WO2018159833A1 - Method for distinguishing cells, method for inspecting cancer, measurement device, device for inspecting cancer, and inspection program - Google Patents

Abstract

This method for distinguishing cells is provided with: a measurement step for measuring a signal that corresponds to the substance or the amount of bonds in one or more selected from the group consisting of prescribed amino acids, prescribed nucleic acid bases, and prescribed inter-atom bonds in some or all target cells; and a distinguishing step for distinguishing, on the basis of the measured signal, whether the target cells are non-epithelial cancer cells.

Description

Cell discrimination method, cancer inspection method, measuring device, cancer inspection device, and inspection program

The present invention relates to a cell discrimination method, a cancer inspection method, a measurement device, a cancer inspection device, and an inspection program.

An examination method has been proposed in which light from a living tissue or the like is analyzed to check whether or not there is a cancerous part in the living tissue or the like (see Patent Document 1). For precise cancer testing, it is desirable to analyze light from tissues and cells based on more appropriate parameters.

Japan Special Table 2009-538156

According to the first aspect of the present invention, 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. 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.
According to the second aspect of the present invention, 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. An information output that outputs a determination result obtained by determining whether or not the target cell is a non-epithelial cancer cell based on the measured signal and a measurement unit that measures a signal corresponding to the amount of binding A section.
According to the third aspect of the present invention, 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. It is a conceptual diagram in case the cell is hold | maintained. 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, and (b) is a diagram showing how the holding unit holds the cells. It is a conceptual diagram in the case of holding cells that have undergone epithelial-mesenchymal transition. 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. 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.

Hereinafter, a cell discrimination method, a cancer inspection method, a measurement device, a cancer inspection device, an inspection program, and the like according to an embodiment will be described with reference to the drawings as appropriate. In the cell discrimination method of the present embodiment, light from a cell is measured, and based on the measurement result, the cell is a non-epithelial cancer cell, or an epithelial cancer cell or a non-cancerous cell. (Hereinafter referred to as “cell discrimination”).
In the present embodiment, “cancer” 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). 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. When a tissue containing epithelial cells becomes cancerous, it usually becomes a malignant tumor having well-differentiated epithelial-like properties (arrow A1).

When a malignant tumor becomes large, for example, the blood vessel wall is damaged by infiltration, and cancer cells in contact with blood may be separated from the malignant tumor and circulate in the blood. The cancer cells circulating in the blood are called circulating cancer cells (CTC), and the degree of progression, malignancy, prognosis and / or metastasis of the tumor can be determined by examining the number per unit volume in the blood. You can get information about.

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.

Therefore, when examining circulating cancer cells in the blood, in addition to epithelial cancer cells, non-epithelial cancer cells with different biochemical markers can be detected more reliably. It is important to obtain information about prognosis such as the possibility of metastasis. 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. In 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.
In addition, although it has the structure which analyzes the backscattered light in FIG. 1, you may make it the structure which analyzes the light scattered forward. If the spectroscopic spectrum which can be analyzed from the light from the flow path structure 90 is obtained, 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.

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). In this embodiment, 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.
In addition, 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 | strength for every wavelength is acquired with the spectrometer 33, the data, such as the said intensity | strength, will be output to the information processing apparatus 40 (the solid line arrow A4 of FIG. 2 showed the flow of information), and will be memorize | stored in the memory | storage part 44 mentioned later. The

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. In the container 81, 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 | recovered based on the result of a Raman measurement.

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. As the predetermined amino acid, an aromatic amino acid, particularly tryptophan and / or phenylalanine can be selected. In particular, cytosine can be selected as the predetermined nucleobase. As 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. As 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. Furthermore, 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. Calculates the intensity of wave number 1659 / cm, the intensity of 1583 / cm corresponding to the double bond between carbon atoms existing in the porphyrin ring of cytochrome c, or the intensity of the C—H bond 1450 / cm of the saturated hydrocarbon. Thus, the discrimination parameter can be obtained, or the discrimination parameter can be calculated by using these intensity values.
Note that 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. In the Stokes scattering region, 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. When calculating the peak intensity, area, and the like, 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. For example, 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 according to the present embodiment 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 ”). Hereinafter, 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. As the predetermined non-cancerous cells, it is preferable to select non-cancerous cells contained in a large proportion in the cell suspension S. For example, 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.

When the discrimination parameter 1 and the discrimination parameter 2 obtained by measuring the target cell C correspond to the point P1 in the discrimination graph 7, the point P1 corresponds to a non-epithelial cancer cell. It is classified as a cancer cell. In particular, when 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.

When the discrimination parameter 1 and the discrimination parameter 2 obtained by measuring the target cell C correspond to the point P2 in the discrimination graph 7, the point P2 corresponds to an epithelial cancer cell. It is classified as a cancer cell.

When the discrimination parameter 1 and the discrimination parameter 2 obtained by measuring the target cell C correspond to the point P3 in the discrimination graph 7, the point P3 corresponds to a predetermined non-cancerous cell. Classified as non-cancerous cells.

When the discrimination parameter 1 and the discrimination parameter 2 obtained by measuring the target cell C correspond to the point P4 in the discrimination graph 7, 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.
Although the above-described discrimination graph 7 is two-dimensional, it can be analyzed under any dimension using any number of discrimination parameters.

It is preferable for precise determination that 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. For example, 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.

A cell suspension S containing the target cell C flows through the cell channel 91. 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. Not. 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 μm or more and 200 μm or less, and 20 μm or more and 100 μm 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 μm or more and 20 μm or less.

(Method of controlling movement of target cell C)
Hereinafter, with reference to FIGS. 6 to 8, a method in which the movement of the target cell C is controlled by the flow rate control unit 53 and the non-epithelial cancer cell Cn is separated from the cell group of the target cell C and collected will be described. To do.

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. In the cell channel 91, 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. According to Bernoulli's theorem, a liquid with a larger flow velocity has a lower pressure, and the target cell C1 is attracted to the holding portion 93a and held in contact with the side surface of the holding portion 93a. In this way, 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. At this time, 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. According to Bernoulli's theorem, 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. In this way, the laser light is irradiated while the target cell Cn is held, and Raman measurement and cell discrimination are performed. When the result of Raman measurement and cell discrimination indicates that the target cell Cn is a non-epithelial cancer cell, the target cell Cn is held in the holding unit 93a until the cell suspension S is completely poured. When the non-epithelial cancer cell Cn is held in the holding part 93a, the irradiation position of light for Raman measurement is moved from the holding part 93a to the holding part 93b, and the holding part 93 performs Raman measurement and cell discrimination in the same manner as described above. I do.
Note that 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. Here, 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. At this time, 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. Thus, the non-epithelial cancer cells Cn are separated and collected from other cell groups contained in the cell suspension S.
Note that 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. Thus, the measuring device 100 can also be a cancer testing device.

(Flow of cancer testing methods)
FIG. 9 is a flowchart showing a flow of a cancer testing method including the cell discrimination method of the present embodiment.

In step S1001, blood is collected from an individual to be examined by a medical worker or the like. When step S1001 ends, the process proceeds to step S1003. In 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. When step S1003 ends, the process proceeds to step S1005.

In 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. When step S1005 ends, the process proceeds to step S1007. In 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. When step S1007 ends, the process proceeds to step S1009.

In 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. When step S1009 ends, the process proceeds to step S1011. In 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.

In 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. When step S1019 ends, the process proceeds to step S1013. In 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.

In 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. . When step S1015 ends, the process proceeds to step S1017. In 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.

According to the above-described embodiment, the following operational effects can be obtained.
(1) 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. A measurement step and a determination step of determining whether the target cell C is a non-epithelial cancer cell Cn based on the measured signal. Thereby, it is possible to accurately determine whether the target cell C is a non-epithelial cancer cell.

(2) In the cell discriminating method of the present embodiment, 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.

(3) In the cell discrimination method of the present embodiment, 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.

(4) In the cell discrimination method of the present embodiment, 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.

(5) In the cell discrimination method of the present embodiment, in the measurement step, 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.

(6) In the cell discrimination method of the present embodiment, in the measurement step, 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.

(7) In the cell discrimination method of the present embodiment, in the measurement step, a signal corresponding to the amount of cytosine is measured. Thereby, it is possible to accurately determine whether the target cell C is a non-epithelial cancer cell associated with the amount of cytosine.

(8) In the cell discrimination method of this embodiment, in the measurement step, 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.

(9) In the cell discrimination method of this embodiment, in the measurement step, 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.

(10) In the cell discrimination method of the present embodiment, 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. As a result, 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.

(11) In the cell discrimination method of the present embodiment, in the measurement step, in the intensity of the Stokes scattering region of the Raman scattered light, a peak including 749 / cm in the half-value range and 782 / cm in the half-value range. The amount of the corresponding substance or bond is calculated based on the statistical values calculated from the data of the peak, the peak including 1583 / cm in the range of half width, or the peak including 1659 / cm in the range of half width. Thereby, it is possible to accurately determine whether the target cell C is a non-epithelial cancer cell without using a biochemical marker.

(12) In the cell discrimination method of the present embodiment, 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.

(13) 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.

(14) The measurement apparatus according to the present embodiment 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.

(15) 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.

(16) 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. As a result, the cells that have been accurately sorted and taken out can be analyzed in more detail.

In the cell discrimination method, cancer inspection apparatus or cancer inspection method, and measurement apparatus according to the above-described embodiment, 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.

The following modifications are also within the scope of the present invention, and can be combined with the above-described embodiment.
(Modification 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. Here, 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. Furthermore, 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. In this case, 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. Further, 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. .

Further, when applied to a personal computer (hereinafter referred to as a PC), the above-described control-related program can be provided through a recording medium such as a CD-ROM or a data signal such as the Internet. 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. Thus, 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)
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.

(Induction of epithelial-mesenchymal transition in cultured ovarian cancer cells)
When the expression of E-cadherin was confirmed, OVCAR-3 (ATCC) was added to the epithelial-mesenchymal transition induction reagent StemXVivo EMT Inducing Media Supplement (R & D Systems), and 5 On the 10th day, the expression of the mesenchymal marker vimentin could be confirmed in some cells. Hereinafter, this cell is referred to as a cell in which epithelial-mesenchymal transition is induced. It was confirmed that the mesenchymal state was maintained even in suspension culture.

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 μm 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. In the same specimen, the ratio of the intensity of 1583 / cm corresponding to the double bond between carbon atoms in the porphyrin ring of cytochrome c to the intensity of 1659 / cm corresponding to the double bond between carbon atoms and the amide bond (“1583 cm ⁻¹ / 1659 cm ⁻¹ ” in the figure: discrimination parameter 2) 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. Epithelial-mesenchymal transition (EMT) -induced cultured ovarian cancer cells and data consisting of cultured ovarian cancer cells (OVCAR-3) and peripheral blood mononuclear cells are separated into clusters. is doing. However, 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. Therefore, when cell discrimination is performed using the discrimination parameter 1-1 and the discrimination parameter 2, it is preferable to discriminate whether 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.

The disclosure of the following priority application is hereby incorporated by reference.
Japanese Patent Application 2017-039727 (filed on March 2, 2017)

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 | region, 90 ... Flow path Structure 91, cell flow path, 92a, 92b, auxiliary flow path, 93a, 93b, holding portion, 94a, 94b, bypass flow path, 100, measuring device, Cn, non-epithelial cancer cell.

Claims (18)

A measurement step of measuring a signal corresponding to an 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 part or all of the target cell;
A determination step of determining whether the target cell is a non-epithelial cancer cell based on the measured signal;
A method for distinguishing cells comprising: The method for discriminating cells according to claim 1,
The cell discrimination method, wherein the target cell is a cell contained in blood of a living body. The method for discriminating cells according to claim 1 or 2,
The discrimination step is a cell discrimination method for discriminating whether the target cell is a non-epithelial cancer cell, an epithelial cancer cell or a non-cancerous cell. In the method for discriminating cells according to any one of claims 1 to 3,
The method for discriminating a cell, wherein the predetermined amino acid is an aromatic amino acid. The method for discriminating cells according to claim 4,
The method for distinguishing cells in which the aromatic amino acid is tryptophan or phenylalanine. In the method for discriminating cells according to any one of claims 1 to 5,
The method for discriminating a cell, wherein the predetermined nucleobase is cytosine. The cell discriminating method according to any one of claims 1 to 6, wherein the predetermined interatomic bond is an interatomic bond that serves as an indicator of the amount of an organic compound in a part or all of the target cell. How to determine. The method for discriminating cells according to claim 7,
The predetermined interatomic bond is selected from the group consisting of a double bond between carbon atoms, an amide bond, a C—H bond in a saturated hydrocarbon, and a double bond between carbon atoms present in the porphyrin ring of cytochrome c. A method for discriminating cells that are at least one interatomic bond. In the method for discriminating cells according to any one of claims 1 to 3,
In the intensity of the Stokes scattering region of Raman scattered light, the measurement step includes a peak including 749 / cm in the half-value range, a peak including 782 / cm in the half-value range, and 1583 / cm in the half-value range. Based on the statistical value calculated from the data of the peak, the peak including 1659 / cm in the range of half width, the peak including 1003 / cm in the range of half width, or the peak including 1450 / cm in the range of half width A cell discrimination method for calculating the amount of substance or binding. In the method for discriminating cells according to any one of claims 1 to 9,
In the measurement step, a signal corresponding to an amount of at least two substances or bonds 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. Measure and
The discrimination step calculates a ratio between the amount of one substance or binding and the amount of the other substance or binding, and discriminates a cell for performing the discrimination of the target cell based on one or more of the ratios. Method. In the method for discriminating cells according to any one of claims 1 to 9,
The measurement step includes
The ratio of the numerical value quantifying the peak size of the Raman spectrum corresponding to cytosine to the numerical value quantifying the peak size of the Raman spectrum corresponding to tryptophan,
The ratio of the numerical value of the Raman spectrum peak corresponding to phenylalanine to the numerical value of the Raman spectrum peak corresponding to the C—H bond of the saturated hydrocarbon, and the porphyrin ring of cytochrome c Quantify the size of the Raman spectrum peak corresponding to the double bond between the carbon atoms or the amide bond to the value quantified the size of the Raman spectrum peak corresponding to the double bond between the carbon atoms present in The ratio of
A cell discrimination method for performing the discrimination of the target cell based on at least one ratio selected from the group consisting of: Using the method for discriminating cells according to any one of claims 1 to 11 to discriminate cells obtained from a test object;
Providing information about the presence or absence, the degree, or the possibility of metastasis of the cancer to be examined based on the result of the discrimination, and a method for examining cancer. A measurement unit that 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 bonds in a part or all of the target cell;
Based on the measured signal, an information output unit that outputs a discrimination result obtained by discriminating whether the target cell is a non-epithelial cancer cell;
A measuring device comprising: The measuring device according to claim 13,
A flow path through which cells move;
A holding part connected to the flow path and holding a part of the cells;
An irradiation unit for irradiating the holding unit with light;
With
The measurement unit has an 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 cells in the holding unit. A measuring device that measures the corresponding signal. The measuring device according to claim 14,
A plurality of the holding portions;
A measuring device comprising a plurality of the irradiation units. In the measuring device according to any one of claims 13 to 15,
A measurement apparatus comprising a separation control unit that controls an operation of separating the cells based on the determination result. A measuring device according to any one of claims 13 to 16, comprising:
The information output unit is a cancer inspection apparatus that outputs information about the presence, degree, or metastasis possibility of a cancer to be examined from which the cells are acquired. 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 in a part or all of the target cell Is a determination process for determining whether or not the cancer cell is a non-epithelial cancer cell,
An inspection program for causing a processing device to perform an output process for outputting information on a result of the discrimination process, or information on the presence / absence, degree, or metastasis possibility of a cancer to be inspected from which the target cell is acquired.

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