WO2018159833A1 - Procédé de distinction de cellules, procédé d'inspection de cancer, dispositif de mesure, dispositif d'inspection de cancer, et programme d'inspection - Google Patents

Procédé de distinction de cellules, procédé d'inspection de cancer, dispositif de mesure, dispositif d'inspection de cancer, et programme d'inspection Download PDF

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Publication number
WO2018159833A1
WO2018159833A1 PCT/JP2018/008115 JP2018008115W WO2018159833A1 WO 2018159833 A1 WO2018159833 A1 WO 2018159833A1 JP 2018008115 W JP2018008115 W JP 2018008115W WO 2018159833 A1 WO2018159833 A1 WO 2018159833A1
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cell
discrimination
predetermined
cells
target cell
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PCT/JP2018/008115
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English (en)
Japanese (ja)
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宗彰 匹田
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株式会社ニコン
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/65—Raman scattering
    • 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
    • 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
    • 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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • 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
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor

Definitions

  • the present invention relates to a cell discrimination method, a cancer inspection method, a measurement device, a cancer inspection device, and an inspection program.
  • the amount of at least one substance or bond selected from the group consisting of a predetermined amino acid, a predetermined nucleobase, and a predetermined interatomic bond in a part or all of the target cell is a measurement step of measuring a corresponding signal, and a determination step of determining whether the target cell is a non-epithelial cancer cell based on the measured signal.
  • the measuring apparatus comprises at least one substance selected from the group consisting of a predetermined amino acid, a predetermined nucleobase, and a predetermined interatomic bond in a part or all of the target cell.
  • the test program includes at least one substance selected from the group consisting of a predetermined amino acid, a predetermined nucleobase, and a predetermined interatomic bond in a part or all of the target cell. Based on a signal corresponding to the amount of binding, a determination process for determining whether the target cell is a non-epithelial cancer cell, and information on the result of the determination process, or the target cell is acquired.
  • the processing device performs an output process for outputting information on the presence or absence, the degree, or the possibility of metastasis of the cancer to be examined.
  • FIG. 1 is a conceptual diagram for explaining circulating cancer cells and epithelial-mesenchymal transition.
  • FIG. 2 is a conceptual diagram for explaining a configuration of a cancer inspection apparatus related to a cell discrimination method according to an embodiment.
  • FIG. 3 is a conceptual diagram illustrating a configuration of an information processing apparatus related to a cell discrimination method according to an embodiment.
  • FIG. 4 is a diagram for explaining a method of discriminating cells based on discrimination parameters.
  • FIG. 5 is a diagram showing the structure of the flow channel structure.
  • FIG. 6 is a diagram showing a flow of Raman measurement in the cancer testing method, (a) is a conceptual diagram showing a case where details are not held in the holding unit, and (b) is shown in the holding unit.
  • FIG. 7 is a diagram showing a flow of Raman measurement in the cancer testing method
  • (a) is a conceptual diagram showing a case where cells are not held in the holding unit
  • (b) is a diagram showing how the holding unit holds the cells.
  • FIG. 8 is a diagram showing the flow of Raman measurement in the cancer testing method, (a) is a conceptual diagram showing a case where all the holding parts are filled with cells that have undergone epithelial-mesenchymal transition, (B) is a conceptual diagram when the cells that have undergone epithelial-mesenchymal transition are released from the holding unit.
  • FIG. 8 is a diagram showing the flow of Raman measurement in the cancer testing method
  • (a) is a conceptual diagram showing a case where all the holding parts are filled with cells that have undergone epithelial-mesenchymal transition
  • (B) is a conceptual diagram when the cells that have undergone
  • FIG. 9 is a flowchart showing the flow of the cancer testing method.
  • FIG. 10 is a diagram for explaining a program related to a cancer testing method.
  • FIG. 11 is a diagram showing the measurement results of Raman scattered light for ovarian cancer cells.
  • FIG. 12 is a diagram showing the measurement results of Raman scattered light for ovarian cancer cells.
  • cell discrimination method includes cancer (carcinoma), sarcoma, blood tumor and other malignant tumors.
  • FIG. 1 is a conceptual diagram for explaining circulating cancer cells (CTC: Circulating Tumor Cell) and epithelial-mesenchymal transition (EMT).
  • CTC Circulating Tumor Cell
  • EMT epithelial-mesenchymal transition
  • Epithelial cells constitute an epithelium that covers the surface of an individual or an organ of an individual, and are usually polar and are arranged in contact with adjacent cells by intercellular bonding.
  • a tissue containing epithelial cells becomes cancerous, it usually becomes a malignant tumor having well-differentiated epithelial-like properties (arrow A1).
  • CTC circulating cancer cells
  • Circulating cancer cells include non-epithelial cancer cells in addition to epithelial cancer cells that circulate in the blood with epithelial-like properties.
  • Non-epithelial cancer cells include cells that have become non-epithelial non-epithelial-like properties due to epithelial-to-mesenchymal transition (arrow A2) from epithelial cancer cells. Then, after forming micrometastases in the blood vessels, it undergoes a mesenchymal epithelial transition and becomes highly differentiated, thereby playing a major role in metastasis of malignant tumors.
  • the cell discrimination method of this embodiment is for discriminating non-epithelial cancer cells, and in addition to the discrimination of cells contained in blood as described above, the purpose is to detect non-epithelial cancer cells. It can be used for various purposes.
  • FIG. 2 is a diagram illustrating a configuration of the measurement apparatus 100 used in the cell discrimination method of the present embodiment.
  • the measuring device 100 measures the spectrum of Raman scattered light.
  • the measuring device 100 includes a measuring unit 2 and an information processing device 40.
  • the measurement unit 2 includes an irradiation optical system 10, an objective optical system 20, a detection optical system 30, and a cell delivery system 80.
  • the irradiation optical system 10 includes a light source device 11, a galvano scanner 12, relay lenses 13a and 13b, and an optical mirror 14.
  • the objective optical system 20 includes an optical mirror 21, an objective lens 22, and a stage 23.
  • the cell delivery system 80 includes a container 81 for containing the cell conditioning solution S, a pump 82, a flow channel structure 90, and a collection container 83 for collecting the cells R.
  • the flow path structure 90 is placed on the stage 23.
  • the detection optical system 30 includes a dichroic mirror 31, relay lenses 32 a and 32 b, and a spectrometer 33. In FIG. 1, the irradiation light and the detection light are schematically shown using a one-dot chain line. Control of the pump 82 by the information processing apparatus 40 is schematically indicated by a broken-line arrow A3.
  • the configuration of the measuring device 100 is not particularly limited.
  • the cell delivery system 80 may be configured as an independently controlled system that is separate from the measurement apparatus 100.
  • the light source device 11 includes a laser oscillation device such as a semiconductor laser.
  • the wavelength of the light oscillated by the light source device 11 is not particularly limited as long as the spectrum of scattered light can be measured, but a wavelength of 532 nm, 785 nm, or the like can be appropriately used from the viewpoint of measurement sensitivity and the like.
  • Light emitted from the light source device 11 enters the galvano scanner 12.
  • the galvano scanner 12 includes a movable mirror (not shown) and the like, and scans by changing the emitting direction of the laser beam.
  • the light emitted from the galvano scanner 12 enters the relay lenses 13a and 13b.
  • the light that has passed through the relay lenses 13 a and 13 b is reflected by the optical mirror 14 and the dichroic mirror 31 and then enters the objective optical system 20.
  • the light incident on the objective optical system 20 is reflected by the optical mirror 21, refracted by the objective lens 22, and, as will be described in detail later, the cells held in the holding portion formed in the flow path in the flow path structure 90. Is incident on.
  • target cells C there are no particular limitations on the type, state, or the like of cells that are to be subjected to Raman measurement (hereinafter referred to as target cells C).
  • the target cell C is a cell contained in the blood of a living body, particularly human blood, and the target cell C is held in the middle of the flow channel while being perfused, and Raman measurement is performed. Determine whether it is circulating cancer cells, epithelial cancer cells, or non-cancerous cells in the blood.
  • the target cell C may be fixed with formalin or the like and Raman measurement may be performed.
  • the light scattered by the target cell C in the flow channel structure 90 is transmitted through the objective lens 22, reflected by the optical mirror 21, and enters the detection optical system 30.
  • the light incident on the detection optical system 30 passes through the dichroic mirror 31, passes through the relay lenses 32a and 32b, and enters the spectroscope 33.
  • the spectroscope 33 is configured to include a detector such as a grating or a CCD, and obtains the intensity for each wavelength by splitting the incident light. If the intensity
  • the spectroscope 33 is configured to include a detector such as a grating or a CCD, and obtains the intensity for each wavelength by splitting the incident light. If the intensity
  • the cell delivery system 80 controls the movement and retention of the target cell C flowing through the flow channel structure 90, while passing the cell suspension S containing the target cell C and the buffer solution, which has been put into the container 81, along the tube. And send it out.
  • the target cell C may contain non-epithelial cancer cells Cn and cells Ce other than non-epithelial cancer cells.
  • the target cell C is collected in the collection container 83 when it is detected that it is a non-epithelial cancer cell Cn after undergoing Raman measurement. In addition, it can set suitably about which cell is collect
  • FIG. 3 is a diagram illustrating the configuration of the information processing apparatus 40.
  • the information processing apparatus 40 includes an input unit 41, a display unit 42, a communication unit 43, a storage unit 44, and a control unit 50.
  • the control unit 50 includes a parameter calculation unit 51, a determination unit 52, a flow rate control unit 53, and an information output unit 54.
  • the input unit 41 is configured by an input device such as a keyboard and a touch panel, and receives input data including a numerical value as a reference when determining the target cell C using a determination parameter described later.
  • the input unit 41 stores the received input data in the storage unit 44 described later.
  • the input data may be acquired via the communication unit 43 described later.
  • the display unit 42 is configured by a display device such as a liquid crystal monitor, and based on the results of cell discrimination such as the number of epithelial and / or non-epithelial cancer cells, and the cell discrimination, tumor progression, malignancy
  • the information (hereinafter referred to as cancer analysis information) obtained by analyzing the prognosis and / or the possibility of metastasis (hereinafter referred to as cancer analysis) is displayed. Further, the display unit 42 appropriately displays a spectrum, a statistical value obtained from the spectrum, and the like.
  • the display unit 42 may be configured to print and output the determination result on a paper medium such as thermal paper.
  • the communication unit 43 is configured by a communication device that performs communication using a communication network such as the Internet, and transmits a determination result of cell discrimination and cancer analysis information, and transmits and receives necessary data as appropriate.
  • the storage unit 44 is configured by a non-volatile memory or the like, and performs a cell discrimination or a cancer analysis using a program that causes the control unit 50 to perform processing, measurement data output from the spectrometer 33, and discrimination parameters. The numerical value used as the reference for the is stored.
  • the control unit 50 is configured by a processing device such as a CPU, functions as a main body for controlling the measurement device 100, and executes a program mounted on the storage unit 44 or a storage medium (not shown), thereby determining the cell. Various processing such as calculation processing and output processing are performed.
  • the parameter calculation unit 51 of the control unit 50 calculates a discrimination parameter used for cell discrimination from the spectral spectrum of the measurement data stored in the storage unit 44.
  • the discrimination parameter of the present embodiment is a numerical value obtained by quantifying a signal corresponding to a predetermined amino acid, a predetermined nucleobase, or a predetermined amount of interatomic bonds, or a numerical value calculated from two or more of the signals. Cell discrimination can also be performed using a plurality of discrimination parameters.
  • the discrimination parameter is selected according to the type of cell to be measured, measurement conditions, and the like.
  • the predetermined amino acid an aromatic amino acid, particularly tryptophan and / or phenylalanine can be selected.
  • cytosine can be selected as the predetermined nucleobase.
  • the predetermined interatomic bond an interatomic bond that serves as an index of the amount of the organic compound in part or all of the target cell C can be selected.
  • the interatomic bond that serves as an index of the amount of the organic compound particularly, a bond between a plurality of atoms including carbon atoms, that is, a double bond and an amide bond between carbon atoms, a CH bond in a saturated hydrocarbon, or the like is selected. be able to.
  • a double bond between carbon atoms present in the porphyrin ring of cytochrome c can be selected as the predetermined interatomic bond.
  • a method for quantifying a signal corresponding to a predetermined amino acid, a predetermined nucleobase, or a predetermined interatomic bond is not particularly limited, and various statistical values can be used.
  • the parameter calculation unit 51 can quantify the signal using the intensity of the wave number of the Stokes region of Raman scattered light corresponding to a predetermined amino acid, a predetermined nucleobase, or a predetermined interatomic bond.
  • the parameter calculation unit 51 corresponds to an intensity of 749 / cm corresponding to tryptophan, an intensity of wave number 1003 / cm corresponding to phenylalanine, an intensity of 782 / cm corresponding to cytosine, a double bond or an amide bond between carbon atoms.
  • the parameter calculation unit 51 not only has the above-mentioned wave number intensity, but also an appropriate wave intensity according to a predetermined amino acid, a predetermined nucleobase, or a predetermined type of interatomic bond related to the calculation of the discrimination parameter. Can be calculated.
  • the parameter calculation unit 51 can quantify the signal using the maximum intensity, area, and the like of a peak corresponding to a predetermined amino acid, a predetermined nucleobase, or a predetermined interatomic bond.
  • the parameter calculation unit 51 includes a peak including 749 / cm in the range of half width, a peak including 1003 / cm in the range of half width, a peak including 782 / cm in the range of half width, A peak including 1659 / cm in the range, a peak including 1583 / cm in the range of half width, or a peak including 1450 / cm in the range of half width, the maximum intensity, area, etc. are calculated and used as discrimination parameters.
  • the discrimination parameter can be calculated using these intensity values.
  • the parameter calculation unit 51 obtains the background by appropriately subtracting backgrounds such as noise and autofluorescence.
  • the parameter calculation unit 51 of the present embodiment calculates a single predetermined amino acid, a predetermined nucleobase, a predetermined amount of interatomic bond, or a parameter based on these amounts as a discrimination parameter.
  • a discrimination parameter is calculated by calculating a ratio between the amount of one predetermined amino acid, predetermined nucleobase or predetermined interatomic bond and the amount of other predetermined amino acid, predetermined nucleobase or predetermined interatomic bond. It can be.
  • the method for calculating the discrimination parameter is not particularly limited.
  • the parameter calculation unit 51 determines the ratio of the numerical value obtained by quantifying the peak size of the Raman spectrum corresponding to cytosine to the numerical value obtained by quantifying the peak size of the Raman spectrum corresponding to tryptophan. -1 (in the figure, it represented as "749cm -1 / 782cm -1") and.
  • the parameter calculation unit 51 of the present embodiment quantifies the Raman spectrum peak size corresponding to phenylalanine with respect to the numerical value obtained by quantifying the Raman spectrum peak size corresponding to the C—H bond of the saturated hydrocarbon.
  • the ratio of the numerical values is set as a discrimination parameter 1-2 (in the figure, expressed as “1003 cm ⁇ 1 / 1450 cm ⁇ 1 ”).
  • the discrimination parameter 1-1 and the discrimination parameter 1-2 are collectively referred to as a discrimination parameter 1.
  • the parameter calculation unit 51 of the present embodiment uses a double bond between carbon atoms or a numerical value obtained by quantifying the peak size of the Raman spectrum corresponding to the double bond between carbon atoms present in the porphyrin ring of cytochrome c. (in the figure, represented as "1583cm -1 / 1659cm -1") size determined ratio of a value obtained by quantifying the parameter 2 of the peak of the Raman spectrum corresponding to the amide bond to.
  • the determination unit 52 of the control unit 50 compares the determination parameter calculated by the parameter calculation unit 51 with a value that is stored in the storage unit 44 and serves as a reference for cell determination (hereinafter referred to as a determination reference value). Thus, it is determined whether the target cell C is a non-epithelial cancer cell, an epithelial cancer cell, or a non-cancerous cell.
  • the discrimination unit according to the present embodiment performs cell discrimination based on whether or not a point corresponding to the combination of the discrimination parameter 1 and the discrimination parameter 2 is included in the cell profile region mapped two-dimensionally based on the discrimination reference value. .
  • the discriminant reference value and the cell profile area indicate the reference value and range of the discriminant parameter that can be taken by non-epithelial cancer cells, epithelial cancer cells, and non-cancerous cells, respectively, based on past data.
  • Discrimination reference value and cell profile area are multivariate analysis of discriminant parameter values obtained by performing Raman measurement on non-epithelial cancer cells, epithelial cancer cells, and non-cancerous cells. It can be set by analyzing using.
  • the range of discrimination parameter values that can be taken by these cells is set based on the statistical values obtained by Raman measurement. For example, a parameter when the range is expressed in the form of an approximate function can be used as the discrimination reference value.
  • FIG. 4 is a conceptual diagram for explaining a method of discriminating the target cell C based on the discrimination parameter and the cell profile region 70.
  • FIG. 4 shows a graph with the discrimination parameter 1 on the horizontal axis and the discrimination parameter 2 on the vertical axis (hereinafter referred to as the discrimination graph 7).
  • the cell profile region 70 shown in the discrimination graph 7 includes a cell profile region 70a corresponding to non-epithelial cancer cells, a cell profile region 70b corresponding to epithelial cancer cells, and predetermined non-cancerous cells. Is provided with a cell profile region 70c.
  • the predetermined non-cancerous cells it is preferable to select non-cancerous cells contained in a large proportion in the cell suspension S.
  • the cell suspension S is a peripheral blood for discrimination of circulating cancer cells. Is obtained from the fraction obtained by centrifugation, it is preferable to set a cell profile region of peripheral blood mononuclear cells as the cell profile region 70c of the predetermined non-cancerous cell.
  • Four or more cell profile regions 70 may be set corresponding to various cells, and cell profile regions 70b and 70c of epithelial cancer cells and / or predetermined non-cancerous cells are provided. It may be omitted.
  • the point P1 corresponds to a non-epithelial cancer cell. It is classified as a cancer cell.
  • the target cell C is collected from blood or the like, the point P1 corresponds to a cancer cell that has undergone epithelial-mesenchymal transition.
  • the point P2 corresponds to an epithelial cancer cell. It is classified as a cancer cell.
  • the point P3 corresponds to a predetermined non-cancerous cell. Classified as non-cancerous cells.
  • the point P4 is an epithelial cancer cell, a non-epithelial cancer cell, Since it does not correspond to any of the non-cancerous cells, the target cell C is classified as not being these cells.
  • the above-described discrimination graph 7 is two-dimensional, it can be analyzed under any dimension using any number of discrimination parameters.
  • the discrimination reference value and the cell profile region 70 are set based on the type of cancer, the site, the gene involved, and the like.
  • the type or site of cancer can be cancer (carcinoma) or cancer of an organ in the body, for example, ovarian cancer.
  • the cell profile region 70 when the subject to be examined is an ovarian cancer patient, it is preferable to set the cell profile region 70 so as to detect cancer cells whose ovarian cancer has undergone epithelial-mesenchymal transition.
  • the “epithelial cancer cell” in the present embodiment is a cell that expresses one or more proteins selected from the group consisting of cytokeratin, EpCAM, and E-cadherin, and the “epithelial-mesenchymal cancer cell” is A cell that expresses vimentin and / or N-cadherin.
  • the flow rate control unit 53 (FIG. 3) of the control unit 50 controls the flow rate of the cell suspension S and phosphate buffered saline (hereinafter referred to as PBS), thereby moving the target cell C in the flow path. And the operation of separating the target cell based on the result of cell discrimination is controlled.
  • FIG. 5 is a conceptual diagram showing the structure of the flow path structure 90.
  • the channel structure 90 includes a cell channel 91, auxiliary channels 92a and 92b, holding portions 93a and 93b, bypass channels 94a and 94b, and a connecting portion 95.
  • the cell channel 91 is connected to the auxiliary channel 92a via the holding part 93a and the bypass channel 94a, and is connected to the auxiliary channel 92b via the holding part 93b and the bypass channel 94b.
  • the connection part 95 connects the flow path inside the flow path structure 90 and the external flow path.
  • the number of holding parts 93 can be set as appropriate. For example, when 20 ml of blood is collected from a person to be examined and the number of circulating cancer cells is examined, the stage is increased as the number of detected circulating cancer cells increases from 0 to several. Changes in cancer progression, malignancy, prognosis and / or metastatic potential.
  • the flow channel structure 90 may include only one holding portion 93, but preferably includes a plurality of holding portions 93.
  • the flow channel structure 90 is preferably provided with a plurality of cell flow channels 91 or two or more and ten or less holding portions 93 so that the flow channel is not too complicated. Thereby, cell discrimination can be performed efficiently.
  • PBS flows through the auxiliary flow paths 92a and 92b.
  • the direction in which the liquid flows inside the cell channel 91 and the auxiliary channels 92a and 92b is schematically indicated by solid line arrows A5, A6, and A7.
  • the flow rate of the liquid flowing through each of the cell flow channel 91 and the auxiliary flow channels 92a and 92b is independently controlled by the flow rate control unit 53.
  • the liquid flowing through the auxiliary flow paths 92a and 92b is not limited to PBS, but preferably has a temperature, osmotic pressure, and the like that are close to the buffer of the cell flow path 91 to the extent that the Raman measurement is not affected.
  • the holding portions 93a and 93b have an anti-ellipsoidal shape opened on the cell flow channel 91 side, but are not particularly limited as long as the flow of the cell flow channel 91 is not directly applied to the target cell C.
  • the width W and the depth L of the opening portions of the holding portions 93a and 93b that are in contact with the cell channel 91 are preferably 10 ⁇ m or more and 200 ⁇ m or less, and 20 ⁇ m or more and 100 ⁇ m or less so that the entire target cell C is accommodated and held. More preferably.
  • the tube diameters of the bypass circuits 94a and 94b are set smaller than the average width of the target cell C so as not to allow the target cell C to pass, and are preferably 0.1 ⁇ m or more and 20 ⁇ m or less.
  • FIG. 6A is a diagram showing a moving state of the target cell C when the cell channel 91 and the auxiliary channels 92a and 92b are set to flow at substantially the same speed.
  • non-epithelial cancer cells Cn and non-epithelial cancer cells Ce flow as target cells C.
  • the target cell C that flows through the cell flow path 91 advances along the cell flow path 91 without being attracted to the holding portions 93a and 93b because the flow rates in the cell flow path 91 and the auxiliary flow path 92 are substantially equal.
  • FIG. 6B is a diagram showing a moving state of the target cell C when the flow rate in the auxiliary flow path 92a is increased after the state of FIG. 6A.
  • a liquid with a larger flow velocity has a lower pressure
  • the target cell C1 is attracted to the holding portion 93a and held in contact with the side surface of the holding portion 93a.
  • the laser beam is irradiated while the target cell C1 is held, and Raman measurement and cell discrimination are performed.
  • FIG. 7 (a) is a diagram showing a moving state of the target cell C when the flow rate in the auxiliary flow path 92a is lowered after the state of FIG. 6 (b). This corresponds to a case where it is determined that the target cell C1 is not a non-epithelial cancer cell as a result of Raman measurement and cell discrimination performed on the target cell C1 in FIG. 6B.
  • the flow rate in the auxiliary channel 92a is lowered, and the cell channel 91 and the auxiliary channels 92a and 92b have substantially the same flow rate.
  • the target cell C ⁇ b> 1 held in the holding portion 93 a due to the flow velocity difference between the cell flow channel 91 and the auxiliary flow channel 92 moves away from the holding portion 93 a and travels along the cell flow channel 91.
  • FIG. 7B is a diagram showing a moving state of the target cell C when the flow rate in the auxiliary flow path 92a is increased after the state of FIG. 7A.
  • a liquid having a higher flow velocity has a lower pressure, and thus the target cell Cn is attracted to the holding portion 93a and held in contact with the side surface of the holding portion 93a.
  • the laser light is irradiated while the target cell Cn is held, and Raman measurement and cell discrimination are performed.
  • the target cell Cn is held in the holding unit 93a until the cell suspension S is completely poured.
  • the measuring apparatus 100 may include a plurality of irradiation units that irradiate light independently to each of the plurality of holding units 93, such as the holding unit 93a and the holding unit 93b. Thereby, a cell can be discriminated more efficiently.
  • the plurality of irradiation units may share a part of the irradiation optical system 10 or the objective optical system 20 of the measurement apparatus 100 as long as it has a plurality of irradiation ports.
  • FIG. 8A shows that both the holding portions 93a and 93b hold the non-epithelial cancer cells Cn, and all the cell suspensions S have been flown, and PBS has flowed into the cell channel 91. Indicates the state. Since the auxiliary flow paths 92a and 92b hold epithelial cancer cells Cn, PBS flows at a higher flow rate than the cell flow path 91.
  • FIG. 8B is a diagram showing a state of movement of epithelial cancer cells Cn when the flow velocity in the auxiliary flow channels 92a and 92b is lowered after FIG. 8A.
  • the flow velocity in the auxiliary flow channels 92a and 92b is lowered, and the flow velocity is substantially equal in the cell flow channel 91 and the auxiliary flow channels 92a and 92b.
  • the target cell C1 held in the holding portions 93a and 93b due to the flow rate difference between the cell flow channel 91 and the auxiliary flow channel 92 is separated from the holding portion 93a and proceeds along the cell flow channel 91.
  • Non-epithelial cancer cells Cn are collected in the collection container 83 when they exit the flow channel structure 90.
  • the non-epithelial cancer cells Cn are separated and collected from other cell groups contained in the cell suspension S.
  • the algorithm for holding the target cell C, separating it, and collecting it is not limited to the method of the present embodiment, and can be appropriately designed according to the number of holding parts 93 required.
  • the information output unit 54 of the control unit 50 includes information that is a result of cell discrimination of each target cell C, the number that is determined as epithelial cancer cells Cn, the number that is determined as non-epithelial cancer cells, and the number A display image including the number of circulating cancer cells obtained by combining the two numbers is created and output to the display unit 42. Furthermore, the information output unit 54 creates a display image including cancer analysis information indicating the degree of progression, malignancy, prognosis, and / or metastasis potential of the tumor, and outputs the display image to the display unit 42.
  • the measuring device 100 can also be a cancer testing device.
  • FIG. 9 is a flowchart showing a flow of a cancer testing method including the cell discrimination method of the present embodiment.
  • step S1001 blood is collected from an individual to be examined by a medical worker or the like.
  • step S1001 ends, the process proceeds to step S1003.
  • step S1003 the cell suspension S containing a part of cells estimated to contain circulating cancer cells is adjusted using centrifugation or the like from the blood collected in step S1001.
  • step S1003 ends, the process proceeds to step S1005.
  • step S1005 the cell suspension S prepared in step S1003 is caused to flow through the flow path, and the flow rate control unit 53 temporarily holds and holds the target cell in the vacant holding unit 93 of the cell flow path 91.
  • the Raman spectrum of the target cell C obtained is acquired.
  • step S1005 ends, the process proceeds to step S1007.
  • step S1007 the parameter calculation unit 51 determines the amount of at least one substance or bond selected from the group consisting of a predetermined amino acid, a predetermined nucleobase, and a predetermined interatomic bond in the Raman spectrum acquired in step S1005.
  • a discrimination parameter is calculated from the signal.
  • step S1007 ends, the process proceeds to step S1009.
  • step S1009 the determination unit 52 determines whether the temporarily retained target cell C is a non-epithelial cancer cell Cn, an epithelial cancer cell, or a non-cancerous cell based on the determination parameter. I do.
  • step S1009 ends, the process proceeds to step S1011.
  • step S1011 the control unit 50 determines whether or not the target cell C is determined as a non-epithelial cancer cell in step S1009. If it is determined as a non-epithelial cancer cell, a negative determination is made in step S1011 and the process proceeds to step S1019. If it is determined as a non-epithelial cancer cell, an affirmative determination is made in step S1011 and the process proceeds to step S1013.
  • step S1019 the flow rate control unit 53 adjusts the flow rate difference between the cell flow path 91 and the auxiliary flow path 92, and temporarily holds the target cell that is determined not to be a non-epithelial cancer cell. C is released from the holding unit 93.
  • step S1019 ends, the process proceeds to step S1013.
  • step S1013 the control unit 50 determines whether or not all of the cell suspension S has been poured. When the flow of the cell suspension S is not finished, a negative determination is made in step S1013, and the process returns to step S1005. When the cell suspension S has been poured, an affirmative decision is made in step S1013 and the process proceeds to step S1015.
  • step S1015 the control unit 50 opens the epithelial cancer cells Cn held in the holding unit 93 to the cell flow channel 91, and separates them from other cell groups in the cell suspension S and collects them.
  • step S1017 the information output unit 54 displays information on the degree of progression, malignancy, prognosis, and / or metastasis of the tumor to be examined from which the target cells C have been collected on the display unit 42.
  • the cell discrimination method of this embodiment measures a signal corresponding to at least one substance selected from the group consisting of a predetermined amino acid, a predetermined nucleobase, and a predetermined interatomic bond, or the amount of binding.
  • the target cell C is a cell contained in blood of a living body, and in the discrimination step, the target cell C is circulated including non-epithelial cancer cells Cn. To determine whether it is a cancer cell or a non-cancerous cell in the blood. Thereby, the number of circulating cancer cells can be accurately calculated.
  • the discrimination step determines whether the target cell C is an epithelial cancer cell or a cell Cn in which the epithelial cancer cell is epithelial-mesenchymal transition. Do. Thereby, information about whether or not non-epithelial cells Cn indicating the prognosis of the tumor such as the possibility of metastasis can be obtained accurately.
  • the target cell C is a cell collected from an ovarian cancer patient, and the epithelial cancer cell is an ovarian cancer cell. Thereby, it is possible to accurately determine whether the target cell C is a non-epithelial cancer cell.
  • a signal corresponding to the amount of aromatic amino acid is measured. This makes it possible to accurately determine whether the target cell C is a non-epithelial cancer cell that is associated with the amount of aromatic amino acid.
  • a signal corresponding to the amount of tryptophan and / or phenylalanine is measured. This makes it possible to accurately determine whether the target cell C is a non-epithelial cancer cell that is associated with the amount of tryptophan and / or phenylalanine.
  • a signal corresponding to the amount of cytosine is measured in the measurement step. Thereby, it is possible to accurately determine whether the target cell C is a non-epithelial cancer cell associated with the amount of cytosine.
  • a signal corresponding to the amount of bonds between a plurality of atoms including carbon atoms is measured. This makes it possible to accurately determine whether or not the target cell C is a non-epithelial cancer cell that is associated with the amount of bonds between a plurality of atoms including carbon atoms.
  • a signal corresponding to the amount of double bonds between carbon atoms in the porphyrin ring of cytochrome c is measured. This makes it possible to accurately determine whether or not the cancer cell is a non-epithelial cancer cell that is associated with the amount of bonds between a plurality of atoms including carbon atoms.
  • the discrimination parameter in the measurement step, a signal corresponding to the amount of the organic compound in part or all of the target cell C is measured.
  • the discrimination parameter can be normalized by the amount of the organic compound, etc., and it can be more accurately discriminated whether it is a non-epithelial cancer cell.
  • the measurement step corresponds to at least two substances selected from the group consisting of a predetermined amino acid, a predetermined nucleobase, and a predetermined interatomic bond, or the amount of binding.
  • a signal is measured, and in the discrimination step, a ratio between the amount of one substance or bond and the amount of another substance or bond is calculated, and the target cell C is discriminated based on one or more of the ratios. . Accordingly, it is possible to accurately determine whether or not the cancer cell is a non-epithelial cancer cell by appropriately combining the determination parameters.
  • the cancer inspection apparatus or cancer inspection method of this embodiment determines whether the target cell C is a non-epithelial cancer cell Cn by the cell determination method of this embodiment. Based on the result of this determination, information on the presence, degree, or metastasis potential of the cancer to be examined is provided. Thereby, non-epithelial cancer cells can be accurately identified, and accurate information on the current prognosis of cancer can be provided.
  • the measurement apparatus outputs an information output that outputs a determination result obtained by determining whether the target cell C is a non-epithelial cancer cell based on the signal measured in the measurement step. A part. As a result, it is possible to provide a result of accurately discriminating non-epithelial cancer cells.
  • the measurement device of this embodiment includes a cell channel 91 in which the target cell C moves, a holding unit 93 that is connected to the cell channel 91 and holds a part of the target cells, and emits light to the holding unit 93.
  • An objective optical system 20 for irradiation Thereby, it is possible to accurately determine whether or not the cells contained in the cell suspension S are non-epithelial cancer cells sequentially.
  • the measurement device of the present embodiment includes a flow rate control unit that controls an operation of separating the target cell C based on the discrimination result of the cell discrimination.
  • the target cell is a non-epithelial cancer cell, an epithelial cancer cell, or Although it is discriminated whether it is a non-cancerous cell, a cell discrimination method, a cancer inspection apparatus or a cancer inspection method, and a measurement apparatus that only determine whether it is a non-epithelial cancer cell are also included in the present invention. Is included.
  • Module 1 A program for realizing the information processing function of the information processing apparatus 40 according to the present embodiment is recorded on a computer-readable recording medium, and the above-described determination parameter calculation processing, determination processing, and the like recorded on the recording medium
  • the program may be read into a computer system and executed.
  • the “computer system” includes an OS (Operating System) and hardware of peripheral devices.
  • the “computer-readable recording medium” refers to a portable recording medium such as a flexible disk, a magneto-optical disk, an optical disk, and a memory card, and a storage device such as a hard disk built in the computer system.
  • the “computer-readable recording medium” dynamically holds a program for a short time like a communication line when transmitting a program via a network such as the Internet or a communication line such as a telephone line.
  • a volatile memory in a computer system serving as a server or a client in that case may be included and a program that holds a program for a certain period of time may be included.
  • the above program may be for realizing a part of the functions described above, or may be realized by a combination with the program already recorded in the computer system. .
  • FIG. 10 is a diagram showing this state.
  • the PC 950 is provided with a program via the CD-ROM 953. Further, the PC 950 has a connection function with the communication line 951.
  • a computer 952 is a server computer that provides the program, and stores the program in a recording medium such as a hard disk.
  • the communication line 951 is a communication line such as the Internet or PC communication, or a dedicated communication line.
  • the computer 952 reads the program using the hard disk and transmits the program to the PC 950 via the communication line 951. That is, the program is transmitted as a data signal by a carrier wave and transmitted via the communication line 951.
  • the program can be supplied as a computer-readable computer program product in various forms such as a recording medium and a carrier wave.
  • the inspection program according to the present modified example is based on a signal corresponding to at least one substance selected from the group consisting of a predetermined amino acid, a predetermined nucleobase, and a predetermined interatomic bond, or the amount of binding.
  • a discrimination process for discriminating between a non-epithelial cancer cell Cn, an epithelial cancer cell or a non-cancerous cell, information on the result of the discrimination process, or a test object from which the target cell C has been acquired causes the processing device to perform output processing that outputs information about the presence, degree, or metastasis potential of cancer. Thereby, it is possible to accurately determine whether the target cell C is a non-epithelial cancer cell.
  • Example 2 Measure the Raman distribution of cultured ovarian cancer cells, cells that induce epithelial-mesenchymal transition by adding an epithelial-mesenchymal transition-inducing reagent to these cultured ovarian cancer cells, and determine the distribution of discriminating parameters. Plotted for each cell.
  • Example 1 Cells obtained by inducing epithelial-mesenchymal transition obtained as described above, cultured ovarian cancer cells (OVCAR-3), and peripheral blood mononuclear cells (Lonza) were fixed using paraffin, and specimens Then, a known deparaffinization treatment was performed. A rough region for light irradiation is set for a sample subjected to deparaffinization treatment, the region is divided into several unit regions, and a laser having a wavelength of 532 nm having a beam diameter of about 1 ⁇ m for each unit region. Irradiated with light, the Raman scattered light was measured.
  • the spectra acquired for each unit region were averaged, and peaks corresponding to glass and autofluorescence were removed by a known algorithm.
  • the determination method of cell of the present embodiment the Stokes scattering zone, the ratio of the intensity of 749 / cm, corresponding to a tryptophan, to the intensity of the corresponding 782 / cm to cytosine (in the figure "749cm -1 / 782cm -1": The discrimination parameter 1-1) was calculated.
  • FIG. 11 is a graph showing the discrimination parameter 1-1 of each cell on the horizontal axis and the discrimination parameter 2 on the vertical axis.
  • the respective data of cultured ovarian cancer cells (OVCAR-3), cultured epithelial-mesenchymal transition (EMT) -induced cultured ovarian cancer cells, and peripheral blood mononuclear cells are divided into clusters. From the measurement values given as shown in FIG. 11, various determination reference values and cell profile regions are determined based on the centroid of the data distribution corresponding to each cell by the multivariate analysis method, the distance of each point from the centroid, and the like. Can be set in various ways.
  • Example 2 The Raman spectrum obtained in Example 1 was further analyzed, and the C—H bond of saturated hydrocarbon having an intensity of 1003 / cm corresponding to phenylalanine in the Stokes scattering region was determined by the cell discrimination method of the present embodiment. ratio intensity of 1450 / cm to (in the figure "1003 cm -1 / 1450 cm -1 ': determining parameter 1-2) were calculated.
  • FIG. 12 is a graph showing the discrimination parameter 1-2 for each cell on the horizontal axis and the discrimination parameter 2 on the vertical axis.
  • EMT Epithelial-mesenchymal transition
  • OVCAR-3 cultured ovarian cancer cells
  • peripheral blood mononuclear cells are separated into clusters.
  • point D in the figure is a point for cultured ovarian cancer cells, but is located closer to a cluster of peripheral blood mononuclear cells than a cluster of cultured ovarian cancer cells.
  • the target cell C is a non-epithelial cancer cell or a cultured ovarian cancer cell or a peripheral blood cell. It is considered that the accuracy is higher than that of determining whether the cell is a nuclear cell.
  • the present invention is not limited to the contents of the above embodiment.
  • Other embodiments conceivable within the scope of the technical idea of the present invention are also included in the scope of the present invention.
  • DESCRIPTION OF SYMBOLS 40 Information processing apparatus 50 ... Control part 51 ... Parameter calculation part 52 ... Discrimination part 53 ... Flow rate control part 54 ... Information output part 70, 70a, 70b, 70c ... Cell profile area

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Abstract

L'invention concerne un procédé pour distinguer des cellules comprenant : une étape de mesure pour mesurer un signal qui correspond à la substance ou la quantité de liaisons dans un ou plusieurs éléments choisis dans le groupe constitué par des acides aminés prescrits, des bases d'acide nucléique prescrites, et des liaisons inter-atomes prescrites dans certaines ou toutes les cellules cibles ; et une étape de distinction pour distinguer, sur la base du signal mesuré, si les cellules cibles sont des cellules cancéreuses non épithéliales.
PCT/JP2018/008115 2017-03-02 2018-03-02 Procédé de distinction de cellules, procédé d'inspection de cancer, dispositif de mesure, dispositif d'inspection de cancer, et programme d'inspection WO2018159833A1 (fr)

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