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WO2011099565A1 - Méthode de détection de cellules cancéreuses dans le sang et programme utilisé à cet effet - Google Patents

Méthode de détection de cellules cancéreuses dans le sang et programme utilisé à cet effet Download PDF

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Publication number
WO2011099565A1
WO2011099565A1 PCT/JP2011/052892 JP2011052892W WO2011099565A1 WO 2011099565 A1 WO2011099565 A1 WO 2011099565A1 JP 2011052892 W JP2011052892 W JP 2011052892W WO 2011099565 A1 WO2011099565 A1 WO 2011099565A1
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Prior art keywords
cell
fluorescence
cells
oncolytic virus
blood
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Japanese (ja)
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佐藤　淳
眞砂　明典
幸夫 辻野
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Sysmex Corp
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Sysmex Corp
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage

Definitions

  • the present invention relates to a method for detecting cancer cells from a blood sample collected from a subject and a program used therefor.
  • Cancer metastasis is considered to occur when cancer cells in the primary lesion spread throughout the body through blood vessels and lymphatic vessels, and some of them engraft in other parts of the organ. Cancer cells circulating in the blood are called CTC (CirculatingirTumor Cell). It has been reported that the number of CTCs in the blood correlates with cancer metastasis and prognosis. Therefore, measurement of the number of CTCs is considered useful as a method for predicting the prognosis and therapeutic effect of metastatic cancer such as metastatic breast cancer.
  • Cancer cells are known to have increased telomerase activity that is hardly detected in most normal cells. Therefore, a virus (Oncolytic Virus) carrying a replication cassette containing a telomerase promoter and a labeled cassette containing a gene for a labeled protein (eg, green fluorescent protein (GFP)) is grown in cancer cells.
  • a technique for specifically labeling cancer cells is known (Patent Document 1).
  • An oncolytic virus having a GFP gene is commercially available as Telomescan (registered trademark) (OBP-401). Telomescan (registered trademark) can specifically grow in cancer cells and produce GFP, whereby the cancer cells can specifically emit fluorescence.
  • telomescan registered trademark
  • OBP-401 telomescan (registered trademark)
  • GFP fluorescence emission of GFP
  • Non-Patent Document 1 a cancer cell in blood, that is, CTC is specifically identified. Can be detected.
  • oncolytic virus infects not only cancer cells but also some normal cells such as leukocytes (some monocytes and lymphocytes) and proliferates. . Therefore, when CTC is detected using an oncolytic virus that expresses a fluorescent protein, the fluorescence intensity may be detected in normal cells other than cancer cells. As a result, cancer cells may not be detected accurately.
  • the present invention uses an oncolytic virus that expresses a fluorescent protein to detect cancer cells in blood that can accurately determine whether cells contained in a blood sample collected from a subject are cancer cells. It is an object to provide a method and a program used therefor.
  • the present inventors have found that it is possible to accurately determine whether a cell is a cancer cell by using an oncolytic virus that expresses a fluorescent protein and using the fluorescence information and cell size information of the cell. Completed the invention.
  • the present invention includes a step of preparing a measurement sample by treating cells contained in a blood sample collected from a subject with an oncolytic virus that expresses a fluorescent protein, and the measurement sample includes Blood, including a step of acquiring fluorescence information and cell size information of a cell, and a step of determining whether or not the cell is a cancer cell based on the fluorescence information and cell size information acquired in the acquisition step A method for detecting a middle cancer cell is provided.
  • the present invention provides a computer based on the information acquired by the acquisition means, the acquisition means for acquiring fluorescence information and cell size information of cells that have been infected and proliferated by an oncolytic virus in a blood sample, Provided is a blood cancer cell detection program for functioning as determination means for determining whether or not a cell is a cancer cell, and display means for displaying the determination result of the determination means.
  • the present invention it is possible to accurately determine whether a cell contained in a blood sample collected from a subject is a cancer cell using an oncolytic virus that expresses a fluorescent protein. It can be detected with high accuracy.
  • FIG. 3 is a scattergram in Example 1 with an integral value on the X axis and a volume on the Y axis.
  • FIG. 2 is a scattergram in Example 1 with a peak value on the X axis and a volume on the Y axis.
  • FIG. 3 is a scattergram in Example 1 with an integral value on the X axis and an area on the Y axis.
  • FIG. 3 is a scattergram in Example 1 with an integral value on the X axis and a diameter on the Y axis.
  • FIG. 2 is a scattergram in Example 1 with an integral value on the X axis and a perimeter on the Y axis.
  • Example 1 it is a three-dimensional frequency distribution diagram which took the area on the X-axis, the peak value on the Y-axis, and the integrated value on the Z-axis.
  • the method for detecting cancer cells in blood is as follows. (1) A step of preparing a measurement sample (preparation step) by treating cells contained in a blood sample collected from a subject with an oncolytic virus expressing a fluorescent protein; (2) a step of acquiring fluorescence information and cell size information of cells contained in the measurement sample (acquisition step); (3) A step of determining whether or not the cell is a cancer cell based on the fluorescence information and the cell size information acquired in the acquisition step (determination step) including.
  • the “blood sample” may be either blood collected from a subject or processed blood obtained by processing blood.
  • the blood sample is preferably a blood sample obtained by removing serum from whole blood, particularly peripheral blood, in that the determination efficiency is further improved.
  • Subjects include patients who are trying to determine whether they have cancer cells, such as patients suspected of suffering from cancer or patients suffering from cancer.
  • a known method can be used as a method for removing serum from whole blood.
  • a method of centrifuging whole blood to which an anticoagulant for example, ethylenediaminetetraacetic acid, sodium citrate, heparin, etc.
  • Centrifugation is preferably performed at 500 rpm to 3500 rpm for 3 to 30 minutes.
  • the fluorescent protein a known protein that is usually used in the biochemical field can be used.
  • fluorescent proteins such as green fluorescent protein (GFP) and variants thereof (for example, Enhanced-humanized GFP (EGFP), red-shiftGFP (rsGFP)), yellow fluorescent protein (YFP), blue fluorescent protein (BFP), etc. are used. be able to.
  • the fluorescent protein is preferably a green fluorescent protein.
  • the “oncolytic virus” is a restricted-proliferating virus that cannot propagate in normal cells but can specifically propagate in cancer cells.
  • the virus in which the promoter which shows a promoter activity specifically in a cancer cell was integrated is mentioned.
  • Examples of promoters that exhibit specific promoter activity in cancer cells include human telomerase promoter, human prostate cancer specific antigen (PSA) promoter, human alpha protein (AFP) promoter, fetus Sex cancer antigen (CEA) promoter and the like.
  • PSA human prostate cancer specific antigen
  • AFP human alpha protein
  • CEA fetus Sex cancer antigen
  • the human telomerase promoter is preferred in that it can exhibit promoter activity in many types of cancer cells.
  • the hTERT promoter which is a gene encoding human telomerase reverse transcriptase, is more preferred.
  • SEQ ID NO: 1 is the nucleic acid sequence of the hTERT promoter.
  • the hTERT promoter the entire 455 bp nucleic acid sequence shown in SEQ ID NO: 1 may be used, but the 181 bp region 5 ′ upstream of SEQ ID NO: 1 is considered to be an important core region for downstream gene expression. Therefore, a nucleic acid sequence containing at least this core region may be used.
  • the above-mentioned oncolytic virus expresses a fluorescent protein. That is, the oncolytic virus has a gene encoding a fluorescent protein.
  • the gene for the fluorescent protein can be placed under the control of the cancer cell-specific promoter.
  • the above-mentioned fluorescent protein gene can be placed under the control of a promoter capable of exhibiting promoter activity in an oncolytic virus.
  • promoters that can control the expression of the fluorescent protein gene include cytomegalovirus (CMV) promoter, SV40 late promoter, MMTV LTR promoter, RSV LTR promoter, SR ⁇ promoter, and the like.
  • CMV cytomegalovirus
  • SV40 late promoter SV40 late promoter
  • MMTV LTR promoter MMTV LTR promoter
  • RSV LTR promoter SR ⁇ promoter
  • the fluorescent protein gene is preferably placed under the control of a CMV promoter or hTERT promoter.
  • the nucleic acid sequences of these promoters are known.
  • An expression cassette comprising at least a gene encoding the above fluorescent protein and a promoter for expressing it (a cancer cell-specific promoter or a promoter capable of exhibiting promoter activity in an oncolytic virus) is a common genetic engineering It can be obtained by a technique. For example, a gene encoding a fluorescent protein and a promoter are amplified by PCR or the like based on a known sequence, each gene obtained is ligated to an appropriate plasmid, and a necessary portion is excised, thereby expressing an expression cassette. Can be obtained. See, for example, International Publication No. 2006/36004.
  • the above-mentioned oncolytic virus may have a replication cassette in which a gene required for virus growth is linked downstream of the above cancer cell-specific promoter.
  • genes necessary for virus growth include early genes (EIA, EIB, etc.), IRES that are protein synthesis initiation signals specific to the Picornaviridae family, and the like. The nucleic acid sequences of these genes are known.
  • the above replication cassette can be prepared by a normal genetic engineering technique.
  • the above cancer cell-specific promoter and, if necessary, a gene necessary for virus growth are amplified by polymerase chain reaction (PCR) or the like based on a known sequence, and each of the obtained genes is converted into an appropriate plasmid.
  • the replication cassette can be obtained by linking to and cutting out the necessary part. See, for example, International Publication No. 2006/36004.
  • virus examples include those derived from adenovirus, herpes simplex virus, Sendai virus, and reovirus.
  • human adenovirus is preferred.
  • Telomescan registered trademark
  • E1A gene E1A gene
  • IRES gene and E1B gene E1B gene
  • an expression cassette containing the CMV promoter and GFP gene OBP-401 (manufactured by Oncoris Bioformer) is preferred.
  • This embodiment is a step of preparing a measurement sample by treating cells contained in a blood sample with an oncolytic virus that expresses a fluorescent protein (hereinafter referred to as “preparation step”). Included).
  • the oncolytic virus expressing the fluorescent protein is infected with cancer cells in the blood sample.
  • fluorescent protein can be expressed in cancer cells.
  • the treatment of the cells contained in the blood sample with the oncolytic virus may be performed by a known method, and is not particularly limited.
  • an oncolytic virus can be infected with cancer cells in a blood sample by incubating the blood sample with an oncolytic virus in the presence of a medium commonly used for animal cell culture. Examples of the medium used for incubating the blood sample and oncolytic virus include RPMI-1640, Dulbecco's modified Eagle medium (DMEM), minimal essential medium (MEM), and the like.
  • the oncolytic virus When incubating a blood sample with an oncolytic virus, it is preferable to incubate the oncolytic virus with the blood sample in an amount of 6 ⁇ 10 4 to 6 ⁇ 10 8 PFU (Plaque Forming Unit) per ml of blood sample. .
  • the time and temperature at which the above blood sample is incubated with the oncolytic virus may be any conditions as long as the oncolytic virus can grow on cancer cells in the blood sample, and can be appropriately adjusted depending on the type of oncolytic virus.
  • the incubation can be performed at a temperature of 25 to 40 ° C. for 1 to 36 hours, more preferably at 30 to 37 ° C. for 12 to 24 hours.
  • the preparation step of the present embodiment may further include mixing a blood sample treated with an oncolytic virus with a nonionic surfactant and a fixing agent.
  • Leukocytes monoocytes, lymphocytes, etc.
  • telomerase enzymes that are specifically expressed in cancer cells such as telomerase. Therefore, when cells contained in a blood sample are treated with an oncolytic virus that expresses a fluorescent protein, the oncolytic virus may grow on normal white blood cells. As a result, fluorescent protein-derived fluorescence may occur from normal white blood cells. Therefore, in the acquisition process to be described later, fluorescence information that becomes noise increases.
  • the order of mixing the blood sample treated with the oncolytic virus with the nonionic surfactant and the immobilizing agent is not particularly limited.
  • the immobilizing agent may be mixed after mixing a nonionic surfactant and a blood sample treated with oncolytic virus.
  • a nonionic surfactant may be mixed after mixing the immobilizing agent and the blood sample treated with the oncolytic virus.
  • a blood sample treated with a nonionic surfactant, an immobilizing agent and an oncolytic virus may be mixed simultaneously.
  • the order of mixing the nonionic surfactant after mixing the immobilizing agent and the blood sample treated with the oncolytic virus is more preferable.
  • the nonionic surfactant may be any one of a polyoxyethylene surfactant and a fatty acid ester surfactant of a polyoxy compound, but a polyoxyethylene nonionic surfactant is more preferable.
  • Polyoxyethylene nonionic surfactants include higher alcohol / ethylene oxide adducts, alkylphenol / ethylene oxide adducts, higher fatty acids / ethylene oxide adducts, higher aliphatic amines / ethylene oxide adducts, higher fatty acid amide / ethylene oxide adducts.
  • the nonionic surfactant in the present embodiment is preferably a higher alcohol / ethylene oxide adduct.
  • the alkyl group of the higher alcohol of the higher alcohol / ethylene oxide adduct may be linear or branched, and preferably has 12 to 20 carbon atoms, more preferably 15 to 20 carbon atoms.
  • the higher alcohol / ethylene oxide adduct preferably has an ethylene oxide polymerization number of 15 to 40, more preferably 20 to 30.
  • polyoxyethylene octyldodecyl ether in which the alkyl group of the higher alcohol is branched, the number of carbon atoms is 18, and the polymerization number of ethylene oxide is 25 is more preferable.
  • the concentration of the nonionic surfactant when mixed with the blood sample and the fixing agent can be appropriately selected depending on the kind of the nonionic surfactant, but is preferably 0.005 to 0.5% by weight, more preferably 0.045 to 0.055% by weight.
  • aldehyde compounds such as paraformaldehyde, glutaraldehyde, and formaldehyde are preferable. Of these, paraformaldehyde is more preferable.
  • the concentration of the fixing agent when mixed with the blood sample and the nonionic surfactant can be appropriately selected depending on the type of the fixing agent, but is preferably 0.005 to 0.5% by weight, and preferably 0.045 to 0.8%. 055% by weight is more preferred.
  • the above mixing is preferably performed at pH 6 to 9 and osmotic pressure 200 to 400 m0sm.
  • This embodiment includes a step of acquiring cell fluorescence information and cell size information contained in the measurement sample obtained in the preparation step (hereinafter also referred to as “acquisition step”).
  • Fluorescence information in the present embodiment is information obtained from fluorescence emitted by a fluorescent protein.
  • the fluorescence intensity detected from the fluorescence emitted by the fluorescent protein the peak value of the fluorescence intensity, the minimum value of the fluorescence intensity, the average value of the fluorescence intensity, the calculated numerical value derived from the fluorescence intensity such as the integrated value of the fluorescence intensity, etc. It is done.
  • fluorescence information in the present embodiment a peak value of fluorescence intensity and an integral value of fluorescence intensity are preferable.
  • Cell fluorescence information can be obtained by a known method. More specifically, the fluorescence information of a cell can be acquired using a fluorescence microscope, a flow cytometer, or the like.
  • a measurement sample obtained in the above-described preparation step is smeared on a slide glass to prepare a smear. At this time, it is preferable to create a slide so that cells do not overlap.
  • the fluorescence in the cells present in the smear prepared as described above is observed with a fluorescence microscope equipped with a CCD camera.
  • the excitation light irradiated by the fluorescence microscope can be appropriately selected depending on the type of fluorescent protein used.
  • fluorescent protein for example, when GFP is used as the fluorescent protein, green light (G excitation / 546 nm) can be used as excitation light.
  • the fluorescence in the above-described cells is imaged using a CCD camera of a fluorescence microscope.
  • the above-described fluorescence information of the cell is acquired by performing image processing on an image obtained by imaging.
  • the image processing for acquiring the fluorescence information can be appropriately performed according to the type of the fluorescence information. More specifically, the fluorescence information can be acquired by processing an image obtained by imaging using a computer in which a computer program for image processing is installed.
  • Computer programs for image processing are well known, and storage media (image processing software) in which such computer programs are stored are generally commercially available. Examples of the image processing software include ImagePro (registered trademark) (manufactured by Roper Industries), WinROOF (registered trademark) (Mitani Corporation), and MetaMorph (registered trademark) (Molecular Devices).
  • the brightness (maximum brightness) of the pixel having the highest brightness among the pixels having the brightness values within the range selected in the above image is set to the fluorescence intensity. Get as the peak value of.
  • the integrated value of the fluorescence intensity a value obtained by integrating the luminance of each pixel having the luminance value within the range selected in the above-described image is acquired as the integrated value of the fluorescence intensity.
  • the flow cytometer which can be used is a flow cytometer which can detect fluorescence.
  • a conventionally known flow cytometer including a flow cell, a light source, a detection unit (fluorescence detection unit) that detects fluorescence, an analysis unit that analyzes data output from the fluorescence detection unit, and the like can be used.
  • ⁇ A measurement sample passing through the flow cell of the flow cytometer is irradiated with light from a light source.
  • the light source of the flow cytometer only needs to have a wavelength suitable for excitation of the fluorescent protein to be used.
  • the light source include a semiconductor laser, an argon laser, and a He—Ne laser.
  • the fluorescence detection unit may include a light receiving element that is generally used in a flow cytometer and can detect fluorescence. Specific examples of the fluorescence detection unit include a photomultiplier tube and an avalanche photodiode.
  • the wavelength of fluorescence detected by the fluorescence detection unit can be appropriately selected depending on the type of fluorescent protein used. For example, when GFP is used as the fluorescent protein, fluorescence at 480 to 580 nm may be detected by the fluorescence detection unit.
  • Fluorescence detected by the fluorescence detection unit is output to the analysis unit as an electrical signal.
  • the analysis unit performs analysis using the electrical signal output from the fluorescence detection unit, and acquires fluorescence information.
  • the analysis unit can analyze the waveform of the fluorescence intensity over time by obtaining the fluorescence intensity when an electrical signal is output from the fluorescence detection unit.
  • An example of the analysis result of the waveform of the fluorescence intensity over time by this analysis unit is shown in FIG. In FIG. 1, the vertical axis indicates the fluorescence intensity, and the horizontal axis indicates the detection time. In this case, for example, the maximum height of the fluorescence intensity waveform can be acquired as the peak value of the fluorescence intensity. Further, the area value of the waveform of the fluorescence intensity can be acquired as an integrated value of the fluorescence intensity.
  • the “cell size information” in the present embodiment is not particularly limited as long as it is information reflecting the cell size.
  • Examples of the cell size information include a cell diameter, a cell area, a cell volume, a cell perimeter, and a roundness.
  • cell diameter, cell area, cell volume, and cell perimeter are preferred.
  • the cell size information can be obtained by a known method. More specifically, cell size information can be acquired using a fluorescence microscope, a flow cytometer, or the like.
  • the area occupied by the cell in the above-mentioned image is measured to calculate an average value and converted to a diameter when the cell is assumed to be spherical.
  • the area consisting of pixels having a luminance value within the range selected in the above-mentioned image is measured.
  • the volume of a cell the three-dimensional image of a cell is constructed
  • the length of the outer periphery (outline) of a cell is measured.
  • examples of the flow cytometer that can be used include conventionally known flow cytometers that can acquire cell size information by electrical or optical measurement. . More specifically, an electric resistance type flow cytometer that can acquire cell size information by electric resistance, an optical flow cytometer that can acquire cell size information by scattered light, and the like can be mentioned. A flow cytometer that can perform both electrical and optical measurements can also be used.
  • An electric resistance type flow cytometer for acquiring cell size information is a measuring unit comprising an orifice for introducing the above-described measurement sample and a pair of electrodes electrically connected by an electrolyte through the orifice. And an analysis unit. The interelectrode electrical resistance generated when the measurement sample passes through the orifice of the flow cytometer is measured by the measurement unit of the flow cytometer.
  • the inter-electrode electrical resistance measured by the measurement unit is output as an electrical signal to the analysis unit of the flow cytometer.
  • the analysis unit performs analysis based on the electrical signal output from the measurement unit, and acquires cell size information.
  • the analysis unit can analyze the waveform of the electrical resistance over time by obtaining the electrical resistance when the electrical signal is output from the measurement unit.
  • An example of the analysis result of the electrical resistance waveform over time by the analysis unit is shown in FIG. In FIG. 2, the vertical axis represents electric resistance, and the horizontal axis represents detection time.
  • the magnitude of electrical resistance strongly correlates with cell volume.
  • the maximum height of the waveform of electric resistance can be acquired as the volume of the cell.
  • the cell diameter, the cell area, and the cell perimeter can be obtained from the obtained cell volume.
  • An optical flow cytometer for acquiring cell size information is a detection unit that detects scattered light in addition to or in addition to the fluorescence detection unit of the flow cytometer used in the above-described fluorescence detection.
  • the scattered light detection unit may include a light receiving element that is generally used in a flow cytometer and can detect scattered light. Specific examples of the scattered light detection unit include a photodiode and an avalanche photodiode. Scattered light generated when the measurement sample passes through the flow cell of the flow cytometer is measured by the scattered light detector. Here, forward scattered light is preferable as the scattered light.
  • Scattered light measured by the scattered light detection unit is output as an electrical signal to the analysis unit of the flow cytometer.
  • the analysis unit performs analysis based on the electrical signal output from the scattered light detection unit, and acquires cell size information.
  • the analysis unit can analyze the waveform of the forward scattered light intensity over time by obtaining the intensity of the forward scattered light when the electrical signal is output from the scattered light detection unit.
  • An example of the analysis result of the waveform of the forward scattered light intensity over time by this analysis unit is shown in FIG.
  • the vertical axis represents the forward scattered light intensity
  • the horizontal axis represents the detection time.
  • the forward scattered light intensity strongly correlates with the cell surface area
  • the waveform width of the forward scattered light intensity strongly correlates with the cell length.
  • the maximum height of the waveform of the forward scattered light intensity can be acquired as the cell surface area.
  • the maximum width of the waveform of the forward scattered light intensity can be acquired as the cell diameter.
  • the cell volume, cell area, and cell perimeter can also be obtained from the obtained cell surface area and cell diameter.
  • This embodiment includes a step of determining whether a cell is a cancer cell based on the fluorescence information and cell size information acquired in the acquisition step (hereinafter also referred to as “determination step”). Including.
  • the fluorescence information and the cell size information obtained in the acquisition step are used to separate cancer cells in the measurement sample from other cells, debris, and the like in the measurement sample. More specifically, a cell in a measurement sample having predetermined fluorescence information and predetermined cell size information is determined as a cancer cell.
  • the threshold value is determined from the accumulation of data related to fluorescence information and cell size information of cancer cells in blood, and data related to fluorescence information and cell size information of cells other than cancer cells contained in blood.
  • a plurality of data including a value related to fluorescence information and a value related to cell size information is acquired from a plurality of measurement samples including only cancer cells based on the procedure of the acquisition step described above.
  • a plurality of data including a value related to fluorescence information and a value related to cell size information is obtained from a measurement sample prepared from a blood sample not containing cancer cells. Based on the acquired data, it is possible to determine whether or not the cell is a cancer cell from cells and debris contained in a measurement sample prepared from a blood sample. Threshold values of a value relating to fluorescence information and a value relating to cell size information, respectively. Can be set.
  • FIG. 4 shows a schematic diagram of a scattergram based on fluorescence information and cell size information obtained in the acquisition step.
  • the X axis is fluorescence information
  • the Y axis is cell information.
  • cancer cells mainly appear in the area A in FIG.
  • oncolytic viruses may grow on leukocytes. Therefore, white blood cells may show values related to fluorescence information similar to cancer cells.
  • the inventors' investigation has revealed that the value relating to the cell size information from leukocytes where the oncolytic virus grows is smaller than that of cancer cells. Therefore, white blood cells mainly appear in the region B in FIG.
  • oncolytic viruses do not grow in cells other than cancer cells and leukocytes and debris. Therefore, the values relating to fluorescence information of cells other than cancer cells and leukocytes and debris are extremely small.
  • cells other than cancer cells and leukocytes, debris, and the like mainly appear in the region C of FIG. That is, the cells in which the fluorescence information and the cell size information obtained in the acquisition step described above appear in the area A in FIG. 4 can be determined as cancer cells.
  • the computer 100 mainly includes a main body 110, a display unit 120, and an input unit 130.
  • a CPU 110a, a ROM 110b, a RAM 110c, a hard disk 110d, a reading device 110e, an input / output interface 110f, and an image output interface 110h are connected to each other via a bus 110i so that data communication is possible.
  • the CPU 110a can execute computer programs stored in the ROM 110b and computer programs loaded in the RAM 110c.
  • the ROM 110b is configured by a mask ROM, PROM, EPROM, EEPROM, or the like, and is recorded with a computer program executed by the CPU 110a and data used therefor.
  • the RAM 110c is configured by SRAM, DRAM, or the like. The RAM 110c is used for reading computer programs recorded in the ROM 110B and the hard disk 110d. Further, when these computer programs are executed, they are used as a work area of the CPU 110a.
  • the hard disk 110d is installed with various computer programs to be executed by the CPU 110a such as operating system and application system program, and data used for executing the computer program.
  • An application program 140a described later is also installed in the hard disk 110d.
  • the reading device 110e is configured by a flexible disk drive, a CD-ROM drive, a DVD-ROM drive, or the like, and can read a computer program or data recorded on the portable storage medium 140.
  • the portable storage medium 140 stores an application program 140a related to the acquisition process and the determination process in a computer.
  • the CPU 110a reads the application program 140a from the portable storage medium 140, and the application program 140a is stored in the hard disk 110d. It is also possible to install.
  • an operating system that provides a graphical user interface environment such as Windows (registered trademark) manufactured and sold by Microsoft Corporation in the United States is installed in the hard disk 110d.
  • Windows registered trademark
  • the application program 140a related to the acquisition process and the determination process described above operates on the operating system.
  • the input / output interface 110f includes, for example, a serial interface such as USB, IEEE1394, RS-232C, a parallel interface such as SCSI, IDE, IEEE1284, and an analog interface including a D / A converter, an A / D converter, and the like. ing.
  • An input device 130 including a keyboard and a mouse is connected to the input / output interface 110f, and the user can input data to the computer main body 110 by using the input device 130.
  • the input / output interface 110f is connected to a fluorescence microscope 200 equipped with a CCD camera. Thereby, the computer main body 110 can acquire the imaged image data from the fluorescence microscope 200.
  • the image output interface 110h is connected to a display unit 120 constituted by an LCD, a CRT, or the like, and outputs a video signal corresponding to the image data given from the CPU 110a to the display unit 120.
  • the display unit 120 outputs image data according to the input video signal. Further, the display unit 120 outputs a determination result given from a CPU 110a described later.
  • FIG. 6 is an example of a flowchart showing processing for determining whether or not the cancer cell is executed by the CPU 110a.
  • image data obtained by imaging cells included in the measurement sample from the fluorescence microscope 200 is acquired by the CPU 110a via the input / output interface 110f and stored in the hard disk 110d (step S11).
  • the CPU 110a reads the application program 140a stored in the hard disk 110d, performs image processing on the image data, and executes processing for calculating the integral value of fluorescence intensity and the volume of cells as parameters (step S12). .
  • the CPU 110a determines whether or not the cell is a cancer cell based on the integrated value of the fluorescence intensity, which is the calculated parameter, and the cell volume (step S13).
  • the hard disk 110d stores the coordinates of the parameter area where the cancer cells appear in the area A of FIG. 7 with respect to the integrated value of the fluorescence intensity and the volume of the cell.
  • the CPU 110a determines whether or not the cell is a cancer cell from the integrated value of the fluorescence intensity, which is the calculated parameter, the volume of the cell, and the parameter area. Specifically, when each parameter regarding a cell enters the parameter area, it is determined that the cell is a cancer cell. If the cell is detached, it is determined that the cell is not a cancer cell. Then, the CPU 110a stores the scattergram shown in FIG. 7 as a determination result in the RAM 110c and outputs it to the display unit 120 via the image output interface 110f (step S14).
  • the scattergram is used as the determination result described above, but the determination result is not limited to this.
  • the determination result include the count number of cells determined to be cancer cells, image data of cells determined to be cancer cells, and a value (positive or negative) indicating the presence or absence of cancer cells in the measurement sample.
  • the determination result output to the display unit is not limited to one. At least one selected from the determination results can be output to the display unit.
  • the parameters of the fluorescence information and the cell size information are not limited thereto. Is not to be done.
  • the parameters of the fluorescence information include a peak value of fluorescence intensity, a minimum value of fluorescence intensity, and an average value of fluorescence intensity.
  • the cell size information parameters include cell area, cell perimeter, cell diameter, and cell roundness.
  • the parameter region of the integrated value of the fluorescence intensity and the cell area is used as the parameter region where cancer cells appear in the determination step performed by the computer. It is not limited to.
  • the parameter area for example, the peak value of the fluorescence intensity shown in the area A of FIG. 8 and the parameter area for the cell volume, the integrated value of the fluorescence intensity shown in the area A of FIG. 9 and the parameter area for the cell area, FIG.
  • the number of parameters used in the parameter area is not limited to two.
  • At least one selected from the parameters of the fluorescence information and at least one selected from the parameters of the cell size information can be used as parameters.
  • three parameters including a peak value of fluorescence intensity, an integrated value of fluorescence intensity, and a cell area can also be used as parameters used in the parameter region.
  • the example in which the fluorescence microscope 200 equipped with the CCD camera is connected to the computer 100 via the input / output interface 110f is shown, but these aspects are not limited thereto.
  • a flow cytometer 300 may be connected instead of the fluorescence microscope 200.
  • the computer main body 110 acquires data relating to fluorescence and data relating to electrical resistance and / or scattered light from the flow cytometer 300.
  • CPU110a calculates the parameter of fluorescence information from the acquired data regarding fluorescence.
  • parameters of cell size information are calculated from data relating to electrical resistance and / or scattered light.
  • the computer 100 and the flow cytometer 300 may be separate or integrated.
  • the measurement sample used in this example was prepared as follows.
  • a mononuclear cell that is one of white blood cells (WBC) was prepared as follows. 1. Into a 12-15 mL centrifuge tube, put 5.0 mL of Polymorphprep TM (Daiichi Kagaku), a reagent for separating mononuclear cells, and collect a vacuum blood collection tube (Benoject II vacuum blood collection tube, 3.8% citric acid) The sample was collected from 4 healthy individuals using 4.5 ml of sodium (manufactured by Terumo), and 5.0 mL of blood within 2 hours after the collection was overlaid. 2.
  • MB468 which is one of the breast cancer cells, was cultured in a culture solution (DMEM-F12 (Sigma) containing 10% FBS (Hyclone)) so as to be 90% confluent in a 125 cm 2 flask.
  • the obtained cultured cells were suspended in 1000 ⁇ l of DMEM-F12 so as to have about 3 ⁇ 10 5 cells to prepare MB468 cell samples.
  • RPMI-1640 was added to the obtained sample to make up to 10 ml.
  • Oncolytic virus Telomescan (registered trademark) (OBP-401) was added to a final concentration of 6 ⁇ 10 6 PFU / ml and cultured at 37 ° C. for 24 hours while rotating. The culture solution was centrifuged at 1500 rpm for 5 minutes with a weak brake, and the supernatant was removed to obtain cells on which oncolytic virus had grown.
  • PFA paraformaldehyde
  • Emulgen 2025G has the following structural formula:
  • n 25.
  • Imaging 7 types of beads (7 Peaks Beads (Cyto-Cal Multifluor Fluorescence Intensity Calibrator, Thermo Scientific, FC3M)) with known fluorescence intensities under exposure times of 20 ms, 50 ms, 100 ms, 200 ms and 400 ms A calibration curve for each exposure time was prepared.
  • the maximum fluorescence intensity (cell peak value (MEFL)) from the treated sample and the integration of the fluorescence intensity Values (MEFL), fluorescence distribution area (cell area (pixel 2 )), and fluorescence distribution perimeter (cell perimeter (pixel)) were digitized.
  • the cell volume (pixel 3 ) was calculated using the fluorescence distribution area assuming that the cell was a true sphere.
  • the cell diameter (pixel) was similarly calculated using the fluorescence distribution area.

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Abstract

La présente invention concerne une méthode de détection de cellules cancéreuses dans le sang qui comprend les étapes consistant à traiter, au moyen d'un virus oncolytique exprimant une protéine fluorescente, les cellules contenues dans un échantillon de sang prélevé chez un sujet ; à obtenir des données de fluorescence sur les cellules et des informations relatives à la taille des cellules ; et à déterminer si les cellules sont des cellules cancéreuses ou pas sur la base des données de fluorescence et des informations relatives à la taille des cellules obtenues. L'invention concerne également un programme utilisé pour la mise en œuvre de ladite méthode.
PCT/JP2011/052892 2010-02-10 2011-02-10 Méthode de détection de cellules cancéreuses dans le sang et programme utilisé à cet effet Ceased WO2011099565A1 (fr)

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WO2018180012A1 (fr) * 2017-03-31 2018-10-04 ソニー株式会社 Dispositif de traitement d'informations, système de traitement d'informations et procédé de traitement d'informations
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US9945783B2 (en) 2012-03-30 2018-04-17 Sysmex Corporation Cervical cancer information providing method and device
JPWO2015174539A1 (ja) * 2014-05-13 2017-04-20 学校法人順天堂 細胞の検出方法
WO2015174539A1 (fr) * 2014-05-13 2015-11-19 学校法人順天堂 Méthode pour la détection de cellules
JP2016174578A (ja) * 2015-03-20 2016-10-06 東ソー株式会社 微小粒子選別装置および前記装置を備えた微小粒子回収装置
JP2016128821A (ja) * 2016-01-20 2016-07-14 シスメックス株式会社 細胞分析方法および細胞分析装置
JPWO2018180012A1 (ja) * 2017-03-31 2020-02-13 ソニー株式会社 情報処理装置、情報処理システム、及び情報処理方法
WO2018180012A1 (fr) * 2017-03-31 2018-10-04 ソニー株式会社 Dispositif de traitement d'informations, système de traitement d'informations et procédé de traitement d'informations
WO2019013137A1 (fr) * 2017-07-10 2019-01-17 株式会社Ｉｄファーマ Composition pour le traitement du cancer
JP2019014696A (ja) * 2017-07-10 2019-01-31 株式会社Ｉｄファーマ 癌の治療組成物
JP7117087B2 (ja) 2017-07-10 2022-08-12 株式会社Ｉｄファーマ 癌の治療組成物
JP2020527274A (ja) * 2017-07-20 2020-09-03 上海睿▲玉▼生物科技有限公司Ｓｈａｎｇｈａｉ Ｒｕｉｙｕ Ｂｉｏｔｅｃｈ Ｃｏ．， Ｌｔｄ． 蛍光画像の蛍光強度確定方法及びシステム
CN113167786A (zh) * 2018-11-29 2021-07-23 希森美康株式会社 免疫细胞的分析方法及细胞分析装置
JPWO2022024564A1 (fr) * 2020-07-30 2022-02-03
WO2022024564A1 (fr) * 2020-07-30 2022-02-03 浜松ホトニクス株式会社 Dispositif d'évaluation, procédé d'évaluation, programme d'évaluation et support d'enregistrement
JP7644458B2 (ja) 2020-07-30 2025-03-12 浜松ホトニクス株式会社 判別装置、判別方法、判別プログラム及び記録媒体

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