STIL-arhgef7 signal detection method and use thereof

Abstract

To provide a method for detecting a STIL-ARHGEF7 signal with high sensitivity on pathological tissue.SOLUTION: The inventors paid attention on the fact that STIL enhances the motility and invasive capacity of cancer cells through ARHGEF7 and PAK1 and developed a method for detecting STIL-ARHGEF7 signals in pathological tissue using the proximity ligation assay method.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a method for detecting a STIL-ARHGEF7 signal. [Background technology]

[0002] Motility of normal cells is tightly controlled, but cancer cells exhibit dysregulated motility, which leads to invasion and metastasis. The Dbl family RhoGEF ARHGEF7 / β-PIX and its binding partner p21-activated kinase PAK1 are overexpressed in various cancers and have been shown to be involved in cancer cell invasion. As an important step in cell migration, the ARHGEF7-PAK1 complex accumulates at the migrating front of cells, leading to efficient cytoskeletal remodeling. However, the molecular mechanism by which the complex accumulates at the migrating front has not been fully elucidated.

[0003] The inventors' group has been analyzing the centriole duplication-associated factor SCL / TAL1-interrupting locus (hereinafter sometimes referred to as "STIL"), which is believed to play an important role in cell division, on the assumption that it also plays an important role in cancer cell invasion. In 2020, the inventors reported that STIL forms a ternary complex with ARHGEF7 and PAK1 and accumulates with these proteins at the lamellipodia protrusions of migrating cells, and that the ternary complex enhances the motility and invasiveness of cancer cells (see Non-Patent Document 1). [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Ito H, et al., “Indispensable role of STIL in the regulation of cancer cell motility through the lamellipodial accumulation of ARHGEF7-PAK1 complex”, Oncogene, 2020, 39:1931-1943 Summary of the Invention [Problem to be solved by the invention]

[0005] Cancer diagnosis involves the use of pathological tests, genetic tests, diagnostic imaging, and clinical tests using blood, either alone or in combination. Among these, pathological tests are important for making a definitive diagnosis of whether or not a cancer is present and the type of cancer. In pathological tests, pathological specimens are prepared mainly from cells taken from a part of the body or from tissues thinly sliced ​​from a part of a lesion, and pathologists make a pathological diagnosis by observing the condition of the tissues and cells in the pathological specimen with the naked eye, a magnifying glass, an optical microscope, or an electron microscope. In pathological diagnoses, hematoxylin-eosin stained (hereinafter sometimes referred to as "HE stained") specimens are often used, and epithelial and non-epithelial tissues are visually identified from images of the HE stained specimens, and a morphological judgment is made to diagnose cancer.

[0006] Although diagnostic technology has advanced, there are many cases where diagnosis is difficult when the specimen is very small or when there is severe inflammation. In addition, when the lesion is large, it may not be possible to fit it all within the observation range of a single field of view of a microscope, and there is a risk of the lesion being overlooked.

[0007] Pathological examination is necessary for a definitive diagnosis of cancer, but if there was a method for detecting the presence of cancer cells or cells that are likely to be cancer (hereinafter sometimes referred to as "cancer cells, etc.") as auxiliary information for cancer diagnosis or treatment, it would be possible to prevent lesions from being overlooked. Therefore, there has been a demand for technology that can detect cancer cells, etc. with high sensitivity even when the specimen is tiny, when there is strong inflammation, or when the lesion is large. [Means for solving the problem]

[0008] In order to solve the above problems, the present inventors have focused on the fact that STIL enhances the motility and invasiveness of cancer cells via ARHGEF7 and PAK1, and have found a method for detecting a signal of the ternary complex in pathological tissue, thereby completing the present invention. That is, the detection method of the present invention may include the following configurations.

[0009] (1) A method for detecting STIL-ARHGEF7 signals in pathological tissues using the Proximity Ligation Assay method. (2) The detection method according to (1), wherein an anti-STIL antibody and an anti-ARHGEF7 antibody are used as primary antibodies in the Proximity Ligation Assay method. (3) The detection method according to (1) or (2), wherein the pathological tissue is tissue derived from a patient with a malignant tumor or a patient suspected of having a malignant tumor. (4) Use of the detection method described in (1) or (2) for obtaining auxiliary information for the diagnosis or treatment of cancer. (5) (a) dividing the entire pathological tissue into a predetermined number of non-overlapping regions and photographing the regions under a microscope; (b) importing the captured images into image analysis software and detecting the STIL-ARHGEF7 signal and the number of cell nuclei in each image; (c) converting each image into numerical data of STIL-ARHGEF7 signal / cell nuclei count; (d) tiling each of the images containing said numerical data to reconstruct a single image representative of the entire pathological tissue; (e) determining that the cell is a cancer cell or has a high possibility of being a cancer cell if the value of STIL-ARHGEF7 signal / cell nucleus number is 0.4 or more; A method for aiding in the detection of cancer cells or cells likely to be cancerous. Effect of the Invention

[0010] The present invention provides a method for detecting STIL-ARHGEF7 signals in pathological tissues with high sensitivity using a Proximity Ligation Assay method. It is known that STIL, ARHGEF7, and PAK1 form a ternary complex and enhance the motility and invasiveness of cancer cells, so detection of STIL-ARHGEF7 signals can be effectively used to discover cancer cells, etc.

[0011] According to the present invention, since the detection sensitivity of the signal is high, the signal can be detected with high sensitivity even when the specimen is minute. Furthermore, when inflammation is strong, a reaction similar to wound healing occurs, and white blood cells such as neutrophils, lymphocytes, and macrophages, and fibroblasts may accumulate. Generally, it becomes difficult to find cancer cells as the number of cells increases, but since the present invention focuses on the STIL-ARHGEF7 signal, it is easy to detect only cancer cells in which the STIL-ARHGEF7 signal is enhanced. Furthermore, by detecting the signal using the Proximity Ligation Assay method, a comprehensive and accurate STIL-ARHGEF7 interaction signal map can be created. Since the presence or absence of expression of cancer cells, etc. can be confirmed comprehensively and accurately in the entire excised tissue, lesions are less likely to be overlooked. In other words, by using the detection method of the present invention, auxiliary information for the diagnosis and treatment of cancer can be obtained. [Brief description of the drawings]

[0012] [Figure 1] Schematic diagram showing that STIL forms a ternary complex with PAK1 via ARHGEF7, enhancing the motility and invasion of cancer cells. [Diagram 2] Schematic diagram of the method for detecting STIL-ARHGEF7 signals in pathological tissues. [Diagram 3] HE staining of pathological tissue. (1) indicates areas diagnosed as non-cancerous, and (2) indicates areas diagnosed as cancerous. [Figure 4] The HE stained image in Figure 3 is overlaid with the area diagnosed as cancerous in a pathological examination (dark color). [Diagram 5]Enlarged views of the area in Fig. 3(1) (A, HE staining) and DAPI-stained view (B). Enlarged views of the area in Fig. 3(2) (C, HE staining) and DAPI-stained view (D). [Figure 6] Mapping diagrams of STIL-ARHGEF7 signals in pathological tissues. (A) is a mapping diagram highlighting the area where STIL-ARHGEF7 signal / nucleus number ≥ 0.4. (B) is a diagram with the area diagnosed as cancerous in the pathological examination overlaid on the mapping diagram of (A). The dark-colored part is the overlapping area of the region where STIL-ARHGEF7 signal / nucleus number ≥ 0.4 and the area diagnosed as cancerous in the pathological examination. (C) is a mapping diagram highlighting the area where STIL-ARHGEF7 signal / nucleus number ≥ 0.5. (D) is a diagram with the area diagnosed as cancerous in the pathological examination overlaid on the mapping diagram of (C). The dark-colored part is the overlapping area of the region where STIL-ARHGEF7 signal / nucleus number ≥ 0.5 and the area diagnosed as cancerous in the pathological examination.

Mode for Carrying Out the Invention

[0013] Hereinafter, embodiments of the present invention will be described in detail, but the scope of the present invention is not limited to these embodiments.

[0014] <Ternary complex of ARHGEF7, PAK1 and STIL> Cancer cells have high motility and invade surrounding tissues, causing organ failure and the like. Motile cells form characteristic protrusions of the cell membrane, such as lamellipodia, at the front of the cell, and the activation of small guanosine Rho family GTPases is involved in their formation. Rac1 is said to be involved in the formation of lamellipodia, and lamellipodia via Rac1 are thought to promote cell migration.

[0015] Rac1 is activated by being converted from an inactive form bound to GDP to an active form bound to GTP through interaction with a Rho-specific guanine nucleotide exchange factor (GEF). Many molecules have been identified that activate Rac1, of which the Dbl family GEF ARHGEF7 / β-PIX (pak-interacting exchange factor) is the most studied. ARHGEF7 accumulates at membrane ruffling sites in motile cells, and previous studies suggest that ARHGEF7 plays an important role in cell motility.

[0016] Many molecules that bind to ARHGEF7 have been identified. Among them, PAK1 and PAK4, which are members of the p21-activated kinase (PAK) family, have been reported to be overexpressed in various cancers and to be involved in nerve outgrowth.

[0017] PAK1 has a p21-binding domain (PBD), an autoinhibitory domain, and a binding site for ARHGEF7. PAK1 forms a homodimer and binds to ARHGEF7. By binding to GTP-bound Rac1 via its PBD, PAK1 acquires kinase activity and autophosphorylates many substrates, including proteins essential for cytoskeleton formation (FILAMIN-A / FLNA). Furthermore, PAK1 enhances the interaction between ARHGEF7 and Rac1, promoting further activation of Rac1 and PAK1 (a simplified diagram showing the relationship between these molecules is shown in Figure 1). Thus, the Rac1 / ARHGEF7 / PAK1 cascade is involved in cytoskeleton remodeling and the directional formation of lamellipodia. However, it remains unclear how the accumulation of the ARHGEF7-PAK1 complex at the cell leading edge is regulated in cancer cells.

[0018] Incidentally, STIL is a protein essential for early embryonic development and is thought to be involved in many aspects of cell function. Increased expression of STIL has been reported in various types of cancers such as lung cancer, prostate cancer, and ovarian cancer. The group of the present inventors discovered that knockdown of STIL inhibits the motility of cancer cells and also reduces PAK1 activity. And it was revealed that STIL is involved in the activation of positive feedback of cytoskeletal remodeling via ARHGEF7 by accumulating the ARHGEF7-PAK1 complex in lamellipodia. That is, since STIL supports the efficient migration of cancer cells, it was speculated that STIL plays an important role in the invasion and metastasis of cancer cells. Incidentally, STIL binds to ARHGEF7 via a coiled-coil domain, and this binary complex forms a ternary complex together with PAK1 (see Non-Patent Document 1).

[0019] Although it is speculated that ARHGEF7, PAK1, and STIL form a ternary complex, it is not clear how the complex is formed. Probably, it is speculated that ARHGEF7 and STIL bind, and PAK1 forms a ternary complex via this binary complex. Thereafter, it is speculated that PAK1 phosphorylates itself and also phosphorylates FilaminA and the like. Phosphorylation leads to the activation of intracellular signal transduction cascades and ultimately to the enhancement of cancer cell motility (invasion) and metastasis.

[0020] <Proximity Ligation Assay method> The present inventors focused on the STIL-ARHGEF7 binary complex, which is considered to be directly bound, among the STIL-ARHGEF7-PAK1 ternary complex. That is, the present invention is a method for detecting STIL-ARHGEF7 signals on pathological tissues using the Proximity Ligation Assay method. The Proximity Ligation Assay method (hereinafter, sometimes referred to as the "PLA method") is also called the proximity ligation assay, and is a method for specifically detecting the binding of target proteins and complex formation on tissues with high sensitivity. This detection method uses rolling circle amplification (RCA) of complementary oligonucleotide probes, and since two types (a pair of) of probes are amplified, it is possible to detect with high sensitivity. In addition, since a pair of oligonucleotide-labeled antibodies generates an amplified signal only when the probes are in close proximity, the STIL-ARHGEF7 interaction can be detected as a clear signal.

[0021] In the present invention, it is preferable to carry out the measurement by the following steps. TM This may be performed according to the PLA method protocol (Sigma-Aldrich).

[0022] (1) Sample preparation The pathological tissue in the present invention is preferably tissue from a patient with a malignant tumor or a patient suspected of having a malignant tumor. The tissue may be from any part of the subject, but is preferably a biological sample obtained by a conventional method from a biopsy or surgically removed sample of various organs, or a peripheral blood or oral mucosa swab. The malignant tumor in the present invention is cancer or sarcoma (including hematological malignant tumors), and the cancer is preferably a solid cancer. A solid cancer is a cancer that is recognized as a clear mass in an organ or tissue, and includes gastric cancer, pancreatic cancer, lung cancer, colon cancer, breast cancer, hepatocellular carcinoma, bile duct cancer, renal cell carcinoma, head and neck cancer, thyroid cancer, uterine cancer (cervical cancer, uterine body cancer), ovarian cancer, and prostate cancer. Gastric cancer is preferable. Sarcoma includes osteosarcoma, chondrosarcoma, rhabdomyosarcoma, leiomyosarcoma, malignant lymphoma, and leukemia.

[0023] The obtained biological sample may be embedded in paraffin. In that case, the sample may be fixed with a fixative before embedding. Fixatives include formalin solution, paraformaldehyde solution, glutaraldehyde solution, osmium tetroxide solution, alcohol acetate, methanol, ethanol, acetone, and the like. Among them, formalin fixation, which is widely used as a fixative for pathological tissues, is preferred. In the case of cells, dry fixation or ethanol fixation is preferred.

[0024] Specifically, the resected pathological tissue is preferably fixed by immersing in a fixative within 3 hours after removal. When formalin is used, it is preferable to use a 10% neutral buffered formalin solution. The fixation may be performed at room temperature, or preferably in a refrigerator at 4°C or below. The fixation is preferably performed for 24 hours or more. The time may be changed as appropriate depending on the size of the tissue, etc. It is then preferable to dehydrate the specimen by immersing it in 70% ethanol (v / v) for 2 hours and then in 100% ethanol for 2 hours 6 to 7 times.

[0025] It is then preferable to replace the ethanol by immersing the tissue in an organic solvent. Xylene is preferable because of its ease of handling. When using xylene, it is preferable to immerse the fixed tissue in xylene for 10 to 60 minutes, and it is preferable to repeat this process three times. It is then preferable to infiltrate the tissue in paraffin solution 3 to 5 times, embed the tissue in hard paraffin dissolved at 60° C., and once the paraffin has solidified, store the tissue in a cool, dark place and slice the tissue to a thickness of about 3 μm. It is then preferable to carry out a deparaffinization treatment with xylene or ethanol, and an antibody activation treatment by heat treatment or enzyme treatment. The deparaffinization treatment and the antibody activation treatment can be carried out by conventional methods.

[0026] (2) Addition of primary antibody The primary antibodies against STIL and ARHGEF7 may be monoclonal or polyclonal antibodies, or a combination of both. Each primary antibody can be obtained by immunizing a mammal (e.g., mouse, rat, hamster, guinea pig, rabbit, cat, dog, pig, goat, cow, etc.) with STIL and ARHGEF7, respectively. The primary antibodies may be chimeric monoclonal antibodies, human monoclonal antibodies, and human antibodies that can be produced using gene recombination technology. There are two PLA methods: direct and indirect. In the direct method, it is preferable to use a primary antibody of an antibody-oligonucleotide conjugate, and in the indirect method, it is preferable to use a secondary antibody of an antibody-oligonucleotide conjugate and an unmodified primary antibody detected by that antibody. In both methods, it is preferable to use antibodies produced in different immunized animals, but if only antibodies produced by immunized animals of the same species are available, it is preferable to use the direct method and directly label the primary antibody for the RCA (Rolling Circle Amplification) reaction.

[0027] In the present invention, the indirect method is preferred. The indirect method is explained below. In the present invention, it is preferred to incubate the above-mentioned biological sample with primary antibodies that specifically recognize STIL and ARHGEF7, respectively. For STIL, it is preferred to use a rabbit antibody as an anti-STIL antibody, and for ARHGEF7, it is preferred to use a mouse antibody as an anti-ARHGEF7 antibody. It is preferred to add the primary antibody to the biological sample and incubate at 37°C for 10 to 120 minutes or at 4°C overnight. It is more preferred to incubate at 37°C for 60 minutes.

[0028] (3) Addition of secondary antibody (PLA probe) Next, it is preferable to bind an anti-STIL antibody pair of secondary antibodies (PLA probes) according to the type of primary antibody to the primary antibody. The secondary antibodies are modified with nucleic acids, and are designed so that when two types of secondary antibodies are brought into close proximity, the modified nucleic acids undergo a ligation reaction to form a circular structure. When STIL and ARHGEF7 immunized animals are of different species, for example, when rabbit antibodies are used for STIL and mouse antibodies are used for ARHGEF7 as primary antibodies, Duolink PLA is used as the secondary antibody. TM Probes: Rabbit PLUS (Sigma-Aldrich) and Duolink PLA TM It is preferable to use the probe mouse MINUS (Sigma-Aldrich). After the primary antibody reaction, the sample is added with a PLA probe pair and incubated at 37° C. for 10 to 120 minutes, preferably 60 minutes at 37° C.

[0029] (4) Ligation It is preferable to add ligase and incubate for circularization of the PLA probe. When STIL and ARHGEF7 interact to form a complex, the secondary antibodies come close to each other, causing a ligation reaction to form a circular DNA template. Incubation is preferably at 37°C for 10 to 60 minutes, more preferably at 37°C for 30 minutes.

[0030] (5) DNA amplification and labeling of the amplified DNA It is preferable to add a polymerase and a fluorescently labeled oligonucleotide probe and then incubate. The PLA probe functions as a primer for DNA polymerase, so that a single-stranded nucleic acid is extended along the circular structure of the formed circular DNA template (RCA method). The extended nucleic acid, that is, the nucleic acid that modifies the secondary antibody, is designed to incorporate a specific repeat sequence. The fluorescently labeled oligonucleotide probe is designed to hybridize to the amplified complementary sequence, which causes the fluorescent dye to aggregate, enabling highly sensitive signal detection by fluorescence microscopy image analysis.

[0031] The fluorescently labeled oligonucleotide probe hybridizes to the amplified complementary sequence and can be visualized by fluorescence microscopy image analysis. Incubation at 37°C for 60 to 180 minutes is preferred, with 120 minutes at 37°C being more preferred.

[0032] (6) Slide preparation After incubation, the sample is washed, nuclear stained with DAPI or the like, and then mounted with a mounting medium containing an anti-fading agent. The treated sample is then placed on a slide and a cover glass is attached to prepare the slide. The nuclear staining is preferably DAPI staining or Hoechst staining, and conventional methods can be used.

[0033] (7) Imaging analysis and mapping using fluorescence microscopy The stained slides are analyzed by the following steps. (a) dividing the entire pathological tissue into a predetermined number of non-overlapping regions and photographing the regions under a microscope; (b) importing the captured images into image analysis software and detecting the STIL-ARHGEF7 signal and the number of cell nuclei in each image; (c) converting each image into numerical data of STIL-ARHGEF7 signal / cell nuclei count; (d) tiling and reconstructing each of the images containing said numerical data into a single image representative of the entire pathological tissue. Since the STIL-ARHGEF7 signal reflects the enhancement of the motility and invasiveness of cancer cells, the STIL-ARHGEF7 signal detection method of the present invention can be used to obtain auxiliary information for cancer diagnosis or treatment. Specifically, after the step (d), the method includes a step (e) of determining that the cells are cancer cells or have a high possibility of being cancer cells if the value of STIL-ARHGEF7 signal / cell nucleus number is 0.4 or more, thereby assisting in the detection of cancer cells, etc.

[0034] A more detailed explanation will be given. In step (a), the entire pathological tissue is divided into a predetermined number of non-overlapping regions and photographed. Instead of photographing only the regions of interest individually, the divided images are photographed one field at a time, and in step (d) they are reconstructed into a unified image representing the entire pathological tissue, making it possible to review the entire range later and preventing lesions from being overlooked. There is no limit to the size of the division or the number of divisions. It is more preferable to divide into a fixed size (for example, a grid with equal intervals).

[0035] It is preferable to use a fluorescence microscope or an image cytometer capable of capturing images of multiple fields of view, and to capture images using a filter that matches the fluorescence wavelength of the signal. The number of images to be captured may be preset in an image capture device such as a microscope, and the image capture device may automatically determine the number of images to be captured according to its own resolution.

[0036] In step (b), the STIL-ARHGEF7 signal and the number of cell nuclei in each image are detected. It is preferable to detect the captured images by importing them into image processing software or the like. As the image analysis software, software capable of performing calculation processing with high reproducibility is preferable, and Image J is preferable.

[0037] A schematic diagram of the method is shown in Figure 2. It is preferable to capture divided images of the pathological tissue in the XY plane, count the STIL-ARHGEF7 signal (referred to as PLA signal in Figure 2) and cell nuclei in each image, and remove non-specific signals.

[0038] In step (c), each image is converted into numerical data by dividing the STIL-ARHGEF7 signal by the number of cell nuclei. If the value of STIL-ARHGEF7 signal / number of cell nuclei is 0.4 or more, the area is highly likely to be a cancer cell, etc., so a program may be written to emphasize images containing numerical data with a STIL-ARHGEF7 signal / number of cell nuclei value of 0.4 or more so that such areas can be identified when tiling each image containing numerical data in step (d).

[0039] In step (d), the images including the numerical data are tiled to reconstruct a single image representing the entire pathological tissue. A spreadsheet software can be used for the tiling operation. When using a spreadsheet software, it is preferable to make the aspect ratio of each cell the same as the aspect ratio of the image captured in step (a), since the tiling diagram reflects the entire pathological tissue diagram. In addition, it is preferable to program the software to highlight the color of cells with a STIL-ARHGEF7 signal / cell nucleus value of 0.4 or more, since this makes it possible to objectively and easily identify areas suspected of cancer.

[0040] In the present invention, since the purpose is to prevent cancer from being overlooked, when the value of STIL-ARHGEF7 signal / cell nucleus number is 0.4 or more, it is determined that the location contains cancer cells or cells that are likely to be cancerous, from the perspective of the balance between sensitivity and specificity. The higher the threshold value of the value of STIL-ARHGEF7 signal / cell nucleus number, the higher the sensitivity and the better the definitive diagnosis, but in consideration of the purpose of the present invention, which is to use it only for obtaining auxiliary information and to prevent cancer from being overlooked, the upper limit is preferably 0.7 or less, more preferably 0.6 or less, and even more preferably 0.5 or less.

[0041] <How to estimate areas likely to be cancerous> By using the method of the present invention, STIL-ARHGEF7 signals can be detected in pathological tissues. If STIL-ARHGEF7 signals / cell nuclei number ≧0.4, it can be inferred that the location is highly likely to contain cancer cells or the like.

[0042] Signal detection using the PLA method is highly sensitive, so even if the specimen is very small or there is strong inflammation, diagnosis can be made with high sensitivity and accuracy. In addition, since the captured images are converted into numerical data, even if multiple images are tiled in step (d), there is no need to align the conditions such as brightness of each tile image, making tiling easy and allowing the entire pathological tissue to be reviewed. Therefore, there is little risk of overlooking areas where cancer cells or cells that are likely to be cancer are present.

[0043] In actual clinical practice, pathological diagnosis is used for the definitive diagnosis of cancer cells. Although the detection method of the present invention cannot be used for the definitive diagnosis of cancer, it can objectively, sensitively, and comprehensively infer the locations where cancer cells or cells highly likely to be cancer are present. Therefore, it can be used for obtaining auxiliary information for cancer diagnosis or treatment.

Examples

[0044] The present invention will be specifically described by the following examples, but the present invention is not limited thereto. All the following operations were carried out at room temperature.

[0045] <Sample preparation> A part of the stomach of a gastric cancer patient was excised as a biological sample. The biological sample was fixed with 10% neutral buffered formalin for 24 hours, then immersed in 70% ethanol water (v / v) (once for 2 hours), and then immersed in 100% ethanol (6 times for 2 hours each) for dehydration. Thereafter, the tissue was immersed in xylene for 60 minutes, and this operation was carried out 3 times. Then, it was immersed in paraffin solution 3 times, embedded in hard paraffin melted at 60 °C, and stored in a cool and dark place after the paraffin solidified. The tissue embedded in paraffin was thinly sliced to 3 μm using a microtome, attached to a silane-coated slide glass, and dried overnight at 37 °C. The slide glass was immersed in a xylene solution for 15 minutes to remove the paraffin. Three chambers were prepared for xylene, and this operation was carried out 3 times. Next, it was immersed in 100% ethanol, 90% ethanol solution (v / v), 80% ethanol solution (v / v), and 70% ethanol solution (v / v) to remove the xylene and the remaining paraffin. Thereafter, it was washed 3 times with PBS to remove the alcohol component. Next, 10 mM citrate buffer was added and heated to 100 °C for 15 minutes for activation treatment. Thereafter, it was washed again with PBS. The surrounding area and the region of interest of the tissue were surrounded with a water-repellent pen (PAP PEN), and when the pen dried, it was quickly returned to PBS.

[0046] <Detection of STIL-ARHGEF7 signal using the PLA method> The following method is Duolink TMThe PLA method protocol was followed (Sigma-Aldrich).

[0047] (1) Addition of primary antibody The area enclosed was blown with a water-repellent pen, and the slide glass was shook to remove moisture without completely drying. Anti-STIL antibody and anti-ARHGEF7 antibody (both Santa Cruz) were prepared as primary antibodies. Each antibody was diluted in a dedicated antibody diluent. Approximately 20-40 μl of diluted primary antibody (anti-STIL antibody: mixed to a concentration of 9.1 μg / mL, anti-ARHGEF7 antibody: 2 μg / mL) was added per slide. The slide was placed in a chamber box and incubated overnight at 4°C.

[0048] (2) Addition of secondary antibody (PLA probe) The next day, remove the primary antibody from the slides and resuspend the antibody in Duolink. TM Wash twice with 1mL of Wash buffer A included in the PLA kit. TM Probe MouseMINUS (1 / 5 volume), Duolink PLA TM 20-30 μL of PLA liquid mixed with Probe Rabbit PLUS (1 / 5 volume) was added, and the mixture was kept in an incubator at 37° C. for 1 hour to prevent drying.

[0049] (3) Ligation reaction After incubation, the supernatant was removed from the dish, and the dish was washed three times with 1 mL of the attached ash buffer B. 20 to 40 μL of ligation buffer containing 1 / 40 the amount of ligase was added thereto, and the dish was left to stand in an incubator at 37° C. for 30 minutes.

[0050] (4) DNA amplification and labeling of the amplified DNA After the incubation, 30-40 μL of an amplification buffer containing 1 / 80 the amount of polymerase was added thereto, and the mixture was allowed to stand in an incubator at 37° C. for 160 minutes. From this point on, all operations were carried out in the dark.

[0051] (5) Imaging analysis and mapping using fluorescence microscopy After washing twice with 1 mL of the attached Wash buffer B, the specimen was washed with 0.01x Wash buffer B and air-dried in the dark. The specimen was then mounted using the attached mounting material containing DAPI (4',6-diamidino-2-phenylindole: nuclear stain), and the treated specimen was placed on a slide, a cover glass was attached to make a slide, and the specimen was photographed using a fluorescence microscope (IN CELL ANALYZER 6000) with an objective lens of ×20 or more. Specifically, when the photographing range was specified on the microscope, the corresponding number of photographs was automatically input, and the photographing device was set to photograph the specified range by automatically moving the camera sideways or back and forth from the upper left corner of the slide. Each image was excited with 405 nm and 561 nm laser light and photographed using 455 / 50 nm and 605 / 52 nm emission filters.

[0052] The captured images were imported into Image j. The images were opened in Image j (NIH), and a threshold that would extract only the signal was selected from Image>Adjust>Threshold. The images were binarized using Process>Binary>Make binary. The number of signals was counted using Analyze>Analyze particles. At this time, noise larger or smaller than the signal was excluded by limiting the particle size to 1.5~4.5μm. After setting these conditions for one image, the same processing was performed on all images using batch processing, and the number of STIL-ARHGEF7 signals in each image was counted. The number of cell nuclei was also counted, with a lower limit of 60μm.

[0053] Numerical data including the number of signals and nuclei in each image were extracted as a CSV file and loaded into Microsoft Excel software. At this time, the aspect ratio of each cell in the Excel software was set to be the same as that of the captured image, and a macro was set to arrange the numerical data in Excel in the same way as the captured image. This allowed the tiling diagram of the numerical data to reflect the entire pathological tissue. The color of cells corresponding to images with a certain number or more of STIL-ARHGEF7 signals / cell nuclei was highlighted.

[0054] The results are shown in Figures 3 to 6. Figure 3 shows HE staining of the pathological tissue, where (1) indicates a region diagnosed as non-cancerous in the pathological examination, and (2) indicates a region diagnosed as cancerous. In fact, the dark-colored regions shown in Figure 4 were diagnosed as cancerous in the pathological examination. The characteristics of cancerous and non-cancerous regions can also be seen in the enlarged view (Figure 5) of the regions (1) and (2). Specifically, in Figure 5(A), which shows an enlargement of the non-cancerous region, morphologically uniform epithelial cells are regularly arranged to form glandular ducts, whereas in Figure 5(C), which shows an enlargement of the cancerous region, atypical epithelial cells with cell nuclei of different sizes are seen to form irregular glandular ducts.

[0055] FIG. 6(A) is a mapping diagram in which the region in the pathological tissue where STIL-ARHGEF7 signal / cell nucleus number ≧0.4 is highlighted. The dark colored parts in FIG. 6(B) are the overlapping parts of the region where STIL-ARHGEF7 signal / cell nucleus number ≧0.4 is overlapping with the region diagnosed as cancerous by pathological examination. The mapping diagram obtained by the detection method of the present invention was 83.9% consistent with the diagram in which the cancerous area was identified by pathological examination, demonstrating that the method has extremely high sensitivity. In other words, the detection method of the present invention can prevent lesions from being overlooked.

[0056] Similarly, when the areas diagnosed as cancerous by pathological diagnosis were overlaid on a mapping diagram (FIG. 6(C)) highlighting the areas where STIL-ARHGEF7 signals / cell nuclei number ≧0.5, the dark areas in FIG. 6(D) are the overlapping areas of the two. The mapping diagram obtained by the detection method of the present invention was 71.2% consistent with the diagram in which the cancerous areas were identified by pathological examination.

[0057] In this way, the detection method of the present invention has been proven to be useful as auxiliary information for the diagnosis or treatment of cancer, since the areas predicted to be likely to be cancer cells by the detection method of the present invention are highly likely to be definitively diagnosed as cancer in pathological diagnosis as well. In addition, since the mapping image shown in Figure 6 shows the STIL-ARHGEF7 signal-positive areas in the entire pathological tissue, cancer cells can be confirmed over a wide area, and are less likely to be overlooked.

Claims

1. A method for detecting STIL-ARHGEF7 signals in pathological tissue using the Proximity Ligation Assay method.

2. The detection method according to claim 1 , wherein an anti-STIL antibody and an anti-ARHGEF7 antibody are used as primary antibodies in the proximity ligation assay method.

3. The detection method according to claim 1 or 2, wherein the pathological tissue is tissue derived from a patient with a malignant tumor or a patient suspected of having a malignant tumor.

4. 3. Use of the detection method according to claim 1 or 2 for obtaining auxiliary information for the diagnosis or treatment of cancer.

5. (a) dividing the entire pathological tissue into a predetermined number of non-overlapping regions and photographing the regions under a microscope; (b) importing the captured images into image analysis software and detecting the STIL-ARHGEF7 signal and the number of cell nuclei in each image; (c) converting each image into numerical data of STIL-ARHGEF7 signal / cell nuclei number; (d) tiling each image containing said numerical data and reconstructing it into a single image representative of the entire pathological tissue; (e) determining that the cell is a cancer cell or has a high possibility of being a cancer cell when the value of STIL-ARHGEF7 signal / cell nucleus number is 0.4 or more; A method for aiding in the detection of cancer cells or cells likely to be cancerous.

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