Method for testing blood samples using nematode migration behavior

Abstract

The present invention provides a blood sample testing method. The present invention provides, for example, a method for testing or analyzing a blood sample from a subject that has cancer or a subject suspected of having cancer, the method involving evaluating the taxis behavior of a nematode with respect to the blood sample. The present invention thereby provides, for example, a method for evaluating whether a subject has cancer.

Description

[Technical field]

[0001] The present invention relates to a method for testing a blood sample using the motility behavior of nematodes. [Background technology]

[0002] It is constructed as a cancer screening test that utilizes the characteristics of nematodes that are attracted to cancer-specific odors in biological substances or processed products thereof. Patent Document 1 discloses a method for determining whether or not a subject from which a sample is derived may have cancer by utilizing the difference in responsiveness of nematodes to a urine sample. [Prior art documents] [Patent documents]

[0003] [Patent Document 1] WO2015 / 088039 Summary of the Invention

[0004] The present invention provides a method for testing a blood sample using the motility behavior of nematodes.

[0005] The present inventors have found that blood samples can be examined or analyzed by the nematode's tactic behavior. The present inventors have found that nematodes can exhibit attractive behavior toward blood samples from cancer patients. The present inventors have also found that nematodes can exhibit repulsive behavior toward blood samples from non-cancer subjects (subjects without cancer). The present inventors have further found that the responsiveness of nematode tactic behavior can be improved by using blood samples with reduced protein content by subjecting blood samples to protein removal treatment. The present invention is based on these findings.

[0006] According to the present invention, the following inventions are provided. [1] A method for testing or analyzing a blood sample from a subject having or suspected of having cancer, comprising assessing the tactic behavior of nematodes toward the blood sample. [2] The method according to [1] above, wherein the blood sample is a serum sample or a plasma sample. [3] The method according to [1] or [2] above, wherein the blood sample is a serum sample or a plasma sample having a reduced protein content. [4] The method according to any one of the above [1] to [3], wherein the blood sample has been subjected to a deproteinization treatment and has a reduced protein content. [5] The method according to [4] above, wherein the deproteinization treatment is a deproteinization treatment using a solution containing one or more selected from the group consisting of methanol, formic acid, and trichloroacetic acid, or a deproteinization treatment by ultrafiltration. [6] The method according to any one of [1] to [5] above, wherein the cancer is a solid cancer. [7] The method according to [6] above, wherein the cancer is a gastrointestinal cancer.

[0007] According to the present invention, a blood sample can be analyzed. [Brief description of the drawings]

[0008] [Figure 1] Figure 1 shows the results of evaluation of the tactic behavior of C. elegans toward urine samples obtained from two cancer patients and four cancer-negative humans (non-cancer subjects). [Diagram 2] Figure 2 shows the results of evaluation of tactic behavior of C. elegans toward plasma samples obtained from two cancer patients and two non-cancer controls, respectively. [Diagram 3] FIG. 3 shows the results of evaluation of tactic behavior of C. elegans toward serum samples obtained from two cancer patients and two non-cancer controls, respectively. [Figure 4] Figure 4 shows the results of evaluating the tactic behavior of nematodes toward the deproteinized plasma samples obtained by subjecting plasma samples obtained from two cancer patients and two non-cancer subjects to a protein removal treatment. [Diagram 5] Figure 5 shows the results of evaluating the tactic behavior of nematodes toward the deproteinized serum samples obtained by subjecting serum samples obtained from two cancer patients and two non-cancer subjects to a protein removal treatment. [Figure 6]Figure 6 is a summary of Figures 4 and 5. Detailed Description of the Invention

[0009] As used herein, the term "subject" refers to a mammal, for example, a human. In addition, as used herein, the term "subject" includes healthy subjects, subjects suspected of having cancer, and subjects suffering from cancer. As used herein, the term "subject suspected of having cancer" includes subjects who have never been specifically suspected of having cancer, in addition to subjects who are clearly suspected of having cancer.

[0010] In this specification, "cancer" refers to a malignant tumor. Cancer can be broadly classified into hematopoietic tumors and solid cancers. Hematopoietic tumors include leukemia, malignant lymphoma, and myeloma. Solid cancers include solid cancers such as gastrointestinal cancer, such as stomach cancer, esophageal cancer, colon cancer, colorectal cancer, gallbladder cancer, and pancreatic cancer, lung cancer, breast cancer, liver cancer, uterine cancer, ovarian cancer, head and neck cancer, and tongue cancer; sarcomas such as osteosarcoma, chondrosarcoma, rhabdomyosarcoma, leiomyosarcoma, fibrosarcoma, liposarcoma, and angiosarcoma; sarcomas such as chondrosarcoma, Ewing's sarcoma, malignant hemangioendothelioma, malignant schwannoma, osteosarcoma, and soft tissue sarcoma, and blastomas such as hepatoblastoma, medulloblastoma, nephroblastoma, neuroblastoma, pancreatic blastoma, pleuropulmonary blastoma, and retinoblastoma.

[0011] In this specification, "detecting cancer" can be interpreted as "detecting cancer cells," "identifying cancer," "determining cancer," "a method for assisting in the diagnosis of cancer," "a method for obtaining preliminary information for the diagnosis of cancer," and the like. The method of the present invention is an industrially applicable method. In one embodiment, the method of the present invention is not a method performed by a physician. In one embodiment, the method of the present invention does not include a medical procedure.

[0012] As used herein, the term "nematode" refers to Caenorhabditis elegans ( Caenorhabditis elegans) means nematodes. Caenorhabditis elegans strains isolated from various environments are registered and published at the Caenorhabditis Genetics Center (CGC) in the Department of Biological Sciences at the University of Minnesota, USA, and are available for distribution. Therefore, those skilled in the art can obtain most of the known strains from the CGC. From the viewpoint of being able to reproduce by self-fertilization, hermaphrodites are preferably used.

[0013] In this specification, "taxis behavior" refers to attraction behavior or repulsion behavior. Attractive behavior refers to behavior that shortens the physical distance from a certain substance, and repulsive behavior refers to behavior that increases the physical distance from a certain substance. A substance that induces attractive behavior is called an attractant, and a substance that induces repulsive behavior is called a repellent. Nematodes have the property of being attracted to attractants and repelled by repellents through their sense of smell. An attractive behavior toward an attractant is called an attractive behavior (sometimes referred to as "positive" in this specification), and a repulsive behavior away from a repellent is called an avoidance behavior (sometimes referred to as "negative" in this specification). In addition, attractive behavior and avoidance behavior are collectively called taxis behavior.

[0014] As used herein, the term "wild strain" refers to a wild strain of nematode, and examples thereof include the N2 Bristol strain, which is a common wild strain.

[0015] As used herein, a "blood sample" refers to a sample containing blood obtained from a subject. Examples of blood samples include serum samples and plasma samples. As used herein, a "test sample" refers to a sample to be tested.

[0016] According to the present invention, there is provided a method for testing (or analyzing) a biological sample (particularly a blood sample) from a subject having or suspected of having cancer. According to the present invention, the method of the present invention may also be a method for detecting (or predicting or diagnosing) cancer in a subject having or suspected of having cancer, or a method for obtaining basic information on the efficacy of a treatment, comprising evaluating the tactic behavior of nematodes toward a urine sample from the subject. The method of the present invention may be an invention that can be industrially applied. The method of the present invention may be a non-medical method.

[0017] As the nematode, a wild-type nematode (e.g., N2 Bristol strain) can be used. Since the nematode strain exhibits an attractive behavior to the urine of a cancer patient, such a nematode exhibiting an attractive behavior to the urine of a cancer patient can be used in the present invention.

[0018] A blood sample can be obtained from a subject. When evaluating a blood sample by the method of the present invention, the blood sample can be diluted with a solvent such as an aqueous solvent such as water and used as a diluted sample. The dilution ratio at this time can be, for example, 5 times or more, 10 times or more, 20 times or more, 30 times or more, 40 times or more, 50 times or more, 60 times or more, 70 times or more, 80 times or more, 90 times or more, 100 times or more, 200 times or more, 300 times or more, 400 times or more, 500 times or more, 600 times or more, 700 times or more, 800 times or more, 900 times or more, 1,000 times or more, 2,000 times or more, 3,000 times or more, 4,000 times or more, 5,000 times or more, 6,000 times or more, 7,000 times or more, 8,000 times or more, 9,000 times or more, or 10,000 times or more. The dilution ratio may be 100 or less, 200 or less, 300 or less, 400 or less, 500 or less, 600 or less, 700 or less, 800 or less, 900 or less, 1,000 or less, 2,000 or less, 3,000 or less, 4,000 or less, 5,000 or less, 6,000 or less, 7,000 or less, 8,000 or less, 9,000 or less, or 10,000 or less. In some embodiments, the blood sample may be diluted at a dilution ratio of 5 to 100,000, for example, 10 to 10,000, 50 to 2,000, 50 to 500, or 500 to 5,000 to be used as a diluted sample.

[0019] A urine sample can be obtained from a subject. When evaluating a urine sample by the method of the present invention, the urine sample may be diluted with a solvent such as water. When diluted, the dilution ratio may be, for example, 5 to 20,000 times, for example, 10 to 10,000 times, for example, 10 to 5,000 times, 10 to 2,000 times, for example, 100 to 1,000 times, and the nematode also exhibits tactic behavior toward the diluted urine sample. In one embodiment of the present invention, the dilution ratio of urine may be, for example, 100 times or 1,000 times, or may be 2 or more times including 100 times and 1,000 times. For example, the dilution ratio of a dog urine sample may be 10 to 2,000 times, for example, 100 to 1,000 times. For example, the dilution ratio of a cat urine sample may be 5 to 20000, or 10 to 10000, for example, 500 to 5000. For example, the dilution ratio of a mouse urine sample may be 5 to 20000, or 10 to 10000, for example, 500 to 5000.

[0020] The dilution ratio of the sample can be determined by a person skilled in the art by preparing a dilution series (e.g., a 2-fold to 10-fold dilution series) of a test sample (e.g., a blood sample) obtained from each of cancer patients and non-cancer subjects, evaluating the nematode's tactics against these dilution series, and appropriately determining a dilution ratio that shows a good difference in responsiveness to urine from cancer patients and non-cancer patients. The dilution ratio can be, for example, 100-fold to 20,000-fold, for example, 500-fold to 10,000-fold, for example, 1,000-fold to 10,000-fold. From the viewpoint of improving the accuracy of the test, the dilution concentration can be appropriately considered, and a dilution ratio that shows a better separation ability can be determined before the test or analysis. The analysis can also be performed using samples with multiple dilution ratios.

[0021] Serum or plasma can be obtained from a sample (e.g., a blood sample) by a conventional method. Serum is the liquid component when blood is coagulated. Plasma is a component in blood other than cells. Specifically, plasma can be the supernatant obtained by centrifuging blood.

[0022] The blood sample may be subjected to the analysis of the present invention after reducing the amount of protein contained therein by a protein removal treatment. Thus, in the present invention, the blood sample may be a blood sample having a reduced protein content. In an embodiment of the present invention, the blood sample may be plasma having a reduced protein content. In an embodiment of the present invention, the blood sample may be serum having a reduced protein content. The reduced protein content is a protein content that is less than the protein content contained in a normal blood sample. A blood sample (e.g., serum and plasma) having a reduced protein content may be obtained, for example, by subjecting a blood sample (e.g., serum and plasma) to a protein removal treatment. The protein removal treatment is not particularly limited, and may be a method that can be normally used by a person skilled in the art. The protein removal treatment may include, for example, a method in which one or more agents (e.g., protein denaturants) selected from the group consisting of acids (e.g., formic acid, perchloric acid, trichloroacetic acid, metaphosphoric acid, etc.) and organic solvents (e.g., acetone, acetonitrile, methanol, ethanol, etc.) are added to the sample to denature and insolubilize the proteins, thereby removing the proteins from the sample. Protein removal treatment also includes a method of removing proteins from a sample by physical methods such as ultrafiltration, for example, ultrafiltration using a membrane filter (such as a centrifugal filter device), dialysis, and ultracentrifugation. The obtained blood sample can be diluted with a solvent such as water and then subjected to the analysis of the present invention. That is, the blood sample can be a dilution of a blood sample (such as plasma and serum) having a reduced protein content.

[0023] The taxis behavior of nematodes can be evaluated by placing a sample (e.g., a urine sample or a blood sample) and a nematode at a certain distance (e.g., about 0 cm to about 5 cm, e.g., 1 cm to 5 cm) and observing whether the nematode shows an attractive or repellent behavior toward the sample (e.g., a urine sample or a blood sample). The observation of taxis behavior can be performed, for example, on a solid medium such as an agar medium.

[0024] Method for analyzing test samples using nematodes The method for analyzing a sample using nematodes can be performed by placing a test sample (e.g., a urine or blood sample) obtained from a subject and the nematode at a certain distance from each other, and observing whether the nematode exhibits an attractive or repulsive behavior toward the test sample. If the nematode exhibits an attractive behavior, the subject can be evaluated as having or possibly having cancer. If the nematode exhibits an repulsive behavior, the subject can be evaluated as not having or possibly having cancer. Examples of the analysis method using human specimens are as disclosed in WO2015 / 088039, the entire contents of which are incorporated herein by reference.

[0025] More specifically, a method for analyzing a test sample (e.g., a urine sample or a blood sample) using nematodes includes, for example, Placing a test sample (e.g., a urine sample or a blood sample) obtained from a subject in a petri dish (e.g., a petri dish containing a solid medium); Placing nematodes at a certain distance from the test sample in a petri dish in which the test sample is placed; After placement, allowing the nematodes to act.

[0026] The method of analyzing a sample (e.g., a urine sample or a blood sample) using nematodes may include, for example, determining that the subject is suffering from cancer or may be suffering from cancer if the nematode exhibits an attractive behavior to the test sample. Also, the method of analyzing a sample (e.g., a urine sample or a blood sample) using nematodes may indicate that the subject from which the sample is derived may be suffering from cancer if the nematode exhibits an attractive behavior to the test sample.

[0027] The taxis behavior of nematodes can be evaluated by the difference or ratio between the number of nematodes approaching the test sample and the number of nematodes receding from the test sample. In the case of evaluation based on the difference in the number of nematodes, if the difference is a positive value, the test sample as a whole can be evaluated as inducing an attractive behavior and being derived from a cancer patient, and / or if the difference is a negative value, the test sample as a whole can be evaluated as inducing a repellent behavior and being derived from a subject without cancer. In the case of evaluation based on the ratio of the number of nematodes, if the ratio is 1 or more, the test sample can be evaluated as inducing an attractive behavior and being derived from a subject with cancer, and / or if the ratio is less than 1, the test sample can be evaluated as inducing a repellent behavior and being derived from a subject without cancer. In addition, the taxis behavior of nematodes can be evaluated, for example, using a taxis index as an index as follows.

[0028]

number

[0029] If the taxis index is a positive value, the test sample as a whole can be evaluated as inducing attractive behavior and being derived from a subject having cancer, and if it is a negative value, the test sample as a whole can be evaluated as inducing repulsive behavior and being derived from a subject not having cancer.

[0030] The taxis index is closer to 1, the higher the ratio of nematodes that show attractive behavior, the closer to -1, the higher the ratio of nematodes that show repellent behavior, and the closer to 0, the higher the ratio of nematodes that show neither attractive nor repellent behavior. The higher the absolute value of the taxis index, the clearer the behavior evaluation result. If the nematodes that showed taxis behavior and the nematodes that showed repellent behavior are comparable, they may be considered positive (if positive, further detailed testing may be performed), or they may be excluded from the evaluation subject. In the present invention, an individual from which a positive test sample is derived can be subjected to further detailed testing to determine whether or not the individual has cancer. When two or more different dilution ratios of test samples are tested, in terms of improving sensitivity, if the sample is positive for any of the dilution ratios, it may be determined to be cancer, or in terms of improving specificity, if all of the samples are positive, it may be determined to be cancer.

[0031] The present invention may involve evaluating a urine sample obtained from a subject having cancer or a subject suspected of having cancer using an evaluation system for the nematode's motility behavior, and then evaluating a blood sample obtained from the subject using the evaluation system for the nematode's motility behavior.

[0032] The subject who is evaluated as having or possibly having cancer by the method of the present invention may then be definitively diagnosed as having cancer by a doctor's diagnosis of cancer. The subject who is evaluated as having or possibly having cancer by the method of the present invention may then be subjected to a detailed examination to receive a definitive diagnosis of cancer. The subject who is evaluated as having or possibly having cancer by the method of the present invention may then be subjected to cancer treatment (e.g., chemotherapy, radiation therapy, and surgical resection, and combinations thereof, etc.).

[0033] According to the present invention, there is provided a composition or kit comprising a nematode or its eggs for use in the analysis method of the present invention, the method for detecting cancer, the method for detecting cancer cells, the method for identifying cancer, the method for determining cancer, the method for assisting in the diagnosis of cancer, or the method for obtaining preliminary information for the diagnosis of cancer. In addition to the nematode or its eggs, the kit may also comprise a behavioral evaluation system for the nematode (e.g., a dish, agar, and an instruction manual) and / or a part thereof.

[0034] According to the present invention, there is provided the use of nematodes in the manufacture of a composition or kit for use in the analytical method of the present invention, the method for detecting cancer, the method for detecting cancer cells, the method for identifying cancer, the method for diagnosing cancer, the method for assisting in the diagnosis of cancer, or the method for obtaining preliminary information for the diagnosis of cancer. EXAMPLES

[0035] Example 1: Chemotaxis analysis of urine or blood samples In this example, based on the procedure "(3) Detection using nematode olfaction" described in WO2015 / 088039, urine samples from two human patients with gastric cancer and four human subjects with negative cancer were analyzed at 10-fold dilution (condition 1) and 100-fold dilution (condition 2) with water. As a result, as shown in FIG. 1, the nematodes exhibited attractive behavior toward the urine of the two gastric cancer patients and repulsive behavior toward the urine of the cancer-negative humans. Blood samples were collected from the two gastric cancer patients (SE-005 and SE-006) and two selected from the four cancer-negative subjects (SE-001 and SE-002), and serum and plasma were obtained by standard methods. The serum and plasma were untreated, treated with an organic solvent (methanol:formic acid = 1000:1), treated with acid (10% trichloroacetic acid), or ultrafiltered (using Amicon Ultra 3K according to the manufacturer's manual), and each sample was diluted with water, after which the nematode tactic behavior toward the diluted sample was evaluated according to the above procedure.

[0036] (1) Taxis of nematodes to untreated plasma and serum <Method> Furthermore, the taxis of nematodes to the untreated plasma and serum of the subjects SE-001, SE-002, SE-005, and SE-006 used in Example 1 were confirmed. The untreated plasma and serum were each diluted with water. The dilution rate was 10 -2 ,10 -3 ,10 -4 , or 10 -5 The four dilution ratios were determined to be the most attractive for cancer patients. <Result> For plasma, as shown in FIG. -2 In the dilution, the bacteria showed repulsion to healthy subjects' samples and attraction to cancer patients' samples, similar to the urine samples in Example 1. In addition, for the plasma of healthy subjects, a change in taxis toward an attractive tendency was observed as the dilution ratio was increased, whereas attraction to the plasma of cancer patients was confirmed at all dilution ratios. For serum, as shown in FIG. -2 or 10 -3 At a dilution rate of 1, the drug repelled healthy subjects and attracted cancer patients. However, at higher dilutions, no common trends were observed.

[0037] (2) Taxis of nematodes to deproteinized plasma and serum <Method> Next, the plasma and serum were deproteinized in three ways: ultrafiltration (Amicon Ultra 3K), methanol / formic acid treatment (1000:1), or 10% trichloroacetic acid (TCA) treatment to obtain blood samples. The obtained blood samples were further diluted with water. The dilution ratio of the blood samples was 10 -2 ,10 -3 ,10 -4 , or 10 -5 The nematode taxis were then evaluated for the diluted samples. However, when the samples were deproteinized by methanol / formic acid treatment, the supernatant was diluted two-fold with water to prepare blood samples. <Result> As shown in Figures 4 and 5, in tests using plasma and serum, the separation ability of nematode responses in samples from cancer and healthy subjects tended to be higher regardless of the deproteinization treatment. In addition, tests using plasma were able to achieve higher resolution than tests using serum. In serum tests, deproteinization with methanol / formic acid or TCA was found to be more effective and superior in terms of separation ability.

Claims

1. 1. A method of testing or analyzing a blood sample from a subject having or suspected of having cancer, comprising assessing the tactic behavior of a nematode towards the blood sample; The blood sample is a serum or plasma sample having a reduced protein content; method.

2. The method of claim 1 , wherein the blood sample is a serum sample or a plasma sample.

3. 3. The method according to claim 1 or 2, wherein the blood sample is a blood sample that has been subjected to a deproteinization treatment and has a reduced protein content.

4. The method according to any one of claims 1 to 3, wherein the deproteinization treatment is a deproteinization treatment with a solution containing one or more selected from the group consisting of methanol, formic acid, and trichloroacetic acid, or a deproteinization treatment by ultrafiltration.

5. The method according to any one of claims 1 to 4, wherein the cancer is a solid cancer.

6. The method of claim 5 , wherein the cancer is a gastrointestinal cancer.

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