JP6700048B2 - Anti-cancer drug evaluation method - Google Patents

Description

  The present invention is a method for evaluating the drug efficacy of an anticancer agent, particularly preferably an anticancer agent acting part of a tumor cell is labeled with a labeling substance, and the anticancer agent effectively acts on the tumor cell from a change in light intensity due to the application of the anticancer agent. It is about how to determine if it is working.

  An anticancer agent that targets a protein such as a receptor possessed by a tumor cell and specifically acts on a tumor cell having the target protein has been developed as a molecular target therapeutic agent. Examples include trastuzumab for HER2/neu of breast cancer cells, cetuximab for pancreatum mab, EGFR of colon cancer cells, rituximab for CD20 of lymphoma, bevacizumab for vascular endothelial growth factor VEGF of non-small cell lung cancer, ovarian cancer cells, etc. (Non-patent document 1). The effect of these anti-cancer agents on tumor cells is judged by the reduction of tumor tissue and the reduction of tumor markers in image diagnosis represented by CT and MRI examinations.

  When evaluating the effects of anti-cancer agents by CT or MRI imaging, it is necessary to wait for a period of about one month until the tumor size increases or decreases, so it is possible to judge the drug efficacy in a short period or in real time. Is not suitable for. In addition, since tumor cells do not directly detect tumor cells themselves, false positives due to factors other than tumors and false negatives that do not correlate with the deterioration of the disease state occur, so that the drug efficacy can be accurately evaluated. I haven't arrived.

  On the other hand, in recent years, clinical application of a cancer diagnosis method and a prognosis determination method using circulating blood tumor cells (Circulating Tumor Cell, CTC) has been proposed (Patent Document 1).

Japanese Patent Publication No. 2008-533487

Andrew M. Scott, et. al. 2012. Antibody therapy of cancer. Nature Reviews Cancer 12.

  By measuring the number of CTCs contained in a certain amount of blood over the period of anti-cancer drug administration and treatment, it has become possible to estimate the drug effect of the anti-cancer drug administered from the increase and decrease. However, in order to estimate the drug effect based on the CTC number, it was necessary to administer the drug for a certain period, and it was not possible to estimate the drug effect before the drug administration. Further, it was difficult to evaluate whether or not the actually administered anticancer drug acts on tumor cells based on only the increase/decrease in the number of CTCs. Therefore, the present invention aims to provide a method for evaluating the drug efficacy of an anticancer drug before the administration of the anticancer drug, and further to accurately evaluate the drug efficacy of the anticancer drug when actually administered. ..

  The present invention completed in view of the above-mentioned object is a method for evaluating the drug efficacy of an anticancer agent, particularly preferably an anticancer agent acting part of a tumor cell is labeled with a labeling substance, from a change in light intensity due to the application of the anticancer agent, It relates to a method for evaluating drug efficacy.

  The method of the present invention can detect rare tumor cells, and can directly detect the drug effect of an anticancer agent on tumor cells, and thus has high sensitivity as compared with image diagnosis and is more accurate for tumor markers. The drug efficacy can be evaluated. The method for evaluating the drug efficacy of the anticancer agent of the present invention can evaluate whether or not the anticancer agent exerts an effective therapeutic effect on each patient to be administered, before administration, or during or after the administration period. To This allows the selection of an appropriate anti-cancer agent and allows treatment with a regimen that minimizes side effects.

It is a figure for demonstrating the separation structure used for the concentration process of this invention. It is a figure for demonstrating the detection structure used for the expansion process and detection process of this invention. It is a figure for demonstrating the detection structure used for the expansion process and detection process of this invention. It is a figure for explaining a detection structure and a detection device used for the present invention. It is a figure for demonstrating the separation concentration method in the concentration process of this invention. It is a figure for demonstrating the separation concentration method in the concentration process of this invention. It is a figure which shows the detection result of the human breast cancer cell in the anticancer agent non-application sample of Example 1 of this invention (A). It is a figure which shows the detection result of the human breast cancer cell in the anticancer drug application sample of Example 1 of this invention (B). It is a figure which shows the light intensity change of a HER2 label|marker with or without application of trastuzumab in Example 1 of this invention. It is a figure which shows the detection result of the human colon cancer cell in the anticancer agent non-application sample of Example 2 of this invention (A). It is a figure which shows the detection result of the human colon cancer cell in the anticancer agent application sample of Example 2 of this invention (B). It is a figure which shows the light intensity change of an EGFR label|marker in the presence or absence of application of panitumumab in Example 2 of this invention.

The present invention is a method for evaluating the efficacy of an anticancer drug on a patient,
An anticancer agent applying step of applying an anticancer agent to a biological sample obtained from a patient,
An enrichment step of enriching tumor cells from a biological sample,
A development step of developing a biological sample contained in the concentrated liquid obtained in the concentration step on a substrate,
A detection step of detecting the light intensity by labeling the action part of the anticancer agent present in the tumor cells spread on the substrate,
An evaluation step of determining the effect of the anticancer agent based on the detected light intensity,
And a method for evaluating an anticancer agent.

Another aspect of the present invention is a method for evaluating the efficacy of an anticancer drug on a patient,
An enrichment step of enriching tumor cells from a biological sample obtained from a patient administered an anti-cancer agent,
A development step of developing a biological sample contained in the concentrated liquid obtained in the concentration step on a substrate,
A detection step of detecting the light intensity by labeling the action part of the anticancer agent present in the tumor cells spread on the substrate,
An evaluation step of determining the effect of the anticancer agent based on the detected light intensity,
And a method for evaluating an anticancer agent.

  INDUSTRIAL APPLICABILITY The present invention can evaluate the drug efficacy of an anticancer agent for patients suffering from or at risk of suffering from individual cancer. Among the anti-cancer agents, the molecular-targeted drug is a drug that targets one or several kinds of proteins contained in cancer cells, and therefore, the effect on the molecular-targeted drug may vary greatly depending on the type or mutation of the cancer cells. .. For example, in the case of an antibody drug containing a monoclonal antibody, if a mutation occurs in the antigenic site of a protein in cancer cells, the action as an antibody drug will be weakened. The method for evaluating the drug efficacy of the anticancer agent of the invention makes it possible to evaluate whether the anticancer agent exerts an effective therapeutic effect for each patient to be administered, before administration, or during or after the administration period. To do. This allows the selection of an appropriate anti-cancer agent or combination of anti-cancer agents as well as the choice of treatment with a regimen that minimizes side effects.

  In another aspect of the present invention, the evaluation method of the present invention evaluates the drug efficacy of an anticancer agent for a patient, selects an anticancer agent or a combination thereof having an excellent drug efficacy according to the evaluation, and comprises administering to the patient. It also relates to the treatment method of. Further, a further aspect of the present invention also relates to a method of diagnosing a patient as to which anticancer agent the cancer afflicted by the patient is susceptible to, which comprises the steps described above.

  In the present invention, the patient may be a patient suffering from cancer or a patient who has undergone cancer treatment such as surgery, medication, or radiation treatment for cancer, or may have metastatic cancer after cancer treatment. It may be a patient. Since the present invention can be used as a diagnostic method, it also includes patients at risk of suffering from cancer. The cancer afflicted by the patient of the present invention may be any cancer, for example, leukemia, lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma, hematopoietic cell malignancies such as multiple myeloma, brain tumor, breast cancer, endometrial cancer. -Uterus, cervical cancer, ovarian cancer, esophageal cancer, gastric cancer, appendix cancer, colon cancer, liver cancer, gallbladder cancer, cholangiocarcinoma, pancreatic cancer, adrenal cancer, gastrointestinal stromal tumor, mesothelioma-oral floor Lung cancer such as cancer, gingival cancer, tongue cancer, buccal mucosa cancer, oral cancer, head and neck cancer, salivary gland cancer, sinus cancer, thyroid cancer, kidney cancer, lung cancer, osteosarcoma, bone cancer, prostate cancer, testicular cancer, It includes but is not limited to kidney cancer, bladder cancer, skin cancer, anal cancer.

  In the present invention, the biological sample refers to any sample as long as it is a sample containing cells, and includes a biological sample obtained from a living body and a culture sample obtained by cell culture, tissue culture and the like. The biological sample is derived from biological samples such as urine, blood, plasma, serum, saliva, semen, feces, sputum, cerebrospinal fluid, ascites fluid, amniotic fluid, cell aggregates, tumors, organs or tissues such as lymph nodes or arteries. Examples include samples. Examples of the culture sample include cell cultures, tissue cultures, and culture solutions thereof. The present invention can be used to evaluate the effect of anticancer agents on tumor cells contained in biological samples. Tumor cells may be cells derived from any cancer, but more specifically, circulating blood tumor cells (CTC), which are cancer cells that metastasize through blood or lymph, such as gastric cancer, colon cancer, Examples thereof include CTC cells derived from esophageal cancer, liver cancer, lung cancer, pancreatic cancer, bladder cancer, uterine cancer (epithelial tumor), and lymphocyte and leukocyte tumor cells (lymphoma, leukemia) contained in blood. In another embodiment, a suspension of cells obtained from a tumor obtained by biopsy can be used as a sample.

  The anticancer agent evaluated in the present invention may be any anticancer agent used for the treatment of cancer. From the viewpoint of labeling the action site of the anticancer agent present in tumor cells, it is preferably a molecularly targeted therapeutic agent, and more preferably an antibody drug.

  Examples of molecular targeted therapeutic agents for treating cancer include any molecular targeted therapeutic agents that are on the market or will be marketed from the market, for example, imanitinib, gefitinib, erlotinib, afatinib, dasatinib, bosutinib, nilotinib, vandetanib, sunitinib, Tyrosine kinase inhibitors such as axitinib, pazopanib, lenvatinib, lapatinib, nintedanib, nilotinib, crizotinib, ceritinib, alectinib, ruxolitinib, tofacitinib, ibrutinib, and K, and sorafenib, vemurafenib, abrafenib, dabrafenib And MEK inhibitors such as trametinib and proteasome inhibitors such as bortezomib and carfilzomib.

  Examples of the antibody drug for treating cancer include any antibody drug that is on the market or is to be marketed, and examples thereof include rituximab, cetuximab, brentuximab vedotin, trastuzumab, bevacizumab, mepolizumab, gemtuzumab ozogamicin, Examples include mogamulizumab, pertuzumab, alemtuzumab, inotuzumab, panitumumab, ofatumumab, ipilimumab, ramucirumab, nivolumab and the like.

  The action site of the anticancer agent present in the tumor cells can be appropriately selected according to the type of the anticancer agent. For example, in the case of a tyrosine kinase inhibitor, a tyrosine kinase such as Bcr-Abl tyrosine kinase, KIT tyrosine kinase, EGFR tyrosine kinase, RET tyrosine kinase, VEGFR tyrosine kinase, PDGFR kinase, anaplastic lymphoma kinase, anaplastic lymphoma kinase, JAK2. Tyrosine kinase, Bruton's tyrosine kinase, etc. can be the action site. In the case of Raf kinase inhibitor, Raf kinase and B-Raf enzyme serve as action sites, CDK inhibitors serve as cyclin-dependent kinases, MEK inhibitors serve as MEK, and proteasome inhibitors serve as proteasomes as action sites. In the case of an antibody drug, the antigen bound by the antibody serves as the action site. From the viewpoint of facilitating detection, the action portion is preferably a membrane protein appearing on the cell surface.

Step of Applying Anticancer Agent The step of applying an anticancer agent is an in vitro step of applying an anticancer agent to a biological sample obtained from a patient. Prior to application of the anti-cancer agent, the biological sample may be subjected to pretreatment such as dilution, concentration and culture. In the present invention, the step of applying the anticancer agent may be carried out at any stage as long as the anticancer agent can be evaluated. As an example, the anticancer agent application step may be performed before the concentration step, the anticancer agent application step may be performed after the concentration step, and the anticancer agent application step may be performed after the development step. The concentration of the applied anti-cancer agent can be arbitrarily set according to the type of the applied anti-cancer agent. For example, it is possible to set a concentration similar to the blood concentration that can be achieved when the anticancer drug is administered to a patient according to a usual prescription, or a concentration higher or higher, but not limited to these concentrations. Not a thing.

  In another aspect of the present invention, in place of the anti-cancer agent application step, may include the step of administering an anti-cancer agent to the patient, instead of not including the anti-cancer agent application step, a biological sample obtained from the patient to whom the anti-cancer agent was administered. It can also be used as an evaluation method. In this case, the efficacy of the administered anticancer drug can be evaluated. The biological sample can be obtained at any timing after the administration of the anticancer agent, but it is preferable to obtain the biological sample before the decrease in tumor size or the decrease in CTC number occurs. Further, by obtaining biological samples over time during the anticancer agent treatment period, it is possible to evaluate the change in drug efficacy against the anticancer agent, that is, the acquisition of cancer resistance. When it is confirmed that the resistance to the anticancer drug is acquired, a new treatment policy such as surgery, radiation treatment, administration of another kind of anticancer drug, etc. can be considered to be useful for the cancer treatment.

Concentration step The concentration step is a step of separating and/or concentrating in order to facilitate detection of live tumor cells in the biological sample. The concentration method is not particularly limited as long as live tumor cells can be more selectively recovered by reducing components other than live tumor cells in the biological sample. For example, a filter method that separates and concentrates by utilizing the size difference between tumor cells and components other than tumor cells, a magnetic bead method that concentrates by antibody magnetic particles by utilizing the difference in antibody expression profile on the cell surface, an inter-cell A specific gravity method utilizing a difference in specific gravity can be exemplified. Among them, the specific gravity method is particularly preferable because it can selectively concentrate live tumor cells in a short time.

Details of the concentration step using the specific gravity method will be described below.
The specific gravity separation in the present invention is not particularly limited as long as tumor cells and components other than tumor cells can be separated by the difference in specific gravity. The density gradient solution is a liquid substance that forms a density gradient by itself or by centrifugation, and if the density (specific gravity) of the target cells is specified and an appropriate one is selected and used for the separation, good. Examples of the selection index include nutritional components, pH, isotonicity, and the like. Specifically, sucrose, glycerol, dextran, metrizamide, iodixanol, a copolymer of sucrose and epichlorohydrin, colloidal silica particles with a polyvinylpyrrolidone coating, sucrose polymer, diatrizoic acid, iohexol, nicodentz. Examples thereof include ionic and nonionic ones. Examples of commercially available density gradient solutions include GE Healthcare Bioscience's trade name Ficoll, Ficoll-Paque or Percoll, and Axis-Shield PoC AS trade name Lymphoprep, Polymorphprep or OptiPrep.

  As an instrument for carrying out the concentration step, for example, the cell separation concentration structure shown in FIG. 1 can be used. The separation/concentration structure 1 is composed of two tubular members 2 and 3. The tubular member 2 forming the upper portion of the separation/concentration structure has an opening, and the tubular member 3 has one end closed to form a bottom portion 5. The tubular members 2 and 3 each have a communication opening 6 provided at the opposite end of the opening or bottom, and when the two members are connected, the internal spaces of the two tubular members communicate with each other, so that one separation/concentration is achieved as a whole. Form a structure.

  The density gradient solution is injected from the bottom portion (closed end 5 of the tubular member 3) of the separation and concentration structure 1 to the vicinity of the separation portion. More specifically, when the separation/concentration structure is left stationary, the liquid level height of the density gradient solution becomes higher than the communication port end of the upper tubular member 2 (toward the tubular member 2 side), that is, When the lower tubular member (the tubular member 3) is separated, the components that have passed through the density gradient solution and moved to the closed end 5 side of the tubular member 3 by the centrifugal separation operation together with most of the density gradient solution. The target component (cells) maintained on the density gradient solution is injected into the tubular member 3 so that the target component (cells) can be separated while being maintained in the tubular member 2, preferably about 1 mm higher. Then, the biological sample solution is overlaid on the density gradient solution, the opening is sealed with the cap 4, and the centrifugation operation is performed. The centrifugation operation may be generally performed at a low speed of about 1000 to 2000×g, but the conditions to be maintained on the density gradient solution in consideration of the density of target cells and the density of the density gradient solution to be used. Select. For example, if the target cells are tumor cells and the centrifugation as described above is performed, the density of the density gradient solution should be set in the range of 1.060 to 1.095 g/mL according to the type of tumor cells. You can From the viewpoint of increasing the concentration rate of tumor cells, the density of the density gradient solution is preferably 1.075 g/ml or more, more preferably 1.080 g/ml or more. From the viewpoint of increasing the concentration rate of tumor cells, 1.100 g/ml or less is preferable, 1.096 or less is more preferable, and 1.093 or less is further preferable. More specifically, the density of the density gradient solution can be set in the range of 1.082 to 1.091 g/mL. The osmotic pressure of the density gradient solution can be set in the range of 200 to 450 mOsm/kg, and more preferably 300 to 400 mOsm/kg. The pH of the solution can be arbitrarily selected within a range where cells are not damaged, and it can be exemplified that the pH is adjusted to a range of 6.8 to 7.8.

  By the centrifugation operation, a component having a density higher than that of the density gradient solution passes through the gradient layer of the density gradient solution and moves into the lower tubular member (tubular member 3). On the other hand, target cells having a smaller density than the density gradient solution are maintained on the density gradient solution in the upper tubular member (cylindrical member 2). Therefore, if the connected tubular members are separated into the state shown in FIG. 1 while maintaining the airtightness of the opening, the upper tubular member (cylindrical member 2) will contain the target cells. Images can be collected. This fraction can be easily collected without requiring special skill by, for example, dropping the cap 4 to open the closed state and dropping the fraction downward. On the other hand, the fraction moved into the lower tubular member (the tubular member 3) can be discarded, for example, together with the tubular member.

  In the present invention, a target cell can be further efficiently separated by adding a substance that specifically binds to a target cell or by adding a substance that specifically binds to a component other than the target cell. .. The apparent density can be reduced by binding a substance that specifically binds to a substance having a relatively low density such as porous silica particles. Thus, as the substance used for the purpose of adjusting the density, in addition to the porous silica particles, for example, polyvinyl compounds such as polyethylene, polypropylene, polyvinyl chloride, polyacrylonitrile, polyacrylate, polymethacrylate, polycarbonate. Representative examples include organic polymers, polystyrene latex, nylon, copolymers such as polyterephthalate, inorganic materials such as glass, silica, zirconia, biopolymers such as cellulose, dextran, agarose, cellulose and sepharose, and biological samples such as red blood cells. As a substance that specifically binds to a target cell or the like, an antibody, an antigen, a peptide, a polypeptide, a growth factor, a cytokine, a biopolymer such as a lectin can be exemplified (hereinafter, referred to as a “specifically binding substance”). A substance that binds to a “substance used for adjusting the density” and adjusts the density of target cells or components other than the target cells is referred to as a “binding agent”).

  The concentration step may further include a selection step for the purpose of concentrating the target cells. As such a selection step, for example, when a blood sample is used, hemolysis treatment is performed. By performing the hemolysis step, the number of red blood cells can be reduced, thereby selectively enriching the target cells. Such a selection step may be performed after centrifugation for concentration or may be performed before centrifugation. Further centrifugation may be performed after the selection step.

  The collected cells can be measured by a method such as diluting or suspending, and then applying to a slide or microscopically observing the cells captured in a well or a flow cytometry method. In particular, the method of capturing and measuring cells one by one in a plurality of holding holes provided on the substrate by the dielectrophoretic force used in the present invention is more sensitive and highly accurate in observing and analyzing each cell. preferable.

Development Step The development step is performed by developing the concentrated liquid obtained through the concentration step on the substrate. By developing the concentrated solution, cells contained in the concentrated solution can be distributed on the substrate at intervals suitable for detection. It is preferable to develop in a non-aggregated state, and it is preferable to sufficiently suspend the concentrated liquid before development. In the developing step, any technique can be used as long as cells can be distributed on the substrate at an interval suitable for detection, and the concentrated solution may simply be applied on the substrate, but if necessary, Further processing may be performed. As an example, cells can be expanded by applying vibration or inductive migration force after applying the concentrated solution onto the substrate. In order to spread cells evenly, it is preferable to have holding holes on the substrate. By placing approximately one cell in each holding hole, it is easy to detect target cells in the subsequent detection step. become. Depending on the desired cell expansion density, the number of cells in the concentrated solution may be counted and diluted so that an appropriate number of cells may be expanded, and the concentrated solution to be expanded may be measured and expanded. You can also

  As the instrument used in the developing step of the present invention, for example, the biological sample detection structure shown in FIG. 2 can be used. This structure is a structure 8 having a plurality of holding holes (through holes) 7 for holding cells in order to detect light emitted by a substance indicating the presence of cells in a subsequent detection step, and is a flat plate shape. Are arranged on the substrate 9. Further, it is preferable that the substrate and the upper lid substrate 10 are made of a translucent material, and that the electrodes provided on the holding hole side of the substrate and on the surface of the upper lid substrate are transparent electrodes such as ITO. This makes it possible to observe the light emitted from the holding hole from above or below the substrate. The holding hole is composed of the insulator film 11, but may be provided with the light shielding film 12. By providing the light-shielding film, it is possible to reduce optical noise such as background noise caused by autofluorescence of the insulator film itself and crosstalk noise caused by leaked light from an adjacent holding hole. Only the light emitted by the substance to be observed inside can be detected with high sensitivity and high accuracy.

In addition, the structure includes an accommodating portion 13 that accommodates the suspension containing the cells above the retaining hole, and the retaining hole is provided so as to communicate with the accommodating portion. Further, the accommodating portion is provided with an inlet 14 for introducing the cell suspension and an outlet 15 for discharging the cell suspension.
A dielectrophoretic force is used as a method for capturing cells in the holding holes. Due to this dielectrophoretic force, living cells can be captured in a large number of holding holes in an extremely short time of about several seconds. In order to apply the dielectrophoretic force to the cells, it is sufficient to apply an AC electric field such that the lines of electric force are concentrated on the holding holes while the containing portion and the holding holes are filled with the suspension. As a structure for applying such an AC electric field, as shown in FIG. 3 in addition to the structure shown in FIG. 2, they are arranged on the surface of the substrate on the side of the holding holes at positions corresponding to different holding holes. The electrodes 16 and 17 forming a pair of electrodes (comb-shaped electrodes 18) are provided, and the holding hole extends from the upper surface of the holding hole to the comb-shaped electrode on the substrate. You can In any of the configurations, by exposing the electrode at the bottom of the holding hole and applying an AC voltage having a predetermined waveform between the two electrodes, the cells in the suspension are held in the holding hole by the dielectrophoretic force. Can be captured. Also, by arranging the holding holes in an array, the electric field generated by the voltage applied between the electrodes is generated almost uniformly in all the holding holes, and the cells are similarly guided and trapped in all the holding holes. can do.

  FIG. 4 is a diagram showing a detection device used in the present invention. As an example of the detection device used in the present invention, the substrate 9 and the upper lid substrate 10 shown in FIG. 2, an AC power supply 19 for applying an AC voltage for generating the dielectrophoretic force 24 to the electrodes, and an AC power supply from the AC power supply. And a detector 21 for detecting the light 20 emitted by the substance showing the presence of the cells 23 trapped in the holding holes of the structure after the voltage is applied. A fluorescence microscope can be illustrated as an example of the detection unit.

  An AC power source is connected to the pair of electrodes of the structure through a conductive wire 22. The AC power supply only needs to be able to apply a voltage sufficient between the electrodes to move the cells to the holding holes and generate an electric field necessary for the dielectrophoretic force to be captured. Specifically, a power supply that can apply an AC voltage having a peak voltage of about 1 V to 20 V and a waveform of a frequency of about 10 kHz to 10 MHz, such as a sine wave, a rectangular wave, a triangular wave, or a trapezoidal wave can be exemplified. In particular, it is particularly preferable to use a rectangular wave having a frequency of 100 kHz to 3 MHz as a frequency and a waveform capable of moving cells and capturing only one cell in one holding hole. As the AC voltage having such a waveform, the rectangular wave instantaneously reaches the set peak voltage as compared with the case where the waveform is a sine wave, a triangular wave, or a trapezoidal wave, so that the cells are moved quickly toward the holding hole. This makes it possible to reduce the probability that two or more cells enter the retention hole so that they overlap (there is a higher probability that only one cell can be captured in one retention hole). Although cells can be electrically regarded as capacitors, as long as the peak voltage of the rectangular wave does not change, it becomes difficult for electric current to flow through the cells trapped in the holding holes, and electric lines of force are less likely to occur. The dielectrophoretic force is less likely to be generated in the holding holes that have captured the cells. Therefore, once a cell is trapped in the holding hole, the probability that another cell is trapped in the same holding hole is low, and instead, electric lines of force are generated and dielectrophoretic force is generated in other holding holes ( The cells are sequentially captured in empty holding holes (where the cells are not captured).

  In addition, in the detection device used in the present invention, it is preferable to employ a power source that generates an AC voltage having no DC component. When an AC voltage with a DC component is applied, the electrostatic force (electrophoretic force) generated by the DC component causes cells to move with a biased force in a specific direction, making it difficult to capture cells by dielectrophoretic force. is there. Also, when an AC voltage with a DC component is applied, the ions contained in the cell-containing suspension generate an electric reaction on the electrode surface to generate heat, causing the cells to undergo thermal motion, making it impossible to control the movement due to dielectrophoretic force. , It becomes difficult to move to the holding hole and capture. The AC voltage having a DC component means a voltage having a frequency duty ratio of not 50%, a voltage having an offset, a voltage having an extremely long cycle (for example, 1 second or more), and the like.

Detecting Step The detecting step is a step of labeling the action portion of the anticancer agent existing in the tumor cells spread on the substrate and detecting the light intensity. In this detection, the action part existing in the tumor cells can be labeled with a labeling substance, and a light source corresponding to the label can be introduced to detect the light intensity. In the detection step, detection for distinguishing tumor cells from cells other than tumor cells may be performed simultaneously. In order to identify tumor cells, they may be labeled with a labeling substance such as a substance that is specifically expressed in tumor cells or a substance that is specifically expressed in non-tumor cells, or the size and shape of tumor cells, etc. It is also possible to identify by the appearance of.

  In the present invention, as the labeling substance, a labeling substance that can bind to a specific substance on the surface or inside of cells and that can be detected in the detection step is used. The specific substance is a substance that specifically exists/is not present on the surface or inside of a cell having a specific property (hereinafter, referred to as “target cell”), and is a substance that enables the identification of the target cell. I mean. To be specifically present in a target cell means that the target cell is present in the target cell and is not present in other than the target cell, or is present in the target cell more than in the cells other than the target cell. Specific substances include nuclear stains such as DAPI, which are used to distinguish certain tumor cells from red blood cells, and leukocyte markers such as CD45, which distinguish tumor cells from white blood cells and lymphocytes. In addition, the action part of the anticancer agent existing in the tumor cells is also included in the specific substance. Specific examples of tumor cells include CD20, CD30, CD33, CD52, HER2, HER3, EGFR, VEGFR, PD-L1, CEA, Mucins, PSMA, cytokeratin (CK) and the like.

  Among the specific substances listed above, CD20 is the action part of ibritumomabuchiuxetane, rituximab, ocrelizumab, etc., CD30 is the action part of brentuximab vedotin, etc., and CD33 is gemtuzumaboo. CD52 is an action part of alemtuzumab, HER2 is an action part of lapatinib, trastuzumab, pertuzumab, etc., EGFR is gefitinib, erlotinib, afatinib, vandetanib, lapatinib, cetuximab, VEGF is an action part of vandetanib, axitinib, sunitinib, pazopanib, nintedanib, sorafenib, bevacizumab, ranibizumab, ramucirumab and the like.

  The specific substance can be observed (detected) by labeling with a labeling substance that specifically binds to the specific substance. That is, a recognition substance that specifically binds to a specific substance and a label that emits an optically detectable signal are combined to form a labeling substance. The labeling substance for detecting the specific substance of the present invention is not particularly limited as long as the target cell can be detected. The labeling substance refers to any substance capable of showing the presence of components constituting cells. As a typical labeling substance, a substance capable of specifically binding to a specific substance such as an antibody, a ligand, a lectin, etc., which can be detected based on properties such as fluorescence, phosphorescence, or luminescence (for example, a fluorescent dye) An example is one in which The labeling substance is a substance that binds a substance capable of specifically binding to a specific substance, which is a substance that catalyzes a reaction that emits light, for example, a reaction that produces a substance that emits fluorescence or phosphorescence, or a luminescence reaction. Good. Examples of the fluorescent dye include FITC (fluorescein isocyanate), PE (phycoerythrin), and rhodamine. Moreover, examples of the substance that catalyzes the reaction include peroxidase, β-galactosidase, alkaline phosphatase, and luciferase.

  As described above, the labeling substance is a substance in which a substance that specifically binds to a specific substance and a label that emits an optically detectable signal are bound, even if it itself binds or reacts with the specific substance. It may be. When binding a label that emits an optically detectable signal to a substance that specifically binds to a specific substance, both may be directly bound by a known chemical method or the like, or specifically to the specific substance. It may be indirectly bound to the substance to be bound via a substance to be bound. For example, a substance that specifically binds to a specific substance may be bound to biotin, and a substance that generates a signal may be bound to avidin or streptavidin. In this case, avidin or streptavidin bound to a substance that generates a signal binds to biotin bound to a substance specifically bound to a specific substance, and as a result, (specific substance)-(specific substance specific The specific substance is indirectly labeled with the labeling substance by forming a complex of (binding substance)-(biotin)-(avidin or streptavidin)-(label generating a signal). Such indirect cases are also included in the labeling substance of the present invention.

  The detection step used in the present invention is not particularly limited as long as the action site existing in tumor cells is labeled with a labeling substance and an increase or decrease in light intensity can be detected. Preferably, a device capable of observing a biological sample may be used by including the substrate and an optical detection unit that irradiates an observation region of the substrate with light and detects an optical signal generated. As the light source, a halogen lamp, a mercury lamp, a metal, a ride lamp, a laser, an LED, or the like can be used, and the light from the light source is observed by an optical means including an optical filter, a mirror, a lens, or the like as necessary. The light may be guided to the area. A fluorescence microscope system is an example of a system capable of performing such a detection step.

  As optical signal information, an image (bright field) composed of light intensity distribution by transmitted light and reflected light in a wide wavelength range, fluorescence intensity detected from fluorescence emitted by a fluorescent substance, peak value of fluorescence intensity, and fluorescence intensity Examples include a minimum value, an average value of fluorescence intensity, a numerical value calculated from fluorescence intensity such as an integrated value of fluorescence intensity, and the like. The bright field information is not particularly limited as long as the information on the cell morphology can be acquired. For example, the diameter of a cell, the area of a cell, the volume of a cell, the perimeter of a cell, the circularity, etc. can be illustrated.

  In a general detection step, the action part existing in the tumor cells may be labeled with a labeling substance to be detected, and at the same time, another labeling substance may be used or the bright field observation may be performed to identify the tumor cells. As an example, tumor cells may be fluorescently labeled using an antibody labeled with FITC or PE, for example, an anti-CK antibody, or a fluorescent labeling substance for identifying cells other than tumor cells, for example, an anti-CD45 antibody or a nuclear staining substance. After fluorescent labeling with (DAPI etc.), observation with a fluorescence microscope or the like can be mentioned. More specifically, in the present invention, in order to detect CTC, nucleated cells are identified (excluding red blood cells) by fluorescence observation at a DAPI wavelength (UV excitation and blue emission), and the presence or absence of expression of CD45 and CK. Identify. The DAPI-positive, CK-positive and CD45-negative cells thus identified are tumor cells (CTC), DAPI-positive, CK-negative and CD45-positive cells are leukocytes, and DAPI-positive, CK-positive and CD45-positive or DAPI Negative and CK positive cells can be distinguished as a noise signal typified by dust. In addition, DAPI-positive, CK-negative, and CD45-negative cells are compared with normal cells such as red blood cells and white blood cells in bright field observation by comparing the cell morphology and size, or by performing Papanicolaou staining or Giemsa staining to obtain intracellular nuclei. CTCs can also be specified by morphological characteristics such as cytoplasm. When a tumor cell (CTC) is identified in the detection step, the identification can be performed manually by a fluorescence microscope, by a fluorescence-labeled image taken, or automatically by software.

Evaluation step In the detection step, after detecting the light intensity of the action site existing in the tumor cell, the effect of the anticancer agent can be determined based on the detected light intensity. This is because the labeling substance that binds to the action site cannot bind to the action site depending on the binding property of the anticancer agent to the action site, and the light intensity from the labeling substance changes. For example, when the anti-cancer agent acts (binds) on the action site existing on the surface of the tumor cell to mask the action site, the labeling substance cannot bind to the action site and the light intensity decreases. In addition, even when the anticancer agent acts (bonds) on the action site existing on the surface of the tumor cell to internalize the specific substance (Internalization) and exhibits a drug effect of suppressing the growth signal, the labeling substance after application of the anticancer agent Cannot act on the action part, and the light intensity decreases. Therefore, after the application of the anti-cancer agent, if the light intensity from the labeling substance bound to the specific substance of the tumor cells is decreased, the anti-cancer agent can act on the action site, and thus it can be determined that the drug efficacy is high. ..

  By comparing the light intensity detected by performing the same concentration, development, and detection steps as those of the present invention on the biological sample to which the anticancer agent is not applied, the change in the light intensity can be determined, thereby determining the effect of the anticancer agent be able to. Therefore, a comparison step may be included instead of or in addition to the evaluation step. Further, the effect of the anticancer agent may be determined by preparing a control or a calibration curve in advance according to the type of cancer and comparing it with the measured light intensity. When compared with a biological sample to which no drug is applied, the light intensity is reduced to 70% or less, preferably to 50% or less, and more preferably 30% as compared with the biological sample to which the drug is not applied. When it decreases below, it can be judged that the tested anticancer agent is effective, while there is almost no change in light intensity, for example, the change is 20% or less, preferably 10% or less, more preferably 5% or less. In some cases, it can be determined that the anticancer drug is not effective.

  In the method for evaluating the efficacy of an anticancer agent in a patient to which an anticancer agent is administered, the biological sample obtained before the administration of the anticancer agent is the same concentration as that of the present invention, the light intensity detected by performing the detection step, and the obtained after the administration of the anticancer agent. By comparing the light intensity detected for the biological sample, the change in light intensity can be determined, and thereby the effect of the anti-cancer agent. Therefore, a comparison step may be included instead of or in addition to the evaluation step. When the biological sample is obtained over time after the administration of the anticancer drug, the change in the efficacy of the anticancer drug can be evaluated by comparing the light intensities among the biological samples thus obtained. it can. Further, the effect of the administered anticancer agent may be determined by preparing a control or a calibration curve in advance according to the type of cancer and comparing it with the measured light intensity. When comparing with a biological sample before administration of an anticancer agent, as compared with a biological sample before administration of an anticancer agent, when the light intensity is reduced to 70% or less, preferably to 50% or less, more preferably 30% The tested anti-cancer agent can be judged to be effective if it falls below. On the other hand, when there is almost no change in the light intensity, for example, when the change is 20% or less, preferably 10% or less, more preferably 5% or less, it can be determined that the anticancer agent is not effective.

  All documents mentioned in this specification are incorporated herein by reference in their entirety. Hereinafter, the present invention will be described in more detail based on examples, but the present invention is not limited to the examples.

Example 1 Method for detecting HER2-expressing strain (SKBR3) As a blood sample, a suspension prepared by adding about 3000 human breast cancer cells (SKBR3) to 3 ml of blood of a healthy person obtained by obtaining informed consent was prepared by applying no anticancer agent. It was used as a sample. Similarly, a suspension prepared by mixing about 3000 human breast cancer cells (SKBR3) treated with 10 μg/mL trastuzumab (Herceptin) for 3 hours in 3 ml of blood of a healthy person was used as a sample for anticancer drug application.

  Tumor cells were enriched from these samples using the isolation construct of the embodiment shown in FIG. In detail, in the separation structure, the cylindrical member 2 is a cylindrical polypropylene member having an inner diameter of 18 mm, a length of 70 mm, and a capacity of 15 mL. The inclination angle of the tapered portion of the tubular member 2 is 30°, and the communication opening with the tubular member 2 is Φ2 mm. The tubular member 3 is a polypropylene member having an inner diameter of 10 mm, a length of 54 mm, and a volume of 3 mL.

  As schematically shown in FIG. 5, 3 mL of the density gradient solution 25 having a density of 1.084 g/mL was injected into the tubular member 3 on the lower side of the separation structure (the white portion in the lower part of the figure represents the density. Part filled with gradient solution). Specifically, the solution was injected so that the liquid level of the density gradient solution was higher than the communication opening 6 of the upper tubular member 2 by about 1 mm (the liquid surface is therefore located inside the upper tubular member). Subsequently, a mixed solution 26 of 3 mL of blood sample, 3 mL of physiological saline, and 75 μL of a binder (trademark RosetteSep, StemCell Technologies Inc) was overlaid on the density gradient solution (mixing in which black-painted portion was overlaid in the figure). Part of the liquid).

  After injecting the sample, the opening of the separation structure was sealed with a cap 4 (made of polypropylene), and the mixture was centrifuged at 2000×g for 10 minutes at room temperature. By the centrifugation operation, the cells were maintained at the interface 27 between the density gradient solution and the sample, as shown on the left side of FIG. After the tubular members 2 and 3 constituting the separation structure are separated at the separating portion without removing the cap, the cap is removed to open the seal as shown on the right side of FIG. A part of the density gradient solution and the cells maintained thereon were allowed to flow out from the communication opening 6 of the above, and were collected with a 50 mL tube installed below, and the inner wall of the upper tubular member was washed to remove the cells attached to the wall. Collected at the same time.

  An erythrocyte lysate containing ammonium chloride as a main component was added to the collected cell suspension to make up to 30 mL, and the mixture was centrifuged at 300×g for 10 minutes at room temperature. After centrifugation, the liquid at the top of the pellet was pipetted off, the cells in the pellet were resuspended in 30 mL of 300 mM mannitol solution and centrifuged at 300 xg for 5 minutes at room temperature. This centrifugation operation is for removing cell debris and platelets and for concentrating tumor cells. The number of normal leukocytes mixed with the tumor cells is 300,000 to 500,000, and the density is increased by binding cells outside the tumor cells (erythrocytes and leukocytes) with a binder to increase the density difference with the tumor cells. Then, by performing the separation using the separation structure, the tumor cells can be separated with high recovery and selectivity.

  For the measurement of the collected cells, a method such as coating on a slide or observing cells captured in a well under a microscope or a flow cytometry method can be used. In the present Example 1, a method was adopted in which the separated cells were captured by the dielectrophoretic force in the holding holes (about 300,000) having a hole diameter of Φ30 μm and a depth of 40 μm, which were provided on the substrate.

The cell suspension was introduced into the accommodating portion of the structure shown in FIG. 2, an AC voltage (voltage 20 Vpp, frequency 1 MHz, rectangular wave) was applied between the substrates, and the cells were trapped in the holding holes by the dielectrophoretic force.
While applying the AC voltage, a 300 mM mannitol aqueous solution containing 0.01% poly-L-lysine was introduced into a container in which approximately one cell was trapped in one holding hole, and after standing for 3 minutes, The application of the alternating voltage was stopped, and the solution in the container was removed by suction. Then, a cell membrane permeation reagent was introduced into the accommodation part, and the cell membrane was permeated by leaving still for 10 minutes. Then, the solution in the container was removed by suction, PBS was introduced, and the cell membrane permeation reagent remaining in the container was washed.

  Next, 800 μL of cell stain (FITC-labeled anti-CK antibody (Miltenyi Biotec), PE-labeled anti-HER2 antibody (R&D systems), APC-labeled anti-CD45 antibody (Beckman-Coulter), DAPI (0.5 μg/mL) A cell staining solution mixed with (Dojindo Laboratories Inc.) was sent to carry out cell labeling (25° C., 30 minutes), followed by washing with an aqueous mannitol solution to detect human breast cancer cells. Since both tumor cells and normal white blood cells have nuclei, they were stained with DAPI, and those without nuclei such as red blood cells and dead cell debris were excluded with detection software. Further, although tumor cells do not express CD45, but there are cells expressing CK and cells not expressing CK, DAPI-positive, CD45-negative, CK-positive and negative cells were extracted by image processing. Then, the light intensity by PE-labeled anti-HER2 was calculated for each human breast cancer cell extracted by the image processing. Since normal white blood cells express CD45 but not CK, they were identified as tumor cells by detecting DAPI-positive, CK-negative, and CD45-positive cells.

An image of the entire holding part was taken in order to observe all the cells captured in the plurality of holding holes. For this, a fluorescence microscope (IX71; Olympus) equipped with a computer-controlled motorized stage and an electron multiplying cooled CCD camera (EMCCD; FLOVEL, ADT-100) was used. LabVIEW (National Instruments) was used as image acquisition and analysis software. The number of normal white blood cells trapped in the holding holes after the treatment was about 100,000 to 500,000, which was an amount that did not interfere with the detection of human breast cancer cells. Moreover, since human breast cancer cells were trapped in the holding holes, the total number of cells could be counted by scanning the holding part. Since human breast cancer cells (SKBR3) added to the blood sample are cell lines in which both CK and HER2 are highly expressed, in the sample to which the anticancer agent is not applied, as shown in FIG. 7A, cells positive for both CK and HER2 (DAPI positive, CD45 negative ) Could be detected.
On the other hand, in the sample to which the anticancer agent was applied, as shown in FIG. 7B, cells in which CK was positive but the light intensity due to HER2 labeling was remarkably reduced were detected.

  The relationship between the light intensity and the abundance of each HER2-labeled tumor cell was measured and shown in the graph (FIG. 8). Since the light intensity of the HER2 label treated with trastuzumab was largely shifted to the low brightness side, it was confirmed that trastuzumab acts on the human breast cancer cells.

Example 2 Standard Detection Method for EGFR-Expressing Strain (WiDr) In the same manner as in Example 1, a sample prepared by mixing approximately 1000 human colon cancer cells (WiDr) with 3 ml of blood of a healthy subject was used as a sample to which an anticancer agent was not applied, A sample prepared by mixing 3 ml of blood of a healthy subject with about 1000 human colon cancer cells (WiDr) treated with panitumumab (Vectivix) at 20 μg/mL for 2 hours was used as an anticancer drug application sample.

  In the same manner as in Example 1, the anticancer agent-untreated sample and the anticancer agent-applied sample were overlaid with a mixed solution of physiological saline and a binder on the density gradient solution, followed by centrifugation and recovery operations.

  The cell suspension was introduced into the accommodating portion of the structure shown in FIG. 2, an AC voltage (voltage 20 Vpp, frequency 1 MHz, rectangular wave) was applied between the substrates, and the cells were trapped in the holding holes by the dielectrophoretic force.

  While applying the AC voltage, a 300 mM mannitol aqueous solution containing 0.01% poly-L-lysine was introduced into a container in which approximately one cell was trapped in one holding hole, and after standing for 3 minutes, The application of the alternating voltage was stopped, and the solution in the container was removed by suction. Then, a cell membrane permeation reagent was introduced into the accommodation part, and the cell membrane was permeated by leaving still for 10 minutes. Then, the solution in the container was removed by suction, PBS was introduced, and the cell membrane permeation reagent remaining in the container was washed.

  Next, 800 μL of cell stain (FITC-labeled anti-CK antibody, PE-labeled anti-EGFR antibody, APC-labeled anti-CD45 antibody, DAPI (0.5 μg/mL)-mixed cell stain) was delivered to the container, and the cells were transferred. After labeling (25° C., 30 minutes), the cells were washed with an aqueous mannitol solution to detect human colon cancer cells. Since both tumor cells and normal white blood cells have nuclei, they were stained with DAPI, and those without nuclei such as red blood cells and dead cell debris were excluded with detection software. Further, although tumor cells do not express CD45, but there are cells expressing CK and cells not expressing CK, DAPI-positive, CD45-negative, CK-positive and negative cells were extracted by image processing. Then, the light intensity by the PE-labeled anti-EGFR was calculated for each human colon cancer cell extracted by the image processing. Since normal white blood cells express CD45 but not CK, they were identified as tumor cells by detecting DAPI-positive, CK-negative, and CD45-positive cells.

  An image of the entire holding part was taken in order to observe all the cells captured in the plurality of holding holes. For this, a fluorescence microscope (IX71; Olympus) equipped with a computer-controlled motorized stage and an electron multiplying cooled CCD camera (EMCCD; FLOVEL, ADT-100) was used. LabVIEW (National Instruments) was used as image acquisition and analysis software. The number of normal white blood cells trapped in the holding holes after the treatment was about 100,000 to 500,000, which was not an obstacle to the detection of human colon cancer cells. Moreover, since human colon cancer cells were trapped in the holding holes, the total number of cells could be counted by scanning the holding part. Since the human colon cancer cell (WiDr) used in Example 2 is a cell line that highly expresses CK and expresses in EGFR, the sample to which the anticancer agent is not applied can be labeled well with CK as shown in FIG. Although the intensity was low, it could be detectably labeled. On the other hand, in the sample to which the anticancer agent was applied, as shown in FIG. 9B, cells in which the CK was positive but the light intensity was decreased by the EGFR labeling were detected. FIG. 10 shows the change in the light intensity of the EGFR label with and without the panitumumab treatment. Although the light intensity of the EGFR-labeled human colon cancer cells not exposed to panitumumab was low, the light intensity analysis showed that the panitumumab treatment group shifted to the low intensity side. It was confirmed that the effect on tumor cells can be detected with high sensitivity.

1 Separation structure 2 Cylindrical member (upper side)
3 Cylindrical member (lower side)
4 Cap 5 Bottom 6 Communication opening end (separation part)
7 Holding hole (through hole)
8 structure 9 substrate 10 upper lid substrate 11 insulating film 12 light-shielding film 13 accommodating portion 14 inlet port 15 outlet port 16 electrode (+)
17 electrodes (-)
18 Comb-shaped electrode 19 AC power supply 20 Light 21 Detection part 22 Conductive wire 23 Cell 24 Dielectrophoretic force 25 Density gradient solution 26 Mixed liquid 27 Interface

Claims (9)

A method of assessing the efficacy of an anti-cancer agent in a patient, comprising:
An anticancer agent applying step of applying an anticancer agent to a biological sample obtained from a patient,
A concentrating step of concentrating tumor cells from the biological sample,
A developing step of developing cells contained in the concentrated solution obtained in the concentrating step on a substrate,
A detection step of detecting the light intensity by labeling the action part of the anticancer agent present in the tumor cells spread on the substrate,
Based on the detected light intensity, an evaluation step of determining the efficacy of the anticancer agent,
The method comprising: A method of assessing the efficacy of an anti-cancer agent in a patient, comprising:
An enrichment step of enriching tumor cells from a biological sample obtained from a patient administered an anti-cancer agent,
A development step of developing a biological sample contained in the concentrated liquid obtained in the concentration step on a substrate,
A detection step of detecting the light intensity by labeling the action part of the anticancer agent present in the tumor cells spread on the substrate,
An evaluation step of determining the effect of the anticancer agent based on the detected light intensity,
The method comprising:   The method for evaluating an anticancer agent according to claim 1 or 2, wherein the concentration step is a specific gravity method.   The concentrating step includes a tubular structure having one end closed to form a bottom and the other end having an opening, and a cap sealing the opening. The structure is composed of two or more tubular members and is separated. The method for evaluating an anticancer agent according to claim 3, wherein a separation structure that is separable in part is used.   The said anticancer agent is a molecular target drug, The evaluation method of the anticancer agent as described in any one of Claims 1-4 characterized by the above-mentioned.   The method for evaluating an anticancer agent according to claim 1, wherein the tumor cells are circulating tumor cells in blood.   The method for evaluating an anticancer agent according to claim 1, wherein the action part is selected from the group consisting of HER2 and EGFR.   The evaluation method according to claim 7, wherein when the action site is HER2, the anticancer drug is one or more drugs selected from the group consisting of lapatinib, trastuzumab, and pertuzumab.   The evaluation method according to claim 7, wherein when the action site is EGFR, the anticancer agent is one or more drugs selected from the group consisting of gefitinib, erlotinib, afatinib, vandetanib, lapatinib, cetuximab, and panitumumab.