Method for detecting circulating tumor cells

Abstract

To provide means that detects circulating tumor cells including deficient APC protein.SOLUTION: A labeled antibody that binds to both wild-type APC protein and deficient APC protein, and a labeled antibody that binds to the wild-type APC protein but does not bind to the deficient APC protein, are used to perform immunostaining of circulating tumor cells, and a signal derived from the label is measured.SELECTED DRAWING: Figure 8

Description

Technical Field

[0001] The present invention relates to a method for detecting circulating tumor cells in blood (hereinafter also referred to as "CTC").

Background Art

[0002] In cancer cells, mutations such as nonsense mutations and frameshift mutations are often observed in genes encoding specific proteins. In transcripts from genes having such mutations, a stop codon exists at the position where protein synthesis is interrupted, or an amino acid sequence different from the original amino acid sequence may be encoded from the middle. In such cases, in cancer cells, a protein lacking a part of the original protein is expressed. For example, Non-Patent Document 1 describes that the colon cancer cell line DLD-1 expressing only the defective type of APC (adenomatous polyposis coli) protein was not detected by an immunostaining method using an antibody that binds to the C-terminal side of APC.

Prior Art Documents

Non-Patent Documents

[0003]

Non-Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0004] Defective proteins in cancer cells are thought to be related to tumor formation, recurrence, metastasis, etc. Also, among cancer cells, there are cells that detach from tumor tissues, infiltrate into blood vessels, and circulate in the blood. Such cancer cells are called CTCs and are considered to be one of the causes of cancer recurrence and metastasis. Since CTCs are also cancer cells, like the defective APC protein in colorectal cancer cells, CTCs may contain defective proteins characteristic of the cancer type from which they originate. Therefore, detecting CTCs having defective proteins is clinically useful. An object of the present invention is to provide a method for detecting CTCs containing defective APC proteins.

Means for Solving the Problems

[0005] The present invention provides the following inventions [1] to

[15] .

[0006] [1] A step of contacting CTCs in a sample with a first labeled antibody containing a first labeling substance and a second labeled antibody containing a second labeling substance different from the first labeling substance, and detecting CTCs containing a defective APC protein to which the first labeled antibody binds and the second labeled antibody does not bind, wherein the first labeled antibody contains an antibody capable of binding to wild-type APC protein and defective APC protein, and the second labeled antibody contains an antibody capable of binding to the wild-type APC protein and not binding to the defective APC protein. A method for detecting CTCs.

[0007] [2] The detection method according to [1] above, further comprising a step of detecting cells containing wild-type APC protein to which the first labeled antibody and the second labeled antibody bind.

[0008] [3] The detection step includes a step of detecting a first signal derived from the first labeling substance and a second signal derived from the second labeling substance, and a step of detecting blood-circulating cancer cells containing the defective APC protein based on the first signal and the second signal. The detection method according to [1] or [2] above.

[0009] [4] The step of detecting includes a step of detecting a first signal derived from the first labeling substance and a second signal derived from the second labeling substance, and a cell in which the value of the ratio of the second signal to the first signal is less than a threshold value, or a cell in which the value of the ratio of the first signal to the second signal is equal to or greater than the threshold value is detected as a blood-circulating cancer cell containing the defective APC protein. The detection method according to any one of [1] to [3] above.

[0010] [5] The step of detecting includes a step of acquiring an image of the cells in the sample and detecting a first signal derived from the first labeling substance and a second signal derived from the second labeling substance in the image of the cells, and a cell containing the first signal and substantially not containing the second signal is detected as a CTC containing the defective APC protein. The detection method according to any one of [1] to [4] above.

[0011] [6] The first labeling substance is a fluorescent substance, the second labeling substance is a fluorescent substance, the first signal is a fluorescent signal, the second signal is a fluorescent signal, and the image is a fluorescent image. The detection method according to [5] above.

[0012] [7] The defective APC protein is a protein lacking the C-terminal region of the wild-type APC protein. The detection method according to any one of [1] to [6] above.

[0013] [8] The first labeling substance and the second labeling substance are fluorescent substances having fluorescence emission maxima in different wavelength ranges. The detection method according to any one of [1] to [7] above.

[0014] [9] Further includes a step of labeling a mesenchymal marker protein of the CTC, and a step of detecting a cell to which the first labeling antibody binds, the mesenchymal marker protein is labeled, and the second labeling antibody does not bind as a mesenchymal CTC containing the defective APC protein. The detection method according to any one of [1] to [8] above.

[0015]

[10] The detection method according to [9] above, wherein the mesenchymal marker protein is at least 1 selected from the group consisting of vimentin, N-cadherin, OB-cadherin, fibronectin, integrin A5b1, Snail, Slug, ETS1, α-SMA, Twist, FAP, FSP-1, SIP1, Goosecoid, LEF-1, and FOXC2.

[0016]

[11] The detection method according to any one of [1] to

[10] above, further comprising a step of labeling the epithelial marker protein of the CTC, and a step of detecting, as epithelial cells containing the wild-type APC protein, cells to which the first labeled antibody and the second labeled antibody are bound and the epithelial marker protein is labeled.

[0017]

[12] The detection method according to

[11] above, wherein the epithelial marker protein is at least 1 selected from the group consisting of cytokeratin, EpCAM, E-cadherin, ZO-1, laminin-1, entactin, MUC1, desmoplakin, and α1 collagen.

[0018]

[13] The detection method according to any one of [1] to

[12] above, wherein the first labeled antibody binds to a site present in both the wild-type APC protein and the defective APC protein, and the second labeled antibody binds to a site present in the wild-type APC protein and not present in the defective APC protein.

[0019]

[14] A method for detecting CTCs containing a defective APC protein to which a first labeled antibody binds and a second labeled antibody does not bind, wherein the CTCs are prepared by contacting CTCs in a sample with the first labeled antibody and the second labeled antibody, the first labeled antibody comprises a first labeling substance and an antibody capable of binding to a wild-type APC protein and a defective APC protein, and the second labeled antibody comprises a second labeling substance different from the first labeling substance and an antibody capable of binding to the wild-type APC protein and not binding to the defective APC protein.

[0020]

[15] The detection method according to any one of [1] to

[14] above, wherein the detection of the CTCs is performed by a fluorescence microscope or a flow cytometer.

Advantages of the Invention

[0021] According to the present invention, CTCs containing a defective APC protein can be detected.

Brief Description of the Drawings

[0022]

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Mode for Carrying Out the Invention

[0023] In the method for detecting CTCs of the present embodiment (hereinafter, also referred to as "the detection method of the present embodiment"), first, CTCs in a sample are brought into contact with a first labeled antibody containing a first labeling substance and a second labeled antibody containing a second labeling substance different from the first labeling substance. The detection method of the present embodiment is performed in vitro.

[0024] Since CTCs generally exist in the blood, the sample is either blood (whole blood) collected from a subject or a preparation from such blood. The whole blood is, for example, peripheral blood. Known anticoagulants such as heparin, EDTA salts, and sodium citrate may be added to the whole blood as necessary. The whole blood may be diluted with an appropriate aqueous medium as necessary. Such an aqueous medium is not particularly limited as long as it does not interfere with the antigen-antibody reaction described below, and examples include water, physiological saline, and buffer solutions. The subject is not particularly limited, and examples include healthy individuals, cancer patients, and those suspected of having cancer. The type of cancer is not particularly limited. Since the defective APC protein is a marker for colorectal cancer, samples from colorectal cancer patients are particularly suitable for the detection method of this embodiment.

[0025] When the sample is a preparation from whole blood, the preparation is preferably a fraction prepared from whole blood obtained from a subject and capable of containing CTCs. As used herein, the term "fraction capable of containing CTCs" includes both fractions containing CTCs and fractions that may contain CTCs. In the art, it is known that even in blood collected from cancer patients, CTCs may not be present in the blood. Examples of fractions capable of containing CTCs include fractions obtained by removing red blood cells from whole blood, fractions obtained by removing red blood cells and / or white blood cells from whole blood, and fractions obtained by selectively recovering CTCs from whole blood. Removal of red blood cells can be performed, for example, by adding a hemolytic agent to whole blood to cause hemolysis. Removal of white blood cells can be performed, for example, by capturing white blood cells using a solid phase (such as magnetic particles, microfluidic channels, etc.) immobilized with an anti-CD45 antibody. Since most of the cells in whole blood are occupied by red blood cells and white blood cells, a fraction capable of containing CTCs can be obtained by removing red blood cells and white blood cells from whole blood. Selective recovery of CTCs from whole blood can be performed, for example, by using commercially available CTC enrichment and recovery devices such as the ClearCell® FX system, the On-Chip® Sort system, and commercially available separation and enrichment chips such as the CTChip® FR1S. According to these, a fraction capable of containing CTCs can be separated and recovered from whole blood. More preferably, a sample is obtained by separating whole blood into a fraction capable of containing CTCs and a fraction containing other cells and recovering the fraction capable of containing CTCs. The fraction capable of containing CTCs, separated and recovered from whole blood in such a manner, is particularly suitable as a sample in the detection method of the present embodiment. It is difficult to completely remove other cells such as white blood cells from the fraction capable of containing CTCs. The fraction capable of containing CTCs may contain a small amount of cells other than CTCs such as white blood cells.

[0026] The sample may contain fixed CTCs. The fixation treatment suppresses the degradation of proteins in CTCs. The fixation treatment can be performed by adding a fixation reagent such as paraformaldehyde (PFA), formaldehyde, etc. to the fraction that may contain CTCs. A commercially available cell fixative may also be used. The sample may contain membrane-permeabilized CTCs. By the membrane permeabilization treatment, the labeling antibody described later can pass through the cell membrane and enter the cell. The membrane permeabilization treatment can be performed by adding a membrane permeabilization reagent such as methanol, acetone, surfactant, etc. to the fraction that may contain CTCs. The fixation treatment and the membrane permeabilization treatment themselves are known in the art. Preferably, the sample contains fixed and membrane-permeabilized CTCs.

[0027] The APC protein is a protein consisting of 2,843 amino acid residues encoded by the APC gene isolated as the responsible gene for familial adenomatous polyposis. The amino acid sequence of the APC protein itself is known and is disclosed in known databases such as NCBI (National Center for Biotechnology Information). The APC protein is usually wild-type, but CTCs may contain a defective APC protein. As one of the functions of the wild-type APC protein, it is known to suppress Wnt signal transduction by binding to β-catenin. As a result, excessive cell proliferation is prevented. Therefore, the normal APC gene is considered a tumor suppressor gene. When a mutation occurs in the APC gene, a defective APC protein may be expressed. Mutations in the APC gene are frequently observed, especially in colorectal cancer. Defective APC proteins exist in various forms depending on the type and location of the gene mutation, and most of them cannot bind to β-catenin. Therefore, defective APC proteins cannot control Wnt signal transduction, and uncontrolled cell proliferation that leads to the development of cancer may occur.

[0028] As used herein, a "deficient APC protein" lacks the amino acid sequence of the C-terminal region of the wild-type APC protein or contains an amino acid sequence different from that of the wild-type APC protein in the C-terminal region. The C-terminal region may be a region of 30% or more and 60% or less of the total amino acid sequence of the wild-type APC protein, including the C-terminal amino acid residue. For example, the C-terminal region may be a region from the 1139th amino acid residue to the C-terminal (2843rd) amino acid residue of the wild-type APC protein. In this case, the C-terminal region is 59.9% of the total amino acid sequence of the wild-type APC protein. Alternatively, the C-terminal region may be a region from the 1991st amino acid residue to the C-terminal (2843rd) amino acid residue of the wild-type APC protein. In this case, the C-terminal region is 30% of the total amino acid sequence of the wild-type APC protein. Preferably, the C-terminal region is a region from the 1200th amino acid residue to the C-terminal (2843rd) amino acid residue of the wild-type APC protein. The positions of gene mutations leading to deletion of the C-terminal region of the APC protein are specifically described in Zhang Z. et al., ONCOLOGY LETTERS 19: 1781-1788, 2020, Zhang L. and Shay J.W., JNCI J Natl Cancer Inst (2017) 109(8): djw332, etc.

[0029] When the amino acid sequence of the C-terminal region is absent, the truncated APC protein does not have a C-terminal region. That is, the truncated APC protein lacks the C-terminal region compared to the wild-type APC protein. When the truncated APC protein contains an amino acid sequence different from that of the wild-type APC protein in the C-terminal region, the C-terminal region of the truncated APC protein has lost the functions carried out by the C-terminal region of the wild-type APC protein. Such functions include, for example, the ability to bind to β-catenin, the ability to bind to microtubules, the ability to bind to EB1 protein, and the like. Naturally, when the truncated APC protein does not have a C-terminal region, the truncated APC protein has also lost such functions. Mutations in the APC gene that can give rise to truncated APC proteins include nonsense mutations due to base substitutions, frameshift mutations due to base insertions or deletions, and the like. Specifically, a mutation in the APC gene may result in the occurrence of a stop codon, and only a part of the transcription product of the APC gene may be translated. Alternatively, a part of the APC gene may be deleted, and the gene itself may be in a state where a part is absent. In the truncated APC proteins generated in these cases, the amino acid sequence of the C-terminal region is absent. Also, when a frameshift mutation occurs in the APC gene, the base sequence downstream of the mutated site may change. In this case, the C-terminal region of the truncated APC protein generated has an amino acid sequence different from that of the wild-type APC protein.

[0030] CTCs in the sample can be at least any one of i) CTCs that contain truncated APC protein and do not contain wild-type APC protein, ii) CTCs that do not contain truncated APC protein and contain wild-type APC protein, and iii) CTCs that contain both truncated APC protein and wild-type APC protein. Preferably, the CTCs of i) and / or iii) are detected as "CTCs containing truncated APC protein". Also, the CTCs of ii) above are detected as "CTCs containing wild-type APC protein". When the sample contains at least one of the CTCs of i) and iii), CTCs containing truncated APC protein can be detected from the sample.

[0031] In the detection method of this embodiment, in order to detect CTCs containing defective APC protein, two labeled antibodies, a first labeled antibody containing a first labeling substance (hereinafter also simply referred to as "first labeled antibody") and a second labeled antibody containing a second labeling substance (hereinafter also simply referred to as "second labeled antibody"), are used. As used herein, a "labeled antibody" is a complex containing a detection antibody and a labeling substance. The detection antibody that binds to the APC protein is specifically an antibody capable of binding to both the wild-type APC protein and the defective APC protein described below, and an antibody capable of binding to the wild-type APC protein and not binding to the defective APC protein. The labeling substances for detecting the APC protein are the first labeling substance and the second labeling substance described below. The labeled antibody can be a detection antibody labeled with a labeling substance. Alternatively, the labeled antibody may be a complex composed of a detection antibody and a secondary antibody that specifically binds to the detection antibody and is labeled with a labeling substance.

[0032] Labeling of an antibody with a labeling substance is known in the art and can be appropriately selected according to the type of the labeling substance. For example, a suitable cross-linking agent can be used to bind the detection antibody and the labeling substance. When the labeling substance is a protein, the first labeled antibody and / or the second labeled antibody can be a fusion protein of the labeling substance and the detection antibody. A detection antibody modified with biotin and a labeling substance modified with avidin may be used. In this case, the labeling substance can be indirectly bound to the detection antibody through the specific binding between biotin and avidin. Biotin includes biotin, as well as biotin analogs such as desthiobiotin and oxybiotin. Avidin includes avidin, as well as avidin analogs such as streptavidin and tamavidin (registered trademark).

[0033] As used herein, the term "antibody" encompasses full-length antibodies and fragments thereof. The full-length antibody may be any of IgG, IgA, IgM, IgD, and IgE, but is preferably IgG. Examples of antibody fragments include Fab, Fab', F(ab')2, Fd, Fd', Fv, light chain, the heavy chain variable region (VHH) of heavy chain antibodies, reduced IgG (rIgG), single-chain antibodies (scFv), and the like. The antibody may be either a monoclonal antibody or a polyclonal antibody, with monoclonal antibodies being preferred. The antibody may be derived from any animal. Such animals are preferably mammals, including, for example, rabbits, mice, alpacas, camels, rats, pigs, sheep, goats, cows, horses, donkeys, and humans.

[0034] The first labeled antibody, as a detection antibody, includes an antibody capable of binding to both wild-type APC protein and defective APC protein. The detection antibody in the first labeled antibody may be an antibody that binds to a site present in both wild-type APC protein and defective APC protein. The second labeled antibody, as a detection antibody, includes an antibody capable of binding to wild-type APC protein and not binding to defective APC protein. The detection antibody in the second labeled antibody may be an antibody that binds to a site present in wild-type APC protein and not present in defective APC protein. That is, the first labeled antibody and the second labeled antibody recognize different epitopes. Preferably, the epitope of the first labeled antibody is present at a site other than the C-terminal region in wild-type APC protein, and the epitope of the second labeled antibody is present within the C-terminal region in wild-type APC protein.

[0035] The contact between CTCs in the sample, the first labeled antibody, and the second labeled antibody can be carried out, for example, by mixing a sample that may contain CTCs, a solution containing the first labeled antibody, and a solution containing the second labeled antibody. Alternatively, after immobilizing CTCs in the sample on a solid phase capable of immobilizing CTCs, a solution containing the first labeled antibody and a solution containing the second labeled antibody may be added to the solid phase. In the contacting step, a solution containing both the first labeled antibody and the second labeled antibody may be used. The order of contacting CTCs with the first labeled antibody and the second labeled antibody is not particularly limited, and they may be mixed simultaneously or sequentially.

[0036] When CTCs contain a defective APC protein and / or a wild-type APC protein, an antigen-antibody reaction occurs due to the contact between the CTCs, the first labeled antibody, and the second labeled antibody. The reaction is usually carried out in an aqueous medium. The aqueous medium is not particularly limited, and examples include water, physiological saline, phosphate-buffered saline (PBS), Tris-buffered saline (TBS), and Good's buffers. Examples of Good's buffers include MES, Bis-Tris, ADA, PIPES, Bis-Tris-Propane, ACES, MOPS, MOPSO, BES, TES, HEPES, HEPPS, Tricine, Tris, Bicine, and TAPS.

[0037] Due to the above antigen-antibody reaction, the APC protein contained in CTCs is labeled with the labeled antibody. With reference to FIGS. 1A to 1C, the labeling of the APC protein contained in CTCs will be described. FIG. 1A is a schematic diagram of a wild-type APC protein to which the first labeled antibody and the second labeled antibody are bound inside a cell. The wild-type APC protein has both the epitope of the first labeled antibody and the epitope of the second labeled antibody. Therefore, when CTCs containing the wild-type APC protein come into contact with the first labeled antibody and the second labeled antibody, both the first labeled antibody and the second labeled antibody bind to the wild-type APC protein. From cells containing the wild-type APC protein, both a signal derived from the first labeling substance possessed by the first labeled antibody and a signal derived from the second labeling substance possessed by the second labeled antibody can be obtained.

[0038] Figure 1B is a schematic diagram of a defective APC protein to which a first labeled antibody is bound inside a cell. In the defective APC protein of this example, the C-terminal region is deleted. As described above, this defective APC protein can be generated by deletion of the APC gene, generation of a stop codon due to mutation of the APC gene, or the like. In this defective APC protein, the epitope of the second labeled antibody is lost. Therefore, the first labeled antibody binds to the defective APC protein, while the second labeled antibody does not bind. From the cells containing the defective APC protein, a signal derived from the first labeling substance of the first labeled antibody can be obtained, but a signal derived from the second labeling substance of the second labeled antibody cannot be obtained.

[0039] Figure 1C is a schematic diagram of a defective APC protein to which a first labeled antibody is bound inside a cell. The defective APC protein of this example contains an amino acid sequence different from that of the wild-type APC protein in the C-terminal region. As described above, this defective APC protein can be generated by a frameshift mutation of the APC gene or the like. In this defective APC protein, the epitope of the second labeled antibody is lost. Therefore, the first labeled antibody binds to the defective APC protein, while the second labeled antibody does not bind. From the cells containing the defective APC protein, a signal derived from the first labeling substance of the first labeled antibody can be obtained, but a signal derived from the second labeling substance of the second labeled antibody cannot be obtained.

[0040] Thus, there are differences in the signals obtained between the defective APC protein and the wild-type APC protein. In the detection method of the present embodiment, based on this difference, it is possible to identify whether the APC protein contained in each cell is a wild-type APC protein or a defective APC protein. That is, in the detection method of the present embodiment, a cell containing an APC protein to which the first labeled antibody binds and the second labeled antibody does not bind can be detected as a cell containing a defective APC protein. Also, in the detection method of the present embodiment, a cell containing an APC protein to which the first labeled antibody and the second labeled antibody bind can be detected as a cell containing a wild-type APC protein. The defective APC protein is known as a marker for colorectal cancer. Therefore, a cell containing the defective APC protein in a sample that may contain CTCs can be determined to be a CTC.

[0041] The first labeled antibody contains a first labeling substance, and the second labeled antibody contains a second labeling substance. The second labeling substance is different from the first labeling substance. Specifically, the first labeling substance and the second labeling substance are substances that generate different signals from each other. The signal derived from the first labeling substance is called the "first signal", and the signal derived from the second labeling substance is called the "second signal". The labeling substance is preferably a substance that generates a signal by itself (hereinafter also referred to as a "signal generating substance"). The first labeling substance and the second labeling substance may be the same type of signal generating substance, but it is preferable that the first signal and the second signal are distinguishable and different. Examples of the signal generating substance include fluorescent substances.

[0042] The fluorescent substance is not particularly limited and can be appropriately selected from, for example, known fluorescent dyes and fluorescent proteins. Examples of the fluorescent dye include fluorescein isothiocyanate (FITC), rhodamine, coumarin, imidazole derivative, indole derivative, allophycocyanin, phycoerythrin (PE), PerCP / Cy5.5 (trademark), Alexa Fluor (registered trademark), Cy3 (registered trademark), Cy5 (registered trademark), Cy5.5 (registered trademark), Cy7 (registered trademark), DyLight (registered trademark) Fluor, and the like. Examples of the fluorescent protein include green fluorescent protein, yellow fluorescent protein, blue fluorescent protein, red fluorescent protein, and the like. When both the first labeling substance and the second labeling substance are fluorescent substances, it is preferable that the first labeling substance and the second labeling substance are fluorescent substances having fluorescence emission maxima in different wavelength ranges from each other. That is, it is preferable that the wavelength of the fluorescence emission maximum of the first labeling substance is different from the wavelength of the fluorescence emission maximum of the second labeling substance. Thereby, each of the first signal and the second signal can be measured.

[0043] In a preferred embodiment, the detection of CTCs containing a defective APC protein is performed by detecting a first signal and a second signal generated from individual cells. Referring to FIGS. 1B and 1C, no second signal is detected from the defective APC protein. Therefore, cells with a second signal below the threshold can be determined as CTCs containing a defective APC protein. Further, cells with a second signal higher than the threshold may be determined as cells containing a wild-type APC protein. In a further embodiment, a value of the ratio of the first signal to the second signal is obtained. Based on the value of the ratio, CTCs containing a defective APC protein may be detected. For example, cells in which the value of the ratio of the second signal to the first signal (second signal / first signal) is less than the threshold, or the value of the ratio of the first signal to the second signal (first signal / second signal) is greater than or equal to the threshold can be detected as CTCs containing a defective APC protein. Further, cells in which the value of the ratio of the second signal to the first signal (second signal / first signal) is greater than or equal to the threshold, or the value of the ratio of the first signal to the second signal (first signal / second signal) is less than the threshold may be determined as cells containing a wild-type APC protein. The threshold for each signal can be set independently with reference to Example 2 described below.

[0044] When the labeling substance is a fluorescent substance, the fluorescence generated from the fluorescent substance can be measured using devices such as a fluorescence microscope, FCM, and IFCM. In this specification, FCM is a device that irradiates light onto individual particles (e.g., cells) in a liquid flowing in a flow cell and acquires optical information from each particle. IFCM is an FCM equipped with an imaging unit such as a CCD camera. IFCM can acquire images of individual cells in a liquid flowing in a flow cell. For example, in a short time of several seconds to several minutes, optical information, fluorescence images, and bright-field images (also referred to as transmitted light images) can be acquired from each of several to several million cells, and quantitative measurement can be performed. Also, information on individual cells can be extracted by image processing.

[0045] The information obtained by measurement with FCM is optical information of individual particles. Examples of the optical information include fluorescence signal information. As the fluorescence signal information, for example, fluorescence intensity based on the waveform of the fluorescence signal for each cell can be obtained. In the present specification, the fluorescence intensity obtained by measurement with FCM can be the peak value, width, area, etc. of the waveform of the fluorescence signal. Here, the "area" of the waveform of the fluorescence signal is calculated by integrating the waveform of the fluorescence signal. In the detection of CTCs containing the defective APC protein, the fluorescence intensity obtained by measurement with FCM can be used as the first signal and the second signal. FCM is not particularly limited, and a commercially available device may be used. Examples of the commercially available device include FACSVerse (trademark) (manufactured by Becton Dickinson Japan).

[0046] The detection of CTCs containing the defective APC protein may be performed based on an image of each cell in the sample. The image of the cell may be a fluorescence image of the cell. The means for obtaining the fluorescence image of the cell is not particularly limited. When the labeling substance is a fluorescent substance, the fluorescence image of the cell can be obtained by devices such as the above-described fluorescence microscope and IFCM capable of obtaining a fluorescence image.

[0047] The fluorescence microscope and IFCM capable of obtaining a fluorescence image are not particularly limited, and a commercially available device may be used. The light source is not particularly limited, and a light source having a wavelength suitable for exciting the fluorescent dye can be appropriately selected. As the light source, for example, a blue semiconductor laser, a red semiconductor laser, an argon laser, a He-Ne laser, a mercury arc lamp, etc. are used. Also, the bright-field light source such as IFCM is not particularly limited, and for example, a white laser light source, an LED lamp, a mercury lamp, a xenon lamp, etc. are used. Examples of the commercially available system include ImageStream MK II (manufactured by Cytek Japan), DeNovo (manufactured by bioview), Metafer (manufactured by Metasystems), MI-1000 (manufactured by Sysmex Corporation), etc.

[0048] When using a fluorescence microscope or an IFCM capable of acquiring fluorescence images, based on the acquired fluorescence image and transmitted light image, for example, parameters including a fluorescence signal area value, total fluorescence signal intensity (hereinafter, the total fluorescence signal intensity acquired by an IFCM or a fluorescence microscope is also simply referred to as "fluorescence intensity"), cell size, aspect ratio value, etc. can be obtained. In the detection of CTCs containing a defective APC protein, it is preferable to use, as the first signal and the second signal, at least the fluorescence intensity obtained by measurement with an IFCM. The fluorescence intensity obtained by measurement with an IFCM is the integrated value of the pixel values of each pixel constituting the area showing the fluorescence signal in the fluorescence image of an individual cell. Particles with a total fluorescence signal intensity smaller than a predetermined threshold can be determined as cells to which the labeled antibody is not bound.

[0049] The first signal can be the fluorescence intensity derived from the first labeling substance obtained by measurement with an IFCM. The second signal can be the fluorescence intensity derived from the second labeling substance obtained by measurement with an IFCM. In this case, as the value of the ratio between the first signal and the second signal, the value of the ratio of their fluorescence intensities may be obtained. As the value of the ratio between the first signal and the second signal, for example, first signal / second signal, second signal / first signal, etc. are obtained. Based on this ratio value, it may be possible to distinguish whether the APC protein is a wild-type APC protein or a defective APC protein. For example, if the second signal / first signal is less than the threshold value, the cells (CTCs) in the sample can be determined to contain a defective APC protein. Also, if the second signal / first signal is greater than or equal to the threshold value, the cells in the sample can be determined to contain a wild-type APC protein. The threshold value may be, for example, 0.1, 0.2, 0.25, 0.3, 0.5, 0.75, 1.0, 1.5, 2.0, or 3.0. The value of the ratio between the first signal and the second signal may be used as an index by itself. Alternatively, the value of the ratio between the first signal and the second signal may be combined with another index or measured value.

[0050] The detection method of this embodiment may further include a step of labeling a mesenchymal marker protein and / or an epithelial marker protein, and a step of detecting CTCs labeled with the mesenchymal marker protein and / or the epithelial marker protein. It is known that CTCs include epithelial CTCs (Epithelial CTC), mesenchymal CTCs (Mesenchymal CTC), and intermediate CTCs (Intermediate CTC) in the process of transformation. It has been pointed out that cancer cells may undergo epithelial-mesenchymal transition (EMT) and change from epithelial properties to mesenchymal properties. Cancer cells that have undergone EMT are known to acquire migratory and invasive properties and circulate in the blood. Therefore, it is considered that mesenchymal cancer cells are more likely to metastasize and have a higher malignancy than epithelial cancer cells. In the detection method of this embodiment including the above steps, by labeling the mesenchymal marker protein and / or the epithelial marker protein, it is possible to determine whether the cells in the sample are mesenchymal, epithelial, or intermediate. The cells to be determined are not limited to CTCs, and all cells contained in the sample can be determined.

[0051] Examples of mesenchymal marker proteins include vimentin, N-cadherin, OB-cadherin, fibronectin, integrin A5b1, snail, slug, ETS1, α-SMA, twist, FAP, FSP-1, SIP1, goosecoid, LEF-1, FOXC2, etc. Examples of epithelial marker proteins include cytokeratin, EpCAM, E-cadherin, ZO-1, laminin-1, entactin, MUC1, desmoplakin, α1 collagen, etc.

[0052] The labeling of mesenchymal marker proteins and epithelial marker proteins can be carried out by known labeling methods. The labeling of mesenchymal marker proteins can be performed, for example, by contacting a labeled antibody capable of binding to the mesenchymal marker protein with the cells in the sample. Similarly, the labeling of epithelial marker proteins can be performed by contacting a labeled antibody capable of binding to the epithelial marker protein with the cells in the sample. Hereinafter, the labeled antibody capable of binding to the mesenchymal marker protein is also referred to as the "third labeled antibody", and the labeled antibody capable of binding to the epithelial marker protein is also referred to as the "fourth labeled antibody". Also, the labeling substance contained in the third labeled antibody is also referred to as the "third labeling substance", and the labeling substance contained in the fourth labeled antibody is also referred to as the "fourth labeling substance". The third labeled antibody includes, as a detection antibody, an antibody capable of binding to the mesenchymal marker protein. The fourth labeled antibody includes, as a detection antibody, an antibody capable of binding to the epithelial marker protein.

[0053] The third labeling substance and the fourth labeling substance only need to be signal generating substances, and can be, for example, fluorescent substances. Hereinafter, the signal derived from the third labeling substance is referred to as the "third signal", and the signal derived from the fourth labeling substance is referred to as the "fourth signal". When labeling the mesenchymal marker protein, the first labeling substance, the second labeling substance, and the third labeling substance may be signal generating substances of the same type, but it is preferable that the first signal, the second signal, and the third signal are distinguishable and different from each other. When labeling the epithelial marker protein, the first labeling substance, the second labeling substance, and the fourth labeling substance may be signal generating substances of the same type, but it is preferable that the first signal, the second signal, and the fourth signal are distinguishable and different from each other. When labeling both the mesenchymal marker protein and the epithelial marker protein, the first labeling substance, the second labeling substance, the third labeling substance, and the fourth labeling substance may be signal generating substances of the same type, but it is preferable that the first signal, the second signal, the third signal, and the fourth signal are distinguishable and different from each other.

[0054] When labeling a mesenchymal marker protein and the first labeling substance, the second labeling substance, and the third labeling substance are fluorescent substances, it is preferable that the wavelength of the fluorescence emission maximum of the first labeling substance, the wavelength of the fluorescence emission maximum of the second labeling substance, and the wavelength of the fluorescence emission maximum of the third labeling substance are different from each other. When labeling an epithelial marker protein and the first labeling substance, the second labeling substance, and the fourth labeling substance are fluorescent substances, it is preferable that the wavelength of the fluorescence emission maximum of the first labeling substance, the wavelength of the fluorescence emission maximum of the second labeling substance, and the wavelength of the fluorescence emission maximum of the fourth labeling substance are different from each other. When labeling both a mesenchymal marker protein and an epithelial marker protein and the first labeling substance, the second labeling substance, the third labeling substance, and the fourth labeling substance are fluorescent substances, it is preferable that the wavelength of the fluorescence emission maximum of the first labeling substance, the wavelength of the fluorescence emission maximum of the second labeling substance, the wavelength of the fluorescence emission maximum of the third labeling substance, and the wavelength of the fluorescence emission maximum of the fourth labeling substance are different from each other.

[0055] The detection of the third signal and / or the fourth signal itself can be carried out in the same manner as the first signal and the second signal. When the third labeling substance and / or the fourth labeling substance is a fluorescent substance, the third signal and / or the fourth signal can be detected by FCM, IFCM, or a fluorescence microscope. In a preferred embodiment, the determination of whether the cells in the sample are mesenchymal, epithelial, or intermediate is carried out by detecting the third signal and / or the fourth signal generated from individual cells. For example, when labeling a mesenchymal marker protein, cells with a third signal equal to or higher than the threshold can be determined as mesenchymal cells. When labeling an epithelial marker protein, cells with a fourth signal equal to or higher than the threshold can be determined as epithelial cells. When labeling both a mesenchymal marker protein and an epithelial marker protein, cells with a third signal equal to or higher than the threshold and a fourth signal lower than the threshold can be determined as mesenchymal cells. Also, cells with a third signal lower than the threshold and a fourth signal equal to or higher than the threshold can be determined as epithelial cells. Furthermore, cells with a third signal equal to or higher than the threshold and a fourth signal equal to or higher than the threshold can be determined as intermediate cells. The threshold for each signal can be set independently with reference to Example 3 described below.

[0056] Mesenchymal marker proteins and epithelial marker proteins can also be expressed in CTCs. Therefore, for one cell, by combining the detection result of APC protein by the first labeled antibody and the second labeled antibody with the detection result of the marker protein by the third labeled antibody and / or the fourth labeled antibody, it is possible to determine whether the one cell is a CTC of mesenchymal, epithelial, or intermediate type. For example, a cell with a second signal below the threshold and a third signal above the threshold can be determined as a mesenchymal CTC containing a defective APC protein. Alternatively, a cell with a second signal higher than the threshold and a third signal above the threshold may be determined as a mesenchymal cell (or CTC) containing a wild-type APC protein. Also, a cell with a second signal below the threshold and a fourth signal above the threshold can be determined as an epithelial CTC containing a defective APC protein. Alternatively, a cell with a second signal higher than the threshold and a fourth signal above the threshold may be determined as an epithelial cell (or CTC) containing a wild-type APC protein. Further, a cell with a second signal below the threshold, a third signal above the threshold, and a fourth signal above the threshold can be determined as an intermediate CTC containing a defective APC protein. Alternatively, a cell with a second signal higher than the threshold, a third signal above the threshold, and a fourth signal above the threshold may be determined as an intermediate cell (or CTC) containing a wild-type APC protein.

[0057] In a further embodiment, a cell in which the value of the ratio of the second signal to the first signal (second signal / first signal) is less than the threshold or the value of the ratio of the first signal to the second signal (first signal / second signal) is greater than or equal to the threshold and the third signal is greater than or equal to the threshold can be determined as a mesenchymal CTC containing a defective APC protein. Alternatively, a cell in which the second signal / first signal is greater than or equal to the threshold or the first signal / second signal is less than the threshold and the third signal is greater than or equal to the threshold may be determined as a mesenchymal cell (or CTC) containing a wild-type APC protein.

[0058] In a further embodiment, cells in which the second signal / first signal is less than a threshold value, or the first signal / second signal is greater than or equal to the threshold value and the fourth signal is greater than or equal to the threshold value can be determined as epithelial CTCs containing a defective APC protein. Alternatively, cells in which the second signal / first signal is greater than or equal to the threshold value, or the first signal / second signal is less than the threshold value and the fourth signal is greater than or equal to the threshold value can be determined as epithelial cells (or CTCs) containing a wild-type APC protein.

[0059] In a further embodiment, cells in which the second signal / first signal is less than a threshold value, or the first signal / second signal is greater than or equal to the threshold value, the third signal is greater than or equal to the threshold value and the fourth signal is greater than or equal to the threshold value can be determined as intermediate CTCs containing a defective APC protein. Alternatively, cells in which the second signal / first signal is greater than or equal to the threshold value, or the first signal / second signal is less than the threshold value, the third signal is greater than or equal to the threshold value and the fourth signal is greater than or equal to the threshold value can be determined as intermediate cells (or CTCs) containing a wild-type APC protein.

[0060] A further embodiment of the present invention is a method for detecting CTCs containing a defective APC protein to which a first labeled antibody binds and a second labeled antibody does not bind. CTCs containing a defective APC protein to which a first labeled antibody binds and a second labeled antibody does not bind are prepared by contacting CTCs in a sample with a first labeled antibody and a second labeled antibody. The first labeled antibody includes a first labeling substance and an antibody capable of binding to a wild-type APC protein and a defective APC protein. The second labeled antibody includes a second labeling substance different from the first labeling substance and an antibody capable of binding to a wild-type APC protein and not binding to a defective APC protein. By measuring the label of the CTCs prepared by contacting the first labeled antibody and the second labeled antibody and detecting a defective APC protein to which the first labeled antibody binds and the second labeled antibody does not bind, it can be found that the sample contains CTCs containing a defective APC protein. The method for detecting the APC protein of CTCs, the first labeled antibody, the labeling substance, CTCs, and the APC protein are as described above.

[0061] In the above detection method, CTCs in the sample may be contacted with the third labeled antibody and / or the fourth labeled antibody. The mesenchymal marker protein, epithelial marker protein, and each labeled antibody are as described above. These antibodies are preferably labeled with a labeling substance. The labeling substance is as described above. By confirming whether an antibody that labels a mesenchymal marker protein or an epithelial marker protein is bound, it is possible to determine whether CTCs are mesenchymal or epithelial.

[0062] In a sample collected from a subject, if the amount of CTCs containing the mutant APC protein is equal to or more than a predetermined amount, the subject or the sample may be determined to be "mutant APC protein positive". In a sample collected from a subject, if the amount of CTCs containing the mutant APC protein is less than the predetermined amount or no CTCs containing the mutant APC protein are detected, the subject or the sample may be determined to be "mutant APC protein negative". In a preferred embodiment, in a sample collected from a subject, if the amount of CTCs containing the mutant APC protein is less than the predetermined amount or no CTCs containing the mutant APC protein are detected, and if the amount of CTCs containing the wild-type APC protein in the sample is equal to or more than the predetermined amount, the subject or the sample may be determined to be "mutant APC protein negative".

[0063] The "predetermined amount", which is a threshold for determining mutant APC protein positive, can be set as appropriate. For example, detection is performed in advance using a plurality of specimens that have been determined to be mutant APC protein positive and specimens that have been determined to be mutant APC protein negative by, for example, nucleotide sequence analysis, etc., and a value that can appropriately classify mutant APC protein positive and negative using indices such as positive predictive value, negative predictive value, sensitivity, and specificity is set as the above "predetermined amount". Detection is performed on a specimen whose mutant APC protein is unknown whether it is negative or positive, and by applying the predetermined amount, it is possible to determine whether the specimen is mutant APC protein negative or positive.

[0064] In a sample collected from a subject, it is also conceivable that CTCs containing a defective APC protein are contained in a predetermined amount or more, and CTCs containing a wild-type APC protein are contained in a predetermined amount or more. In this case, the genotype of the APC gene of the subject may be determined to be a heterozygote of the defective type and the wild-type.

[0065] The reagents used in the above detection method can be provided to the user in the form of a reagent kit. The reagent kit contains a first labeled antibody and a second labeled antibody. The first labeled antibody and the second labeled antibody are as described above.

[0066] The reagent kit is composed of a container containing each reagent and a box containing the container. An attached document may be included in the box. The attached document may describe the composition of the reagent kit, the composition of each reagent, the usage method, etc. An example of the reagent kit of this embodiment is shown in FIG. 2A. In FIG. 2A, 10 indicates the reagent kit, 11 indicates the container containing the reagent containing the first labeled antibody and the second labeled antibody, 12 indicates the packing box, and 13 indicates the attached document. In this example, the first labeled antibody and the second labeled antibody are contained in the same container as one reagent.

[0067] Another example of the reagent kit is shown in FIG. 2B. In FIG. 2B, 20 indicates the reagent kit, 21 indicates the first container containing the reagent containing the first labeled antibody, 22 indicates the second container containing the reagent containing the second labeled antibody, 23 indicates the packing box, and 24 indicates the attached document. In this example, the first labeled antibody and the second labeled antibody are contained in separate containers.

[0068] The reagent kit may further include a reagent containing a third labeled antibody that labels a mesenchymal marker protein or a fourth labeled antibody that labels an epithelial marker protein. An example of the reagent kit is shown in FIG. 2C. In FIG. 2C, 30 indicates the reagent kit, 31 indicates a first container containing a reagent containing a first labeled antibody, 32 indicates a second container containing a reagent containing a second labeled antibody, 33 indicates a third container containing a reagent containing a third labeled antibody or a fourth labeled antibody, 34 indicates a packing box, and 35 indicates an attached document.

[0069] The reagent kit may further include a reagent containing a third labeled antibody and a reagent containing a fourth labeled antibody. An example of the reagent kit is shown in FIG. 2D. In FIG. 2D, 40 indicates the reagent kit, 41 indicates a first container containing a reagent containing a first labeled antibody, 42 indicates a second container containing a reagent containing a second labeled antibody, 43 indicates a third container containing a reagent containing a third labeled antibody, 44 indicates a fourth container containing a reagent containing a fourth labeled antibody, 45 indicates a packing box, and 46 indicates an attached document.

[0070] Hereinafter, the present invention will be described in detail with reference to examples, but the present invention is not limited to these examples.

Example

[0071] Example 1: Detection of Cultured Cells Expressing Defective APC Protein To establish a measurement system for detecting CTCs expressing defective APC protein, first, a measurement system capable of detecting cultured cells expressing defective APC protein was examined.

[0072] (1) Cells and Antibodies As cell lines expressing the APC protein, the lung cancer-derived cell line A549, the colorectal cancer-derived cell lines HCT116 and DLD1 were purchased from ATCC. A549 cells and HCT116 cells were known to express the wild-type (i.e., full-length) APC protein. DLD1 cells were known to express a truncated APC protein lacking the amino acid residues from the 1427th amino acid residue of wild-type APC to the C-terminal amino acid residue. In the Western blot described below, the anti-APC-N antibody (Santacruz: sc-53165 AF488) was used as an antibody recognizing the N-terminal side of the wild-type APC protein. In the immunostaining described below, the Alexa 488-labeled anti-APC-N antibody (Santacruz: sc-53165 AF488) was used as a labeled antibody recognizing the N-terminal side of the wild-type APC protein. Also, as a labeled antibody recognizing the C-terminal side of the wild-type APC protein, a combination of the unlabeled anti-APC-C antibody (Millipore: MAB3786) and the Alexa 647-labeled secondary antibody recognizing the unlabeled anti-APC-C antibody (abcam: ab150115) was used. The site recognized by the anti-APC-C antibody was not present in the truncated APC protein expressed by DLD1 cells. That is, it was predicted that the anti-APC-C antibody would not bind to the truncated APC protein expressed by DLD1 cells.

[0073] (2) Confirmation of APC protein expression in each cell line Each of the A549 cells, HCT116 cells, and DLD1 cells was lysed with a solubilizer containing a surfactant. The lysed cells were centrifuged to obtain the supernatant. A commercially available sample buffer was added to the supernatant, and the resulting mixture was boiled for 5 minutes. The heat-denatured mixture was separated by SDS-PAGE by a conventional method, and the proteins in the gel were transferred to a nitrocellulose membrane. This membrane was blocked with a blocking buffer (Nacalai Tesque: 03953-95), and Western blotting was performed using an anti-APC-N antibody and an HRP-labeled anti-mouse IgG antibody (MLB Life Science: 330). The results are shown in Figure 3. As can be seen from Figure 3, a band derived from the anti-APC-N antibody was confirmed in each lane. The APC protein detected in DLD1 cells had a smaller molecular weight compared to the APC protein detected in A549 cells and HCT116 cells. This result was consistent with the report that A549 cells and HCT116 cells express wild-type APC protein and DLD1 cells express a defective APC protein.

[0074] (3) Cell culture and immunostaining The above cells were cultured by a conventional method. The cells were collected, washed with PBS, and then fixed by adding a 4% paraformaldehyde (PFA) solution. The fixed cells were washed with PBS containing 0.2% Pluronic F-127. The fixed cells were subjected to membrane permeabilization treatment with cooled methanol at a final concentration of 90%. The cells subjected to membrane permeabilization treatment were washed with PBS containing 0.2% Pluronic F-127. Immunostaining was performed on the fixed and membrane-permeabilized cells using three kinds of antibodies. The procedure for immunostaining was as follows.

[0075] First, 5% BSA-containing PBS containing an unlabeled anti-APC-C antibody was added and incubated at room temperature for 1 hour. After incubation, the cells were washed twice with PBS containing 0.2% Pluronic F-127. Next, 5% BSA-containing PBS containing an Alexa 647-labeled secondary antibody (abcam: ab150115) that recognizes the unlabeled anti-APC-C antibody was added and further incubated at room temperature for 30 minutes. After incubation, the cells were washed twice with PBS containing 0.2% Pluronic F-127. Then, 5% BSA-containing PBS containing an Alexa 488-labeled anti-APC-N antibody was added to the washed cells and incubated at room temperature for 1 hour. After incubation, the cells were washed twice with PBS containing 0.2% Pluronic F-127. Thereby, a sample containing immunostained cells was obtained. In the following experiments, the unlabeled anti-APC-C antibody bound to the Alexa 647-labeled secondary antibody that recognizes the unlabeled anti-APC-C antibody is referred to as the "Alexa 647-labeled anti-APC-C antibody".

[0076] (4) Measurement by IFCM A sample containing immunostained cells was measured by ImageStream MK II (manufactured by Cytek Japan), which is IFCM. By the measurement, bright-field images and fluorescence images were acquired for each individual particle in the sample. The acquired image data was analyzed with IDEAS Application v6.2 (manufactured by MERCK MILLIPORE), which is software for image analysis. Images of 5000 to 10000 particles were acquired from the images obtained by ImageStream MK II. The particles in the acquired images were set as the analysis targets. The fluorescence images of the analysis target particles were analyzed with the Intensity feature of IDEAS, and the total fluorescence signal intensities (fluorescence intensities) of Alexa 488 and Alexa 647 were acquired.

[0077] (5) Detection of APC protein An example of the images obtained for each of A549 cells, HCT116 cells, and DLD1 cells is shown in Fig. 4. In Fig. 4, "APC-N" shows the fluorescence image derived from Alexa 488-labeled anti-APC-N antibody, and "APC-C" shows the fluorescence image derived from Alexa 647-labeled anti-APC-C antibody. As can be seen from Fig. 4, in the fluorescence images of A549 cells and HCT116 cells, fluorescence derived from each labeled antibody was detected within the cell contours confirmed in the bright-field image. On the other hand, in the fluorescence image of DLD1 cells, fluorescence derived from Alexa 488-labeled anti-APC-N antibody was detected within the cell contours confirmed in the bright-field image, but fluorescence derived from Alexa 647-labeled anti-APC-C antibody was not detected. This suggested that the anti-APC-C antibody could not bind to the APC protein expressed in DLD1 cells. From this result and the result of the Western blot in (2) above, it was shown that cells expressing wild-type APC protein and cells expressing defective APC protein could be detected by measuring cells immunostained with labeled anti-APC-N antibody and anti-APC-C antibody by IFCM.

[0078] To analyze the tendency of APC protein expression in the whole cells contained in the sample, for each individual cell in the sample, the value of the ratio of the fluorescence intensity derived from Alexa 647-labeled anti-APC-C antibody to the fluorescence intensity derived from Alexa 488-labeled anti-APC-N antibody (also referred to as the "APC-C / APC-N ratio value") was calculated. The APC-C / APC-N ratio values were compared among the samples containing A549 cells, HCT116 cells, and DLD1 cells, respectively. The results are shown in Fig. 5. The dots in the figure indicate the APC-C / APC-N ratio values of individual cells. As can be seen from Fig. 5, a large difference was observed in the APC-C / APC-N ratio values between the samples containing A549 cells and HCT116 cells and the sample containing DLD1 cells. From this result, it was shown that cells expressing wild-type APC protein and cells expressing defective APC protein could be distinguished based on the fluorescence intensities derived from each of the fluorescently labeled anti-APC-N antibody and anti-APC-C antibody.

[0079] (6) Measurement by FCM HCT116 cells and DLD1 cells were immunostained following the same procedure as (3) above. A sample containing the immunostained cells was measured by FACSVerse (manufactured by BD Japan), which is a flow cytometer. The measurement data were analyzed by a conventional method to obtain the intensity of the fluorescence signal (fluorescence intensity) generated from each of Alexa 488 and Alexa 647. A scattergram was created with the fluorescence intensity derived from the Alexa 647-labeled anti-APC-C antibody on the vertical axis and the fluorescence intensity derived from the Alexa 488-labeled anti-APC-N antibody on the horizontal axis.

[0080] The scattergram of immunostained DLD1 cells is shown in Fig. 6A, and the scattergram of immunostained HCT116 cells is shown in Fig. 6B. In Fig. 6, "APC-N (Area)" indicates the fluorescence intensity (area) derived from the Alexa 488-labeled anti-APC-N antibody, and "APC-C (Area)" indicates the fluorescence intensity (area) derived from the Alexa 647-labeled anti-APC-C antibody. By comparing the scattergrams of Figs. 6A and B, thresholds were set for the fluorescence intensities derived from the Alexa 647-labeled anti-APC-C antibody and the Alexa 488-labeled anti-APC-N antibody, respectively. In each scattergram, the vertical line represents the threshold of the Alexa 488-labeled anti-APC-N antibody, and the horizontal line represents the threshold of the Alexa 647-labeled anti-APC-C antibody. As can be seen from Fig. 6A, in the scattergram of DLD1 cells, the fluorescence intensity derived from the Alexa 488-labeled anti-APC-N antibody was strongly detected, but mainly weakly fluorescent cells derived from the Alexa 647-labeled anti-APC-C antibody were detected (lower right of Fig. 6A. 86.2% of the total). On the other hand, referring to Fig. 6B, in the scattergram of HCT116 cells, mainly cells in which the fluorescence intensity derived from the Alexa 647-labeled anti-APC-C antibody and the fluorescence intensity derived from the Alexa 488-labeled anti-APC-N antibody both exceeded the threshold were detected (upper right of Fig. 6B. 87.6% of the total). Thus, the scattergram of immunostained DLD1 cells and the scattergram of immunostained HCT116 cells showed a large difference in cell distribution. From this result, it was shown that even using FCM, cells expressing wild-type APC protein and cells expressing defective APC protein could be distinguished based on the fluorescence intensities derived from the Alexa 647-labeled anti-APC-C antibody and the Alexa 488-labeled anti-APC-N antibody. In addition, in the determination based on the measurement results of FCM, the sensitivity was 89% and the specificity was 88%.

[0081] Example 2: Detection of primary cultured cells expressing defective protein Primary cultured cells prepared from tumor tissues were subjected to the same immunostaining and IFCM measurement as in Example 1 to examine whether cells containing APC protein could be detected.

[0082] (1) Cells and antibodies Tumor tissues were collected from each of 13 colorectal cancer patients (see Fig. 7). Primary cultured cells (also referred to as isolated tumor-derived Cancer Cells: "iCCs") were prepared from the tumor tissues of each patient by a conventional method. Genomic DNA was extracted from each iCCs and subjected to nucleotide sequence analysis to confirm whether there was a mutation causing a C-terminal deletion of the APC protein in the APC gene. By nucleotide sequence analysis, a stop codon was detected within the gene due to a mutation in the APC gene in Donor 1, Donor 2, Donor 6, Donor 7, Donor 11, Donor 12, and Donor 14. A frameshift was detected due to a mutation in the APC gene in Donor 3. In Donor 4, both the wild-type nucleotide sequence and a stop codon were detected. In Donor 8, both the wild-type nucleotide sequence and a frameshift were detected. The genotypes of the APC genes of Donor 4 and Donor 8 were considered to be heterozygotes of the defective type and the wild-type. In Donor 10, both a stop codon and a frameshift were detected. In Donor 13 and 15, the wild-type nucleotide sequence was detected. In the immunostaining described below, the same labeled antibody as in Example 1 was used.

[0083] (2) Immunostaining, IFCM measurement, and detection of APC protein In the same manner as in Example 1, iCCs of each patient were immunostained to prepare samples. In the immunostaining, in addition to the above antibodies, nuclei were stained with Hoechst (registered trademark) 33342. In the same manner as in Example 1, samples containing immunostained iCCs were measured by IFCM. For samples containing iCCs of each patient, the value of the APC-C / APC-N ratio was calculated. The results are shown in FIG. 7. The cut-off of the value of the APC-C / APC-N ratio was set to 0.25, and iCCs less than the cut-off were determined to be CTCs containing a defective APC protein, and iCCs equal to or higher than the cut-off value were determined to be cells containing a wild-type APC protein. As can be seen from FIG. 7, in the sample containing iCCs of Donor 15, most iCCs had an APC-C / APC-N ratio value higher than the cut-off and were determined to be cells containing a wild-type APC protein. This was consistent with the results of the nucleotide sequence analysis. In the samples containing iCCs of Donor 4 and Donor 8, both cells containing a wild-type APC protein and CTCs containing a defective APC protein were detected. This was consistent with the results of the nucleotide sequence analysis. In samples containing iCCs of other Donors, most iCCs had an APC-C / APC-N ratio value lower than the cut-off and were determined to be CTCs containing a defective APC protein. Except for Donor 13, this was consistent with the results of the nucleotide sequence analysis.

[0084] Furthermore, except for Donors 4 and 8 which were made heterozygotes, the cut-off of the average value of the APC-C / APC-N ratio of the particles in the sample was set to 0.25, and samples less than the cut-off were determined to be "defective type", and samples equal to or higher than the cut-off were determined to be "wild-type". In this case, the samples of Donors 1, 2, 3, 6, 7, 10, 11, 12, 13 and 14 were determined to be of the defective type, and the sample of Donor 15 was determined to be of the wild-type. Except for Donor 13, this was consistent with the results of the nucleotide sequence analysis. From the above results, it was shown that based on the fluorescence intensities derived from the fluorescently labeled anti-APC-N antibody and anti-APC-C antibody respectively, it was possible to distinguish between patient samples of the type expressing the wild-type APC protein and patient samples of the type expressing the defective APC protein.

[0085] An example of the acquired images of iCCs from patients determined to have the defective type and iCCs from patients determined to have the wild type by IFCM measurement is shown in Fig. 8. As can be seen from Fig. 8, in the fluorescence image of the wild type, fluorescence derived from each labeled antibody was detected. On the other hand, in the fluorescence image of the defective type, fluorescence derived from the Alexa 488-labeled anti-APC-N antibody was detected, but fluorescence derived from the Alexa 647-labeled anti-APC-C antibody was not detected. Therefore, it was shown that iCCs immunostained with the labeled anti-APC-N antibody and anti-APC-C antibody can be detected by IFCM, and iCCs expressing wild-type APC protein and iCCs expressing defective-type APC protein can be detected.

[0086] Example 3: Discrimination of epithelial, mesenchymal or intermediate cells A method for discriminating whether a cell is epithelial, mesenchymal or intermediate based on the signal values of epithelial cell markers and mesenchymal cell markers was established.

[0087] (1) Cells and antibodies As the cells used in the preliminary experiment for determining the threshold value, the lung adenocarcinoma-derived cell line HCC827 was used. HCC827 cells are normally epithelial cells, but it was known that EMT was induced by stimulation with TGF-β and they became intermediate or mesenchymal cells. In addition, iCCs from 15 colorectal cancer patients prepared in Example 2 were also used. In the Western blot described below, an anti-vimentin antibody (BD Pharmingen: 550513) and an anti-GAPDH antibody (TREVIGEN: 2275-PC-100) were used. In the immunostaining described below, an eFluor 615-labeled anti-cytokeratin antibody (Thermo Fisher: 42-9003-82) and a PE-labeled anti-vimentin antibody (Santacruz: sc-6260 PE) were used.

[0088] (2) Confirmation of epithelial-mesenchymal transition in HCC827 cells HCC827 cells were cultured by a conventional method, and TGF-β was added to the medium to induce EMT. HCC827 cells cultured without the addition of TGF-β were also prepared. Each cell was lysed with a solubilizer containing a surfactant. In the same manner as in Example 1, samples were prepared from the lysed cells, separated by SDS-PAGE, and the proteins were transferred to a nitrocellulose membrane. Western blotting was performed using an anti-vimentin antibody and an HRP-labeled anti-mouse IgG antibody (MLB Life Sciences, 330). The results are shown in Fig. 9. As can be seen from Fig. 9, it was confirmed that the band derived from vimentin increased by TGF-β treatment. From this result, it was shown that EMT was induced in HCC827 cells by TGF-β treatment.

[0089] (3) Immunostaining, IFCM measurement, and determination of threshold value of HCC827 cells HCC827 cells were cultured by a conventional method, and TGF-β was added to the medium to induce EMT. HCC827 cells cultured without adding TGF-β were also prepared. The cells were collected, washed with PBS, and then fixed by adding a 4% PFA solution. The fixed cells were washed with PBS containing 0.2% Pluronic F-127. The fixed cells were subjected to membrane permeabilization treatment with cooled methanol at a final concentration of 90%. The cells treated with membrane permeabilization were washed with PBS containing 0.2% Pluronic F-127. For the fixed and membrane-permeabilized cells, PBS containing 5% BSA containing eFluor 615-labeled anti-cytokeratin antibody and PE-labeled anti-vimentin antibody was added and incubated at room temperature for 1 hour. Then, the cells were washed twice with PBS. Thereby, a sample containing immunostained cells was obtained. The sample was measured with ImageStream MK II (manufactured by Cytek Japan). By the measurement, bright-field images and fluorescence images were acquired for each particle in the sample. The data of the acquired images were analyzed with IDEAS Application v6.2 (manufactured by MERCK MILLIPORE). Images of 5000 to 10000 particles were acquired from the images obtained with ImageStreem Mk II. The particles in the acquired images were used as the analysis targets. The fluorescence images of the particles to be analyzed were analyzed with the Intensity feature of IDEAS, and the intensities of the fluorescence signals (fluorescence intensities) generated from eFluor 615 and PE, respectively, were obtained. A scattergram was created with the fluorescence intensity derived from the eFluor 615-labeled anti-cytokeratin antibody on the vertical axis and the fluorescence intensity derived from the PE-labeled anti-vimentin antibody on the horizontal axis.

[0090] The scattergram of the cells not treated with TGF-β is shown in Fig. 10A, and the scattergram of the cells treated with TGF-β is shown in Fig. 10B. By comparing the scattergrams of Figs. 10A and B, thresholds were set for the fluorescence intensities derived from the labeled anti-vimentin antibody and the labeled anti-vimentin antibody, respectively. In each scattergram, the vertical line represents the threshold of vimentin, and the horizontal line represents the threshold of cytokeratin. Thereby, the particles on the scattergram are epithelial cells (vimentin-negative and cytokeratin-positive: Vim - / CK+ ) and mesenchymal cells (vimentin-positive and cytokeratin-negative: Vim + / CK - ) and intermediate cells (vimentin-positive and cytokeratin-positive: Vim + / CK + ) were shown to be classifiable into three groups. Specifically, referring to FIG. 10A, 95.9% of the cells without TGF-β treatment were classified as epithelial cells. On the other hand, referring to FIG. 10B, in the cells treated with TGF-β, the cells classified as epithelial cells decreased to 3.75%, the cells classified as intermediate cells increased to 69.1%, and the cells classified as mesenchymal cells increased to 25.4%. The threshold value set in this study was used for the examination of iCCs and the discrimination of cells in the examples described later.

[0091] (4) Immunostaining, IFCM measurement, and cell discrimination of iCCs Patient-derived iCCs determined to express the truncated APC protein in Example 2 were immunostained in the same manner as in (3) above using an eFluor 615-labeled anti-cytokeratin antibody and a PE-labeled anti-vimentin antibody. Then, a sample containing the immunostained iCCs was measured by IFCM to create a scattergram. Based on the threshold values set in (3) above, the iCCs of each patient were classified into epithelial cells, mesenchymal cells, and intermediate cells. As an example, the scattergrams for Donor 1, Donor 2, Donor 3, and Donor 12 are shown in FIGS. 11A to D, respectively. FIG. 11A shows the results for Donor 1, FIG. 11B shows the results for Donor 2, FIG. 11C shows the results for Donor 12, and FIG. 11D shows the results for Donor 3. Referring to FIGS. 11A to D, it was found that epithelial cells were the most abundant in the iCCs of Donor 1, intermediate cells were the most abundant in the iCCs of Donor 2, and mesenchymal cells were the most abundant in the iCCs of Donor 12. Also, in the iCCs of Donor 3, mesenchymal cells were the most abundant, but 12% of epithelial cells were also present. From this, it was suggested that the iCCs of Donor 3 are of a type that contains both epithelial cells and mesenchymal cells. Thus, it was shown that based on the expression of cytokeratin and vimentin, it is possible to discriminate whether iCCs expressing the truncated APC protein are epithelial, mesenchymal, or intermediate.

[0092] (5) Immunostaining, FCM measurement, and cell discrimination of HCC827 cells In the same manner as in (3) above, HCC827 cells induced to undergo EMT and HCC827 cells cultured without the addition of TGF-β were prepared. These cells were treated in the same manner as in (3) above to obtain a sample containing immunostained cells. The sample was measured by FCM using a FACSVerse (Becton Dickinson Japan). The measurement data were analyzed by a conventional method to obtain the intensity of the fluorescence signal (fluorescence intensity) generated from each of eFluor 615 and PE. A scattergram was created with the fluorescence intensity derived from the eFluor 615-labeled anti-cytokeratin antibody on the vertical axis and the fluorescence intensity derived from the PE-labeled anti-vimentin antibody on the horizontal axis.

[0093] The scattergram of cells without TGF-β treatment is shown in Fig. 12A, and the scattergram of cells with TGF-β treatment is shown in Fig. 12B. In the figures, "Vimentin (Area)" indicates the fluorescence intensity (area) derived from the PE-labeled anti-vimentin antibody, and "Cytokeratin (Area)" indicates the fluorescence intensity (area) derived from the eFluor 615-labeled anti-cytokeratin antibody. By comparing the scattergrams of Figs. 12A and B, thresholds were set for the fluorescence intensities derived from each of the labeled anti-vimentin antibody and the labeled anti-vimentin antibody. In each scattergram, the vertical line represents the threshold of vimentin, and the horizontal line represents the threshold of cytokeratin. From Figs. 12A and B, in the scattergram of cells without TGF-β treatment, the fluorescence intensity derived from the eFluor 615-labeled anti-cytokeratin antibody is strongly detected, but cells with weak fluorescence intensity derived from the PE-labeled anti-vimentin antibody are mainly detected (upper left of Fig. 12A. 96.5% of the total), whereas in the scattergram of cells with TGF-β treatment, cells with strong fluorescence intensity derived from the PE-labeled anti-vimentin antibody are mainly detected (right side of Fig. 12B. 95.6% of the total when the upper right and lower right are combined). Thus, the cell distributions were significantly different between the scattergram of cells without TGF-β treatment and the scattergram of cells with TGF-β treatment. From this result, it was shown that even using FCM, based on the expression of cytokeratin and vimentin, it is possible to discriminate whether iCCs are epithelial or mesenchymal, and whether cells are undergoing EMT.

[0094] Example 4: Detection of Circulating Cancer Cells Expressing Defective APC Protein The same immunostaining and IFCM measurement as in Example 1 were performed on CTCs derived from colorectal cancer patients to examine whether CTCs containing APC protein could be detected. Also, by the immunostaining and IFCM measurement performed in Example 3, it was discriminated whether CTCs derived from colorectal cancer patients were epithelial, mesenchymal, or intermediate.

[0095] (1) Cells and Antibodies Blood was collected from each of the same colorectal cancer patients as in Example 2. CTCs were separated and concentrated from the blood of each patient using the ClearCell (registered trademark) FX system (Biolidics), a CTC enrichment and recovery device, and the separation and concentration chip CTChip (registered trademark) FR1S (Biolidics). In the immunostaining described below, the same fluorescently labeled anti-APC-N antibody and anti-APC-C antibody as in Example 1, and the same fluorescently labeled anti-cytokeratin antibody and anti-vimentin antibody as in Example 3 were used.

[0096] (2) Immunostaining and IFCM measurement A 4% PFA solution was added to the CTCs of each patient for fixation. The fixed CTCs were washed with PBS containing 0.2% Pluronic F-127. The fixed CTCs were subjected to membrane permeabilization with cooled methanol at a final concentration of 90%. The cells subjected to membrane permeabilization were washed with PBS containing 0.2% Pluronic F-127. PBS containing Alexa 488-labeled anti-APC-N antibody, Alexa 647-labeled anti-APC-C antibody, eFluor 615-labeled anti-cytokeratin antibody, PE-labeled anti-vimentin antibody, and Hoechst (registered trademark) 33342 was added to the fixed and membrane-permeabilized CTCs, and the mixture was incubated at room temperature for 1 hour. Thereafter, the CTCs were washed twice with PBS containing 0.2% Pluronic F-127. Thereby, a sample containing immunostained CTCs was obtained. The sample was measured with an ImageStream MK II (Cytek Japan). By the measurement, bright-field images and fluorescent images were acquired for each individual particle in the sample. The data of the acquired images were analyzed with IDEAS Application v6.2 (MERCK MILLIPORE). The fluorescent images of the particles to be analyzed were analyzed with the Intensity feature of IDEAS, and the intensities of the fluorescent signals (fluorescent intensities) generated from Alexa 488, Alexa 647, eFluor 615, and PE were acquired.

[0097] (3) Detection of APC protein and discrimination of cells For samples containing CTCs of each patient, the value of the APC-C / APC-N ratio was calculated. CTCs containing the defective APC protein were detected from the samples of Donors 1, 2, 3, 6, 7, 10, 11, 12, and 14. For any of these Donors, since the average value of the APC-C / APC-N ratio of the particles in the sample was 0.25 or less, they were considered "positive for defective APC protein". From the samples of the other Donors, cells containing the wild-type APC protein were detected, and the CTCs containing the defective APC protein were less than a predetermined amount or not detected.

[0098] A scattergram was created with the fluorescence intensity derived from the eFluor 615-labeled anti-cytokeratin antibody on the vertical axis and the fluorescence intensity derived from the PE-labeled anti-vimentin antibody on the horizontal axis. Based on the threshold values set in Example 3, the CTCs of each patient were classified into epithelial cells, mesenchymal cells, and intermediate cells. An example of an image obtained by IFCM of the epithelial CTCs, intermediate CTCs, and mesenchymal CTCs of the patients determined to be defective by IFCM measurement is shown in FIG. 13. Referring to FIG. 13, in epithelial CTCs, fluorescence derived from the eFluor 615-labeled anti-cytokeratin antibody was detected, but fluorescence derived from the PE-labeled anti-vimentin antibody was not detected. In mesenchymal CTCs, fluorescence derived from the PE-labeled anti-vimentin antibody was detected, but fluorescence derived from the eFluor 615-labeled anti-cytokeratin antibody was not detected. In intermediate CTCs, fluorescence derived from the eFluor 615-labeled anti-cytokeratin antibody and the PE-labeled anti-vimentin antibody was detected. In any of the CTCs, fluorescence derived from the Alexa 488-labeled anti-APC-N antibody was detected, but fluorescence derived from the Alexa 647-labeled anti-APC-C antibody was not detected. Therefore, it was shown that CTCs expressing the defective APC protein could be detected by measuring CTCs immunostained with the labeled anti-APC-N antibody and anti-APC-C antibody by IFCM. Also, by performing immunostaining with anti-cytokeratin antibody and anti-vimentin antibody and IFCM measurement, it was shown that it was possible to discriminate which of epithelial cells, mesenchymal cells, and intermediate cells the CTCs expressing the defective APC protein were.

[0099] Example 5: Detection of Circulating Cancer Cells Expressing Defective APC Protein (2) CTC derived from colorectal cancer patients different from those in Example 2 were analyzed by the immunostaining and IFCM measurement performed in Example 4, and the association with the disease stage of colorectal cancer was examined.

[0100] (1) Cells and Antibodies Blood was collected from each of 79 colorectal cancer patients at stages I to IV. CTCs were separated and concentrated from the blood of each patient in the same manner as in Example 4. In the immunostaining described below, the same fluorescently labeled anti-APC-N antibody and anti-APC-C antibody as in Example 1, and the same fluorescently labeled anti-cytokeratin antibody and anti-vimentin antibody as in Example 3 were used.

[0101] (2) Immunostaining and IFCM Measurement In the same manner as in Example 4, CTCs of each patient were immunostained and IFCM measurement was performed. Based on the acquired images, for each patient, CTCs in which fluorescence derived from the Alexa 488-labeled anti-APC-N antibody was detected but fluorescence derived from the Alexa 647-labeled anti-APC-C antibody was not detected were counted as CTCs expressing defective APC protein. A scattergram was created with the fluorescence intensity derived from the eFluor 615-labeled anti-cytokeratin antibody on the vertical axis and the fluorescence intensity derived from the PE-labeled anti-vimentin antibody on the horizontal axis. Based on the threshold value set in Example 3, CTCs of each patient were classified into epithelial cells, mesenchymal cells, and intermediate cells. Based on the acquired images, mesenchymal CTCs expressing defective APC protein among the mesenchymal CTCs of each patient were counted. The results are shown in Table 1.

[0102] [Table 1]

[0103] As can be seen from Table 1, the proportion of patients in whom CTCs expressing the defective APC protein were detected increased as the disease stage progressed. Also, the proportion of patients in whom mesenchymal CTCs expressing the defective APC protein were detected increased as the disease stage progressed. In general, the CTC concentration in the blood is said to increase in correlation with the cancer stage. Furthermore, it is said that the higher the malignancy of the cancer, the more likely mesenchymal CTCs are to be detected. Also, mutations in the APC gene that cause colorectal cancer are mostly known to result in defective APC proteins. The results shown in Table 1 were consistent with those known reports.

[0104] Example 6: Evaluation of Storage Stability of Specimens It was examined whether cells containing the APC protein and cells that had undergone EMT could be detected from samples prepared from blood stored at room temperature for 24 hours or 48 hours.

[0105] (1) Detection of APC in Blood Stored at Room Temperature Blood was collected from healthy individuals and dispensed into multiple 15-ml tubes. HCT116 cells or DLD-1 cells suspended in PBS containing 0.2% Pluronic F-127 were added to each tube. Then, these tubes were left standing at room temperature and collected after 24 hours and 48 hours had passed. For comparison, samples that were not left standing at room temperature (blood immediately after addition of cells; hereinafter referred to as "samples after 0 hours") were also prepared. To each tube, a hemolytic agent (G-BIOSCIENCE) four times the volume of the blood was added, and hemolysis treatment was performed by shaking and stirring at room temperature for 10 minutes. Cell pellets were collected by centrifugation and washed with PBS containing 0.2% Pluronic F-127. A 4% PFA solution was added to the washed cells for fixation, and the cells were washed with PBS containing 0.2% Pluronic F-127. These fixed cells were subjected to membrane permeabilization treatment with cooled methanol at a final concentration of 90%. Then, the cells were washed with PBS containing 0.2% Pluronic F-127. The added cells and white blood cells were immunostained for the fixed and membrane-permeabilized cells in the same manner as in the procedure of (3) of Example 1. The same fluorescently labeled anti-APC-N antibody and anti-APC-C antibody as in Example 1 were used for immunostaining. Samples containing the immunostained cells were measured with an ImageStream MK II. By the measurement, fluorescent images were obtained for individual particles in the samples. The data of the obtained images were analyzed with IDEAS Application v6.2. The fluorescent images of the particles to be analyzed were analyzed with the Intensity feature of IDEAS, and the intensities of the fluorescent signals (fluorescent intensities) generated from Alexa 488 and Alexa 647, respectively, were obtained.

[0106] The results of plotting the fluorescence intensity derived from Alexa 488-labeled anti-APC-N antibody for each sample are shown in Fig. 14A. Also, the results of plotting the fluorescence intensity derived from Alexa 647-labeled anti-APC-C antibody for each sample are shown in Fig. 14B. From Fig. 14A, it can be seen that the fluorescence intensity derived from Alexa 488-labeled anti-APC-N antibody could be stably measured in all samples after 0 hours, 24 hours, and 48 hours. From Fig. 14B, there was no change in the fluorescence intensity derived from Alexa 647-labeled anti-APC-C antibody in the samples after 0 hours and 24 hours, but there was a change in the fluorescence intensity in the samples after 48 hours. From these results, it was shown that for samples stored at room temperature for up to 24 hours after blood collection, it is possible to detect cells containing the defective APC protein by immunostaining and IFCM measurement using fluorescently labeled anti-APC-C antibody and anti-APC-N antibody.

[0107] (2) Detection of cells that have undergone EMT using HCC827 cells In the same manner as in (3) of Example 3, HCC827 cells induced to undergo EMT by TGF-β and HCC827 cells cultured without adding TGF-β were prepared. EMT-induced HCC827 cells or HCC827 cells cultured without adding TGF-β suspended in PBS containing 0.2% Pluronic F-127 were added to normal human blood aliquoted into a plurality of 15 ml tubes. Then, these tubes were allowed to stand at room temperature and collected after 24 hours and 48 hours had elapsed. For comparison, a sample after 0 hours was also prepared. To each tube, a hemolytic agent (G-BIOSCIENCE) four times the volume of the blood was added, and hemolysis treatment was performed by shaking and stirring at room temperature for 10 minutes. The cell pellet was recovered by centrifugation and washed with PBS containing 0.2% Pluronic F-127. A 4% PFA solution was added to the washed cells for fixation, and the cells were washed with PBS containing 0.2% Pluronic F-127. The fixed cells were subjected to membrane permeabilization treatment with cooled methanol at a final concentration of 90%. Then, the cells were washed with PBS containing 0.2% Pluronic F-127. The added cells were immunostained for the fixed and membrane-permeabilized cells in the same manner as the procedure in (3) of Example 3 above. For immunostaining, the same eFluor 615-labeled anti-cytokeratin antibody and PE-labeled anti-vimentin antibody as in Example 1 were used. Samples containing the immunostained cells were measured with an ImageStream MK II. By the measurement, fluorescence images were obtained for individual particles in the sample. The data of the obtained images were analyzed with IDEAS Application v6.2. The fluorescence images of the particles to be analyzed were analyzed with the Intensity feature of IDEAS, and the intensities of the fluorescence signals (fluorescence intensities) generated from eFluor 615 and PE, respectively, were obtained.

[0108] The results of plotting the fluorescence intensity derived from the PE-labeled anti-vimentin antibody for each sample are shown in Fig. 15A. Also, the results of plotting the fluorescence intensity derived from the eFluor 615-labeled anti-cytokeratin antibody for each sample are shown in Fig. 15B. In the figures, "EMT(-)" indicates HCC827 cells cultured without adding TGF-β, and "EMT(+)" indicates HCC827 cells in which EMT was induced. From Fig. 15A, at 0 hours, 24 hours, and 48 hours, in all samples, in EMT(+), the fluorescence intensity derived from the PE-labeled anti-vimentin antibody was higher than that in EMT(-). Therefore, it can be seen that the HCC827 cells in which EMT was induced and the HCC827 cells cultured without adding TGF-β could be distinguished. From Fig. 15B, it can be seen that the fluorescence intensity derived from the eFluor 615-labeled anti-cytokeratin antibody could be stably measured in all samples at 0 hours, 24 hours, and 48 hours. From these facts, it was shown that even in samples stored at room temperature for 48 hours after blood collection, it was possible to detect EMT cells by immunostaining and IFCM measurement using fluorescently labeled anti-cytokeratin antibody and anti-vimentin antibody.

Explanation of symbols

[0109] 10, 20, 30, 40: Reagent kit 11: Container 12, 23, 34, 45: Packing box 13, 24, 35, 46: Enclosed document 21, 31, 41: First container 22, 32, 42: Second container 33, 43: Third container 44: Fourth container

Claims

1. A step of contacting circulating cancer cells in a sample with a first labeled antibody containing a first labeling substance and a second labeled antibody containing a second labeling substance different from the first labeling substance; A step of detecting circulating cancer cells containing a defective APC protein to which the first labeled antibody is bound and the second labeled antibody is not bound; comprising The first labeled antibody includes an antibody capable of binding to wild-type APC protein and defective APC protein, The second labeled antibody includes an antibody capable of binding to the wild-type APC protein and not binding to the defective APC protein, A method for detecting circulating cancer cells.

2. The detection method according to claim 1, further comprising a step of detecting cells containing wild-type APC protein to which the first labeled antibody and the second labeled antibody are bound.

3. The step of detecting A step of detecting a first signal derived from the first labeling substance and a second signal derived from the second labeling substance; A step of detecting circulating cancer cells containing the defective APC protein based on the first signal and the second signal; The detection method according to claim 1, comprising

4. The step of detecting A step of detecting a first signal derived from the first labeling substance and a second signal derived from the second labeling substance; A step of detecting, as circulating cancer cells containing the defective APC protein, cells in which the value of the ratio of the second signal to the first signal is less than a threshold value, or cells in which the value of the ratio of the first signal to the second signal is greater than or equal to the threshold value; The detection method according to claim 1, comprising

5. The step of detecting A step of acquiring an image of cells in the sample and detecting a first signal derived from the first labeling substance and a second signal derived from the second labeling substance in the image of the cells; A step of detecting, as circulating cancer cells containing the defective APC protein, cells containing the first signal and substantially not containing the second signal; The detection method according to claim 1, comprising

6. The detection method according to claim 5, wherein the first labeling substance is a fluorescent substance, the second labeling substance is a fluorescent substance, the first signal is a fluorescent signal, the second signal is a fluorescent signal, and the image is a fluorescent image.

7. The detection method according to claim 1, wherein the defective APC protein is a protein lacking the C-terminal region of the wild-type APC protein.

8. The detection method according to claim 1, wherein the first labeling substance and the second labeling substance are fluorescent substances having fluorescence emission maxima in different wavelength ranges.

9. A step of labeling a mesenchymal marker protein of the circulating cancer cells in the blood; A step of detecting, as mesenchymal circulating cancer cells containing the defective APC protein, cells to which the first labeled antibody binds, the mesenchymal marker protein is labeled, and the second labeled antibody does not bind; The detection method according to claim 1, further comprising the above.

10. The detection method according to claim 9, wherein the mesenchymal marker protein is at least one selected from the group consisting of vimentin, N-cadherin, OB-cadherin, fibronectin, integrin A5b1, snail, slug, ETS1, α-SMA, twist, FAP, FSP-1, SIP1, goosecoid, LEF-1, and FOXC2.

11. A step of labeling an epithelial marker protein of the circulating cancer cells in the blood; A step of detecting, as epithelial cells containing the wild-type APC protein, cells to which the first labeled antibody and the second labeled antibody bind and the epithelial marker protein is labeled; The detection method according to claim 1, further comprising the above.

12. The detection method according to claim 11, wherein the epithelial marker protein is at least one selected from the group consisting of cytokeratin, EpCAM, E-cadherin, ZO-1, laminin-1, entactin, MUC1, desmoplakin, and α1 collagen.

13. The detection method according to claim 1, wherein the first labeled antibody binds to a site present in both the wild-type APC protein and the defective APC protein, and the second labeled antibody binds to a site present in the wild-type APC protein and not present in the defective APC protein.

14. A method for detecting circulating cancer cells in the blood containing a defective APC protein to which a first labeled antibody binds and a second labeled antibody does not bind, wherein the circulating cancer cells in the blood are prepared by contacting the circulating cancer cells in the sample with the first labeled antibody and the second labeled antibody. The first labeled antibody includes a first labeling substance and an antibody capable of binding to a wild-type APC protein and a defective APC protein. The second labeled antibody includes a second labeling substance different from the first labeling substance and an antibody capable of binding to the wild-type APC protein and not binding to the defective APC protein. A method for detecting circulating cancer cells in blood.

15. The detection method according to any one of claims 1 to 14, wherein the detection of the circulating cancer cells in blood is performed by a fluorescence microscope or a flow cytometer.

Images