WO2006115243A1

WO2006115243A1 - Method of producing cancer stem cell

Info

Publication number: WO2006115243A1
Application number: PCT/JP2006/308560
Authority: WO
Inventors: Nobuyuki Takakura; Yoshihiro Yamada; Hiroyasu Kidoya
Original assignee: National University Corporation Kanazawa University
Priority date: 2005-04-25
Filing date: 2006-04-24
Publication date: 2006-11-02
Also published as: JP4752051B2; JP2006296364A

Abstract

A method of producing a cancer stem cell comprising the step of in vitro cell fusion of a tumor cell and a blood cell (for example, blood cells including a hematopoietic stem cell and/or a precursor cell) and a method of producing a cancer-bearing model animal comprising the step of administering a mixture of the cancer stem cell obtained by the cell fusion of a tumor cell and a blood cell and the tumor cell to a non-human mammal.

Description

How to create cancer stem cells

[0001] The present invention relates to a method for producing cancer stem cells.

Background art

[0002] Modern cancer research and treatment treats cancer as a homogeneous cell population. In the development of cancer therapeutics, the development of therapeutics is based solely on the cell death of proliferating cancer cells. It is powerful. Due to these circumstances, many cancer drugs and treatments have been developed. The current situation is that the cancer has not been completely cured. On the other hand, recently, cancer tissues are sensitive to cancer stem cells that are resistant to anti-cancer drugs and pile cancer drugs that are separated from the cancer stem cells and multiply excessively. The concept of cancer recurring unless it induces cell death of cancer stem cells has been proposed and has received much attention.

[0003] For example, according to Non-Patent Document 1, it is cultured in a test tube! Speaking tumor cell lines do not have uniform cell growth Some cells belong to a group of stem cells called SP (side populatio n) that have high drug efflux ability, and these cells differentiate into various cells It is said to have pluripotency. In Non-Patent Documents 2 and 3, there are tumor cells having cell surface markers that are partly different from other tumor cells among breast cancer and brain tumor cells that have developed in individuals. Has been shown to have a higher ability to engraft individuals than other tumor cells!

[0004] In these reports, tumors are not biochemically homogeneous, but some cells have stem cell-like multipotency and high engraftment potential by transplantation, which are cancer stem cells It is explained. However, it is currently unknown how these cancer stem cells have acquired different traits from other cancer cells, and how to obtain cancer stem cells. However, there is no suggestion or teaching in these documents. Once a method for experimentally obtaining cancer stem cells has been developed and the cause of cancer stem cell development has been elucidated, treatments that suppress the stem cell transformation of cancer cells and treatments that target cancer stem cells Expected to be able to develop methods, cancer malignant and prognostic methods, or cancer is inhabited-it is expected to develop into diagnosis and treatment methods that eradicate cancer by elucidating the pinch . Therefore, development of a method for obtaining cancer stem cells experimentally with good reproducibility is eagerly desired. Non-patent literature l: Kondo, T "et al., Proc. Natl. Acad. Sci. USA" 101 (3), pp.781-786, 2004

Non-Patent Document 2: AH "Iajj, M. et al" Proc. Natl. Acad. Sci., U S A "100 (7), pp.3983-39 88, 2003

Non-Patent Document 3: Singh, S.K., et al., Nature, 432 (7015), pp.396-401, 2004

Disclosure of the invention

Problems to be solved by the invention

[0005] An object of the present invention is to provide a method for obtaining cancer stem cells experimentally with good reproducibility.

Means for solving the problem

[0006] The inventors of the present invention have conventionally attempted a detailed analysis of the involvement of hematopoietic cells in the molecular mechanism of angiogenesis in cancer tissues. As a result, during tumor growth, hematopoietic stem cells and hematopoietic progenitor cells invading only the mature blood cells of the myeloid and lymphocyte lineages invaded the tumor (Okamoto, R., et al. , Blood, 105 (7), pp.275 7-2763, 2005), and many of these leaked out of the blood vessels within the tumor. They found that these blood cells secrete angiogenesis-promoting factors and contribute greatly to the formation of new blood vessels formed in the tumor, that is, tumor angiogenesis.

[0007] Further, since suppression of blood cell invasion into a tumor does not cause an increase in tumor, the present inventors are involved in the malignant transformation of tumors in addition to promoting blood vessel formation in blood cells. Based on the hypothesis that a mechanism exists, we analyzed the correlation between these blood cells and tumor cells. As a result, by co-culturing blood cells containing hematopoietic stem cell Z progenitor cells and tumor cells, the tumor cells and blood cells fuse, and the above-mentioned tumor cells fused with blood cells are not fused. It was found that the expression of CD133 (AC133) and c-kit, which are surface antigens of stem cells with strong expression, was induced. In addition, a small amount of tumor cells fused with blood cells and a small amount of tumor cells fused with blood cells. It was found that mixing tumor cells (which cannot form tumors even when transplanted into individuals) and transplanting them into mice induces very high malignant tumor formation. . This is considered to have been experimentally reproduced that the growth of cancer is promoted in a mosaic state in which normal cancer cells and cancer stem cells are mixed in the formation of cancer thread and tissue. The present invention has been completed based on the above findings.

[0008] That is, according to the present invention, there is provided a method for producing cancer stem cells, comprising a step of in vitro cell fusion of tumor cells and blood cells. According to a preferred embodiment of the present invention, there is provided the above method for performing the above cell fusion by co-culturing blood cells containing hematopoietic stem cells and Z or progenitor cells and tumor cells. In addition, the present invention provides a cancer stem cell that can be obtained by the above method.

Another aspect of the present invention is a method for producing a model tumor tissue according to the present invention, wherein a mixture of cancer stem cells obtained by cell fusion of tumor cells and blood cells and the tumor cells is cultured. A method comprising the steps is provided. The present invention also provides a model tumor tissue that can be obtained by the above method.

From another point of view, according to the present invention, there is provided a method for preparing a cancer-bearing model animal, wherein a mixture of a cancer stem cell obtained by cell fusion of a tumor cell and a blood cell and the tumor cell is used as an animal. There is provided a method comprising the step of administering to: The present invention also provides a cancer-bearing model animal that can be obtained by the above method.

Brief Description of Drawings

[0009] [Fig. 1] B16 tumor cells (alone) or a mixture of B16 cells and the cancer stem cells obtained in Example 1 of Example were implanted subcutaneously in mice, and the size of the formed tumors was measured over time. It is a figure showing the measurement results.

FIG. 2 shows a flow cytometric analysis of a tumor formed when a B16 tumor cell labeled with a red fluorescent dye is transplanted into a mouse that expresses GFP throughout the body. In the figure, (A) shows the result of confirming a cell group that simultaneously expresses DS-RED and GFP formed by cell fusion in vivo in the tumor (R2), and (B) shows the cell of this R2 The result of having analyzed the expression of the stem cell marker (C-Kit or CD133) is shown.

BEST MODE FOR CARRYING OUT THE INVENTION [0010] The method of the present invention is a method for producing cancer stem cells, and includes a step of cell fusion of tumor cells and blood cells in vitro (in vitro).

The type of tumor cell line to be fused with blood cells is not particularly limited, and includes, for example, any cancer cells of mammals other than humans (including cancer cells such as solid cancer and blood cancer).

) Can be used. For example, tumor cell lines (such as B16 cells that are mouse melanoma cell lines), sublines thereof, and primary tumor cells can be used. The tumor cell line may be introduced with a gene for expressing a fluorescent chromoprotein in order to facilitate cell fusion and subsequent confirmation of the behavior of cells derived from tumor cells. For this purpose, for example, a plasmid capable of expressing a red fluorescent chromoprotein (DS-RED) can be introduced. The gene transfer can be performed by a known method, and any method available to those skilled in the art, such as an electroporation method and a DEAE-DEXTRAN method, can be employed.

[0011] The type of blood cell is not particularly limited as long as it is a blood cell derived from an animal species that can be fused with a tumor cell. For example, blood cells derived from any blood or tissue such as peripheral blood or fetal liver may be used. For example, blood cells containing hematopoietic stem cells and Z or progenitor cells can be preferably used. For example, cells expressing CD11b containing hematopoietic stem cells as blood cells on the cell surface can be collected from the bone marrow of mammals. Blood cells can be collected by a person skilled in the art, such as a method using a cell sorter, a vaning method using an antibody, a magnetic cell recovery method using magnetic beads, or a method using an automatic fluorescent cell recovery device other than EPICS. Any method can be employed. In any case, blood cells are not limited to CD lib-positive cells including hematopoietic stem cells, monocytes, macrophages, and neutrophils, but are blood cells that can turn tumor cells into cancer stem cells. For example, you can use some cells.

[0012] In order to facilitate confirmation of cell fusion and subsequent behavior of cells derived from blood cells, blood cells may be introduced with a gene for expressing a fluorescent chromoprotein. For this purpose, it is preferable to use a gene that expresses a protein having a color tone different from that of a fluorescent chromoprotein expressed from a gene introduced into a tumor cell. For example, when a plasmid capable of expressing red fluorescent chromoprotein (DS-RED) is introduced into tumor cells, blood cells Blood cells that also control the bone marrow strength of mice expressing green 'fluorescent protein (GFP) or related proteins in the whole body (green mice) can be used, and fused cells can be easily identified. Is possible. However, for the above purpose, any means that can confirm DNA derived from tumor cells and blood cells can be adopted, for example, deletion or insertion into cell DNA. Cells that have been marked with may be used, or cells with the surface labeled with a different fluorescent dye may be used for short-term culture.

[0013] For example, cell fusion can be performed by simultaneously seeding and co-culturing about 10 ⁴ tumor cells and about 10 ⁶ blood cells, for example. The medium used for the culture is not particularly limited, and can be appropriately selected by those skilled in the art. For example, in addition to DMEM, RPΜΙ, αΜΕΜ, F12 medium and the like can be used. In culturing, a medium in which an appropriate concentration, for example, about 10% of urine serum is added to the basic medium, or a medium in which one or more appropriate cytodynamic ins are added can also be used. As the site force-in, for example, a force that can use stem cell factor, M-CSF, etc. The type and concentration are not limited to these, and those skilled in the art can appropriately select them. Co-culture for cell fusion can be performed, for example, at a temperature of about 37 ° C. in the presence of 5% CO for about 1 to 10 days.

2

A fusion cell of about 1 to 10%, preferably about 0.1 to several% is obtained. As described above, when tumor cells and Z or blood cells themselves have cell fusion ability, cell fusion can be performed by co-cultivation, but polyethylene glycol can be used without using the cell fusion ability of the cells themselves. Cell fusion can also be carried out by a general cell fusion method such as a fusion method using PEG or a cell fusion method using electric pulses. Furthermore, cell fusion is also possible by mechanically injecting cell nuclei extracted from blood cells into tumors, or conversely, injecting tumor cell nuclei into blood cells. When tumor cells are labeled with a red fluorescent dye and blood cells are labeled with a green fluorescent dye, the fused cells are stained yellow and can be separated using a cell sorter or the like. In this way, the cancer stem cells of the present invention can be isolated.

[0014] By using the cancer stem cells obtained as described above, a model tumor tissue can be formed in vivo or in vitro. As explained earlier, during tumor formation, cancer Stem cells and cancer stem cell strength Cancer cells that have been separated are considered to be in a mosaic pattern. The presence of cancer stem cells promotes, for example, the growth of tumor cells, or the cancer is engrafted in the individual to form cancer tissue. To produce a model tumor tissue that is closer to a real tumor tissue by mixing and culturing the fused cells that are stem cells and the tumor cells used to generate the fused cells, or transplanting the cell mixture to an animal. Is possible.

[0015] For example, the model tumor tissue is obtained by mixing and culturing the fusion cells, which are cancer stem cells obtained as described above, and the tumor cells used for fusion in a ratio of, for example, about 1:10. Can be generated in vitro. An appropriate medium as exemplified above can be used for the culture. In addition, for example, by transplanting a cell mixture containing a fusion cell that is a cancer stem cell and the tumor cell used for fusion at the above-mentioned ratio into a mammal other than a human, a tumor tissue can be formed at the transplant site. This animal can be used as a tumor model animal. Examples of animals include, but are not limited to, the ability to use mice, rats, rabbits, guinea pigs, monkeys, and the like. In general, it is desirable to select an animal species that can engraft the tumor cells used for fusion. When transplanting the above cell mixture into an animal, the cell mixture may be suspended in, for example, phosphate buffered saline (PBS), or a semi-solid medium such as collagen or matrigel, or other medium may be used. Oh ,. For example, when the cell mixture is implanted subcutaneously, the tumor tissue formation can be evaluated by measuring the size of the tumor over time, but the cell mixture is intravenously or intraperitoneally. When transplanted, tumors formed in the lungs, liver, etc. may be observed non-visually.

[0016] V, not bound by any particular theory, but tumor cells fused with blood cells are transformed into highly malignant cancer cells that increase the tumor, and the phenotype of the cells It can be concluded that the cancer stem cells have been transformed. This process occurs in vivo, and tumor cells generated by transformation in vivo become cancer stem cells by cell fusion with immature blood cells such as hematopoietic stem cells, and together with surrounding tumor cells, mosaic cells It is thought to contribute to the development of cancer by forming a tumorous tissue.

[0017] By using the cancer stem cells of the present invention, the cancer stem cell generation mechanism and the cancer stem It becomes possible to study the formation process of cancer involving cells in detail. In the cancer stem cell generation method of the present invention, cell fusion is performed in the presence of a test substance, and the degree of inhibition of cell fusion is confirmed, thereby screening a substance that inhibits the generation of cancer stem cells. It becomes possible to do. In addition, a substance that induces apoptosis of the cancer stem cell of the present invention can be screened. Substances screened in this way are expected to be useful as anti-cancer drugs. Furthermore, the model tumor tissue or animal model tumor provided by the present invention reproduces a malignancy closer to that of a real tumor than that of a tumor cell alone without cancer stem cells. Therefore, it is extremely useful for detailed elucidation of mechanisms such as cancer development and growth, or cancer metastasis, and is extremely useful for the development of antitumor agents effective in vivo.

Example

[0018] Example 1: Cell fusion of tumor cells and blood cells in vitro

B16 cells, which are mouse melanoma cell lines, were used as tumor cell lines to be fused with blood cells. B16 melanoma cells were subcultured in normal medium supplemented with 10% urine serum in DMEM (Sigma) medium. A plasmid capable of expressing red fluorescent chromoprotein (DS-RED) (Betaton's Dickinson) was introduced into B16 melanoma cells using lipofuctamine (Invitrogen). Separately, cells that express green 'fluorescent' protein (GFP) in the whole body (green mouse) bone marrow force CD lib (cell population including hematopoietic stem cells) on the cell surface are cell sorters (EPICS, Coulter) (Manufactured).

[0019] 10 ⁴ B16 tumor cells and 10 ⁶ CDllb-positive blood cells were seeded simultaneously in a 12 weU culture dish and stem cell factor (Gibconnet) made in DMEM basal medium containing 10% urine serum And M-CSF (manufactured by Gibconnet) were co-cultured using a medium to which final concentrations of 20 ng / ml and lOng / ml were added, respectively. When co-cultured at 37 ° C under 5% CO for 1-10 days, red and green

2

The presence of fused cells stained yellow by co-expressing fluorescent protein was observed, and the ratio was 0.1-1.0% of all cells. Fusion cells (DS-RED positive and GFP positive fusion cells) that generate red and green fluorescence simultaneously were separated using a cell sorter (EPICS) to obtain the cancer stem cells of the present invention. [0020] Example 2: Tumor formation model with cancer stem cells

In the field of tumor formation by cancer stem cells, cancer stem cells and cancer stem cells are considered to be mosaics. The cancer stem cells obtained in Example 1 actually have a positive effect on the occurrence of cancer, because the cancer stem cells are thought to engraft in the individual to form cancer tissue. This was confirmed by the following experiment. A mixture of B16 tumor cells 1.1 x 10 ⁴ (B16 alone) or B16 tumor cells 1.0 x 10 ⁴ and cancer stem cells 0.1 x 10 ⁴ obtained in Example 1 was suspended in PBS, respectively, and C57B1 / 6 strain mice were administered and transplanted under the back skin. Tumor formation was evaluated by measuring tumor size over time. The results are shown in Fig. 1. Tumor formation was not observed in individuals transplanted with 1.1 x 10 ⁴ B16 tumor cells, whereas individuals transplanted with the same amount of B16 tumor cells mixed with 0.1 x 10 ⁴ cancer stem cells. Significant tumor formation was observed, and it was confirmed that tumor cells including cancer stem cells changed to a cell population with high tumorigenicity.

[0021] In addition, 1 × 10 ⁶ B16 tumor cells fluorescently labeled with DS-RED obtained in Example 1 (this number of cells is the number of cells that can form tumors alone) in PBS. Suspended, transplanted into C57B1 / 6 lineage green mouse, and tumor cells that simultaneously express GFP and DS-RED in the formed tumor (cells derived from the host green mouse and tumor cells fused together) We examined whether or not there exists. 14 days after transplantation of B16 tumor cells expressing DS-RED, the tumor was removed from the mouse, and the tumor tissue was minced with surgical scissors, and then disperse (Boehringer) was added to the minced tumor tissue. The cells were allowed to stand at room temperature for 10 minutes, and then the cell suspension was filled into a syringe equipped with a syringe needle, and the cells were dispersed by repeating aspiration and discharge several times. These cells were analyzed by flow cytometry. As a result, as shown in FIG. 2 (A), the presence of fusion cells expressing both DS-RED and GFP was recognized. In addition, when this fused cell was stained with a monoclonal antibody against c-Kit and CD133 (both manufactured by Pharmingen) and analyzed by flow cytometry, c-Kit was almost 100% and CD133 was 50-60% was found to be expressed in the fused cells (Fig. 2 (B)). Since c-Kit and AC133 are known to be expressed in immature stem cell levels, it was confirmed that the cancer stem cell generation process obtained in Example 1 occurred in the actual tumor formation process. did it. Industrial applicability

According to the present invention, cancer stem cells can be efficiently generated with high reproducibility in vitro. Using these cancer stem cells, a model tumor tissue that is closer to the actual tumor tissue can be generated in vitro, or a tumor-bearing model animal that reproduces the tumor development process in vivo can be created.

Claims

The scope of the claims

[1] A method for producing cancer stem cells, comprising the step of in vitro cell fusion of tumor cells and blood cells.

[2] The method according to [1], wherein the cell fusion is performed by co-culturing blood cells containing hematopoietic stem cells and Z or progenitor cells with tumor cells.

[3] A cancer stem cell obtainable by the method according to claim 1.

[4] A method for producing a model tumor tissue, comprising a step of culturing a cancer stem cell obtained by cell fusion of tumor cells and blood cells and a mixture of the tumor cells.

[5] A method for preparing a cancer-bearing model animal, comprising a step of administering a cancer stem cell obtained by cell fusion of tumor cells and blood cells and a mixture of the tumor cells to a mammal other than a human. Including methods.

[6] A cancer-bearing model animal obtainable by the method according to claim 5.