells. However, since such proliferation and/or metastasis require a large amount of nutrients, cancer cells sometimes increase the expression levels of transporters for glucose and amino acids. For example, L-type amino acid transporter LAT1 is known to be highly expressed in various human cancer tissues (prostate cancer, colon cancer, bladder cancer, Barrett's esophagus adenocarcinoma, oral squamous cell carcinoma, liver cancer) (Non-Patent Document 2). However, transporters reported so far, including LAT1, are expressed in small amounts in normal cells, and it is difficult to say that they are cancer-specific transporters (Non-Patent Documents 3 and 4).

As described above, no marker highly specific to esophageal cancer or a marker showing a high positive rate for early-stage esophageal cancer has been found so far. Esophageal cancer not only lacks subjective symptoms, but also has the characteristics of easy metastasis after onset and poor prognosis. Therefore, it is necessary to identify tumor markers that are highly specific for esophageal cancer and can realize early detection of esophageal cancer.

prior art literature

patent documents

Patent Document 1: Japanese Patent Application No. 11-248546

Patent Document 2: Japanese Patent Application No. 2004-76282

Patent Document 3: Japanese Patent Application No. 10-126648

Patent Document: International Publication No. 2008/096416 Pamphlet

non-patent literature

Non-Patent Document 1: Imai H, et al.Histopathology.2009 Jun;54(7):804-13.

Non-Patent Document 2: Kondoh N, et al.Int J Oncol.2007 Jul;31(1):81-7.

Non-Patent Literature 3: del Amo EM, et al. Eur J Pharm Sci.2008 Oct 2;35(3):161-74. Epub 2008 Jul 5.

Non-Patent Literature 4: Mackenzie B, Erickson JD. Pflugers Arch. 2004 Feb;447(5):784-95. Epub 2003 Jul 4. Review.

Non-Patent Document 5: Sugawara M, et al. Biochim Biophys Acta. 2000 Dec 20;1509(1-2):7-13.

Non-Patent Document 6: Hatanaka T, et al. Biochim Biophys Acta. 2001 Feb 9; 1510(1-2): 10-7.

Non-Patent Document 7: Gu S, et al.Genomics.2001 Jun 15;74(3):262-72.

Non-Patent Literature 8: Sundberg BE, et al.J Mol Neurosci.2008 Jun;35(2):179-93.Epub 2008 Apr 17.

Non-Patent Document 9: Desforges M, et al.J Physiol.2009 Jan 15;587(Pt 1):61-72.Epub 2008 Nov 17.

Non-Patent Document 10: Gao H, et al. Biol Reprod. 2009 Jun; 80(6): 1196-208. Epub 2009 Jan 28.

Non-Patent Document 11: Desforges M, et al.Am J Physiol Cell Physiol.2006Jan;290(1):C305-12.Epub 2005 Sep 7.

Non-Patent Literature 12: Song B, et al. World J Gastroenterol. 2005 Mar 14; 11(10): 1463-72.

Contents of the invention

The problem to be solved by the invention

The present invention was made in view of the above circumstances, and an object of the present invention is to provide a tumor marker that is highly specific for esophageal cancer and enables early detection of esophageal cancer. Furthermore, the object of the present invention is to provide: an examination method for esophageal cancer (especially early esophageal cancer) targeting the tumor marker, a composition for detection of esophageal cancer comprising a molecule for detecting the tumor marker, the combination A method for producing a substance, a kit for detecting esophageal cancer comprising the composition, and a method for screening a drug for treating esophageal cancer using the tumor marker.

method used to solve the problem

The SLC38A4 protein (alias: SNAT4) belongs to the sodium ion-dependent amino acid transporter (non-patent document 4), and its gene was first cloned from the muscle of rats in 2000, and for humans in 2001, it was cultured from the liver. cloned in cells (Non-Patent Documents 5 to 7). The SLC38A4 protein is structurally classified into the glutamine transporter superfamily, but actually has a low affinity for glutamine, and its transport capacity is much lower than other glutamine transporters. On the other hand, L-alanine and asparagine are mainly transported (Non-Patent Document 4).

Regarding the localization of SLC38A4 in adults, it was detected from rat muscle and liver by Northern blotting at the mRNA level, and the liver had the highest content in human tissues (Non-Patent Documents 6 to 8). Genes were amplified from skeletal muscle other than liver, brain, lung, heart, small intestine, kidney, pancreas, placenta, and uterus by RT-PCR (Non-Patent Documents 9 and 10). Information on the localization of the protein level of SLC38A4 was detected in the liver and placenta by Western blotting, and in the placenta by immunohistostaining (Non-Patent Documents 9 and 11). Regarding the relationship with cancer, mRNA expression was confirmed in the hepatocellular carcinoma cell line JHH4, but it was at the same level as in normal liver tissue, and expression enhancement was not confirmed experimentally (Patent Document 2). In addition, in mice, the results of gene expression analysis using microarrays for highly metastatic strained hepatocellular carcinoma Hca-F and low metastatic hepatocellular carcinoma Hca-P reported that the expression of SLC38A4 gene in Hca-F Higher expression, but the causal relationship with liver cancer metastasis is unclear (Non-Patent Document 12). Thus, the relationship between the expression of SLC38A4 and the esophagus or esophagus cancer has not been known so far.

In view of the above background, in order to explore cancer-specific markers, the present inventors performed immunohistostaining on human cancer pathological sections and normal tissue sections for various marker candidates, and found that in the self-developed 80 transporter Among the antigen-specific antibodies to the protein, the antibody against the SLC38A4 protein reacted specifically and strongly to esophageal cancer tissue (especially early esophageal cancer tissue). Furthermore, based on the knowledge that SLC38A4 protein is highly expressed in esophageal cancer and hardly detected in normal tissues, the present inventors found that esophageal cancer can be examined using the expression of SLC38A4 protein as an index, and an immunological method using an anti-SLC38A4 protein antibody It is useful in the examination of esophageal cancer. Furthermore, the present inventors have found that the SLC38A4 protein can be used to screen drugs for treating esophageal cancer.

That is, the present invention relates to a method for examining esophageal cancer (especially early esophageal cancer) using the expression of the SLC38A4 protein as an index, a composition for detecting esophageal cancer comprising an anti-SLC38A4 protein antibody, and a method for producing the composition comprising an anti-SLC38A4 protein antibody. A kit for detecting esophageal cancer with an antibody to the SLC38A4 protein, and a method for screening a drug for treating esophageal cancer using the SLC38A4 protein. More specifically, the present invention provides the following (1) to (14).

(1) A method for examining esophageal cancer, comprising: detecting the expression of SLC38A4 protein in cells or tissues isolated from a subject.

(2) The method according to (1), wherein the expression of the SLC38A4 protein is detected using an antibody.

(3) The method according to (2), wherein the antibody recognizes a region including the amino acid sequence described in SEQ ID NO: 2 in the SLC38A4 protein.

(4) The method according to any one of (1) to (3), wherein the esophageal cancer is early-stage esophageal cancer.

(5) A composition for detecting esophageal cancer, comprising an anti-SLC38A4 protein antibody.

(6) The composition according to (5), wherein the antibody recognizes a region including the amino acid sequence described in SEQ ID NO: 2 in the SLC38A4 protein.

(7) The composition according to (5) or (6), wherein the esophageal cancer is early-stage esophageal cancer.

(8) The method for producing the composition described in (5), including the following steps:

(a) the process of immunizing the SLC38A4 protein or an immunogenic portion thereof, and

(b) A step of isolating and/or purifying an antibody that binds to the SLC38A4 protein.

(9) The method for producing the composition described in (6), which includes the following steps:

(a) a step of immunizing a peptide comprising the amino acid sequence described in SEQ ID NO: 2 in the SLC38A4 protein, and

(b) A step of isolating and/or purifying an antibody that binds to a region including the amino acid sequence described in SEQ ID NO: 2 in the SLC38A4 protein.

(10) The production method according to (9) or (10), wherein the composition is a composition for detection of early esophageal cancer.

(11) A kit for detecting esophageal cancer, comprising the composition described in (5) to (7).

(12) A screening method for a drug for treating esophageal cancer, which includes the following steps:

(a) providing a SLC38A4 protein or a portion thereof,

(b) a process of contacting the candidate compound with the SLC38A4 protein or a portion thereof, and

(c) A step of selecting a compound that binds to the SLC38A4 protein or a part thereof.

(13) A screening method for a drug for treating esophageal cancer, which includes the following steps:

(a) a step of administering a candidate compound or a control to an esophageal cancer model animal (other than human),

(b) the process of collecting the tissue of the esophagus of the model animal, and

(c) A step of detecting the expression of the SLC38A4 protein in the collected tissue, and selecting a compound that reduces the expression of the SLC38A4 protein compared with the control.

(14) The screening method according to (12) or (13), wherein the therapeutic drug is a therapeutic drug for early esophageal cancer.

The effect of the invention

In the present invention, it was found that the expression of the SLC38A4 protein was specifically detected in esophageal cancer, and was particularly detected at a high positive rate in esophageal cancer at an early, well-differentiated stage. Therefore, according to the examination method for esophageal cancer of the present invention using the expression of SLC38A4 protein as an index, esophageal cancer can be detected early with high accuracy. Thus, treatment of esophageal cancer can be performed at an early stage of progression of esophageal cancer, and can greatly contribute to the treatment of patients and the improvement of prognosis of patients. In addition, according to the screening method of the therapeutic drug for esophageal cancer of the present invention using the SLC38A4 protein, it is possible to efficiently identify a candidate compound for a therapeutic drug. The composition for detection of esophageal cancer and the kit for detection of esophageal cancer of the present invention containing the anti-SLC38A4 protein antibody are extremely useful for the detection of esophageal cancer and the screening of therapeutic drugs for esophageal cancer in the present invention.

Description of drawings

Fig. 1 is a diagram showing the full-length amino acid sequence of human-derived SLC38A4 protein and the amino acid sequence (boxes: 29th to 47th) artificially used as an immunogen.

Fig. 2 is a graph showing the results of analyzing the antibody titers of antisera obtained after immunizing rabbits, antigen-affinity-purified antibodies, and normal rabbit sera by antigen solid-phase ELISA. The X-axis represents the antigen concentration (μg/ml), and the Y-axis represents the absorbance (OD450).

Fig. 3 is a graph showing the results of evaluating the performance of an anti-SLC38A4 protein antibody by flow cytometry using cells forcibly expressing SLC38A4. The X-axis represents the amount of fluorescence of the green fluorescent protein Azami-Green as an indicator of target gene expression. The Y-axis represents the reactivity of PE-labeled secondary antibodies.

Fig. 4 is a micrograph showing the results of evaluating the performance of an anti-SLC38A4 protein antibody by cell immunostaining using cells forcibly expressing SLC38A4. The left panel shows reactivity to 293T cells not expressing SLC38A4 protein, and the right panel shows reactivity to 293T cells transiently expressing SLC38A4 protein.

Fig. 5 is a micrograph showing the results of immunohistostaining to confirm the stainability of an anti-SLC38A4 protein antibody using an esophageal cancer patient sample. Use typical 2 cases. The left figure is the result using the normal part of the patient, and the right figure is the result using the patient's tissue including the cancerous part.

Fig. 6 is a graph showing the positive rate of each grade of esophageal cancer samples using an anti-SLC38A4 protein antibody.

Fig. 7 is a photomicrograph showing the results of immunohistostaining with an anti-SLC38A4 protein antibody using WHO grade I and grade I-II esophageal cancer samples. Four typical cases of grade I are shown in the figure.

Fig. 8 is a photomicrograph showing the results of immunohistostaining with an anti-SLC38A4 protein antibody using WHO grade II and II-III esophageal cancer samples. Typical 4 cases of grade II are shown in the figure.

Fig. 9 is a micrograph showing the results of immunohistostaining with an anti-SLC38A4 protein antibody using a WHO grade III esophageal cancer sample. Four typical cases of grade III are shown in the figure.

Fig. 10 is a graph showing scores of tissue staining for each grade of esophageal cancer samples using an anti-SLC38A4 protein antibody.

Fig. 11 is a microscope showing the results of immunohistostaining of 6 samples of digestive system cancers (gastric adenocarcinoma, colorectal adenocarcinoma, rectal cancer, pancreatic cancer, hepatocellular carcinoma, and kidney cancer) using an anti-SLC38A4 protein antibody photo. The figure shows a typical example of tissue staining of each cancer sample.

Detailed ways

<Examination method for esophageal cancer>

The present invention provides a method for examining esophageal cancer, which includes the step of detecting the expression of SLC38A4 protein in cells or tissues isolated from a subject.

In the present invention, "cell or tissue" refers to a cell or tissue that becomes a sample (object) when detecting the expression of SLC38A4 protein in the test method of the present invention. The present invention is applied to cells or tissues isolated from a living body. "Isolation from a subject" refers to a state in which cells or tissues are completely isolated from the organisms from which they originate by removing cells or tissues from a living body. The subjects from which cells or tissues are extracted are not limited to cancer patients, and may be healthy people (including those who may suffer from cancer). A part of an organ and/or tissue of a subject collected in a biopsy (Biopsy) is supplied to the invented inspection method.

The pathological tissue is usually prepared in the state existing in the living body, that is, in the state of being combined with the surrounding cells (as a tissue sheet), and used in the method of the present invention, but it may also be after the pathological tissue is separated from the surrounding cells used in the method of the invention.

When the detection result of the expression of the SLC38A4 protein is used for the diagnosis of esophageal cancer, as the pathological tissue, it is preferable to use a tissue judged to be cancer by other diagnostic methods, a tissue with a high probability of being judged to be cancer, or Tissue that may be cancerous. The tissue used is preferably a tissue judged to be cancer by other diagnostic methods, or a tissue judged to have a high probability of being cancer. Here, examples of other diagnostic methods include diagnostic methods using X-ray contrast examination, endoscopy, ultrasonography, CT examination, MRI examination, PET examination, and tumor markers. Usually, tissue suspected of being cancer by one or more of the above diagnostic methods is used.

In the present invention, the "SLC38A4 protein" whose expression is detected is typically a protein comprising the amino acid sequence described in SEQ ID NO: 1. However, the amino acid sequence of a protein may be mutated in nature (ie, not artificially) due to mutation of its encoding gene and the like. Therefore, in the present invention, such natural mutants of the SLC38A4 protein can also be detected.

In the present invention, "detecting the expression of the SLC38A4 protein" includes both the detection of the presence or absence of the expression of the SLC38A4 protein and the detection of the degree of expression. The expression amount of SLC38A4 protein can be grasped as an absolute amount or a relative amount. When the relative amount is known, for example, it can be judged by comparing it with the expression amount of SLC38A4 protein in a prepared standard sample. A "standard sample" is a sample whose expression of the SLC38A4 protein is determined in advance. For example, a pathological tissue at a site where esophageal cancer has been determined can be used as a standard sample in the present invention. In addition, non-cancerous tissues (normal tissues) can also be used as standard samples in the present invention.

The detection of the expression of SLC38A4 protein in the present invention is preferably performed by immunological methods. Examples of immunological methods include immunohistochemical staining, ELISA, radioimmunoassay, FCM, immunoprecipitation, and western blotting. In the immunological method, an anti-SLC38A4 protein antibody is used, the antibody is brought into contact with the SLC38A4 protein, and the SLC38A4 protein is detected using the binding property (bound amount) of the antibody to the SLC38A4 protein as an index. Here, "contacting" refers to placing the antibody and the SLC38A4 protein under physiological conditions under which the anti-SLC38A4 protein antibody can recognize the SLC38A4 protein. For example, in the case of using the antibody to stain the SLC38A4 protein on the cell surface, it refers to infiltrating cells or tissues isolated from a subject into a solution containing the antibody, or fully dripping the solution containing the antibody, or Spray into the cells or tissues, and place under the physiological conditions that the antibody can recognize SLC38A4 present in the cells or tissues. According to the immunological detection method, rapid and sensitive detection is possible, and the operation is also simple. The detection method of the present invention is also advantageous in that it places less burden on the patient's body.

The type and/or source of the anti-SLC38A4 protein antibody used in the detection method of the present invention is not particularly limited as long as it has specific binding to the SLC38A4 protein. Preferably, it is an antibody that recognizes the amino acid sequence "GIGNSEKAAMSSQFANEDT" (SEQ ID NO: 2) at positions 29 to 47 of the SLC38A4 protein (SEQ ID NO: 1).

If an antibody bound to a labeled substance is used as the anti-SLC38A4 protein antibody, the amount of the antibody bound to the SLC38A4 protein can be directly measured by detecting the label, which is convenient. On the other hand, this method also has problems in that it is troublesome to prepare antibodies bound to the labeled substance, and the detection sensitivity is generally low. Therefore, in the present invention, indirect detection methods such as a method using a secondary antibody bound to a labeled substance, and/or a method using a polymer obtained by binding a secondary antibody to a labeled substance are preferred. Here, the "secondary antibody" is an antibody that specifically binds to the anti-SLC38A4 protein antibody. For example, when an anti-SLC38A4 protein antibody is prepared as a rabbit antibody, an anti-rabbit IgG antibody can be used as the secondary antibody. For antibodies derived from various biological species such as rabbits, goats, and mice, commercially available secondary antibodies can be used, and appropriate secondary antibodies can be selected according to the source species of the anti-SLC38A4 protein antibody and used in the present invention. Instead of the secondary antibody, protein A, protein G, etc. to which a labeling substance is bound can also be used.

Examples of labeling substances include peroxidase, β-D-galactosidase, microperoxidase, horseradish peroxidase (HRP), fluorescein isothiocyanate (FITC), isothiocyanate Rhodamine (RITC), alkaline phosphatase, biotin and radioactive substances, etc. In the present invention, by using biotin as a labeling substance and reacting it with avidin peroxidase, antibodies bound to the SLC38A4 protein can be detected with high sensitivity.

Immunohistochemical staining of living tissue is generally performed through the following steps (1) to (10). Among them, regarding the immunohistochemical staining method of living tissues, various literatures and written books can be referred to (for example, "Enzyme Antibody Method, Revised Third Edition", edited by Keiichi Watanabe and Kazuho Nakane, inter-scholastic project).

(1) Fixed and embedded in paraffin

A tissue sample surgically collected from a living body is fixed with formalin or paraformaldehyde, absolute ethanol, or the like. Then embedded in paraffin. Generally, after alcohol dehydration, it is treated with xylene, and finally embedded in paraffin. The paraffin-embedded specimen is cut into thin sections of a desired thickness (for example, 3 to 5 μm thick), and stretched on a glass slide. In addition, alcohol-fixed specimens, dry-sealed specimens, frozen specimens, etc. can sometimes be used instead of paraffin-embedded specimens.

(2) Deparaffinization

Generally, it is treated sequentially with xylene, alcohol and purified water.

(3) Pretreatment (Antigen Retrieval)

Enzyme treatment, heat treatment, and/or pressure treatment, etc. are performed for antigen retrieval as necessary.

(4) Removal of endogenous peroxidase

In the case of using peroxidase as a labeling substance at the time of staining, the endogenous peroxidase activity is removed by treating with aqueous hydrogen peroxide in advance.

(5) Inhibition of non-specific reactions

The section is treated with a bovine serum albumin solution (for example, 1% solution) for several minutes to several tens of minutes to suppress non-specific reactions. In addition, if the subsequent primary antibody reaction is performed using an antibody solution containing bovine serum albumin, this step can be omitted.

(6) Antibody response

Antibody diluted to an appropriate concentration is dropped onto the section on the glass slide, and reacted for tens of minutes to several hours. After the reaction, wash with an appropriate buffer such as phosphate buffer.

(7) Addition of labeling reagents

Peroxidases are often used as labeling substances. In the above antibody reaction, an anti-SLC38A4 protein antibody conjugated with peroxidase can also be used. In the case where the anti-SLC38A4 protein antibody is not labeled, a secondary antibody conjugated with peroxidase or a Enzyme protein G, protein A, etc. For example, in the case of using a secondary antibody, the peroxidase-conjugated secondary antibody is dropped onto a section on a glass slide, and then reacted for tens of minutes to several hours. After the reaction, wash with an appropriate buffer such as phosphate buffer.

(8) Color reaction

Dissolve DAB (3,3'-diaminobenzidine) in Tris buffer. Then add hydrogen peroxide water. The color-developing solution prepared in this way is soaked into the section for several minutes (for example, 5 minutes) to develop color. After color development, the sections were washed extensively with tap water to remove DAB.

(9) Nuclear staining

Nuclear staining was carried out by allowing Mayer's hematoxylin to react for several seconds to several tens of seconds. Rinse with running water to develop color (usually a few minutes).

(10) Dehydration, clarification, sealing

After dehydration with alcohol, clear with xylene, and finally seal with synthetic resin or glycerin, mounting medium, etc.

The probability that a cell or tissue is cancerous and/or the cancerous site in the tissue is strongly correlated with the staining intensity of immunostaining and/or the proportion of cells stained by immunostaining. Therefore, in the present invention, by detecting the expression of the SLC38A4 protein, it is possible to evaluate the probability that a cell or tissue isolated from a subject is cancerous and/or identify a cancerous site in the tissue. Thereby, the location and status of esophageal cancer (including the location and/or status of metastasis, the presence of cancer cells infiltrating into new tissues, etc.) can be visually confirmed. In particular, since the expression of SLC38A4 protein is detected at a high positive rate in esophageal cancer at an early stage of well-differentiated stage, according to the method of the present invention, the stage of pathological tissue (well-differentiated stage) can be determined. Compared with advanced cancer with grade development, cancer cells at the highly differentiated stage have more similarities in cell morphology with normal squamous epithelial cells, and it is difficult to distinguish cancer from normal cells. However, the tissue of the present invention using an anti-SLC38A4 protein antibody Staining is extremely useful in the detection of early-stage cancers that are difficult to distinguish from normal ones visually. In the present invention, "early esophageal cancer" specifically refers to esophageal cancer that has not reached grade III, preferably grade I or grade II esophagus cancer. The information obtained by carrying out the above methods on cancer patients can be used in the evaluation or understanding of the patient's condition, evaluation of therapeutic effect, and the like. For example, if the method of the invention is carried out in parallel with the treatment, the efficacy of the treatment can be evaluated on the basis of the resulting information. Specifically, by implementing the method of the present invention after administration of the drug, changes in staining properties in pathological tissues can be studied, and the therapeutic effect can be judged from changes in the increase or decrease of stained sites. This makes it possible to use the method of the invention as a monitor of the effect of the treatment. On the other hand, the information obtained for persons other than patients, that is, persons not recognized as cancer, can be used for determination and evaluation of the presence or absence of esophageal cancer. According to the method of the present invention, cancer diagnosis can be performed based on an objective indicator such as stainability.

The diagnosis of cancer in the subject is usually performed by a doctor (including a person instructed by the doctor. The same below.), and the data on the expression level of the SLC38A4 protein in the pathological tissue obtained by the method of the present invention is of great importance to the doctor. Diagnosis comes into play. Therefore, the method of the present invention can also be said to be a method of collecting and presenting data that contribute to a doctor's diagnosis.

<Composition for detection of esophageal cancer and its production method>

The present invention provides a composition for detecting esophageal cancer, which contains an anti-SLC38A4 protein antibody. The type and/or source of the anti-SLC38A4 protein antibody is not particularly limited as long as it has specific binding property to the SLC38A4 protein. The antibody used in the composition for detecting esophageal cancer of the present invention may be a monoclonal antibody or a polyclonal antibody. In addition, the antibody used in the composition for detecting esophageal cancer of the present invention may be a functional fragment of the antibody and/or a multimer form of the functional fragment (for example, dimer, trimer, tetramer, polymer). Such functional fragments and/or multimers thereof include, for example, Fab, Fab', F(ab')2, Fv, scFv, sc(Fv)2, dsFv, and diabodies. Here "Fab" refers to a monovalent antigen-binding fragment of an immunoglobulin comprising a light chain and part of a heavy chain. Can be obtained by papain digestion of antibodies, as well as by recombinant methods. "Fab'" comprises one or more than two cysteines of the hinge region of the antibody and differs from Fab by the addition of a small number of residues at the carboxyl terminus of the CH1 domain of the heavy chain. "F(ab')2" is a bivalent antigen-binding fragment of an immunoglobulin comprising two light chains and portions of two heavy chains. "Fv" is the smallest antibody fragment with a complete antigen recognition and binding site. Fv is a dimer composed of a heavy chain variable region and a light chain variable region firmly linked by non-covalent bonds. A "scFv" comprises the heavy and light chain variable regions of an antibody, which regions are present in a single polypeptide chain. "sc(Fv)2" is obtained by combining two heavy chain variable regions and two light chain variable regions into a single chain through a linker. "dsFv" is a disulfide bond stabilized Fv. A "diabody" is a small antibody fragment with two antigen-binding sites, the fragment comprising a heavy chain variable region joined to a light chain variable region in the same polypeptide chain, each region forming a complementary region with the other chain pair.

The anti-SLC38A4 protein antibody is preferably an antibody that recognizes the amino acid sequence "GIGNSEKAAMSSQFANEDT" (SEQ ID NO: 2) at positions 29 to 47 of the SLC38A4 protein (SEQ ID NO: 1).

The method for producing the composition for detecting esophageal cancer of the present invention includes a step of immunizing the SLC38A4 protein or an immunogenic part thereof, and a step of isolating and/or purifying an antibody that binds to the SLC38A4 protein. Further, a step of mixing other components allowed as a composition may be included.

The antibody preparation method used in the composition for detecting esophageal cancer of the present invention is known in the art, for example, described in Harlow and Lane, Antibodies: A Laboratory Manual (NewYork: Cold Spring Harbor Laboratory Press, 1988). Antibodies can be prepared by usual immunological methods and phage surface display methods.

The preparation of polyclonal antibodies can be carried out according to the following steps. First, an antigen (for example, the full length or a part of the SLC38A4 protein described in SEQ ID NO: 1) is prepared, and then animals such as rabbits are immunized with the antigen. The SLC38A4 protein as an antigen can be prepared by separating and purifying it from a biological sample. In addition, SLC38A4 protein prepared as a recombinant protein can also be used as an antigen. The recombinant protein is prepared by inserting the gene encoding the SLC38A4 protein or a part thereof into a vector in an expressible state, introducing the vector into an appropriate host, and then isolating and purifying the protein expressed in the host. The SLC38A4 protein, preferably the peptide region comprising the 29th to 47th amino acid sequence "GIGNSEKAAMSSQFANEDT" (SEQ ID NO. 2) can be prepared to combine with GST, β-galactosidase, maltose binding protein, histidine ( A fusion protein of a His) tag or the like is used as an antigen. Such fusion proteins can be easily isolated and purified by general methods.

Immunization is repeated as necessary, blood is collected when the antibody titer rises sufficiently, and serum is obtained by centrifugation or the like. The obtained serum can be prepared as an IgG fraction by affinity chromatography using protein G, protein A, or the like. Antibodies that bind to the immunogen SLC38A4 protein or a part thereof can be further isolated and purified from antiserum and IgG fractions by affinity purification using the SLC38A4 protein or a part thereof.

The polyclonal antibody used in the composition for detecting esophageal cancer of the present invention is particularly preferably immune to a peptide containing the amino acid sequence "GIGNSEKAAMSSQFANEDT" (SEQ ID NO: 2) from the 29th to 47th positions of the SLC38A4 protein, as the peptide that binds to the peptide. Polyclonal antibody obtained by isolation and purification of antibody.

On the other hand, monoclonal antibodies can be prepared according to the following procedure. Firstly, the immunization operation is carried out according to the same steps as above. Repeat the immunization as needed, and extract antibody-producing cells from the immunized animal when the antibody titer rises sufficiently. Next, the resulting antibody-producing cells are fused with myeloma cells to obtain hybridomas. Clones producing antibodies with high specificity for the protein of interest are then selected. The antibody of interest can be obtained by purifying the culture fluid of the selected clone after monocloning the hybridoma. On the other hand, hybridomas can be grown to a desired number or more, transplanted into the peritoneal cavity of an animal (for example, a mouse) to proliferate in ascites, and then the ascites can be purified to obtain the target antibody. In the purification of the above-mentioned culture fluid or ascites fluid, affinity chromatography using protein G or protein A is preferably used. Alternatively, affinity chromatography in which an antigen is immobilized can also be used. In addition, methods such as ion exchange chromatography, gel filtration chromatography, ammonium sulfate fractionation, and centrifugation can also be used. These methods can be used alone or in any combination.

Based on the thus obtained antibody or its gene, functional fragments of antibodies such as Fab, Fab', F(ab')2, Fv, scFv, sv(Fv)2, dsFv and diabodies and/or their Multimers (eg, dimers, trimers, tetramers, and multimers).

As described above, in the case of directly detecting the amount of antibody bound to SLC38A4 protein, the obtained anti-SLC38A4 protein antibody can be directly labeled with enzyme, fluorescence, or the like and used. On the other hand, when performing an indirect detection method using a secondary antibody or the like to detect the amount of antibody bound to the SLC38A4 protein, the anti-SLC38A4 protein antibody (primary antibody) may not be labeled, but the secondary antibody that recognizes the antibody may be labeled.

The composition for detecting esophageal cancer of the present invention may contain other components permitted as a composition in addition to the anti-SLC38A4 protein antibody. Such other components include, for example, carriers, excipients, disintegrants, buffers, emulsifiers, suspending agents, stabilizers, preservatives, preservatives, physiological salts, labeling compounds, secondary antibodies, and the like. As excipients, lactose, starch, sorbitol, D-mannitol, sucrose and the like can be used. As the disintegrant, starch, carboxymethylcellulose, calcium carbonate and the like can be used. As a buffer, phosphate, citrate, acetate, etc. can be used. As the emulsifier, gum arabic, sodium alginate, tragacanth, etc. can be used. As the suspending agent, glycerin monostearate, aluminum monostearate, methylcellulose, hydroxymethylcellulose, hydroxymethylcellulose, sodium lauryl sulfate and the like can be used. As a stabilizer, propylene glycol, diethylaminosulfite, ascorbic acid, and the like can be used. As the preservative, phenol, benzalkonium chloride, benzyl alcohol, chlorobutanol, methylparaben, and the like can be used. As preservatives, benzalkonium chloride, p-hydroxybenzoic acid, chlorobutanol and the like can be used.

<Esophageal cancer detection kit>

The present invention also provides a kit for detecting esophageal cancer comprising the above composition for detecting esophageal cancer. In the kit of the present invention, in addition to the composition (antibody standard) for detection of esophageal cancer, a substrate required for the detection of the label, a positive control and/or a negative control, or in the dilution and/or washing of the reagent can be combined. Buffer used in etc. When an unlabeled antibody is used as an antibody standard, the kit of the present invention may contain a labeled substance that binds to the antibody (eg, secondary antibody, protein G, protein A, etc.). Furthermore, the kit of the present invention may comprise instructions for use of the kit. The kit of the present invention is useful, for example, in the diagnosis of esophageal cancer.

[Screening of Esophageal Cancer Therapeutic Drugs]

The invention also provides a method for screening drugs for treating esophagus cancer. One of the methods is a screening method targeting the SLC38A4 protein, which includes the steps of providing the SLC38A4 protein or a part thereof, contacting a candidate compound with the SLC38A4 protein or a part thereof, and selecting an SLC38A4 protein or a part thereof. A part of the process of combining compounds. Through this method, a compound that binds to the SLC38A4 protein can be obtained for screening of drugs for treating esophageal cancer. As candidate compounds, chemically synthesized or natural low-molecular-weight compounds, natural or synthetic proteins, peptides, antibodies (including the antibodies of the present invention), cell extracts, culture supernatants, and the like can be used. When an antibody is used as a candidate compound, the binding activity of the candidate compound can be measured by EIA, ELISA, or the like. The use of high-throughput methods by combinatorial chemistry techniques to screen synthetic compounds that bind to proteins is also well known to those skilled in the art (Verdine GL. Nature (ENGLAND) 1996 Nov 7; 384: 11-13, Hogan JCJr. Nature (ENGLAND) 1996 Nov 7;384:17-19). The compounds thus obtained can also be used in targeted screening of esophageal cancer.

Another embodiment is a screening method using the expression of the SLC38A4 protein as an indicator, which includes the steps of administering a candidate compound or a control to an esophageal cancer model animal (other than human), and isolating the esophagus tissue of the model animal and a step of detecting the expression of SLC38A4 protein in the isolated tissue and selecting a compound that reduces the expression of SLC38A4 protein compared with the control. This method can be implemented in combination with the above-mentioned screening method targeting the SLC38A4 protein, and can also be implemented independently.

In this method, an animal model of esophageal cancer is first prepared. For example, ethanol or acetaldehyde can be subcutaneously injected into an ALDH2 knockout mouse to induce squamous cell carcinoma and obtain an animal model of esophageal cancer (for example, refer to Japanese Patent Laid-Open 2005-110601 Bulletin). In addition, an animal model of esophageal cancer can also be produced by transplanting human esophageal cancer cells into immunodeficient mice.

Next, a candidate compound or a control is administered to the model animal. As candidate compounds, chemically synthesized or natural low-molecular-weight compounds, natural or synthetic proteins, peptides, antibodies (including the antibodies of the present invention), cell extracts, culture supernatants, and the like can be used. As controls, positive controls and/or negative controls can be used. Here, physiological saline or the like is usually used as a negative control. It may also be a substance that has been found to have no therapeutic effect. As a positive control, a substance or the like that has been found to have a therapeutic effect is used. The method of administration is well known to those skilled in the art, and can be appropriately selected from oral, intravenous, intradermal or subcutaneous, and the like.

Next, the tissue of the esophagus of the model animal was isolated. When the isolated tissue is subjected to immunostaining, it can be fixed, paraffin-embedded, etc. as described above.

Finally, the expression of SLC38A4 protein in the isolated tissue was detected, and the compound that decreased the expression of SLC38A4 protein compared with the control was selected. In the case of detecting the expression of the SLC38A4 protein by immunostaining, by comparing the staining image of the positive control-administered group with the staining image of the candidate compound-administered group, the staining of the candidate compound-administered group is selected to be more negative (for example, staining weak intensity, or a small proportion of strongly stained cancer cells). As the staining image of the control, it is also possible to prepare the staining image of the positive control administration group (the staining image of the administration group of the substance having a therapeutic effect: standard staining image) instead of the staining image of the negative control administration group, and select the same or more. Compounds that stain images negatively.

Example

The present invention will be described in more detail below by way of examples, but the present invention is not limited to these examples.

(1) Production of antibodies

According to the amino acid sequence information of the SLC38A4 protein, the secondary structure was predicted by computer, and all factors including its contact rate with solvent, softness, surface exposure, antigenicity, hydrophilicity and polarity were integrated, and the results were judged from 29th to 29th. The 47th amino acid sequence "GIGNSEKAAMSSQFANEDT" (SEQ ID NO: 2) has the highest antigenicity (Fig. 1). Artificially synthesized peptides containing amino acid sequences from 29th to 47th, combined with KLH, then mixed with complete Freund's adjuvant, and immunized more than two rabbits 6 times every other week. Antigen-specific IgG was purified from the antiserum obtained after immunization using an agarose bead column immobilized with the antigen peptide. For purified IgG, ELISA was performed using a 96-well plate immobilized with antigen peptides, and when the titer (absorbance OD450) was 1.0 or higher at an antigen concentration of 0.4 μg/ml, it was used in subsequent experiments. The titer of the antigen-specific IgG fraction obtained in this example was 1.2 (Fig. 2).

(2) Confirmation of antibody performance

The resulting antibody preferably shows reactivity in cell staining and flow cytometry (FCM) to cultured 293T cells forced to express the full length of the SLC38A4 protein. As the expression vector, pcDNA3.1 (Invitrogen) was used. An IRES sequence serving as a ribosome entry site was loaded downstream of the SLC38A4 gene and a gene for green fluorescent protein Azami-Green (Amalgam Corporation) was loaded immediately downstream of the IRES sequence. The expression vector was transiently introduced into 293T cells using a lipofectamine transfection reagent (Invitrogen), and the cells that Azami-Green fluoresced were judged as cells into which the SLC38A4 gene had been introduced.

First, in order to open pores in the cell membrane of 293T cells (1×10 ⁵ cells) after gene transfer, they were fixed with 4% paraformaldehyde/phosphate buffer solution (PBS) at 4°C for 15 minutes, and then fixed with 0.1 % TritonX-100/PBS for 15 minutes. Next, a 1 μg/ml antibody solution (diluted in 100 μl of PBS containing 1% BSA and 0.1% TritonX-100) was reacted with the cells for 1 hour at room temperature, and then a PE-labeled anti-rabbit IgG antibody was used as a secondary antibody. (manufactured by BECKMANCOULTER, 200-fold dilution. The diluent is the same as the primary antibody diluent) to react. When the antibody was reacted with the antigen-expressing cells, cells that glowed due to Azami-Green (green) simultaneously glowed due to PE (red light) (ie, the scatter point moved up and to the right in the FCM data). When the stained cells were developed on a scatter plot, the scatter points did not shift to the upper right when the rabbit IgG used as a negative control was used. On the other hand, when the myc-tag antibody (manufactured by MBL: PL14) was used as a positive control , and in the case of using the anti-SLC38A4 protein antibody, the scatter points shifted to the upper right ( FIG. 3 ). In addition, 293T cells into which the mock gene was introduced and 293T cells into which the SLC38A4/SNAT4 gene was introduced as controls were treated in the same manner as above, and then observed using a UV fluorescence microscope (OLYMPUS IX71 fluorescence microscope system from OLYMPUS). As a result, almost no fluorescence (red) was observed in the control cells, but fluorescence (red) was detected in the 293T cells into which the SLC38A4/SNAT4 gene was introduced ( FIG. 4 ). Judging from the results in Figs. 2 to 4, the prepared antibody specifically recognized the SLC38A4 protein.

(3) Staining of tissue sections (paraffin-embedded) using antibodies

In order to uniformly source the samples from Asians, all tissue sections (paraffin-embedded) were purchased from Shanghai Outo Company (China). First, stainability was confirmed using 2 cases each of normal esophagus (left) and squamous cell carcinoma of esophagus (right). From this staining, the progression rate of esophageal squamous cell carcinoma was evaluated based on WHO's tumor classification (Pathology and Genetics of Tumors of the Digestive System Edited by Stanley R. Hamilton Lauri A. Aaltonen IARCPress Lyon, 2000, p16). In this evaluation, "well differentiated" in the WHO tumor classification was classified as grade I, "moderately differentiated" as grade II, and "poorly differentiated" as grade III (the same applies hereinafter). . Patient numbers Com01-D7 and Com01-D8 were both WHO grade I squamous cell carcinoma of the esophagus. For deparaffinization of tissue sections, 3 xylene treatments for 5 minutes, 100% ethanol treatments for 5 minutes twice, 95% ethanol treatments once for 5 minutes, A 5 min treatment in 90% ethanol was performed once, a 5 min treatment in 80% ethanol was performed once, a 5 min treatment in 70% ethanol was performed once, followed by a 5 min treatment in PBS Treatments were performed 3 times (all treatments were performed at room temperature). Next, for antigen retrieval, the tissue sections were soaked in 10 mM citrate buffer (pH 6) containing 0.05% Tween20, and treated in an autoclave at 125° C. for 5 minutes. In order to eliminate endogenous peroxidase activity, treat in PBS containing 3% hydrogen peroxide for 10 minutes at room temperature, and then replace with PBS containing 5% normal goat serum and 0.5% BSA (blocking solution). ) at room temperature for 30 minutes. Wipe off the excess solution with a cloth, add an appropriate amount of anti-SLC38A4 protein antibody diluted to 1 μg/ml with blocking solution (enough to soak the tissue section), and react at room temperature for 2 hours. Wash 3 times with PBS containing 0.05% Tween20 at room temperature, 5 minutes each time, then add an appropriate amount of Histostar (Ms+Rb) (MBL company) stock solution as a secondary antibody (to the extent that the tissue section is fully soaked), at room temperature The reaction was carried out for 60 minutes. Wash with PBS containing 0.05% Tween20 at room temperature 3 times, 5 minutes each time, and then react with DAB substrate solution (MBL company) for 10 minutes. The reaction was stopped by washing the tissue sections with water. After hematoxylin staining, dehydration was performed with ethanol and xylene, and specimens were prepared with specimen preparation solution (Matsunami Glass). Microscopic examination and recording were performed under a bright-field microscope (OLYMPUS IX71), and the results confirmed that the anti-SLC38A4 protein antibody did not react with normal esophagus, but reacted with esophageal squamous cell carcinoma (Figure 5).

Next, 96 cases of esophageal cancer patient samples recorded in Table 1 and Table 2 were used by the same method as above (27 cases of grade I of well-differentiated type in the WHO pathological classification, between well-differentiated and moderately differentiated) - 4 cases of grade II, 36 cases of moderately differentiated grade II, 3 cases of moderately differentiated and poorly differentiated grade II-III, and 26 cases of poorly differentiated grade III) for tissue staining.

Table 1

Table 2

Among them, the degree of staining is divided into 4 stages according to its staining intensity and distribution. That is, strong positive (++) when the entire examined cancer tissue is very strongly stained, positive (+) when the entire examined cancer tissue is stained, and weakly stained when a part of the entire examined cancer tissue is stained It is weakly positive (+-), and it is negative (-) if the cancer tissue is not stained at all. As a result, among the biopsied tissue samples from patients with squamous cell carcinoma of the esophagus under investigation, 10 cases were strongly positive, 24 cases were positive, 18 cases were weakly positive, and 44 cases were negative. The total number of positive cases obtained by adding all the weak positive cases was 52, equivalent to 54% of the total. In addition, according to the classification of the results according to the WHO pathological classification level, it was judged that 26 cases were positive (83.9% positive rate) among the 31 cases classified as grade I and grade I-II (Figure 6 and Figure 7), and they were classified as grade II 22 of the 39 cases classified as grade II-III were positive (positive rate 56.4%) (Figure 6 and Figure 8), and 4 of the 26 cases classified as grade III were positive (positive rate 15.4%) (Figure 6 and Figure 9). From these results, it was found that the positivity rate by the anti-SLC38A4 protein antibody was high in esophageal cancer at an earlier, well-differentiated stage ( FIG. 6 ). In addition, if the positive cases in each grade are classified according to the staining intensity, in Grade I, 25.8% are strongly positive, 32.3% are positive, and 25.8% are weakly positive; in Grade II, 5.1% are strong positive, and 5.1% are positive. 30.8%, weak positive was 20.5%, in addition, in grade III, strong positive was 0%, positive was 7.7%, weak positive was 7.7%, found that the lower the grade, the higher the staining intensity in tissue staining (Fig. 10). From these results, it is considered that the staining of esophageal cancer tissue with an anti-SLC38A4 protein antibody is extremely useful for the detection of early-stage cancer that is visually indistinguishable from normal.

In addition, in order to evaluate the staining property of the anti-SLC38A4 protein antibody in other digestive system cancers, gastric adenocarcinoma, colorectal adenocarcinoma, rectal adenocarcinoma, pancreatic cancer, hepatocellular carcinoma, and renal carcinoma were stained according to the above-mentioned method in FIG. 5 ( 4 cases of each type of cancer). These samples were all purchased from Shanghai OutDo Company. As a result, staining was not confirmed in these 6 types of carcinoma ( FIG. 11 ). Staining with an anti-SLC38A4 protein antibody was shown to be a specific reaction for esophageal squamous cell carcinoma. Industrial availability

As described above, according to the present invention, it is possible to detect esophageal cancer at an early stage with high precision using the expression of the SLC38A4 protein as an indicator. Thus, treatment of esophageal cancer can be performed at an early stage of development of esophageal cancer, the treatment success rate of the patient can be increased, and the prognosis of the patient can be improved. Furthermore, according to the present invention, screening of a drug for treating esophageal cancer can be efficiently performed by targeting the SLC38A4 protein and using the expression of the SLC38A4 protein as an index. Thus, the present invention can greatly contribute to the diagnosis and treatment of esophageal cancer, the development of therapeutic drugs, and the like.

Claims (9)

1. Use of an anti-SLC38A4 protein antibody for producing a composition for detecting esophageal cancer. 2. The use according to claim 1, wherein the esophageal cancer is early esophageal cancer. 3. A composition for detecting esophageal cancer, comprising an antibody that recognizes a region including the amino acid sequence described in SEQ ID NO: 2 in the SLC38A4 protein. 4. The composition according to claim 3, wherein the esophageal cancer is an early stage esophageal cancer. 5. The manufacture method of composition described in claim 3, wherein, comprise the following steps: (a) a step of immunizing a peptide comprising the amino acid sequence described in SEQ ID NO: 2 in the SLC38A4 protein, and (b) A step of isolating and/or purifying an antibody that binds to a region including the amino acid sequence described in SEQ ID NO: 2 in the SLC38A4 protein. 6. The production method according to claim 5, wherein the composition is a composition for detection of early esophageal cancer. A kit for detecting esophageal cancer, comprising the composition according to claim 3 or 4. 8. Use of an anti-SLC38A4 protein antibody for the manufacture of a kit for detecting esophageal cancer. 9. The use according to claim 8, wherein the esophageal cancer is early esophageal cancer.