Method for detecting circulating cancer cells in blood

Abstract

The purpose of the present invention is to provide a method for detecting circulating cancer cells in blood containing a defective APC protein. The above-mentioned problem is solved by immunostaining circulating cancer cells in blood using a labeled antibody that binds to both a wild-type APC protein and a defective APC protein and a labeled antibody that binds to a wild-type APC protein but does not bind to a defective APC protein, and measuring a signal derived from the label.

Description

Technical Field

[0001] The present invention relates to a method for detecting circulating cancer cells (hereinafter also referred to as “CTCs”) in blood. Background Art

[0002] In cancer cells, nonsense mutations, frameshift mutations and other mutations are often seen in genes encoding specific proteins. In the transcription products from genes with such mutations, there is sometimes a stop codon at the position where protein synthesis is interrupted, or an amino acid sequence different from the original amino acid sequence is encoded from the middle. In this case, in cancer cells, a protein with a defective portion of the original protein is expressed. For example, in non-patent literature 1, it is recorded that the defective colorectal cancer cell line DLD-1 that only expresses APC (adenomatous polyposis coli) protein was not detected in the immunostaining method using an antibody that binds to the C-terminal side of APC.

[0003] Prior art literature Non-patent literature Non-patent document 1: Neufeld KL and White RL, Nuclear and cytoplasmic localizations of adenomatous polyposis coli protein, Proc. Natl. Acad. Sci. USA, vol.94, pp.3034-3039, April 1997 Summary of the invention It is believed that defective proteins in cancer cells are associated with the formation, recurrence, metastasis, etc. of tumors. In addition, among cancer cells, there are cells that are free from tumor tissues and 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 recurrence or metastasis of cancer. Since CTCs are also cancer cells, CTCs may contain defective proteins characteristic of the cancer species of origin, such as the defective APC protein in colorectal cancer cells. Therefore, it is clinically useful to detect CTCs with defective proteins. The subject of the present invention is to provide a method for detecting CTCs containing defective APC proteins.

[0004] The present invention provides the following inventions [1] to

[15] .

[0005] [1] A method for detecting CTCs, comprising: a step of bringing CTCs in a sample 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; and a step of detecting CTCs containing a defective APC protein that is bound to the first labeled antibody and not bound to the second labeled antibody, wherein the first labeled antibody contains an antibody that can bind to a wild-type APC protein and a defective APC protein, and the second labeled antibody contains an antibody that can bind to the wild-type APC protein and not to the defective APC protein.

[0006] [2] The detection method according to [1] above, further comprising the step of detecting cells containing the wild-type APC protein bound to the first labeled antibody and the second labeled antibody.

[0007] [3] A detection method according to the above-mentioned [1] or [2], wherein the detection process comprises: a process of detecting a first signal derived from the first marker substance and a second signal derived from the second marker substance; and a process of detecting circulating cancer cells in the blood containing the defective APC protein based on the first signal and the second signal.

[0008] [4] A detection method according to any one of [1] to [3] above, wherein the detection step comprises: a step of detecting a first signal derived from the first marker substance and a second signal derived from the second marker substance; and a step of detecting cells in which the ratio of the second signal to the first signal is below a threshold value, or cells in which the ratio of the first signal to the second signal is above a threshold value, as circulating cancer cells in the blood containing the defective APC protein.

[0009] [5] A detection method according to any one of [1] to [4] above, wherein the detection process includes: obtaining an image of cells in the sample, detecting a first signal derived from the first marker substance and a second signal derived from the second marker substance in the image of the cells; and detecting cells containing the first signal and substantially free of the second signal as CTCs containing the defective APC protein.

[0010] [6] The detection method according to the above [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.

[0011] [7] The detection method according to any one of [1] to [6] above, wherein the defective APC protein is a protein in which the C-terminal region of the wild-type APC protein is missing.

[0012] [8] A detection method according to any one of [1] to [7] above, wherein the first labeling substance and the second labeling substance are fluorescent substances having maximum fluorescence emission in different wavelength regions.

[0013] [9] A detection method according to any one of [1] to [8] above, further comprising: a step of labeling the interstitial marker protein of the CTC; and a step of detecting cells that are bound to the first labeling antibody, labeled with the interstitial marker protein and not bound to the second labeling antibody as interstitial CTCs containing the defective APC protein.

[0014]

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

[0015]

[11] A 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 cells that are bound to the first labeling antibody and the second labeling antibody and labeled with the epithelial marker protein as epithelial cells containing the wild-type APC protein.

[0016]

[12] The detection method according to the above-mentioned [1], wherein the epithelial marker protein is at least one selected from cytokeratin, EpCAM, E-cadherin, ZO-1, laminin-1, nestin, MUC1, desmoplakin, and α1 collagen.

[0017]

[13] A 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 but not in the defective APC protein.

[0018]

[14] A method for detecting CTCs, comprising detecting CTCs containing a defective APC protein that is bound to a first marker antibody and is not bound to a second marker antibody, wherein the CTCs are prepared by bringing the CTCs in a sample into contact with the first marker antibody and the second marker antibody, wherein the first marker antibody contains a first marker substance and an antibody that can bind to a wild-type APC protein and a defective APC protein, and the second marker antibody contains a second marker substance different from the first marker substance and an antibody that can bind to the wild-type APC protein and does not bind to the defective APC protein.

[0019]

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

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

[0020] According to the present invention, CTCs containing defective APC proteins can be detected. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1A Schematic representation of wild-type APC protein bound to a primary labeled antibody and a secondary labeled antibody.

[0022] Figure 1B is a schematic diagram of a defective APC protein bound to a primary labeled antibody.

[0023] Figure 1C is a schematic diagram of a defective APC protein bound to a primary labeled antibody.

[0024] Figure 2A This is a schematic diagram showing an example of the kit according to the present embodiment.

[0025] Figure 2B This is a schematic diagram showing an example of the kit according to the present embodiment.

[0026] Figure 2C This is a schematic diagram showing an example of the kit according to the present embodiment.

[0027] Figure 2D This is a schematic diagram showing an example of the kit according to the present embodiment.

[0028] Figure 3 The graph shows the results of Western blotting detection of APC protein in A549 cells, HCT116 cells, and DLD1 cells.

[0029] Figure 4The image is an example of a bright field image obtained by immunostaining A549 cells, HCT116 cells, and DLD1 cells and measuring them using an imaging flow cytometer (IFCM), a fluorescent image derived from an Alexa 488-labeled anti-APC-N antibody (APC-N), a fluorescent image derived from an unlabeled anti-APC-C antibody (Alexa647-labeled anti-APC-C antibody) bound to an Alexa 647-labeled secondary antibody that recognizes an APC-C antibody (APC-C), and an image in which APC-N and APC-C are superimposed (Merge).

[0030] Figure 5 The graph shows the ratio of the fluorescence intensity of APC-C to the fluorescence intensity of APC-N (APC-C / APC-N ratio) obtained by measuring immunostained A549 cells, HCT116 cells, and DLD1 cells using IFCM.

[0031] Fig. 6A This is a scattergram obtained by immunostaining DLD1 cells and measuring them using a flow cytometer (FCM). The vertical axis represents the fluorescence intensity (area) derived from APC-C, and the horizontal axis represents the fluorescence intensity (area) derived from APC-N.

[0032] Figure 6B This is a scattergram obtained by immunostaining HCT116 cells and measuring them by FCM. The vertical axis is the fluorescence intensity (area) derived from APC-C, and the horizontal axis is the fluorescence intensity (area) derived from APC-N.

[0033] Figure 7 The graph shows the APC-C / APC-N ratios obtained by immunostaining primary cultured cells (iCCs) derived from 13 colorectal cancer patients (donors 1 to 4, 6 to 8, and 10 to 15) and measuring them using IFCM.

[0034] Figure 8 This is an example of bright field images and fluorescent images acquired for iCCs of colorectal cancer patients determined to be APC protein-deficient by IFCM measurement and iCCs of colorectal cancer patients determined to be APC protein-wild type.

[0035] Fig. 9 The graph shows the results of Western blotting detection of vimentin and GAPDH in HCC827 cells.

[0036] Fig. 10A This is a scattergram obtained by immunostaining HCC827 cells not treated with TGF-β and measuring them using IFCM. The vertical axis represents the fluorescence intensity derived from the labeled anti-cytokeratin antibody, and the horizontal axis represents the fluorescence intensity derived from the labeled anti-vimentin antibody.

[0037] Fig. 10B This is a scattergram obtained by immunostaining HCC827 cells treated with TGF-β and measuring them using IFCM. The vertical axis represents the fluorescence intensity derived from the labeled anti-cytokeratin antibody, and the horizontal axis represents the fluorescence intensity derived from the labeled anti-vimentin antibody.

[0038] Fig.11A This is a scattergram obtained by immunostaining and measuring iCCs derived from donor 1 using IFCM. The vertical axis represents the fluorescence intensity derived from the labeled anti-cytokeratin antibody, and the horizontal axis represents the fluorescence intensity derived from the labeled anti-vimentin antibody.

[0039] Fig. 11B This is a scattergram obtained by immunostaining and measuring with IFCM the iCCs derived from donor 2. The vertical axis represents the fluorescence intensity derived from the labeled anti-cytokeratin antibody, and the horizontal axis represents the fluorescence intensity derived from the labeled anti-vimentin antibody.

[0040] Fig. 11C This is a scattergram obtained by immunostaining iCCs derived from donor 12 and measuring them using IFCM. The vertical axis represents the fluorescence intensity derived from the labeled anti-cytokeratin antibody, and the horizontal axis represents the fluorescence intensity derived from the labeled anti-vimentin antibody.

[0041] Fig.11D This is a scattergram obtained by immunostaining and measuring iCCs derived from donor 3 using IFCM. The vertical axis represents the fluorescence intensity derived from the labeled anti-cytokeratin antibody, and the horizontal axis represents the fluorescence intensity derived from the labeled anti-vimentin antibody.

[0042] Fig. 12A This is a scattergram obtained by immunostaining HCC827 cells that were not treated with TGF-β and measuring them using FCM. The vertical axis is the fluorescence intensity (area) derived from the labeled anti-cytokeratin antibody, and the horizontal axis is the fluorescence intensity (area) derived from the labeled anti-vimentin antibody.

[0043] Fig. 12B This is a scattergram obtained by immunostaining HCC827 cells treated with TGF-β and measuring them using FCM. The vertical axis is the fluorescence intensity (area) derived from the labeled anti-cytokeratin antibody, and the horizontal axis is the fluorescence intensity (area) derived from the labeled anti-vimentin antibody.

[0044] Fig.13 This is an example of bright field images and fluorescent images acquired for epithelial CTCs, intermediate CTCs, and stromal CTCs of a colorectal cancer patient determined to be defective by IFCM measurement.

[0045] Fig.14AThe graph shows the fluorescence intensity of APC-N measured by IFCM after peripheral blood mononuclear cells (PBMCs) were stored at room temperature for 0, 24, or 48 hours, immunostained with Alexa 488-labeled anti-APC-N antibody and Alexa 647-labeled anti-APC-C antibody.

[0046] Fig. 14B The graph shows the fluorescence intensity of APC-C measured by immunostaining PBMCs with Alexa 488-labeled anti-APC-N antibody and Alexa 647-labeled anti-APC-C antibody after storing PBMCs at room temperature for 0, 24, or 48 hours and then measuring the fluorescence intensity by IFCM.

[0047] Fig.15A The graph shows the fluorescence intensity of HCC827 cells in which epithelial-mesenchymal transition (EMT) was induced and HCC827 cells in which EMT was not induced, which were stored at room temperature for 0, 24, or 48 hours, immunostained with a labeled anti-vimentin antibody, and measured by IFCM.

[0048] Fig. 15B The graph shows the fluorescence intensity of HCC827 cells induced with EMT and HCC827 cells not induced with EMT stored at room temperature for 0, 24, or 48 hours, immunostained with a labeled anti-cytokeratin antibody, and measured by IFCM.

[0049] Description of Reference Numerals 10, 20, 30, 40: Test kit 11: Container 12, 23, 34, 45: Packing box 13, 24, 35, 46: Attached documents 21, 31, 41: First container 22, 32, 42: Second container 33, 43: The third container 44: The fourth container DETAILED DESCRIPTION In the detection method of CTC of the present embodiment (hereinafter also referred to as "the detection method of the present embodiment"), first, CTC in a sample, 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 are brought into contact. The detection method of the present embodiment is performed in vitro.

[0050] CTC is usually present in blood, so the sample is blood (whole blood) collected from the subject, or a modulated substance from the blood. Whole blood is, for example, peripheral blood (peripheral blood). In whole blood, known anticoagulants such as heparin, EDTA salt, sodium citrate, etc. can also be added as needed. Whole blood can also be diluted with an appropriate aqueous medium as needed. This aqueous medium is not particularly limited as long as it does not hinder the antigen-antibody reaction described later, and examples include water, physiological saline, buffer, etc. The subject is not particularly limited, and examples include healthy people, cancer patients, suspected cancer patients, etc. The type of cancer is not particularly limited. Since the defective APC protein is a marker for colorectal cancer, samples derived from colorectal cancer patients are particularly suitable for the detection method of this embodiment.

[0051] In the case where the sample is a modulator from whole blood, the modulator is preferably a fraction modulated by whole blood obtained from the subject and may contain CTCs. In this specification, "a fraction that may contain CTCs" includes both a fraction containing CTCs and a fraction that may contain CTCs. In this technical field, it is known that even blood collected from cancer patients sometimes does not contain CTCs in the blood. As a fraction that may contain CTCs, for example, a fraction obtained by removing red blood cells from whole blood, a fraction obtained by removing red blood cells and / or white blood cells from whole blood, and a fraction obtained by selectively recovering CTCs from whole blood can be cited. The removal of red blood cells can be carried out, for example, by adding a hemolytic agent to whole blood and hemolyzing. The removal of white blood cells can be carried out, for example, by using a solid phase (magnetic particles, microfluidics, etc.) fixed with anti-CD45 antibodies to capture white blood cells. Since most of the cells in whole blood are occupied by red blood cells and white blood cells, a fraction that may contain CTCs can be obtained by removing red blood cells and white blood cells from whole blood. The selective recovery of CTCs from whole blood can be performed, for example, by using commercially available CTC concentration and recovery devices such as the ClearCell (registered trademark) FX system and the On-Chip (registered trademark) Sort system and commercially available separation and concentration tips such as the CTChip (registered trademark) FR1S. Accordingly, fractions that may contain CTCs can be separated and recovered from whole blood. More preferably, a sample is obtained by separating whole blood into fractions that may contain CTCs and fractions that contain other cells and recovering the fractions that may contain CTCs. The fractions that may contain CTCs separated and recovered from whole blood are particularly suitable as samples in the detection method of this embodiment. It is difficult to completely remove other cells such as leukocytes from the fractions that may contain CTCs. In the fractions that may contain CTCs, cells other than leukocytes and CTCs may be contained in small amounts.

[0052] The sample may also contain CTCs that have been fixed. The decomposition of proteins in CTCs is suppressed by the fixation. The fixation can be performed by adding a fixative agent such as paraformaldehyde (PFA) or formaldehyde to the fraction that may contain CTCs. Commercially available cell fixatives may also be used. The sample may also contain CTCs that have been permeabilized. Through the permeabilization, the labeled antibodies described later can enter the cells through the cell membrane. The permeabilization can be performed by adding a permeabilization agent such as methanol, acetone, or a surfactant to the fraction that may contain CTCs. The fixation and permeabilization themselves are well known in the technical field. Preferably, the sample contains CTCs that have been fixed and permeabilized.

[0053] APC protein is a protein composed of 2843 amino acid residues encoded by the APC gene extracted as the responsible gene for familial adenomatous polyposis. The amino acid sequence of APC protein itself is well known and is disclosed in well-known databases such as NCBI (National Center for Biotechnology Information). APC protein is usually wild-type, but CTCs may contain defective APC protein. As one of the functions of wild-type APC protein, it is known to inhibit Wnt signaling by binding to β-catenin. As a result, excessive cell proliferation is prevented. Therefore, the normal APC gene is considered to be a tumor suppressor gene. If the APC gene mutates, defective APC protein can be expressed. Mutations in the APC gene are particularly common in colorectal cancer. There are various defective APC proteins depending on the type or position of the gene mutation, but most of them cannot bind to β-catenin. Therefore, defective APC protein cannot control Wnt signaling and can cause uncontrolled cell proliferation that leads to the onset of cancer.

[0054] In the present specification, "defective APC protein" means that the amino acid sequence of the C-terminal region of the wild-type APC protein does not exist, or the C-terminal region contains an amino acid sequence different from that of the wild-type APC protein. The C-terminal region can be a region containing more than 30% and less than 60% of the amino acid residues at the C-terminus in the full amino acid sequence of the wild-type APC protein. For example, the C-terminal region can be a region from the 1139th amino acid residue of the wild-type APC protein to the amino acid residue of the C-terminus (No. 2843). The C-terminal region in this case is a region of 59.9% of the full amino acid sequence of the wild-type APC protein. Alternatively, the C-terminal region can be a region from the 1991st amino acid residue of the wild-type APC protein to the amino acid residue of the C-terminus (No. 2843). The C-terminal region in this case is a region of 30% of the full amino acid sequence of the wild-type APC protein. Preferably, the C-terminal region is a region from the 1200th amino acid residue of the wild-type APC protein to the amino acid residue of the C-terminus (No. 2843). The location of the gene mutation that causes the defect in the C-terminal region of the APC protein is specifically recorded in Zhang Z. et al., ONCOLOGY LETTERS 19: 1781-1788, 2020, Zhang L. and Shay JW, JNCI J Natl Cancer Inst (2017) 109(8): djw332, etc.

[0055] In the absence of the amino acid sequence of the C-terminal region, the defective APC protein does not have a C-terminal region. That is, the defective APC protein lacks the C-terminal region compared to the wild-type APC protein. In the case where the defective 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 defective APC protein loses the function of the C-terminal region of the wild-type APC protein. As such functions, for example, the ability to bind to β-catenin, the ability to bind to microtubules, the ability to bind to EB1 protein, etc. can be cited. Of course, in the case where the defective APC protein does not have a C-terminal region, the defective APC protein also loses this function. As mutations of the APC gene that can produce defective APC proteins, there are nonsense mutations caused by base substitutions, frameshift mutations caused by base insertions or deletions, etc. Specifically, due to mutations in the APC gene, a stop codon is generated, and only a portion of the transcription product of the APC gene may be translated. Alternatively, it may be a state where a portion of the APC gene is missing and the gene itself does not exist. In these cases, the amino acid sequence of the C-terminal region does not exist in the defective APC protein produced. In addition, if a frameshift mutation occurs in the APC gene, the base sequence downstream of the mutation site may change. In this case, the C-terminal region of the defective APC protein produced has an amino acid sequence different from that of the wild-type APC protein.

[0056] The CTCs in the sample may be at least any one of: i) CTCs containing defective APC protein and not containing wild-type APC protein, ii) CTCs containing wild-type APC protein and not containing defective APC protein, and iii) CTCs containing both defective APC protein and wild-type APC protein. Preferably, the CTCs of i) and / or iii) are detected as "CTCs containing defective APC protein". In addition, the CTCs of ii) above are detected as "CTCs containing wild-type APC protein". In the case where the sample contains at least one of the CTCs of i) and iii), the CTCs containing the defective APC protein can be detected from the sample.

[0057] In the detection method of the present embodiment, in order to detect CTCs containing defective APC proteins, two types of labeled antibodies are used, namely, a first labeled antibody containing a first labeling substance (hereinafter, also referred to as "first labeled antibody") and a second labeled antibody containing a second labeling substance (hereinafter, referred to as "second labeled antibody"). In the present specification, "labeled antibody" refers to a complex containing a detection antibody and a labeling substance. The detection antibody that binds to the APC protein is specifically an antibody that can bind to both the wild-type APC protein and the defective APC protein described later, and an antibody that can bind to the wild-type APC protein and not to the defective APC protein. The labeling substances used to detect the APC protein are the first labeling substance and the second labeling substance described later. The labeled antibody can be a detection antibody labeled with a labeling substance. Alternatively, the labeled antibody can be a complex composed of a detection antibody and a second antibody that specifically binds to the detection antibody and is labeled with a labeling substance.

[0058] The labeling of antibodies based on marker substances is well known in the art and can be appropriately selected according to the type of marker substance. For example, the detection antibody and the marker substance can be combined using an appropriate cross-linking agent. When the marker substance is a protein, the first marker antibody and / or the second marker antibody can be a fusion protein of the marker substance and the detection antibody. It is also possible to use a detection antibody modified with biotin and a marker substance modified with avidin. In this case, the marker substance can be indirectly bound to the detection antibody via the specific binding of biotin and avidin. Biotin includes biotin, and biotin analogs such as desthiobiotin and oxobiotin. Avidin includes avidin, and avidin analogs such as streptavidin and Tamavidin (registered trademark).

[0059] In this specification, the term "antibody" includes full-length antibodies and fragments thereof. Full-length antibodies may also be any of IgG, IgA, IgM, IgD and IgE, preferably IgG. Antibody fragments include, for example, Fab, Fab', F(ab') 2 , Fd, Fd', Fv, light chain, heavy chain variable region (VHH) of heavy chain antibody, reduced IgG (rIgG), single chain antibody (scFv), etc. The antibody may also be any of a monoclonal antibody and a polyclonal antibody, preferably a monoclonal antibody. The antibody may also be an antibody derived from any animal. As such an animal, a mammal is preferred, and examples thereof include rabbits, mice, alpacas, camels, rats, pigs, sheep, goats, cattle, horses, donkeys, humans, etc.

[0060] The first labeled antibody includes an antibody that can bind to both the wild-type APC protein and the defective APC protein as a detection antibody. The detection antibody in the first labeled antibody can be an antibody that binds to sites present in both the wild-type APC protein and the defective APC protein. The second labeled antibody includes an antibody that can bind to the wild-type APC protein and not to the defective APC protein as a detection antibody. The detection antibody in the second labeled antibody can be an antibody that binds to a site present in the wild-type APC protein and not present in the 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 in a site other than the C-terminal region in the wild-type APC protein, and the epitope of the second labeled antibody is present in the C-terminal region in the wild-type APC protein.

[0061] The contact of CTC in the sample with the first labeled antibody and the second labeled antibody can be carried out, for example, by mixing a sample that can contain CTC, a solution containing the first labeled antibody, and a solution containing the second labeled antibody. Alternatively, after the CTC in the sample is fixed on a solid phase that can fix CTC, a solution containing the first labeled antibody and a solution containing the second labeled antibody can be added to the solid phase. In the contacting process, a solution containing both the first labeled antibody and the second labeled antibody can also be used. The order of contacting CTC with the first labeled antibody and the second labeled antibody is not particularly limited, and they can be mixed simultaneously or sequentially.

[0062] In the case where the CTC contains defective APC protein and / or wild-type APC protein, an antibody-antigen reaction occurs by contacting the CTC with 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 thereof include water, saline, phosphate buffered saline (PBS), Tris buffered saline (TBS), Good's buffer, and the like. Examples of Good's buffer include MES, Bis-Tris, ADA, PIPES, Bis-Tris-Propane, ACES, MOPS, MOPSO, BES, TES, HEPES, HEPPS, Tricine, Tris, Bicine, TAPS, and the like.

[0063] Through the above-mentioned antigen-antibody reaction, the APC protein contained in CTC is labeled with the labeled antibody. Figure 1A to Figure 1C , the labeling of APC protein contained in CTC is described. Figure 1ASchematic diagram of a wild-type APC protein bound to a first marker antibody and a second marker antibody in a cell. The wild-type APC protein has both the epitope of the first marker antibody and the epitope of the second marker antibody. Therefore, if a CTC containing a wild-type APC protein is contacted with the first marker antibody and the second marker antibody, both the first marker antibody and the second marker antibody bind to the wild-type APC protein. Both the signal derived from the first marker substance possessed by the first marker antibody and the signal derived from the second marker substance possessed by the second marker antibody can be obtained from a cell containing a wild-type APC protein.

[0064] Figure 1B Schematic diagram of a defective APC protein bound to a first marker antibody in a cell. In the defective APC protein of this example, the C-terminal region is missing. As described above, the defective APC protein can be produced by the generation of a stop codon caused by the deletion of the APC gene, the mutation of the APC gene, etc. In the defective APC protein, the epitope of the second marker antibody is lost. Therefore, in the defective APC protein, the first marker antibody is bound, and the second marker antibody is not bound. A signal derived from the first marker substance possessed by the first marker antibody is obtained from a cell containing the defective APC protein, but a signal derived from the second marker substance possessed by the second marker antibody cannot be obtained.

[0065] Figure 1C Schematic diagram of a defective APC protein bound to a first marker antibody in a cell. The defective APC protein in this example contains an amino acid sequence different from that of a wild-type APC protein in the C-terminal region. As described above, the defective APC protein can be produced by a frameshift mutation of the APC gene, etc. In the defective APC protein, the epitope of the second marker antibody is lost. Therefore, in the defective APC protein, the first marker antibody is bound, but the second marker antibody is not bound. A signal derived from the first marker substance possessed by the first marker antibody is obtained from a cell containing the defective APC protein, but a signal derived from the second marker substance possessed by the second marker antibody cannot be obtained.

[0066] In this way, 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, cells containing an APC protein bound to a first marker antibody and not bound to a second marker antibody can be detected as cells containing a defective APC protein. In addition, in the detection method of the present embodiment, cells containing an APC protein bound to a first marker antibody and a second marker antibody can be detected as cells containing a wild-type APC protein. Defective APC protein is known as a marker for colorectal cancer. Therefore, it is possible to determine that cells containing defective APC protein in a sample that may contain CTCs are CTCs.

[0067] The first labeled antibody contains a first labeled substance, and the second labeled antibody contains a second labeled substance. The second labeled substance is different from the first labeled substance. Specifically, the first labeled substance and the second labeled substance are substances that generate signals that are different from each other. The signal derived from the first labeled substance is referred to as the "first signal", and the signal derived from the second labeled substance is referred to as the "second signal". The labeled substance is preferably a substance that generates a signal (hereinafter also referred to as a "signal generating substance"). The first labeled substance and the second labeled substance can be the same type of signal generating substance, but it is preferred that the first signal and the second signal are distinguishably different. As a signal generating substance, for example, a fluorescent substance can be cited.

[0068] The fluorescent substance is not particularly limited, for example, it can be appropriately selected from known fluorescent pigments and fluorescent proteins. As fluorescent pigments, for example, fluorescein isothiocyanate (FITC), rhodamine, coumarin, imidazole derivatives, indole derivatives, 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, etc. can be cited. As fluorescent proteins, for example, green fluorescent protein, yellow fluorescent protein, blue fluorescent protein, red fluorescent protein, etc. can be cited. In the case where both the first labeling substance and the second labeling substance are fluorescent substances, the first labeling substance and the second labeling substance are preferably fluorescent substances having maximum fluorescence emission in different wavelength regions. That is, it is preferred that the wavelength of the maximum fluorescence emission of the first labeling substance is different from the wavelength of the maximum fluorescence emission of the second labeling substance. Thus, the first signal and the second signal can be measured separately.

[0069] In a preferred embodiment, the detection of CTCs containing defective APC protein is performed by detecting the first signal and the second signal generated by each cell. Figure 1B and Figure 1C , the second signal cannot be detected from the defective APC protein. Therefore, cells with a second signal below the threshold can be judged as CTCs containing defective APC proteins. Furthermore, cells with a second signal above the threshold can also be judged as cells containing wild-type APC proteins. In a further embodiment, the ratio of the first signal to the second signal is obtained. Based on the ratio, CTCs containing defective APC proteins can be detected. For example, cells whose ratio of the second signal to the first signal (second signal / first signal) is lower than the threshold, or whose ratio of the first signal to the second signal (first signal / second signal) is above the threshold can be detected as CTCs containing defective APC proteins. Furthermore, cells whose ratio of the second signal to the first signal (second signal / first signal) is above the threshold, or whose ratio of the first signal to the second signal (first signal / second signal) is lower than the threshold can be judged as cells containing wild-type APC proteins. The thresholds for each signal can be set independently with reference to Example 2 described later.

[0070] When the labeling substance is a fluorescent substance, the fluorescence generated by the fluorescent substance can be measured using a fluorescence microscope, FCM, IFCM and other devices. In this specification, FCM is a device that irradiates light to each particle (e.g., cell) in a liquid flowing in a flow cell and obtains optical information from each particle. IFCM refers to an FCM with a shooting unit such as a CCD camera. IFCM can obtain an image of each cell in the liquid flowing in the flow cell, for example, it can obtain optical information, fluorescence images and bright field images (also called transmitted light images) from several to millions of cells in a short time of several seconds to several minutes and perform quantitative measurements. In addition, by image processing, the information of each cell can be extracted.

[0071] The information obtained by the measurement under FCM is the optical information of each particle. As optical information, for example, fluorescence signal information can be cited. As fluorescence signal information, for example, the fluorescence intensity based on the waveform of the fluorescence signal of each cell can be obtained. In this specification, the fluorescence intensity obtained by the 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 defective APC protein, the fluorescence intensity obtained by the measurement with FCM can be used as the first signal and the second signal. FCM is not particularly limited, and commercially available devices can be used. As a commercially available device, for example, FACSVerse (trademark) (Becton Dickinson, Japan) can be cited.

[0072] The detection of CTCs containing defective APC proteins can also be performed by obtaining an image of each cell in the sample and based on the image of the cell. The image of the cell can also be a fluorescent image of the cell. The method of obtaining the fluorescent image of the cell is not particularly limited. When the labeling substance is a fluorescent substance, the fluorescent image of the cell can be obtained using the above-mentioned fluorescence microscope, IFCM and other devices that can obtain fluorescent images.

[0073] Fluorescence microscope and IFCM capable of obtaining fluorescence images are not particularly limited, and commercially available devices can be used. The light source is not particularly limited, and a light source with a wavelength suitable for the excitation of the fluorescent pigment 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. In addition, bright field light sources such as IFCM are not particularly limited, and for example, white laser light sources, LED lamps, mercury lamps, xenon lamps, etc. are used. As commercially available systems, for example, ImageStream MK II (SITECH JAPAN), DeNovo (bioview), Metafer (Metasystems), MI-1000 (Sysmex Corporation), etc. can be cited.

[0074] When a fluorescence microscope or IFCM capable of obtaining a fluorescence image is used, based on the obtained fluorescence image and transmitted light image, for example, parameters including the fluorescence signal area value, the total fluorescence signal intensity (hereinafter, the total fluorescence signal intensity obtained by IFCM or fluorescence microscope is also referred to as "fluorescence intensity"), the size of the cell, the value of the aspect ratio, etc. can be obtained. In the detection of CTCs containing defective APC protein, it is preferred to use at least the fluorescence intensity obtained by the measurement using IFCM as the first signal and the second signal. The fluorescence intensity obtained by the measurement using IFCM is the integral value of the pixel value of each pixel constituting the area representing the fluorescence signal in the fluorescence image of each cell. It can be judged that the particles whose total fluorescence signal intensity is less than the specified threshold value are cells that are not bound to the labeled antibody.

[0075] The first signal may be a fluorescence intensity derived from a first marker substance obtained by measurement using IFCM. The second signal may be a fluorescence intensity derived from a second marker substance obtained by measurement using IFCM. In this case, as a ratio of the first signal and the second signal, the ratio of these fluorescence intensities may also be obtained. As a ratio of the first signal and the second signal, for example, the first signal / second signal, the second signal / first signal, etc. may be obtained. Based on this ratio, it is 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 lower than a threshold, it is possible to determine that the cells (CTC) in the sample contain a defective APC protein. In addition, if the second signal / first signal is above a threshold, it is determined that the cells in the sample contain a wild-type APC protein. The threshold 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 ratio of the first signal to the second signal may be used as an indicator itself. Alternatively, the ratio of the first signal to the second signal may be combined with other indicators or measurement values.

[0076] The detection method of this embodiment may also include: a step of marking a mesenchymal marker protein and / or an epithelial marker protein, and a step of detecting CTCs marked by mesenchymal marker proteins and / or epithelial marker proteins. It is known that CTCs include epithelial CTCs, mesenchymal CTCs, and intermediate CTCs located in the middle of the transformation. Cancer cells are pointed out to be likely to cause epithelial-mesenchymal transition (EMT), changing from epithelial properties to mesenchymal properties. It is known that cancer cells that cause EMT acquire migration and invasiveness and circulate in the blood. Therefore, it is believed that compared with epithelial cancer cells, mesenchymal cancer cells are prone to metastasis and have a high degree of malignancy. In the detection method of this embodiment including the above-mentioned steps, by marking mesenchymal marker proteins and / or epithelial marker proteins, 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.

[0077] 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, and FOXC2. Examples of epithelial marker proteins include cytokeratin, EpCAM, E-cadherin, ZO-1, laminin-1, entactin, MUC1, desmoplakin, and α1 collagen.

[0078] The labeling of mesenchymal marker proteins and the labeling of epithelial marker proteins can be labeled by a known labeling method. The labeling of mesenchymal marker proteins can be performed, for example, by bringing a labeled antibody that can bind to mesenchymal marker proteins into contact with cells in a sample. Similarly, the labeling of epithelial marker proteins can be performed by bringing a labeled antibody that can bind to epithelial marker proteins into contact with cells in a sample. Hereinafter, the labeled antibody that can bind to mesenchymal marker proteins is also referred to as a "third labeled antibody", and the labeled antibody that can bind to epithelial marker proteins is also referred to as a "fourth labeled antibody". In addition, the labeling substance contained in the third labeled antibody is also referred to as a "third labeled substance", and the labeling substance contained in the fourth labeled antibody is also referred to as a "fourth labeled substance". The third labeled antibody contains an antibody that can bind to a mesenchymal marker protein as a detection antibody. The fourth labeled antibody contains an antibody that can bind to an epithelial marker protein as a detection antibody.

[0079] The third marker substance and the fourth marker substance can be any signal-generating substance, for example, a fluorescent substance. Hereinafter, the signal derived from the third marker substance is referred to as the "third signal", and the signal derived from the fourth marker substance is referred to as the "fourth signal". In the case of marking a mesenchymal marker protein, the first marker substance, the second marker substance, and the third marker substance can be the same signal-generating substance, but preferably the first signal, the second signal, and the third signal are respectively distinguishably different. In the case of marking an epithelial marker protein, the first marker substance, the second marker substance, and the fourth marker substance can be the same signal-generating substance, but preferably the first signal, the second signal, and the fourth signal are respectively distinguishably different. In the case of marking both a mesenchymal marker protein and an epithelial marker protein, the first marker substance, the second marker substance, the third marker substance, and the fourth marker substance can be the same signal-generating substance, but preferably the first signal, the second signal, the third signal, and the fourth signal are respectively distinguishably different.

[0080] When the mesenchymal marker protein is labeled, and the first marker substance, the second marker substance, and the third marker substance are fluorescent substances, it is preferred that the wavelength of the maximum fluorescence emission of the first marker substance, the wavelength of the maximum fluorescence emission of the second marker substance, and the wavelength of the maximum fluorescence emission of the third marker substance are different from each other. When the epithelial marker protein is labeled, and the first marker substance, the second marker substance, and the fourth marker substance are fluorescent substances, it is preferred that the wavelength of the maximum fluorescence emission of the first marker substance, the wavelength of the maximum fluorescence emission of the second marker substance, and the wavelength of the maximum fluorescence emission of the fourth marker substance are different from each other. When both the mesenchymal marker protein and the epithelial marker protein are labeled, and the first marker substance, the second marker substance, the third marker substance, and the fourth marker substance are fluorescent substances, it is preferred that the wavelength of the maximum fluorescence emission of the first marker substance, the wavelength of the maximum fluorescence emission of the second marker substance, the wavelength of the maximum fluorescence emission of the third marker substance, and the wavelength of the maximum fluorescence emission of the fourth marker substance are different from each other.

[0081] The detection of the third signal and / or the fourth signal itself can be detected in the same manner as the first signal and the second signal. In the case where the third marker substance and / or the fourth marker substance is a fluorescent substance, the third signal and / or the fourth signal can be detected using 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 performed by detecting the third signal and / or the fourth signal generated by each cell. For example, in the case of marking a mesenchymal marker protein, cells with a third signal above a threshold value can be determined as mesenchymal cells. In the case of marking an epithelial marker protein, cells with a fourth signal above a threshold value can be determined as epithelial cells. In the case of marking both mesenchymal marker proteins and epithelial marker proteins, cells with a third signal above a threshold value and a fourth signal below a threshold value can be determined as mesenchymal cells. In addition, cells with a third signal below a threshold value and a fourth signal above a threshold value can be determined as epithelial cells. Furthermore, cells with a third signal above a threshold value and a fourth signal above a threshold value can be determined as intermediate cells. The threshold value for each signal can be independently set with reference to Example 3 described later.

[0082] Mesenchymal marker proteins and epithelial marker proteins can also be expressed in CTCs. Therefore, for cells 1, by combining the detection results of APC proteins based on the first marker antibody and the second marker antibody with the detection results of marker proteins based on the third marker antibody and / or the fourth marker antibody, it is possible to determine which type of CTC the cell 1 is among the mesenchymal type, epithelial type and intermediate type. For example, cells whose second signal is below the threshold and whose third signal is above the threshold can be determined as mesenchymal CTCs containing defective APC proteins. Alternatively, cells whose second signal is above the threshold and whose third signal is above the threshold can be determined as mesenchymal cells (or CTCs) containing wild-type APC proteins. In addition, cells whose second signal is below the threshold and whose fourth signal is above the threshold can be determined as epithelial CTCs containing defective APC proteins. Alternatively, cells whose second signal is above the threshold and whose fourth signal is above the threshold can be determined as epithelial cells (or CTCs) containing wild-type APC proteins. In addition, cells in which the second signal is below the threshold, the third signal is above the threshold, and the fourth signal is above the threshold can be determined as intermediate CTCs containing defective APC protein. Alternatively, cells in which the second signal is above the threshold, the third signal is above the threshold, and the fourth signal is above the threshold can be determined as intermediate cells (or CTCs) containing wild-type APC protein.

[0083] In a further embodiment, a cell in which the ratio of the second signal to the first signal (second signal / first signal) is lower than a threshold value, or a cell in which the ratio of the first signal to the second signal (first signal / second signal) is higher than a threshold value and the third signal is higher than a threshold value can be determined as a stromal CTC containing a defective APC protein. Alternatively, a cell in which the second signal / first signal is higher than a threshold value, or the first signal / second signal is lower than a threshold value and the third signal is higher than a threshold value can be determined as a stromal cell (or CTC) containing a wild-type APC protein.

[0084] In a further embodiment, a cell in which the second signal / first signal is below a threshold or the first signal / second signal is above a threshold and the fourth signal is above a threshold can be determined as an epithelial CTC containing a defective APC protein. Alternatively, a cell in which the second signal / first signal is above a threshold or the first signal / second signal is below a threshold and the fourth signal is above a threshold can be determined as an epithelial cell (or CTC) containing a wild-type APC protein.

[0085] In a further embodiment, a cell in which the second signal / first signal is below a threshold value or the first signal / second signal is above a threshold value, the third signal is above a threshold value, and the fourth signal is above a threshold value can be determined as an intermediate CTC containing a defective APC protein. Alternatively, a cell in which the second signal / first signal is above a threshold value or the first signal / second signal is below a threshold value, the third signal is above a threshold value, and the fourth signal is above a threshold value can be determined as an intermediate cell (or CTC) containing a wild-type APC protein.

[0086] A further embodiment of the present invention is a method for detecting CTCs containing defective APC proteins that are bound to a first labeled antibody and not bound to a second labeled antibody. CTCs containing defective APC proteins that are bound to a first labeled antibody and not bound to a second labeled antibody are modulated by contacting CTCs, first labeled antibodies, and second labeled antibodies in a sample. The first labeled antibody contains a first labeled substance, and an antibody that can bind to a wild-type APC protein and a defective APC protein. The second labeled antibody contains a second labeled substance different from the first labeled substance, and an antibody that can bind to a wild-type APC protein and does not bind to a defective APC protein. The label of the CTC modulated by contacting the first labeled antibody and the second labeled antibody is measured, and the defective APC protein that is bound to the first labeled antibody and not bound to the second labeled antibody is detected, thereby knowing that the sample contains CTCs containing defective APC proteins. The detection method of the APC protein of CTCs, the first labeled antibody, the labeled substance, the CTC, and the APC protein are as described above.

[0087] The above detection method can also contact the CTC in the sample with a third marker antibody and / or a fourth marker antibody. The mesenchymal marker protein, the epithelial marker protein and each marker antibody are as described above. These antibodies are preferably labeled by a marker substance, respectively. The marker substance is as described above. By confirming whether an antibody that marks a mesenchymal marker protein or an epithelial marker protein is combined, it can be determined whether the CTC is mesenchymal or epithelial.

[0088] When a sample collected from a subject contains a predetermined amount or more of CTCs containing a defective APC protein, the subject or the sample can be determined to be "defective APC protein positive". When a sample collected from a subject contains less than a predetermined amount of CTCs containing a defective APC protein or when no CTCs containing a defective APC protein are detected, the subject or the sample can be determined to be "defective APC protein negative". In a preferred embodiment, when a sample collected from a subject contains less than a predetermined amount of CTCs containing a defective APC protein or when no CTCs containing a defective APC protein are detected, that is, when the sample contains a predetermined amount or more of CTCs containing a wild-type APC protein, the subject or the sample can be determined to be "defective APC protein negative".

[0089] The threshold value for determining whether the defective APC protein is positive, i.e., the "predetermined amount", can be appropriately set. For example, a plurality of specimens determined to be positive for the defective APC protein and a plurality of specimens determined to be negative for the defective APC protein by base sequence analysis or the like can be used for pre-testing, and a value that can appropriately classify the defective APC protein positive and negative using the positive accuracy rate (positive median rate), negative accuracy rate (negative median rate), sensitivity, specificity, etc. as an index can be set as the above-mentioned "predetermined amount". By testing a specimen whose defective APC protein is unknown to be negative or positive, it can be determined whether the specimen is negative or positive for the defective APC protein by applying the predetermined amount.

[0090] The sample collected from the subject may contain a predetermined amount or more of CTCs containing defective APC protein and a predetermined amount or more of CTCs containing wild-type APC protein. In this case, the genotype of the APC gene of the subject may be determined to be a heterozygous defective type and wild-type.

[0091] The reagents used in the above detection method can be provided to the user in the form of a kit. The kit comprises a first labeled antibody and a second labeled antibody. The first labeled antibody and the second labeled antibody are as described above.

[0092] The kit is composed of containers for accommodating each reagent and a box for accommodating the containers. The box may contain accompanying documents. The accompanying documents may record the composition of the kit, the composition of each reagent, the method of use, etc. An example of the kit of this embodiment is shown in Figure 2A .exist Figure 2A , 10 indicates a reagent kit, 11 indicates a container containing a reagent containing a first labeled antibody and a second labeled antibody, 12 indicates a packaging box, and 13 indicates accompanying documents. In this example, the first labeled antibody and the second labeled antibody are contained in the same container as the reagent of 1.

[0093] Another example of the kit is shown in Figure 2B .exist Figure 2B 20 indicates a reagent kit, 21 indicates a first container containing a reagent containing a first labeled antibody, 22 indicates a second container containing a reagent containing a second labeled antibody, 23 indicates a packaging box, and 24 indicates accompanying documents. In this example, the first labeled antibody and the second labeled antibody are contained in different containers.

[0094] The kit may also include a reagent containing a third marker antibody for labeling a mesenchymal marker protein or a fourth marker antibody for labeling an epithelial marker protein. Figure 2C .exist Figure 2C In the figure, 30 indicates a 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 packaging box, and 35 indicates accompanying documents.

[0095] The kit may also include a reagent containing a third labeled antibody and a reagent containing a fourth labeled antibody. An example of a kit is shown in Figure 2D .exist Figure 2D In the figure, 40 indicates a 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 packaging box, and 46 indicates accompanying documents.

[0096] Hereinafter, the present invention will be described in detail by way of examples, but the present invention is not limited to these examples.

[0097] Example Example 1: Detection of cultured cells expressing defective APC protein In order to establish an assay system for detecting CTCs expressing a defective APC protein, first, an assay system capable of detecting cultured cells expressing a defective APC protein was studied.

[0098] (1) Cells and antibodies As cell lines expressing APC protein, lung cancer cell line A549, colon cancer cell line HCT116 and DLD1 were purchased from ATCC. A549 cells and HCT116 cells are known to express wild-type (i.e., full-length) APC protein. DLD1 cells are known to express defective APC protein in which amino acid residues from amino acid residue 1427 to the C-terminus of wild-type APC are missing. In the protein blotting described below, anti-APC-N antibody (Santacruz: sc-53165 AF488) is used as an antibody that recognizes the N-terminal side of wild-type APC protein. In the immunostaining described below, Alexa 488-labeled anti-APC-N antibody (Santacruz: sc-53165AF488) is used as a labeled antibody that recognizes the N-terminal side of wild-type APC protein. In addition, as a labeled antibody that recognizes the C-terminal side of the wild-type APC protein, a combination of an unlabeled anti-APC-C antibody (Millipore: MAB3786) and an Alexa 647-labeled secondary antibody (abcam: ab150115) that recognizes the unlabeled anti-APC-C antibody was used. The site recognized by the anti-APC-C antibody does not exist in the defective APC protein expressed in DLD1 cells. In other words, it is speculated that the anti-APC-C antibody does not bind to the defective APC protein expressed in DLD1 cells.

[0099] (2) Confirmation of APC protein expression in each cell line A549 cells, HCT116 cells, and DLD1 cells were dissolved separately using a solubilizing agent containing a surfactant. The dissolved cells were centrifuged to obtain a 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 using a conventional method, and the proteins in the gel were transferred to a nitrocellulose membrane. The membrane was blocked with a blocking buffer (NACALAI TESQUE: 03953-95), and protein 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 .according to Figure 3 It can be seen that bands derived from anti-APC-N antibodies were confirmed in all lanes. Compared with the APC protein detected in A549 cells and HCT116 cells, the APC protein detected in DLD1 cells has a smaller molecular weight. This result is consistent with reports that A549 cells and HCT116 cells express wild-type APC protein, and DLD1 cells express defective APC protein.

[0100] (3) Cell culture and immunostaining The above cells were cultured by conventional methods. The cells were recovered, washed with PBS, and then fixed by adding 4% paraformaldehyde (PFA) solution. The fixed cells were washed with PBS containing 0.2% Pluronic F-127. The fixed cells were permeabilized with cooled methanol at a final concentration of 90%. The permeabilized cells were washed with PBS containing 0.2% Pluronic F-127. The fixed and permeabilized cells were immunostained using 3 antibodies. The immunostaining steps are as follows.

[0101] First, add PBS containing 5% BSA containing unlabeled anti-APC-C antibody and incubate at room temperature for 1 hour. After incubation, wash the cells twice with PBS containing 0.2% Pluronic F-127. Next, add PBS containing 5% BSA containing Alexa 647 labeled secondary antibody (abcam: ab150115) that recognizes unlabeled anti-APC-C antibody, and incubate at room temperature for 30 minutes. After incubation, wash the cells twice with PBS containing 0.2% Pluronic F-127. Then, add PBS containing 5% BSA containing Alexa 488 labeled anti-APC-N antibody to the washed cells, and incubate at room temperature for 1 hour. After incubation, wash the cells twice with PBS containing 0.2% Pluronic F-127. Thus, a sample containing immunostained cells is 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 “Alexa 647-labeled anti-APC-C antibody”.

[0102] (4) IFCM-based determination The sample containing immunostained cells was measured using ImageStream MK II (SITECH JAPAN) as IFCM. Through the measurement, bright field images and fluorescent images were obtained for each particle in the sample. The data of the obtained image was analyzed using IDEAS Application v6.2 (MERCK MILLIPORE) as image analysis software. Images of 5000 to 10000 particles were obtained from the images obtained by ImageStream MK II. The particles in the obtained image were set as the analysis object. The fluorescent image of the particles of the analysis object was analyzed using the Intensity feature of IDEAS to obtain the total fluorescence signal intensity (fluorescence intensity) of Alexa 488 and Alexa 647.

[0103] (5) Detection of APC protein An example of images obtained for A549 cells, HCT116 cells, and DLD1 cells is shown in FIG. Figure 4 .exist Figure 4 In the figure, "APC-N" indicates the fluorescence image derived from the anti-APC-N antibody labeled with Alexa 488, and "APC-C" indicates the fluorescence image derived from the anti-APC-C antibody labeled with Alexa 647. Figure 4 It can be seen that in the fluorescence images of A549 cells and HCT116 cells, fluorescence derived from each labeled antibody was detected within the outline of the cell confirmed by the bright field image. On the other hand, in the fluorescence image of DLD1 cells, fluorescence derived from the Alexa 488 labeled anti-APC-N antibody was detected within the outline of the cell confirmed by the bright field image, but fluorescence derived from the Alexa 647 labeled anti-APC-C antibody was not detected. This suggests that the anti-APC-C antibody cannot bind to the APC protein expressed in DLD1 cells. According to this result and the results of the protein blotting in (2) above, it is possible to detect cells expressing wild-type APC protein and cells expressing defective APC protein by measuring cells immunostained with labeled anti-APC-N antibody and anti-APC-C antibody using IFCM.

[0104] In order to analyze the tendency of APC protein expression in the entire cells contained in the sample, the ratio of the fluorescence intensity derived from the Alexa 647 labeled anti-APC-C antibody to the fluorescence intensity derived from the Alexa 488 labeled anti-APC-N antibody (also referred to as "APC-C / APC-N ratio") was calculated for each cell in the sample. The APC-C / APC-N ratio was compared between samples containing A549 cells, HCT116 cells, and DLD1 cells. The results are shown in Figure 5 The points in the figure represent the APC-C / APC-N ratio of each cell. Figure 5 It can be seen that a large difference was observed in the APC-C / APC-N ratio between the sample containing A549 cells and HCT116 cells and the sample containing DLD1 cells. This result shows that cells expressing wild-type APC protein and cells expressing defective APC protein can be distinguished based on the fluorescence intensity derived from the fluorescently labeled anti-APC-N antibody and anti-APC-C antibody, respectively.

[0105] (6) FCM-based assay HCT116 cells and DLD1 cells were immunostained in the same manner as in (3) above. The sample containing the immunostained cells was measured using FACSVerse (Becton Dickinson, Japan) as FCM. The measurement data were analyzed by conventional methods to obtain the intensity (fluorescence intensity) of the fluorescence signal generated by Alexa 488 and Alexa 647, respectively. A scatter plot was prepared with the vertical axis being the fluorescence intensity derived from the Alexa 647 labeled anti-APC-C antibody and the horizontal axis being the fluorescence intensity derived from the Alexa 488 labeled anti-APC-N antibody.

[0106] The dot plots of immunostained DLD1 cells are shown in Fig. 6A The scatter plot of immunostained HCT116 cells is shown in Figure 6B In Figure 6, "APC-N (Area)" indicates the fluorescence intensity (area) derived from the anti-APC-N antibody labeled with Alexa 488, and "APC-C (Area)" indicates the fluorescence intensity (area) derived from the anti-APC-C antibody labeled with Alexa 647. Fig. 6A The scatter plots of A and B were compared to set thresholds for the fluorescence intensity derived from Alexa 647 labeled anti-APC-C antibody and Alexa 488 labeled anti-APC-N antibody, respectively. In each scatter plot, the vertical line represents the threshold of Alexa 488 labeled anti-APC-N antibody, and the horizontal line represents the threshold of Alexa 647 labeled anti-APC-C antibody. Fig. 6A It can be seen that in the scatter plot of DLD1 cells, cells with strong fluorescence intensity derived from Alexa 488-labeled anti-APC-N antibody but weak fluorescence intensity derived from Alexa 647-labeled anti-APC-C antibody were mainly detected ( Fig. 6A 86.2% of the total). On the other hand, referring to Figure 6B In the scatter plot of HCT116 cells, cells whose fluorescence intensity derived from Alexa 647-labeled anti-APC-C antibody and fluorescence intensity derived from Alexa 488-labeled anti-APC-N antibody exceeded the threshold were mainly detected ( Figure 6B 87.6% of the whole). Thus, the distribution of cells in the scatter plot of immunostained DLD1 cells and the scatter plot of immunostained HCT116 cells is very different. According to this result, using FCM, it is also possible to distinguish between cells expressing wild-type APC protein and cells expressing defective APC protein based on the fluorescence intensity derived from Alexa 647 labeled anti-APC-C antibody and Alexa 488 labeled anti-APC-N antibody. In addition, in the determination of the measurement results based on FCM, the sensitivity was 89% and the specificity was 88%.

[0107] Example 2: Detection of primary cultured cells expressing defective proteins The 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 the APC protein could be detected.

[0108] (1) Cells and antibodies Thirteen patients with colorectal cancer (ref. Figure 7 ) Tumor tissues were collected from each patient. Primary culture cells (isolated tumor-derived Cancer Cells: also called "iCCs") were prepared from the tumor tissues of each patient by conventional methods. Genomic DNA was extracted from each iCCs and subjected to base sequence analysis to confirm whether there was a mutation in the APC gene that produced a C-terminal defect in the APC protein. By base sequence analysis, in donors 1, 2, 6, 7, 11, 12, and 14, a stop codon was detected in the gene due to a mutation in the APC gene. In donor 3, a frameshift was detected due to a mutation in the APC gene. In donor 4, both the wild-type base sequence and the stop codon were detected. In donor 8, both the wild-type base sequence and the frameshift were detected. It is believed that the genotype of the APC gene of donors 4 and 8 is a heterozygous defective type and wild type. In donor 10, both the stop codon and the frameshift were detected. In donors 13 and 15, wild-type base sequences were detected. In the immunostaining described below, the same labeled antibody as in Example 1 was used.

[0109] (2) Immunostaining, IFCM assay, and APC protein detection The iCCs of each patient were immunostained in the same manner as in Example 1 to prepare a sample. In the immunostaining, Hoechst (registered trademark) 33342 was used to stain the nucleus in addition to the above-mentioned antibodies. The sample containing the immunostained iCCs was measured by IFCM in the same manner as in Example 1. The APC-C / APC-N ratio was calculated for the sample containing the iCCs of each patient. The results are shown in Figure 7 The cut-off value of the APC-C / APC-N ratio was set at 0.25, and iCCs below this cut-off value were judged as CTCs containing defective APC protein, and iCCs above this cut-off value were judged as cells containing wild-type APC protein. Figure 7It can be seen that in the sample containing iCCs from donor 15, most of the iCCs became APC-C / APC-N ratios higher than the critical value, and were judged to be cells containing wild-type APC protein. This is consistent with the results of base sequence analysis. In the sample containing iCCs from donor 4 and the sample containing iCCs from donor 8, both cells containing wild-type APC protein and CTCs containing defective APC protein were detected. This is consistent with the results of base sequence analysis. In the samples containing iCCs from other donors, most of the iCCs became APC-C / APC-N ratios lower than the critical value, and were judged to be CTCs containing defective APC protein. Except for donor 13, it is consistent with the results of base sequence analysis.

[0110] Furthermore, except for donors 4 and 8, which were set as heterozygotes, the critical value of the average value of the APC-C / APC-N ratio of the particles in the sample was set to 0.25, and the samples below the critical value were judged as "defective type", and the samples above the critical value were judged as "wild type". In this case, the samples of donors 1, 2, 3, 6, 7, 10, 11, 12, 13 and 14 were judged as defective type, and the sample of donor 15 was judged as wild type. Except for donor 13, the results were consistent with the base sequence analysis. According to the above results, it is possible to distinguish between the patient sample of the type expressing the wild-type APC protein and the patient sample of the type expressing the defective APC protein based on the fluorescence intensity derived from the fluorescently labeled anti-APC-N antibody and the anti-APC-C antibody, respectively.

[0111] An example of images obtained for iCCs of a patient determined to be defective type and iCCs of a patient determined to be wild type by IFCM measurement is shown in FIG. Figure 8 .according to Figure 8 It can be seen that in the wild-type fluorescence image, fluorescence derived from each labeled antibody was detected. On the other hand, in the defective fluorescence image, 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 expressing wild-type APC protein and iCCs expressing defective APC protein can be detected by measuring iCCs immunostained with labeled anti-APC-N antibody and anti-APC-C antibody using IFCM.

[0112] 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 the epithelial cell marker and the mesenchymal cell marker is established.

[0113] (1) Cells and antibodies As cells used in preliminary experiments for determining threshold values, HCC827, a cell line derived from lung adenocarcinoma, was used. HCC827 cells are known to be epithelial cells in general, but they become intermediate or mesenchymal cells by inducing EMT through stimulation with TGF-β. In addition, iCCs derived from 15 colorectal cancer patients prepared in Example 2 were also used. In the protein blotting described later, anti-vimentin antibodies (BD Pharmingen: 550513) and anti-GAPDH antibodies (TREVIGEN: 2275-PC-100) were used. In the immunostaining described later, eFluor 615-labeled anti-cytokeratin antibodies (ThermoFisher: 42-9003-82) and PE-labeled anti-vimentin antibodies (Santacruz: sc-6260 PE) were used.

[0114] (2) Confirmation of epithelial-mesenchymal transition in HCC827 cells HCC827 cells were cultured by conventional methods, and TGF-β was added to the culture medium to induce EMT. In addition, HCC827 cells cultured without the addition of TGF-β were also prepared. Each cell was dissolved with a solubilizing agent containing a surfactant. The sample was prepared from the dissolved cells in the same manner as in Example 1, separated by SDS-PAGE, and the protein was transferred to a nitrocellulose membrane. Protein blotting was performed using an anti-vimentin antibody and an HRP-labeled anti-mouse IgG antibody (MLB LIFE SCIENCE, 330). The results are shown in Fig. 9 .according to Fig. 9 It was found that the band derived from vimentin was enlarged by TGF-β treatment. This result showed that EMT was induced in HCC827 cells by TGF-β treatment.

[0115] (3) Immunostaining, IFCM assay and threshold determination of HCC827 cells HCC827 cells were cultured by conventional methods, and TGF-β was added to the culture medium to induce EMT. In addition, HCC827 cells cultured without adding TGF-β were also prepared. The cells were recovered, washed with PBS, and then fixed by adding 4% PFA solution. The fixed cells were washed with PBS containing 0.2% Pluronic F-127. The fixed cells were permeabilized with cooled methanol at a final concentration of 90%. The permeabilized cells were washed with PBS containing 0.2% Pluronic F-127. PBS containing 5% BSA containing eFluor 615-labeled anti-cytokeratin antibody and PE-labeled anti-vimentin antibody was added to the fixed and permeabilized cells and incubated at room temperature for 1 hour. Then, the cells were washed twice with PBS. Thus, a sample containing immunostained cells was obtained. The sample was measured using ImageStream MK II (SITECH JAPAN). Through the measurement, bright field images and fluorescent images were obtained for each particle in the sample. The acquired image data was analyzed using IDEAS Application v6.2 (MERCK MILLIPORE). Images of 5,000 to 10,000 particles were acquired from the images acquired by ImageStream MK II. The particles in the acquired images were set as the analysis object. The fluorescence image of the particles to be analyzed was analyzed using the Intensity feature of IDEAS to obtain the intensity (fluorescence intensity) of the fluorescence signals generated by eFluor 615 and PE, respectively. A scatter plot was made with the vertical axis being the fluorescence intensity derived from the anti-cytokeratin antibody labeled with eFluor 615 and the horizontal axis being the fluorescence intensity derived from the anti-vimentin antibody labeled with PE.

[0116] The scatter plot of cells not treated with TGF-β is shown in Fig. 10A The scatter plot of cells treated with TGF-β is shown in Fig. 10B .Will Fig. 10A The scatter plots of A and B were compared, and thresholds were set for the fluorescence intensity derived from the labeled anti-vimentin antibody and the labeled anti-cytokeratin antibody, respectively. In each scatter plot, the vertical line represents the threshold of vimentin, and the horizontal line represents the threshold of cytokeratin. This shows that the particles on the scatter plot can be classified as epithelial cells (vimentin negative and cytokeratin positive: Vim - / CK + ), mesenchymal cells (vimentin positive and cytokeratin negative: Vim + / CK - ) and intermediate cells (vimentin positive and cytokeratin positive: Vim + / CK + ) These three groups. Specifically, refer to Fig. 10A , among the cells not treated with TGF-β, 95.9% of the total were classified as epithelial cells. Fig. 10B In cells treated with TGF-β, the number of cells classified as epithelial cells decreased to 3.75%, the number of cells classified as intermediate cells increased to 69.1%, and the number of cells classified as mesenchymal cells increased to 25.4%. The threshold value set in this study was used for the study of iCCs described below and for the discrimination of cells in the examples.

[0117] (4) Immunostaining, IFCM assay and cell identification of iCCs In Example 2, iCCs derived from patients that were determined to express defective APC protein were immunostained using eFluor 615-labeled anti-cytokeratin antibody and PE-labeled anti-vimentin antibody in the same manner as in (3) above. Then, IFCM measurement was performed on the sample containing the immunostained iCCs to create a scatter plot. Based on the threshold value set in (3) above, the iCCs of each patient were classified into epithelial cells, mesenchymal cells, and intermediate cells. As an example, the scatter plots for donor 1, donor 2, donor 3, and donor 12 are shown in FIG. Fig.11A ~D. Fig.11A The results for donor 1 are shown. Fig. 11B The results for donor 2 are shown. Fig. 11C The results for donor 12 are shown. Fig.11D The results for donor 3 are shown. Fig.11A ~D It can be seen that in the iCCs of donor 1, epithelial cells are the most numerous, in the iCCs of donor 2, intermediate cells are the most numerous, and in the iCCs of donor 12, mesenchymal cells are the most numerous. In addition, in the iCCs of donor 3, mesenchymal cells are the most numerous, but epithelial cells also exist in 12%. This suggests that the iCCs of donor 3 are of a type that contains both epithelial cells and mesenchymal cells. In this way, it is shown that the iCCs expressing defective APC protein can be judged based on the expression of cytokeratin and vimentin to determine whether they are epithelial, mesenchymal, or intermediate.

[0118] (5) Immunostaining, FCM assay and cell identification of HCC827 cells As in (3) above, HCC827 cells induced with EMT and HCC827 cells cultured without adding 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 using FACSVerse (Becton Dickinson, Japan) as FCM. The measurement data was analyzed by conventional methods to obtain the intensity (fluorescence intensity) of the fluorescence signal generated by eFluor 615 and PE, respectively. A scatter plot was made with the vertical axis being the fluorescence intensity derived from the anti-cytokeratin antibody labeled with eFluor 615 and the horizontal axis being the fluorescence intensity derived from the anti-vimentin antibody labeled with PE.

[0119] The scatter plot of cells not treated with TGF-β is shown in Fig. 12A The scatter plot of cells treated with TGF-β is shown in Fig. 12B In the figure, "Vimentin (Area)" indicates the fluorescence intensity (area) derived from PE-labeled anti-vimentin antibody, and "Cytokeratin (Area)" indicates the fluorescence intensity (area) derived from eFluor 615-labeled anti-cytokeratin antibody. Fig. 12A The scatter plots of A and B were compared to set thresholds for the fluorescence intensity derived from the labeled anti-vimentin antibody and the labeled anti-cytokeratin antibody, respectively. In each scatter plot, the vertical line represents the threshold for vimentin, and the horizontal line represents the threshold for cytokeratin. Fig. 12A As shown in Figure 1 and B, in the scatter plot of cells not treated with TGF-β, cells with strong fluorescence intensity derived from the eFluor 615-labeled anti-cytokeratin antibody but weak fluorescence intensity derived from the PE-labeled anti-vimentin antibody were mainly detected ( Fig. 12A 96.5% of the total), whereas in the scatter plot of cells treated with TGF-β, cells with strong fluorescence intensity derived from PE-labeled anti-vimentin antibody were mainly detected ( Fig. 12B The upper right and lower right are combined to form 95.6% of the total). Thus, the distribution of cells in the scatter plot of cells not treated with TGF-β and the scatter plot of cells treated with TGF-β is very different. According to this result, using FCM, it is also possible to determine whether iCCs are epithelial or mesenchymal, or whether cells induce EMT, based on the expression of cytokeratin and vimentin.

[0120] Example 4: Detection of circulating cancer cells in the blood expressing defective APC protein The CTCs derived from colorectal cancer patients were subjected to the same immunostaining and IFCM measurement as in Example 1 to investigate whether CTCs containing APC protein can be detected. In addition, the immunostaining and IFCM measurement performed in Example 3 were used to determine whether the CTCs derived from colorectal cancer patients were epithelial, mesenchymal, or intermediate.

[0121] (1) Cells and antibodies Blood was collected from 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) and the separation and concentration tip CTChip (registered trademark) FR1S (Biolidics) as a CTC concentration and recovery device. 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.

[0122] (2) Immunostaining and IFCM assay 4% PFA solution was added to the CTC of each patient for fixation. The fixed CTC was washed with PBS containing 0.2% Pluronic F-127. The fixed CTC was permeabilized with cooled methanol at a final concentration of 90%. The permeabilized cells 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 permeabilized CTC and incubated at room temperature for 1 hour. Then, the CTC was washed twice with PBS containing 0.2% Pluronic F-127. Thus, a sample containing immunostained CTC was obtained. The sample was measured with ImageStream MK II (SITECH JAPAN). Through the measurement, bright field images and fluorescent images were obtained for each particle in the sample. The acquired image data was analyzed using IDEAS Application v6.2 (MERCK MILLIPORE). The fluorescence image of the particles to be analyzed was analyzed using the Intensity feature of IDEAS to obtain the intensity (fluorescence intensity) of the fluorescence signals generated by Alexa 488, Alexa 647, eFluor 615, and PE, respectively.

[0123] (3) Detection of APC protein and cell identification The APC-C / APC-N ratio was calculated for the samples containing CTCs of each patient. CTCs containing defective APC protein were detected from samples of donors 1, 2, 3, 6, 7, 10, 11, 12, and 14. For any of these donors, the average value of the APC-C / APC-N ratio of particles in the sample was less than 0.25, and therefore, it was considered "defective APC protein positive". Cells containing wild-type APC protein were detected from samples of other donors, and CTCs containing defective APC protein were less than the specified amount or not detected.

[0124] A scatter plot was prepared with the fluorescence intensity of the anti-cytokeratin antibody labeled with eFluor 615 as the vertical axis and the fluorescence intensity of the anti-vimentin antibody labeled with PE as the horizontal axis. Based on the threshold value 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 for epithelial CTCs, intermediate CTCs, and mesenchymal CTCs of a patient determined to be defective by IFCM measurement is shown in Fig.13 . Reference Fig.13 In epithelial CTCs, fluorescence from eFluor 615 labeled anti-cytokeratin antibodies was detected, but fluorescence from PE labeled anti-vimentin antibodies was not detected. In interstitial CTCs, fluorescence from PE labeled anti-vimentin antibodies was detected, but fluorescence from eFluor 615 labeled anti-cytokeratin antibodies was not detected. In interstitial CTCs, fluorescence from eFluor615 labeled anti-cytokeratin antibodies and PE labeled anti-vimentin antibodies were detected. In all CTCs, fluorescence from Alexa 488 labeled anti-APC-N antibodies was detected, but fluorescence from Alexa 647 labeled anti-APC-C antibodies was not detected. Therefore, it is shown that CTCs expressing defective APC proteins can be detected by measuring CTCs immunostained with labeled anti-APC-N antibodies and anti-APC-C antibodies with IFCM. In addition, it was shown that immunostaining and IFCM measurement using anti-cytokeratin antibodies and anti-vimentin antibodies can distinguish whether CTCs expressing defective APC protein are epithelial cells, mesenchymal cells, or intermediate cells.

[0125] Example 5: Detection of circulating cancer cells expressing defective APC protein in the blood (2) CTCs derived from a colorectal cancer patient different from the patient in Example 2 were analyzed by immunostaining and IFCM measurement performed in Example 4, and their relationship with the stage of colorectal cancer was examined.

[0126] (1) Cells and antibodies Blood was collected from patients with colorectal cancer of stage I to IV (79 patients). CTCs were isolated 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.

[0127] (2) Immunostaining and IFCM assay The same operation as in Example 4 was performed, and the CTCs of each patient were immunostained and measured by IFCM. Based on the images obtained, for each patient, CTCs in which fluorescence derived from Alexa 488 labeled anti-APC-N antibodies was detected but fluorescence derived from Alexa 647 labeled anti-APC-C antibodies was not detected were counted as CTCs expressing defective APC proteins. A scatter plot was made with the vertical axis being the fluorescence intensity derived from eFluor 615 labeled anti-cytokeratin antibodies and the horizontal axis being the fluorescence intensity derived from PE labeled anti-vimentin antibodies. Based on the threshold value set in Example 3, the CTCs of each patient were classified into epithelial cells, mesenchymal cells, and intermediate cells. Based on the images obtained, the interstitial CTCs expressing defective APC proteins in the interstitial CTCs of each patient were counted. The results are shown in Table 1.

[0128] [Table 1]

[0129] According to Table 1, the more the disease progresses, the higher the proportion of patients in which CTC expressing defective APC protein is detected. In addition, the more the disease progresses, the higher the proportion of patients in which interstitial CTC expressing defective APC protein is detected. It is generally believed that the concentration of CTCs in the blood increases in correlation with the stage of cancer. It is also believed that the higher the malignancy of the cancer, the more interstitial CTCs are detected. In addition, mutations in the APC gene, which is known to be the cause of colorectal cancer, produce defective APC protein in most cases. The results shown in Table 1 are consistent with these known reports.

[0130] Example 6: Evaluation of storage stability of specimens The purpose was to investigate whether cells containing APC protein and cells inducing EMT could be detected from samples prepared from blood stored at room temperature for 24 hours or 48 hours.

[0131] (1) Detection of APCs in blood stored at room temperature Blood was collected from healthy subjects 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 to stand at room temperature and collected after 24 hours and 48 hours. For comparison, samples that were not left to stand at room temperature were also prepared (blood just after the cells were added. Hereinafter, referred to as "samples after 0 hours"). A hemolytic agent (G-BIOSCIENCE) was added to each tube in an amount 4 times the amount of blood, and hemolysis was performed by shaking and stirring at room temperature for 10 minutes. Cell pellets were recovered by centrifugation and washed with PBS containing 0.2% Pluronic F-127. 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 permeabilized with cooled methanol at a final concentration of 90%. Then, the cells were washed with PBS containing 0.2% Pluronic F-127. For the fixed and permeabilized cells, the added cells and leukocytes were immunostained in the same manner as in step (3) of Example 1. In the immunostaining, the same fluorescently labeled anti-APC-N antibody and anti-APC-C antibody as in Example 1 were used. The sample containing the immunostained cells was measured using ImageStream MK II. Through the measurement, a fluorescent image was obtained for each particle in the sample. The data of the obtained image was analyzed using IDEAS Application v6.2. The fluorescent image of the particles to be analyzed was analyzed using the Intensity feature of IDEAS to obtain the intensity (fluorescence intensity) of the fluorescent signals generated by Alexa 488 and Alexa 647, respectively.

[0132] The results of plotting the intensity of fluorescence derived from the Alexa 488-labeled anti-APC-N antibody for each sample are shown in FIG. Fig.14A In addition, for each sample, the intensity of the fluorescence derived from the Alexa 647-labeled anti-APC-C antibody is plotted and the results are shown in FIG. Fig. 14B .according to Fig.14A It was found that the intensity of fluorescence derived from the Alexa488-labeled anti-APC-N antibody could be stably measured in all samples after 0 hour, 24 hours, and 48 hours. Fig. 14BIt can be seen that the intensity of fluorescence derived from the Alexa 647-labeled anti-APC-C antibody did not change in the samples after 0 hours and 24 hours, but changed in the samples after 48 hours. This shows that cells containing defective APC proteins can be detected by immunostaining and IFCM measurement using fluorescently labeled anti-APC-C antibodies and anti-APC-N antibodies as long as the sample is stored at room temperature for 24 hours after blood collection.

[0133] (2) Detection of EMT-inducing cells using HCC827 cells In the same manner as in Example 3 (3), HCC827 cells induced with EMT by TGF-β and HCC827 cells cultured without adding TGF-β were prepared. HCC827 cells induced with EMT or HCC827 cells cultured without adding TGF-β suspended in PBS containing 0.2% Pluronic F-127 were added to the blood of healthy subjects divided into multiple 15 ml tubes. Then, these tubes were left to stand at room temperature and recovered after 24 hours and 48 hours. For comparison, samples after 0 hours were also prepared. A hemolytic agent (G-BIOSCIENCE) in an amount 4 times the amount of blood was added to each tube, and hemolysis was performed by shaking and stirring at room temperature for 10 minutes. The cell pellets were 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 permeabilized with cooled methanol at a final concentration of 90%. The cells were then washed with PBS containing 0.2% Pluronic F-127. For the fixed and permeabilized cells, the same operation as step (3) of Example 3 above was performed to immunostain the added cells. In the immunostaining, the same eFluor 615-labeled anti-cytokeratin antibody and PE-labeled anti-vimentin antibody as in Example 1 were used. The sample containing the immunostained cells was measured using ImageStreamMK II. Through the measurement, a fluorescent image was obtained for each particle in the sample. The data of the obtained image was analyzed using IDEAS Application v6.2. The fluorescent image of the particles to be analyzed was analyzed using the Intensity feature of IDEAS to obtain the intensity (fluorescence intensity) of the fluorescent signals generated by eFluor 615 and PE, respectively.

[0134] The results of plotting the fluorescence intensity derived from the PE-labeled anti-vimentin antibody for each sample are shown in FIG. Fig.15AIn addition, for each sample, the fluorescence intensity derived from the eFluor 615-labeled anti-cytokeratin antibody is plotted and shown in FIG. Fig. 15B In the figure, "EMT(-)" indicates HCC827 cells cultured without the addition of TGF-β, and "EMT(+)" indicates HCC827 cells induced with EMT. Fig.15A It can be seen that in all samples after 0 hours, 24 hours, and 48 hours, the fluorescence intensity derived from the PE-labeled anti-vimentin antibody was higher in EMT (+) than in EMT (-). Therefore, it can be seen that HCC827 cells induced with EMT and HCC827 cells cultured without the addition of TGF-β can be distinguished. Fig. 15B It can be seen that the fluorescence intensity derived from the anti-cytokeratin antibody labeled with eFluor 615 can be stably measured in all samples after 0 hours, 24 hours, and 48 hours. This shows that even samples stored at room temperature for 48 hours after blood collection can be used to detect EMT cells by immunostaining and IFCM measurement using fluorescently labeled anti-cytokeratin antibodies and anti-vimentin antibodies.

Claims

1. A method for detecting circulating cancer cells in blood, comprising: A step of contacting circulating cancer cells in the blood of a sample, 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; as well as a step of detecting cancer cells circulating in the blood containing a defective APC protein to which the first labeled antibody is bound and to which the second labeled antibody is not bound, The first labeled antibody contains an antibody that can bind to wild-type APC protein and defective APC protein, The second labeled antibody contains an antibody that can bind to the wild-type APC protein but not to the defective APC protein.

2. The detection method according to claim 1, wherein: Also includes: A step of detecting cells containing the wild-type APC protein to which the first labeled antibody and the second labeled antibody are bound.

3. The detection method according to claim 1, wherein: The detection process 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 cancer cells circulating in the blood containing the defective APC protein based on the first signal and the second signal.

4. The detection method according to claim 1, wherein: The detection process 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 cells in which the ratio of the second signal to the first signal is below a threshold value, or cells in which the ratio of the first signal to the second signal is above a threshold value, as blood-circulating cancer cells containing the defective APC protein.

5. The detection method according to claim 1, wherein: The detection process includes: 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; and A step of detecting cells containing the first signal and substantially containing the second signal as blood-circulating cancer cells containing the defective APC protein.

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 in which the C-terminal region of the wild-type APC protein is missing.

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

9. The detection method according to claim 1, wherein Also includes: a step of labeling a mesenchymal marker protein of cancer cells circulating in the blood; as well as A step of detecting cells to which the first labeled antibody is bound, which are labeled with the mesenchymal marker protein and to which the second labeled antibody is not bound, as mesenchymal blood circulating cancer cells containing the defective APC protein.

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

11. The detection method according to claim 1, wherein Also includes: a step of labeling the epithelial marker protein of the cancer cells circulating in the blood; as well as A step of detecting cells to which the first labeled antibody and the second labeled antibody are bound and to which the epithelial marker protein is labeled as epithelial cells containing the wild-type APC protein.

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

13. The detection method according to claim 1, wherein: The first labeled antibody binds to sites 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 but not in the defective APC protein.

14. A method for detecting circulating cancer cells in the blood, comprising detecting circulating cancer cells in the blood containing a defective APC protein bound to a first labeled antibody and not bound to a second labeled antibody, wherein: The circulating cancer cells in the blood are prepared by contacting the circulating cancer cells in the blood in a sample with the first labeled antibody and the second labeled antibody. The first labeled antibody contains a first labeling substance and an antibody that can bind to a wild-type APC protein and a defective APC protein. The second labeled antibody contains a second labeling substance different from the first labeling substance and an antibody that can bind to the wild-type APC protein but not to the defective APC protein.

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

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