JP2008011797A - Method for evaluating anticancer action - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To properly evaluate an anticancer action in vivo in a method for evaluating an anticancer action in vitro. <P>SOLUTION: The method for evaluating an anticancer action on cancer cells of a medicine associated with an anticancer action comprises: a process (a) for sticking and supporting drop lump 20 of collagen gel embedding cancer cells 40 on the inside of a culture vessel 10; a process (b) for supplying a culture solution 30 containing a medicine 50 associated with an anticancer action and immune cells 60 coexisting with the cancer cells 40 in an in vivo environment to the culture vessel 10 and culturing the cancer cells 40; and a process (c) for evaluating the culture result of the cancer cells 40 in the collagen drop lump 20. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a method for evaluating an anticancer effect, and more specifically, is used for selection of an anticancer agent used for cancer treatment or development of an anticancer agent, and is involved in an anticancer effect such as an anticancer agent. The present invention is directed to a method for evaluating the anticancer effect of a drug on a cancer cell by culturing the cancer cell in the presence of the drug.

Cancer cells are cultured in vitro in the presence of anticancer agents for the development of anticancer agents or the selection of appropriate anticancer agents for cancer treatment. Evaluation of the anticancer effect of anticancer drugs on the skin is being carried out.
However, in conventional methods for evaluating anticancer effects, drugs that can exert excellent anticancer effects when administered in vivo are not used because only low evaluations can be obtained in the evaluation of anticancer effects in vitro. In some cases, such as in some cases, in vitro evaluation results do not necessarily lead to actual clinical effects. Therefore, in the conventional evaluation method, it is not possible to select a drug such as an anticancer agent suitable for cancer treatment, and the results of cancer treatment may not be improved.

  Even in the research and development of anticancer drugs, even if the effectiveness of a drug is determined by a conventional evaluation method from a large number of drugs, it often does not match the actual clinical results, which is a major obstacle to research and development. It was.

  Therefore, an object of the present invention is to reproduce the in-vivo environment and appropriately evaluate the anticancer action in vivo in the above-described method for evaluating the anticancer action in vitro.

The present inventor has made various studies in order to solve the above problems. In the process, if the in vivo environment is reproduced in the anticancer action evaluation test, the state in which the subject is actually acting in vivo can be reproduced. I noticed that it was possible to avoid the occurrence of a problem that the result was not. Specifically, when supplying drugs that are involved in anticancer effects to culture vessels, I realized that it would be sufficient to supply immune cells that coexist with cancer cells in the environment in vivo. .
By the way, as an evaluation method that looks similar to the idea of the present inventor, there is a method disclosed in JP-A-11-174053. In the method disclosed in JP-A-11-174053, a lymphocyte medium obtained by adding an anticancer drug or the like to a solution of lymphocytes separated from blood and a target cell solution containing cancer cells are mixed, and immunization is performed. It is a technology that measures and evaluates performance. This technology is intended to evaluate the anticancer effect of lymphocyte immunity in the presence of anticancer drugs, but since it is trying to evaluate cancer cells by monolayer culture, it is clinically It has been found that it is necessary to carry out the culture at a concentration of the anticancer agent 10 to 100 times higher than the concentration of the anticancer agent to be administered. However, in such a high-concentration environment of anticancer agents, immune cells may be killed, so that it is difficult to culture in the presence of immune cells, and the culture method described in JP-A-11-174053 In the end, it was impossible to say that anti-cancer action was evaluated in vitro by reproducing the internal environment.

Therefore, in conducting an evaluation test based on reproduction of the in vivo environment, a culture method that allows the sensitivity of an anticancer drug to be observed even if the anticancer drug is used at a concentration similar to the concentration of the anticancer drug administered to the human body. It became necessary to adopt.
The inventor is able to see the sensitivity of an anticancer agent even when an anticancer agent is used at a concentration similar to the concentration of an anticancer agent administered clinically to the human body in the evaluation of the anticancer effect. Various studies were repeated on the law. In the process, attention was paid to a culture method using a droplet-like collagen gel, which was previously developed by the present applicant. In this culturing method, as described in detail in International Publication No. WO95 / 18216, a droplet of a collagen solution in which animal cells are dispersed is gelled on the surface of the support to form a droplet-like collagen gel. This is a technique of forming and fixing on the surface and bringing the culture solution into contact with the droplet-shaped collagen gel.

As described in International Publication No. WO95 / 18216, this culture technique previously developed by the present inventor can be applied to a droplet consisting of a gel of collagen even if only a small amount of animal cells can be recovered. By culturing in an embedded state, an accurate and sufficient sensitivity test can be performed, and whether or not this technology can evaluate clinically the concentration of physiological anticancer drugs administered to the human body. There was no knowledge about.
As the inventor made various studies focusing on this culture technique, the following has been found.
With this technology, even if the anticancer drug concentration in the anticancer drug susceptibility test is approximately the same as the anticancer drug concentration clinically administered to the human body, the anticancer effect can be observed. . Moreover, in utilizing this culture technique, as a method of reproducing the internal environment, if immune cells are allowed to coexist in the culture solution, activation of immune cells by using cytokines as activators is not required. That is to say. In order to evaluate immune therapy in vitro using immune cells, it is necessary to evaluate immune cells that have been activated by culturing for a long time with cytokines such as IL-12 and co-cultured with cancer cells. It was conventional technical common sense to say that. Activation of immune cells through the use of cytokines takes a long time and is therefore difficult to match the time needs of cancer patients. Moreover, there was an economic problem of using expensive cytokines. However, when immune cells were allowed to coexist in the culture medium, it was found that a cytokine network was spontaneously generated and it was not necessary to activate immune cells in advance by using cytokines as activators.

As described above, the present inventors have found that, according to the technology for three-dimensional culture of cancer cells in a collagen gel, it can be evaluated with a physiological anticancer agent concentration that is clinically administered to the human body, By coexisting immune cells, it was found that the body environment can be reproduced in vitro and the anticancer action can be evaluated at low cost and without being restricted by the time schedule, and the present invention has been completed. It was.
That is, the method for evaluating anticancer activity according to the present invention is a method for evaluating the anticancer activity of a drug involved in anticancer activity on cancer cells, wherein the collagen embeds the cancer cells. A step (a) of fixing a gel droplet to an inner surface of a culture container; and a drug involved in the anticancer action in the culture container and an immune cell coexisting with the cancer cell in an in vivo environment. A step (b) of culturing cancer cells by supplying a culture solution containing the step, and a step (c) of evaluating the culture results of the cancer cells in the collagen droplets.

Each configuration will be specifically described.
〔cancer cell〕
Cancer cells to be evaluated for anticancer activity are used for cancer treatment research and development of anticancer drugs, in addition to primary cancer cells collected from cancer patients who perform cancer treatment. In addition, there are subcultured cancer cells prepared in advance. In addition to human cancer cells, animal cancer cells and artificially cultured cancer cells may also be used.
[Drugs involved in anticancer effects]
The drug involved in the anticancer action according to the present invention is an anticancer agent that directly acts on cancer cells, and does not directly attack cancer cells, but is an immune cell in vivo. And drugs that function to inhibit the growth of cancer cells, to slow down the activity of cancer cells, or to kill cancer cells by cooperative action with other drugs.

Examples of the anticancer agent include, but are not limited to, alkylating agents, platinum derivatives, antimetabolites typified by 5FU anticancer agents, topoisomerase inhibitors, microtubule inhibitors, and the like. It is done.
The above-mentioned drugs that do not directly attack cancer cells and are involved in the anti-cancer action other than anti-cancer drugs are not particularly limited, for example, anti-drug prodrugs, anti-cancer drugs or Examples thereof include agents that modulate intracellular metabolic enzyme activities related to the metabolism of the prodrug (hereinafter referred to as “intracellular enzyme modulators”), immunotherapeutic agents, and the like.
The prodrug of the anticancer agent is converted into an active form by intracellular enzymes of organs such as liver and cancer cells, but the amount of active substance increases by increasing the enzymatic activity of the cytokine network, Since it brings about the enhancement of the antitumor effect, it is mentioned as a drug involved in the anticancer action.

As the intracellular enzyme regulator, for example, a single agent does not have a direct antitumor effect, but is involved in an anticancer effect by inhibiting a degrading enzyme (DPD) of a 5FU anticancer agent. , Gimeracil and the like.
Examples of the immunotherapeutic agent include drugs used for biological response modifier therapy such as krestin, lentinan and OK-432 (hereinafter abbreviated as “BRM preparation”), cytokine preparations such as interleukins and interferons, and the like. Can be mentioned.
In addition, a drug that finally participates in the anticancer action can be used by enhancing the function of the anticancer agent or improving the in vivo immune function.

These drugs can be used alone, or those that show anticancer activity for the first time when combined with a specific drug can be used in a predetermined combination. Even a drug that can be used alone is used in such a combination when the anticancer action in combination with another drug is evaluated.
[Basic culture technology]
Basically, culture techniques employed in normal methods for evaluating anticancer effects are applied.
There are no particular limitations on basic culture techniques such as culture apparatus, culture vessel, supply / exchange of culture solution, and adjustment and maintenance of the culture environment.

The culture techniques described in the above-mentioned International Publication WO95 / 18216 and JP-A-2003-9853 can be used.
[Culture container]
In a normal culture technique, a culture container that can hold a culture solution with cancer cells, which is a culture sample, is used. If the surface which can fix a collagen gel is provided, the specification and structure will not be limited. For example, a culture dish such as a petri dish or a multi-dish, a flask or the like can be used. Culture plates such as so-called cover slips or cell disks, which are in the form of glass or plastic flakes, can be used by being housed in a separate container for storing the culture solution. In this case, the surface of the culture plate corresponds to the inner surface of the culture vessel.

The inner surface of the culture vessel that firmly supports and supports the collagen gel droplets means the inner wall surface or the inner bottom surface, particularly the inner bottom surface, and may be smooth or spread of the supplied collagen solution droplets. It is also possible to provide an image line composed of a frame-like protrusion or groove for regulating These culture vessels and supports are preferably optically transparent in order to facilitate observation and evaluation after culture.
[Collagen gel droplets]
The technique of the international publication WO95 / 18216 specification can be applied.
<Collagen solution>
A conventional collagen material used in various cell embedding culture methods, a polymer material such as a polysaccharide or other extracellular matrix, and a liquid material are used. As the collagen, acid-soluble type I collagen can be used.

In addition to collagen and cancer cells, various components necessary for culture can be added to the collagen solution. For example, there are nutritional components and bactericidal components. A part of immune cells can be added to the collagen solution. Various cells that are normal cells, not cancer cells, and compounds that act on the cells, for example, inorganic salts such as calcium and calcium phosphate, lipids, carbohydrates, and proteins can also be added.
The collagen solution preferably contains a buffer solution composition that is the same as or close to the physiological condition of the target cancer cell. In cancer cells derived from animals including humans, the buffer strength is pH 6.2 to 7.6, preferably pH 6.8 to 7.4, and the salt strength when converted to salt is set to 100 to 180 mmol. It is preferable to leave. More preferably, it is set to 140 to 160 mmol.

As a method of blending a culture sample containing cancer cells with a collagen solution, a conventional method is applied. The density of the cancer cells seeded in the collagen solution can be set to about 10 ³ to 10 ⁷ cells / ml.
The collagen concentration and viscosity of the collagen solution affect the structure of the collagen gel droplet formed. Specific numerical conditions vary depending on conditions such as the purpose of the test, but the collagen concentration is preferably 0.1 to 2.0% by weight. If the concentration is too high, the viscosity becomes high, and if the concentration is too low, it is difficult to maintain the droplet shape. The viscosity is preferably 50 to 2000 centipoise, more preferably 100 to 1000 centipoise. If the viscosity is too low, the cells settle in the collagen solution and come into contact with the inner surface of the culture vessel and grow into a single layer. Therefore, the effects of anticancer agents and the like cannot be accurately evaluated. If the viscosity is too high, handling of the collagen solution is difficult. The gel strength when the collagen solution is gelled also has an effect on the performance. Collagen having a gel strength of about 50 to 1000 grams or about 50 to 700 grams is preferable, and more desirably, the gel strength is 100 to 500. Use grams. The numerical value of gel strength is a value measured according to JIS. If the gel strength is too low, the collagen gel droplets may peel off during the culture, or the gel may easily expand and contract. If the gel strength is too high, the growth of cancer cells is suppressed. .

<Formation of collagen gel droplets>
When the collagen solution is supplied to the inner surface of the culture vessel in the form of drops, the collagen solution swells on the surface of the support due to the action of the surface tension of the collagen solution. Specifically, it is possible to employ a method in which a collagen solution is dropped directly from a collagen solution container, or a collagen solution in a form of water droplets is gently placed using an instrument such as a spoid.
The supplied collagen gel solution gels as it is. A collagen gel having a substantially hemispherical shape is formed. In some cases, the upper end of the hemisphere is flat, or the planar shape is an ellipse or ellipse other than a circle. It depends on various conditions such as viscosity and temperature of collagen solution, amount of collagen solution, wettability of inner surface. It can be set to a preferred shape according to the purpose of the culture and the evaluation method after the culture. For example, a shape that allows easy imaging or image analysis of a collagen gel droplet is selected.

One or a plurality of collagen gel droplets of the same form made of the same type of collagen solution can be provided in the culture container. Plural types of collagen gel droplets having different collagen solutions and different cancer cells contained therein can be provided in the same culture vessel. The plurality of collagen gel droplets may be arranged at intervals from each other, and the plurality of collagen gel droplets may be in contact or connected in part.
Since the collagen gel droplet is gelled after the droplet of the collagen solution is arranged on the inner surface of the culture vessel, the collagen gel droplet is surely fixed to the support surface. Therefore, the culture result of the target cancer cell can be easily identified and observed or photographed after culturing, rather than in the case of a collagen gel spreading in layers.

Under normal test conditions, the size of one drop-like collagen gel can be set to 3 to 300 microliters in terms of the amount of collagen solution drops to be placed. Preferably it is 3 to 150 microliters. Practically, those in the range of 5 to 100 microliters are preferred. More preferably, about 15 to 50 microliters is used. When primary cells are cultured, those of around 30 microliters are preferred. The height of the collagen gel droplet lump is preferably set to be about 2 mm or less.
The number and arrangement interval of the droplet-shaped collagen gel formed in the culture container can be arbitrarily set according to the size of the droplet-shaped collagen gel and the structure of the support.

Conventionally, a collagen gel used for microscopic observation and image analysis has to have a high transparency, but even a relatively low transparency can be used in the method of the present invention. Specifically, those having a transmittance of 1 to 95% with respect to 400 nm light can be used. Those having a low transmittance within the above range can be satisfactorily tested without being affected by turbidity by carrying out a dyeing treatment described later.
The formed collagen gel droplets need to be maintained in a gel state at least until the culture is completed. When the three-dimensional collagen gel droplets are also dried, the water is lost, and a flat dry product is formed. Once dried, it is difficult to return to the original three-dimensional shape even if it is brought into contact with moisture. Therefore, the collagen gel droplet can be kept in a water-containing state so as not to be excessively dried after the collagen gel droplet is formed and before the culture solution is supplied. There is no concern that the collagen gel droplets will dry after contacting the culture. Fixing and drying the collagen gel droplet after the culture has no problem in evaluating the culture result.

<Advantages of collagen gel droplets>
By using collagen gel droplets, cancer cells can be cultured in a state close to the in vivo environment.
In the living body, anticancer agents and the like are transported to the site where cancer cells exist together with nutrients and the like by blood and lymph, and the anticancer agents and the like act from the entire periphery of the cancer cells.
As in the prior art, the state in which the anticancer agent, nutrients and waste are in and out only on the flat surface of the collagen gel layer formed on the entire inner bottom surface of the culture dish is the above-mentioned in vivo environment. Due to the large difference, the anticancer effect could not be evaluated accurately.

Collagen gel droplets are much smaller in volume than layered collagen gels and are adequate and sufficient even if only a small amount of cancer cells can be obtained for the purpose of culturing from living tissue. Sowing at a high density. Moreover, in the case of a droplet-like collagen gel, the large area of the convex surface comes into contact with the culture solution. The uptake of nutrients into the cells and the discharge of waste from the cells is carried out very efficiently. Moreover, the quantity of the culture solution which contacts a collagen gel can be increased relatively. A culture environment very close to the in vivo environment is established.
[Culture medium]
Basically, techniques common to culture media used in normal culture methods can be applied.

The culture solution contains a drug involved in the anticancer effect and immune cells that coexist in the in vivo environment with the target cancer cells.
In addition, nutrient components and pH adjusting components necessary for culture are included as necessary.
It is preferable to mix the components of the culture solution so as to be suitable for the growth of the target cancer cells and to suppress the growth of other cells. As a culture solution, a serum culture solution containing serum and a serum-free culture solution not containing serum are known, and any of them can be used in the present invention. A serum-free culture medium is characterized in that a culture medium used for normal cell culture contains serum as a component, but does not contain serum. Therefore, the serum-free culture solution is configured by combining various chemical substances other than serum necessary for culture. Specific blending components and their proportions can be set as necessary, but use a serum-free culture solution that has a composition that ensures the growth of the desired cancer cells and inhibits the growth of other cells. . In particular, it is preferable to use a serum-free culture medium composed of a compounding component that suppresses excessive proliferation of fibroblasts. In serum culture fluids, those with a serum concentration of about 0.001 to 5.0% by weight are usually used, but good results can be obtained even when a serum concentration of about 5 to 20% by weight is used. In some cases. The amount of the culture solution only needs to cover the collagen gel droplet lump so that the culture can be performed satisfactorily. When the number of cancer cells embedded in collagen gel droplets is large, it is necessary to increase the amount of the culture solution or shorten the replacement interval of the culture solution during the culture. .

<Immune cells>
It is an immune cell that coexists with the cancer cell that is the subject of culture in the in vivo environment.
Specifically, PBMC (peripheral blood mononuclear cells in autologous plasma) can be used. PBMC includes lymphocytes and monocytes. Monocytes include macrophages. Lymphocytes include NK cells, B cells, and T cells. These components are used alone or in combination.
The immune cell may be collected from a living body, or may be synthesized or cultured in vitro.

There may be multiple types of immune cells, such as the ABO blood group, which have slightly different properties even with the same component. If necessary, any one type of immune cells can be used, or multiple types of immune cells can be used in combination.
When used for cancer treatment, immune cells contained in bodily fluids such as blood and lymph collected from a living body of a cancer patient can be used. In this way, it is possible to construct a culture environment that is closer to the in vivo environment, including the types of immune cells described above.
It is preferable that immune cells (E; effector cells) are contained in the culture solution in an amount of 0.01 to 100 in terms of the ratio of the number of cells to cancer cells (T; Tumor) (hereinafter referred to as “E / T ratio”). In this range, an evaluation result with particularly good detection sensitivity can be obtained. More preferably, it is 0.1-100, More preferably, it is 0.5-10.

[Culture of cancer cells]
In a state where the culture solution is supplied to the culture vessel in which the collagen gel droplet lump is arranged, the culture operation is performed for a certain period.
After the culture medium is supplied in the first stage of the culture work, it may be left until the culture work is completed, or a new culture liquid may be additionally supplied in the middle, or replaced with a culture liquid having a different composition component. You can also Some of the components of the culture solution can be added later. For example, it is also possible to prepare a culture solution containing necessary components excluding immune cells in advance, supply this culture solution to the culture vessel, and then add only immune cells later. Different immune cells can be added to a plurality of culture vessels.

In order to bring the culture solution into contact with the collagen gel droplets in the culture vessel, it is sufficient that the culture solution is simply poured into a culture dish or the like so that the liquid level is high enough to cover the collagen gel droplets. When the culture plate is placed in the culture container, the culture solution may be injected while the culture plate is accommodated. As the culture solution, various known culture solutions are used according to the test conditions.
Culturing is carried out by maintaining a predetermined period of time under environmental conditions such as a constant temperature culture apparatus or a carbon dioxide gas culture apparatus in a state where the droplet-like collagen gel fixed on the support surface is in contact with the culture solution. What is necessary is just to make an anticancer agent contact the collagen gel droplet fixed to the inner surface of a culture container before the culture | cultivation start or the suitable stage in culture | cultivation. Specifically, an anticancer agent may be added to the culture solution or replaced with a culture solution to which the anticancer agent has been added. The same applies to drugs other than anticancer drugs. Even when testing the influence of temperature changes such as heating or radiation, the collagen gel droplets may be exposed to such environmental conditions for a certain period of time.

[Evaluation of culture results]
Evaluate the results of culturing cancer cells in collagen droplets. In the collagen gel droplet lump that has been cultured, cancer cells grow or die according to the culture conditions.
Basically, a normal technique for evaluating anticancer activity may be applied.
For example, the number of colonies and cells formed by visual observation with a microscope can be measured, or an image obtained by taking a photograph or video can be analyzed. When the culture plate is accommodated in the culture container and cultured, the culture plate can be taken out of the culture container and observed or photographed. In image analysis, a captured image is electronically processed and discriminated by analyzing it with a computer or the like, and the growth state of a target cancer cell can be accurately evaluated.

  By selectively staining only live cells in collagen gel droplets and evaluating the results, cancer cells can be clearly distinguished from live cells and dead cells. It is possible to accurately evaluate the proliferation state of cancer cells. As a staining method, a normal staining method for various cells can be employed as long as it is a method that can selectively stain live cells compared to dead cells and other objects. Specific staining agents and staining conditions may be according to ordinary methods. For example, NR (neutral red) staining method, latex particle staining method, FDA (fluorescein diacetate) staining method using intracellular enzyme activity, or other staining methods using cell phagocytosis A staining method using a fluorescent reagent can be employed. For the colorimetric analysis by NR staining, the technique of “image colorimetric quantification method” described in Japanese Patent Publication No. 3222785 can be used, and the image absorbance measured by this image colorimetric quantification method is: It has been confirmed that it exhibits good linearity with respect to the number of viable cancer cells in an NR-stained collagen gel drop (literature Jpn. J. Cancer. Res. 92, p203-210, 2001).

As another method for evaluating the culture results, colorimetric analysis can be performed using a color reagent that selectively causes a color reaction due to the metabolic activity of living cells. If a water-soluble color reagent is used, as with FDA staining, cancer cells are not adversely affected, and evaluation over time can be performed. As the color reagent, AB (Alamar Blue, trade name, manufactured by Alamar Bioscience) colorant, WST-1 colorant, XT colorant, MTT dye reducing agent, and the like can be used.
A method of selectively staining a substance contained in cells and evaluating the result can also be applied. For example, a staining reagent used for tissue staining can be used for staining. Specific stains include dyes such as hematoxylin, Giemsa solution, crystal violet, ethidium bromide that stains nucleic acids, reagents that stain components such as proteins, carbohydrates, and lipids, reagents that stain cell membrane substances, cells Examples thereof include reagents for staining specific sites such as an internal skeleton, antigens for specific antigens, DNA probes, and the like.

Staining of live cells and cell components can be performed in double and triple layers. By multiple staining of cells, various effects of cells can be evaluated on the same sample.
The culture result can also be evaluated after the collagen gel droplet lump is dissolved and separated from the support surface. Specifically, there is a method of quantifying a substance contained in cells in a collagen gel droplet lump and evaluating the result. For example, a Coomassie brilliant blue coloration method or a Raleigh method for quantifying proteins, a DABA fluorescence coloring method for quantifying nucleic acids, a luciferin luminescence method for measuring ATP, and various methods for quantifying saccharides can be used. There is also a method for quantifying substances secreted from cells into the culture solution and evaluating the results. For example, a method for quantifying lactic acid, which is a representative metabolite (waste product), and the like can be mentioned.

  The method for evaluating anticancer activity according to the present invention is a method in which a culture solution supplied to a culture vessel is resistant to a culture solution supplied in a state where cancer cells are embedded in a droplet of collagen gel fixedly supported on the inner surface of the culture vessel. By including a drug involved in cancer action and immune cells that coexist with cancer cells in the in vivo environment, the anticancer action of the drug against cancer cells can be achieved in a culture environment very close to the in vivo environment. Can be evaluated.

[Evaluation of anticancer drugs]
The embodiment shown in FIG. 1 schematically shows a basic method for evaluating an anticancer agent.
A hemispherical collagen gel droplet, that is, a collagen gel drop 20 is fixedly supported on the inner bottom surface of the culture vessel 10. The collagen gel drop 20 is formed by dropping a droplet of a collagen solution containing cancer cells 40 onto the inner bottom surface of the culture vessel 10 and gelling it in a hemispherical state.
The culture solution 30 is supplied to the culture vessel 10 from above the collagen gel drop 20. The culture solution 30 contains nutrient components necessary for culturing the cancer cells 40 and is added with PBMC (peripheral blood mononuclear cells in autologous plasma) such as lymphocytes.

In the culture environment thus prepared, the anticancer agent 50 is added to the culture solution 30. The anticancer agent 50 enters the collagen gel drop 20 from the culture solution 30 and attacks the cancer cell 40 to kill it, stop its activity, or suppress its growth.
After finishing the culture test for a certain period, the proliferation state or the decrease state of the cancer cells 40 in the collagen gel drop 20 is evaluated by a conventional method.
When the anticancer agent 50 is used for cancer treatment, PBMC60 which is an immune cell in the living body also exists in the in vivo environment including the cancer cell 40 to which the anticancer agent 50 is administered. The PBMC 60, which is an immune cell, usually works in cooperation with the anticancer agent 50 to enhance the function of the anticancer agent 50. Depending on the anticancer agent 50, the PBMC 60 may suppress the function.

In any case, the culture environment described above is an environment very close to the in vivo environment in combination with the support of the cancer cells 40 in the three-dimensional structure of the collagen gel drop 20. The performance of the cancer agent 50 or the sensitivity of the cancer cells 40 to the anticancer agent 50 will be evaluated.
[Evaluation of intracellular enzyme regulators]
The embodiment shown in FIG. 2 is a method for evaluating an anticancer action when an anticancer agent and an intracellular enzyme regulator are used in combination, and specifically, a DPD inhibitor is used as the intracellular enzyme regulator. This is an example. Since it is basically the same as the above embodiment, the differences will be mainly described.

The collagen gel drop 20 that embeds the cancer cells 40 is fixedly supported on the inner bottom surface of the culture vessel 20, and the culture solution 30 containing the PBMC 60 is supplied from the same to the above embodiment. .
In such a culture environment, CDHP as the DPD inhibitor 52 is added together with 5FU as the anticancer agent 50. It is known that the DPD inhibitor 52: CDHP has an action of inhibiting the metabolism of the anticancer agent 50: 5FU and enhancing the effect of 5FU.
The anticancer agent 50 acts in cooperation with the PBMC 60 and the DPD inhibitor 52 to perform functions such as attack on the cancer cells 40 and growth suppression.

If another drug is used as the intracellular enzyme adjusting agent instead of the DPD inhibitor 52, the intracellular enzyme adjusting function of the drug can be compared or evaluated. If the combination of the anticancer agent 50 and the DPD inhibitor 52 is variously changed and cultured and evaluated, an appropriate combination for the cancer cell 40 can be found.
[Evaluation of immunotherapeutic agents]
The embodiment shown in FIG. 3 is a method for evaluating the anticancer effect of an immunotherapeutic agent, and is specifically an example when a BRM preparation is used as the immunotherapeutic agent. The BRM preparation 54 acts on the cancer cell 40 via immune cells in the living body, unlike the normal anticancer agent 50 that acts directly on the cancer cell 40. Since it is basically the same as the above embodiment, the differences will be mainly described.

The collagen gel drop 20 that embeds the cancer cells 40 is fixedly supported on the inner bottom surface of the culture vessel 20, and the culture solution 30 containing the PBMC 60 is supplied from the same to the above embodiment. .
In such a culture environment, PRM (product name: Krestin) which is the BRM preparation 54 is administered. The BRM preparation 54 acts in cooperation with the PBMC 60 to perform functions such as attack on the cancer cells 40 and growth inhibition.
In addition to the BRM preparation 54, it does not act directly on the cancer cells 40, but in the presence of immune cells in the living body, the growth and activity of the cancer cells 40 are suppressed or killed. It is also possible to evaluate the functions and effects of other immunotherapeutic agents that are effective in doing so.

[Evaluation of other anticancer effects]
In addition to the above-described embodiments, the present invention can also be used for applications in which a more appropriate anticancer effect can be evaluated by a culture environment close to the in vivo environment.
For example, a prodrug of an anticancer agent that is not an anticancer active substance, such as 5'DFUR, can be evaluated. 5'DFUR is underestimated for its anticancer effect in a normal anticancer drug sensitivity test. However, in vivo, when the enzyme in the tumor is activated by the cytokine network, 5′DFUR is changed into an active form in the tumor and exhibits an anticancer effect. If it is a culture environment in which PBMC 60 is present in the culture solution 30, the environmental conditions are the same as those in the living body described above, and thus the anticancer effect of 5′DFUR can be appropriately evaluated. Of course, the same evaluation can be performed for a drug having the same function as 5′DFUR.

  If the BRM preparation 54 or 5′DFUR and the anticancer agent 50 are simultaneously administered to the culture solution 30, these synergistic anticancer effects can be evaluated.

The anticancer activity evaluation method according to the present invention was specifically carried out and its performance was evaluated.
Example 1
In Example 1 shown below, when an anticancer agent 5FU and an intracellular enzyme regulator CDHP (DPD inhibitor) are three-dimensionally cultured in a collagen gel drop, immune cells are allowed to coexist as in the present invention. It is clarified that the immune system in the body can be reproduced, but when 5FU and CDHP are used in combination, the conventional evaluation method may be underestimated than the actual anticancer action in the clinical setting. It has been known.
[Immune cells]
10 ml of blood was collected from a human peripheral blood vessel into a blood collection tube containing heparin. The same amount of the above blood sample was added with a pipette to 10 ml of PBS (phosphate buffer solution, manufactured by Invitrogen) at room temperature and mixed.

Dispense 25 ml of Ficoll solution (manufactured by GE Healthcare) into each 50 ml centrifuge tube, and add 20 ml of the collected blood dilution solution on the liquid surface so as not to break the density gradient of Ficoll solution. Poured carefully and layered on the Ficoll liquid surface.
This was centrifuged with a centrifuge at room temperature, 1800 rpm, and brake-off for 20 minutes, and the white intermediate layer in the centrifuge tube was sucked with a pipette to recover a PBMC (plasma peripheral blood mononuclear cell) layer. Subsequently, after centrifugation at 4 ° C., 1700 rpm, brake on for 10 minutes, the supernatant is discarded, and the sediment is tapped and pulverized, and then RPMI1640 (manufactured by Invitro) is added to this sediment while pipetting. The total volume was made up to 40 ml to obtain a suspension.

The suspension is further centrifuged at 4 ° C., 1500 rpm, brake-on for 10 minutes, the supernatant is discarded, and the sediment is tapped again and pulverized. Then, RPMI1640 (manufactured by Invitro) is added to the sediment. 5 ml was added, pipetted and suspended, and the number of cells was measured.
Then, after centrifuging the suspension with a centrifuge at 4 ° C., 1300 rpm, and brake-on for 10 minutes, RPMI1640 was added to prepare a cell suspension of 1 to 5 × 10 ⁷ cells / ml. .
The recovered cell suspension was cryopreserved as follows.
Mix the above recovered cell suspension with the same amount of cryopreservation solution, dispense 1 to 2 ml each into multiple freezing tubes (2 ml volume), place in refrigerator, -20 ° C freezer, -80 ° C deep freezer and place The solution was gradually cooled and frozen, and finally stored frozen under liquid nitrogen. At this time, the cryopreservation solution used for cryopreservation of the cell suspension was prepared by mixing 3.4 ml of CP-1 (manufactured by Kyokuto Pharmaceutical Co., Ltd.) and 1.6 ml of 25% by weight serum albumin (manufactured by Yoshitsugu Pharmaceutical Co., Ltd.) in ice. We used what we did.
〔cancer cell〕
Human colorectal cancer cell line HCT-116 was peeled off with 0.1% by weight trypsin-containing Hanks solution (manufactured by Invitrogen; Ca and Mg free), collected at 1300 rpm × 3 minutes using a centrifuge, and subjected to blood cell count The number of cells was measured with a board, and it was confirmed that this cancer cell suspension could secure the necessary number of cells.
[Collagen solution]
1 volume of Ham F12 (made by Nitta Gelatin Co., Ltd.) with 10 times the concentration of 8 volumes of acid-soluble type I collagen 0.3 wt% solution (Nitta Gelatin Co., Ltd., “Cell Matrix Type CD”) Then, 1 volume of reconstitution buffer solution (50 mM NaOH solution containing 260 mM sodium bicarbonate and 200 mM Hepes) (manufactured by Nitta Gelatin Co., Ltd.) was placed in each ice-cooled 50 ml centrifuge tube and mixed in the tube. The following operations were performed using the collagen mixture thus obtained.

That is, the cell suspension of HCT-116 is centrifuged at 1300 rpm × 3 minutes, and the resulting precipitate is suspended in DME medium (manufactured by Invitrogen), and then the cell density of cancer cells is 2 × 10. ^The cancer cell suspension and the collagen mixture were placed in an ice-cooled 50 ml centrifuge tube so as to be ⁵ cells / ml and mixed in the tube.
[Preparation of collagen gel drop]
Next, using a micropipette, 3 drops (total 90 μl) of the collagen mixture containing the above cancer cells per well of a 6-well plate are placed in a hemisphere and placed in a 37 ° C. carbon dioxide incubator. Gelled for 1 hour to produce a collagen gel drop.
[Culture medium and culture work]
To the collagen gel drop in which the above cancer cells were embedded, 4 ml of a serum-free culture solution (manufactured by Nitta Gelatin Co., “PCM2”) was added, overlaid, and cultured overnight. To this, as shown below, immune cells, anticancer agents and the like were added, and the culture operation was continued.
[Addition of immune cells]
Estimate the number of cells necessary to prepare 2 × 10 ⁵ cells / ml of the above-mentioned immune cells derived from blood samples, thaw the required number of immune cells in liquid nitrogen frozen storage in a 37 ° C. water bath, Some cells are sampled and mixed with an equal amount of 0.4% by weight trypan blue solution (manufactured by Invitrogen). Immediately after mixing, the number of cells is measured, and the number of living cells excluding dead cells is confirmed. The number of immune cells obtained in this work was calculated.

The number of necessary immune cells was calculated so that the E / T ratio in one well of the 6-well plate was 1, and an immune cell suspension for all wells (for all data) was prepared. In addition, since the immune cell suspension is added to 4 ml of each medium of a 6-well plate overlaid with a collagen gel drop containing cancer cells, the density of the immune cell suspension is also considered in consideration of the dilution factor. It was determined. Based on the above, a fixed amount of immune cell suspension was added to one well of a 6-well plate in which cancer cells were embedded with collagen gel.
[Addition of anticancer drugs, etc.]
Prepare 5FU (Kyowa Hakko Kogyo) at 30 μg / ml and DPD inhibitor CDHP (manufactured by Taiho Pharmaceutical Co., Ltd., “Gimeracil”) at 60 μg / ml. 5FU and CDHP were brought into contact with cancer cells embedded in the collagen gel drop by adding to each 4 ml of each medium of a 6-well plate overlaid with a collagen gel drop. 5FU and CDHP were added overnight after the drop was prepared, and contact was continued for 6 days under contact conditions of final concentration of 5FU of 0.3 μg / ml and CDHP of 0.6 μg / ml.
[Evaluation of anticancer effect]
After 6 days of continuous contact, Neutral Red staining solution (Nitta Gelatin Co., Ltd.) was added in an amount of 40 μl per well of 4 ml of each medium, and living cells were stained at 37 ° C. in a carbon dioxide incubator. Performed for 3 hours under% CO ₂ concentration. Subsequently, 10 ml by weight of neutral buffered formalin solution was added in an amount of 3 ml per well of a 6-well plate, and the dye was fixed in the cells over 1 hour. The plate on which live cells were stained and fixed was washed with water in a vat filled with tap water for 30 minutes to remove excess fixing solution. Finally, in order to dry the collagen gel, the washed plate was drained, and then the collagen gel drop was air-dried in a clean bench to obtain a sample for image colorimetric determination.

The culture plate sample was measured in the following manner according to “Image Colorimetric Determination Method” described in Japanese Patent Publication No. 3222785.
Light transmitted from below to each collagen gel drop of the untreated group and the contact-treated group (group treated with drugs and / or immune cells involved in anticancer action) in the culture plate sample. Was irradiated.
For each sample (collagen gel drop) in the untreated group and the contact treated group, from the sample image information input to the computer, on the image between the image when there is no sample (blank image) and the image when there is (sample image) Absorbance was calculated. The image absorbance was calculated for all samples.

X% (contact treatment group OD / non-treatment group OD × 100%) was calculated from the image absorbance value of the contact treatment group when the image absorbance value of the non-treatment group was 100%.
The evaluation was performed by calculating the growth inhibition rate; 100-X%, and showing how many cancer cells in the contact treatment group inhibited growth compared to the untreated group (0%).
As a result of the above steps, the growth inhibition rate against human colon cancer cell line HCT-116 was 15% in the group treated with drugs (5FU and CDHP) alone, which were involved in anticancer action, as compared to the untreated group. However, in the group treated with anticancer drugs (5FU and CDHP) and immune cells (treated group under reproduction of the body environment), it shows 80% of the untreated group, and shows a remarkable inhibitory effect. I understood.

In the treatment group with drugs alone involved in anticancer effects, the anticancer effects were underestimated rather than the actual clinical results, whereas treatment groups with drugs involved in anticancer effects and immune cells were actually It can be seen that the anticancer activity is excellent as well as the clinical results.
Therefore, in the presence of immune cells in PBMC, the action of CDHP, which enhances the effect by inhibiting the enzyme (DPD) that degrades anticancer drug 5FU, is a drug alone involved in anticancer action. It can be seen that it is further remarkably enhanced. This is considered to be a phenomenon recognized by the interaction of CDHP with components derived from immune cells secreted from PBMC, and it can be seen that the in vivo diversity of anticancer activity can be reproduced.
-Example 2-1
Example 2-1 shown below shows that when an anticancer drug prodrug 5′DFUR (manufactured by Chugai Pharmaceutical Co., Ltd., “Flutulon”) is three-dimensionally cultured in a collagen gel drop, immune cells are used as in the present invention. However, when 5'DFUR is used, the conventional evaluation method shows almost no anticancer effect, and the actual clinical system can be reproduced. It is known to be underestimated than the anticancer activity in the field.

Preparation of cell suspension of cell line HCT-116, preparation of collagen gel drop, co-culture with immune cells derived from PBMC, cell staining and fixation, and image colorimetric method The operation of the quantitative method is the same as in Example 1.
5′DFUR, which is a prodrug of an anticancer agent, was prepared to 300 μg / ml with physiological saline. By adding this 5 ′ DFUR to each medium of 4 ml per well of the 6-well plate overlaid with the cancer cell-containing collagen gel drop in Example 1, the 5 ′ DFUR was added to the collagen gel drop. Contacted with embedded cancer cells. 5'DFUR was added overnight after the drop was prepared, and contact was continued for 6 days under the contact conditions of 5'DFUR at a final concentration of 3 µg / ml.

As a result of the above steps, the growth inhibition rate against the human colon cancer cell line HCT-116 was 13% in the drug (5′DFUR) alone treatment group involved in the anticancer action compared to the untreated group. However, in the group treated with the drug (5′DFUR) and immune cells involved in the anticancer activity, 29% of the untreated group showed an increase in the suppression effect.
Since 5′DFUR is a 5FU prodrug and not an active form, it has been considered that the antitumor effect is hardly exhibited in the anticancer drug sensitivity test. However, in vivo, an enzyme in a tumor is activated by a cytokine network, and is converted into an active form in the tumor, thereby exhibiting an antitumor effect.

In the above evaluation results, the drug alone treatment group involved in the anticancer action is underestimated for the anticancer action compared to the actual clinical result, while the drug and immunity involved in the anticancer action are not evaluated. As can be seen from the fact that the treatment group with cells shows a superior anticancer effect as in the actual clinical results, the method according to the present invention reproduces the cytokine network and is very close to the actual in vivo environment. It can be seen that a culture environment has been established.
-Example 2-2
In Example 2-2 shown below, a prodrug 5′DFUR (manufactured by Chugai Pharmaceutical Co., Ltd., “Flutulon”) and a BRM preparation PSK (manufactured by Kureha Co., Ltd., “Krestin”) are combined in a collagen gel drop. In the original culture, if immune cells coexist as in the present invention, it is clarified that even the immune system in the body can be reproduced. However, when 5′DFUR and PSK are used together, It is known that this would be underestimated than the actual anticancer effect in the clinical setting.

Preparation of cell suspension of cell line HCT-116, preparation of collagen gel drop, co-culture with immune cells derived from PBMC, cell staining and fixation, and image colorimetric method The operation of the quantitative method is the same as in Example 1.
5 ′ DFUR was prepared in the same manner as in Example 2-1. By adding this 5′DFUR and PSK as a BRM preparation to each medium of 4 ml per 6-well plate overlaid with the collagen gel drop containing cancer cells of Example 1, 5 ′ DFUR and PSK were contacted with cancer cells embedded in collagen gel drops. 5′DFUR and PSK were added overnight after the drop was prepared, and contact was continued for 6 days under the contact conditions of 5′DFUR at a final concentration of 3 μg / ml and PSK at 100 μg / ml.

As a result of the above steps, the growth inhibition rate against human colon cancer cell line HCT-116 was 15% in the group treated with drugs (5′DFUR and PSK) alone, which were involved in anticancer action, as compared to 15% in the untreated group. However, in the group treated with drugs (5′DFUR and PSK) and immune cells involved in the anticancer action, 45% of the group treated with no treatment was markedly enhanced.
In the treatment group with drugs alone involved in anticancer effects, the anticancer effects were underestimated rather than the actual clinical results, whereas treatment groups with drugs involved in anticancer effects and immune cells were actually It can be seen that, as with the clinical results, the anticancer activity is more excellent.
Looking at the above evaluation results, the drug-only treatment group involved in anti-cancer activity is underestimated for anti-cancer activity than the actual clinical results. It can be seen that the group treated with cells shows a better anticancer effect as in the actual clinical results. From this, it can be seen that also in Example 2-2, the cytokine network is reproduced and a culture environment very close to the actual in vivo environment is constructed as in Example 2-1.
Example 3
In Example 3 shown below, when the BRM preparation PSK (manufactured by Kureha, “Krestin”) is three-dimensionally cultured in a collagen gel drop, the immune system in the body can be obtained by coexisting immune cells as in the present invention. However, when 5'DFUR and PSK are used in combination, the conventional evaluation method underestimates the anticancer effect rather than the actual anticancer effect in the clinical setting. It is known that

Preparation of cell suspension of cell line HCT-116, preparation of collagen gel drop, co-culture with immune cells derived from PBMC, cell staining and fixation, and image colorimetric method The operation of the quantitative method is the same as in Example 1.
By adding PSK, which is a BRM preparation, to each medium of 4 ml per 6-well plate overlaid with the collagen gel drop containing cancer cells of Example 1, PSK was embedded in the collagen gel drop. Contacted with cancer cells. PSK was added one night after the drop was prepared, and contact was continued for 6 days under the contact condition of PSK of 500 μg / ml at the final concentration.

As a result of the above steps, the growth inhibition rate for human colorectal cancer cell line HCT-116 was 18% in the treatment group with a drug (PSK) alone, which was involved in anticancer action, compared to the untreated group, In the group treated with the drug (PSK) and immune cells involved in the anti-cancer effect, 32% of the untreated group showed an increase in the suppressive effect.
In the treatment group with drugs alone involved in anticancer effects, the anticancer effects were underestimated rather than the actual clinical results, whereas treatment groups with drugs involved in anticancer effects and immune cells were actually It can be seen that, as with the clinical results, the anticancer activity is more excellent.
In general, PSK does not act directly on tumors, but is thought to act via immune cells, and it has been difficult to evaluate in vitro. However, as described above, in the group treated with the drug and immune cells involved in the anticancer action, the cancer cell growth inhibitory effect is enhanced. From this, it can be seen that according to the method of the present invention, a culture environment very close to the actual in vivo environment can be constructed, and the drug effect via PBMC can be evaluated.
Example 4
Example 4 below is a conventional evaluation method for the expression level of an anticancer drug metabolizing enzyme, particularly TP gene mRNA, in cancer cells under immune cell co-culture using a collagen gel embedded culture method. Comparison was made between layer culture (two-dimensional culture) and collagen gel-embedded culture method (three-dimensional culture), which is a method according to the present invention.

Here, TP (thymidine phosphorylase) is an enzyme that converts 5′DFUR, which is a prodrug of an anticancer agent, into 5FU, and is present in a large amount in the cancer tissue to efficiently exert an anticancer effect. It is believed that. Therefore, the result shown in Example 4 is suggested about the relationship with Example 2-1 and 2-2 that 5'DFUR showed evaluation in the body and the effect enhancement.
In the same manner as in Example 1, preparation of a cell suspension of the cell line HCT-116, production of a collagen gel drop, and co-culture with immune cells derived from PBMC were performed.
The number of cultured cancer cells was measured, 3 ml of trypsin was added to peel off the cells, 7 ml of a mixed solution of DMEM (manufactured by Invitro) and F-12 medium was added, and the mixture was centrifuged at 3000 rpm × 5 minutes. After washing with PBS, RNA extraction was performed using Rneasy Mini Kit (manufactured by QIAGEN).

Next, the absorbance of the extracted RNA was measured.
The concentration of RNA was determined from the measured absorbance, and DNA was removed from the RNA sample using DNA-free ^™ (Ambion).
From the RNA sample from which the DNA was removed, cDNA synthesis was performed using SuperScript ^™ III First-Strand Synthesis System for RT-PCR (manufactured by Invitrogen).
Real-Time PCR was performed under the conditions shown in Table 1 using the target primer of TP from the synthesized cDNA, using LightCycler First-Strand DNA Master Plus SYBR GreenI (Roche).

  Table 2 shows the results of three-dimensional culture of cancer cells and PBMC in collagen gel drop according to the present invention, compared with the results of no treatment and two-dimensional culture. The comparison was made by expressing the gene mRNA of TP, which is one of the tumor 5FU metabolizing enzymes.

It was considered that increased TP activity was induced in the tumor, and as a result, the sensitivity to 5′DFUR was enhanced. In the three-dimensional culture, it was found that the expression of the enzyme in the tumor was more remarkable.
In other words, Table 2 shows that the body environment cannot be sufficiently reproduced only by two-dimensionally culturing cancer cells and immune cells, but only by three-dimensionally culturing cancer cells and immune cells in a collagen gel. It shows that the culture environment very close to the internal environment can be reproduced, and that the evaluation results shown in Examples 2-1 and 2-2 were obtained. It can be considered that the evaluation results shown in each example were obtained by three-dimensionally culturing cancer cells and immune cells in a collagen gel and reproducing a culture environment very close to the internal environment.
-Example 5
In the same manner as in Example 1, preparation of a cell suspension of the cell line HCT-116, production of a collagen gel drop, and co-culture with immune cells derived from PBMC were performed.

And what made anticancer agent 5FU contact, what made 5'DFUR contact, what made PSK contact, what made 5'DFUR and PSK contact, 5'DFUR and cytokine system preparation TNF (R & D The anti-cancer activity was evaluated by changing the E / T ratio of the system contacted with “Recombinant Human TNF-α / TNFSF1A” manufactured by SYSTEMS.
Drug contact with cancer cells is 3 μg / ml for 5′DFUR contact with 500 μg / ml contact with PSK at the final concentration, and 5′DFUR with PSK contact In each case, 3 μg / ml, 500 μg / ml, and 5′DFUR and TNF were brought into contact with each other at 3 μg / ml and 30 Unit / ml, respectively.

  The results are shown in FIG. 4 and FIG. 5 for each PBMC collected from the blood of the subject A or B. In FIGS. 4 and 5, “T / C (%)” on the vertical axis represents “relative proliferation ratio”. A specific calculation method is to conduct a test of growing and culturing cancer cells for a certain period of time under conditions where a drug to be evaluated is added and conditions where no drug is added, and the degree of proliferation (number of cancer cells) under the condition where the drug is added. ): T and the degree of proliferation (number of cancer cells) under the condition where no drug is added: the ratio (T / C) to C is calculated and multiplied by 100 to obtain the “T / C (%)” value. did. Therefore, the higher the “T / C (%)”, the weaker the medicinal effect, and the lower the “T / C (%)”, the higher the medicinal effect.

4 and 5, it can be seen that the E / T ratio has an effect on the anticancer effect. In 5FU and PSK, the larger the PBMC ratio, the lower the sensitivity. It can be seen that the combination of 'DFUR and PSK increases the sensitivity as the PBMC ratio increases.
Thus, by changing the E / T ratio, the detection sensitivity is improved, and when there are some drug candidates that are determined to be effective, it is easy to determine which drug is optimal. You can do it.
In addition, it can also be seen that the drug selection and dosage can be determined very appropriately in consideration of the ratio of the number of cancer cells and immune cells in the patient.

  The method for evaluating an anticancer effect according to the present invention is, for example, to construct a culture environment close to the in vivo environment containing cancer cells and appropriately evaluate the sensitivity of cancer cells to anticancer agents and the like. it can. As a result, it becomes possible to efficiently conduct research and development of anticancer drugs and selection of drugs to be administered in cancer treatment.

The schematic diagram explaining the evaluation method of the anticancer effect | action used as embodiment of this invention Schematic diagram illustrating an evaluation method for anticancer action according to another embodiment Schematic diagram illustrating an evaluation method for anticancer action according to another embodiment Diagram showing the effect of E / T ratio on anticancer effect Diagram showing the effect of E / T ratio on anticancer effect

Explanation of symbols

DESCRIPTION OF SYMBOLS 10 Culture container 20 Collagen gel drop 30 Culture solution 40 Cancer cell 50 Anticancer agent 52 DPD inhibitor 54 BRM formulation 60 PBMC

Claims (5)

A method for evaluating the anticancer activity of a drug involved in anticancer activity against cancer cells,
A step (a) of fixing and supporting a collagen gel droplet embedding the cancer cells on the inner surface of the culture vessel;
A step of culturing cancer cells by supplying a culture solution containing the drug involved in the anti-cancer effect and immune cells coexisting with the cancer cells in an in vivo environment to the culture vessel (b) )When,
A step (c) of evaluating the result of culturing the cancer cells in the collagen droplets;
A method for evaluating an anticancer effect, comprising:   The drug involved in the anticancer action is an anticancer drug, a prodrug of the anticancer drug, a drug that adjusts intracellular metabolic enzyme activity related to metabolism of the anticancer drug or the prodrug, and an immunotherapeutic agent The method for evaluating an anticancer effect according to claim 1, which is selected from the group consisting of:   The anticancer activity according to claim 1 or 2, wherein the immune cell is PBMC selected from the group consisting of lymphocytes including NK cells, B cells and T cells, and monocytes including macrophages. Evaluation methods.   The method for evaluating an anticancer effect according to any one of claims 1 to 3, wherein the immune cells are contained in the culture solution in an amount of 0.01 to 100 in a ratio of the number of cells to cancer cells.   A step (a-1) in which the step (a) prepares a collagen solution containing the cancer cells, a step (a-2) in which the collagen solution is dropped onto the inner surface of the culture vessel and gelled; The evaluation method of the anticancer action in any one of Claim 1 to 4 containing this.

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