JP4986449B2 - Method for examining floating cells - Google Patents

Description

  The present invention relates to a method for examining floating cells such as cancer cells circulating in blood.

  It is considered that cancer metastasis is caused by cancer cells falling from solid cancer tissues, entering peripheral blood, and being carried to other places in the body to proliferate. Cancer cells floating in the peripheral blood are called CTC (Circulating Tumor Cell). The number of CTCs in blood has been reported to correlate with the possibility of cancer metastasis and prognosis, and the number of CTCs in blood was measured to serve as an index for the diagnosis of cancer and the determination of the effects of cancer treatments. It is known to do. In particular, it is clinically performed in the United States to diagnose the degree of progression, malignancy, and prognosis of breast cancer by measuring the number of blood CTCs. Kits for measuring are commercially available.

  A method for measuring the number of CTCs in the blood using a commercially available kit is a surface antigen (EpCAM) that is specifically expressed on the cell surface of epithelial cells (cancer cells are epithelial cells, usually not present in peripheral blood). (Epithelial Cell Adhesion Molecule)) is contacted with a magnetic bead immobilizing an antibody specific to blood and blood, and cancer cells captured on the magnetic bead are separated from an epithelial specific surface antigen (site). Fluorescence staining with a fluorescently labeled antibody against keratin) and counting the number of fluorescently stained cells. At this time, both the nucleus and the cell membrane were fluorescently stained so that a fluorescent reagent that specifically binds to the nucleic acid was also brought into contact with the specimen at the same time so that unreacted fluorescently labeled antibody that was not bound to cells was not measured. Count cells. Furthermore, in order not to measure cells other than CTC, fluorescent labeled CD45 antibody is also allowed to act simultaneously, the nucleus is stained, the cell membrane is stained with fluorescent labeled anti-cytokeratin antibody, and cells that are not stained with fluorescent labeled anti-CD45 antibody Count as CTC. These three types of fluorescent dyes used have different fluorescence wavelengths so that they can be clearly distinguished from each other.

  On the other hand, it is known that oncogenes proliferate in cancer cells, and it is known that the number of oncogenes in cancer cells correlates with the malignancy and prognosis of cancer. For this reason, the number of oncogenes in cancer cells is measured in order to diagnose cancer malignancy and predict prognosis. For example, in breast cancer, growth of an oncogene called HER-2 / neu and an EGFR (epidermal growth factor receptor) gene has been examined. As a means for detecting genes in living cells, fluorescent in situ hybridization (FISH) is widely used. The FISH method is performed by contacting a fluorescently labeled DNA probe that hybridizes with an oncogene with a test cell and measuring the fluorescently labeled DNA probe that has hybridized with the oncogene in the cell. In this case as well, the cell nucleus is stained with a fluorescent reagent that specifically reacts with the nucleic acid so as not to measure anything other than the cell nucleus, such as an unreacted probe. In addition, a fluorescently labeled DNA probe that hybridizes with a gene other than an oncogene is also allowed to act simultaneously, and the number of oncogenes is determined based on the number of light-emitting sites of the oncogene detection DNA probe. It is calculated by dividing by the number of light emitting sites Thus, three different fluorescent dyes are also used in the FISH method. As these three types of fluorescent dyes, those having different fluorescence wavelengths to the extent that they can be clearly distinguished are selected as in the case of CTC counting described above. It is known that the FISH method is performed on CTC collected from peripheral blood (Non-patent Document 1).

  As described above, in the CTC counting and the FISH method, three types of fluorescent dyes having different fluorescence wavelengths to the extent that they can be clearly identified are used. For this reason, the wavelength range of the fluorescent dye used for CTC counting and the fluorescent wavelength of the fluorescent dye used for the FISH method overlap or are close to each other. Therefore, when performing the CTC counting and the FISH method for CTC, samples for each measurement are prepared separately, and both measurements are performed completely (Non-patent Document 1).

Sondong Meng et al., PNAS, June 22, 2004, vol.101, no.25, 9393-9398

  As described above, when performing CTC counting and the CTC FISH method for one blood sample, it is necessary to collect a necessary amount of blood for both the CTC counting and the FISH method. In addition, although the same blood sample is used, the CTC in which the number of oncogenes is measured by the FISH method is different from the CTC counted by the CTC count, and the carcinogenesis in the cells actually counted as CTC You cannot know the number of genes.

  Therefore, an object of the present invention is to provide both a method including fluorescent staining of the surface antigen of floating cells and a FISH method, such as when performing both CTC counting and FISH method for CTC for one specimen. When performing, it is providing the test | inspection method which can reduce the quantity of the sample to be used conventionally. Another object of the present invention is to provide a test method that can detect genes in cells that are actually fluorescently stained, such as counted CTCs, by the FISH method.

  The inventors of the present application can reduce the amount of specimen to be used and can actually be fluorescently stained if the FISH method can be carried out using cells after fluorescent staining as a material, such as the counted CTC. It was conceived that genes in cells can be detected by FISH method. However, as described above, the cell surface and nucleus are fluorescently stained for CTC counting, while fluorescence is also observed in the FISH method. The fluorescent wavelength ranges of the fluorescent dyes used for both are overlapping or close to each other. For this reason, of course, even if the FISH method is performed on the counted CTC after fluorescence staining, both fluorescence by fluorescence staining and fluorescence by FISH method are observed, and detection by the FISH method is accurate. It is considered impossible. In fact, there is no known example of performing the FISH method on floating cells after fluorescent staining.

  The inventors of the present application fluorescently stained the cell nucleus in the FISH method, while the surface antigen stained in the CTC measurement is present on the cell surface. At the same time, it was thought that the fluorescence of the nucleus could be observed by observing the nucleus with a fluorescence microscope, or without being disturbed by the fluorescently stained surface antigen. Actually, the FISH method was performed on the CTC after fluorescence staining, which was subjected to CTC counting, and when the nucleus was focused on the nucleus and observed with a fluorescence microscope, surprisingly, for CTC counting, The present inventors have found that the FISH method can be performed without being disturbed by the fluorescent dye used, and have completed the present invention.

That is, the present invention provides a floating cell test comprising subjecting a floating cell fluorescently stained with a cell surface antigen to a fluorescence in situ hybridization method, then focusing on the cell nucleus and observing the nucleus with a fluorescence microscope. In the method , the floating cells are cancer cells circulating in blood, the floating cells subjected to the fluorescence in situ hybridization method are those in which nuclei are also fluorescently stained, and the fluorescence Provided is a method for examining floating cells, wherein observation in an in situ hybridization method is performed after 15 hours or more have passed after fluorescent staining of a cell surface antigen .

  According to the method of the present invention, since the FISH method is performed using cells after fluorescent staining as a material, the total amount of specimens used for both measurements can be reduced as compared with the conventional method. Therefore, when CTC measurement and FISH method are performed by the method of the present invention, the amount of blood collected from a patient can be reduced as compared with the conventional case, and a valuable blood sample can be used effectively. In addition, according to the method of the present invention, genes in cells that are actually fluorescently stained can be detected by FISH. Therefore, when performing the CTC measurement and the FISH method according to the method of the present invention, the number of oncogenic genes in the actually counted CTC can be measured, compared with the case where the conventional CTC measurement and the FISH method are performed separately. In addition, the diagnosis of cancer progression and malignancy, the possibility of metastasis and prognosis can be made more accurately.

  In the method of the present invention, the floating cells to be subjected to the FISH method are those in which cell surface antigens are fluorescently stained, and can include CTCs counted. The “floating cell” in the present invention means a cell that has been suspended in a liquid such as blood or culture solution before the cell surface is subjected to fluorescent staining. When stained in CTC counting, it is bound to a magnetic bead, and is attached to a glass slide by the FISH method, but what was a floating cell before the start of examination is called a floating cell in the present invention.

  As an example of fluorescent staining, a CTC counting method will be described. As described above, a kit for counting CTC is commercially available (trade name “ECKIT”, importer: Ortho Clinical Diagnostics Inc.). This kit contains magnetic beads immobilized with a monoclonal antibody against the surface antigen (EpCAM) specifically expressed on the cell surface of epithelial cells and a monoclonal antibody against cytokeratin (CK), which is a surface antigen specific to epithelial cells. PE-labeled anti-CK monoclonal antibody labeled with the fluorescent dye phycoerythrin (PE) and APC-labeled anti-CD45 monoclonal labeled with the fluorescent dye allophycocyanin (APC) for the monoclonal antibody against CD45, the lymphocyte surface antigen. An antibody and 4′-6-diamidino-2-phenylindole (DAPI), which is a fluorescent dye that specifically binds to nucleic acid. APC fluorescence is red, PE fluorescence is orange, and DAPI fluorescence is blue.

  When counting CTC, first, a blood sample is brought into contact with anti-EpCAM monoclonal antibody-immobilized magnetic beads, and CTC is bound onto the magnetic beads. Magnetic beads are collected using magnetic force, and PE-labeled anti-CK monoclonal antibody, APC-labeled anti-CD45 monoclonal antibody and DAPI are reacted with cells on the magnetic beads. The cell surface of CTC is stained with PE, and the nucleus is stained with DAPI but not with APC. Among the cells immobilized on the magnetic beads, the number of cells stained with PE and DAPI but not stained with APC is counted as CTC by an automatic analyzer. Thereby, the number of CTCs contained in a predetermined amount of blood is counted.

  In the method of the present invention, the FISH method is performed using, as a material, cells after the surface of floating cells such as CTC after counting have been fluorescently stained. The FISH method itself is well known, and kits for that purpose are also commercially available. For example, a kit for measuring the HER-2 / neu gene, which is an oncogene in breast cancer cells, by the FISH method is commercially available from Astellas Pharma Inc. under the trade name “Path Vision HER-2 DNA probe kit”. The FISH method can be performed using such a commercially available kit. Similarly, a kit for measuring EGFR (Epidermal Growth Factor Receptor) gene (also referred to as HER-1 / erb-B1 gene) useful for diagnosis of solid cancer such as breast cancer by FISH method is described as “LSI EGFR / CEP 7 It is commercially available from VYSIS under the trade name "Dual Color Probe" (Bredel, M., et al. (1999) Clin Cancer REs 5, 1786-92). The gene detected by the FISH method is not limited to the HER-2 / neu gene and EGFR gene.For example, TOP2A, c-myc, ZNF217 CyclinD1, p53, p16, c-myc, chromosome 5, chromosome 6. Genes such as androgen receptor, 1p36, 1q25, 19q13, 19p13, SYT, EWSR1, FUS, BCR, CHOP, and FKHR can be exemplified, but are not limited thereto.

  As an example of the FISH method, the FISH method for detecting the HER-2 / neu gene using the commercially available kit will be described. In the method of the present invention, the cell to be subjected to the FISH method is a cell whose surface has been fluorescently stained, such as CTC after counting as described above, but the FISH method itself is similar to the FISH method using normal cells as a material. Can be done. In addition, when using CTC counted using the above-mentioned ECKIT (trade name) as a material, CTC is in a state of being bound to magnetic beads, but CTC is still bound to magnetic beads and subjected to the FISH method. be able to.

  Magnetic beads in the form of suspension and bound with fluorescently stained cells can be deposited on a glass slide using commercially available cytospin. Cell dehydration and fixation can be performed by conventional methods. Detection of the HER-2 / neu gene in CTC using the above-mentioned commercially available kit can be performed as follows. The kit includes a HER-2 / neu DNA probe having a base sequence complementary to a partial region in the HER-2 / neu gene. This HER-2 / neu DNA probe is a fluorescent dye spectrum orange. It is labeled with. The kit also contains a CEP17 DNA probe having a base sequence complementary to a partial region of the alpha satellite DNA sequence in the centromere region of chromosome 17, and this CEP17 DNA probe is a fluorescent dye spectrum green. It is labeled. In addition, as in the case of CTC counting, DAPI for staining nuclei is included. In the FISH method, a genome having a base sequence complementary to each DNA probe is reacted with the above-mentioned two types of fluorescently labeled DNA probes in a state where chromosomal DNA is denatured by heating fixed cells according to a conventional method. Each DNA probe is hybridized to the DNA region. The HER-2 / neu DNA probe hybridizes with a region of the oncogenic HER-2 / neu gene (17q11.2-q12), and the CEP17 DNA probe is an alpha satellite DNA sequence in the centromere region of chromosome 17 ( It hybridizes with 17p11.1-q11.1). After hybridization, the nucleus is fluorescently stained with DAPI. The fluorescent label of the HER-2 / neu DNA probe emits orange fluorescence, the fluorescent label of the CEP17 DNA probe emits green fluorescence, and DAPI emits blue fluorescence, so these three types of fluorescent dyes are clearly distinguished. it can. After treatment with each DNA probe and DAPI, the cells are observed with a fluorescence microscope, and the number of regions where the HER-2 / neu DNA probe is hybridized (spots where orange fluorescence is observed) is hybridized with the CEP17 DNA probe. The number of HER-2 / neu genes can be measured by dividing by the number of regions (spots where green fluorescence is observed). In addition, since nuclei are stained with DAPI in CTC counting, staining of nuclei with DAPI may be omitted in the FISH method, but staining with DAPI may be performed again.

  Similarly, a commercially available kit for detecting the EGFR gene by the FISH method (commercially available from VYSIS under the trade name “LSI EGFR / CEP 7 Dual Color Probe” described above) is an EGFR located on chromosome 7p12. A DNA probe that hybridizes with a gene, labeled with approximately 300 kb of DNA with a fluorescent dye spectrum orange, and a DNA probe labeled with a fluorescent dye spectrum green with a DNA that hybridizes with the CEP7 gene (7p11.1-q11.1) The others are the same as the above-described kit for detecting the HER-2 / neu gene. Using these DNA probes, the EGFR gene in the nucleus can be detected in the same manner as in the kit for detecting the HER-2 / neu gene described above.

  In addition, when the floating cells stained with fluorescence are those whose nuclei are also stained with a fluorescent reagent that specifically binds to a nucleic acid such as DAPI described above, as a fluorescent label of a nucleic acid probe used in the FISH method, In addition, a fluorescent dye that emits fluorescence of a color different from that of the fluorescent reagent used for staining the nucleus is used. For nuclear staining, DAPI is used in commercially available kits for both CTC counting and FISH methods. Therefore, in the commercially available FISH kit, a nucleic acid probe that emits fluorescence of a color that can be visually distinguished from DAPI is selected. This requirement is satisfied.

Further, when the FISH method is performed following the fluorescent staining of the cell surface antigen, the FISH method is preferably performed after 15 hours or more, preferably about 18 to 48 hours after the fluorescent staining of the surface antigen. By setting this time, the fluorescence of the fluorescent dye used for fluorescent staining of the surface antigen becomes somewhat weak, so that the fluorescence microscope observation after the FISH method can be performed more accurately.

  The cells after FISH are observed with a fluorescence microscope. At this time, focus on the nucleus and observe. Since the cell surface was fluorescently stained, even if the cells were observed with a fluorescence microscope after the FISH method, it was thought that accurate observation was not possible due to interference with the fluorescence of the cell surface. Surprisingly, however, when the nucleus is focused and observed, the fluorescent dye on the cell surface, which appears in the same plane as the nucleus, is not a hindrance because it is located at a different position from the nucleus, The fluorescent dye that exists in a three-dimensionally overlapping position with the nucleus (that is, the fluorescent dye located on the front side of the objective lens relative to the nucleus and the fluorescent dye located on the back side of the nucleus as viewed from the objective lens) is out of focus. It was found that there was no problem in detecting the fluorescently labeled nucleic acid probe used in the FISH method. The present invention is based on this surprising new finding.

  Observation with a fluorescence microscope is preferably performed at the maximum magnification. The greater the magnification, the less focused the object is, but the out-of-focus area becomes more out of focus compared to low magnification, making it less susceptible to the effects of fluorescent dyes on the cell surface. Thus, the result of the FISH method can be observed more accurately. Since the maximum magnification of the fluorescence microscope is usually 1000 times, it is preferable to observe at 1000 times.

  Hereinafter, the present invention will be described more specifically based on examples. However, the present invention is not limited to the following examples.

1. Measurement of CTC number Using the above-mentioned commercially available ECKIT (trade name), the operation as described in the package insert was performed to measure the CTC number in peripheral blood. That is, 10 ml of peripheral blood was collected from a breast cancer patient using a dedicated blood collection tube included in the kit. Next, 7.5 ml of the collected blood was transferred to a tube, and 6.5 ml of dilution buffer included in the kit was added. Thereafter, the mixture was inverted and centrifuged at 800 g for 10 minutes at room temperature. After centrifugation, the tube was applied to a CellTracks (trade name) AutoPrep System instrument. The operation of the device was in accordance with the device instruction manual. Here, the above-described binding of CTC to the magnetic beads, fluorescence staining of the cell surface with PE-labeled anti-cytokeratin monoclonal antibody and APC-labeled anti-CD45 monoclonal antibody, and nuclear staining with DAPI are automatically performed. Next, using CellTracks (trade name) Analyzer II, it was operated according to the instrument procedure and the number of CTCs was measured. Here, cells that bind on magnetic beads and are stained with PE-labeled anti-cytokeratin monoclonal antibody, not stained with APC-labeled anti-CD45 monoclonal antibody, and nuclei stained with DAPI are automatically counted. .

2. Slide fixing method
After CTC counting, a sample was pipetted from a dedicated cartridge and fixed by the cytospin method. That is, after CTC counting, a sample was pipetted from a dedicated cartridge and placed in a cytospin dedicated cartridge. Centrifugation was performed at 1400 rpm for 10 minutes at room temperature, and the cells were affixed on a glass slide. After centrifugation, the cells were dried with a dryer.

3. FISH method
The FISH method was performed using the above-mentioned commercially available kit “LSI EGFR / CEP 7 Dual Color Probe” (trade name) for detecting the EGFR gene, according to the package insert of the kit. That is, the slide on which the cells were fixed was placed in a 70% formamide / 2 × SSC solution at 73-74 ° C. and allowed to stand for 2 minutes. Immediately, the slide was placed in 70% ethanol at ice temperature and allowed to stand for 5 minutes. Next, the slide was replaced with ice-cold 100% ethanol and allowed to stand for 5 minutes or longer. The slide was dried with a dryer. For each slide, 2 μL of the EGFR DNA probe and CEP7 DNA probe solution included in the kit was added to a 0.5 ml microtube, and heated in a thermostat at 75 ° C. for 5 minutes. The microtube was immediately transferred to ice and allowed to stand for 5 minutes or more. 2 μL of this probe solution was dropped on the cells fixed on the slide. A cover glass was placed with care to prevent bubbles from entering. Next, a parafilm (trade name) that was slightly larger than the cover glass was placed and sealed with bonds. The slide was transferred to a moist chamber and allowed to react overnight in a 37 ° C. incubator. The parafilm (trade name) was peeled off, placed in a 2x SSC solution at room temperature, and waited for the cover glass to peel off naturally. Washed with 50% Formamid / 2x SSC solution at 45-47 ° C for 15 minutes. Subsequently, the plate was washed with a 2 × SSC solution at 45 to 47 ° C. for 10 minutes and with a 2 × SSC / 0.1% Tween 20 solution at 45 to 47 ° C. for 5 minutes. After washing, it was dried with a dryer. The dried slide was encapsulated with DAPI mounting medium. The FISH method was started after 24 hours from the CTC count.

4). Fluorescence microscope observation The analysis was performed by observing with a fluorescence microscope at a magnification of 1000 times. The nuclei were stained in weak blue by DAPI, and the position where the EGFR DNA probe and CEP7 DNA probe were hybridized could be clearly observed without being disturbed by the fluorescent label on the cell surface.

Claims (6)

A method for inspecting a floating cell , comprising subjecting a floating cell fluorescently stained with a cell surface antigen to a fluorescence in situ hybridization method, then focusing on the cell nucleus and observing the nucleus with a fluorescence microscope, The floating cells are cancer cells circulating in the blood, the floating cells subjected to the fluorescence in situ hybridization method are those in which the nucleus is also fluorescently stained, and observation in the fluorescence in situ hybridization method Is a method for examining floating cells, which is carried out after 15 hours or more have passed after fluorescent staining of a cell surface antigen .
. The method of claim 1 wherein said cancer cells are breast cancer cells. The method according to claim 2 , wherein the surface antigen is cytokeratin. The fluorescence in situ probe used in the hybridization method and hybridized with EGFR gene, EGFR / gene detection probe or HER-2 / neu gene hybridized claim a HER-2 / neu gene detection probe 2 Or the method of 3 . The method according to any one of claims 1 to 4 , wherein the observation with the fluorescence microscope is performed at a maximum magnification of the fluorescence microscope. 6. The method according to claim 5 , wherein the magnification is 1000 times.