Cancer cell staining method

Abstract

To provide methods for staining cancer cells, which can inactivate the endogenous peroxidase in blood without dissolving a hydrophilic polymer when capturing cancer cells in blood on a hydrophilic polymer layer and staining them by the immunoperoxidase method.SOLUTION: The present invention relates to a method for staining cancer cells, which comprising capturing cancer cells in blood on a hydrophilic polymer layer, inactivating the endogenous peroxidase in blood using hydrogen peroxide and sodium azide, and staining the cancer cells by the immunoperoxidase method.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a method for staining cancer cells in blood. [Background technology]

[0002] It is known that once cancer cells develop, they eventually appear in the blood and body fluids, and these cancer cells that appear in the blood are called circulating tumor cells (CTCs). By examining these circulating tumor cells in the blood, it is expected that they will be useful for confirming the effectiveness of cancer treatment, predicting prognosis and life expectancy, predicting the effectiveness of anticancer drugs before administration, and examining treatment methods using the genetic analysis of cancer cells.

[0003] As a method for capturing CTCs, for example, Patent Document 1 discloses a method for capturing CTCs in a hydrophilic polymer layer. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] JP 2018-50548 A Summary of the Invention [Problem to be solved by the invention]

[0005] In Patent Document 1, cancer cells captured in a hydrophilic polymer layer are stained and observed by the fluorescent antibody technique (Patent Document 1,

[0053] ). However, the fluorescent antibody technique has room for improvement in that the staining disappears within about 10 days to 2 weeks, the background during observation is significantly affected, and it is difficult to observe background tissues because a dark-field microscope is used.

[0006] Therefore, the present inventors investigated other staining methods and focused on the immunoperoxidase method, which is a type of enzyme antibody method. In the immunoperoxidase method, an antibody labeled with peroxidase (enzyme) is bound to cancer cells (antigens), and then a substrate such as diaminobenzidine (DAB) is reacted with the peroxidase to stain the cancer cells. Compared to the fluorescent antibody method, this method has the advantages of being able to retain staining for a long time, having less background influence during observation, and using a bright-field microscope to easily observe background tissue. However, the present inventors further investigated the method and found the following problems.

[0007] When cancer cells in blood are stained by the immunoperoxidase method, it is necessary to inactivate (block) peroxidase (endogenous peroxidase) in blood in advance. This inactivation is usually performed using hydrogen peroxide and methanol, but hydrophilic polymers such as poly 2-methoxyethyl acrylate (PMEA) described in Patent Document 1 are dissolved in methanol. Therefore, when the immunoperoxidase method is applied instead of the fluorescent antibody method in the method of Patent Document 1, the hydrophilic polymer is dissolved by methanol, and the cancer cells captured in the hydrophilic polymer layer flow out, leaving room for improvement.

[0008] The present invention aims to solve the above-mentioned problems and provide a method for staining cancer cells, which can inactivate endogenous peroxidase in blood without dissolving the hydrophilic polymer when cancer cells in blood are captured in a hydrophilic polymer layer and stained by an immunoperoxidase method. [Means for solving the problem]

[0009] As a result of further investigations conducted by the present inventors to solve the above problems, they discovered that by using sodium azide instead of methanol, endogenous peroxidase in blood can be inactivated without dissolving hydrophilic polymers, and thus arrived at the present invention. That is, the present invention relates to a method for staining cancer cells, comprising the steps of capturing cancer cells in blood in a hydrophilic polymer layer, inactivating endogenous peroxidase in the blood using hydrogen peroxide and sodium azide, and staining the cancer cells by an immunoperoxidase method.

[0010] The immunoperoxidase method is preferably a direct immunoperoxidase method.

[0011] The blood is preferably one that has been subjected to a concentration treatment.

[0012] Preferably, the blood is taken from the patient prior to treatment.

[0013] The hydrophilic polymer layer is preferably formed from at least one hydrophilic polymer selected from the group consisting of a polymer represented by the following formula (I) and poly(meth)acryloylmorpholine. [ka] (In the formula, R 1 is a hydrogen atom or a methyl group, R 2 represents an alkyl group, m is 1 to 5, and n is the number of repetitions.

[0014] The hydrophilic polymer layer is preferably formed from a copolymer of at least one hydrophilic monomer selected from the group consisting of a compound represented by the following formula (I-1) and (meth)acryloylmorpholine, and another monomer. [ka] (In the formula, R 1 , R 2 , m is the same as above.) Effect of the Invention

[0015] According to the present invention, the method for staining cancer cells includes the steps of capturing cancer cells in blood in a hydrophilic polymer layer, inactivating endogenous peroxidase in the blood using hydrogen peroxide and sodium azide, and staining the cancer cells by an immunoperoxidase method, so that endogenous peroxidase in the blood can be inactivated without dissolving the hydrophilic polymer. [Brief description of the drawings]

[0016] [Figure 1] 13 is a microscopic image of stained cancer cells in an embodiment. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0017] The present invention is a method for staining cancer cells, comprising a step of capturing cancer cells in blood in a hydrophilic polymer layer (capture step), a step of inactivating endogenous peroxidase in the blood using hydrogen peroxide and sodium azide (inactivation step), and a step of staining the cancer cells by an immunoperoxidase method (staining step).

[0018] In the above method, sodium azide is used instead of methanol, which has been used conventionally, to inactivate endogenous peroxidase in blood. This allows endogenous peroxidase to be inactivated without dissolving hydrophilic polymers. Furthermore, by staining cancer cells using the immunoperoxidase method, it is easier to observe cancer cells and their background tissues than with the fluorescent antibody method, and samples that can be stored for a long time can be prepared.

[0019] Each step of the above method will be explained below.

[0020] (Capturing process) In this step, cancer cells in the blood are captured by the hydrophilic polymer layer, which can be achieved, for example, by contacting the blood with the hydrophilic polymer layer.

[0021] Cancer cells are captured by the hydrophilic polymer layer, but blood cells such as red blood cells, white blood cells, and platelets are not easily captured (adsorbed) by the hydrophilic polymer layer. Therefore, by contacting blood with the hydrophilic polymer layer and leaving it for a predetermined time, and then washing with phosphate buffered saline (PBS) or the like as necessary, cancer cells can be selectively captured by the hydrophilic polymer layer.

[0022] The method for bringing blood into contact with the hydrophilic polymer layer is not particularly limited, but examples thereof include injection, dripping, and spraying of blood onto the hydrophilic polymer layer.

[0023] It is preferable to subject the blood to a concentration treatment before contacting it with the hydrophilic polymer layer. The concentration treatment reduces blood cells and increases the proportion of cancer cells in the blood, allowing more cancer cells to be captured by the hydrophilic polymer layer. The method of concentration treatment is not particularly limited, but centrifugation is preferable, and concentration gradient centrifugation is more preferable. Concentration gradient centrifugation can be performed, for example, using OncoQuick (registered trademark) from Greiner Bio-One.

[0024] In addition, after treatment such as surgery or chemotherapy, the number of cancer cells in the blood usually decreases. Therefore, in order to capture more cancer cells, it is preferable to use blood collected from the patient before treatment.

[0025] The hydrophilic polymer layer (layer formed by a hydrophilic polymer) can be formed on a predetermined substrate, such as acrylic resins (polyacrylic resins) such as polymethyl acrylate, polymethyl methacrylate, polyacrylic acid, and polymethacrylic acid, cycloolefin resins (polycycloolefins), carbonate resins (polycarbonate), styrene resins (polystyrene), polyester resins such as polyethylene terephthalate (PET), polydimethylsiloxane, and glass such as soda-lime glass and borosilicate glass.

[0026] The thickness of the hydrophilic polymer layer (layer formed of a hydrophilic polymer) is preferably 10 to 500 nm, more preferably 30 to 400 nm, and even more preferably 50 to 350 nm. By adjusting the thickness within the above range, the selective capture of cancer cells is improved.

[0027] The hydrophilic polymer can be appropriately selected from those having hydrophilicity. For example, homopolymers and copolymers of one or more hydrophilic monomers, copolymers of one or more hydrophilic monomers and other monomers, etc. can be mentioned. Examples of the homopolymers and copolymers include polyacrylic acid, polyacrylic acid esters, polymethacrylic acid, polymethacrylic acid esters, polyacryloylmorpholine, polymethacryloylmorpholine, polyacrylamide, polymethacrylamide, etc.

[0028] The hydrophilic monomer may be any of various monomers having a hydrophilic group, such as known hydrophilic groups including an amide group, a sulfate group, a sulfonic acid group, a carboxyl group, a hydroxyl group, an amino group, an amide group, and an oxyethylene group.

[0029] Specific examples of hydrophilic monomers include (meth)acrylic acid, (meth)acrylic acid esters, (alkoxyalkyl (meth)acrylates such as methoxyethyl (meth)acrylate, hydroxyalkyl (meth)acrylates such as hydroxyethyl (meth)acrylate), (meth)acrylamide, (meth)acrylamide derivatives having a cyclic group ((meth)acryloylmorpholine, etc.), etc. Among these, (meth)acrylic acid, (meth)acrylic acid esters, alkoxyalkyl (meth)acrylates, and (meth)acryloylmorpholine are preferred, alkoxyalkyl (meth)acrylates are more preferred, and 2-methoxyethyl acrylate is particularly preferred.

[0030] The other monomers may be appropriately selected within a range that does not impair the action and effect of the hydrophilic polymer, and examples thereof include aromatic monomers such as styrene, vinyl acetate, and N-isopropylacrylamide that can impart temperature responsiveness.

[0031] Among them, the hydrophilic polymer is preferably at least one selected from the group consisting of a polymer represented by the following formula (I) and poly(meth)acryloylmorpholine. [ka] (In the formula, R 1 is a hydrogen atom or a methyl group, R 2 represents an alkyl group, m is 1 to 5, and n is the number of repetitions.

[0032] R 2 The number of carbon atoms in the alkyl group of R is preferably 1 to 10, and more preferably 1 to 5. 2 is particularly preferably a methyl group or an ethyl group. m is preferably an integer of 1 to 3. n (the number of repeating units) is preferably an integer of 15 to 1,500, and more preferably an integer of 40 to 1,200.

[0033] In addition, as the hydrophilic polymer, a copolymer of at least one hydrophilic monomer selected from the group consisting of a compound represented by the following formula (I-1) and (meth)acryloylmorpholine and other monomers can also be suitably used. [ka] (In the formula, R 1 , R 2 , m is the same as above.)

[0034] A substrate having a hydrophilic polymer layer formed thereon can be produced by coating all or part of the substrate surface with a hydrophilic polymer solution or dispersion by a known method, such as the method described in JP 2018-50548 A. Examples of such substrates include multi-well plates, slide chambers (slide glasses with chambers), etc.

[0035] Fibronectin may be preliminarily adsorbed onto the hydrophilic polymer layer. By contacting blood with the hydrophilic polymer layer to which fibronectin has been adsorbed, it becomes possible for a larger number of cancer cells to be captured by the hydrophilic polymer layer.

[0036] After the capture step, if necessary, the cancer cell tissue may be fixed using formaldehyde or the like, or the cell membrane of the cancer cell may be permeabilized using a non-ionic surfactant such as Triton X (octylphenol ethoxylate), before moving on to the inactivation step.

[0037] (Inactivation process) In this step, endogenous peroxidase in the blood is inactivated using hydrogen peroxide and sodium azide. This can be carried out, for example, by contacting the hydrophilic polymer layer that has been subjected to the capture step with a mixed solution of hydrogen peroxide and sodium azide and reacting for a predetermined time (about 5 to 30 minutes). The reaction is preferably carried out in the dark at room temperature (about 1 to 30°C).

[0038] The method of bringing the hydrophilic polymer layer into contact with the mixed liquid is not particularly limited, and examples of the method include pouring, dripping, spraying, etc. of the mixed liquid into the hydrophilic polymer layer. Alternatively, the substrate on which the hydrophilic polymer layer is formed may be immersed in the mixed liquid.

[0039] The mixture can be prepared, for example, by dissolving sodium azide in distilled water and then mixing it with an aqueous hydrogen peroxide solution. The concentrations of hydrogen peroxide and sodium azide in the mixture are not particularly limited and can be appropriately changed within a range in which inactivation can be achieved.

[0040] After the inactivation step, washing, serum blocking, etc. are carried out as necessary, and then the staining step is started.

[0041] (dying process) In this process, the cancer cells are stained using the immunoperoxidase method. There are two types of immunoperoxidase method: the direct immunoperoxidase method (direct method) and the indirect immunoperoxidase method (indirect method). The direct immunoperoxidase method is a method in which an antigen-antibody reaction is carried out only once using a primary antibody labeled with peroxidase, while the indirect immunoperoxidase method is a method in which an antigen-antibody reaction is carried out using an unlabeled primary antibody, and then a further antigen-antibody reaction is carried out using another antibody labeled with peroxidase.

[0042] The indirect immunoperoxidase method can amplify the color signal, but requires washing each time an antigen-antibody reaction is performed, which may cause the cancer cells captured in the hydrophilic polymer layer to detach, and also requires a long processing time. On the other hand, the direct immunoperoxidase method requires less washing because the antigen-antibody reaction is performed only once, and can be processed in a shorter time than the indirect immunoperoxidase method. Therefore, in the above method, it is preferable to stain with the direct immunoperoxidase method.

[0043] The direct immunoperoxidase method can be carried out, for example, by adding a peroxidase-bound antibody (primary antibody) to the hydrophilic polymer layer that has been inactivated, reacting for a predetermined time (about 15 to 60 minutes), washing, and then adding a color-developing substrate and reacting for a predetermined time (about 1 to 10 minutes). These reactions are preferably carried out in the dark at room temperature (about 1 to 30°C).

[0044] The antibody may be any antibody that reacts with cancer cells and does not react with blood cells, and may be EpCAM, CK, etc. EpCAM is preferred because it does not require permeabilization of the cell membrane. Furthermore, the peroxidase that can be bound to the antibody may be horseradish peroxidase (HRP), etc.

[0045] As the color-developing substrate, diaminobenzidine (DAB), Co-DAB, etc. can be used.

[0046] After the staining process, washing, counterstaining with hematoxylin or the like (poststaining, nuclear staining), dehydration, mounting, etc. can be performed as necessary to obtain a sample in which cancer cells have been stained.

[0047] The obtained samples are easy to observe for cancer cells and their background tissues, and can be stored for a long time, so by observing these samples, a definitive diagnosis of cancer cells can be easily performed. EXAMPLES

[0048] The present invention will be specifically described based on examples, but the present invention is not limited to these examples.

[0049] (Production of testing equipment) Using AIBN (azobisisobutyronitrile), 2-methoxyethyl acrylate was thermally polymerized at 80°C for 6 hours to produce poly(2-methoxyethyl acrylate) (molecular weight Mn: approximately 15,000, Mw: approximately 50,000). A 0.25 W / V% methanol solution of the obtained poly(2-methoxyethyl acrylate) was then prepared. The prepared poly(2-methoxyethyl acrylate) solution (0.25 W / V%) was poured into a slide glass with a chamber and dried to prepare a hydrophilic polymer layer. The thickness of the hydrophilic polymer layer measured by TEM observation was 135 nm. Next, fibronectin was adsorbed onto the poly (2-methoxyethyl acrylate) coated portion (hydrophilic polymer layer), and the resultant was left at 40°C for 1 hour and then washed with PBS to prepare a test device.

[0050] (Example) Blood samples taken from colon cancer patients were centrifuged using OncoQuick to separate (concentrate) cancer cells. The fraction containing the cancer cells was then injected into the test device and left at room temperature overnight, after which it was washed with PBS, allowing the cancer cells to be selectively captured in the hydrophilic polymer layer.

[0051] After drying the test equipment, it was immersed in a 10% formaldehyde solution at room temperature for 10 minutes, and then washed with PBS.

[0052] 200mg of sodium azide was dissolved in 200ml of distilled water, and then mixed with 30V / V% hydrogen peroxide solution. The test device was immersed in the resulting mixture and left to react at room temperature for 10 minutes in the dark to inactivate endogenous peroxidase. It was then washed with PBS.

[0053] Serum prepared using the VECTASTAIN Elite Kit from Vector Laboratories was dropped onto the test device to perform serum blocking.

[0054] EpCAM (antibody) bound to HRP (label) was diluted with PBS and 200 μl of each was dropped onto the test device. After dropping for 30 minutes at room temperature, the device was washed with PBS. Next, DAB (coloring substrate) was dropped onto the test device and left for 4 minutes to react, staining the cancer cells. After that, the device was washed with running water for 2 minutes.

[0055] For post-staining (nuclear staining), hematoxylin was dropped onto the specimen and left for 15 seconds, after which it was washed with running water for 3 minutes.

[0056] The samples were prepared by mounting with a water-soluble mounting medium.

[0057] The obtained sample was observed under a microscope, and the microscopic image shown in Figure 1 was obtained. The area indicated by the arrow in Figure 1 is a cancer cell stained brown.

Claims

1. A step of capturing cancer cells in the blood in a hydrophilic polymer layer; Fixing the cancer tissue with formaldehyde; inactivating endogenous peroxidase in the blood using hydrogen peroxide and sodium azide; and staining the cancer cells by an immunoperoxidase method. A method for staining cancer cells, wherein the hydrophilic polymer layer is formed from the hydrophilic polymer described below in (b). (b) a polymer represented by the following formula (I): 【Chemistry 1】 (In the formula, R １ is a hydrogen atom or a methyl group, R ２ represents an alkyl group, m is 1 to 5, and n is the number of repetitions.)

2. 2. The method for staining cancer cells according to claim 1, wherein the immunoperoxidase method is a direct immunoperoxidase method.

3. 3. The method for staining cancer cells according to claim 1, wherein the blood is concentrated.

4. 4. The method for staining cancer cells according to claim 1, wherein the blood is collected from a patient before treatment.

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