JP5809782B2 - Method for evaluating drug or radiosensitivity of cancer tissue-derived cell mass or cancer cell aggregate - Google Patents

Description

  The present invention relates to a drug or radiosensitivity evaluation method using a cancer tissue-derived cell mass or a cancer cell aggregate. More specifically, the present invention relates to a drug or radiosensitivity evaluation method using a cancer tissue-derived cell mass or cancer cell aggregate that can reconstruct cancer in vitro and retains proliferative ability.

  In recent years, as a result of various studies accumulated to overcome cancer, the results of treatment for early-stage cancer have improved dramatically. However, the treatment of advanced cancer remains difficult and cancer continues to be the leading cause of death among Japanese. According to the 2007 vital statistics by the Ministry of Health, Labor and Welfare, more than 340,000 people die from cancer each year.

  In cancer research so far, especially when examining the behavior in vitro, experiments using cancer cell lines established by subculture under conditions optimized for culture are the mainstream. Such cancer cell lines include human breast cancer cell lines (MDF7, NCI / ADR HS578T, MDA-MB-22231 / ATCC, MDA-MB-4335, MDA-N, BT-549, T-47D), human offspring Cervical cancer cell line (HeLa), human lung cancer cell lines (A549, EKVX, HOP-62, HOP-92, NCI-H23, NCI-H226, NCI-H322M, NCI-H460, NCI-H522) and human colon cancer cells Strains (Caco-2, COLO 205, HCC-2998, HCT-15, HCT-116, HT29, KM12, SW-620) human prostate cancer cell lines (DU-145, PC-3, LNCaP), etc. In fact, it is widely used for research.

  In order to realize diagnosis and treatment for each cancer patient, primary culture of cancer cells is considered promising and research is ongoing. For example, a CD-DST method (Collagen gel droplet embedded drug sensitivity test) using primary cultured cells has been developed. This in vitro test method is a drug sensitivity test in which an isolated tissue or cell from a patient is embedded in a collagen gel droplet and verified by combining three-dimensional culture and image colorimetry (for example, non-patented) Reference 1). However, primary culture cells are difficult to handle because no culture method has been established.

  As a result of research on cancer cells, cancer cells that make up cancer may be composed of multiple subpopulations, which are called “tumor progenitor cells” or “tumor stem cells”, but are small populations that are self-replicating. There are a number of reports that support the existence of subpopulations that are possible and that can be the source of the majority of cancer cells by differentiation (eg, Non-Patent Documents 2 and 3). Such stem cells can be obtained, for example, by separating a tumor extracted from a living body into single cells and sorting them, and some of them are said to show proliferative ability even in vitro ( Non-patent document 4). However, there is a negative report on the theory of explaining the origin of cancer by stem cells in this way (Non-Patent Document 5), and it does not go beyond the hypothesis.

  Even in the current state of cancer research, there are many unknowns about cancer.

Takamura Y et al. (2002) Prediction of chemotherapeutic response by collagen gel droplet embedded culture-drug sensitivity test in human breast cancers. Int. J. Cancer, 98, 450-455 Vermeulen L et al. (2008) Single-cell cloning of colon cancer stem cells reveals a multi-lineage differentiation capacity. PNAS Vol.105 No.36 13427-13432 Ricci-Vitiani L et al. (2007) Identification and expansion of human colon-cancer-initiating cells. Nature Vol.445 111-115 Todaro M et al. (2007) Colon cancer stem cells dictate tumor growth and resist cell death by production of interleukin-4.Cell Stem Cell 1: 389-402 Shmelkov S V et al. (2008) CD133 expression is not restricted to stem cells, and both CD133 + and CD133- metastatic colon cancer cells initiate tumors. The Journal of Clinical Investigation Vol. 118 2111-2120

  The object of the present invention is to be able to reproduce the behavior of cancer cells in vivo in vitro and to be used as a sample for cancer analysis and treatment research, which can accurately verify the effects in vivo. An object of the present invention is to provide an evaluation method such as a drug sensitivity test or a radiosensitivity test using a novel cancer tissue-derived cell mass or cancer cell aggregate.

  The present inventors intend to conduct a therapeutic sensitivity test for individual cancer patients, and considering the possibility that the cell line that has been used as a research material for cancer research is different from patient cancer, As a result of intensive studies on the culture of cancer cells as research materials in order to solve the above problems, a novel cancer tissue-derived cell mass or cancer cell aggregate can be prepared and used for drug or radiosensitivity evaluation As a result, the present invention has been completed.

  That is, the present invention provides a novel drug sensitivity evaluation or radiosensitivity using a novel cancer tissue-derived cell mass or cancer cell aggregate that can accurately reflect the behavior of cancer cells in vivo in an individual even in vitro. The purpose is to provide an evaluation method.

  The present invention includes a step of allowing a drug to act on a cancer tissue-derived cell mass or a cancer cell aggregate mass derived from a patient in vitro and evaluating the action of the drug on the cancer tissue-derived cell mass or the cancer tissue-derived cell mass. It is related with the evaluation method of the influence.

  Assessing the effect can include comparing the growth status of the cancer tissue-derived cell mass or cancer cell aggregate in the presence or absence of different concentrations of the drug.

  Assessing the effect can include making a life or death determination of the cells in the cancer tissue-derived cell mass or cancer cell aggregate in the presence or absence of different concentrations of the drug.

Assessing the effect can include analyzing intracellular signaling in cells in the cancer tissue-derived cell mass or cancer cell aggregate in the presence or absence of the agent.

The agent may be EGF (epidermal growth factor) or IGF (insulin-like growth factor), and the analysis of intracellular signaling may be an analysis of phosphorylation of Akt and / or ERK1 / 2.

The above drugs include antibodies against EGFR (epidermal growth factor receptor), EGFR inhibitors, antibodies against HER2 (human EGFR-related substance 2), HER2 inhibitors, αIR3 (insulin-like growth factor I receptor α neutralizing antibody: mouse monoclonal) Antibody), and selected from the group consisting of IGF-IR inhibitors, the analysis of intracellular signaling can be an analysis of phosphorylation of Akt and / or ERK1 / 2.

  The evaluation method may be to evaluate in advance the effect of the drug on the patient from whom it originated.

  In the evaluation method, genes of the cancer tissue-derived cell mass or cancer cell aggregate mass can be evaluated in advance to obtain genetic information, and a drug to be acted on can be selected based on the genetic information.

  The above-mentioned cancer tissue-derived cell mass or cancer cell aggregate mass may have undergone a storage state by freezing.

  The present invention also includes a step of allowing radiation to act on a cancer tissue-derived cell mass or cancer cell aggregate from a patient in vitro and evaluating the effect of the radiation on the cancer tissue-derived cell mass or cancer cell aggregate. It is related with the evaluation method of the influence.

  Assessing the effect may comprise comparing the growth state under radiation at different intensities or the growth state of the cancer tissue-derived cell mass or cancer cell aggregate in the absence of radiation.

  Evaluating the effect may include performing a life / death determination of cells in a cancer tissue-derived cell mass or cancer cell aggregate in the presence or absence of radiation at different intensities.

  The evaluation method may be to evaluate in advance the influence of radiation on the patient derived from the evaluation method.

  In the above evaluation method, genes of the cancer tissue-derived cell mass or cancer cell aggregate mass can be evaluated in advance to obtain genetic information, and irradiation can be selected based on the genetic information.

  The above-mentioned cancer tissue-derived cell mass or cancer cell aggregate mass may have undergone a storage state by freezing.

  The present invention also includes a step of allowing a drug candidate compound to act on a cancer tissue-derived cell mass or a cancer cell aggregate in vitro, and evaluating the action of the drug candidate compound on the cancer tissue-derived cell mass or cancer cell aggregate. The present invention relates to a method for screening a drug.

  Assessing the effect may include comparing the growth state of the cancer tissue-derived cell mass or cancer cell aggregate in the presence of the drug candidate compound with the growth state in the absence of the drug candidate compound.

  Assessing the above-described action can include determining whether a cell in a cancer tissue-derived cell mass or a cancer cell aggregate in the presence or absence of a drug candidate compound is alive or dead.

  Assessing the action can include analyzing intracellular signal transduction of cells in the cancer tissue-derived cell mass or cancer cell aggregate in the presence or absence of the drug candidate compound.

The agent may be EGF or IGF, and the intracellular signaling analysis may be an analysis of Akt and / or ERK1 / 2 phosphorylation.

The agent is selected from the group consisting of an antibody against EGFR, an EGFR inhibitor, an antibody against HER2, a HER2 inhibitor, αIR3, and an IGF-IR inhibitor, and the analysis of intracellular signaling is performed using Akt and / or ERK1 / Analysis of the phosphorylation of two.

  In the screening method described above, genes of cancer tissue-derived cell mass or cancer cell aggregate mass can be evaluated in advance to obtain genetic information, and drug candidate compounds to be acted on can be selected based on the genetic information.

  The above-mentioned cancer tissue-derived cell mass or cancer cell aggregate mass may have undergone a storage state by freezing.

  By using a drug or radiosensitivity evaluation method using a cancer tissue-derived cell mass or cancer cell aggregate of the present invention, it is possible to know in advance the same behavior in vivo as before in vivo administration or application. The effect can be predicted. Therefore, it is possible to quickly and accurately establish an optimal treatment method corresponding to each patient, not uniform.

  Furthermore, in the present invention, a candidate drug such as an anticancer drug is applied to a cancer tissue-derived cell mass or a cancer cell aggregate, and the sensitivity can be examined to screen the anticancer drug. Effective anti-cancer agents can be efficiently screened for individual patients or for specific populations with certain common characteristics.

It is a figure which shows the cancer tissue origin cell mass of this invention. In one embodiment of the cancer tissue-derived cell mass of the present invention, it is a diagram showing that cells express the surface antigens CD133, CD44, CD166 and the like. It is a figure showing the change of the shape of the cell mass derived from the cancer tissue of the present invention and the proliferation ability in the in vitro culture process. It is a figure which shows the result of the drug sensitivity test by 5-FU in vitro using the cell mass derived from the cancer tissue of this invention. It is the figure which compared the tumor tissue obtained by transplanting the cancer tissue origin cell mass of this invention to a mouse | mouth, and the tumor tissue extracted from the biological body which is the origin of a cancer tissue origin cell mass. It is a figure which shows the result of the in vitro radiation sensitivity test using the cell mass derived from the cancer tissue of this invention. It is a figure which shows the cancer tissue origin cell cluster of this invention obtained from various cancer tissues. It is a figure showing the result of a hormone sensitivity culture test using a breast cancer tissue origin cell mass. It is a figure which shows the cell mass derived from the cancer tissue of this invention obtained from the mouse islet tumor. It is the figure which compared the state before and behind cryopreserving the cancer tissue origin cell mass of this invention. It is a figure which shows the cancer cell aggregate lump derived from a cancer tissue origin cell lump. It is a figure which shows the cancer cell aggregate derived from a human colon cancer operation specimen. It is a figure which shows the state after freeze-preserving and thaw | melting the cancer tissue origin cell mass after trypsin treatment. It is a figure which shows the result of the drug sensitivity test by the in vitro doxorubicin using a cancer cell aggregate. It is a figure which shows the result of the drug sensitivity test by a cisplatin by the life-and-death determination in vitro using the cell mass derived from a cancer tissue. It is a figure which shows the influence of EGF and cetuximab by in vitro signal transduction analysis using a cancer tissue-derived cell mass. It is a figure which shows the influence of IGF and (alpha) IR3 by the in vitro signal transduction analysis using a cancer tissue origin cell mass.

  The cancer tissue-derived cell mass of the present invention is an isolate or a culture thereof separated as a mass containing 3 or more cancer cells from a cancer tissue obtained from an individual, and retains proliferative ability in vitro. Can be such that

  Here, the “separate separated from a cancer tissue obtained from an individual as a mass containing three or more cancer cells” is obtained by treating a cancer tissue obtained from a cancer generated in a living body. Or an isolate containing 3 or more, preferably 8 or more cancer cells. Such isolates do not include those that have been separated into single cells, nor do they include constructs that have been separated into single cells and then reconstituted. However, this separated product includes not only a product immediately after being separated from a living body but also a product that has been kept in physiological saline for a certain period of time, or a product that has been frozen or refrigerated.

  “Cancer tissue obtained from an individual” refers to cancer tissue obtained by excision by surgery or the like, as well as cancer tissue obtained so that it can be handled in vitro for histological examination with an injection needle or endoscope. Point to.

  “A culture of an isolate separated from a cancer tissue obtained from an individual as a mass containing three or more cancer cells” is obtained by treating a cancer tissue obtained from a cancer generated in vivo. In addition, it refers to those obtained by culturing in vitro an isolated product separated as a mass containing 3 or more cancer cells. The culture time is not particularly limited as long as it is present in the medium even for a short time. Such a culture often exhibits a substantially spherical shape or an elliptical sphere shape by culturing for a certain period, preferably 3 hours or more. The culture here includes a substantially spherical or elliptical spherical culture after elapse of a certain period of time, and an amorphous culture up to that. Further, an indefinite shape obtained by further dividing such a substantially spherical or elliptical spherical culture, and a substantially spherical or elliptical spherical product by further culture are also cultures referred to herein.

  In the present invention, “drug” refers to any physiologically active substance capable of stimulating a living body or cells in addition to those used for the purpose of treating cancer.

In vitro, the cancer tissue-derived cell mass of the present invention is “capable of maintaining proliferation ability” at a temperature of 37 ° C. and 5% CO ₂ incubator at least 10 days or more, preferably 13 It means that the growth ability can be maintained for a period of more than 30 days, more preferably more than 30 days.

  Such a cancer tissue-derived cell mass can retain its proliferative ability for a period of 10 days or more, preferably 13 days or more, more preferably 30 days or more by continuing the culture as it is. By performing the mechanical division, the proliferation ability can be maintained substantially indefinitely.

  The machine division can be performed by using a scalpel, a knife, scissors, an ophthalmic sword or the like. Alternatively, it can also be performed by attaching an injection needle to the syringe and repeating the suction and discharge of the cancer tissue-derived cell mass together with the culture solution. For example, a 1 ml syringe and a 27G needle are preferably used in the present invention, but are not limited thereto.

  Here, the medium for culturing the cell mass derived from the cancer tissue of the present invention is not particularly limited, but an animal cell culture medium is preferably used. Particularly preferably, a serum-free medium for stem cell culture is used. Such a serum-free medium is not limited as long as it is used for culturing stem cells. A serum-free medium refers to a medium that does not contain unprepared or unpurified serum, and can be used after adding purified blood-derived components or animal tissue-derived components (for example, growth factors).

  The serum-free medium of the present invention can be prepared using a medium used for animal cell culture as a basal medium. As the basal medium, for example, BME medium, BGJb medium, CMRL 1066 medium, Glasgow MEM medium, Improved MEM Zinc Option medium, IMDM medium, Medium 199 medium, Eagle MEM medium, αMEM medium, DMEM medium, RPMI 1640 medium, Fischer's medium , And combinations thereof.

  The serum tissue-derived cell mass of the present invention can be cultured by adding a serum substitute to such a serum-free medium. Serum substitutes contain, for example, albumin, amino acids (eg, non-essential amino acids), transferrin, fatty acids, insulin, collagen precursors, trace elements, 2-mercaptoethanol or 3 ′ thiolglycerol, or equivalents thereof as appropriate. Can be.

  In the culture method of the present invention, a commercially available serum substitute can also be used. Examples of such commercially available serum substitutes include knockout serum replacement (KSR), chemically-defined lipid concentrated fatty acid concentrate (manufactured by Gibco), and glutamax (manufactured by Gibco).

  The medium for culturing the cancer tissue-derived cell mass of the present invention may also contain vitamins, growth factors, cytokines, antioxidants, pyruvate, buffers, inorganic salts and the like.

  In particular, any serum-free medium such as a serum-free medium containing EGF and bFGF, such as a serum-free medium containing a serum substitute such as knockout serum replacement (KSR, manufactured by Invitrogen) and bFGF can be preferably used. . The content of serum substitute or EGF is preferably 10-30% w / v of the whole medium.

  Such a medium is not limited, but a commercially available product includes a serum-free medium (Gibco) for STEMPRO human ES cells.

  The incubator used for culturing the cell mass derived from cancer tissue is not particularly limited as long as it can generally cultivate animal cells. For example, flask, tissue culture flask, dish, petri dish, tissue culture A dish, a multi-dish, a microplate, a microwell plate, a multiplate, a multiwell plate, a chamber slide, a petri dish, a tube, a tray, a culture bag, and a roller bottle can be mentioned.

  The incubator is preferably non-cell-adhesive and is three-dimensionally cultured in the presence of a cell support substrate such as an extracellular matrix (ECM) in the medium. The cell support matrix may be intended for adhesion of cell mass derived from cancer tissue. Examples of such a cell supporting substrate include matrigel using an extracellular matrix, such as collagen gel, gelatin, poly-L-lysine, poly-D-lysine, laminin, and fibronectin. Such conditions are suitably used particularly when it is desired to grow the cancer tissue-derived cell mass of the present invention.

Other culture conditions can be appropriately set. For example, the culture temperature is not limited, but is preferably about 30 to 40 ° C. Most preferably, it is 37 degreeC. The CO ₂ concentration is, for example, about 1 to 10%, preferably about 2 to 5%.

  The cancer tissue-derived cell mass of the present invention can be cultured in such a medium and culture conditions. Furthermore, depending on the individual nature of the cell mass derived from the cancer tissue, co-culture with other cells may be preferable, or the presence of additional special supplements such as hormones may be necessary.

  Specifically, co-culture may be performed together with feeder cells. As feeder cells, stromal cells such as fetal fibroblasts can be used. Specifically, although not limited, NIH3T3 or the like is preferable.

  Alternatively, for specific types of breast cancer, uterine cancer, and prostate cancer, it is preferable to culture in the presence of hormones. Specifically, estrogen for breast cancer, progesterone for uterine cancer, testosterone for prostate cancer, and the like, but not limited thereto, various hormones can be added to conveniently adjust the culture conditions. Furthermore, by investigating how the behavior of cancerous tissue-derived cell mass changes after culturing due to the presence of such hormones, it is possible to determine the hormone dependence of the cancer in the patient from which it is derived, and effective antihormonal treatment There is a possibility that gender can be predicted.

  The cancer tissue-derived cell mass of the present invention can also be cultured in suspension culture. In suspension culture, a cancer tissue-derived cell mass is cultured in a medium under conditions that are non-adherent to the incubator. Examples of such suspension culture include embryoid body culture method (Keller et al., Curr. Opin. Cell Biol. 7, 862-869 (1995)), and SFEB method (eg, Watanabe et al., Nature Neuroscience 8, 288- 296 (2005); International Publication No. 2005/123902). Although not particularly limited, it can be used, for example, when forming or maintaining a stable cell mass derived from a cancer tissue having a substantially spherical shape and sometimes a basement membrane.

  The cancer tissue-derived cell mass of the present invention includes a product immediately after separation from an individual cancer tissue-derived cell mass, a product after refrigeration and freezing storage, and a culture thereof. The culture can be performed for a period of preferably 3 hours or longer, more preferably 10 hours to 36 hours, and even more preferably 24 hours to 36 hours or longer.

  The number of cancer cells constituting the cancer tissue-derived cell cluster is at least 3 or more, preferably 8 or more, more preferably 10 or more, still more preferably 20 or more, and most preferably 50 or more. When the cancer tissue-derived cell mass of the present invention is a separated substance, it is preferably 1000 or less, more preferably about 500 or less. In the case of a culture after culturing the isolate, the number can be increased by culturing. However, even if it is a culture, it is preferably 10,000 or less, more preferably 5000 or less.

  In the present invention, the term “cancer cell” is used in a commonly used meaning, and refers to a disordered cell-ordered cell such as unlimited division / proliferation and deviation from apoptosis in vivo. More specifically, it refers to a cell that has lost its cell growth control function or is extremely attenuated, and typically has acquired infinite growth ability with a frequency of 80% or more, many of which also have invasive metastatic ability. It often means that it is a cell that is positioned as a malignant neoplasm that leads to death, including humans, especially mammals.

  In the present invention, the type of cancer tissue derived is not particularly limited, and lymphoma, blastoma, sarcoma, liposarcoma, neuroendocrine tumor, mesothelioma, schwannoma, meningioma occurring in animals including mammals Adenomas, melanomas, leukemias, lymphoid malignancies, and the like, and cancers that occur in mammalian epithelial cells are particularly preferable. Non-small cell lung cancer, hepatocellular carcinoma, biliary tract cancer, esophageal cancer, stomach cancer, colorectal cancer, pancreatic cancer, cervical cancer, ovarian cancer, endometrial cancer, bladder cancer, Examples include pharyngeal cancer, breast cancer, salivary gland cancer, renal cancer, prostate cancer, labial cancer, anal cancer, penile cancer, testicular cancer, thyroid cancer, and head and neck cancer. There are no particular restrictions on animals including mammals, but animals belonging to primates including monkeys and humans, animals belonging to rodents such as mice, squirrels and rats, animals belonging to rabbits, cats such as dogs and cats, etc. An animal belonging to the eye is exemplified.

  Among them, in the present invention, in particular, colon cancer tissue origin, ovarian cancer tissue origin, breast cancer tissue origin, lung cancer tissue origin, prostate cancer tissue origin, kidney cancer tissue origin, bladder cancer tissue origin, pharyngeal cancer tissue origin, or pancreatic cancer Although it is especially preferable that it is derived, it is not limited.

  In the case of a cancer tissue-derived cell mass derived from colorectal cancer tissue, the contained cancer cells are not particularly limited, but may express CD133.

  The separation treatment of cancer tissue obtained from cancer that has occurred in vivo includes, but is not limited to, enzymatic treatment of cancer tissue obtained from an individual.

  The enzyme treatment can be treatment with one of collagenase, trypsin, papain, hyaluronidase, C. histolyticum neutral protease, thermolysin, and dispase, or a combination of two or more thereof. Enzymatic treatment conditions may include isotonic salt solutions buffered to physiologically acceptable pH, eg about 6-8, preferably about 7.2-7.6, eg, PBS or Hanks balanced salt solution, For example, at about 20-40 ° C., preferably about 25-39 ° C., for a time sufficient to degrade the connective tissue, for example about 1-180 minutes, preferably 30-150 minutes, a concentration sufficient for that, for example about It may be 0.0001-5% w / v, preferably about 0.001% -0.5% w / v.

  The enzyme treatment conditions include, but are not limited to, treatment with a mixed enzyme containing collagenase. For example, a mixture comprising one or more proteases selected from the group consisting of C. histolyticum neutral protease, thermolysin, and dispase; and one or more collagenases selected from the group consisting of collagenase I, collagenase II, and collagenase IV Enzymatic treatment is included.

  Such mixed enzymes include, but are not limited to, Liberase Blendzyme 1 (registered trademark) and the like.

  The cancer tissue-derived cell mass of the present invention may alternatively include three or more cancer cell aggregates and exhibit a substantially spherical shape or an elliptical spherical shape.

  Although not limited, it may include a basement membrane-like substance existing on the outer peripheral surface of the cancer cell aggregate.

  Here, although not particularly limited, the cancer cells forming the aggregate may have one or more surface antigens selected from the group consisting of CD133, CD44, CD166, CD117, CD24, and ESA on the cell surface. Many. CD133, CD44, CD166, CD117, CD24, and ESA are generally surface antigens expressed on cells such as leukocytes such as lymphocytes, fibroblasts, epithelial cells, and tumor cells. These surface antigens are involved in various signal transductions in addition to their function as cell-cell and cell-matrix adhesion, but are also surface markers for various stem cells.

  In the present invention, when a cell group “expresses” a surface antigen such as CD133, 80% or more, preferably 90% or more, more preferably substantially all of the cells present in the cell group are surface antigens. Indicates the state.

  In the present specification, the “basement membrane-like substance” is not limited, but preferably contains at least one of collagen, laminin, nidogen, proteoglycan such as heparan sulfate proteoglycan, and glycoprotein such as fibronectin. Refers to a substance. In the present invention, a basement membrane-like material containing laminin is preferable.

  Laminin is a high-molecular glycoprotein that constitutes the basement membrane. The functions of laminin are diverse and are involved in cell functions such as cell adhesion, signal transduction, proliferation of normal cells and cancer cells. Laminin has a structure in which each of three different subunits is linked by a disulfide bond, and 11 types are found depending on the different types of each subunit.

  Among these, laminin 5 is usually produced only from epithelial cells, and is known as a component having an activity of promoting the adhesion of epithelial cells to the basement membrane and the motor function. This laminin 5 has a structure in which each one of α3 chain, β3 chain, and γ2 chain forms a complex. In particular, γ2 chain is considered to be unique to LN5 and is not included in other LN molecular species. Absent.

  The cancer tissue-derived cell mass of the present invention may have a configuration in which the outer periphery of an aggregate of cancer cells is entirely wrapped in a film formed by such a basement membrane-like substance. Such morphology can be analyzed by observing a cancer tissue-derived cell cluster with an electron microscope, immunostaining of basement membrane components, or a combination of both.

  The presence of laminin can be detected, for example, by contacting an antibody recognizing laminin, for example, a mouse laminin-derived rabbit antibody of Sigma-Aldrich and a cell mass derived from cancer tissue, and measuring the antibody antigen reaction.

  It is also possible to use a specific antibody that specifies the type of laminin. For example, the presence of laminin 5 can be detected by, for example, contacting an antibody having reactivity to the above-described unique γ2 chain or a fragment thereof with a cell mass derived from cancer tissue and measuring the reaction of the antibody. it can.

  In the cancer tissue-derived cell mass of the present invention, it is preferable that a thin membrane-like basement membrane-like material is formed on the order of several μm, preferably about 40 to 120 nm, although there is no limitation.

  The size of the cell mass derived from the cancer tissue of the present invention is not limited, and includes an irregular shape having a particle size or a volume average particle size of about 8 μm to 10 μm. Also included. The diameter is preferably 40 μm to 1000 μm, more preferably 40 μm to 250 μm, and still more preferably 80 μm to 200 μm.

  The cancer tissue-derived cell mass of the present invention often has one or more sequences selected from the group consisting of a shelf-like array, a sheet-like array, a multilayered array, and a syncytial array, but is not particularly limited.

  The cancer tissue-derived cell mass of the present invention is typically a step of subjecting a fragment of cancer tissue excised from a living body to an enzyme treatment; and a mass containing 3 or more cancer cells among the enzyme-treated products. Can be prepared by a method comprising the steps of:

  Furthermore, although not limited, the cancer tissue-derived cell mass of the present invention can be prepared by a method including a step of culturing the components thus collected for 3 hours or more.

  First, cancer tissue excised from a living body can be fragmented as it is, and first, it can be maintained in an animal cell culture medium before fragmentation. Such animal cell culture media include, but are not limited to, Dulbecco MEM (such as DMEM F12), Eagle MEM, RPMI, Ham's F12, Alpha MEM, Iskov modified Dulbecco and the like. At this time, suspension culture is preferably performed in a cell non-adhesive incubator.

  It is also preferred that the cancer tissue be washed prior to stripping. Such washing includes, but is not limited to, acetate buffer (acetate + sodium acetate), phosphate buffer (phosphate + sodium phosphate), citrate buffer (citric acid + sodium citrate), boric acid A buffer solution such as a buffer solution, a tartrate buffer solution, a Tris buffer solution, or a phosphate buffered saline can be used. In the present invention, it is particularly preferable that the tissue can be washed in HBSS. The appropriate number of washings is 1 to 3 times.

  The fragmentation can be performed by dividing the tissue after washing with a knife, scissors, a cutter (manual, automatic) or the like. The size and shape after the fragmentation are not particularly limited and can be performed randomly, but preferably 1 mm to 5 mm square, more preferably 1 mm to 2 mm square.

  Next, the fragmented product thus obtained is subjected to an enzyme treatment. Such enzyme treatment may be treatment with one of collagenase, trypsin, papain, hyaluronidase, C. histolyticum neutral protease, thermolysin, and dispase, or a combination of two or more thereof. Enzymatic treatment conditions may include isotonic salt solutions buffered to physiologically acceptable pH, eg about 6-8, preferably about 7.2-7.6, eg, PBS or Hanks balanced salt solution, For example, at about 20-40 ° C., preferably about 25-39 ° C., for a time sufficient to degrade the connective tissue, for example about 1-180 minutes, preferably 30-150 minutes, a concentration sufficient for that, for example about It may be 0.0001-5% w / v, preferably about 0.001% -0.5% w / v.

  Although not limited, the conditions for the enzyme treatment may be, for example, treatment with a mixed enzyme containing collagenase. More preferably, one or more proteases selected from the group consisting of C. histolyticum neutral protease, thermolysin, and dispase; and one or more collagenases selected from the group consisting of collagenase I, collagenase II, and collagenase IV, Treatment with a mixed enzyme containing.

  Such mixed enzymes include, but are not limited to, Liberase Blendzyme 1 (registered trademark) and the like.

  Next, it is preferable to select and collect a mass containing three or more cancer cells from the enzyme-treated product thus obtained. The method of sorting and collecting is not particularly limited, and any method known to those skilled in the art for distributing the size can be used.

  Among the size distribution methods, a simple method is visual, fractionation using a phase-contrast microscope, or screening, but is not particularly limited as long as it is a fractionation method based on particle diameters available to those skilled in the art. When using a sieve, it is preferable to collect components that pass through a sieve mesh size of 20 μm and do not pass through 500 μm. More preferably, components that pass through a sieve mesh size of 40 μm and do not pass through 250 μm are recovered.

  Here, the mass containing three or more cancer cells to be selected is the cancer tissue-derived cell mass of the present invention, and has a certain range of sizes. The size within a certain range includes small particles having a volume average particle diameter of about 8 μm to 10 μm, but in the case of a nearly spherical shape, the diameter is 20 μm to 500 μm, preferably 30 μm to 400 μm, more preferably 40 μm to 250 μm, In the case of an elliptical shape, the major axis is 20 μm or more and 500 μm or less, preferably 30 μm or more and 400 μm or less, more preferably 40 μm or more and 250 μm or less. More preferably, it is 40 μm or more and 250 μm or less. The volume average particle diameter can be measured by evaluating the particle size distribution and particle shape using a phase contrast microscope (IX70; manufactured by Olympus Corporation) with a CCD camera.

  Any of the separation-treated product and its culture obtained as a selective recovery component thus obtained is the cancer tissue-derived cell mass of the present invention. The culture may be a product in which the separated and collected components are present in the medium in a short time, for example, at least 3 hours, preferably 10 hours to 36 hours, more preferably 24 hours. By culturing for a period of ˜36 hours or longer, it may have a substantially spherical shape or a substantially elliptical spherical shape. The culture time may exceed 36 hours, and several days, 10 days or more, 13 days or more, or 30 days or more may have elapsed.

  Cultivation can be performed as it is for a long time in the medium, but preferably, the ability to proliferate can be maintained substantially infinitely by performing mechanical division periodically during the culture.

  For example, the cancer tissue-derived cell mass of the present invention has a high degree of colonization in transplantation into a heterologous animal even if it is 10 or less cancer cell-derived cell clusters having a diameter of 100 micrometers (corresponding to 1000 cells or less). Therefore, the cancer tissue-derived cell mass of the present invention is useful for easy preparation of cancer model animals such as mice, and more rigorous verification of cancer tissues, evaluation of drug sensitivity, or treatment including radiotherapy. Evaluation of an aspect is attained.

  The cancer tissue-derived cell mass of the present invention can be stored frozen and can retain its proliferative ability under normal storage conditions.

  The cancer cell aggregate of the present invention is a cancer cell-derived cell mass or a cancer tissue obtained from an individual, and is converted into single cells, and then the individual cells in the single cell product are completely separated from each other or completely into individual cells. Formed by aggregation of several cells that have not been separated, or individual cells and some cells that have not been completely separated into individual cells, to aggregate to a total of 3 or more cells Aggregates or cultures thereof, such that they can retain their proliferative ability in vitro.

  Here, “to make a cancer tissue-derived cell mass or a cancer tissue obtained from an individual into a single cell” means that at least a part of the cancer tissue-derived cell mass or the obtained cancer tissue is separated into single cells in vitro. It means to perform the processing. Thus, typically, after such treatment, there may be cells that have separated into individual single cells, but some cells may not be separated into individual cells, and Even in this case, it corresponds to “single cell” as used herein. In this case, those that are not separated until they are mixed include aggregates of up to 10 cells, preferably aggregates of 2 to 3 cells.

  “Aggregating to 3 or more cells” means individual cancer cells obtained from cancerous tissue generated in vivo or cancer tissue-derived cell masses found by the present inventors as single cells. It refers to a state in which a group of cells that have not been separated from each other or individual cells, or a combination thereof, gathered to contain at least three or more cells.

  In the case of subjecting a cancer tissue derived from a cancer tissue-derived cell mass or a cancer tissue obtained from a living body to a single cell treatment, it is not limited, but includes an enzyme treatment of a cancer tissue obtained from an individual. .

  The enzyme treatment is typically a treatment with trypsin, dispase, and optionally one of collagenase, papain, hyaluronidase, C. histolyticum neutral protease, thermolysin, and dispase, or a combination of two or more thereof. possible. Enzymatic treatment conditions may include isotonic salt solutions buffered to physiologically acceptable pH, eg about 6-8, preferably about 7.2-7.6, eg, PBS or Hanks balanced salt solution, For example, at about 20-40 ° C., preferably about 25-39 ° C., for a time sufficient to degrade the connective tissue, for example about 1-180 minutes, preferably 30-150 minutes, a concentration sufficient for that, for example about It may be 0.0001-5% w / v, preferably about 0.001% -0.5% w / v.

  Without limitation, the enzyme treatment typically may be trypsin or dispase treatment alone.

  Individual cells are usually obtained after the single cell treatment. However, cells that are not completely separated individually are also included.

  Such cells may be aggregated as they are, but preferably, for example, a ROCK inhibitor is present and aggregated immediately after the single cell treatment.

  ROCK is a Rho-associated coiled-coil kinase (ROCK: GenBank accession number: NM_005406) and is one of the main effector molecules of Rho GTPase and is known to control various physiological phenomena. (Also called Rho-binding kinase). As a ROCK inhibitor, Y27632 etc. are illustrated, for example. In addition, Fasudil (HA1077), H-1152, Wf-536 (these are all available from Wako Pure Chemical Industries, Ltd.), and derivatives thereof, as well as antisense nucleic acids, RNA interference-inducing nucleic acids for ROCK, and these Vector containing.

Prior to aggregation, the 96-well culture plate was used to treat the treatment separated into trypsin treatment (eg, but not limited to, 0.25% trypsin-EDTA, treatment at 37 ° C. for 5 minutes) to a single cell or a population of 10 cells or less. Seed at low density (eg, 500 / 0.32 cm ² , medium volume of about 0.15 ml). Immediately or after culturing for several days, the ROCK inhibitor can be added at a concentration of about 1 to 100 μM, preferably about 10 μM.

  Such aggregates can be cultured in vitro. The culture time is not particularly limited as long as it is present in the medium even for a short time. Such a culture often exhibits a substantially spherical shape or an elliptical sphere shape by culturing for a certain period, preferably 3 hours or more. The culture here includes a substantially spherical or elliptical spherical culture after elapse of a certain period of time, and an amorphous culture up to that. Further, an indefinite shape obtained by further dividing such a substantially spherical or elliptical spherical culture, and a substantially spherical or elliptical spherical product by further culture are also cultures referred to herein.

In vitro, the cancer cell aggregate of the present invention “can retain growth ability” means at least 10 days or more, preferably 13 days under a cell culture condition of a temperature of 37 ° C. and a 5% CO ₂ incubator. As described above, it means that the growth ability can be maintained for a period of 30 days or more.

  Such cancer cell agglomerates can maintain their proliferative ability for a period of 10 days or more, preferably 13 days or more, and more preferably 30 days or more by continuing the culture as they are. The ability to proliferate can be maintained substantially indefinitely by performing general division or by further unicellularization and aggregation.

  Here, the culture medium for culturing the cancer cell aggregate of the present invention is the same as the culture medium for culturing the cancer tissue-derived cell mass.

  The cancer cell aggregate of the present invention can be cultured in such a medium and culture conditions. Furthermore, the culturing of cancer cell aggregates may require co-culture with other cells due to their individual nature, or may require the presence of additional specialized supplements such as hormones.

  Specifically, co-culture may be performed together with feeder cells. As feeder cells, stromal cells such as fetal fibroblasts can be used. Specifically, although not limited, NIH3T3 or the like is preferable.

  Alternatively, for specific types of breast cancer, uterine cancer, and prostate cancer, it is preferable to culture in the presence of a hormone, as in the case of a cancer tissue-derived cell mass.

  The cancer cell aggregate of the present invention can also be cultured in suspension culture, similar to the cancer tissue-derived cell cluster.

  The number of cancer cells constituting the cancer cell aggregate is at least 3 or more, preferably 8 or more, more preferably 10 or more, still more preferably 20 or more, and there is no particular upper limit in the number. When the cancer cell aggregate of the present invention is a separated product, the number is preferably 1000 or less, more preferably 500 or less. In the case of a culture after culturing the isolate, the number can be increased by culturing. However, even if it is a culture, it is preferably 10,000 or less, more preferably 5000 or less.

  The size of the cancer cell aggregate of the present invention is not limited, and includes those having an irregular shape having a particle size or a volume average particle size of about 8 μm to 10 μm, and those having a particle size of 1 mm or more that grows greatly after culturing. Is also included. The diameter is preferably 40 μm to 1000 μm, more preferably 40 μm to 250 μm, and still more preferably 80 μm to 200 μm.

  The cancer cell aggregate of the present invention often has one or more sequences selected from the group consisting of a shelf-like array, a sheet-like array, a multilayered array, and a syncytial array, but is not particularly limited.

  The cancer cell aggregate of the present invention typically includes a step of making a cancer tissue excised from a living body into a single cell; and a step of aggregating cells in the single cell product into three or more cells. It can be prepared by a method.

  Furthermore, although not limited thereto, the cancer cell aggregate of the present invention can be prepared by a method including a step of culturing the aggregated component for 3 hours or more.

  First, when the cancer cell aggregate of the present invention is obtained from a cancer tissue-derived cell mass, it is directly subjected to the enzyme treatment, but the cancer tissue removed from the living body is converted into a single cell by being subjected to the enzyme treatment as it is. On the other hand, it is preferable to make a fragment before the enzyme treatment. Prior to fragmentation, it can be maintained in animal cell culture media. Such animal cell culture media include, but are not limited to, Dulbecco MEM (such as DMEM F12), Eagle MEM, RPMI, Ham's F12, Alpha MEM, Iskov modified Dulbecco and the like. At this time, suspension culture is preferably performed in a cell non-adhesive incubator.

  It is also preferred that the cancer tissue be washed prior to stripping. Such washing includes, but is not limited to, acetate buffer (acetate + sodium acetate), phosphate buffer (phosphate + sodium phosphate), citrate buffer (citric acid + sodium citrate), boric acid A buffer solution such as a buffer solution, a tartrate buffer solution, a Tris buffer solution, or a phosphate buffered saline can be used. In the present invention, it is particularly preferable that the tissue can be washed in HBSS. The appropriate number of washings is 1 to 3 times.

  The fragmentation can be performed by dividing the tissue after washing with a knife, scissors, a cutter (manual, automatic) or the like. The size and shape after the fragmentation are not particularly limited and can be performed randomly, but preferably 1 mm to 5 mm square, more preferably 1 mm to 2 mm square.

  Next, the fragmented product thus obtained is subjected to an enzyme treatment. Such an enzyme treatment can be mainly a trypsin treatment as described above. Enzyme treatment conditions can be 20 ° C. to 45 ° C., minutes to hours.

Next, the cells in the single cell product thus obtained are aggregated to 3 or more cells. Prior to aggregation, preferably a ROCK inhibitor can be quickly added to a single cell product.

  Here, the aggregate containing 3 or more cancer cells obtained by aggregation is the cancer cell aggregate of the present invention and has a certain range of sizes. The size within a certain range includes small particles having a volume average particle diameter of about 8 μm to 10 μm, but in the case of a nearly spherical shape, the diameter is 20 μm to 500 μm, preferably 30 μm to 400 μm, more preferably 40 μm to 250 μm, In the case of an elliptical shape, the major axis is 20 μm or more and 500 μm or less, preferably 30 μm or more and 400 μm or less, more preferably 40 μm or more and 250 μm or less. More preferably, it is 40 μm or more and 250 μm or less. The volume average particle diameter can be measured by evaluating the particle size distribution and particle shape using a phase contrast microscope (IX70; manufactured by Olympus Corporation) with a CCD camera.

  Any of the thus obtained aggregates or cultures thereof is the cancer cell aggregate of the present invention. The culture may be one in which the separated and collected components are present in the medium for a short time, for example, at least 3 hours or more, preferably 10 hours or more and 36 hours, more preferably 24 hours. By culturing for a period of ˜36 hours or longer, it may have a substantially spherical shape or a substantially elliptical spherical shape. The culture time may exceed 36 hours, and several days, 10 days or more, 13 days or more, or 30 days or more may have elapsed.

Cultivation can be performed as it is for a long time in the medium, but preferably, the ability to proliferate can be maintained substantially infinitely by performing mechanical division periodically during the culture.

  Furthermore, the cancer cell aggregate of the present invention has a high degree of colonization in transplantation into a heterologous animal even when, for example, the cancer cell aggregate is 10 micrometers or less (corresponding to 1000 cells or less) having a diameter of 100 micrometers. Therefore, the cancer cell aggregate of the present invention is useful for easy preparation of cancer model animals including mice, and treatment modes including more strict cancer tissue verification, drug sensitivity evaluation, or radiotherapy. Can be evaluated.

  The cancer cell aggregate of the present invention can be stored frozen and can retain its growth ability under normal storage conditions.

  The thus obtained cancer tissue-derived cell mass or cancer cell aggregate of the present invention behaves in vitro in the same manner as cancer tissue in vivo, can be stably cultured, and retains the ability to grow. To do.

  Therefore, it is useful, for example, for identifying the types of existing drugs to which the obtained tumor derived from cancer tissue is sensitive, or for confirming the sensitivity to radiation individually for each patient. For the drug or radiosensitivity, any known method can be used and is not limited.

  Specifically, drug sensitivity is determined by culturing cell masses derived from cancer tissue or cancer cell aggregates in the presence or absence of different concentrations of drugs in vitro and measuring their growth rate or survival rate. Can be done by doing. Such measurements include, for example, visually observing the number of viable cells several hours or days after addition of the test drug together with a control example, image analysis after photographing with a CCD camera, or proteins contained in each cell. Colorimetric measurement of the amount of protein by staining with a binding dye (for example, sulforhodamine B) is included.

Alternatively, drug sensitivity can be examined in vitro by culturing cancer tissue-derived cell mass or cancer cell aggregate in the presence or absence of the drug and analyzing intracellular signal transduction.

The analysis of intracellular signal transduction is, but not limited to, analyzing the presence and extent of phosphorylation of Akt and / or ERK1 / 2 in cells. Akt is a serine / threonine kinase and is activated by phosphorylation itself. On the other hand, extracellular signal-regulated kinase (ERK) belongs to the mitogen-activated protein kinase family. AKT and ERK are involved in the growth and survival of cancer cells.

As a specific method for analyzing the presence and extent of phosphorylation of such Akt and / or ERK1 / 2, it is known that it is located upstream of these enzymes in the cell signaling pathway. And then detecting the presence and extent of phosphorylation of Akt and / or ERK1 / 2. For example, a cancer tissue-derived cell mass or a cancer cell aggregate is cultured in the presence of an epidermal growth factor (EGF), an EGFR antibody, or an EGFR inhibitor, and then cell lysis treatment is performed. For example, it can be performed by Western blot. Alternatively, it can be cultured in the presence of an antibody against HER2 having a structure similar to that of EGFR, and then subjected to Western blot analysis in the same manner.

  In this way, the influence of the drug on the patient from which the cancer tissue-derived cell mass or cancer cell aggregate is derived can be examined before administration.

  Such cancer tissue-derived cell aggregates or cancer cell aggregates are also useful for screening unknown drugs. Such unknown drug sensitivities can also be performed by measuring the proliferation rate of cancer tissue-derived cell mass or cancer cell aggregate mass in vitro, measuring the reduction rate, determining cell viability, or analyzing intracellular signaling. For the measurement of the growth rate, for example, the number of viable cells after several hours or days after the addition of the test drug is visually observed together with a control example, image analysis is performed after taking a CCD camera, or included in each cell. Colorimetric measurement as protein amount by staining with protein-binding dye sulforhodamine B, measurement of SD (Succinyl dehidrogenase) activity, and the like are included.

Test compound susceptibility measurement data for all human cultured cells, ie, the concentration that inhibits cell growth by 50% (GI ₅₀ ), the concentration that apparently inhibits cell growth (TGI), and the number of cells is reduced to 50% at the time of seeding. It is also possible to perform information processing by calculating the concentration (LC ₅₀ ) and the like. The GI ₅₀ , TGI, and LC ₅₀ values are values specific to the cancer cell aggregate to be tested. The overall average GI ₅₀ , TGI, and LC ₅₀ values are obtained, the difference between this average value and the Log GI ₅₀ value in each individual cell is obtained, and they are converted into absolute values based on the average Log GI ₅₀ value and made positive or negative. Is written. The larger the positive value, the more sensitive the drug can be judged.

  As a radiosensitivity test using the cancer tissue-derived cell mass or cancer cell aggregate of the present invention, X-rays, γ rays using a radioactive isotope of cobalt as a radiation source, and particle beams obtained by accelerating electron beams with a linear accelerator Or a known test in which a heavy particle beam such as α-ray extracted by a cyclotron or the like is used alone or in combination with a radiosensitizer.

  In a radiosensitivity test, cell masses derived from cancer tissue or cancer cell aggregates are exposed to radiation or cultured without exposure, and their growth rate, reduction rate, or survival rate is measured and compared. Can be broken. Such measurements include, for example, visual observation of the number of viable cells several hours or days after irradiation with a control example, image analysis after taking a CCD camera, or binding of proteins contained in each cell. And colorimetric measurement as a protein amount by staining with an organic dye (for example, sulforhodamine B).

  In this way, it is possible to examine the influence of radiation on a patient from which a cancer tissue-derived cell mass or a cancer cell aggregate is derived before irradiation.

  The cancer tissue-derived cell mass or cancer cell aggregate used in such a drug sensitivity test or radiation sensitivity test may be one that has been stored by freezing.

  If such cryopreservation is possible, further collect the genetic information of the cancer tissue-derived cell mass or cancer cell aggregate of the patient from which it was derived or other genetic information of the patient in advance, drug selection based on that information, It will also be possible to examine the possibility of radiation irradiation.

EXAMPLES The present invention will be specifically described below with reference to examples, but the present invention is not limited to these examples. In addition, all the parts and% in each example are based on weight. The culture conditions not specifically defined below are all under 37 ° C. and 5% CO ₂ incubator conditions. Centrifugation conditions are 4 ° C., 1000 rpm, and 5 minutes unless otherwise specified.

(Example 1)
(Preparation of cancer tissue-derived cell mass from human colon cancer mouse transplanted tumor)
Tumors transplanted with human colon cancer mice were prepared by the xenotransplantation method as follows.

  First, a surgically removed specimen of a human tumor (colorectal cancer) is cut into approximately 2 mm cubes under aseptic operation. Next, a small incision of about 5 mm is made on the back of severely immunodeficient mice (nude mice, preferably NOD / SCID mice), and the subcutaneous tissue is exfoliated. After the prepared tumor piece is inserted subcutaneously, it is closed with a skin suture clip. Some transplanted tumors are observed as subcutaneous tumors after about 14 days to 3 months.

  The obtained colon cancer mouse was bred under SPF (specific pathogen free) breeding conditions. When the tumor became 1 cm in size, the tumor was removed and 20 ml of DMEM (Gibco; 11965-092) + 1% Pen Strep ( Gibco; 15140-022) (both at a final concentration of 100 units / ml penicillin, 100 μg / ml) were collected in a 50 ml centrifuge tube (IWAKI; 2345-050).

  Next, 20 ml HBSS (Gibco; 14025-092) was added, and the tumor was washed by inversion mixing. Next, 20 ml of new HBSS was added, and this operation was repeated twice, and the tumor tissue was transferred to a 10 cm tissue culture dish (tissue culture dish) (IWAKI; 3020-100). On this culture dish, the necrotic tissue was removed using a surgical knife.

  The tumor pieces from which the necrotic tissue was removed were transferred to a new 10 cm dish containing 30 ml of HBSS. Next, the tumor piece was cut into about 2 mm square using a surgical knife.

  Tumor debris together with HBSS was transferred to a new 50 ml centrifuge tube, centrifuged, and the supernatant was discarded and washed with 20 ml HBSS by inversion mixing.

  Centrifugation and washing were repeated. Thereafter, 20 ml of DMEM + 1% Pen Strep + 0.28 U / ml (final concentration) Blendzyme 1 (Roche; 11988417001) was added and mixed. This was transferred to a 100 ml Erlenmeyer flask and treated with Liberase Blendzyme 1 (Roche Diagnostics) for 2 hours while rotating the stirrer at low speed in a 37 ° C constant temperature bath.

  Next, the enzyme-treated product was collected in a 50 ml centrifuge tube, centrifuged, the supernatant was discarded, and 20 ml HBSS was added and mixed. The components that passed through the stainless steel mesh (500 μm) and passed through the filter were collected in a 50 ml centrifuge tube, and further subjected to centrifugation. Discard the supernatant, add 1 mg / ml DNaseI solution (Roche; 1284932) (100 mg of 10 mg / ml stock + 900 μl of PBS), mix and stand at 4 ° C for 5 minutes, add 20 ml HBSS and mix. Centrifugation was performed and the supernatant was discarded. After mixing with 20 ml HBSS, it was sieved in steps of 500-250-100 μm and then passed through a 40 μm cell strainer (BD; 352340). The cell strainer was immersed in a 10 cm tissue culture dish (tissue culture dish) containing 30 ml of HBSS and gently shaken to remove single cells, small cell clusters of 40 μm or less, and waste. The cell strainer was transferred to another 10 cm tissue culture dish (tissue culture dish) containing 30 ml of HBSS, and the cell mass captured by the cell strainer was collected by pipetting.

  Further, the same centrifugation operation as described above was performed several times, and the resulting component was mixed with 4 ml StemPro hESC SFM (Gibco; A10007-01) + 8 ng / ml bFGF (Invitrogen; 13256-029) + 0.1 mM 2-mercapto Ethanol (Wako; 137-06862) + 1% PenStrep + 25 μg / ml Amphotericin B (Wako; 541-01961) was added and mixed, and transferred to a 6 cm non-treated dish (EIKEN CHEMICAL; AG2000).

This was cultured at 37 ° C. for 36 hours in a 5% CO ₂ incubator (Sanyo MCO-17AIC).

  As a result, as shown in FIG. 1, it changes from an irregular shape to a well-formed spherical shape with the passage of time, is substantially spherical after at least 3 to 6 hours, and is perfectly spherical after 24 hours. A derived cell mass was obtained.

(Example 2)
(Preparation of cancer tissue-derived cell mass from human colorectal cancer surgical specimen)
A cancer tissue-derived cell mass was obtained in the same manner as in Example 1 except that a colorectal cancer surgical specimen was used. As a result, as shown in FIG. 7, a substantially spherical cancer tissue-derived cell cluster similar to that in FIG. 1 was obtained after at least 12 hours.

(Example 3)
(Preparation of cancer tissue-derived cell mass from human ovarian cancer surgical specimen)
A cancer tissue-derived cell mass was obtained in the same manner as in Example 2 except that the ovarian cancer surgical specimen was used. As a result, as shown in FIG. 7, a substantially spherical cancer tissue-derived cell cluster similar to that in FIG. 1 was obtained after at least 12 hours.

Example 4
(Preparation of cancer tissue-derived cell mass from human pancreatic cancer surgical specimen)
A cancer tissue-derived cell mass was obtained in the same manner as in Example 2 except that a pancreatic cancer surgical specimen was used. As a result, as shown in FIG. 7, a substantially spherical cancer tissue-derived cell cluster similar to that in FIG. 1 was obtained after at least 12 hours.

(Example 5)
(Preparation of cancer tissue-derived cell mass from human small cell carcinoma surgical specimen)
A cancer tissue-derived cell mass was obtained in the same manner as in Example 2 except that a small cell cancer surgical specimen which is a type of lung cancer was used. As a result, as shown in FIG. 7, a substantially spherical cancer tissue-derived cell cluster similar to that in FIG. 1 was obtained after at least 12 hours.

(Example 6)
(Preparation of cancer tissue-derived cell mass from human renal cancer surgical specimen)
A cancer tissue-derived cell mass was obtained in the same manner as in Example 2 except that a renal cancer surgical specimen was used. As a result, as shown in FIG. 7, a substantially spherical cancer tissue-derived cell cluster similar to that in FIG. 1 was obtained after at least 12 hours.

(Example 7)
(Preparation of cancer tissue-derived cell mass from human bladder cancer surgical specimen)
A cancer tissue-derived cell mass was obtained in the same manner as in Example 2 except that a bladder cancer surgical specimen was used. As a result, as shown in FIG. 7, a substantially spherical cancer tissue-derived cell cluster similar to that in FIG. 1 was obtained after at least 12 hours.

(Example 8)
(Preparation of cancer tissue-derived cell mass from human breast cancer surgical specimen)
A cancer tissue-derived cell mass was obtained in the same manner as in Example 2 except that a breast cancer surgical specimen was used. As a result, as shown in FIG. 7, a substantially spherical cancer tissue-derived cell cluster similar to that in FIG. 1 was obtained after at least 12 hours.

Example 9
(Preparation of cancer tissue-derived cell mass from human prostate cancer surgical specimen)
A tissue-derived cell mass was obtained in the same manner as in Example 2 except that a prostate cancer surgical specimen was used. Dihydrotestosterone (DHT) at a concentration of 10 ⁻⁸ mol / L was added to the culture medium and cultured in the same manner as in Example 1. As a result, as shown in FIG. 7, a substantially spherical cancer tissue-derived cell cluster similar to that in FIG. 1 was obtained after at least 12 hours.

(Example 10)
(Preparation of cancer tissue-derived cell mass from human pharyngeal cancer surgical specimen)
A cancer tissue-derived cell mass was obtained in the same manner as in Example 2 except that the pharyngeal cancer surgical specimen was used. As a result, as shown in FIG. 7, a substantially spherical cancer tissue-derived cell cluster similar to that in FIG. 1 was obtained after at least 12 hours.

(Example 11)
(Hormone sensitivity test of breast cancer-derived cancer tissue-derived cell mass)
Under the same medium conditions as in Example 8, it was investigated how the state of cell mass derived from breast cancer tissue obtained from a plurality of patients differed with or without estradiol. As a result, as shown in FIG. 8, it was found that there were cases where growth was promoted by the addition of estradiol and cases that did not respond to estradiol. It was found that it can be applied as a susceptibility test when hormonal therapy is performed on the patient from whom it originated.

(Example 12)
(Preparation of cancer tissue-derived cell mass from mouse islet tumor)
RipTag is a transgenic mouse in which SV40-T antigen is forcibly expressed under the control of the rat insulin promoter. Tumors develop in islets. A cancer tissue-derived cell mass was obtained in the same manner as in Example 2 except that the islet tumor of the RipTag mouse was used. As a result, at least 12 hours later, a substantially spherical cancer tissue-derived cell mass similar to that shown in FIG. 1 was obtained (FIG. 9).

(Example 13)
The cell mass derived from the cancer tissue obtained in Example 2 and cultured in the culture shown in FIG. 7 was taken out 24 ml after culturing with 5 ml of the medium, centrifuged at 1000 rpm at 4 ° C., and the supernatant was discarded. The collected cancer tissue-derived cell mass was suspended in a cell banker (BLC-1, manufactured by Mitsubishi Chemical Medicine), and further 10 μM Y27632 (manufactured by Wako Pure Chemical Industries, Ltd.) was added, and a cryopreservation tube (Cryogenic vials 2.0 ml, Nalge Nunc) and stored in a -80 ° C deep freezer.

  After 7 days from storage, the mixture was briefly reheated in a 37 ° C water bath. This was suspended in PBS, further centrifuged at 1000 rpm at 4 ° C., and the supernatant was discarded. The obtained precipitate was suspended in StemPro (manufactured by Invitro) and cultured. As shown in FIG. 10, the state of the cells 24 hours after thawing was good.

Furthermore, the survival of the obtained cancer tissue-derived cell mass was confirmed by transplanting into NOD-SCID mice as a mass containing about 1000 cells.

(Example 14)
(Preparation of cancer cell aggregates from cancer tissue-derived cell clusters)
Using the cancer tissue-derived cell mass obtained by the same method as in Example 2, the following treatment was performed. First, collagen gel (Cell Matrix type IA: 5x DMEM: buffer for gel reconstitution = 7: 2: 1) 50 μL / well was spread on the center of a 24-well plate (untreated dish). The collagen gel was solidified by standing at 37 ° C. for 30 minutes. Cell masses derived from cancer tissue in suspension culture (100 per well) are collected in a 1.5 mL tube. This was centrifuged for about 5 seconds, and the supernatant was removed. The cell mass derived from cancer tissue was suspended in collagenase gel (30 μL per well), and 30 μL each was placed on the previously solidified gel. The mixture was allowed to stand at 37 ° C. for 30 minutes to solidify, and StemPro (EGF 50 ng / mL) 600 μL / well was added. The culture was performed for 10 days while changing the medium once every 2-3 days.
Next, the medium was replaced with 1 mL / well of DMEM (Gibco; 11965-092, containing collagenase IV 200 mg / mL) and cultured at 37 ° C. for about 5 hours.
After incubation, transfer to a 1.5 mL Eppendorf tube, centrifuge (approximately 5 seconds), remove the supernatant, add 1 mL of PBS to suspend, centrifuge (Chibitan, approximately 5 seconds), and remove the supernatant. Repeated twice. 1 mL of Trypsin / EDTA (0.05%) was added and suspended, and the mixture was allowed to stand at 37 ° C. for 8 minutes. After suspending several times, it was confirmed that there was no large mass like a cell mass derived from cancer tissue. This was transferred to a 15 mL tube and suspended by adding 2 mL of DMEM (Gibco; 11965-092).
Next, the suspension was centrifuged (1000 rpm, 5 minutes), and the supernatant was removed. It was suspended in 2 mL StemPro (EGF 50 ng / mL, Y-27632 10 μM) and transferred to a φ35 mm non-treated dish (Iwaki: 1000-035). This was cultured overnight at 37 ° C.
After 12 hours, formation of a cell mass derived from a cancer tissue having a diameter of about 40 μm was confirmed. The medium was replaced with StemPro (EGF 50 ng / mL).

As a result, as shown in FIG. 11, after 4 days, completely arranged spherical cancer cell aggregates were obtained.

(Example 15)
(Preparation of cancer cell aggregates from human colon cancer surgical specimens)
A cancer cell aggregate was obtained in the same manner as in Example 14 except that the colorectal cancer surgical specimen was used. As a result, as shown in FIG. 12, an almost spherical cancer cell aggregate similar to FIG. 1 was obtained after at least 12 hours.

(Example 16)
Cell preservation of the cancer tissue-derived cell mass obtained by the same method as in Example 2 was performed. The cancer tissue-derived cell mass was treated with trypsin in the same manner as in Example 14 to obtain a single cell. The cryopreservation solution used was Cell Banker 1 (Juji Field) with Y-27632 added.

Single cells that were frozen and stored for 10 days were then reconstituted briefly in a 37 ° C water bath. This was suspended in PBS, further centrifuged at 1000 rpm at 4 ° C., and the supernatant was discarded. The obtained precipitate was suspended in StemPro (manufactured by Invitro) and cultured. As shown in FIG. 13, the state of the cells at 24 hours after thawing was good, and the cell mass derived from cancer tissue was reformed after thawing.

(Comparative Example 1)
Using human colorectal cancer surgical specimens, single cells were prepared according to the method described in the literature (Todaro M et al. (2007) Colon cancer stem cells dictate tumor growth and resist cell death by production of interleukin-4. Cell Stem Cell 1: 389-402). Samples treated up to were prepared. However, CD133 positive cells selected by single cell treatment could not be found to grow in vitro.

  Evaluation items in Examples and the like were measured as follows.

<Identification of surface antigen>

The cell mass derived from the cancer tissue obtained in Example 1 was dispersed into single cells using trypsin / EDTA. These cells were reacted with a surface antigen-specific antibody labeled with fluorescence, and then analyzed by flow cytometry. As a result, as shown in FIG. 2, the presence of cells that uniformly and simultaneously express the surface antigen was observed.

<Confirmation of basement membrane-like material>
The cell mass derived from the cancer tissue obtained in Example 1 was cultured for 3 days in 1 cc of a serum-free medium (Gibco) for STEMPRO human ES cells under the culture conditions of a temperature of 37 ° C. and a 5% CO ₂ incubator. This was fixed in formalin, embedded in paraffin, sliced, and stained with anti-laminin antibody (Sigma-Aldrich, mouse laminin-derived rabbit antibody) according to the manufacturer's instructions. Laminin antigenicity was observed in the cytoplasm of the cells near the periphery. Accordingly, it was found that the cancer tissue-derived cell mass of the present invention was surrounded by laminin around the cancer cell aggregate. On the other hand, the expression of laminin could not be confirmed 24 hours after the surgical specimen treatment.

<Detection of hypoxia>
Example of detection of hypoxia using pimonidazole The nitroimidazole compound, pimonidazole, has the property of forming adducts with proteins and nucleic acids in the absence of oxygen. The hypoxic region of the tissue treated with pimonidazole under hypoxia can be recognized using an antibody that specifically recognizes pimonidazole. In the cancer tissue, a hypoxic region appears about 100 micrometers away from the blood vessel, but the cancer tissue-derived cell mass obtained in Example 1 also has a hypoxic region on the inside with a boundary of about 100 micrometers from the outer edge. Cell death was observed.

<Evaluation of in vitro proliferation ability>
The proliferation ability of the cancer tissue-derived cell mass in vitro was verified as follows. The cell mass derived from the cancer tissue obtained in Example 1 was treated with collagen gel (CellMatrix type IA (Nitta Gelatin): 5x DMEM (Gibco; 12100-038): Gel reconstitution buffer (50 mM NaOH, 260 mM NaHCO3, 200 mM HEPES) = 7: 2: 1) were embedded at 10 × each, and cultured in 1 cc of a serum-free medium (Gibco) for STEMPRO human ES cells under the culture conditions of a temperature of 37 ° C. and a 5% CO ₂ incubator. The state of the cells was regularly observed, and the size was measured with a phase contrast microscope (40 times magnification) equipped with a CCD camera. As a result, as shown in FIG. 3, the proliferation ability could be maintained for at least 13 days without mechanical division. Furthermore, when mechanical division was performed on the 13th day, it was confirmed that the proliferation ability was maintained for at least 13 days. The mechanical division was performed by dividing a cancer tissue-derived cell mass having a diameter of 500 micrometers into four with an ophthalmic sword.

<Confirmation of cell number>
In the same manner as in Example 1, a cell mass derived from a cancer tissue of 100 to 250 μm was treated with 0.25% trypsin and 2.6 mM EDTA for 3 minutes, and mechanically decomposed by pipetting about 30 times. This solution was diluted and dispensed so that one cell would enter one well of a 96-well culture plate. For cell clusters that were not unicellularized, the number of constituent cells was counted and recorded. Thereafter, culture (conditions same as above) was carried out, the increase in the number of cells in each well was recorded, and the culture was observed for 30 days. As a result, it was confirmed that if there are three cells, some cells can grow to a cell mass.

<Drug sensitivity test>
A drug susceptibility test using the sample of Example 2 was performed using 5-FU, which is known to bind to thymidylate synthase, which is a metabolic process necessary for DNA synthesis, and inhibit DNA synthesis. In the test, a cell mass derived from a cancer tissue was treated with a collagen gel (CellMatrix type IA (Nitta Gelatin): 5x DMEM (Gibco; 12100-038): Gel reconstitution buffer (50 mM NaOH, 260 mM NaHCO3, 200 mM HEPES) = 7: 2: Each 10 × was embedded in 1) and cultured in 1 cc of serum-free medium (Gibco) for STEMPRO human ES cells under the culture conditions of a temperature of 37 ° C. and a 5% CO ₂ incubator. Further, 5-FU was applied at concentrations of 0.01 μg / ml, 0.1 μg / ml, 1 μg / ml, 10 μg / ml, and 100 μg / ml, and the conditions on day 0 and day 8 of the culture were compared and evaluated. The result is shown in FIG. About the increase rate regarding the area | region of a cancer tissue origin cell mass, the increase rate regarding the area in a medicine non-application culture was relatively described as 1. In FIG. 4, cancer cell proliferation on the 8th day of culture is suppressed depending on the concentration of 5-FU, and it has been proved that the cancer tissue-derived cell mass of the present invention is useful in a drug sensitivity test. It was.

<Transplantation test to different animals>
Ten cells of cancer tissue-derived cell mass of about 100 micrometers in diameter obtained in Example 2 and cultured for 3 days according to the present invention were suspended in Matrigel (BD) and administered subcutaneously to the back of NOD-SCID mice. . Tumor formation was evaluated by measuring tumor size over time. As a result, significant tumor formation was observed in the mouse individuals transplanted with the cancer tissue-derived cell mass of Example 2 of the present invention, and it was confirmed that the cancer tissue-derived cell mass of the present invention has a high tumor-forming ability. . When this tissue was analyzed, it was found that a similar tissue type was obtained between a tumor formed by transplanting into a mouse and a tumor existing in the living body (FIG. 5).

<Radiation irradiation test>
The cancer tissue-derived cell mass having a diameter of about 100 micrometers used in the present invention obtained in Example 2 was converted into a collagen gel (CellMatrix type IA (Nitta Gelatin): 5x DMEM (Gibco; 12100-038): buffer for gel reconstitution) Embedded in (50 mM NaOH, 260 mM NaHCO3, 200 mM HEPES) = 7: 2: 1) in 1 cc of serum-free medium (Gibco) for STEMPRO human ES cells under the conditions of 37 ° C. and 5% CO ₂ incubator. × 10 pieces were inoculated and cultured. This was irradiated with gamma rays using cobalt radioisotope as a radiation source, and the state of the lump was confirmed. The result is shown in FIG. In FIG. 6, the growth of cancer cells up to the 8th day of culture is suppressed depending on the irradiation dose, and it was actually proved that the cancer tissue-derived cell mass of the present invention is useful in the radiation irradiation test. .

<Drug sensitivity test>
Doxorubicin is known to exert an antitumor effect by inserting between the base pairs of tumor cell DNA, inhibiting the DNA polymerase, RNA polymerase, and topoisomerase II reactions and suppressing the biosynthesis of both DNA and RNA. Was used to conduct a drug sensitivity test using the sample of Example 12. In the test, cancer cell aggregates were treated with collagen gel (CellMatrix type IA (Nitta Gelatin): 5x DMEM (Gibco; 12100-038): Gel reconstitution buffer (50 mM NaOH, 260 mM NaHCO3, 200 mM HEPES) = 7: 2: 1. ) Was embedded in 10 × and cultured in 1 cc of a serum-free medium (Gibco) for STEMPRO human ES cells under the culture conditions of a temperature of 37 ° C. and a 5% CO ₂ incubator. Furthermore, doxorubicin was applied at a concentration of 0.1 μM, 1 μM, and 10 μM, and the conditions on day 0 and day 8 of culture were compared and evaluated. The result is shown in FIG. About the increase rate regarding the area of a cancer cell aggregate, the increase rate regarding the area in the medicine non-application culture was relatively described as 1. In FIG. 14, cancer cell proliferation on the 8th day of culture was suppressed depending on the concentration of doxorubicin, and it was proved that the cancer cell aggregate of the present invention was useful in the drug sensitivity test.

<Drug sensitivity test>
The cancer tissue-derived cell mass having a diameter of about 100 micrometers used in the present invention obtained in Example 7 was subjected to a drug sensitivity test using the platinum anticancer drug cisplatin to induce cell death by DNA crosslinking. In the test, a cell mass derived from a cancer tissue was treated with Matrigel (BD): 5x DMEM (Gibco; 12100-038): Gel reconstitution buffer (50 mM NaOH, 260 mM NaHCO ₃ , 200 mM HEPES) = 7: 2: 1) Each of the cells was embedded in 5 ×, and cultured in 1 cc of a serum-free medium (Gibco) for STEMPRO human ES cells under a culture condition of a temperature of 37 ° C. and a 5% CO ₂ incubator for 48 hours. Subsequently, staining was performed with a phase contrast microscope, propidium iodide (PI), Calcein, Hoechst33342, and the shape of CTOS, cell death, cell metabolic activity, and nucleus were examined. The result is shown in FIG. Indicated. In FIG. 15, cancer cell death by cisplatin was observed in a concentration-dependent manner after 48 hours of culture. The cancer tissue-derived cell mass of the present invention reacted in a drug-specific manner in a drug sensitivity test, and was proved to be very useful for detecting cell death in a short time using PI staining.

<Drug sensitivity test>
In the same manner as in Example 2, cancer tissue-derived cell clusters having a diameter of about 100 micrometers obtained from a plurality of patients were used for evaluation of cell responses to EGF and cetuximab. Cetuximab is a monoclonal antibody that binds to epidermal growth factor receptor (EGFR) and inhibits the action of EGFR. Used clinically as an anticancer agent. A cell mass derived from cancer tissue is cultured overnight in DMEM / F12 supplemented with 1% BSA, and then 10 ng / ml EGF (manufactured by Sigma Aldlich) or 10 μg / ml cetuximab (Arbitux, Bristol-Myers, Inc.) And both were added to the medium and incubated for 15 minutes. Next, the cell mass derived from the cancer tissue cultured in this manner was each lysed with RIPA buffer, and the lysate was subjected to Western blot analysis to examine the state of phosphorylation of Akt and ERK1 / 2. As a result, it became clear that the activation of intracellular signals by EGF stimulation differs among the tumors. (FIG. 16). The effect of cetuximab is expected in patients with samples whose phosphorylation was suppressed by cetuximab.

<Drug sensitivity test>
In the same manner as in Example 2, cancer tissue-derived cell clusters having a diameter of about 100 micrometers obtained from a plurality of patients were used for evaluation of cell responses to IGF and αIR3. αIR3 is a monoclonal antibody that binds to insulin-like growth factor receptor (IGF-IR) and inhibits the action of IGF-IR. A cell mass derived from cancer tissue is cultured overnight in DMEM / F12 supplemented with 1% BSA, and then treated for 1 hour in the presence or absence of 1 μg / ml αIR3 (Merck). The cells were cultured for 15 minutes in the presence or absence of ng / ml IGF-I (R & D). Next, the cell mass derived from the cancer tissue cultured in this manner was each lysed with RIPA buffer, and the lysate was subjected to Western blot analysis to examine the state of phosphorylation of Akt and ERK1 / 2. As a result, it was found that the pattern of AKT activation can be divided into three categories. Sensitivity pattern (phosphorylated in response to IGF stimulation, αIR3 gives dephosphorylation results), resistance pattern (phosphorylated in response to IGF stimulation, but αIR3 does not give dephosphorylation results) ), And an independent pattern (not responsive to IGF stimulation or αIR3). (FIG. 17). It was found that these three patterns are associated with the growth of the corresponding cell mass derived from cancer tissue.

  The cancer tissue-derived cell mass or cancer cell aggregate of the present invention can be used for a wide range of applications in vitro. And it can be made to proliferate by culture | cultivation and the cancer cell proliferation from a trace amount specimen is enabled. Furthermore, the cancer tissue-derived cell mass of the present invention can be widely used in drug sensitivity tests or radiation sensitivity tests, and can be used for the production of simple tumor-forming animals. For this reason, the cancer tissue-derived cell mass of the present invention can bring about a dramatic improvement with respect to anticancer agents and radiotherapy currently used generally in trial and error or cocktail therapy. That is, before performing such therapy, it is possible to predict the effects of drugs and radiotherapy in advance on the cancer tissue-derived cell mass or cancer cell aggregate obtained from each patient, and only effective drugs are given to patients. It becomes possible to administer. Furthermore, since the cancer tissue-derived cell mass or the cancer cell aggregate of the present invention may be of a size that can be collected with an injection needle or cultured, it can also be obtained from a patient before surgery. It is also possible to predict the effects of anticancer drugs and radiation therapy in a state where there is little burden on the patient. The cancer tissue-derived cell mass or cancer cell aggregate of the present invention can also be used for screening for selection of an unknown drug as an anticancer agent.

Claims (15)

Treating a fragment of cancer tissue excised from a living body with an enzyme containing collagenase;
From enzyme treatment was prepared using the method of dividing the size, diameter, major axis or the volume average step of the mass containing the cancer cells 500μm hereinafter more particle size 20μm sorting recovered;
Culturing the collected mass for at least 3 hours to obtain a substantially spherical or oval spherical culture;
And a method for evaluating the influence of a drug, comprising the steps of causing a drug to act on the culture and evaluating the effect of the drug on the culture.   The enzyme comprises one or more proteases selected from the group consisting of C. histolyticum neutral protease, thermolysin and dispase; and one or more collagenases selected from the group consisting of collagenase I, collagenase II, and collagenase IV The method for evaluating the influence of a drug according to claim 1, which is a mixed enzyme.   Furthermore, the evaluation method of the influence of the chemical | medical agent of Claim 1 or 2 including the process of dividing | segmenting the said substantially spherical shaped or elliptical spherical shaped culture mechanically.  4. The method of any one of claims 1 to 3, wherein the step of evaluating the action comprises comparing the growth state of the culture in the presence or absence of different concentrations of the drug or performing cell viability determination. The evaluation method of the influence of the description medicine. Treating a fragment of cancer tissue excised from a living body with an enzyme containing collagenase;
From enzyme treatment was prepared using the method of dividing the size, diameter, major axis or the volume average step of the mass containing the cancer cells 500μm hereinafter more particle size 20μm sorting recovered;
Culturing the collected mass for at least 3 hours to obtain a substantially spherical or oval spherical culture;
And a method for evaluating the influence of radiation, comprising the steps of causing radiation to act on the culture and evaluating the effect of the radiation on the culture.   The enzyme comprises one or more proteases selected from the group consisting of C. histolyticum neutral protease, thermolysin and dispase; and one or more collagenases selected from the group consisting of collagenase I, collagenase II, and collagenase IV 6. The method for evaluating the influence of radiation according to claim 5, which is a mixed enzyme.   Furthermore, the evaluation method of the influence of the radiation of Claim 5 or 6 including the process of dividing | segmenting the said substantially spherical shaped or elliptical spherical shaped culture mechanically.  8. The method of any one of claims 5 to 7, wherein the step of assessing the effect comprises comparing the growth state of the cultures under radiation with or without radiation at different intensities or performing cell viability determination. The evaluation method of the influence of the radiation of description. Treating a fragment of cancer tissue excised from a living body with an enzyme containing collagenase;
From enzyme treatment was prepared using the method of dividing the size, diameter, major axis or the volume average step of the mass containing the cancer cells 500μm hereinafter more particle size 20μm sorting recovered;
Culturing the collected mass for at least 3 hours to obtain a substantially spherical or oval spherical culture;
And a method for screening a drug, comprising the steps of causing a drug candidate compound to act on the culture and evaluating the action of the drug candidate compound on the culture.   The enzyme comprises one or more proteases selected from the group consisting of C. histolyticum neutral protease, thermolysin and dispase; and one or more collagenases selected from the group consisting of collagenase I, collagenase II, and collagenase IV The method for screening a drug according to claim 9, which is a mixed enzyme.   The method for screening a drug according to claim 9 or 10, further comprising a step of mechanically dividing the substantially spherical or elliptical spherical culture.   12. The method according to any one of claims 9 to 11, wherein evaluating the action comprises comparing the growth state of a culture in the presence or absence of a drug candidate compound, or performing cell viability determination. The screening method of the description medicine. The strip product of excised cancerous tissue from a living organism is treated with enzymes including collagenase, from enzyme treated, the mass was screened collected containing in diameter, major axis, or the cancer cell a volume average particle diameter of 20μm or more 500μm hereinafter, A method for evaluating the influence of a drug, comprising a step of allowing a drug to act on a substantially spherical or oval spherical culture obtained by culturing the collected mass and evaluating the effect of the drug on the culture. The strip product of excised cancerous tissue from a living organism is treated with enzymes including collagenase, from enzyme treated, the mass was screened collected containing in diameter, major axis, or the cancer cell a volume average particle diameter of 20μm or more 500μm hereinafter, A method for evaluating the influence of radiation, comprising a step of causing radiation to act on a substantially spherical or elliptical spherical culture obtained by culturing the collected mass, and evaluating the effect of the radiation on the culture. The strip product of excised cancerous tissue from a living organism is treated with enzymes including collagenase, from enzyme treated, the mass was screened collected containing in diameter, major axis, or the cancer cell a volume average particle diameter of 20μm or more 500μm hereinafter, Drug screening comprising the steps of allowing a drug candidate compound to act on a substantially spherical or oval spherical culture obtained by culturing the collected mass, and evaluating the action of the drug candidate compound on the culture Method.