Cancer detection method using olfactory sense of nematodes

Abstract

To provide a method for identifying an odorant receptor in a nematode.SOLUTION: Provided is a method for identifying an odorant receptor in a nematode, comprising identifying an odorant receptor using a nematode.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a method for detecting cancer using the olfactory sense of nematodes. [Background technology]

[0002] Malignant neoplasms, including cancer, have been the leading cause of death among Japanese people since 1981. World Health Organization According to the World Health Organization, in 2005, the death rate from malignant neoplasms in the world was 13% (7.6 million people), and It is predicted to continue to increase in the future. The earlier cancer is diagnosed, the less mental, physical, economic and social burdens are placed on the patient. The more the disease progresses, the lower the chance of a cure, and the more likely it is to progress to an unresectable recurrence. In the case of cancer, there is a huge burden and loss, and the reality is that treatment is life-prolonging. Cancer is caused by the genes of cells. Most cancers are caused by abnormalities in the blood, so the incidence rate for each age group per 100,000 people is The incidence rate increases proportionally to the 4-6 power (Cancer Patterns in Canada, 1982). If cancer can be detected and treated early, it will be beneficial in various situations, regardless of age or society. It is clear that the burden and losses can be reduced.

[0003] However, early-stage cancer generally has no symptoms, and patients have little motivation to undergo cancer screening. In fact, the cancer screening rate in Japan is 10-35%. % of the population, and the Basic Plan for Cancer Control in Japan aims to have a cancer screening rate of 50% or higher by 2017. Japan is well below its target. Japan is a world leader in early-stage cancer treatment technologies, including endoscopy and surgical techniques. In Japan, where the number of patients is increasing, it is less painful, simple, inexpensive, can be performed on many people, and is highly effective. If a new, accurate cancer screening method is developed and applied, it will revolutionize cancer treatment worldwide. It's no exaggeration.

[0004] Several teams, including the present inventors, have been using trained dogs (cancer detection dogs) to detect cancer. has a distinctive odor and has a high sensitivity and specificity of over 90% for specimens containing early-stage cancer. It has been reported that it is possible to detect it with high accuracy (Non-Patent Document 1: Sonoda, H. et al., Colorectal cancer screening with odor material by canine scent detection. Gut, 60, 814-81 9, 2011). Based on this, we believe that if we can detect the odor specific to cancer, we can build a highly accurate cancer detection system. can be done.

[0005] However, the abilities of cancer detection dogs vary from dog to dog, and their concentration declines during the summer when temperatures rise. In addition, there is no set methodology for training detection dogs. Even when things are going well, five tests a day is the limit. Continuing to test diagnostic samples is a mistaken act by cancer detection dogs. This results in a decrease in accuracy because the detection dog is rewarded with the opportunity to "play with a ball." Therefore, cancer screening using detection dogs cannot be carried out commercially.

[0006] In addition, analytical equipment such as GC / MS (gas chromatography / mass spectroscopy) analysis is used to analyze the A method for identifying cancer-causing substances and diagnosing cancer has been reported (Non-Patent Document 2: Y. Hanai, et al. al., Urinary volatile compounds as biomarkers for lung cancer. Biosci. Biotechno l. Biochem. 76, 679-84, 2012) (Non-patent document 3: Khalid et al., A pilot study combi ning a GC-sensor device with a statistical model for the identification of bladd er cancer from urine headspace. PLoS ONE, 8, e69602, 2013). However, There is a high possibility that volatile substances present in the sample will become noise, and it will take a lot of money to develop and manufacture the equipment to detect them. It requires funding and analytical skills. [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Sonoda, H. et al., Colorectal cancer screening with odour material by canine scent detection. Gut, 60, 814-819, 2011 [Non-patent document 2] Y. Hanai, et al. Urinary volatile compounds as biomarkers for lung cancer. Biosci. Biotechnol. Biochem. 76, 679-84, 2012 [Non-patent document 3] Khalid et al., A pilot study combining a GC-sensor device with a statistical model for the identification of bladder cancer from urine headspace. PLoS ONE, 8, e69602, 2013 Summary of the Invention [Problem to be solved by the invention]

[0008] An object of the present invention is to provide a method for detecting cancer using the olfactory sense of nematodes. [Means for solving the problem]

[0009] As a result of intensive research to solve the above problems, the present inventors have discovered a chemical reaction based on the olfactory sense of nematodes. We have found that cancer can be detected by the response of olfactory nerves or by taxis, and have completed the present invention. Ta. That is, the present invention is as follows.

[0010] (1) The response of nematodes to the odor of biological substances derived from the subject or their processed products is used as an indicator. A method for detecting cancer, comprising detecting cancer. (2) The nematode is Caenorhabditis elegans (1). The method described below. (3) The nematode is a wild-type nematode, a mutant nematode, or a transgenic nematode (1) or ( The method described in 2). (4) The nematodes showed a positive response to the odor of the biological substance or its processed product derived from the subject. If the test result is negative, the subject is determined to have cancer or to be at risk of cancer, and 1. The method according to claim 1.

[0011] (5) The response of the nematode's olfactory nerve to the odor of a biological substance or its processed product derived from the subject. If the difference is large, the subject is determined to have cancer or be at risk of cancer. (1) to (4) 10. The method according to any one of claims 1 to 9. (6) The biological substance or its processed product is a body fluid, a cell, a tissue, or a culture of a cell or tissue. The method according to any one of (1) to (5), wherein the preparation is a substance or a preservative solution. (7) The method according to (6), wherein the body fluid is urine. (8) The method according to (6), wherein the preservative solution is physiological saline. (9) An odorant receptor in a nematode, characterized by identifying an odorant receptor using a nematode Identification method. (10) Inhibiting the expression or function of a gene encoding the receptor, and The method according to (9), which tests the reaction to an odor in a subject.

[0012] (11) The method according to (10), wherein the expression or function of the receptor gene is inhibited by RNAi. . (12) The method according to any one of (9) to (11), wherein the odor is the odor of a cancer. (13) The types of receptors identified vary depending on the cancer type or the concentration of the odorant. The method according to any one of (9) to (12), wherein (14) The nematode is Caenorhabditis elegans (9 2. The method according to claim 1, wherein the first step is to form a first cavity. (15) The reaction of nematodes to the odor of a biological substance derived from a subject or its processed product is used as an indicator. A method for identifying a type of cancer, comprising: identifying a type of cancer by using a method for identifying a type of cancer;

[0013] (16) The method according to (15), comprising the following steps: (a) detecting cancer by the method according to any one of (1) to (8); (b) for a sample detected as being cancerous in the step (a), (14) A modified nematode in which a receptor identified by the method according to any one of (14) is modified is used. and examine their reaction to odors. (c) The response to the odor is different between the modified nematode and the nematode used in step (a). If they are different, the cancer type corresponding to the identified receptor is determined to be the target cancer type to be identified. (17) Receptor modification includes receptor deletion, inhibition of receptor expression or function, and increased receptor activity. The method according to (16), wherein the method is at least one selected from the group consisting of expression and functional enhancement. (18) A kit for detecting or identifying cancer, comprising nematodes. (19) The nematode is Caenorhabditis elegans (1 7) The kit according to claim 7). (20) The nematode is a wild-type nematode, a mutant nematode, or a transgenic nematode. (18) Or (19) A kit according to (19). (21) Nematodes and a storage unit for storing a biological substance or a processed product thereof and the nematode; A detection unit that detects the reaction of the nematodes in the storage unit to the odor; A cancer detection system comprising: [Effects of the Invention]

[0014] The present invention provides a method for detecting cancer using nematodes. The method of the present invention can detect the presence of β-glucan in a sample at high sensitivity and low cost. Furthermore, the method of the present invention can detect early stage cancer. Therefore, the method of the present invention is extremely useful for clinical testing of cancer. [Brief explanation of the drawings]

[0015] [Figure 1] FIG. 1 shows the results of a test of the reaction of nematodes to urine from healthy individuals and cancer patients. [Figure 2] FIG. 1 shows the format of a Petri dish used in the method of the present invention. [Figure 3] FIG. 1 shows the measurement principle when the Yellow Cameleon gene is used as the indicator gene. [Figure 4] FIG. 1 shows the measurement principle when the GCaMP gene is used as the indicator gene. [Figure 5] FIG. 1 shows a chip having microchannels for positioning nematodes. [Figure 6] FIG. 1 is a diagram showing switching of channels in a chip having microchannels. [Figure 7] FIG. 1 shows the results of testing the response of the AWC olfactory nerves of C. elegans to urine from cancer patients. [Figure 8] FIG. 1 shows the results of testing the response of the AWC olfactory nerves of C. elegans to urine from cancer patients. [Figure 9] FIG. 1 shows the results of testing nematode chemotaxis using urine samples from which sediment and solids had been removed. [Figure 10] FIG. 1 shows the results of testing the response of AWA olfactory nerves of C. elegans to urine from cancer patients. [Figure 11] FIG. 1 shows the results of testing the response of AWA olfactory nerves of C. elegans to urine from cancer patients. [Figure 12] FIG. 1 shows an assay plate. [Figure 13] FIG. 1 shows the attraction behavior of nematodes to culture media of cancer cells. [Figure 14] FIG. 1 shows the results of a medium-scale study of the method of the present invention. [Figure 15] FIG. 1 shows the results of a medium-scale test conducted using the method of the present invention and other tumor markers, and comparing the sensitivities. [Figure 16A] FIG. 1 is a block diagram of a system of the present invention. [Figure 16B] FIG. 2 is a configuration diagram of a processing unit of the system of the present invention. [Figure 17] FIG. 1 shows the taxis of C. elegans towards various concentrations of fibroblast culture medium. [Figure 18] This figure shows the taxis of nematodes to various concentrations of cancer cell culture media. colo205 = colon cancer, MKN1 = gastric cancer. [Figure 19]FIG. 1 shows the taxis of C. elegans to cancerous tissues and healthy tissues of sigmoid colon cancer patients. [Figure 20] FIG. 1 shows the taxis of nematodes to dilutions of physiological saline after human cancer tissue slices were stored in the physiological saline. [Figure 21] FIG. 1 shows the results of testing the taxis of nematodes using urine at different concentrations. [Figure 22] Figure 1 shows RNAi screening for olfactory receptors involved in responses to specific odorants. (A) Confirmation that the RNAi screening strategy was effective. The effect of RNAi targeting odr-10 in eri-1 mutants on the chemotactic response to a 10-3 dilution of diacetyl or a 10-3 dilution of pyrazine is shown. A significant difference from the control is shown (P<0.001, Student's t-test). (B) The number of candidate olfactory receptor genes associated with odorant chemotaxis obtained after the tertiary screening. (C) Expression patterns of fluorescent reporters driven by the srx-47 or sra-17 promoter. Green indicates expression of the fluorescent protein Venus, which is driven by the promoters of these genes. Magenta indicates expression of mCherry in AWA, AWB, and AWC olfactory neurons. srx-47 expression was observed in AWA and ASH neurons. sra-17 expression was detected in AWA neurons. Scale bar, 10 μm. [Figure 23] Figure 1 shows the expression patterns of candidate olfactory receptor genes. (Left) Green indicates Venus expression, which is driven by the promoter of the candidate olfactory receptor gene. (Center) Magenta indicates mCherry expression in AWA, AWB, and AWC neurons (A) or dye-stained sensory neurons (ASH, ASJ, AWB, ASK, ADL, ASI, PHA, and PHB neurons) (BM). (Right) Overlaid images. All images are left-side lateral images of the head region of the worm, except for the tail region (C, bottom). Arrows and arrowheads indicate the cell bodies of neurons expressing the candidate receptor genes. Scale bar = 10 μm. [Figure 24]SRI-14 functions in the response to high concentrations of diacetyl in ASH neurons. (A) Chemotaxis to low and high concentrations of diacetyl in wild-type (WT), odr-10, and sri-14 mutants. Diacetyl concentrations are indicated at the bottom (n = 5). (B) Effect of RNAi targeting sri-14 on the repellent response to high concentrations of diacetyl (5 μl undiluted) (n = 8). (C) Chemotaxis of sri-14 mutants to high concentrations of diacetyl (5 μl undiluted, Da), isoamyl alcohol (5 μl undiluted, Iaa), and benzaldehyde (1 μl undiluted, Bz), as well as the repellents octanol (1 μl undiluted, Oct) and nonanone (1 μl undiluted, Nona) (n = 6). (D) Expression pattern of a fluorescent reporter (green) driven by the sri-14 promoter. Arrowheads indicate AWC or ASH cell bodies identified by mCherry or fluorescent dye (magenta), respectively. (E) Effect of ASH- or AWC-specific RNAi of sri-14 in wild-type worms on chemotaxis to high concentrations of diacetyl (5 μl undiluted) (n = 5). (F) Effect of neuron-specific expression of sri-14 cDNA on the defective response of sri-14 mutants to high concentrations of diacetyl (5 μl undiluted) (n = 5). (G) Localization of SRI-14::GFP in ASH sensory cilia. Scale bar, 10 μm (D and G). Error bars represent SEM. *P < 0.05, **P < 0.01, ***P < 0.001, Student's t test (B and C) or Dunnett's test (A, E, and F). [Figure 25] This figure shows the results of repeated assays of chemotaxis to high diacetyl concentrations in RNAi-treated nematodes. In addition to sri-14, RNAi of srh-25, srh-79, srh-216, or srh-281, among candidate receptor genes for the response to high diacetyl concentrations (5 μl of undiluted diacetyl) obtained after the third screening, caused significant and reproducible defects in diacetyl avoidance behavior. Error bars represent SEM. Significant differences compared to the control are indicated (*P<0.05, **P<0.01; Student's t-test with Bonferroni correction). [Figure 26]Figure 1 shows the structure of sri-14. sri-14 encodes a seven-transmembrane protein. (A) Structure of sri-14. The deleted region of the ok2685 strain is shown. (B) Predicted amino acid sequence of SRI-14. The seven transmembrane domains predicted by a hidden Markov model are shown. (C) Hydrophobicity plot of SRI-14. The plot is derived from the hydropathy parameters defined by Kyte and Doolittle. (D) The sri-14 mutant exhibited normal avoidance behavior toward a high osmotic stimulus (4 M NaCl). Error bars represent SEM. Significant differences compared to the control are indicated (**P<0.01, Dunnett's test). [Figure 27] Neurons involved in the response to high concentrations of diacetyl. (A) Chemotaxis to high concentrations of diacetyl (5 μl undiluted) in wild-type worms with specific sensory neuron disruption (n≧8). (B) Chemotaxis to a 10-3 dilution of diacetyl in AWA-depleted worms. (C) Schematic diagram of the neural wiring between AWA and ASH sensory neurons and four layer I interneurons. (D) Chemotaxis to high concentrations of diacetyl (5 μl undiluted) in wild-type worms with specific interneuron inhibition (n≧5). (E) Chemotaxis to a 10-3 dilution of diacetyl in wild-type worms with specific interneuron inhibition (n≧5). Error bars represent SEM. **P<0.01, ***P<0.001, Dunnett's test; †††P<0.001, Student's t-test. In (A), asterisks indicate statistically significant differences compared to wild-type control strains in which no neurons were ablated or inhibited. [Figure 28]Figure 1 shows the response of AWA neurons to various diacetyl concentrations. (A) Calcium response of AWA neurons after stimulation with a low concentration of diacetyl (10-5 dilution) in nematodes of the indicated genotypes. The shaded area around the curve represents the SEM (n≥8 for all genotypes). Black bars indicate the presence of diacetyl stimulation. (B) Mean fluorescence change 10 s after addition of a low concentration of diacetyl (10-5 dilution). Error bars represent the SEM. **P<0.01, Dunnett's test (n≥8 for each genotype). Black represents WT, red represents sri-14 mutant, and blue represents odr-10 mutant. (C) Calcium response of AWA neurons after stimulation with a high concentration of diacetyl (10-3 dilution) in nematodes of the indicated genotypes. Data are shown as in (A) (n≥8 for all genotypes). (D) Mean fluorescence change 10 s after addition of a high concentration of diacetyl (10-3 dilution). Error bars represent SEM (n≧8 for all genotypes). [Figure 29] Responses of ASH neurons to various diacetyl concentrations. (A) Calcium responses of ASH neurons after stimulation with low (10-5 dilution) and high (10-3 dilution) diacetyl concentrations in wild-type worms (n ≥ 11). (B) Calcium responses of ASH neurons in sri-14 mutants (n = 26), odr-10 mutants (n = 28), sri-14 mutants with ASH-specific expression of sri-14 cDNA (sri-14 rescue, n = 9), and wild-type worms with ASH-specific RNAi of sri-14 (n = 20). Shaded areas around the curves represent SEM. Black bars indicate the presence of diacetyl stimulation. (C) Mean fluorescence change 10 seconds after addition of high diacetyl concentration (10-3 dilution). Error bars represent SEM. *P < 0.05, Dunnett's test (n ≥ 11). [Figure 30](A) Calcium imaging of ASH neurons in unc-13 mutants and AWA-disrupted worms. Calcium responses of ASH neurons after stimulation with a high concentration of diacetyl (10-3 dilution) in wild-type (A, N=14), unc-13 mutants (B, N=14), and AWA-disrupted worms (C, N=11). Larger and longer-lasting calcium responses were observed in unc-13 mutants and AWA-disrupted worms compared to wild-type worm neurons. The shaded areas around the curves represent SEM. Black bars indicate the presence of diacetyl stimulation. (D) Mean fluorescence change 10 seconds after the addition of a high concentration of diacetyl. Error bars represent SEM. Significant differences from controls are indicated by *P<0.05, Dunnett's test. [Figure 31] Figure 1 shows that AWC neurons respond to the removal of high concentrations of diacetyl. Calcium responses of AWC neurons after the removal of high concentrations of diacetyl (10-3 dilution) in wild-type neurons (N=10). Shaded areas around the curves represent SEM. Black bars indicate that diacetyl was present. [Figure 32] Responses of AWB neurons to the removal of high or low concentrations of diacetyl. (A) Calcium responses of AWB neurons after removal of low (10-5 dilution, left) or high (10-3 dilution, right) concentrations of diacetyl in wild-type worms (brown, n = 10) or worms ectopically expressing sri-14 in AWB neurons (orange, n = 11). Shaded areas around the curves represent SEM. Black bars indicate the presence of diacetyl. (B) Mean fluorescence change 10 s after removal of diacetyl. White bars, wild-type; orange bars, strains ectopically expressing sri-14 in AWB. Error bars represent SEM. ***P < 0.001, Student's t-test (n ≥ 10). [Figure 33]This is a model diagram of the odorant concentration-dependent switching of olfactory receptors. In response to low concentrations of diacetyl, ODR-10 in AWA neurons, but not SRI-14 in ASH neurons, functions as a diacetyl receptor, resulting in AWA activation and attraction behavior. In contrast, high concentrations of diacetyl are sensed by SRI-14 in ASH neurons but not by ODR-10 in AWA neurons. ASH neurons are activated only by high concentrations of diacetyl, inducing aversion behavior. AWA also responds to high concentrations of diacetyl, indicating that olfactory receptors other than ODR-10 respond to AWA neurons. [Figure 34] FIG. 1 shows the taxis of the N2 strain and gene mutant strains to urine from various cancer patients. [Figure 35] FIG. 1 shows the taxis of the N2 strain and gene mutant strains to various chemicals. [Figure 36] FIG. 1 shows the taxis of olfactory receptor knockdown strains to the urine of breast cancer patients. [Figure 37] FIG. 1 is a schematic diagram of a method for identifying cancer types using a nematode taxis test. DETAILED DESCRIPTION OF THE INVENTION

[0016] The present invention will be described in detail below. The documents cited in this specification, as well as published patent applications, patent publications, and other patent documents, are The present application claims priority from the same references filed in the preceding paragraphs. Patent Application No. 2013-255145 (filed December 10, 2013) and US 61 / 982,341 (filed April 22, 2014) ) includes the contents of the specification.

[0017] 1. Overview (1) Cancer detection The present invention relates to a method for cultivating nematodes in the presence of a biological substance derived from a subject or a processed product thereof, for example. A method for detecting cancer characterized by detecting cancer using olfactory chemotaxis of nematodes as an indicator. It is the law. The present inventors have proposed a method for detecting whether a subject has cancer by using a sample derived from the subject as one embodiment. We focused on the olfactory chemotaxis of the nematode C. elegans to samples. The nematode Caenorhabditis elegans (hereinafter referred to as C. elegans) ans) is widely bred and studied in laboratories around the world as a model organism for biological research. It is a popular creature that is easy to raise and has an excellent sense of smell.

[0018] Since nematodes exhibit chemotaxis to odorants, i.e., they are attracted to or flee from them, in the present invention, In this study, we will use this behavior as an indicator to examine the nematode's response to cancer odors. When the reaction of nematodes to the urine of healthy individuals and cancer patients was investigated, it was found that they were averse to the urine of healthy individuals. They showed avoidance behavior towards urine from cancer patients, but attracted behavior towards urine from cancer patients. 30 samples were examined and the accuracy was 100%. (Figure 1). In addition, the drug reacted to all types of cancer, including early-stage cancers, including stomach cancer, colorectal cancer, and pancreatic cancer, making it a promising treatment for cancer detection. Similar to the behavior of familiar dogs, it was shown that they reacted to the specific smell of cancer, which is common to various types of cancer.

[0019] Therefore, in the present invention, gastric cancer, colorectal cancer, esophageal cancer, pancreatic cancer, prostate cancer, bile duct cancer, , breast cancer, malignant lymphoma, gastrointestinal mesenchymal tumor, appendicitis cancer, lung cancer, and other cancer types are detected. It is possible. This cancer diagnostic system using nematodes can solve many of the conventional problems as follows.

[0020] (i) It is possible to detect early cancer. It is possible to detect early stage cancers (stages 0 and 1) with high accuracy. In the past, specimens that were judged negative by existing tumor markers were found to be positive in this test. This patient developed cancer during the two years of follow-up. The present invention makes it possible to detect cancers that cannot be detected by conventional methods. (ii) The presence of multiple cancer types can be diagnosed with a single test, i.e., in a single screening test. It can diagnose many types of cancer. So far, it has been used to diagnose stomach cancer, colorectal cancer, and , esophageal cancer, pancreatic cancer, prostate cancer, bile duct cancer, breast cancer, malignant lymphoma, gastrointestinal mesenchymal tumor, cecal cancer It has been confirmed that lung cancer can be detected using this method. (iii) High sensitivity. The test of 30 specimens was able to detect the disease with 100% sensitivity and specificity. Even when a total of 242 samples were tested, cancer patients were detected with 100% sensitivity and 95% specificity. It was.

[0021] (iv) Samples are easy to collect. There are no special conditions, such as dietary restrictions, for collecting urine samples, and they are collected during regular checkups. The collected urine sample can be analyzed, which is painless for the subject and is different from other urine tests. The required amount of urine is only a few microliters.

[0022] (v) Analysis is cheap and easy. (v-1) Fast Analysis can be done in a short time. Analysis of chemotaxis in nematodes can be done in about an hour and a half. Analysis of olfactory nerve responses using transgenic nematodes can be completed in approximately 30 minutes. (v-2) Inexpensive For example, for one specimen, two petri dishes for rearing nematodes (each dish costs about 10 yen) and one petri dish for taxis analysis are required. 3-5 plates (approximately 10 yen per plate). Agar is 2.5 yen and 10 yen per plate. Other Even including reagents, the cost per sample is about 100 yen. Even including labor costs, analysis is very inexpensive. Cut. (v-3) Analysis is easy and does not require specialized skills. Analysis of nematode taxis is very easy and can be done by anyone. C. elegans are also easy to raise. No special training in nematodes is required, and analysis can be performed using conventionally reared nematodes. Cut. (v-4) Multiple sample analysis is possible One experimenter can perform about 150 taxis per day. If the taxis is analyzed three times, 50 samples can be analyzed per day. This process can also be automated. It is possible.

[0023] (vi) Easy to implement and can be introduced worldwide (vi-1) The entire system is inexpensive and easy to implement No special room is required for raising nematodes. All you need is a 20°C incubator and a The system requires only a microscope and can be constructed inexpensively and quickly. (vi-2) Can be introduced worldwide Since it does not require expensive measuring equipment, it can be introduced in all countries, not just developed countries. do.

[0024] (vii) Applicability to the diagnosis of recurrence after cancer treatment Since it can detect cancer in all parts of the body, it can be used to determine the possibility of postoperative recurrence.

[0025] As described above, the present invention can reduce pain to the subject, and the operation can be performed simply and inexpensively. This method can be performed on a large number of people and is useful as a new, highly accurate cancer screening method.

[0026] (2) Identification of olfactory receptors Odors are received by olfactory receptors on the olfactory nerve. There are approximately 350 types of olfactory receptors in humans. It is said that there are approximately 10,000 different smells that can be distinguished. It is unclear how the vast variety of odors can be distinguished by receptors that lack them. Therefore, in order to clarify this, it is necessary to clarify the correspondence between odors and receptors. However, such attempts have only been partially carried out, and the in vivo correspondence in particular has not been fully understood. was hardly understood.

[0027] The nematode C. elegans is excellent for in vivo analysis, as it has only about 10 olfactory nerves (compared to about 5 million in humans). The fact that there are only a few individuals, that all of the neural circuits have been identified, and the mechanism by which we sense smell Since the olfactory sense is almost the same as that of mammals, they are considered a model organism for olfactory research. Insect olfactory receptors are seven-transmembrane G protein-coupled receptors, the same as mammalian olfactory receptors, and there are 1,200 of them in the genome. However, the correspondence between odors and receptors is known for only one The diacetyl receptor ODR-10 is a key receptor for odor signals. It was unclear whether

[0028] Therefore, the inventors have used RNAi to inhibit the function of olfactory receptor genes in the nematode body, and We conducted a comprehensive screening to examine how the mice react to 11 different odors. The genes that caused changes in the response to odor were selected and analyzed repeatedly. As a result, we succeeded in obtaining candidate genes for all the odors examined (Figure 22B).

[0029] Next, we investigated whether the candidate genes obtained actually function as olfactory receptors in vivo. In order to clarify this, the inventors investigated whether the preference (likes or dislikes) of the same odor changes depending on the concentration. In humans, preference changes depending on the concentration of an odor. It is empirically known that the fragrance of indole changes depending on the concentration of the compound. For example, indole produces the scent of jasmine at low concentrations. At high concentrations, it gives off the smell of feces and urine. A similar phenomenon has been observed in nematodes, and their activity changes depending on the concentration of the odor. The inventors have previously discovered that the type of olfactory nerve that changes changes, and this changes the preference. Yoshida, K. et al.: Nature Communications 3, 739 (20 12)) So, does the receptor that responds to the same odor change depending on the concentration? To analyze this interesting question, the inventors investigated the effects of high concentrations of diacetyl on the We focused on the sri-14 gene obtained as a result.

[0030] The sri-14 loss-of-function mutant showed normal responses to low concentrations of diacetyl, but not to high concentrations of diacetyl. On the other hand, a known mutant of the diacetyl receptor ODR-10 showed abnormalities only in response to low concentrations of Only the response to diacetyl in ODR-10 was reduced (Fig. 24A). It has already been reported that it functions in the nervous system (Sengupta, P. et al.: Cell 84, 899-9 09 (1996)), and by analyzing the expression of sri-14, phenotype recovery experiments, and experiments inhibiting neural-specific gene expression. This indicates that SRI-14 functions in ASH sensory neurons that perceive unpleasant odors (Fig. 24). SRI-14 was also observed to be localized to sensory cilia, where olfactory receptors are present (Figure 24G).

[0031] Here, the gene expression inhibition experiment can be carried out using, for example, RNAi or antisense nucleic acids. These include inhibition by the expression of dominant-negative mutant genes, and inhibition by the expression of dominant-negative mutant genes. Inhibition using Ai is preferred.

[0032] Next, calcium imaging was used to investigate the response of AWA and ASH sensory neurons to diacetyl. AWA neurons responded to both low and high concentrations of diacetyl, but the odr-10 mutant In the sri-14 mutant, no response to low concentrations of diacetyl was observed (Fig. 28). On the other hand, ASH sensory neurons responded only to high concentrations of diacetyl, and this response was not observed in the sri-14 mutant. The activity of sri-14 was significantly reduced in the odr-10 mutant, but was normal in the odr-10 mutant (Fig. 29). When ectopically expressed in another sensory neuron, AWB, which does not respond to diacetyl, the AWB was resistant to high concentrations of diacetyl. This indicates that SRI-14 functions as a diacetyl receptor in vivo. These results strongly suggest that the same odor activates receptors depending on the concentration. They are used differently, and low concentrations of diacetyl are perceived by the ODR-10 in the AWA nerve, which makes people feel like they like it. It was found that high concentrations of diacetyl are perceived as disliked by SRI-14 in ASH neurons (Tanig uchi, G. et al.: Science Signaling 7, ra39 (2014)) (Figure 33).

[0033] Nematodes have an excellent sense of smell, with almost the same number of olfactory receptors as dogs, making them ideal for use as drug detection dogs. It is possible that they have a high sensitivity to the smell of harmful and beneficial substances. The results of this research will enable us to identify the receptors for these odors. If the binding relationship is understood, it is expected that it will be possible to develop an artificial odor sensor based on that binding. In the future, olfactory analysis using nematodes will be useful and contribute widely to society.

[0034] 2. Detection Method (1) Nematodes The nematode used in the method of the present invention is a type of soil-dwelling nematode, and is used as a model organism for biological research. The nematodes used in the method of the present invention may be either male or female, but are self-fertile. Hermaphrodites are preferred because they can be propagated by C. elegans can be easily reared by feeding them E. coli in a petri dish. If they are placed in a petri dish, within four days, the eggs will hatch and grow into adults, increasing in number by 50 to 100 times. During this time, you can just leave them in the incubator and no special operations are required. If you use hermaphrodites, you do not need to breed them. The necessary equipment is a 20°C incubator and a stereo microscope, and the system can be set up in a short time. , and can be done inexpensively.

[0035] The nematodes used in the present invention include, for example, wild-type nematodes such as caenorhabdites. Caenorhabditis elegans is an example, and the C. elegans Briostol N2 strain It is preferable to use hermaphrodites, but it is also possible to use genetic mutants that lack various genes. These nematodes can be obtained from, for example, the Caenorhabditis Genetic Center (CGC). This can be done.

[0036] In the present invention, in addition to the above-mentioned nematodes (wild-type nematodes), mutant nematodes and transgenic nematodes are also used. Transgenic nematodes can be used. The olfactory nerves AWC and AWA of the nematode are expressed in the transgenic nematode. , nematodes with introduced indicator genes, genes involved in the perception of cancer odors (receptor genes) Inhibition of the expression or function of odor receptor genes in nematodes and cancer C. elegans expressing or functionalizing markers, and fluorescent proteins are added to each cell to facilitate behavioral analysis of C. elegans. Examples include, but are not limited to, nematodes expressing proteins. This applies to all transgenic strains into which foreign genes have been introduced.

[0037] In the present invention, a DNA is constructed in which a target gene is linked directly downstream of a specific promoter of a nematode. This is then microinjected into wild-type or genetically mutant nematodes (e.g., into the gonads). It is possible to create transgenic nematodes that can stably inherit genes. An increase in calcium concentration indicates that nerves are activated, so for example, calcium in the nerves increases. Using an indicator gene that can measure calcium concentration, changes in calcium concentration are used as an indicator. Cancer can be detected by

[0038] The promoter used to express AWC is, for example, the odr-1 promoter (Yu , S., Avery, L., Baude, E. & Garbers, DL Guanylyl cyclase expression in speci fic sensory neurons: a new family of chemosensory receptors. Proc Natl Acad Sci USA 94, 3384-3387 (1997).) odr-1 is a olfactory receptor that binds to the AWC and AWB (another olfactory receptor that is different from the AWC). It induces expression in the nervous system. In addition, examples of promoters used for expressing AWA include the odr-10 promoter. Sengupta, P., Chou, JH & Bargmann, CI odr-10 encodes a seven transme mbrane domain olfactory receptor required for responses to the odorant diacetyl. Cell 84, 899-909 (1996).) It is known that odr-10 induces expression only in AWA. It is being done.

[0039] The nucleotide sequence information of these promoters is available, for example, under accession numbers Z68118 and FO080931. The promoter can also be obtained by purifying nematode genomic DNA and using it as a template. It is also possible to obtain the DNA fragment by amplifying it using PCR. The calcium indicator gene is the Yellow Cameleon (YC) gene (Nagai, T., Yamada, S., Tominaga, T., Ichikawa, M. & Miyawaki, A. Expanded dynamic range of fluorescent indicators for Ca(2+) by circularly permuted yellow fluorescent proteins. Proc Natl Acad Sci USA 101, 10554-10559 (2004).), GCaMP gene (Naka i, J., Ohkura, M. & Imoto, K. A high signal-to-noise Ca2+ probe composed of a si ) and other proteins. These genes are identified by the nucleotide sequences, for example, under accession numbers AB178712 and HM143847. Sequence information is available. These genes are also available through addgene. It is Noh.

[0040] Methods such as linking an indicator gene directly under the promoter and microinjection Such methods are well known in the art and are described, for example, in Molecular Cloning: A Laboratory Manual (4th Edition)" (Cold Spring Harbor Laboratory Press (2012)) Alternatively, a DNA solution can be injected into the gonads of nematodes by known methods (M ello, CC, Kramer, JM, Stinchcomb, D. & Ambros, V. Efficient gene transfer i n C.elegans: extrachromosomal maintenance and integration of transforming sequence ces. EMBO J 10, 3959-3970 (1991).). Mutant nematodes, for example, nematodes with polymorphisms in the genome of wild-type nematodes, can detect the odors of various cancer types. The term "olfactory receptor deletion mutant" refers to a mutant lacking the corresponding olfactory receptor (explained in the Examples below). .

[0041] (2) Biological substances derived from subjects or their processed products The samples used in the present invention are obtained from subjects (healthy individuals, cancer patients, patients suspected of cancer, etc.), animals, etc. "Bio-related substances" are biological samples collected from subjects. For example, body fluids (urine, sweat, saliva, stool), cells (biopsy cells, etc.), cancer tissue (biopsy tissue, tissue In the present invention, these biological samples themselves are used. However, it is preferable to use a processed product of a biologically related substance. " refers to a sample in which a biologically relevant substance has been physically and / or chemically treated.

[0042] Body fluid samples such as urine contain solids and sediment. It can be used as is, but it needs to be given to the nematodes through a thin tube, so It is preferable to carry out a filter removal treatment (e.g., pore size 0.22 μm, MillexGP, Merck Millipore) Urine samples that have been treated to remove solids using such filters are In addition, the present inventors have confirmed through preliminary experiments that nematodes can be easily treated with a filter. It was confirmed that the reaction (attractive behavior toward urine) of the mice was unchanged (Figure 9).

[0043] As mentioned above, when using cells (e.g., cancer cells obtained by biopsy) as biologically-related materials, After cell culture, solids such as cell debris are removed from the culture by centrifugation or filtering. The solid-removed culture supernatant is obtained as a treated product. In addition to the above samples, the present invention also includes cancer cells or cancer tissues (biopsy tissues, tissue sections, etc.). Examples of preservative solutions include physiological saline, buffer solution, formalin, etc. Preservation solutions include, but are not limited to, phosphate, DMSO, etc. It contains a commonly used cryopreservation solution, and after cryopreservation, it can be used by thawing. can.

[0044] (3) Detection using olfactory sense of nematodes First, nematodes necessary for detection are grown. Place several adult nematodes in a petri dish (NGM medium containing E. coli) and incubate for 3 to 6 days. The nematodes are then cultured for 4 days at 15 to 25°C, preferably 20°C. This allows the next generation of nematodes to grow to 300 to 500. They grow to about 100 adults. Next, a petri dish is prepared for the actual testing. Create a 4-point solution and add sodium azide (NaN3) to it. The amount of acetaminophen added is 0.2 to 3 μl at a concentration of 1 M, and is preferably 0.2 to 3 μl. or 0.5 μl.

[0045] The E. coli bacteria in the dish were removed with a washing buffer, and the dish was filled with a biological substance or its equivalent. If urine is used as the sample, place (add) a sample of the treated product. The collected urine can be diluted with, for example, sterile water or a buffer solution for 1.5 to 1 The urine may be diluted 1000 times. The dilution ratio is preferably 10 times. The sample volume is 0.5 to 10 μl, preferably 1 μl.

[0046] The nematodes are placed in the center of the prepared dish. Raise (allow the nematodes to swim) for the specified time (approximately 1 hour). The room temperature should be 23°C ± 1°C. After a certain time has passed, the number of individuals on the positive side and the negative side are counted and the chemotaxis index (formula below) is calculated. Let the number of individuals on the positive side be N(+) and the number of individuals on the negative side be N(-). chemotaxis index= N(+) - N(-) / total number of individuals This becomes:

[0047] A positive or negative response is then used as an indicator to detect cancer. A "positive response" means that the worms "like" or are "interested" in the sample. A "negative response" means that the worms "dislike" or "are not interested" in the sample. It means "yes." When chemotaxis is used as an index, a positive value (+) indicates a positive response (positive chemotaxis, like), a negative value (-) indicates a negative response (positive chemotaxis, like), A value (-) represents a negative response (negative chemotaxis, aversion).

[0048] The chemotaxis index ranges from +1 to -1, with a positive value indicating attraction and a negative value indicating repulsion. If so, it takes a negative value. Although one analysis per specimen is acceptable, multiple analyses are also recommended, and the average chemotaxis index value should be calculated. The accuracy of the values ​​can be improved by calculating the average value. If the mean value is positive, there is cancer or a risk of cancer. The determination of "cancer" can be made, for example, by a definitive diagnosis of cancer or It can be used as an auxiliary material for preliminary diagnosis, and the determination of "there is a risk of cancer" is For example, it can be used as a supplementary material to detect cancer during health checkups and initial cancer diagnosis. .

[0049] The response of nematodes to odors can be detected using behaviors and biological reactions other than chemotaxis as indicators. For example, the weather vane behavior of nematodes (Iino & Yoshida, The Journal of Neuroscience, 2009) and the timing of the decrease in odor concentration. behavior (Pierce-Shimomura et al., The Journal of Neuroscience, 1999), body flexion These include the degree of stimulation (Luo et al., Journal of Neurophysiology, 2008), and neural response. In weathervane behavior, a "positive response" is when the worm turns toward the sample. In addition, in the case of turning behavior, when the sample changed from a high concentration to a low concentration, If the worm turns to the opposite side, it can be said to have made a "negative response." If the distance between the sections is long, it can be said to be a "positive response."

[0050] (4) Detection using genetically modified nematodes (i) Detection using genetically modified nematodes can also be carried out as described above, but An indicator gene that can measure intraneuronal calcium concentration was inserted into the insect's olfactory neurons AWC and AWA. Using transgenic nematodes expressing the protein, changes in calcium concentration (neuronal response) were used as an indicator. This method can be used to analyze several nematodes, so it is not necessary to cultivate nematodes. This has the advantage that the cost is low and it can be done in a short time.

[0051] Figure 3 shows the measurement principle when the Yellow Cameleon gene is used as the indicator gene. show. The indicator proteins used for the measurement are calcium-binding protein CaM and CaM The target M13 is linked to the fusion protein, which has CFP and YFP attached to both ends. (It is encoded by a gene and can be genetically expressed in vivo.) When the sodium concentration is low, CaM and M13 are far apart, and CFP and YFP are also far apart (left image). Therefore, when CFP is excited by light, it emits blue light. When the calcium concentration increases and CaM and M13 bind, CFP and YFP approach each other, and a fluorescence resonance emission occurs between them. Energy transfer (or Förster resonance energy transfer) (FRET) occurs. Even if light is applied to excite the YFP, yellow light is emitted from the YFP (right image). By measuring blue and yellow light simultaneously and calculating the ratio, changes in calcium concentration can be determined.

[0052] Figure 4 shows the measurement principle when the GCaMP gene is used as the indicator gene. The indicator protein is a fusion protein consisting of CaM and M13 linked to GFP. This protein is also encoded by a gene and is genetically determined in vivo. When calcium concentration increases and binds to CaM, the GFP fluorescence intensity increases. Therefore, by measuring GFP fluorescence, changes in calcium concentration can be determined.

[0053] In the present invention, the olfactory sense of a nematode is stimulated by a biological substance derived from a subject or a processed product thereof. If the response to the blood is large, i.e., if the calcium concentration change is large, the subject has cancer. When the olfactory nerve response is large and when the olfactory nerve response is large, the risk of cancer is judged to be high. When the calcium concentration change is large, the "large" refers to the control (a biological sample derived from a healthy person). stimulate the subject's biologically-related substance or its processed product compared to the subject's biologically-related substance or its processed product The change in the fluorescence intensity ratio (ratio = YFP / CFP) or the change in the fluorescence intensity of GFP when This means that is significantly larger.

[0054] (ii) C. elegans expressing calcium indicators in the olfactory neurons were placed in a resin container (e.g. The chip is made of dimethylpolysiloxane (PDMS) resin (Figure 5). The plastic chip has a part for clamping the nematode and four flow channels.

[0055] Switch between channels 1 to 4 using a perfusion device (WPI Multi Channel Perfusion System MPS-2, etc.) By holding the device in place (Fig. 6), the urinary stimulation is turned on and off (Chalasani, SH et al. Dissecting ga circuit for olfactory behavior in Caenorhabditis elegans. Nature 450, 63-70 (2007); Chronis, N., Zimmer, M. & Bargmann, CI Microfluidics for in vivo im aging of neuronal and behavioral activity in Caenorhabditis elegans. Nat Methods 4, 727-731 (2007).

[0056] Figure 6 shows the switching of channels within the chip. Channels 1, 2, and 4 are filled with buffer, Put urine in flow path 3. Flow paths 2 and 3 are always ON. When flow path 1 is ON and flow path 4 is OFF, When channel 1 is turned OFF and channel 4 is turned ON, the worms are not exposed to the urine stimulus. can be done.

[0057] The AWC olfactory nerve is a nerve that responds when an odor is present and then absent (olfactory-OFF response). We look at the reaction when urine is changed from being present to being absent. Because this is a nerve that responds from "nothing" to "yes" (olfactory-ON response), it changes from a state where there is no urine to a state where there is urine. See the reaction when you change it to another state. By the way, the olfactory nerves of nematodes react weakly to urine from healthy people, so the control urine (from healthy people) The same nematode individual was given the control urine and the sample urine in turn, and their reaction was observed. It is preferable to detect cancer based on the difference in the intensity of the signal.

[0058] (iii) Using a fluorescence microscope (e.g., Leica DMI3000B), take a fluorescence image using an objective lens (40x magnification). Images are acquired every 200 ms, for example, but this can be changed depending on the microscope, lens, etc. In the case of Yellow Cameleon, it is necessary to acquire images at two wavelengths separately. Therefore, the camera must be capable of capturing images of two wavelengths simultaneously, such as the ORCA-D2 digital camera. a (Hamamatsu Photonics) is preferable (other cameras with similar functions are also available). In the case of GCaMP, it is sufficient to acquire an image of one wavelength, so it is not necessary to use a general method that can acquire GFP images. All that is needed is a microscope camera.

[0059] (iv) For the acquired images, the cell body of the olfactory nerve (the area where changes are most easily observed) is selected as the ROI (region of interest). The fluorescence intensity of each pixel is measured by software (e.g., Molecular device All calculations and video creation are performed using Metamorph software (manufactured by S Corporation). Other software that supports this can also be used. In the case of Yellow Cameleon, YFP for each pixel Calculate the fluorescence / CFP ratio and calculate the average value within the ROI. In the case of GCaMP, The mean value of the intensities is calculated.

[0060] 3. Receptor Identification In the present invention, a method for identifying odor receptors using nematodes is provided. The present invention provides a method for identifying odor receptors in the mammalian genome. The identification method of the present invention inhibits the expression or function of a gene encoding a receptor, and The method includes testing the response of the nematodes to the odor. As mentioned above, the expression or function of olfactory receptor genes can be inhibited by RNAi or antisense oligonucleotides. Examples include inhibition using nucleic acids and inhibition by expression of dominant-negative mutant genes. However, inhibition using RNAi is preferred.

[0061] Using nematodes in which the expression of the receptor gene was inhibited, the responses of samples derived from various cancers to odors were investigated. If a cancer type does not respond to odors, the receptors are not able to detect the odors of that cancer type. It can be determined that the receptor for In addition, the type of receptor identified varies depending on the concentration of the odorant. The test was conducted to determine whether the receptor reacts to samples of high and low concentrations. The receptor for the sample can be identified.

[0062] The olfactory system detects and responds to a variety of odorants. In most organisms, olfactory receptors , a G protein (heterotrimeric guanine nucleotide-binding protein)-coupled receptor It directly binds to volatile or soluble odorants. Compared to mammalian genomes, the C. elegans genome The Caenorhabditis elegans genome contains more putative olfactory These genes are involved in the synthesis of odorant receptors, which in turn are involved in the synthesis of odorant receptors. This suggests that there may be complexities in the response to specific odorants. RNA interference (RNAi) screening to identify olfactory receptors in C. elegans required for the response This screening yielded 194 candidate odorants associated with 11 odorants. We identified the sensory receptor gene.

[0063] Furthermore, the present inventors identified SRI-14 as being involved in sensing high concentrations of diacetyl. Rescue and neuron-specific RNAi experiments demonstrated that SRI-14 regulates specific chemosensory neurons. In ASH neurons (sensory neurons in nematodes that detect aversive odors and chemicals), Calcium imaging demonstrated that ASH neurons function and induce a repellent response. chemosensory neurons respond only to high concentrations of diacetyl, whereas other types of chemosensory neurons A certain AWA neuron (an olfactory sensory neuron in C. elegans that mainly receives the preferred odor) Loss of SRI-14 function resulted in a response to both high and low concentrations of diacetyl. Loss of ODR-10 function prevented the ASH response to low concentrations of diacetyl, whereas loss of ODR-10 function abrogated the AWA response to low concentrations of diacetyl. Chemosensory neurons ectopically expressing SPI-14 were sensitive to high concentrations of diacetyl. Thus, the worms responded to the olfactory receptors and the sensory neurons. They have a concentration-dependent odor sensitivity mechanism that is differentiated.

[0064] In general, animals detect and respond to a variety of odorants through their olfactory system. The majority of these compounds are volatile and are sensed by olfactory receptor neurons (ORNs). In ORNs, odor molecules directly bind to olfactory receptors and then initiate intracellular signaling pathways. In mammals, olfactory receptors are composed of seven transmembrane G-proteins. Protein (heterotrimeric guanine nucleotide-binding protein)-coupled receptor (GPCR) member of the Milli ( 2 ), and only one olfactory receptor type is present in any individual ORN ( 3 ). However, despite various studies aimed at identifying the correspondence between odors and receptors, The relationship between individual odors and receptors remains largely unknown (4, 5). n)) is a similar process but involves the detection of soluble chemicals.

[0065] The nematode Caenorhabditis elegans is a serovar that expresses odors and Analysis of taste-related chemosensory processes (olfaction, detection of volatile signals, and taste, soluble It is a model organism used for the detection of chemical signals and is known to respond to chemicals. It senses and responds to a variety of chemical cues through approximately 13 sensory neurons. (6, 7). For convenience, we refer to this chemosensory process and odorants as olfaction. The C. elegans genome contains over 1200 GPCR-encoding genes. GPCRs are predicted to be present in 11 of the chemosensory neurons, including putative olfactory receptor genes. These findings are distinct from those of olfaction (3), but are consistent with those of mammals and Multiple types of olfactory receptors (9) are expressed in each ORN, similar to taste recognition in the brain (10). However, it is unclear whether the receptors and odorants or other chemicals are involved. The relationship between the diacetyl-specific receptor ODR-10(11) and the pheromone receptor (which are also GPCRs (12-14)) have only been identified in C. elegans How are odorants sensed by these combinations of receptors in ORNs of the cerebellum? It is not known whether

[0066] Like many animals, the nematode C. elegans also shows preferences for certain odorants and When these are detected by AWA or AWC olfactory neurons, attraction behavior (at traction behavior) and the substance was detected by AWB, ASH, or ADL sensory neurons. When exposed to a stimulant, they exhibit aversion behavior (6, 15, 16). However, some neurons are involved in the aversion behavior. These neurons are involved in both motor and attraction behaviors, for example, AWB neurons (16). We have previously investigated the attraction response or the olfactory response to the same odorant in the nematode C. elegans. showed that the aversion response depends on the concentration of the odorant (17). However, it does raise the possibility that different olfactory receptors function depending on the concentration.

[0067] Here, the inventors have demonstrated that different concentrations of diacetyl are mediated by different olfactory receptors, which By screening odor-receptor pairs, we were able to show that preference changes depending on the odor. As a result, the inventors identified 194 candidate olfactory receptor genes for 11 odorants. Among these, the present inventors have identified SRI-14 as a specific regulator of diacetyl activity. Our results suggest that diacetyl reception is mediated by ODR-1 in AWA neurons. SRI-14 mediates an attractive response at low concentrations, whereas SRI-14 mediates an aversion at high concentrations in ASH neurons. It has been shown to mediate avoidance responses.

[0068] 4. Identifying the type of cancer In the present invention, it is possible to identify the type of cancer by the nematode taxis test. In the present invention, the reaction of nematodes to the odor of a biological substance derived from a subject or a processed product thereof is The present invention provides a method for identifying a type of cancer, which is characterized by using the above as an indicator.

[0069] Research on cancer detection dogs has predicted that the scent varies depending on the type of cancer. We will identify the olfactory receptors for each type of cancer and develop modified versions of these receptors. The modified strains include those lacking the receptor gene (deletion mutants), and those lacking the receptor gene expression. Alternatively, strains with inhibited functions, or strains with highly expressed or highly functional receptor genes may be included. .

[0070] Deletion mutants can be generated using the CRISPR / Cas9 method (Friedland et al., Heritable genome editing). (Iting in C. elegans via a CRISPR-Cas9 system, Nature Methods, 2013) In addition, strains in which the expression or function of the receptor gene is inhibited, strains in which the receptor gene is highly expressed or We will create highly functional modified nematodes. To inhibit expression or function, we will use RNAi. Inhibition using antisense nucleic acids, inhibition by expression of dominant-negative mutant genes To enhance the expression or function of a receptor gene, Methods for linking motors in tandem, introducing enhancers, and multi-coupling receptor genes methods for introducing receptors, methods for modifying the binding sites of receptors with odorants and G proteins, and receptor These include methods that involve modifying the parts of the body that control activation, localization, and affinity for odors.

[0071] The identification method of the present invention includes, for example, the following steps. (a) First, in STEP 1, cancer is detected by the detection method of the present invention. For example, N2 strain It is used to check for the presence or absence of cancer. (b) Next, in STEP 2, we will investigate the receptor mutations and receptor gene expression and function of each cancer type. The cloned strains are used to identify the cancer type. For a sample detected as being cancerous in the step (a), the identified olfactory Using mutant nematodes lacking the receptor and strains with altered receptor gene expression and function, Test the reaction to the

[0072] (c) The response to an odor differs between the modified nematode and the nematode used in the step (a). If so, the cancer type corresponding to the identified receptor is determined to be the target cancer type for identification. For example, among the mutant nematodes and nematodes in which the expression or function of the receptor gene is inhibited, The cancer types corresponding to the receptors identified in nematodes that did not respond to the Alternatively, among nematodes in which the receptor gene is highly expressed or highly functional, Cancer types corresponding to receptors identified in nematodes with enhanced odor responses were identified as targets for identification. It is determined to be an elephant carcinoma. For example, if the odorant receptor mutants in colon cancer do not show any attraction behavior, it can be determined that the cancer is colon cancer. (Diagnosis) is possible (Figure 37).

[0073] 5. Kits and Systems The present invention provides a kit for detecting cancer, which comprises a nematode. However, it may contain one or more components necessary for carrying out the detection method of the present invention. Such components include, for example, buffer solutions, culture media, sodium azide, E. coli, petri dishes, etc. The kit of the present invention may include a part containing only some of the necessary components. It may also be a partial kit, in which case the user can provide the other components. The kit of the present invention may also include instructions for use that explain the detection or identification method. Cut. The present invention also provides a container for accommodating a nematode, a biological substance or a processed product thereof, and the nematode. and a detection unit that detects the behavior of nematodes in the container.

[0074] FIG. 16A is a block diagram of the system of the present invention. The system includes a storage section 30 for storing a biological substance or a processed product thereof and the nematode, and a storage section 30 The device includes a detection unit 10 that detects the reaction of nematodes to odors and a processing unit 20 that processes the detected information. Furthermore, the system of the present invention includes a storage device for storing data processed by the processing unit 20. The storage unit 40 may include a program and a database for cancer detection. It is prepared.

[0075] The storage section 30 may be exemplified by a petri dish, a culture dish, a chip having a microchannel, or the like. There is no limitation as long as it can accommodate the insects and the sample. The system of the present invention is capable of capturing moving images of at least one nematode in real time. The detector 10 includes at least one detector 10 capable of detecting images, population counts, movement trajectories, etc. of nematodes. A device for acquiring data, such as a microscope or camera, e.g., a fluorescent microscope, a digital microscope, The microscope and camera are equipped with a microscope and a digital video camera that can track the movements of the nematodes. It can be equipped with an automatic tracking system that can track individual nematodes or multiple nematodes simultaneously. The distance traveled by the nematodes is then measured from their tracks. The gathered nematodes are photographed and the number of individuals is counted. The microscope and camera are equipped with a It may also be equipped with a sensor that can detect this.

[0076] The system of the present invention can measure the movement of nematodes using real-time images, and also measure the movement of still nematodes using real-time images. It is also possible to measure the movement of nematodes using images (photographs). By taking pictures, it is possible to dynamically locate the location of the nematodes.

[0077] 16B is a block diagram of the processing unit 20 of the system of the present invention. The processing unit 20 includes a calculation means 110 and a database 120. The calculation means 110 includes: (i) an inspection condition setting means 111; (ii) a nematode (iii) a reaction testing means 112, (iii) a cancer determination means 113, and (iv) a test result display means 114.

[0078] (i) Inspection condition setting means 111 The inspection condition setting means 111 sets the conditions necessary for calculation by a GUI (Graphical User Interface). It is a means to input data from a mouse or keyboard, and the input data is displayed in a graph. This can be confirmed.

[0079] The inspection condition setting means stores predetermined conditions in accordance with the inspection purpose in the system of the present invention. The conditions can be, for example, the number of nematodes, the characteristics of the nematodes, chemotaxis These include whether or not the index is accepted and the measurement time. The characteristics of the nematode are that it is a deletion mutant, that it is a strain in which gene expression etc. is inhibited, and that the measurement To achieve this, genes encoding fluorescent proteins (e.g., GFP gene, RFP gene, etc.) are introduced. However, the conditions are not limited to these and may vary depending on the purpose of the inspection. It can be set appropriately.

[0080] (ii) Nematode reaction testing means 112 The nematode reaction inspection means 112 detects or detects cancer from the inspection condition setting means 111 or the database 120. The formula for identification (e.g., chemotaxis index) is selected, and each calculation This is a method for calculating the response of nematodes based on a formula. Measurement of the number of nematodes, measurement of the total distance traveled by a single nematode, and detection of the fluorescence intensity emitted by a single nematode The behavior of the nematodes that react to the test sample is recorded. For example, focusing on one nematode, The distance traveled by each nematode was measured and the total distance traveled was calculated by adding up the distances traveled by each nematode. Or, when a fluorescent protein gene is introduced into a nematode, the sample reacts to the The fluorescence intensity of the nematodes is measured. The fluorescence intensity per nematode is also measured to calculate the total number of nematodes. The sum of the fluorescence intensity emitted from all the nematodes gathered in a certain area can be calculated as It can also be measured by.

[0081] (iii) Means for determining cancer 113 The cancer determination means 113 detects the presence or absence of cancer based on the reaction of the nematode to the odor, or determines the type of cancer. It is a means of identifying species. When the number of individuals is used as the test condition, the number of nematodes that moved to the control area is The ratio or difference of the number of nematodes that have moved to the test area is calculated. When using the above method, what percentage of the distance traveled compared to the control nematode was required to indicate a reaction? If a certain value of this difference or ratio is set as a boundary value in advance, The boundary value is used as a criterion for determining cancer. Also, fluorescence intensity is used as an inspection condition. If so, what percentage increase in fluorescence intensity compared to the control nematode is required to indicate a reaction? Similar to the travel distance, a boundary value can be set. The measured data, boundary values, and sample information (such as information on cancer type) are compared to determine the predetermined Determine whether it is cancer.

[0082] (iv) Judgment result output means 114 The determination result output means 114 outputs the information based on the detected or identified cancer type or the presence or absence of cancer. It is an output means that displays the type of cancer and its probability (risk). It can be displayed in either a graph or a table. It is also possible to display animations of the nematode behavior calculated by (ii) above.

[0083] (v) Data storage means The entered inspection conditions and inspection results are linked and stored in a database. It will be saved at 120. The saved inspection conditions and calculation results can be retrieved from the database 120 again or by setting the inspection conditions. It can be read from the means 111 and the determination result display means 114. The present invention will be explained in more detail below with reference to examples. However, the present invention is not limited to these examples. It is not limited to: [Example]

[0084] Cancer detection (i) C. elegans rearing Five to six wild-type nematode N2 adults were placed in a 6 cm petri dish (NGM medium with E. coli) and incubated for 4 days. The next generation of nematodes was grown to adulthood, with a total of approximately 300 to 500 nematodes.

[0085] (ii) Tactic analysis The format shown in Figure 2 was created in a 9 cm Petri dish, and sodium azide (NaN3) was added to four points. 0.5 μl was placed. Add 1 ml of wash buffer to the nematode rearing plate and transfer the floating nematodes into a tube. After leaving it for a while, the nematodes sink to the bottom, and the supernatant was discarded. Then, 1 ml of wash buffer was added to the tube, and once the nematodes had sunk to the bottom, the supernatant was discarded. This washing was repeated three times to remove E. coli.

[0086] Place 1 μl of urine sample diluted 10 times with sterile water on the "+" mark on a 9 cm petri dish. I placed it. Next, about 100 nematodes were placed in the center of the dish and allowed to swim for an hour. The room temperature was 23°C ± 1°C. After 1 hour, the number of individuals on the + side and the - side was counted and the chemotaxis index was calculated. Each sample was analyzed five times, and the mean chemotaxis index values ​​for the five analyses were calculated. .

[0087] (iii) Results The results are shown in Figure 1. As shown in Figure 1, all of the control urine samples (c1 to c10) from healthy individuals were negative (-) c The hemotaxis index (avoidance reaction) was observed, whereas the urine samples from cancer patients (p1-p20) were all positive. The chemotaxis index was (+) and cancer was detected with 100% accuracy. In FIG. 1, the error bars represent SEM. [Example]

[0088] Calcium imaging In imaging experiments using microfluidics, urine samples are flowed through thin tubes. Therefore, sediment and solids in the urine were removed by centrifugation and filtration (pore size). size 0.22 μm, MillexGP, Merck Millipore). Therefore, the Yellow Cameleon gene (YC3.60) was expressed by the odr-1 and odr-10 promoters, respectively. Calcium imaging was performed according to a known method (Uozumi, T. et al. Temporally-regulated quick activation and inactivation of Ras is important for olfactory behavior. Sci Rep 2, 500 (2012); Shinkai, Y. et al. Behavioral choic e between competing alternatives is regulated by a receptor guanylyl cyclase, GCY-28, and a receptor tyrosine kinase, SCD-2, in AIA interneurons of Caenorhabd itis elegans. J Neurosci 31, 3007-3015 (2011)).

[0089] The nematode was placed in the microchannel so that its head could be brought out of the microchannel. The same line was obtained for both the control urine and the urine from the cancer patient. The response was tested using insects. Fluorescence images of YC3.60 were taken with a Leica DMI3000B microscope (40x objective) and an ORCA-D2 digital microscope. All images were acquired using a camera (Hamamatsu). Exposure times were 200 ms. Alternatively, the fluorescence intensity of CFP and YFP in AWA neurons was measured, and the ratio of the fluorescence intensity of YFP to the fluorescence intensity of CFP was calculated. The ratio was analyzed using Metamorph software (Molecular devices). The ratio was calculated as YFP intensity / CFP intensity (= R), and the average of the ratios over a 10-s window (-10-0 s) was defined as R0. It was set as.

[0090] The results of testing the response of the AWC and AWA olfactory nerves of C. elegans to urine from cancer patients are shown in Figures 7 and 8. show. In Figure 7, the two left panels show the results of tests using control urine, and the two right panels show the results of tests using urine from gastric cancer patients. Figure 7 shows the calcium concentration in the AWC olfactory nerve in response to urine stimulation (with urine → without urine). This figure shows the change in the YFP / CFP ratio of Yellow Cameleon. In comparison, the reaction was significantly stronger with the urine of cancer patients. Figure 8 shows the average fluorescence intensity ratio (YFP / CFP The change in the ratio of the two groups is shown. *** indicates significance at p<0.001. In this example, sediments and solids in the urine were removed by centrifugation and filtration. However, this treatment did not affect the chemotaxis of the nematodes (Fig. 9). A response was also observed in the AWA olfactory nerves upon addition of urine (Figs. 10 and 11).

[0091] These results suggest that the olfactory nerves of C. elegans are important for distinguishing between control urine and urine from cancer patients. Figures 7 to 11 show that the olfactory nerves of the nematode are used to 8, 9 and 11, the error bars indicate that cancer can be detected. indicates SEM. [Example]

[0092] In this example, established cancer cell lines and Cancer detection experiments were carried out using the culture medium or preservation solution. (1) Cancer detection using cancer cell lines To detect cancer using culture supernatant of human cancer cells, colon cancer (colorectal cancer) cells were used. SW480, COLO201 and COLO205, breast cancer cells MCF7, gastric cancer cells NUGC4, MKN1 and MKN7 was used. SW480, COLO201, and COLO205 were obtained from the JCRB Cell Bank (Japanese Collection of Research Bioresources Cell Bank (Tokyo, http: / / cellbank.nibio.go.j p)), and other cells were obtained from the Cell Resource Center, Institute of Development, Aging and Cancer, Tohoku University (Cel l Resource Center for Biomedical Research, Institute of Development, Aging and C All cell lines were obtained from Cancer (Tohoku University, Sendai, Japan). The cells were cultured in RPMI 1640 medium supplemented with BS at 37 °C under 5% CO2 with aeration until they reached subconfluent. The clear culture medium at the top of the medium was used for the test. The medium was spotted at the "+" position on the assay plate (Figure 12). To eliminate the influence of the spotting, the same concentration of Control cultures diluted at different times were spotted (Fig. 12).

[0093] As a result of testing the chemotaxis of nematodes on an assay plate in the same manner as in Example 1, the wild-type nematode ( C. elegans) was cultured in the culture medium of cancer cells (1 / 10 6 ~1 / 10 7 Diluted to 100 mg / kg, the spores showed attractive behavior to the Figure 13). In Figure 13, the bar on the left side of each cell is 1 / 10. 6 Dilution, right bar is 1 / 10 7 Dilution of cancer cell culture medium The results are from the nutrient medium. * indicates significance at p<0.05, ** at p<0.01, and *** at p<0.001. (Dunnett's test). In FIG. 13, the error bars represent SEM.

[0094] The same test was also conducted on the culture medium or preservation medium of fibroblasts (non-cancerous cells). As a result of the experiment, it was shown that the nematodes did not show any attractive behavior (weak repellency) (Figure 13). This does not mean that nematodes are attracted to the secretions of human cells, but rather that they are attracted to the secretions of cancer cells. This means that they exhibit attractive behavior towards "secretion." MEM, EMEM, and RPMI are medium only. KMST-6 and CCD-112CoN are fibroblasts (obtained from RBCs). , ATCC).

[0095] (2) Chemotaxis to fibroblast culture medium and cancer cell culture medium In addition, the chemotaxis to various concentrations of fibroblast culture medium and cancer cell culture medium, as well as human We investigated the taxis of nematodes to cancerous tissues using the following method. Fibroblast culture medium and cancer cell culture medium were diluted with water to various concentrations (from the original solution to 10-9), and the corresponding We observed the chemotaxis of wild-type nematodes to human cancer tissue and healthy tissue. After obtaining the concept, the tumor was excised from a cancer patient and cut into pieces with a diameter of 0.1 to 0.8 mm. As a result, no attraction was observed for fibroblasts at any concentration, whereas the For the vesicles, significant attraction behavior was observed at concentrations of 10-6 and 10-7 (Figures 17 and 18).

[0096] Regarding the taxis of nematodes to human cancer tissue, they exhibit attractive behavior toward cancer tissue fragments. However, they showed aversion to healthy tissues (tissues furthest from the cancerous tissues) from the same patient (Figure 1). 19) It was also found that when cancerous tissue was placed on one side and healthy tissue on the other, nematodes were attracted to the cancerous tissue. It was.

[0097] (3) Chemotaxis of cancer tissue sections to saline preservation solution Human cancer tissue fragments were placed in physiological saline and stored at -20°C (storage period: 3 months). The taxis of nematodes to dilute saline solutions were examined. After obtaining an informed consent from the cancer patient, a 0.5 cm diameter piece of cancer tissue was excised and 20 ml of biopsy fluid was extracted. The saline solution was diluted with water to a concentration of 10-2 to 10-4, and the wild type The chemotaxis of nematodes was observed. As a result, the mice showed attraction to saline containing cancerous tissue, but not to healthy tissue. They showed an aversion to the physiological saline solution that was added (Figure 20). The odr-3 mutant did not show any attraction to the saline solution in the cancer tissue, suggesting that the nematode does not sense odor. It can be said that. [Example]

[0098] Medium-scale testing To confirm the accuracy of the method of the present invention, 242 urine samples (218 control samples) were analyzed. The study was conducted using a pool of 24 cancer patient samples (Table 1). Shows. [Table 1]

[0099] All urine samples were diluted 10-fold, and the chemotaxis test using nematodes was performed on each sample. This was carried out three times.

[0100] As a result, C. elegans showed an attractive response to all urine samples (24 / 24) from cancer patients, and the detection sensitivity was On the other hand, nematodes were detected in most of the control urine samples (207 / 218). In Figure 14, the orange bars (1, 2, 41, 44, 54, 5 6, 90, 157, 196, 202, 208, 213, 220, 226, 232-239, and 241-242) were derived from cancer patients. The blue bars (numbers other than those mentioned above) indicate the control samples. represents SEM.

[0101] The present inventors also analyzed other tumor markers in the same subjects. The tumor markers analyzed were serum CEA, serum anti-p53 antibody (Anti-p53 Ab), and urinary N 1 ,N 12 -diacetylspermine (DiAcSpm) was used. The sensitivity of the method of the invention (NSDT) was extremely high (Figure 15, Table 2). , the percentage of positive samples from cancer patients. [Table 2] Table 2 includes cancer patients with stages 0 and 1. This means that the method of the present invention can be used early. This means that it is also useful for detecting early stage cancer. [Example]

[0102] Consideration of optimal urine concentration method: Three urine samples from healthy individuals (c1, c2, c3) and five urine samples from cancer patients (p2, p5, p8, p17) , p18) were diluted with water to various concentrations (stock solution ~ 10-5) and the chemical properties of wild-type nematodes were compared with them. The mobility was investigated.

[0103] result: Figure 21 shows that 10-fold dilution is preferable. The three graphs on the left are from urine of healthy individuals, and the five graphs on the right are from urine of cancer patients. The results are shown below. [Example]

[0104] Receptor identification (1) Materials and Methods Nematode culture and strains C. elegans, except for the eri-1 mutant, was cultured with E. coli OP50. , nematode growth medium (NGM) plates containing Escherichia coli NA22 as a food source (3 6) and cultured at 20°C under standard conditions. The wild-type nematodes used were the Bristol N2 strain. Other nematode strains included GR1373:eri-1(mg366), VC2123:sri-14(ok2865), and CX3410: odr-10(ky225) and MT7929:unc-13(e51) were used.

[0105] RNA interference and chemotaxis assays RNAi assays were performed using the Ahringer library (37) against eri-1(mg366) (19). This was carried out using the feeding RNAi method. Nine adult eri-1 mutants were cultured in isopropyl β-D-1-thiogalactopyranoside (0.19 Place the plates on NGM plates containing ATP (60 mg / L), ampicillin (60 mg / L), and E. coli. The adult worms were then cultured for 4 days. The worms were then used in a chemotaxis assay. The chemotaxis assay was performed as previously reported (6, 17). 1 μl of each 10 -3 or 10 -4 Diluted odorants (low concentrations), or 1 and 5 μL, respectively In each experiment using 100 ml of undiluted odorant (high concentration), 30 to 50 individuals were used. Ta. Statistical analysis of the RNAi screening results was performed as follows.

[0106] For all days (Table 3) or each day, the z-scores of each single test from 2 SD and the control value were calculated. The threshold for significant difference was z-score (-1.96 and 1.96, P<0.05). However, blocking one receptor did not appear to have a significant effect. , we used a z-score of ±1 (-0.96 and 0.96, P<0.33) as a weaker threshold.

[0107] Osmotic repellency The present inventors have previously reported on osmophobic behavior using 4M NaCl as an osmotic stimulus. Assays were performed as described (38).

[0108] Cell-specific knockdown of sri-14 function Construction of a transgene to knock down sri-14 function in specific neurons The target region of sri-14 (1.6 kb genomic sequence) was determined as previously described (24). was amplified using the following two primers: Tf, 5'-ggcgccgatataattgctaa-3' (SEQ ID NO: 1), and Tr, 5'-ctgctgcgtttttcgtatca-3' (SEQ ID NO: 2) Gene expression was driven by the sra-6(20) promoter for ASH and the ceh promoter for AWC. -36 ( 39 ) and srd-17 promoters.

[0109] Genetic ablation and inhibition of neurons The present inventors have used mouse caspase- 1 (mCasp1), which are the odr-10 (11), str-1 (15), and ceh-36 (39) genes, respectively. The expression was driven by the sra-6(20) promoter. unc-103(gf) was used to detect AIA-, AIB-, AIY-, or AIZ-specific expression of unc-103(gf). The present results are, respectively, gcy-28.d (29), npr-9 (40), ttx-3 (41, 42), or lin-11 (43, 44 ) promoter.

[0110] Preparation and amplification of sri-14 cDNA Total RNA was isolated using the PureLink RNA Mini Kit (Ambion) according to the manufacturer's instructions. The PCR was performed using ReverTra Ace qPCR Master Mix with gDNA Remover (Toyobo) according to the instructions. The sri-14 cDNA was amplified using the following two primers, Nhe I and Kpn I. and inserted into the pPD-DEST vector (Invitrogen). 5'-gagaGCTAGCaaaaaatgcctgcaggtccac-3' (SEQ ID NO: 3) 5'-gagaGGTACCttattgaattctcggttg-3' (SEQ ID NO: 4)

[0111] Calcium imaging To monitor the responses of AWA, AWB, AWC, and ASH neurons, the inventors used and YC3.6 using the odr-10, str-1, odr-3, and sra-6 promoters ( 11 , 15 , 20 , 45 ). A strain expressing 0 was generated. Calcium imaging was performed as previously reported (3 3, 46, 47), which was carried out using a microfluidic device. In the experiment, the nose of the nematode was exposed to diacetyl[10 -5 Dilution (low concentration) and 10 -3 Dilution (high concentration)] or C. elegans were trapped in a microchannel so that they were exposed to flowing water containing an odorless solution. The room temperature was set at 20 to 23°C. Images were taken with a 40× objective lens and a 3CCD digital camera (C7780, Hamamatsu Photonics). Acquired using a Zeiss Axioplan 2. All images were collected with an exposure time of 200 ms. , AWB, AWC, and ASH cell body time stacks were captured and analyzed using AquaCosmos software (ver. 2.6, Hamamatsu Photonics) to detect yellow fluorescent protein (YFP) and cyan fluorescent protein (CFP). The emission ratio of the YEP protein (CFP) was analyzed. This ratio was calculated as YEP intensity / CFP intensity (R). The average ratio in a 10-second window (-10 to 0 seconds) was set as R0.

[0112] (2) Results RNA interference screening of odorant-receptor pairs In order to comprehensively identify the olfactory receptors required for responses to specific odorants, The inventors performed a systematic RNA interference (RNAi) screen. Neuronal RNAi in s) demonstrates the low susceptibility of C. elegans neurons to RNAi. Because of its ineffectiveness in wild-type nematodes (18), the RNAi-enhanced nematode strain eri-1(m g366)(19) was used.

[0113] The present inventors have demonstrated that knockdown of odr-10 by RNAi in eri-1 mutants is suppressed at low concentrations (10- 3 dilution), this nematode strain exhibits a specific defect in response to diacetyl. We confirmed that this method can be effectively used for RNAi screening of olfactory receptor genes (Figure 2 2) 822 genes encoding putative olfactory receptors, including the SRH family (all genes We screened RNases (genes encoding GPCRs) and found that the RNases for 11 odorants The response of C. elegans treated with Ai was examined. The nematode (C. elegans) responded to the odorant concentration depending on the odorant. Because palatability may change (17), we further tested high concentrations (1 μl and 5 μl of undiluted odorant, respectively). Responses to 10 μl of odorant (which induces attractive behavior at low concentrations) were also tested. The quality consisted of six attractants at low concentrations (6), three high-concentration attractants that induce repellency at high concentrations (17). ), and high concentrations of two repellents (6, 20) (Figure 22).

[0114] Regarding RNAi-treated nematode strains that exhibited abnormalities in chemosensory-induced locomotor responses to odorants, Repeated testing was performed (see Materials and Methods) (Table 3). RNAi targeting genes encoding putative olfactory receptors induces the expression of one or more odorants. The olfactory receptors were found to encode putative olfactory receptors, eliciting weaker responses to the olfactory receptors than the control. It was considered that this was the case (Figure 22 and Table 4). Genes expressed in sensory neurons may function as olfactory receptors, The expression patterns of these genes were investigated.

[0115] The present inventors have synthesized fluorescent ligands linked to the promoters of genes encoding individual olfactory receptors. Using a porter, knockdown of the gene caused a severe defect in chemosensory-induced movement. We analyzed the expression patterns of the genes encoding the 16 putative olfactory receptors shown (Fig. 2 2C and Figure 23, A-L). Furthermore, the expression patterns of 19 olfactory receptor genes were analyzed using the WormBase The information provided in (http: / / www.wormbase.org) was used. Of these, 30 were expressed in neurons. 16 genes analyzed by reporter expression Fifteen of the offspring were expressed in sensory neurons involved in olfactory sensation (Table 5).

[0116] Identification of the olfactory receptor SRI-14 that responds to high concentrations of diacetyl ODR-10 is a diacetyl receptor, and odr-10 mutants exhibit a chemical response to low diacetyl concentrations. As previously reported (11), the present inventors have investigated the odr-10 mutant. We confirmed that the cells showed normal chemotaxis to high concentrations of diacetyl (Fig. 24A). It is possible that other receptors exist for diacetyl and that ODR-10 is specific to low concentrations. (21) RNAi screening revealed that five candidate olfactory receptor genes The molecules (srh-25, srh-79, srh-216, srh-281, and sri-14) are involved in high diacetyl concentrations. The results were as follows (Table 3 and Figure 25).

[0117] Therefore, the present inventors performed expression analysis using the upstream promoter, and found that srh-79 and sr Expression of h-216 was not detectable, and expression of srh-25 and srh-281 indicated an insensitivity to high concentrations of diacetyl. This was observed in ADL sensory neurons that were not involved in avoidance behavior (Table 5). How do different receptors produce concentration-dependent responses to a single odorant? To understand how this gene functions, we focused on sri-14. RNAi targeting this gene caused a significant defect in avoidance from high concentrations of diacetyl. This is because (Figure 24B).

[0118] SRI-14 is encoded by a gene with seven exons and, according to WormBase, is ok2 685 is a deletion mutant predicted to be loss of function (Fig. 26A). The amino acid sequence (SEQ ID NO: 5) indicates that the protein has seven putative transmembrane domains. The results showed that the sri-14(ok2865) mutant was able to grow under high osmotic pressure (Fig. 26, B and C). Dynamic analysis showed that worms exhibited normal hyperosmotic avoidance behavior. The mutants showed a repellent response to high concentrations of diacetyl, similar to sri-14 RNAi-treated worms. Furthermore, compared to the odr-10 mutant, the sri-14 mutant showed a defect in the expression of α-glucan at high concentrations. The sri-14 mutant showed a defect in chemotaxis specific to diacetyl (Fig. 24A). The mice showed normal responses to the substance and high concentrations of other attractants (Fig. 24C). Of the odorants detected, SRI-14 was shown to be involved in sensing only high concentrations of diacetyl.

[0119] Reporter expression analysis revealed that sri-14 is expressed in AWC and ASH chemosensory neurons. AWC neurons were found to be resistant to the 10-fold dilution of diacetyl. ASH neurons are required for sensing attractants (22, 23), and ASH neurons are required for sensing repellents (9) and high concentrations of SRI-14 is involved in the avoidance of isoamyl alcohol (17) in the mouse. To clarify whether the AWC or ASH neurons function as a signaling pathway, the present inventors investigated the AWC and ASH neurons. We performed neuron-specific knockdown of sri-14 in ASH neurons (24). ASH-specific knockdown of ri-14 caused defects in avoidance from diacetyl, but AWC-specific knockdown did not reveal any abnormalities (Fig. 24E).

[0120] Furthermore, the defect of sri-14 mutants in avoiding high concentrations of diacetyl was due to the ASH gene of sri-14 cDNA. Rescued by specific expression of α-glucan in AWA, AWB, or AWC olfactory neurons Heterologous expression either partially or not rescued (Fig. 24F). These results suggest that the function of SRI-14 in ASH sensory neurons is related to the response to high concentrations of diacetyl. This indicates that repellency to hyperosmotic conditions is both necessary and sufficient to mediate repellency. The response is mediated by ASH neurons (25, 26), and this response is reduced in sri-14 mutants. The ASH neurons were normal in detecting and responding to other aversive stimuli (Fig. 26D). In addition, SRI-14-green fluorescent protein (GFP) fusion protein inhibits ASH. SRI-14 was localized to the sensory cilia of the neurons (Fig. 24G). This suggests that SRI-14 is involved in the sensory cilia in ASH. These results suggest that it functions as a factor in olfactory signaling.

[0121] Identification of sensory neurons and interneurons involved in concentration-dependent preference changes of odorants fixed Low concentrations of diacetyl (10 -4 Dilute solutions) are sensed by AWA neurons (6), while intermediate concentrations degrees (10 -1 The dilute solution is sensed by AWC neurons (22, 23). However, no sensory neuron can tolerate high concentrations of diacetyl (undiluted). It is unknown whether ASH and AWB are involved in the aversion response. Neurons have been shown to detect and mediate aversion to high concentrations of isoamyl alcohol (17). Furthermore, AWC and AWB neurons are involved in both attraction and avoidance (17, 27).

[0122] Therefore, the present inventors investigated the effects of ASH, AWB, and AWC on the aversion response to high concentrations of diacetyl. The involvement of sensory neurons was investigated. C. elegans in which AWB or AWC were genetically deleted were exposed to high concentrations of diacetyl. However, ASH neurons showed no chemotaxis defects in response to α-glucan (Fig. 27A). Removal of the ASH neurons inhibited this aversion behavior (Fig. 27A). This indicates that SRI-14 sensed high diacetyl concentrations in ASH neurons. This is consistent with results showing that it functions in

[0123] Whether AWA neurons mediate diacetyl avoidance and attraction to diacetyl To investigate this, the inventors analyzed the behavioral responses of nematodes in which AWA neurons were specifically ablated. Nematodes deprived of AWA showed reduced aversion to high concentrations of diacetyl (Figure 24A) and low The AWC concentration decreased the attraction to the repellent mosquitoes (Fig. 27B). and AWB neurons (17, 27). Dual ablation of both AWA and ASH is comparable to the single This may be due to the fact that AWA and ASH are more sensitive to high concentrations of diacetyl than the removal of acetyl. These results indicate that these two proteins function in parallel (Figure 27A).

[0124] To clarify the contribution of interneurons to odor concentration-dependent preference changes, The inventors have demonstrated that direct synaptic connections to AWA or ASH sensory neurons or both. The involvement of AIA, AIB, AIY, and AIZ interneurons with junctions or gap junctions was investigated (Figure 1). 27C). These interneurons were then subjected to the neuron-specific expression of unc-103(gf), which results in hyperpolarization. The functional inhibition of AIA, AIY, or AIZ interneurons was observed by the expression of α- and β-actin. Inhibition changed the response from repellent to attractive in nematodes exposed to high concentrations of diacetyl ( Inhibition of AIB attenuated the aversive response (Fig. 27D). This result suggests that these interneurons It has been shown that AIB and AIY play an important role in the concentration-dependent change in odor preference. and AIZ are important for the different behavioral responses to high and low concentrations of isoamyl alcohol This is consistent with previous reports that

[0125] In contrast, attraction to low concentrations of diacetyl inhibited these interneurons except for AIY. This suggests that the serovars mediate the attraction to low concentrations of diacetyl. This suggests that the neural circuitry mediating this response is different from that mediating aversion to high concentrations of diacetyl. is doing.

[0126] Differential use of olfactory receptors depending on odor concentration The results of the behavioral experiments of nematodes conducted by the present inventors and previous research results (11) show that the effects of diacetyl Concentration-dependent preference changes were mediated by two types of receptors, ODR-10 and ASH in AWA neurons. These results suggest that this is mediated by SRI-14 in neurons. The genetically encoded calcium markers were expressed in wild-type strains, odr-10 mutants, and sri-14 mutants. By calcium imaging using the indicator Yellow Cameleon (YC) 3.60 (30), The responses of AWA and ASH neurons to various concentrations of diacetyl were monitored.

[0127] AWA neurons in wild-type worms or sri-14 mutants exhibit low intracellular calcium concentrations. It increased after exposure to diacetyl (29) (Fig. 28, A and B). However, the odr-10 mutant showed no response to low concentrations of diacetyl (Fig. 28, A and B). This is consistent with the finding that it functions as a specific receptor for low concentrations of diacetyl in neurones. In contrast, AWA neurons are highly expressed in odr-10 mutants and wild-type worms. The odr-10 cells responded normally to high concentrations of diacetyl (Fig. 28, C and D). This is consistent with the observation that the mutants exhibited normal chemotaxis to high concentrations of diacetyl (Figure 24 A) because ablation of these neurons reduced both attractive and aversive responses (Fig. 27, A and B). A) and B) Taken together, these results suggest that receptors other than ODR-10 are present in AWA neurons and are highly concentrated. It is suggested that diacetyl is detected to some extent.

[0128] The present inventors monitored transient Ca2+ responses in ASH neurons. In neurons, Ca2+ responses were detected only at high concentrations of diacetyl (Fig. 29A). We investigated whether the response of ASH to high concentrations of diacetyl is influenced by other neurons. To clarify, the present inventors have identified a neurotransmitter with a defect in synaptic vesicle exocytosis, We analyzed the Ca2+ response in ASH of the unc-13(e51) mutant (31). The calcium response to high concentrations of diacetyl in the acetylcholinesterase inhibitor was similar to that of wild-type worms. The evoked signals were significantly larger and longer-lasting than those of other neurotransmitters (Fig. 30, A, B, and D). This indicates that the signal from ASH inhibits the activity of ASH toward diacetyl.

[0129] In AWA-deprived nematodes, the response to diacetyl was observed to be altered (Figure 27 , A and B), and the inventors have further demonstrated that ASH neurons respond to high concentrations of diacetyl. We tested the effect of AWA ablation on calcium responses and found that ablation of AWA neurons increased the activity of ASH neurons. We found that AWA enhanced the calcium response (Fig. 30, A, C, and D). This indicates that it is involved in the inhibitory circuit of the H response.

[0130] Next, the present inventors investigated the Ca2+ response to high concentrations of diacetyl in ASH neurons of mutant nematodes. The Ca2+ response was monitored. Ca2+ responses were normally evoked in ASH neurons of odr-10 mutants. However, the response was significantly reduced in ASH neurons of sri-14 mutants (Fig. 29, B and C) The defective response to high diacetyl concentrations in ASH of sri-14 mutants was comparable to that of wild-type sri-14. ASH-specific expression of the gene rescued the ASH-mediated phenotype (Fig. 29, B and C). ASH-specific knockdown of sri-14 in C. elegans inhibits ASH response to high concentrations of diacetyl. These results suggest that SRI-14 reduces the Ca2+ response of ASH neurons. It has been shown that it functions as a key component for the reception of high concentrations of diacetyl in is doing.

[0131] Since sri-14 expression was observed in AWC neurons as well as ASH neurons (Fig. 24D ), we monitored the AWC response to high concentrations of diacetyl. The increase in concentration occurs due to odor removal. (32) Therefore, the inventors After the removal of the chill, the response of AWC neurons was examined, and a Ca2+ response occurred in AWC neurons. This result indicates that diacetyl is perceived by AWC and AWA. This is consistent with previous reports that AWC removal was significantly reduced at high diacetone concentrations. This suggests that the response of AWC neurons was not affected by the aversion from the chill (Fig. 27A). The behavioral responses to high concentrations of diacetyl were not significant for the avoidance of acetyl. Neither AWC-specific expression of sri-14 nor AWC-specific knockdown of sri-14 affected Therefore, SRI-14 mediates the response of AWC to high concentrations of diacetyl (Fig. 24, E and F). It is thought that other receptors exist.

[0132] AWB neurons are commonly associated with aversion behavior (15). Previous studies have shown that AWB neurons Ca2+ responses in neurons after removal of nonanone or high concentrations of isoamyl alcohol Since it has been reported that sri-14 cDNA is involved in the expression of sri-14 in AWB neurons (17, 33), we investigated its role in the expression of sri-14 in AWB neurons. We performed ectopic expression of β-diacetyl and monitored the Ca2+ response to various concentrations of diacetyl.

[0133] In wild-type AWB neurons, the inventors have demonstrated that either low or high concentrations of diacetyl We also observed a slight Ca2+ response after odor removal in wild-type AWB neurons. Ca2+ responses in AWB neurons to the removal of high concentrations of diacetyl compared with weak responses The ectopic expression of SRI-14 significantly enhanced the cytotoxicity of EGFR-1. This result indicates that SRI-14 did not alter the response to the removal of the chimpanzee (Fig. 32, A and B). This supports our conclusion that the ATPase activity contributes to the perception of high concentrations of diacetyl. These findings suggest that ODR-10 in AWA neurons and SRI-14 in ASH neurons are involved in the regulation of These receptors mediate attractive and aversion behaviors in response to low and high concentrations of diacetyl, respectively. This suggests that it works (Figure 33).

[0134] (3) Discussion The present inventors have used RNAi scripts to comprehensively identify olfactory receptors for specific odorants. C. elegans is a worm in which olfactory receptors and olfactory signaling are expressed in a similar way to mammals. (23, 34) and is therefore considered a model organism for olfactory analysis. Furthermore, we have developed a whole-nerve network that can trace the pathway along which olfactory signals are transmitted in neural circuits. However, most odorants are not specifically targeted to specific receptors or The correspondence between odorants and receptor oligomers is unknown, and the interaction between odorants and olfactory receptors is unclear. The mechanism by which odor signals are input is unknown. how olfactory signals are transmitted along neural circuits; how olfactory signals are transmitted along neural circuits; Understanding how a small number of ORNs in C. elegans can discriminate between so many odorants Further analysis of the receptor candidates obtained by the RNAi screen will reveal the specific odorant receptors. This will help identify olfactory receptors for these substances and provide an understanding of their mechanisms.

[0135] The present inventors have previously reported that different sensory neurons function depending on the odor concentration. In this study, the present inventors investigated the role of ODR-10 and SRI-10 in the reception of diacetyl. 14 function as receptors specific for low or high concentrations within specific ORNs, respectively. It was found that SRI-14 has a lower affinity for diacetyl than ODR-10 (Fig. 33). However, these receptors have homologous sequences within the odorant recognition site. does not possess the cytoplasmic receptors that underlie the ability of these receptors to distinguish between different concentrations of the same chemical. The mechanism by which this occurs remains unknown.

[0136] Calcium imaging experiments showed that AWA sensory neurons reacted to low and high concentrations of diacetyl Genetic ablation of AWA neurons showed that they respond to both high concentrations of diacetyl and These results caused defects in both repellency and attraction to low concentrations of diacetyl. The results showed that AWA neurons detect diacetyl over a wide range of concentrations and react to different concentrations. These findings, as well as those of the AWB Previously reported findings on the neurites (16) and AWC neurons (26) in C. elegans ans), indicating that these ORNs can mediate both attractive and repulsive behaviors. is present in AWA neurons (11), and odr-10 mutants show reduced attraction to low concentrations of diacetyl. However, aversion to high concentrations of diacetyl was normal, suggesting that AWA neurons It has been suggested that the plant has multiple diacetyl receptors, especially at high diacetyl concentrations. do.

[0137] The inventors' RNAi screening results showed that the activators of SRI-14 reacted with high concentrations of diacetyl. There are candidate receptors. Analysis of these other candidates will lead to the identification of other diacetyl receptors, odor receptors, and This may lead to additional strategies in which different receptors act depending on the concentration of [Table 3] JPEG0007770483000004.jpg204161

[0138] Table 3. Raw data from RNAi screening of srh olfactory receptor family genes. Primary (A), R for srh family genes in the second (B) and third (C) screening Chemotaxis index of NAi-treated nematodes to 11 odorants. The 11 odorants were: Low concentrations of six attractants [isoamyl alcohol (Iaa), benzaldehyde (Bz), pig Non (Bu), pentanedione (Pd), pyrazine (Pz), and trimethylthiazole (Tmt )], two repellents [nonanone (Nona) and octanol (Oct)], and a high concentration of 3 Three attractants [isoamyl alcohol (high Iaa), benzaldehyde (high Bz), and diamine cetyl (high Da)]. Blue, orange, and green shading represent the same-day control, respectively. The values, the average of the control values ​​for all days, and the statistical difference compared to both of these are shown (Materials and Methods). (See Law). [Table 4] JPEG0007770483000006.jpg22289JPEG0007770483000007.jpg22289JPEG0007770483 000008.jpg22389JPEG0007770483000009.jpg18289JPEG0007770483000010.jpg6389

[0139] Table 4. Raw data from the RNAi screen of 194 candidate olfactory receptor genes. After the screening, a total of 194 candidate olfactory receptor genes were obtained. and chemotaxis of nematodes treated with RNAi for these genes in a tertiary screen. Some genes have been shown to be involved in the detection of multiple odorants. It was. [Table 5]

[0140] Table 5. Expression patterns of 35 candidate receptor genes and associated odorants. Sensory neurons Only the neurons listed by name are included. Other neurons are listed as a group of several neurons. See Figure 23 for a photograph of the expression of the analyzed genes. The gene, when removed, conferred chemosensory responsiveness to diacetyl in neurons. Those that showed expression (based on reporter gene expression). Bu, butanone; Bz, benzyl alcohol; Da, diacetyl; Iaa, isoamyl alcohol; Nona, nonanone; Oct, octyl alcohol Pd, pentanedione; Pz, pyrazine; Tmt, trimethylthiazole. [Example]

[0141] The present inventors carried out the above-mentioned examples using C. elegans strains collected from various locations, and found that various cancers A strain was found that did not show any attraction to urine from patients. It was found that the genome contains many single base substitutions. Therefore, in this example, the accuracy of cancer detection was evaluated using the wild-type strain and the above-mentioned gene mutant strain. We considered the following.

[0142] method: Breast cancer, esophageal cancer, bile duct cancer, rectal cancer, appendicitis cancer, prostate cancer, pancreatic cancer, lung cancer, and gastrointestinal mesenchymal tumors Urine samples from cancer patients, healthy individuals, and healthy individuals who showed false positives in a medium-scale experiment. The chemotaxis of wild-type and gene mutant strains was examined for pull.

[0143] result: The gene mutant strains did not show any attraction to urine from almost all cancer types (Figure 34). The mutant strain showed normal tactile response to other odors, suggesting that its basic sense of smell is not functioning. Therefore, it can be said that the gene mutant strains lack the receptors that perceive the odor of cancer types. This is expected (Figure 35).

[0144] Furthermore, the mutant strain exhibited normal attraction behavior to the false-positive samples. The samples that showed positive results in the gene mutant strain were analyzed, and the results were negative in the gene mutant strain. If the test is positive for a gene mutation, it can be diagnosed as cancer, and if the test is positive for a gene mutation, it can be diagnosed as a false positive. Therefore, the use of gene mutants can improve the accuracy of cancer detection (Figure 35). [Example]

[0145] Identification of candidate receptors involved in odor perception in various cancer types Using a next-generation sequencer (Illumina), the entire gene mutant strain obtained in Example 7 was analyzed. The genome was decoded and compared with the genome sequence of N2 to search for mutated receptor genes.

[0146] As a result, it was found that the receptor gene contained a strong mutation (Table 6). [Table 6]

[0147] These genes were analyzed by olfactory neuron AWC-specific RNAi (Esposito et al., Efficient and ce ll specific knock-down of gene function in targeted C. elegans neurons. Gene 395 , 170-176, 2007), and the taxis of each cancer type to urine were measured. As a result, it was found that the receptors that reacted differed depending on the type of cancer. This makes it possible to identify the type of cancer by the nematode taxis test. The results of investigating the taxis of receptor knockdown strains to the urine of breast cancer patients are shown in Figure 36. .

[0148] Research on cancer detection dogs has predicted that the scent varies depending on the type of cancer. We identified the olfactory receptors for each type of cancer and developed mutants that lacked those receptors. The method for generating mutants is the CRISPR / Cas9 method (Friedland et al., Heritable genome Examples include gene editing in C. elegans via a CRISPR-Cas9 system (Nature Methods, 2013). It can be obtained.

[0149] First, in STEP 1, the presence or absence of cancer is examined using the N2 strain. Next, in STEP 2, the receptor variants of each cancer type are used to identify the cancer type. If the odorant receptor mutants of intestinal cancer do not show any attraction behavior, colon cancer can be diagnosed (Figure 37).

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[0155] 10: detection unit, 20: processing unit, 30: storage unit, 40: storage unit 110: Calculation means, 120: Database [Sequence List Free Text]

[0156] SEQ ID NO: 1: Synthetic DNA SEQ ID NO: 2: Synthetic DNA SEQ ID NO: 3: Synthetic DNA SEQ ID NO: 4: Synthetic DNA

Claims

1. A container for containing a urine sample and nematodes; A detection unit that detects the chemotaxis of the nematodes in the storage unit to the odor or the response of the AWA olfactory nerve or the AWC olfactory nerve, and is capable of capturing moving images of at least one nematode in real time; a processing unit that detects the presence or absence of cancer or identifies the type of cancer based on the information detected by the detection unit; A cancer detection system comprising:

2. The system described in claim 1, wherein the detection unit includes a microscope or camera that acquires at least one data selected from an image of the nematodes, a population number, and a movement trajectory.

3. The system described in claim 2, wherein the microscope or camera is equipped with a sensor capable of detecting fluorescence intensity.

4. A system described in any one of claims 1 to 3, comprising a cancer determination means for detecting the presence or absence of cancer or identifying the type of cancer based on measurement data of the nematode's response to odor.

5. A system described in any one of claims 1 to 4, wherein the detection unit detects the chemotaxis or the response of the AWA olfactory nerve or the AWC olfactory nerve using changes in calcium concentration as an indicator.

6. The system described in claim 5, wherein the change in calcium concentration is measured using a fusion protein as an indicator protein, which links the calcium-binding protein CaM to the target M13 to which CaM binds, and has CFP and YFP attached to both ends of the protein.

7. A system described in any one of claims 1 to 6, wherein the storage section contains a test sample and a control sample.

8. A system described in any one of claims 1 to 7, including a processing unit that detects the presence or absence of cancer based on information detected by the detection unit, or identifies the type of cancer.

Images