HK1185910A

HK1185910A - Cancer stem cell mass and process for production thereof

Info

Publication number: HK1185910A
Application number: HK13113120.1A
Authority: HK
Inventors: 山崎达美; 冈部尚文; 小林慎太; 陈玉昭; 加藤淳彦; 铃木雅实; 松原亨一
Original assignee: Chugai Seiyaku Kabushiki Kaisha
Priority date: 2010-10-06
Filing date: 2011-10-06
Publication date: 2014-02-28

Description

Cancer stem cell population and method for preparing same

Technical Field

The present invention relates to a cancer stem cell population substantially free of cells having no carcinogenic potential and having a characteristic of reproducing a hierarchical structure of a cancer tissue, and a method for producing the cancer stem cell population. The present invention also relates to a method for searching for a target molecule of a drug, a method for evaluating a drug, or a method for screening a drug, using a non-human animal model into which the cancer stem cell population is transplanted or a culture system for the cancer stem cell population under in vitro conditions.

Background

Conventionally, cancer having a hierarchical structure is considered to be cancer cells in various differentiation stages that self-replicate to form a large tumor mass. However, in recent years, it has become clear that cancer is formed from limited early-stage differentiated cancer cells having both self-replication ability and multi-differentiation ability. This is called a cancer stem cell model, and the presence of cancer stem cells has been reported in various cancers such as hematological cancer, brain tumor, breast cancer, and colorectal cancer. In cancer stem cell models, it is proposed that the cancer be composed of heterogeneous populations of differentiated cells, and only limited cells, i.e., cancer stem cells, have the ability to form new cancers.

On the other hand, cancer-forming cells such as CML and hematological cancer, which are formed from a monoclonal cell population and do not have a hierarchical structure, or epithelial poorly differentiated cancer are also called cancer stem cells, but they have no hierarchical structure-forming ability (multi-differentiation ability) although they have a self-replication ability, and therefore they deviate from the above-mentioned cancer stem cell model and cause confusion about the definition of cancer stem cells. For example, in 2008, Quintana et al reported that almost all human melanoma cells have tumorigenicity by experiments using hyper-immune deficient mice lacking B cells, T cells, and NK cells (non-patent document 1), but these cells should not be included in cancer stem cells because they do not have a multipotentiality and should be referred to as only cancer forming cells.

Hitherto, the isolation and concentration of cancer stem cells forming a hierarchical structure have been performed by either flow cytometry using a cancer stem cell marker such as CD133 or CD44, or by a method in which cancer cells are cultured in suspension using a stem cell culture medium containing FGF, EGF, or the like. The flow cytometry method damages cells in the procedure, and CD133 or CD44 used as a cancer stem cell marker is not a surface marker specific to cancer stem cells, and therefore, there is still a problem as a method for preparing undamaged cancer stem cells at high purity. In fact, it is known that, in a cancer stem cell population collected from a cancer having a hierarchical structure by a flow cytometry method, if the carcinogenesis ability is evaluated by limiting dilution, the frequency of cancer stem cells is only about 1/262, and a large number of cells other than cancer stem cells are included (non-patent document 2).

In addition, there are reports that: spheroids (cell masses) formed by suspension culture contain cancer stem cells forming cancers with a hierarchical structure, but the cell group constituting the spheroids is heterogeneous, the frequency of cancer stem cells is about 1/240, and a large number of cells other than cancer stem cells coexist (non-patent document 3). Therefore, since the purity of cancer stem cells is low, it is difficult to clarify the properties of cancer stem cells having a hierarchical structure using these cell populations. Further, a method of concentrating cancer stem cells by repeating passage of transplanted human cancer tissues into immunodeficient animals has been reported (non-patent document 4), but the frequency of cancer stem cells most concentrated in pancreatic cancer by this method is about 1/180.

In addition, the following examples are reported: using p75NTR of stem cell marker, p75NTR positive cell is separated from cell strain immortalized by introducing human papilloma virus oncogene into human uterine cervical epithelial cell, and TGF is contained in the cell strainβAnd TNFαHowever, these cells do not form a hierarchical structure and are cancer-forming cells that do not belong to the category of cancer stem cells (patent document 1).

Therefore, a method for producing a cancer stem cell having a hierarchical structure in a high purity and in a large amount has not been known so far, and there is a strong demand for the development thereof.

Documents of the prior art

Patent document

Patent document 1: japanese patent laid-open No. 2008-182912;

non-patent document

Non-patent document 1: quintana E. et al, Nature, 2008, 12/4, 456(7222) 593-8;

non-patent document 2: o' Brien CA. et al, Nature, 2007, 1, 4, 445(7123) 106-10;

non-patent document 3: vermeulen L, et al, Nat Cell biol. 5 month 2010, (12) (5) 468-76;

non-patent document 4: ishizawa K, et al, Cell Stem Cell, 9/3/2010, 7(3) 279-82.

Disclosure of Invention

Problems to be solved by the invention

The present invention addresses the problem of providing a cancer stem cell population that is substantially depleted of cells that are not capable of forming cancer and that has the characteristic of reproducing the hierarchical structure of cancer tissues. Another object of the present invention is to provide a method for producing a cancer stem cell population from which cells having no cancer-forming ability have been substantially removed, the method comprising the step of adherently culturing a cell population containing cancer stem cells. Another object of the present invention is to provide a method for searching for a target molecule of a drug, a method for evaluating a drug, or a method for screening a drug, using a non-human animal model into which the cancer stem cell population is transplanted or a culture system of the cancer stem cell population under in vitro conditions.

Means for solving the problems

The present inventors have conducted intensive studies in order to solve the above problems.

The present inventors established cancer cell lines using hyperimmune deficient mice and compared and analyzed cancers that have a hierarchical structure and cancers that do not have a hierarchical structure using the established cell lines. Furthermore, in cancers having a hierarchical structure that belongs to a cancer stem cell model, since there is no method of separating, concentrating, homogenizing, or culturing the cancer stem cells in large quantities, analysis, screening of a drug using the cancer stem cells, or the like is hindered, and the present inventors have attempted to solve this problem.

The present inventors have transplanted human cancer tissues into NOD/SCID/γ cells that do not have functional T cells, B cells, or natural killer cells_c ^nullMouse (Fujii E. et al, Pathol int 2008; 58: 559-^nullMouse) referred to as "NOG mouse" in the present application), a plurality of human cancer cell lines were established. Since these strains still constructed cancer tissues having the same morphology as the original cancer tissues even when they were subcultured repeatedly in NOG mice, it was considered that human cancer stem cells could be preserved in the cell population. Therefore, human cancer stem cells that can be passaged through NOG mice are very useful research tools.

The present inventors have compared various culture methods after separating cancer cells by repeatedly growing human cancer tissues using NOG mice. As a result, a cancer stem cell composition which is homogeneous and in which cells having carcinogenicity and cells having no carcinogenesis do not substantially coexist was successfully obtained by an adherent culture method using a stem cell culture medium containing no serum, not by a suspension culture method which is generally employed, and the present invention was completed.

The cancer stem cells obtained by the method can be stably maintained by subculture adherent culture using a serum-free stem cell medium, and do not show phenotypic change even after 1 month or more. This cell expresses various large intestine cancer stem cell markers (CD 133, CD44, EpCAM, CD166, CD24, CD26, and CD 29) reported so far, and shows carcinogenesis at a frequency of almost 100%, reconstructing a tumor having the same histopathological characteristics (hierarchical structure) as the original primary tumor. The cells were characterized by exhibiting high proliferation under adherent culture conditions and being positive for Lgr5, a cell surface marker. Moreover, highly proliferative and Lgr 5-positive cancer stem cells have been shown to play an important role in cancer metastasis because tumor masses are formed in organs such as the lung and liver when injected intravenously from a mouse tail.

On the other hand, by subjecting cancer stem cells showing high proliferation under adherent culture conditions and positive for Lgr5 as a cell surface marker to suspension culture or treatment with an anticancer drug such as irinotecan or 5-FU, it is possible to isolate low-proliferation cancer stem cells negative for Lgr 5. The cells also show high carcinogenic potential. And, it shows: by isolating cancer stem cells that are low in proliferation and negative for Lgr5 and culturing again under adherent culture conditions, the cancer stem cells are changed to highly proliferative ones that are positive for Lgr 5. This shows that: cancer stem cells that are highly proliferative and positive for Lgr5 and cancer stem cells that are less proliferative and negative for Lgr5 can be transformed into each other, and have a self-alternating function.

More specifically, the present invention provides the following [ 1] to [ 33 ].

A cancer stem cell population which is substantially depleted of cells that do not have carcinogenic potential and which has the characteristic of reproducing the hierarchical structure of cancer tissues.

[ 2] the cancer stem cell population according to [ 1], wherein the cancer stem cells are derived from human tumor tissue.

The cancer stem cell population according to [ 3] or [ 2], wherein the human tumor tissue is a tumor tissue derived from an epithelial cancer.

The cancer stem cell population according to [ 4] or [ 3], wherein the epithelial cancer is pancreatic cancer, prostate cancer, breast cancer, skin cancer, cancer of the digestive tract, lung cancer, hepatocellular cancer, cervical cancer, uterine body cancer, ovarian cancer, fallopian tube cancer, vaginal cancer, liver cancer, bile duct cancer, bladder cancer, ureteral cancer, thyroid cancer, adrenal cancer, kidney cancer, or cancer of other glandular tissues.

The cancer stem cell population according to any one of [ 5] to [ 1] to [ 4], wherein the cancer stem cell population is substantially homogeneous.

The cancer stem cell population according to any one of [ 6] to [ 1] to [ 5], wherein the frequency of cancer stem cells in limiting dilution analysis is 1/20 or more.

The cancer stem cell population according to any one of [ 1] to [ 6] of [ 7], which comprises 1x10⁴More than one cancer stem cell.

The cancer stem cell population according to any one of [ 8] [ 1] to [ 7], which is prepared by a method comprising the step of adherently culturing a cell population containing cancer stem cells.

The cancer stem cell population according to any one of [ 1] to [ 8] of [ 9], which is prepared by a method comprising the following steps (1) to (3):

(1) a step of preparing a cancer cell mass by transplanting a cell population containing cancer stem cells into a non-human animal belonging to the same or different species;

(2) a step of subdividing the prepared cancer cell mass; and

(3) a step of performing adherent culture of the cell population obtained by the step (2) using a stem cell culture medium.

The cancer stem cell population according to any one of [ 10] [ 1] to [ 9], wherein the non-human animal is any one of a nude mouse, a SCID mouse, a NOD-SCID mouse, a NOG mouse, or a nude rat.

A method for preparing a cancer stem cell population substantially depleted of non-carcinogenic cells, comprising: a step of culturing a cell population comprising cancer stem cells adherently.

The method according to [ 12] or [ 11], wherein the cell population containing cancer stem cells is a cell population having a reconstructed cancer tissue grade structure.

[ 13] the method according to [ 12], wherein the cell population that reproduces a cancer tissue-grade structure is a cancer cell line or spheroid established in a non-human animal, or a cell positive for at least one marker selected from the group consisting of cancer stem cell markers CD24, CD29, CD34, CD44, CD49f, CD56, CD90, CD117, CD133, CD135, CD166, CD184, CD271, CD326, Aldefluor, ABCG2, ABCG5, LGR5 and Msi 1.

The method according to any one of [ 14] to [ 11] to [ 13], wherein the population of cells containing cancer stem cells is proliferated before adherent culture.

The method according to [ 15] or [ 14], which comprises proliferating a cell population containing cancer stem cells by spheroid culture.

[ 16] the method according to [ 14], which comprises proliferating a cell population by transplanting it into a non-human animal and passaging it.

The method according to any one of [ 11] to [ 16] of [ 17], wherein the cancer stem cells are derived from human tumor tissue.

The method according to [ 18 ] to [ 17], wherein the human tumor tissue is a tumor tissue derived from an epithelial cancer.

The method according to [ 19] or [ 18 ], wherein the epithelial cancer is cancer of pancreatic cancer, prostate cancer, breast cancer, skin cancer, digestive tract cancer, lung cancer, hepatocellular carcinoma, cervical cancer, uterine body cancer, ovarian cancer, fallopian tube cancer, vaginal cancer, liver cancer, bile duct cancer, bladder cancer, ureter cancer, thyroid cancer, adrenal cancer, kidney cancer, or other glandular tissues.

The method according to any one of [ 20] to [ 11] to [ 19], wherein the non-human animal is any one of a nude mouse, a SCID mouse, a NOD-SCID mouse, a NOG mouse, or a nude rat.

A method for searching for a target molecule of a drug, which is characterized in that a hierarchical structure formed by cancer stem cells, a cancer progression process from cancer stem cells, or a biological characteristic of cancer stem cells is evaluated using as an index the non-human animal model into which a cancer stem cell population according to any one of [ 1] to [ 10] is transplanted or a culture system of the cancer stem cell population under in vitro conditions.

[ 22] the method for searching for a target molecule of a drug according to [ 21 ], which comprises the steps of (1) to (4) below:

(1) a step of preparing a non-human animal model by transplanting the cancer stem cell population according to any one of [ 1] to [ 10] into a non-human animal;

(2) a step of collecting a tissue piece showing a tissue structure characteristically seen in the cancer progression process of the cancer stem cell population or showing a biological property thereof;

(3) a step of examining the expression of DNA, RNA, protein, peptide or metabolite in the tissue piece collected in (2); and

(4) a step of identifying DNA, RNA, protein, peptide or metabolite in the tissue piece that is altered depending on the hierarchical structure formed by the cancer stem cells, the cancer progression process starting from the cancer stem cells, or the biological characteristics of the cancer stem cells.

The method for searching for a target molecule of a drug according to [ 23] or [ 21 ], which comprises the steps of (1) to (3) below:

(1) culturing the cancer stem cell population according to any one of [ 1] to [ 10] under in vitro conditions, and reconstructing a characteristic structure of a cancer progression process from the cancer stem cells or a biological characteristic of the cancer stem cells;

(2) a step of studying the expression of DNA, RNA, protein, peptide or metabolite of the cultured cells reproducing the characteristic structure; and

(3) identifying DNA, RNA, proteins, peptides and metabolites in the cultured cells that vary depending on the hierarchical structure formed by the cancer stem cells, the progress of cancer from the cancer stem cells, or the biological properties of the cancer stem cells.

The method according to any one of [ 24 ] [ 21 ] to [ 23], wherein the drug is an anticancer drug.

The method according to any one of [ 25 ] to [ 21 ] to [ 24 ], wherein the target molecule is a cancer cell marker.

A method for evaluating a drug, which comprises evaluating a grade structure formed by cancer stem cells, a cancer progression process from cancer stem cells, or a biological property of cancer stem cells in a non-human animal model into which a cancer stem cell population according to any one of [ 1] to [ 10] has been transplanted or in a culture system in which the cancer stem cell population is cultured under in vitro conditions.

The method for evaluating a drug according to [ 27] or [ 26 ], which comprises the steps of (1) to (5) below:

(2) administering a test substance to the non-human animal model of (1);

(3) a step of collecting a tissue piece showing a tissue structure characteristically seen in a cancer progression process starting from cancer stem cells or showing a biological property thereof;

(4) observing a change with time, a cancer progression process, or a biological property thereof of the cancer stem cells in the tissue piece; and

(5) identifying the formation of a hierarchical structure formed by the cancer stem cells, the progression of cancer from the cancer stem cells, or the biological properties of the cancer stem cells, which are inhibited by the test substance.

The method for evaluating a drug according to [ 28] or [ 26 ], which comprises the steps of (1) to (4) below:

(2) treating the cultured cells of (1) with a test substance;

(3) a step of observing a change in the hierarchical structure formed by the cancer stem cells, a cancer progression process starting from the cancer stem cells, or a biological property of the cancer stem cells; and

(4) identifying the formation of a hierarchical structure formed by the cancer stem cells, the progression of cancer from the cancer stem cells, or the biological properties of the cancer stem cells, which are inhibited by the test substance.

A method for screening a drug characterized in that a grade structure formed by cancer stem cells, a cancer progression process from cancer stem cells, or a biological property of cancer stem cells is evaluated as an index in a non-human animal model into which a cancer stem cell population according to any one of [ 1] to [ 10] has been transplanted or in a culture system of the cancer stem cell population under in vitro conditions.

[ 30 ] the method for screening a drug according to [ 29 ], which comprises the steps of (1) to (5) below:

(2) administering a test substance to the non-human animal model of (1);

(5) a step of identifying a test substance that inhibits the formation of a hierarchical structure formed by a specific cancer stem cell, the progression of cancer starting from a cancer stem cell, or a biological property of a cancer stem cell.

The method for screening a drug according to [ 31] or [ 29 ], which comprises the steps of (1) to (4) below:

(2) treating the cultured cells of (1) with a test substance;

(4) a step of identifying a test substance that inhibits the formation of a hierarchical structure formed by a specific cancer stem cell, the progression of cancer starting from a cancer stem cell, or a biological property of a cancer stem cell.

The method according to any one of [ 32 ] [ 26 ] to [ 31], wherein the drug is an anticancer drug.

[ 33] [ 21 ], [ 23], [ 26 ], [ 28], [ 29 ] and [ 31], wherein the culture system under in vitro conditions is a culture of spheroids.

Drawings

FIG. 1 shows photographs of tissue specimens stained by HE. The morphological structures of the colon cancer strains PLR123, PLR59, and PLR325 transplanted into mice were similar to those of human tissues, and were shown to be unaffected by mice. On the other hand, in the colon cancer strain PLR357 transplanted into NOG mice, morphological changes were observed, and it was revealed that it was not suitable for the transplantation experiments into mice.

FIG. 2 is a graph showing the results of flow cytometry analysis of mouse MHC class I negative and 7-AAD Viability Dye (7-AAD Viability Dye) negative cells by EPICS ALTRA using a cancer stem cell marker. Among cancer cell masses formed from colorectal cancer strain PLR123, colorectal cancer strain PLR59, and colorectal cancer strain PLR325, positive cancer stem cell markers were observed. In the differentiated colon cancer strains PLR123 and PLR59, marker-positive cells and marker-negative cells coexisted and were heterogeneous cell populations. On the other hand, in the poorly differentiated colon cancer strain PLR325, all cells were positive to EpCAM, AC133 and Aldefluor, and all cells were negative to CD 44.

FIG. 3 is a graph showing the results of flow cytometry analysis of cells from which mouse cells were removed, by EPICS ALTRA. It was revealed that 95% or more of the cancer cell masses formed by the colorectal cancer strains PLR123, PLR59 and PLR325 were mouse MHC class I negative in mice after removal of mouse cells.

FIG. 4 shows photographs of tissue specimens stained by HE. The hierarchical structure was observed in the differentiated colon cancer strain PLR123 and colon cancer strain PLR 59. On the other hand, no hierarchical structure was observed in the poorly differentiated colon cancer strain PLR 325.

FIG. 5 is a graph showing the expression of LGR5 protein detected by Western blotting. LGR5 protein was detected in the differentiated colon cancer strain PLR123 and colon cancer strain PLR59, indicating the presence of cells positive for the normal intestinal stem cell marker. On the other hand, LGR5 protein was not detected in the poorly differentiated colon cancer strain PLR 325.

FIG. 6 is a graph showing the results of flow cytometry analysis of 7-AAD viability dye-negative cells using cancer stem cell markers by EPICS ALTRA. The commercially available colon cancer strain HCT116 is a cell population showing homogeneity that almost all cells are positive for a stem cell marker.

FIG. 7 shows photographs of tissue specimens stained by HE. No hierarchical structure was found in the commercial colon cancer strain HCT 116.

FIG. 8 is a graph showing the expression of LGR5 protein detected by Western blotting. LGR5 protein was not detected in the commercial colorectal cancer strain HCT 116.

FIG. 9 shows photographs of LGR5 positive cells detected by in situ hybridization. LGR 5-positive cells were not found in the commercial colorectal cancer strain HCT 116.

FIG. 10 is a photograph showing the morphology of a differentiated colon cancer strain PLR123 cultured in vitro. Only in the suspended state, the formation of a cell mass called spheroids is visible.

FIG. 11 is a graph showing the results of flow cytometry analysis of 7-AAD viability dye-negative cells using cancer stem cell markers by EPICS ALTRA. In differentiated colorectal cancer strains PLR123 and PLR59 cultured adherent in vitro, all cells were homogeneous and positive for cancer stem cell markers.

FIG. 12 is a graph showing the expression of LGR5 protein detected by Western blotting. An increase in LGR5 protein was detected in a differentiated colon cancer cell line cultured adherently in vitro in stem cell medium. Indicating that the cells positive for normal intestinal stem cell markers were concentrated by in vitro culture with stem cell culture medium.

FIG. 13 shows photographs of LGR5 positive cells detected by in situ hybridization. It can be seen that all of the well-differentiated colon cancer strains PLR123 cultured adherently in vitro in stem cell culture medium were positive for the LGR5 probe. It was shown that by in vitro culture with stem cell medium, cells positive for the normal intestinal stem cell marker were concentrated, which showed that all cells were homogeneous cells positive for the normal intestinal stem cell marker LGR 5.

FIG. 14 shows photographs of tissue specimens stained by HE. The same hierarchical structure as that of human tissues and NOG established cancer cell lines was observed in cancer cell masses consisting of differentiated colorectal cancer strains PLR123 and PLR59 cultured by in vitro adherent culture of 10 cells. In addition, the same hierarchical structure as that of human tissues and NOG established cancer cell lines was observed in cancer cell masses formed from a differentiated colorectal cancer strain PLR123 cultured by in vitro adherent culture for 1 month or more. It was revealed that the differentiated colon cancer strains PLR123 and PLR59 obtained by adherent culture in vitro all have cancer stem cells with multi-differentiation potential.

FIG. 15 is a graph showing the results of flow cytometry analysis of mouse MHC class I negative and 7-AAD viability dye negative cells by EPICS ALTRA using a cancer stem cell marker. Among cancer cell masses formed by differentiated colorectal cancer strain PLR123 and colorectal cancer strain PLR59 cultured by in vitro adherent culture of 10 cells, cells negative for cancer stem cell markers were observed, and it was shown that differentiated cells not having cancer-forming ability were produced from cancer stem cells.

FIG. 16 shows photographs of HE-stained tissue specimens. The same hierarchical structure as that of human tissues and original colon cancer strains was observed in the second-generation cancer cell masses formed from the differentiated colon cancer strain PLR123 cultured adherent in vitro.

FIG. 17 shows the frequency of formation of cancer cell masses by 100 or less cells in the cancer stem cell population of the present invention and the literature. For this cancer stem cell population, the formation and hierarchical structure of the cancer was seen in all transplants.

[ FIG. 18 ]]It was revealed that in DG44 cells transfected with Lgr4, 5 or 6 cDNA, the specificity of anti-human Lgr5 monoclonal antibodies (mAbs), i.e., 2U2E-2 and 2T15E-2, was confirmed by immunofluorescence microscopyPhotographs of sexual results. Fixing of non-transfected mother cells and transfectants with 5μg/mL antibody treatment. Strong fluorescence (green signal on the right) was observed in cells containing Lgr5 cDNA, but not in mother cells and cells containing Lgr4 or Lgr6 cDNA. Scale bar is 5μm。

FIG. 19 shows the results of flow cytometry to confirm the specificity of 2T15E-2, which is an anti-human Lgr5 monoclonal antibody (mAb), in DG44 cells transfected with Lgr4, 5, or 6 cDNA. Non-transfected progenitor cells and transfectants were co-incubated with monoclonal 2T15E-2 antibody and analyzed by FACS. The 2T15E-2 antibody reacted with cells containing Lgr5 cDNA, but not with the mother cells and cells containing Lgr4 or Lgr6 cDNA. Regarding the expression of Lgr4, Lgr5, and Lgr6 of the transfectants, it was confirmed by western blot analysis.

[ FIG. 20] is a photograph showing the results of Western blot analysis of primary cells of PLR123 cells, β -catenin, TCF1, TCF3, TCF4 and phosphorylated c-JUN protein of suspension cancer stem cells and adherent cancer stem cells. In adherent cancer stem cells positive for Lgr5, the expression of all proteins is upregulated compared to primary cells. GAPDH was also visualized as a reference protein for protein loading.

[ FIG. 21 ]]Photographs showing the histopathological results of xenograft tumors derived from 1 and 10 Lgr5 positive cells from PLR123 and 10 Lgr5 negative cells from PLR 123. All tumors showed hierarchical structure, while their histopathological features were very similar to the original tumors. Scale bar 100μm。

FIG. 22 is a graph showing the results of flow cytometry analysis of reported cancer stem cell markers by culturing adherent cancer stem cells from PLR59 and PLR123 xenografts for 1 month. Adherent cancer stem cells from PLR59 and PLR123 were positive for all cancer stem cell markers that had been reported, even after 1 month of in vitro culture. Gray shows the fluorescence intensity or ALDH activity after staining cells with the indicated antibodies, white shows the fluorescence intensity after staining cells with control isotype antibodies or treating cells for ALDH activity by ALDH inhibitors.

FIG. 23 adherent cancer stem cells from PLR59 and PLR123 xenografts were cultured for 1 month, analyzed by flow cytometry (FIG. 22), and injected into NOG mice. The tumor-forming activity of NOG mice was studied by injecting subcutaneously the number of adherent cancer stem cells shown in the figure into the flank of the NOG mice. The figure shows the results of tumor formation judged 47 days after inoculation. Even by injecting 10 adherent cancer stem cells subcutaneously, tumors were generated at all injection sites, and the histopathological morphology of the tumors was highly similar to the original tumors.

[ FIG. 24 ]]Showing primary cells, suspension cancer stem cells and adherent cancer stem cells of PLR59 cellsβPhotographs of the results of Western blot analysis of catenin, TCF1, TCF3, TCF4 and phosphorylated c-JUN protein. Expression of all proteins is upregulated in Lgr5 positive adherent cancer stem cells compared to primary cells. GAPDH as a reference for protein loading was also visualized.

[ FIG. 25 ]]Showed in PLR123 cells by FH535 (50)μM) and cardamomin (50)μM) inhibition of proliferation of Lgr5 positive adherent cancer stem cells. The number of viable cells after 3 days of co-culture with FH535 (grey bars) and cardamonin (black bars) is shown as a percentage relative to the number of DMSO alone (white bars). Results are the average of 3 experiments. The bars at the top of each bar show the standard deviation.

[ FIG. 26 ]]Showed in PLR59 cells by FH535 (50)μM) and cardamomin (50)μM) inhibition of proliferation of Lgr5 positive adherent cancer stem cells. The number of viable cells after 3 days of co-culture with FH535 (grey bars) and cardamonin (black bars) is shown as a percentage relative to the number on day 0 (white bars).

FIG. 27 shows a graph of cell proliferation of PLR123 cells in the presence or absence of EGF and FGF. Adherent cancer stem cells were cultured for 3 days in the presence or absence (black bars). The number of viable cells is shown as a percentage relative to the number on day 0 (white bars). Results are the average of 3 experiments. The bars at the top of each bar show the standard deviation.

FIG. 28 is a graph showing cell proliferation of PLR59 cells in the presence and absence of EGF and FGF. Adherent cancer stem cells were cultured for 3 days in the presence or absence (black bars). The number of viable cells is shown as a percentage relative to the number on day 0 (white bar).

[ FIG. 29 ]]Shows the effect of chemotherapeutic agents on proliferation of adherent cancer stem cells positive for Lgr5 and suspended cancer stem cells negative for Lgr5 in PLR123 cells. Through 5-FU (10)μg/mL, gray column) or irinotecan (10)μg/mL, black bars) the number of viable cells after treatment is shown as a percentage of the number of viable cells cultured without chemotherapeutic agents (white bars). Results are the average of 3 experiments. The bars at the top of each bar show the standard deviation.

[ FIG. 30 ]]5-FU (10) in PLR59 cellsμg/mL) and irinotecan (10)μg/mL) on proliferation of adherent cancer stem cells positive for Lgr5 and suspension cancer stem cells negative for Lgr 5. The number of viable cells after treatment with 5-FU (grey bar) or irinotecan (black bar) is shown as a percentage relative to the case of culture in the absence of the above drug (white bar).

[ FIG. 31] is a graph showing changes in expression of Lgr5 in adherent cancer stem cells of PLR123 cells after the cells were treated with a chemotherapeutic agent. The results of flow cytometry are shown. The upper panel shows no chemotherapeutic agent (control), the middle 5-FU treated cells, and the lower panel shows irinotecan treated cells. The gray color shows the fluorescence intensity or ALDH activity after staining the cells with the antibody, and the white color shows the fluorescence intensity or ALDH activity under an ALDH inhibitor after staining the cells with the control isotype antibody.

[ FIG. 32 ]]Show that the treatment is carried out by culture conditions and chemotherapyPhotographs showing the change in phenotype of agent-treated large intestine cancer stem cells. Sensitivity of Lgr5 positive cancer stem cells to 5-FU and irinotecan was studied. Both 5-FU and irinotecan significantly inhibited the proliferation of cancer stem cells positive for Lgr 5. After 3 days of exposure to 5-FU or irinotecan, cells resistant to these chemotherapeutic agents appeared. The drug-resistant cells are aggregated in high density in morphology. Scale bar is 25μm。

FIG. 33 is a graph showing the results of flow cytometry analysis of cancer stem cell markers by 5-FU or irinotecan treatment of adherent cancer stem cells of PLR59 cells. The upper part shows 5-FU treated cells and the lower part shows irinotecan treated cells. The gray color shows the fluorescence intensity or ALDH activity after staining the cells with the antibody, and the white color shows the fluorescence intensity or ALDH activity under an ALDH inhibitor after staining the cells with the control isotype antibody.

FIG. 34 shows graphs showing the levels of Lgr5 mRNA of cells before (shown as 1) and after transfer of adherent and suspension cultures of PLR123 cells. F → A shows the transition from suspension culture to adherent culture, and A → F shows the transition from adherent culture to suspension culture. Results are the average of 3 experiments. The bars at the top of each bar show the standard deviation.

[ FIG. 35 ]]Photographs showing the mutual change in morphology of cancer stem cells. When Lgr 5-negative large intestine cancer stem cells were dissociated and cultured on a flat-bottom culture plate, some of the cells adhered to the bottom of the plate and became positive for Lgr5, showing a mesenchymal-cell-like morphology (left side). On the other hand, when Lgr 5-positive adherent large intestine cancer stem cells were cultured on an ultra-low attachment culture plate, some of the cells stopped growing, and a spheroid-like structure was formed. Scale bar is 10μm。

FIG. 36 is a photograph showing the results of Western blot analysis of E-cadherin and Snail protein (Snail) in PLR123 cells, Lgr 5-negative suspension cancer stem cells and Lgr 5-positive adherent cancer stem cells. Suspension cancer stem cells express high levels of E-cadherin, while adherent cancer stem cells express high levels of snail protein. GADPH was used as a loading control.

[ FIG. 37 ] A]Shown in PLR123 cells by E-cadherin antibodies, glusulin antibodies andβphotograph of immunocytochemistry results of legr 5-negative suspension cancer stem cells and legr 5-positive adherent cancer stem cells by catenin antibody. Suspending cancer stem cell to highly express E-cadherin on cell surfaceβEpithelial-like cells of catenin, whereas adherent cancer stem cells are mesenchymal-like cells with snail protein and β -catenin localized in the nucleus. Scale bar is 25μm。

FIG. 38 shows the results of Western blot analysis of E-cadherin and glusulin of Lgr 5-negative suspension cancer stem cells and Lgr 5-positive adherent cancer stem cells in PLR59 cells. Suspension cancer stem cells express high levels of E-cadherin, while adherent cancer stem cells express high levels of snail protein. GADPH was used as a loading control.

[ FIG. 39 ]]Photographs showing the results of immunohistochemistry of xenograft tissues (from PLR123 cells) against Lgr5 antibody and snail protein antibody. Simultaneous expression of nuclear glusulin and cytoplasmic Lgr5 was detected in EMT-like cells in the budding region (left panel), but not in the tubes (right panel). Arrows show Lgr5 positive budding cells. Scale bar is 10μm。

[ FIG. 40 ]]Graph showing tumor forming activity of adherent cancer stem cells of multiple organs. Will be 5X 10⁵Individual adherent cancer stem cells of PLR123 were injected into the tail vein (n = 5). The incidence of tumor formation was shown at day 40 after administration of various organs.

[ FIG. 41]Photographs showing the results of histopathological experiments on tumors of lung, liver, lymph nodes and subcutaneous tissues. In the lung, tumor cells show undifferentiated tumor nests, while in the liver and other organs, tumor cells show tubular structures with multiple stages of differentiation. Scale bar 100μm。

Detailed Description

The present invention relates to a cancer stem cell population that can be effectively used in the development of a drug for the treatment or prevention of human cancer diseases. In particular, it relates to a cancer stem cell population substantially depleted of cells without carcinogenesis, said cancer stem cell population being characterized by a reproducible cancer tissue hierarchy.

The origin of the cancer stem cell population of the present invention is not particularly limited, but is preferably derived from human tumor tissue.

As a first aspect of the present invention, there is provided a cancer stem cell population substantially depleted of cells that are not carcinogenic, the population reproducing the hierarchical structure of cancer tissue.

In the present invention, "cancer" refers to a physiological state of a mammal, or a related physiological state, generally characterized by uncontrolled cellular proliferation. In the present invention, the type of cancer is not particularly limited, and the following cancers may be mentioned: examples of the carcinoma (epithelial cancer) include pancreatic cancer, prostate cancer, breast cancer, skin cancer, cancer of the digestive tract, lung cancer, hepatocellular cancer, cervical cancer, uterine body cancer, ovarian cancer, fallopian tube cancer, vaginal cancer, liver cancer, bile duct cancer, bladder cancer, ureter cancer, thyroid cancer, adrenal cancer, kidney cancer, and other cancers of glandular tissues. As sarcomas (non-epithelial carcinoma) there may be mentioned liposarcoma, leiomyosarcoma, rhabdomyosarcoma, synovial sarcoma, angiosarcoma, fibrosarcoma, malignant peripheral nerve tumor, gastrointestinal stromal tumor, desmoid tumor, Ewing's sarcoma, osteosarcoma, chondrosarcoma, leukemia, lymphoma, myeloma, other solid organ tumors such as melanoma or brain tumor (Kumar V, Abbas AK, Fausio N. Robbins and Cotran Pathological Basis of disease. 7th Ed. Unit I: General Pathology, 7: Neoplasia, Biology of tomor growth: Beni and malignan neoplasms. 269-342, 2005). The cancer stem cell group of the present invention may be a group derived from a tumor tissue derived from the epithelial cancer.

In the present invention, the cancer stem cell refers to a cell having the ability of i) and/or ii) below.

i) It retains self-replication ability. Self-replicating energy refers to the ability of a cell of either or both of the 2 dividing daughter cells to produce cells that maintain the same ability and degree of differentiation in the cell lineage as the parent cell.

ii) capable of differentiating into a plurality of cancer cells constituting the cancer cell mass. Many cancer cells differentiated from cancer stem cells form a hierarchical structure with cancer stem cells as the top in the cell lineage, as in normal stem cells. By producing a plurality of cancer cells from cancer stem cells in stages, a cancer cell mass having various characteristics is formed.

Cancer stem cells are cancer cells that have a cancer-forming ability and, like normal stem cells, have a multi-differentiation ability and a self-replication ability. In addition, at least 1 or more markers selected from the cancer stem cell markers CD24, CD29, CD34, CD44, CD49f, CD56, CD90, CD117, CD133, CD135, CD166, CD184, CD271, CD326, Aldefluor, ABCG2, ABCG5, LGR5, and Msi1 are positive in some cases, but need not necessarily be positive. Preferably at least 1 or more markers selected from CD326, CD133, CD44, ALDH are positive, more preferably at least 2 or more markers selected from CD326, CD133, CD44, ALDH are positive, even more preferably at least 3 or more markers selected from CD326, CD133, CD44, ALDH are positive, most preferably all CD326, CD133, CD44, ALDH are positive.

Furthermore, the ability to transform epithelial mesenchymal, which is a biological activity that cancer stem cells can exhibit, may be one of the properties possessed by cancer stem cells. Normal cells and tumor cells exist in a variously differentiated state in vitro and in vivo. These differentiation states are regulated, at least in part, by the integration of complex signals generated by the tissue microenvironment to which the cells belong. Cells (e.g., cancer cells) can be disrupted by various genes, e.g., via specific factors (e.g., Twist, glusulin, TGF-βOr MMPs) or by inhibiting the adhesive bonding of E-cadherin or the likeProtein, induced to undergo Epithelial Mesenchymal Transition (EMT). Towards EMT-induced cells, similar phenotypic traits and protein markers (e.g., biomarkers) are expressed regardless of the induction method used, indicating that EMT is the primary differentiation program. The present invention relates to the discovery that cells induced towards EMT share multiple properties of cancer stem cells, including: expression of cell surface markers associated with cancer stem cells, proliferation in suspension culture, formation of tumors in vivo with reduced cell numbers, and resistance to specific standard chemotherapeutic drugs. Thus, the differentiation state exhibited by epithelial mesenchymal transformed cells, also known as mesenchymal transition of differentiation or epithelial mesenchymal transition of differentiation, can be used to identify treatments that have cancer stem cells as specific targets. In a non-limiting aspect of the present invention, there is provided a method for inducing epithelial mesenchymal transition, for example, for the purpose of generating a test cell.

The cancer stem cells form a hierarchical structure with the cancer stem cells as vertexes. By producing a variety of cancer cells from cancer stem cells in stages, a cancer cell mass having diverse characteristics is formed. On the other hand, a cancer-forming cell refers to a cancer cell that does not have a multi-differentiation ability but has a cancer-forming ability. In the carcinogenesis assay, carcinogenesis and self-replication can be evaluated, and in the pathological analysis or analysis using differentiation markers, multidifferentiation energy can be evaluated. In order to prove that the stem cells are cancer stem cells, it is necessary to evaluate not only carcinogenesis but also self-replication ability and multi-differentiation ability.

The cancer cell mass is a mass in which cells and the like are not separated but adhered to each other, as in the case of a human tumor tissue, and is constructed of cells other than cancer cells such as cancer cells, interstitial cells, blood cells and the like, and extracellular matrix such as collagen, laminin and the like.

The hierarchical structure is a structure in which a part of a characteristic intrinsic structure appearing in a normal tissue is detected pathologically and histologically in a tumor structure originating in the tissue, and is generally reproduced more highly in a highly differentiated cancer, and for example, in the case of a tumor of a glandular cavity-forming organ (e.g., gastric cancer, colorectal cancer, pancreatic cancer, liver cancer, bile duct cancer, breast cancer, lung adenocarcinoma, prostate cancer), occurrence of a luminal cavity, occurrence of a mucous cell, or the like is observed; in the case of tumors having a squamous epithelial structure (squamous epithelial carcinoma of the lung, skin, vaginal mucosa, etc.), the formation of a multilayered structure of the epithelium, a tendency to keratinization, and the like are observed. On the other hand, it is considered that the reconstruction of this hierarchical structure is insufficient and is mostly atypical in poorly differentiated carcinomas (Kumar V, Abbas AK, Fausio N. Robbins and Cotran Pathological Basis of disease. 7th Ed. Unit I: General Pathology, 7: Neopalaia, Biology of tumor growth: Benign and malignan neoplases. 272. 281, 2005). Since this hierarchical structure is considered to be a structure that is reproduced as a result of various biological reactions of cancer, the value of a non-human animal model that reproduces this hierarchical structure is considered to be high.

The reconstructed hierarchical structure is an inherent structure of a characteristic property possessed by a cancer tissue of a cancer patient from which transplantation is performed, and is similarly observed when transplanted into a non-human animal.

Having carcinogenesis potential means that the cells or cell population transplanted into the non-human animal form a cancer cell mass, preferably a cancer cell mass having a hierarchical structure.

By non-carcinogenic is meant that the cells or cell populations transplanted into the non-human animal are incapable of forming a cancer cell mass.

Cells that are not carcinogenic are substantially removed, which can be confirmed by transplanting a limiting dilution of the cell population into an immunodeficient animal. In the present invention, "cells that do not have carcinogenic potential are substantially eliminated" means that the frequency of formation of cancer cell masses formed when 10 cells per site are subcutaneously transplanted into an immunodeficient animal, preferably an NOG mouse, is 60% or more, preferably 70% or more, more preferably 80% or more, further preferably 90% or more, and most preferably 100%. Further, the methods described in Hu Y & Smyth GK., J immunological methods, 2009, 8/15, 347(1-2): 70-8, Ishizawa K & Rasheed ZA., Cell Stem Cell, 2010, 9/3, 7(3): 279-82, and the like can be used. In this method, 1000 cells, 100 cells, 10 cells were transplanted into an immunodeficient animal, and the frequency of cancer cell mass formation was analyzed using limiting dilution analysis (Hu Y & Smyth GK., J immunological methods, 8/15/2009; 347(1-2): 70-8). In the present invention, the "homogeneous cancer stem cell population" in which cells having no carcinogenic potential are substantially removed means that the frequency of cancer stem cells in the analysis method is 1/20 or more, preferably 1/10 or more, more preferably 1/5 or more, further preferably 1/3 or more, further preferably 1/2 or more, and most preferably 1/1.

Limiting Dilution Analysis (ELDA) is a software application for Limiting Dilution Analysis (LDA) based on the necessity of stem cell assays. ELDA is limiting dilution analysis software that provides meaningful confidence intervals for data sets of all LDAs, including 0% or 100% responses, including tests of the validity of one hit hypothesis, tests for significant differences in frequency among multiple data sets, and the like. For the analysis method according to ELDA advocated by Hu et al (J immunological methods. (2009) 347(1-2), 70-78), the skilled person can also carry out the analysis using the on-line analysis tool provided in http:// bio if.

By using the present invention, cancer stem cells can be produced in large quantities. By increasing the number of culture flasks for adherent culture, a desired number of cancer stem cells can be obtained in principle. For example, when T150 culture flask is used, 4X 10 can be obtained in general by culturing until confluency⁷More than one cell; when 5 flasks are used for the culture, 2X 10 cells can be prepared⁸More than one cell. Thus, the present invention enables the preparation of a desired number of cells.

The cancer stem cell population of the present invention preferably has substantially no cells that are incapable of carcinogenesis, and the population may contain 1 × 10⁴More than one, more preferably 1x10⁵More than one, more preferably 1x10⁶More than one, more preferably 1x10⁷More than one, more preferably 1x10⁸More than one, most preferably 1x10⁹More than one cancer stem cell.

As a second aspect of the present invention, there is provided a method for producing a cell population of the present invention.

The cell population of the present invention can be prepared, for example, by performing adherent culture on a cell population containing cancer stem cells.

As the cell group containing cancer stem cells, cells into which a cancer virus gene such as SV40 or an oncogene such as Ras has been introduced, cells transformed into a cancer tissue strain, or the like can be used.

As the cell population containing cancer stem cells, a cell population reproducing the hierarchical structure of cancer tissue is preferably used, and for example, a collected cancer tissue can be used, but an established cancer cell line prepared by transplanting cancer into a non-human animal and passaging the cancer is preferably used, an established cancer cell line prepared by transplanting cancer into an immunodeficient animal and passaging the cancer is more preferably used, and an NOG established cancer cell line prepared by transplanting cancer tissue into a NOG mouse and passaging the cancer cell line is most preferably used.

The cell population containing cancer stem cells may be spheroids formed by spheroid culture, or may be a cell population containing cells positive for at least 1 or more markers selected from the group consisting of cancer stem cell markers CD24, CD29, CD34, CD44, CD49f, CD56, CD90, CD117, CD133, CD135, CD166, CD184, CD271, CD326, Aldefluor, ABCG2, ABCG5, LGR5, and Msi 1.

The origin of the cell population is not particularly limited, and a cell population derived from a mammal such as a human, monkey, chimpanzee, dog, cow, pig, rabbit, rat, or mouse can be used, but a cell population derived from a human is preferable.

The NOG established cancer cell line can be established by a method known to those skilled in the art, for example, by physically cutting a surgically excised human colon cancer, stomach cancer, lung cancer, breast cancer, pancreatic cancer, etc. with scissors and then subcutaneously transplanting the cut cells into NOG mice for passaging, using the method described in Pathol int 2008, 58: 559-. For NOG established cancer cell lines, the characteristics of the original human cancer tissue were maintained even after passage.

The adherent culture step is not particularly limited as long as it is an adherent culture, and a serum-free stem cell culture medium is preferably used.

Adherent culture refers to culture in which cells are inoculated into adherent culture bottles, plates, and dishes, and then cultured and passaged in an adherent state, thereby removing suspended cells. For cells that proliferated to confluence, they were detached using Accutase, passaged in new adherent flasks, plates, dishes, and cultured continuously.

The culture solution that can be used in the present invention is not particularly limited as long as it can be used for culturing cancer stem cells, and for example, EGF, bFGF, hLIF, HGF, NGF, NSF-1, and TGF can be addedβ、TNFαConventionally known basal media such as heparin, BSA, insulin, transferrin, putrescine, selenite, progesterone, hydrocortisone, D- (+) -glucose, sodium bicarbonate, HEPES, L-glutamine, and N-acetylcysteine, or a mixture thereof are used as the culture medium. The concentration of EGF is not particularly limited, but is 0.1 to 100ng/mL, preferably 0.5 to 50ng/mL, and more preferably 1 to 20 ng/mL. The concentration of bFGF is not particularly limited, but is 0.1 to 100ng/mL, preferably 0.5 to 50ng/mL, and more preferably 1 to 20 ng/mL. The concentration of hLIF is not particularly limited, but is 0.1 to 100ng/mL, preferably 0.5 to 50ng/mL, and more preferably 1 to 20 ng/mL. The concentration of HGF is not particularly limited, but is 0.1 to 100ng/mL, preferably 1 to 50 ng/mL. The concentration of NGF is not particularly limited, but is 0.1 to 100ng/mL, preferably 1 to 50 ng/mL. The concentration of NSF-1 is not particularly limited, but is 0.1 to 100ng/mL, preferably 1 to 50 ng/mL. For TGFβThe concentration of (b) is not particularly limited, but is 0.1 to 100ng/mL, preferably 1 to 50 ng/mL. For TNFαThe concentration of (b) is not particularly limited, but is 0.1 to 100ng/mL, preferably 1 to 50 ng/mL. The concentration of heparin is not particularly limited, but is 10 ng/mL-10μg/mL, preferably 2 to 5μg/mL. The concentration of BSA is not particularly limited, but is 0.1 to 10mg/mL, preferably 1 to 8 mg/mL. The concentration of insulin is not particularly limited, but is 1 to 100μg/mL, preferably 10 to 50μg/mL. The concentration of transferrin is not particularly limited, but is 10 to 500μg/mL, preferably 50 to 200μg/mL. The concentration of putrescine is not particularly limited, but is 1 to 50μg/mL, preferably 10 to 20μg/mL. The concentration of selenite is not particularly limited, but is 1 to 50nM, preferably 20 to 40 nM. The concentration of progesterone is not particularly limited, but is 1 to 50nM, preferably 10 to 30 nM. The concentration of hydrocortisone is not particularly limited, but is 10 ng/mL-10μg/mL, preferably 100 ng/mL-1μg/mL. The concentration of D- (+) -glucose is not particularly limited, but is 1 to 20mg/mL, preferably 5 to 10 mg/mL. The concentration of sodium bicarbonate is not particularly limited, but is 0.1 to 5mg/mL, preferably 0.5 to 2 mg/mL. The concentration of HEPES is not particularly limited, but is 0.1 to 50mM, preferably 1 to 20 mM. The concentration of L-glutamine is not particularly limited, but is 0.1 to 10mM, preferably 1 to 5 mM. The concentration of N-acetylcysteine is not particularly limited, but is 1 to 200μg/mL, preferably 10 to 100μg/mL. The known basic culture solution is not particularly limited as long as it is suitable for culturing cancer cells to be a source of cancer stem cells, and examples thereof include: DMEM/F12, DMEM, F10, F12, IMDM, EMEM, RPMI-1640, MEM, BME, Mocoy's 5A, MCDB131, and the like. Among them, DMEM/F12 is preferable.

The most preferred stem cell culture medium includes: to D-MEM/F12 medium, EGF was added at a final concentration of 20ng/mL, bFGF was added at a final concentration of 10ng/mL, and 4μg/mL heparin, 4mg/mL BSA, 25μInsulin 100 g/mLμg/mL transferrin, 16μA culture medium of putrescine in g/mL, selenite in 30nM, progesterone in 20nM, and D- (+) -glucose in 2.9 mg/mL.

Preferably, the population of cells comprising cancer stem cells is expanded prior to adherent culture.

The proliferation of the cell population is, for example, a proliferation by culturing a spheroid or transplanting it into a non-human animal and passaging it, but is not particularly limited thereto.

Spheroid culture is a culture medium capable of culturing the above-mentioned stem cells, and is a culture medium in which cells are seeded in a non-adherent culture flask, plate, or dish, and then the cells are cultured in a suspended state, and a cell mass formed by culturing in such a suspended state is called a spheroid.

As the non-human animal, an immunodeficient animal can be used in that it is difficult to generate a rejection reaction. As the immunodeficient animal, a non-human animal lacking functional T cells such as a nude mouse or a nude rat, a non-human animal lacking functional T cells and B cells such as a SCID mouse or a NOD-SCID mouse are suitably used, and among them, a non-human animal lacking T cells, B cells and NK cells having excellent transplantability (e.g., a SCID mouse, RAG2KO or RAG1KO mouse and IL-2Rg^nullHighly immunodeficient mice from combinations of mice: which comprises NOD/SCID/gammac^nullMouse, NOD-scid, IL-2Rg^nullMouse or BALB/c-Rag2^null,IL-2Rg^nullMouse, etc.).

When the non-human animal is a athymic nude mouse, SCID mouse, NOD/SCID mouse, or NOG mouse, for example, the week-old is preferably 4 to 100 weeks old.

NOG mice can be prepared, for example, by The method described in WO2002/043477, or can be obtained from The Central institute for Laboratory animals or The Jackson Laboratory (NSG mice).

The cells to be transplanted may be any of cell masses, tissue pieces, cells dispersed into individual cells, cells which are isolated and cultured, cells which are separated from another animal again after the process of transplantation into the animal, and the like, but dispersed cells are preferable. The number of cells to be transplanted may be 10⁶The followingThe above number of cells may be transplanted.

The subcutaneous transplantation is an appropriate transplantation site because of the ease of the transplantation technique, but the transplantation site is not particularly limited, and it is preferable to select an appropriate transplantation site according to the animal to be used. The transplantation procedure of the NOG established cancer cell line is not particularly limited, and can be performed according to a conventional transplantation procedure.

The cell population of the present method can be prepared, for example, by adherent culture of cancer tissue collected from a patient in a serum-free stem cell culture medium. In addition, in order to allow additional proliferation of the cell population, it is not considered what kind of culture has been performed before the adherent culture is performed. For example, it can also be prepared by culturing a spheroid in a cancer tissue collected from a patient and then performing adherent culture using a serum-free stem cell culture medium. Alternatively, the cells can be prepared by transplanting cancer tissues collected from a patient into a non-human animal, passaging the tissue, and then performing adherent culture using a serum-free stem cell medium. Furthermore, from the viewpoint of efficiently obtaining a large amount of the cell population of the present invention, a method for preparing a NOG established cancer cell line prepared by transplanting cancer tissues collected from a patient into a NOG mouse and passaging the cancer cells is most preferably performed by adherent culture using a serum-free stem cell culture medium.

The cell population of the present invention can be used in a method for searching for a target molecule of a drug and a method for evaluating a drug. Examples of the method for evaluating a drug include a method for screening a drug, a method for screening an anticancer drug, and the like.

Examples of the method for searching for a target molecule include: a method of identifying a gene such as DNA or RNA highly expressed in cancer stem cells (for example, a marker for cancer stem cells) using gene chip analysis; a method for identifying a protein, peptide or metabolite highly expressed in cancer stem cells using proteomics, but the method is not limited to these methods.

Examples of the screening method for searching for a target molecule include: and a method of screening a substance inhibiting the proliferation of cancer stem cells from a low molecular library, an antibody library, a microRNA library, or an RNAi library by a cell growth inhibition assay (cell growth inhibition assay). After obtaining the inhibitory substance, the target can be defined.

After obtaining antibodies from the immune cancer stem cells, the antibodies can be screened for binding by ELISA or for inhibition of proliferation by cytostatic assays. After the binding antibody and the functional antibody are obtained, the target molecule can be determined by identifying the antigen.

By using the cancer stem cell population of the present invention, the effect on cancer stem cells can be evaluated using conventional agents or agents under development, and a new drug effect can be found.

By concentrating the homogeneous cancer stem cells, the following items can be performed, for example.

1. Nucleic acids (DNA, RNA), proteins, and the like specifically expressed in cancer stem cells are efficiently identified, and the functions of these molecules are elucidated.

2. Nucleic acids (DNA, RNA), proteins, and the like that are specifically expressed in cancer stem cells are efficiently identified, and drug candidates that inhibit the expression are searched and screened.

3. The concentrated cancer stem cells are used for prognosis and the like of cancer by using the results of biological function analysis (infiltrability, division rate, and the like) as an index.

4. Cancer is reclassified using the results of biological function analysis (infiltrability, division rate, etc.) of each concentrated cancer stem cell as an index, and the method is applied to search and screening of anticancer drugs for each classification.

5. Research on the process of generating a large amount of differentiated cells occupying most of cancer tissues from concentrated cancer stem cells, and search and screening for anticancer drugs (cancer silencing genes) for suppressing the process.

6. The factors responsible for the durability of cancer stem cells were investigated by adjusting the culture conditions (oxygen partial pressure, nutrient conditions, anticancer drug treatment, etc.) of cancer stem cells in a severe direction and detecting a characteristic biological response in cancer stem cells.

7. By adjusting the culture conditions (oxygen partial pressure, nutrient conditions, anticancer drug treatment, etc.) of cancer stem cells in a severe direction, a biological response characteristic to cancer stem cells is detected, and anticancer drugs for suppressing the durability of cancer stem cells are searched and screened.

A method for searching target molecules of a drug.

The present invention relates to a method for searching for a target molecule of a drug, the method being characterized in that: in the non-human animal model into which the cancer stem cell population of the present invention is transplanted or in the culture system of the cancer stem cell population of the present invention under in vitro conditions, the grade structure formed by the cancer stem cells, the progress of cancer starting from the cancer stem cells, or the biological characteristics of the cancer stem cells are evaluated as indicators.

In the case of using a non-human animal model into which a cancer stem cell population is transplanted in the method, target molecules of a drug can be searched for by the following steps (1) to (4).

(1) A step of preparing a non-human animal model by transplanting a cancer stem cell population into a non-human animal;

In the present method, when a culture system of a cancer stem cell population under in vitro conditions is used, target molecules of a drug can be searched for by the following steps (1) to (3).

(1) Culturing the cancer stem cell population under in vitro conditions to reproduce a characteristic structure of a cancer progression process from the cancer stem cells or a biological characteristic of the cancer stem cells;

(2) a step of studying the expression of DNA, RNA, and proteins, peptides or metabolites of the cultured cells reproducing the characteristic structure; and

In the present invention, the observation of the structure of a tissue or cell line characteristic to the progress of cancer can be performed by preparing a thin-cut tissue specimen by a known specimen preparation method such as the AMeX method, and then performing HE staining and immunohistochemical staining (IHC). When the above-mentioned hierarchical structure specific to human tumor tissue, the progress of cancer, or the biological properties thereof are confirmed in the tissue to be tested, the tissue to be tested is regarded as a cancer-associated tissue, and the expression of DNA, RNA, protein, peptide, and metabolite is confirmed. The expression of DNA, RNA, protein, peptide and metabolite is not particularly limited, and can be confirmed by a conventional expression confirmation method. Examples of RNA include: microRNA, siRNA, tRNA, snRNA, mRNA, or non-coding RNA. For example, the transcription level of each gene can be measured by extracting the mRNA of each gene as usual, and performing an RNA hybridization method or an RT-PCR method using the mRNA as a template. Furthermore, the expression level of each gene can also be measured using a DNA array technique. Furthermore, the translation level of a gene can also be measured by recovering fractions containing proteins encoded by the respective genes as usual and detecting the expression of the respective proteins by electrophoresis such as SDS-PAGE. Furthermore, the expression of each protein can be detected by western blotting using an antibody against each protein, and the translation level of the gene can also be measured. By these methods, target molecules of anticancer drugs can be searched.

As a non-limiting example of the target molecule of the above drug, there may be mentioned: examples of the molecule specifically expressed by the cancer stem cells of the present invention include CD133, CD44, EpCAM, CD166, CD24, CD29, and LGR 5.

In the present invention, the above-mentioned drug is not particularly limited, and examples thereof include: anti-inflammatory agents, immunosuppressive agents, antiviral agents, angiogenesis inhibitors, steroid agents, enzyme inhibitors, antibiotics, antihistamines, anticoagulants, anti-infective agents, analgesics, diabetes remedies, arthritis remedies, anti-asthma agents, anti-insomnia agents, antiemetics, migraine remedies, antispasmodics, antidepressants, antipsychotic agents, antipyretics, Parkinson's disease remedies, Alzheimer's disease remedies, sympathetic nerve agents, arrhythmia remedies, hypotensive agents, diuretics, antidiuretic agents, anti-coagulants, vasodilators, sedatives, and the like, preferably anticancer agents. The above-mentioned drugs are not particularly limited, and include: protein agents, nucleic acid agents, low molecular weight agents, cellular agents, and the like. In the present invention, the target molecule is not particularly limited, and examples thereof include: membrane receptors, enzymes, ion channels, transcription factors or nuclear receptors, etc., preferably cancer cell markers.

Method for evaluating drug

The present invention relates to a method for evaluating a drug, the method being characterized in that: in the non-human animal model into which the cancer stem cell population of the present invention is transplanted or in the culture system of the cancer stem cell population under in vitro conditions, the grade structure formed by the cancer stem cells, the progress of cancer starting from the cancer stem cells, or the biological properties of the cancer stem cells are evaluated as indicators.

In the method, when a non-human animal model into which the cancer stem cell population of the present invention has been transplanted is used, the drug can be evaluated by the following steps (1) to (5).

(2) administering a test substance to the non-human animal model of (1);

In the case of using a culture system of a cancer stem cell population under in vitro conditions in the method, the drug can be evaluated by the following steps (1) to (3).

(2) treating the cultured cells of (1) with a test substance;

Method for screening drug

The invention relates to a method for screening drugs, which is characterized in that: in the non-human animal model into which the cancer stem cell population of the present invention is transplanted or in the culture system of the cancer stem cell population under in vitro conditions, the grade structure formed by the cancer stem cells, the progress of cancer starting from the cancer stem cells, or the biological properties of the cancer stem cells are evaluated as indicators.

In the method, when a non-human animal model into which the cancer stem cell population of the present invention has been transplanted is used, drug screening can be performed by the following steps (1) to (5).

(2) administering a test substance to the non-human animal model of (1);

(5) a step of identifying a test substance that inhibits the formation of a hierarchical structure formed by a specific cancer stem cell, the progression of cancer from a cancer stem cell, or a biological property of a cancer stem cell.

In the present method, when a culture system of a cancer stem cell population under in vitro conditions is used, drug screening can be performed by the steps described in the following (1) to (4).

(1) Culturing the cancer stem cell population under in vitro conditions to reproduce the characteristic structure of each cancer progression process or the biological characteristics thereof;

(2) treating the cultured cells of (1) with a test substance;

(3) observing a change with time in the hierarchical structure of cancer stem cells, a cancer progression process, or a biological property thereof in the cultured cells; and

(4) a step of identifying the hierarchical structure formation, the cancer progression process, or a biological property thereof of the cancer stem cells inhibited by the test substance.

The screening method enables screening of an anticancer drug.

The "analyte" in the method of the present invention is not particularly limited, and examples thereof include: natural compounds, organic compounds, inorganic compounds, proteins, antibodies, peptides, amino acids, and the like, as well as compound libraries, expression products of gene libraries, cell extracts, cell culture supernatants, fermentation microorganism products, marine organism extracts, plant extracts, prokaryotic cell extracts, eukaryotic single cell extracts, or animal cell extracts, and the like. These may be purified products, or may be crude purified products such as extracts of plants, animals, microorganisms, and the like. The method for producing the substance to be measured is not particularly limited, and may be a substance isolated from a natural product, a chemically synthesized or biochemically synthesized substance, or a substance produced by genetic engineering.

The test sample can be appropriately labeled as necessary. Examples of the label include a radioactive label and a fluorescent label. In addition to the above-mentioned test samples, the test samples also include mixtures obtained by mixing a plurality of these test samples.

In the method, the method of administering the test substance to the non-human animal model is also not particularly limited. Oral administration or parenteral administration, subcutaneous, intravenous, topical, transdermal, or enteral (rectal) administration, etc., can be suitably selected according to the kind of the substance to be administered.

In the present method, the method for treating the cultured cells of the cancer stem cell population with the test substance is also not particularly limited. The treatment can be performed by adding a sample to be tested to a culture solution of cells or an extract solution of the cells. In the case where the test sample is a protein, for example, the test sample may be introduced into the cancer stem cell population by introducing a vector containing DNA encoding the protein, or may be added to a cell extract of the cancer stem cell population. In addition, for example, a double hybridization method using yeast, animal cells, or the like can also be used.

The evaluation of the test substance can be carried out by removing the transplanted tissue (tissue into which the cancer stem cell population has been transplanted) from the model animal, observing the histological characteristics of the transplanted tissue, or measuring the histological characteristics in a cell culture system.

Specifically, the evaluation of the test substance can be performed as follows: in a culture system of a non-human animal model or a cancer stem cell population under in vitro conditions, the formation of a hierarchical structure formed by a transplanted tissue or a cancer stem cell of the culture system is observed, and it is confirmed whether a hierarchical structure specific to a human cancer cell is formed or not, or the influence on a cancer progression process characteristic to a human cancer disease is exerted. The observation of the structure of a tissue or cell line characteristic to the progress of cancer can be performed by preparing a thin-cut tissue specimen by a known specimen preparation method such as the AMeX method, and then performing HE staining and immunohistochemical staining (IHC).

More specifically, the evaluation of the test substance can be performed as follows: the hierarchical structure of cancer stem cells in a transplanted tissue or culture system is similarly observed for a non-human animal model or a cancer stem cell group to which a control is not administered with a test substance, and it is confirmed whether or not a hierarchical structure specific to human cancer cells is formed. In this case, when the graft tissue or culture system to which the test substance is administered does not have a hierarchical structure specific to human cancer cells or when the ratio thereof is reduced, as compared with the case of the control animal, the test substance can be selected as an effective substance having an effect of treating or preventing human cancer diseases.

More specifically, the evaluation of the test substance can be performed as follows: the cancer progression process of the transplanted tissue or culture system is similarly observed in the non-human animal model or cancer stem cell group of the control to which the test substance is not administered, and it is confirmed whether or not the cancer progression process specific to the human cancer cell is visible. In this case, in comparison with the case of the control animal, in the case where the cancer progression process specific to human cancer cells is not found in the transplanted tissue or culture system to which the test substance is administered, the test substance can be selected as an effective substance having the effect of treating or preventing human cancer diseases.

More specifically, the evaluation of the test substance can be performed as follows: biological characteristics of cancer stem cells in transplanted tissues or culture systems were observed in the same manner in non-human animal models or cancer stem cell groups to which a control was administered without administering a test substance, and whether the characteristics peculiar to human cancer cells were observed or not was confirmed. In this case, in comparison with the case of the control animal, in the case where the characteristic peculiar to the human cancer cell is not found in the transplanted tissue or culture system to which the test substance is administered, the test substance can be selected as an effective substance having the effect of treating or preventing the human cancer disease.

In addition, according to the screening method of the present invention, it is possible to screen a more effective and highly practical prophylactic or therapeutic substance for a human cancer disease by further performing other drug efficacy tests, safety tests, and the like, as necessary, and further performing clinical tests on human cancer patients, as the prophylactic or therapeutic substance for the selected human cancer disease.

The prophylactically effective substance or the therapeutically effective substance thus selected can be industrially produced by chemical synthesis, biochemical synthesis (fermentation), or genetic manipulation, further based on the results of structural analysis thereof.

Furthermore, all prior art documents cited in the present specification are incorporated herein by reference.

Examples

The present invention will be described in detail below with reference to examples, but the present invention is not limited to these examples.

Example 1 preparation and evaluation of cancer Stem cells and cancer-Forming cells Using human cancer cell lines

(1) Morphological evaluation of colorectal cancer Strain transplanted into mouse

Colorectal cancer specimens were obtained from patients with informed consent under approval from the ethical committee of pharmaceuticals Research (singapore) and park Laboratory Services (singapore). The tumor pieces were finely minced with scissors and transplanted into the flank of NOG mice. Human colon cancer xenografts were maintained by passage in NOG mice supplied by the central institute of laboratory animals (japan). For the mice used in this experiment, the treatment was performed according to the animal experimental guidelines of pharmacy Research. A cancer cell mass was prepared by subcutaneously transplanting a large intestine cancer cell line established in an NOG mouse or SCID mouse into the NOG mouse. After the cancer cell mass is picked out, the cancer cell mass is fixed in 4% paraformaldehyde at 4 ℃ for 16-24 hours, and embedded by an AMeX method to prepare a thin-cut tissue specimen. HE staining was performed on the tissue specimens. The results are shown in FIG. 1. The morphological structures of colon cancer strain PLR123, colon cancer strain PLR59, and colon cancer strain PLR325 transplanted into NOG mice were similar to those of human tissues, and were shown to be unaffected by the NOG mice. On the other hand, in the colon cancer strain PLR357 transplanted into NOG mice, morphological changes were observed, and it was revealed that it was not suitable for the transplantation experiments in NOG mice.

(2) Preparation of cells from cancer cell masses

Cancer cell masses were excised from NOG mice and physically minced with scissors. Then, the cells were suspended several times with DPBS (Invitrogen, Cat. No. 14190144), and the tissue was transferred to an enzyme solution containing collagenase/dispase (Roche, Cat. No. 10269638001) and DNase I (Roche, Cat. No. 11284932001), followed by stirring at 37 ℃ for 3 hours. Then, lysis buffer (BD, catalog No. 555899) was added to the cells subdivided by repeating the pipetting, and the mouse erythrocytes were removed. Finally, make it pass through 40μThe cell fluid was prepared by suspending m-cell filters (BD, Cat. No. 352340) several times with DPBS.

(3) Detection of cancer stem cell marker positive cells

Cells prepared from the cancer cell mass were suspended in FACS buffer (2% fetal bovine serum/DPBS), and a rat anti-mouse MHC class I mAb (Abcam, cat. No. ab 15680) was added to the suspension to react at 4 ℃ for 30 minutes. After the cells were washed 1 time with FACS buffer, PE-labeled goat anti-rat IgG2a Ab (BioLegend, Cat. No. 405406) or APC-labeled goat anti-rat IgG2a Ab (BioLegend, Cat. No. 405407) as a class 2 antibody, 7-AAD viability dye (Beckman Coulter, Cat. No. A07704) as a dead cell stain, FITC-labeled mouse anti-human CD326 (EpCAM) mAb (Miltenyi Biotec, Cat. No. 130-. The cells were then washed once with FACS buffer and provided for flow cytometry analysis. For ALDEHYDE DEHYDROGENASE (ALDEHYDE DEHYDROGENASE: ALDH) activity, it was examined by performing the procedure recommended by the manufacturer using AldeFluor kit (Stemcell Technologies, Cat. No. 01700). Analysis of cancer stem cell markers was performed on mouse MHC class I negative and 7-AAD viability dye negative cells using EPICS ALTRA (Beckman Coulter) in flow cytometry analysis. The results are shown in fig. 2. Among cancer cell masses formed from colorectal cancer strain PLR123, colorectal cancer strain PLR59, and colorectal cancer strain PLR325, positive cancer stem cell markers were observed. In the differentiated colon cancer strain PLR123 and colon cancer strain PLR59, marker-positive cells and marker-negative cells were present together, and the cells were heterogeneous cell groups. On the other hand, the poorly differentiated colon cancer strain PLR325 was a cell population showing a homogeneous characteristic that all cells were positive for EpCAM, AC133 and ALDH activities and negative for CD 44.

(4) Removal of mouse cells from cell sap

Cells prepared from the cancer cell mass were suspended in FACS buffer, and a rat anti-mouse MHC class I mAb was added to the suspension, followed by reaction at 4 ℃ for 15 minutes. After washing the cells 1 time with FACS buffer, PE-labeled goat anti-rat IgG2a Ab was added as a grade 2 antibody, and the reaction was carried out at 4 ℃ for 15 minutes. After the cells were washed again 1 time with FACS buffer, the mouse cells were removed by the manufacturer's recommended procedures using cell sorting of EPICS ALTRA or EasySep mouse PE positive selection kit (Stemcell Technologies, catalog No. 18554). The purity of the cells was analyzed using EPICS ALTRA. The results are shown in fig. 3. It was revealed that 95% or more of the cancer cell masses formed by the colorectal cancer strains PLR123, PLR59 and PLR325 were mouse MHC class I negative in mice after removal of mouse cells.

(5) Test for formation of carcinoma of large intestine cancer

After mouse cells were removed from the cell fluid prepared in (2), the number of cells was counted by confirming single cells of cancer cells under a microscope. Cell sap of 10000 cells/mL, 1000 cells/mL, 100 cells/mL was prepared using a Matrigel basal Membrane Matrix (BD, Cat. No. 354234) diluted to 50% with Hank's Balanced Salt Solution (Hank's Balanced Salt Solution, Invitrogen, Cat. No. 24020-117). The respective cell fluids were mixed at 100 deg.CμL/site, i.e., 1000 cells/site, 100 cells/site, 10 cells/site, was subcutaneously transplanted into NOG miceThe number of tumors formed was evaluated. The results are shown in table 1. In the differentiated colon cancer strain PLR123 and the differentiated colon cancer strain PLR59, cancer formation was observed in the transplantation of 100 cells/site or more, and in the poorly differentiated colon cancer strain PLR325, cancer formation was observed in the transplantation of 10 cells/site or more. In addition, the frequency of cells with carcinogenic potential was analyzed using limiting dilution analysis. It was shown that cells having carcinogenic activity were contained at frequencies of 1/161, 1/195 and 1/14 in colon cancer strain PLR123, colon cancer strain PLR59 and colon cancer strain PLR325, respectively.

[ Table 1]

(6) Histological evaluation of hierarchical Structure formation in colorectal cancer Strain

After cancer cell masses consisting of 100 cells and 10 cells were picked out, they were fixed in 4% paraformaldehyde at 4 ℃ for 16 to 24 hours, and embedded by an AMeX method to prepare thin-cut tissue specimens. HE staining was performed on the tissue specimens. The results are shown in fig. 4. The hierarchical structure was observed in the differentiated colon cancer strain PLR123 and colon cancer strain PLR59, and it was shown that colon cancer strain PLR123 and colon cancer strain PLR59 were cancer stem cells. On the other hand, no hierarchical structure was observed in the poorly differentiated colon cancer strain PLR325, and it was shown that the colon cancer strain PLR325 was a cancer-forming cell.

(7) Detection of normal intestinal stem cell marker LGR5 protein

After removing mouse cells from the cell fluid prepared in (2) above, cell lysates of colon cancer strain PLR123, colon cancer strain PLR59, and colon cancer strain PLR325 were prepared using RIPA buffer (Sigma, catalog No. R0278), subjected to SDS-PAGE, and subjected to western blotting. For the detection of LGR5 protein, a rabbit anti-human GPR49 mAb (Abcam, cat # ab 75850) and a mouse anti-human GAPDH mAb (Santa Cruz, cat # Sc-69778) were used for the positive control. The results are shown in fig. 5. LGR5 protein was detected in the differentiated colon cancer strain PLR123 and colon cancer strain PLR59, indicating the presence of cells positive for the normal intestinal stem cell marker. On the other hand, in the poorly differentiated colon cancer strain PLR325, LGR5 protein was not detected.

EXAMPLE 2 characterization of commercially available in vitro cancer cell lines

(1) Culture of commercially available in vitro cancer cell lines

The commercial in vitro cancer cell lines were cultured by a general method (e.g., 37 ℃ C., 5% CO)₂In the presence) was used as a basis, using the media recommended by ATCC (http:// www.atcc.org /). Note that fetal bovine serum in all the media was inactivated by incubation at 56 ℃ for 30 minutes or more.

(2) Detection of cancer stem cell marker positive cells

Cancer cells cultured in vitro were detached from the culture flask using Accutase (ICT, cat 104), suspended in FACS buffer, and added with 7-AAD viability dye (Beckman Coulter, cat a 07704) as a dead cell stain, FITC-labeled mouse anti-human CD326 (EpCAM) mAb as a cancer stem cell marker, PE-labeled mouse anti-human CD133/1 (AC 133) mAb, or PE-labeled mouse anti-human CD44 mAb, respectively, and reacted AT 4 ℃ for 30 minutes. The cells were then washed once with FACS buffer and provided for flow cytometry analysis. ALDEHYDE DEHYDROGENASE (ALDEHYDE DEHYDROGENASE: ALDH) activity was detected by performing the manufacturer's recommended procedure using the AldeFluor kit. Cells that were 7-AAD viability dye negative were analyzed for cancer stem cell markers using EPICS ALTRA (Beckman Coulter) in flow cytometry analysis. The results are shown in fig. 6. In HCT116, a cell population showing a homogeneous characteristic of positive stem cell markers was found for almost all cells.

(3) Test for carcinogenesis of commercially available in vitro cell lines

The cancer cells cultured in vitro were detached from the culture flask by using Accutase, and the number of cells was counted by confirming that the cancer cells were single cells under a microscope. Using Matrigel basement membrane matrix diluted to 50% by Hank's balanced salt solution, cell sap of 10000 cells/mL, 1000 cells/mL, 100 cells/mL was prepared. The respective cell fluids were mixed at 100 deg.CμL/site, i.e., 1000 cells/site, 100 cells/site, 10 cells/site, were subcutaneously transplanted into NOG mice, and the number of tumors formed was evaluated. The results are shown in table 2. In HCT116, cancer formation was seen in transplants of 10 cells/site or more. In addition, the frequency of cells with carcinogenic potential was analyzed using limiting dilution analysis. HCT116 shows 1/9-frequency containing cells with carcinogenic potential.

[ Table 2]

(4) Histological evaluation of hierarchal Structure formation in commercially available in vitro cell lines

After the cancer cell mass formed by 10 cells was picked out, it was fixed in 4% paraformaldehyde at 4 ℃ for 16 to 24 hours, and embedded by the AMeX method to prepare a thin-cut tissue specimen. HE staining was performed on the tissue specimens. The results are shown in fig. 7. No hierarchical structure was seen in HCT116, indicating that HCT116 is a cancer-forming cell.

(5) Detection of normal intestinal stem cell marker LGR5 protein

HCT116 cell lysates were prepared using RIPA buffer, subjected to SDS-PAGE, and subjected to Western blotting. Rabbit anti-human GPR49 mAb was used for detection of LGR5 protein and mouse anti-human GAPDH mAb was used for positive control. The results are shown in fig. 8. LGR5 protein was not detected in HCT 116.

(6) Detection of normal intestinal stem cell marker LGR5 positive cells

HCT116 was seeded at 5000 cells/well in Lab-Tek Chamber slides (Lab-Tek Chamber Slide, Thermo Scientific, Cat. No. 177402) and cultured. After about 24 hours, it was used for in situ hybridization. In situ hybridization was analyzed by performing manufacturer's recommended procedures using a QuantiGene ViewRNA plate-based assay kit (Panomics, cat No. QVP 0010), a QuantiGene ViewRNA plate-based signal amplification kit (Panomics, cat No. QVP 0200), a QuantaGene ViewRNA GPR49 (LGR 5) probe set (Panomics, cat No. VA 1-10587). In addition, DAPI (Invitrogen, catalog No. D21490) was used in nuclear staining. The results are shown in fig. 9. Cells positive for the LGR5 probe were not seen in HCT 116.

EXAMPLE 3 in vitro Strain of cancer Stem cells

(1) Adherent culture of cancer stem cells in vitro (hereinafter, this culture may be abbreviated as adherent culture)

The cells are cultured in the usual manner (e.g., 37 ℃ C., 5% CO)₂In the presence) as a basis, the following stem cell culture medium was used. Stem cell culture media were prepared as follows: to DMEM/F12 (Invitrogen, Cat. No. 11330057) medium was added N-2 supplement (Invitrogen, Cat. No. 17502014), Recombinant Human EGF (Invitrogen, Cat. No. 11330032) at a final concentration of 1X, Recombinant Human EGF (Invitrogen, Cat. No. 11330032) at a final concentration of 20ng/mL, and fiber Growth Factor-basic Human Recombinant (Sigma, Cat, No. F0291) at a final concentration of 10ng/mL, respectivelyμg/mL heparin sodium salt (Sigma, cat # H3149), 4mg/mL Albumax lipid-rich BSA (Invitrogen, cat # 11010021), 20μG/mL of zinc solution of recombinant human insulin (Invitrogen, Cat. No. 12585014), 2.9mg/mL of D- (+) -glucose solution (45%) (Sigma, Cat. No. G8769), 1X of antibiotic-antimycotic agent (Invitrogen, Cat. No. 15240062). Cancer cells containing cancer stem cells were cultured in a 6-well plate for adherent culture (BD, catalog No. 353046) using a stem cell culture medium. After a few days, it can be seenCells attached to the plate and cells in suspension, cells in suspension were removed and only adherent cells were cultured. Adherent cells grown to confluence were detached by Accutase, and cultured using a new 6-well plate for adherent culture, a T25 flask for adherent culture (BD, catalog No. 353109), a T75 flask (BD, catalog No. 356485), and a T150 flask (BD, catalog No. 355001). The passaging was continued by the method shown in (2) below until all adherent cells became positive for a cancer stem cell marker. A portion of the cells were suspended in Bambanker (Wako, Cat. No. 302-14681) as a cell preservation solution and stored at-80 ℃ or lower. FIG. 10 shows the morphology of a differentiated colon cancer strain PLR123 cultured in vitro. The formation of a mass of cells called spheroids is only visible in suspension.

(2) Detection of cancer stem cell marker positive cells

Cancer cells subjected to in vitro adherent culture using a stem cell culture medium were detached from the culture flask by Accutase, suspended in FACS buffer, and added with 7-AAD viability dye as a dead cell stain, FITC-labeled mouse anti-human CD326 (EpCAM) mAb as a cancer stem cell marker, PE-labeled mouse anti-human CD133/1 (AC 133) mAb, or PE-labeled mouse anti-human CD44 mAb, respectively, and reacted at 4 ℃ for 30 minutes. The cells were then washed once with FACS buffer and provided for flow cytometry analysis. ALDEHYDE DEHYDROGENASE (ALDEHYDE DEHYDROGENASE: ALDH) activity was detected by performing the manufacturer's recommended procedure using the AldeFluor kit. EPICS ALTRA was used in flow cytometry analysis to analyze cancer stem cell markers for 7-AAD viability dye negative cells. The results are shown in FIG. 11. In differentiated colorectal cancer strain PLR123 and PLR59 cultured adherent in vitro, all cells were homogeneous and positive as a cancer stem cell marker.

(3) Detection of normal intestinal stem cell marker LGR5 protein

After removing mouse cells from the cell fluid, cell lysates of the medium-differentiated colon cancer strain PLR123, the medium-differentiated colon cancer strain PLR59, the colon cancer strain PLR123 cultured by in vitro adherence using a stem cell culture medium, and the colon cancer strain PLR59 cultured by in vitro adherence using a stem cell culture medium were prepared using RIPA buffer, and subjected to SDS-PAGE, followed by western blotting. Rabbit anti-human GPR49 mAb was used for detection of LGR5 protein and mouse anti-human GAPDH mAb was used for positive control. The results are shown in fig. 12. An increase in LGR5 protein was detected in colorectal cancer strains cultured adherently in vitro in stem cell culture medium. Indicating that the cells positive for normal intestinal stem cell markers are concentrated by in vitro culture with stem cell culture medium.

(4) Detection of normal intestinal stem cell marker LGR5 positive cells

A differentiated colon cancer strain PLR123 cultured in an in vitro adherent manner in a stem cell culture medium was seeded at 50000 cells/well on a Lab-Tek chamber slide and cultured. After about 24 hours, it was used for in situ hybridization. In situ hybridization was analyzed by manufacturer's recommended procedures using a QuantiGene ViewRNA plate-based assay kit, a QuantiGene ViewRNA plate-based signal amplification kit, and a QuantaGene ViewRNA GPR49 (LGR 5) probe set. In addition, DAPI was used in nuclear staining. The results are shown in fig. 13. It was found that all of the differentiated colon cancer strains PLR123 cultured in vitro adherent culture using the stem cell medium were positive to the LGR5 probe. By in vitro culture with stem cell medium, cells that showed positive for the normal intestinal stem cell marker were concentrated, which showed that all cells were homogeneous cells positive for the normal intestinal stem cell marker LGR 5.

(5) Assay for cancer formation in cells cultured adherent in vitro

The cancer cells subjected to in vitro adherent culture were detached from the flask using Accutase, and suspended several times using DPBS. Passing the cell sap through 40μThe number of cells was counted by confirming single cells by m-cell filtration under a microscope. 10000 cells/mL, 1000 cells/mL were prepared using Matrigel basement membrane matrix diluted to 50% by Hank's balanced salt solution100 cells/mL of cell sap. The respective cell fluids were mixed at 100 deg.CμL/site, i.e., 1000 cells/site, 100 cells/site, 10 cells/site, were subcutaneously transplanted into NOG mice, and the number of tumors formed was evaluated. The results are shown in table 3. In the differentiated colorectal cancer strains PLR123 and PLR59 cultured adherent in vitro, cancer formation was observed in all transplants. It was revealed that the differentiated colon cancer strain PLR123 and colon cancer strain PLR59 obtained by adherent culture in vitro were cells having carcinogenic activity.

[ Table 3]

(6) Evaluation of in vitro Strain of cancer Stem cells

The differentiated colorectal cancer strain PLR123 cultured adherent in vitro was further cultured for 1 month or more, and the cancer-forming ability was compared. The cells subjected to in vitro adherent culture were peeled off from the flask using Accutase, and suspended several times using DPBS. Passing the cell sap through 40μThe number of cells was counted by confirming single cells by m-cell filtration under a microscope. Using Matrigel basement membrane matrix diluted to 50% by Hank's balanced salt solution, cell sap of 10000 cells/mL, 1000 cells/mL, 100 cells/mL was prepared. The respective cell fluids were mixed at 100 deg.CμL/site, i.e., 1000 cells/site, 100 cells/site, 10 cells/site, were subcutaneously transplanted into NOG mice, and the number of tumors formed was evaluated. The results are shown in table 4. The mid-differentiated colon cancer strain PLR123 cultured for 1 month or more showed cancer formation in all transplants. The cancer-forming ability by in vitro culture was maintained at 100%, indicating that in vitro strain formation was successful.

[ Table 4]

(7) Histological evaluation of the hierarchical Structure formation of cancer cell clusters formed by 10 cancer cells in adherent culture in vitro

Cancer cell masses formed by 10 cells of differentiated colorectal cancer strain PLR123 and colorectal cancer strain PLR59 cultured in vitro adherent were removed, fixed in 4% paraformaldehyde at 4 ℃ for 16 to 24 hours, and embedded by AMeX method to prepare thin-cut tissue specimens. HE staining was performed on the tissue specimens. The results are shown in fig. 14. The same hierarchical structure as that of human tissues and established cancer cell lines was observed in cancer cell masses formed from 10 cells of differentiated colorectal cancer strains PLR123 and PLR59 cultured adherent to each other in vitro. In addition, the same hierarchical structure as that of human tissues and NOG established cancer cell lines was observed in cancer cell masses formed from a differentiated colorectal cancer cell line PLR123 cultured by in vitro adherent culture for 1 month or more. It was revealed that differentiated colon cancer strains PLR123 and PLR59, which were cultured adherent in vitro, were cancer stem cells having multipotentiality.

(8) Analysis of cancer Stem cell markers in cells prepared from human cancer cell masses formed from 10 cancer cells cultured adherent in vitro

Cells prepared from cancer cell masses formed by differentiated colorectal cancer strain PLR123 and colorectal cancer strain PLR59 cultured by in vitro adherent culture of 10 cells were suspended in FACS buffer, and rat anti-mouse MHC class I mAb was added to the suspension to react at 4 ℃ for 30 minutes. After the cells were washed 1 time with FACS buffer, PE-labeled goat anti-rat IgG2a Ab or APC-labeled goat anti-rat IgG2a Ab as a grade 2 antibody, 7-AAD viability dye as a dead cell stain, FITC-labeled mouse anti-human CD326 (EpCAM) mAb as a cancer stem cell marker, PE-labeled mouse anti-human CD133/1 (AC 133) mAb, or PE-labeled mouse anti-human CD44 mAb were added, respectively, and reacted at 4 ℃ for 30 minutes. The cells were then washed 1 time with FACS buffer and provided for flow cytometry analysis. ALDEHYDE DEHYDROGENASE (ALDEHYDE DEHYDROGENASE: ALDH) activity was detected by performing the manufacturer's recommended procedure using the AldeFluor kit. EPICS ALTRA was used in the flow cytometry analysis to analyze mouse MHC class I negative and 7-AAD viability dye negative cells for cancer stem cell markers. The results are shown in fig. 15. Cancer stem cell marker-negative cells were observed in cancer cell masses formed from differentiated colorectal cancer strain PLR123 and colorectal cancer strain PLR59 cultured by in vitro adherent culture of 10 cells, and it was shown that cancer stem cell marker-negative cells were produced from cancer stem cell marker-positive cells.

(9) Test for carcinogenesis of cells prepared from a human cancer cell Mass (first Generation) formed from 10 cancer cells cultured adherent in vitro (second Generation)

Mouse cells were removed from a cell liquid prepared from a cancer cell mass (first generation) formed from a differentiated colorectal cancer strain PLR123 cultured by in vitro adherent culture of 10 cells, and then the number of human cancer cells was counted by confirming single cells under a microscope. Using Matrigel basement membrane matrix diluted to 50% by Hank's balanced salt solution, cell sap of 10000 cells/mL, 1000 cells/mL, 100 cells/mL was prepared. The respective cell fluids were mixed at 100 deg.CμL/site, i.e., 1000 cells/site, 100 cells/site, 10 cells/site, were subcutaneously transplanted into NOG mice, and the number and morphology of tumors formed were evaluated. The results are shown in table 5. Since the formation of cancer having a hierarchical structure is observed at10 or more, it is shown that the cells contained in the cancer cell mass have the self-replication ability as cancer stem cells. Further, since the frequency of cancer stem cells contained in the cancer cell mass was 1/95, it was shown that differentiated cells not having carcinogenesis ability were produced from cancer stem cell cancer subjected to in vitro adherent culture.

[ Table 5]

(10) Histological evaluation of hierarchical Structure formation of second Generation cancer cell clumps formed by adherent culture of cancer cells in vitro

And (3) picking out a second-generation cancer cell mass formed by in-vitro adherent culture of cancer cells, fixing the second-generation cancer cell mass in 4% paraformaldehyde at 4 ℃ for 16-24 hours, and embedding by using an AMeX method to prepare a thin-cut tissue specimen. HE staining was performed on the tissue specimens. The results are shown in fig. 16. The hierarchical structure similar to that of human tissues and NOG established cancer cell lines was observed in the second generation cancer cell mass formed from a differentiated colorectal cancer cell line PLR123 cultured adherent in vitro.

[ reference example 1] protein expression analysis by Lgr5

(1) Establishment of cells expressing full-length human Lgr4, 5, and 6

Full-length human Lgr4, 5 and 6 cDNAs were cloned by PCR based on the sequences of NM-018490 (Lgr 4), NM-001017403 (Lgr 5) and NM-003667 (Lgr 6). The cloned gene was expressed with or without an HA tag attached to the N-terminus. The expression plasmid was transfected into the Chinese hamster ovary cell line CHO DG44 (Invitrogen) using a Gene Pulser (Gene Pulser, BioRad). HA-Lgr4/DG, HA-Lgr5/DG and HA-Lgr6/DG were selected as stable cell lines using G418.

(2) Preparation of soluble Lgr5-Fc protein

Soluble Lgr5 (amino acids 1-555) protein was expressed as a fusion protein with the Fc portion of mouse IgG2a from CHO DG 44. Transfectants were screened by sandwich ELISA using goat anti-mouse IgG2a (Bethyl labratories) and HRP rat anti-mouse IgG2a mab (serotec). The clone that produced sLgr5-Fc most abundantly was named 2D 3. The 2D3 culture supernatant was recovered, and the Lgr5-Fc protein was affinity-purified by passing through a protein A Sepharose column (Pharmacia). Lgr5-Fc functions as an antigen for protein immunization and ELISA screening.

(3) Generation of anti-Lgr 5 monoclonal antibodies using Lgr5-Fc protein immunization (WO 2009063970)

Using 50 emulsified in complete fischer-tropsch adjuvantμg of Lgr5-Fc, was used to immunize Balb/c mice (Charles River Japan) subcutaneously. After 2 weeks, injections were repeated up to 2 weeks 1 time per week using the same amount in freund's incomplete adjuvant. Mice were injected intravenously with 25 days before cell fusionμg Lgr5 Fc. Spleen lymphocytes from immunized mice were fused with P3-X63Ag8U1 mouse myeloma cells (ATCC) by the current method (Kremer L and Marquez G (2004) Methods mol., 239, 243-260). Hybridoma culture supernatants were screened for antibodies reactive with sLgr5-Fc using ELISA. Lgr5 specific mouse mAbs 2T15E-2 and 2U2E-2 were established.

(4) Immunofluorescent staining for cultured cells and xenograft tissues

For immunofluorescence cytochemistry, cells fixed with 4% paraformaldehyde and methanol were combined with mouse anti-human E-cadherin antibody (Abcam), rabbit anti-human glusulin antibody (Abcam), or rabbit anti-humanβCatenin antibody (Sigma) was co-incubated and then visualized using goat anti-mouse IgG antibody or goat anti-rabbit IgG antibody, respectively, labelled with AlexaFluor 488. For immunofluorescent histochemistry, thin slices of paraffin blocks from the above xenograft tumors were incubated with mouse anti-human Lgr5 antibody (2U 2E-2) or rabbit anti-human glusulin antibody (Abcam). After incubation with primary antibody, Lgr5 protein was detected by goat anti-mouse antibody conjugated to polymer-hrp (dako), visualized by AlexaFluor 488 labeled tyramide (tyramide) (Invitrogen). Snail proteins were detected by biotinylated goat anti-rabbit antibody (VECTOR) and visualized by AlexaFluor 568-labeling streptavidin (Invitrogen). These cells and specimens were also stained with dapi (invitrogen).

(5) Flow cytometry analysis

CSCs were incubated with labeled antibodies and analyzed using EPICS ALTRA (Beckman Coulter) and facscalibur (becton dickinson). The antibodies used were a PE-labeled mouse anti-human CD133 antibody (Miltenyi Biotec), a PE-labeled mouse anti-human CD44 antibody (BD Pharmingen), a FITC-labeled mouse anti-human CD326 (EpCAM) antibody (Miltenyi Biotec), a PE-labeled mouse anti-human CD166 antibody (R & D Systems), a PE-labeled mouse anti-human CD24 antibody (BD Pharmingen), a PE-labeled mouse anti-human CD26 antibody (BD Pharmingen) and a PE-labeled mouse anti-human CD29 antibody (BD Pharmingen).

To stain Lgr5, CSCs were incubated with mouse anti-human Lgr5 antibody (2T 15E-2) followed by PR-labeled mouse anti-mouse IgG antibody (Invitrogen). The activity of aldehyde dehydrogenase was measured using an AldeFluor kit (Stemcell Technologies). Mouse cells and human CSCs were distinguished by staining with anti-mouse MHC class I antibodies (Abcam) and goat anti-human IgG2a antibodies (BioLegend) labeled with PE or APC. Dead cells were also removed by 7-AAD viability dye (Beckman Coulter).

These antibodies were highly specific for Lgr5, and did not cross-react with both Lgr4 and 6, which have high homology to Lgr5 (fig. 18 and 19). By using these antibodies, the present inventors confirmed that adherent cancer stem cells express Lgr 5.

[ reference example 2] tumor reconstitution Capacity of Lgr 5-positive and 5-negative Large intestine CSCs

If the population of stem cells for colorectal cancer is characterized by Wnt signaling, only Lgr 5-positive adherent cells are able to form tumors in vivo. To confirm its authenticity, the inventors investigated the tumorigenicity of both Lgr 5-positive adherent cells and Lgr 5-negative suspension cells.

As a result, it was confirmed that although adherent cells having a tumor formation activity of Lgr 5-positive were stronger than those of Lgr 5-negative suspension cells, both Lgr 5-positive cells and Lgr 5-negative cells maintained tumor formation ability in NOG mice. Tumors were generated at all injection sites (6 out of 6) by subcutaneous injection of 10 Lgr5 positive cells, whereas in Lgr5 negative cells tumors formed at 2 out of 6 injection sites (cells from PLR 123) or 1 (cells from PLR 59) (table 6).

[ Table 6]

It is to be noted that, even with only 1 cell injected per inoculation site for Lgr5 positive cells, tumors were reconstructed at 2 (cells from PLR 123) or 1 (cells from PLR 59) of the 12 injection sites (fig. 20), and the histopathological morphology of tumors from Lgr5 positive and Lgr5 negative cells was substantially the same as the original tumor (fig. 21). And the expression of cell surface markers and tumorigenic activity of Lgr 5-positive CSCs did not change even after 1 month of subculture (fig. 22, 23).

From these results, it was confirmed that Lgr5 positive cells and Lgr5 negative cells derived from PLR59 and PLR123 are high-purity large intestine CSCs, and that Lgr5 positive and Lgr5 negative cells are CSCs of 2 different states of large intestine cancer.

[ reference example 3]TCF andβeffect of catenin

Western blot analysis was performed by the following method. Proteins were extracted using RIPA buffer (Sigma) supplemented with Complete Mini protease inhibitor cocktail (Roche). Proteins were separated on NuPAGE gel (Invitrogen) and transcribed onto PVDF membrane. Blocking with PBS containing 1% skim milk, and subjecting the membrane to rabbit antihuman treatmentβCatenin antibody (Sigma), rabbit anti-human c-JUN antibody (Sigma), rabbit anti-human TCF1 antibody (Cell Signaling), rabbit anti-human TCF3 antibody (Cell Signaling), rabbit anti-human TCF4 antibody (Cell Signaling), rabbit anti-human Lgr5 antibody (Abcam), mouse anti-human E cadherin antibody (Abcam), rabbit anti-human glusulin antibody (Abcam), and mouse anti-human GAPDH antibody (Santa Cruz) detection. The bands of reactivity were detected using BCIP/NBT matrix (KPL).

Consistent with expression of Lgr5, in Lgr5 positive cellsβThe levels of-catenin, TCF1, TCF3, and TCF4 proteins were up-regulated, but not seen in Lgr5 negative cells (fig. 20 and 24).

On the other hand, phosphorylation of the N-terminal region of c-Jun was not detected in Lgr5 positive cancer stem cells, compared with Lgr5 negative cancer stem cells (fig. 20 and 24).

To solve the question whether Wnt signaling drives the proliferation of large intestine cancer stem cells, the present inventors studied as a method for determining whether Wnt signaling drives the proliferation of large intestine cancer stem cellsβFH535 and as Wnt/TCF inhibitorβCardamomin (Induction) of catenin inhibitorsβDegradation of catenin) on the proliferation of large intestine cancer stem cells. Cell proliferation was evaluated by the following method. Suspending cancer stem cells and adherent cancer stem cells to suspend about 100 cancer stem cells and 1 × 10 adherent cells per well, respectively⁴One was inoculated in a 96-well plate. On days 0 and 3, the number of viable cells was determined by Cell Counting Kit-8 assay (Doujindo) according to the manufacturer's protocol. The average absorbance on day 0 was expressed as 100%. For chemosensitivity analysis, suspended cancer stem cells and adherent cancer stem cells were cultured at approximately 100 and 1 × 10 adherent cells per well, respectively⁴One was inoculated in a 96-well plate, incubated for 24 hours, and 10 was addedμg/mL of 5-FU (Hospira), 10μg/mL irinotecan (Hospira), 50mM TCF inhibitor FH535 (Merck), and 50mM as a combined preparationβCardiganine (Merck), a catenin inhibitor. After 3 days of culture in the presence of the drug, cell counting kit-8 was added to the cells. The average absorbance of cells exposed to DMSO or medium only was expressed as 100%. All experiments were performed 3 times each.

As a result, 50μFH535 by M significantly reduced the proliferation of Lgr5 positive large intestine cancer stem cells, while it did not affect the proliferation of Lgr5 negative large intestine cancer stem cells (fig. 25 and fig. 3)26). On the other hand, 50μCardamomin M reduced the number of viable cells to 70% in large intestine cancer stem cells positive to Lgr5 and to about 50% in large intestine cancer stem cells negative to Lgr5 (fig. 25 and 26).

The results show that: TCF-mediated proliferation of Lgr5 positive cells andβcatenin is involved in the survival of large bowel cancer stem cells. Interestingly, Lgr5 positive cells proliferated even without EGF and FGF supply (fig. 27 and 28), showing: large bowel cancer stem cells contain endogenous/autocrine mechanisms that are involved in their proliferation and are used to activate Wnt signaling.

[ reference example 4] ability of Large intestine cancer Stem cells to alternate from Lgr 5-positive state to Lgr 5-negative state

Since one of the characteristics of cancer stem cells is resistance to chemotherapeutic agents, the present inventors investigated the sensitivity of large intestine cancer stem cells to 5-FU and irinotecan. As described above, while Lgr 5-positive cells proliferate in about 2.5 days of doubling time, Lgr 5-negative cancer stem cells are in a quiescent state from the viewpoint of proliferation. In both cases, the treatment with 5-FU (10 μ g/ml) and irinotecan (10 μ g/ml) significantly inhibited the proliferation of large intestine cancer stem cells positive to Lgr5, while the proliferation and survival of large intestine cancer stem cells negative to Lgr5 were not affected (fig. 29 and 30). After exposure of Lgr5 positive large intestine cancer stem cells to 5-FU (10. mu.g/ml) or irinotecan (10. mu.g/ml) for 3 days, cells resistant to these chemotherapeutic agents appeared. Surprisingly, the drug resistant cells were negative for Lgr5 and their morphology changed (fig. 31, 32 and 33), showing: the change from the positive status of Lgr5 to the negative status of Lgr 5.

In order to confirm whether or not an Lgr 5-negative large intestine cancer stem cell has changed to an Lgr 5-positive state, the present inventors have started cell growth when an Lgr 5-negative large intestine cancer stem cell prepared by irinotecan treatment is cultured adherent again in a serum-free stem cell culture medium, and the cell becomes Lgr 5-positive and shows a mesenchymal-cell-like morphology (fig. 34 and 35). On the other hand, when Lgr 5-positive adherent large intestine cancer stem cells were cultured in ultra-low attachment plates, the present inventors observed that several cells stopped growing, forming a spheroid-like structure, showing a very low level of Lgr5 mRNA (fig. 34 and 35).

The mRNA of Lgr5 was evaluated by the quantitative real-time polymerase chain reaction described below. That is, cDNA was synthesized using a first strand cDNA synthesis Kit (SABiosciences) using total RNA isolated using RNeasy Mini Kit (RNeasy Mini Kit inclusion DNA enzyme procedure, Qiagen) including dnase treatment as a template. Quantitative real-time PCR (QRT-PCR) analysis was performed in Mx3005P real-time PCR system (Stratagene) using SYBR Green/Rox qPCR (SAbiosciences). The value of the induction magnification was calculated by the 2-. DELTA.Ct method. GAPDH and ACTB were used as references. All experiments were performed 3 times each.

As primers for quantitative real-time PCR analysis, the following primers were used for amplification of the reactive transcription products.

Lgr5：

A forward primer 5'-AGTTTATCCTTCTGGTGGTAGTCC-3' (SEQ ID NO: 1),

Reverse primer 5'-CAAGATGTAGAGAAGGGGATTGA-3' (SEQ ID NO: 2),

GAPDH：

A forward primer 5'-CTCTGCTCCTCCTGTTCGAC-3' (SEQ ID NO: 3),

Reverse primer 5'-ACGACCAAATCCGTTGACTC-3' (SEQ ID NO: 4),

ACTB：

A forward primer 5'-AAGTCCCTTGCCATCCTAAAA-3' (SEQ ID NO: 5),

Reverse primer 5'-ATGCTATCACCTCCCCTGTG-3' (SEQ ID NO: 6)

From the above results, the present inventors concluded that: large intestine cancer stem cells alternate between Lgr 5-positive and Lgr 5-negative states, and such changes do not require extrinsic factors and niche environments.

[ reference example 5] EMT of Lgr 5-Positive Large intestine cancer Stem cells in vitro and in vivo

Expression nucleusβMesenchymal-like cells of catenin are considered to be EMT-experienced, migratory Cancer stem cells and metastatic forming Cancer stem cells (Brabletz T, Jung A, SpadernaS, Hlubek F, Kirchner T (2005) Opinion: migratory Cancer cells-an integrated concentration of a macromolecular tumor progression. Nat Rev Cancer 5: 744-749.). Since the morphology of the large intestine cancer stem cells positive for Lgr5 is similar to that of mesenchymal cells, the present inventors have conducted experiments as to whether or not the large intestine cancer stem cells positive for Lgr5 correspond to metastatic cancer stem cells. By western blot analysis, low levels of cell surface E-cadherin, high levels of glusulin, and nuclear localization in Lgr5 positive large intestine cancer stem cells were confirmedβExpression of catenin, a feature of EMT (fig. 36, 37 and 38). In contrast, Lgr 5-negative large intestine cancer stem cells did not show any evidence of EMT, i.e., high expression of cell surface E-cadherin, low expression of glusulin, and none of them were foundβNuclear localization of catenin. Also, it was observed that: in xenograft tumor tissues, snail protein and Lgr5 were expressed simultaneously in cells undergoing EMT in the budding region (fig. 39), which supports the notion that large intestine cancer stem cells positive for Lgr5 are equivalent to migratory stem cells.

Also, the present inventors showed that: large intestine cancer stem cells positive for Lgr5 form tumors in a variety of tissues including lung, liver, lymph nodes, and subcutaneous tissue. Interestingly, tumors that held epithelial tubular structures in the liver, lymph nodes and subcutaneous tissue were reconstituted from intravenous injection of tumor cells until at least 40 days later, but failed to reconstitute in the lung (fig. 40, 41).

Industrial applicability of the invention

The present invention provides a cancer stem cell composition which is homogeneous, does not substantially coexist with cells having carcinogenic activity and cells having no carcinogenic activity, and reproduces the hierarchical structure of cancer tissues, and a method for producing the same. By performing gene expression analysis or proteomics analysis using this homogeneous cell population, it is expected that a target specifically expressed in cancer stem cells can be identified or an activated signal transduction system can be identified. Furthermore, continuous mass production of homogeneous cancer stem cells is possible, and the probability of finding a drug or a diagnostic marker effective for the recurrence or metastasis of cancer, which is the most serious result for cancer patients, is expected to be remarkably improved by high throughput (high throughput) analysis using a drug candidate for cancer stem cells.

Claims

1. A cancer stem cell population which is substantially depleted of cells that are not carcinogenic, and which is characterized by a reproducible cancer tissue hierarchy.

2. The population of cancer stem cells of claim 1, wherein said cancer stem cells are from human tumor tissue.

3. The cancer stem cell population of claim 2, wherein the human tumor tissue is tumor tissue from an epithelial cancer.

4. The population of cancer stem cells of claim 3, wherein the epithelial cancer is pancreatic cancer, prostate cancer, breast cancer, skin cancer, cancer of the digestive tract, lung cancer, hepatocellular cancer, cervical cancer, uterine corpus cancer, ovarian cancer, fallopian tube cancer, vaginal cancer, liver cancer, bile duct cancer, bladder cancer, ureter cancer, thyroid cancer, adrenal cancer, kidney cancer, or other cancers of glandular tissue.

5. The cancer stem cell population of any one of claims 1 to 4, wherein: the population of cancer stem cells is substantially homogeneous.

6. The cancer stem cell population according to any one of claims 1 to 5, wherein the frequency of cancer stem cells in limiting dilution analysis is 1/20 or more.

7. The cancer stem cell population of any one of claims 1 to 6, wherein the cancer stem cell population comprises 1x10⁴More than one cancer stem cell.

8. The cancer stem cell population of any one of claims 1 to 7, wherein the cancer stem cell population is prepared by a method comprising the step of adherently culturing a cell population comprising cancer stem cells.

9. The cancer stem cell population of any one of claims 1 to 8, which is prepared by a method comprising the following steps (1) to (3):

(2) a step of subdividing the prepared cancer cell mass; and

10. The cancer stem cell population of any one of claims 1-9, wherein the non-human animal is any one of a nude mouse, a SCID mouse, a NOD-SCID mouse, a NOG mouse, or a nude rat.

11. A method of preparing a population of cancer stem cells substantially depleted of cells that are not carcinogenic, the method comprising the step of adherently culturing a population of cells comprising cancer stem cells.

12. The method according to claim 11, wherein the cell population containing cancer stem cells is a cell population that reproduces the hierarchical structure of a cancer tissue.

13. The method according to claim 12, wherein the cell population that reproduces the hierarchical structure of cancer tissues is a cell that is positive for a cancer cell line, a spheroid, or at least one or more markers selected from the group consisting of cancer stem cell markers CD24, CD29, CD34, CD44, CD49f, CD56, CD90, CD117, CD133, CD135, CD166, CD184, CD271, CD326, Aldefluor, ABCG2, ABCG5, LGR5, and Msi1 established in a non-human animal.

14. The method of any one of claims 11 to 13, wherein the population of cells comprising cancer stem cells is expanded prior to performing adherent culture.

15. The method according to claim 14, wherein the cell population containing cancer stem cells is proliferated by spheroid culture.

16. The method of claim 14, wherein the population of cells is expanded by transplantation into a non-human animal and passaging.

17. The method of any one of claims 11 to 16, wherein the cancer stem cells are from human tumor tissue.

18. The method of claim 17, wherein the human tumor tissue is tumor tissue from an epithelial cancer.

19. The method of claim 18, wherein the epithelial cancer is pancreatic cancer, prostate cancer, breast cancer, skin cancer, cancer of the digestive tract, lung cancer, hepatocellular cancer, cervical cancer, uterine corpus cancer, ovarian cancer, fallopian tube cancer, vaginal cancer, liver cancer, bile duct cancer, bladder cancer, ureter cancer, thyroid cancer, adrenal cancer, kidney cancer, cancer of other glandular tissues.

20. The method of any one of claims 11 to 19, wherein the non-human animal is any one of a nude mouse, a SCID mouse, a NOD-SCID mouse, a NOG mouse, or a nude rat.

21. A method for searching for a target molecule of a drug, characterized in that a hierarchical structure formed by cancer stem cells, a cancer progression process from cancer stem cells, or a biological characteristic of cancer stem cells is evaluated as an index in a non-human animal model into which a cancer stem cell group according to any one of claims 1 to 10 has been transplanted or in a culture system of the cancer stem cell group under in vitro conditions.

22. The method for searching for a target molecule of a drug according to claim 21, comprising the steps of (1) to (4) below:

(1) a step of preparing a non-human animal model by transplanting the cancer stem cell population according to any one of claims 1 to 10 into a non-human animal;

23. The method for searching for a target molecule of a drug according to claim 21, comprising the steps of (1) to (3) below:

(1) culturing the cancer stem cell population according to any one of claims 1 to 10 under in vitro conditions, and reconstructing a characteristic structure of a cancer progression process from the cancer stem cells or a biological characteristic of the cancer stem cells;

24. The method of any one of claims 21 to 23, wherein the drug is an anti-cancer drug.

25. The method of any one of claims 21 to 24, wherein the target molecule is a cancer cell marker.

26. A method for evaluating a drug, which comprises evaluating a hierarchical structure formed by cancer stem cells, a cancer progression process from cancer stem cells, or a biological characteristic of cancer stem cells in a non-human animal model transplanted with a cancer stem cell population according to any one of claims 1 to 10 or in a culture system of the cancer stem cell population under in vitro conditions.

27. The method for evaluating a drug according to claim 26, comprising the steps of (1) to (5) below:

(2) administering a test substance to the non-human animal model of (1);

28. The method for evaluating a drug according to claim 26, comprising the steps of (1) to (4) below:

(2) treating the cultured cells of (1) with a test substance;

29. A method for screening a drug, which comprises evaluating a hierarchical structure formed by cancer stem cells, a cancer progression process from cancer stem cells, or a biological characteristic of cancer stem cells in a non-human animal model transplanted with a cancer stem cell population according to any one of claims 1 to 10 or in a culture system of the cancer stem cell population under in vitro conditions.

30. The method for screening a drug according to claim 29, which comprises the steps of (1) to (5) below:

(2) administering a test substance to the non-human animal model of (1);

31. The method for screening a drug according to claim 29, which comprises the steps of (1) to (4) below:

(2) treating the cultured cells of (1) with a test substance;

32. The method of any one of claims 26 to 31, wherein the drug is an anti-cancer drug.

33. The method of any one of claims 21, 23, 26, 28, 29 and 31, wherein the culture system under in vitro conditions is a spheroid culture.