JP6284147B2 - Establishment method of mouse bone marrow breast cancer cell line with high bone marrow metastasis of breast cancer - Google Patents

Description

  The present invention relates to a method of establishing a bone marrow highly metastatic mouse breast cancer cell of breast cancer, the established cell, an animal model mouse for bone marrow metastasis analysis of breast cancer transplanted with the cell, and a screening for a bone marrow metastasis inhibitor of breast cancer comprising the cell. The present invention relates to cell material and screening method.

Breast cancer, which has recently increased in number among Japanese women, causes metastasis to the bone marrow, lungs, liver, etc. at a high rate, which causes significant distress to the patient, lowers the patient's quality of life (QOL), and becomes a major social problem. Nevertheless, the elucidation of the mechanism of breast cancer metastasis is partly due to the absence of animal models of metastasis to these tissues. Such animal models of metastasis are also indispensable for the development of screening for novel breast cancer bone marrow metastasis inhibitors.
As a metastasis model of breast cancer that has existed so far, a model in which human breast cancer (MDA-231) is transplanted into nude mice has been widely used (Non-patent Document 1).
However, since nude mice have a low immune mechanism, it is not possible to analyze the interaction between cancer cells and the host immunocompetent cells (Non-Patent Document 2), which has recently been attracting attention. Although this model is widely used, bone marrow metastasis does not occur unless the mouse is anesthetized and cells are administered to the left ventricle. Therefore, there is a problem that administration is very skillful.
On the other hand, 4T1 breast cancer cells often used in mouse models are known to have properties similar to human breast cancer (Non-patent Document 3), but their ability to metastasize to other tissues of mice is not necessarily high ( Non-patent document 3).

The present inventors previously established 4T1E / M3 breast cancer cells having high metastatic potential to the lung and / or liver together with the bone marrow using the 4T1 breast cancer cells as a parent strain, and transplanted the 4T1E / M3 breast cancer cell line to the bone marrow of breast cancer. A mouse model for lung and / or liver metastasis analysis was prepared (Patent Document 1, Non-Patent Document 4). And, by comprehensive analysis of the expressed genes for this 4T1E / M3 strain, the expression of ICAM-1 adhesion molecule and bone morphogenetic factor (BMP7) was significantly enhanced compared to the 4T1 breast cancer cell line used as the parent strain, It has been found that the expression level of monocyte chemotactic factor (CCL2 = MCP1) is decreased, and reports that these factors play an important role in cancer cell metastasis (Non-patent Document 4). ~ 6).
However, when this 4T1E / M3 strain was administered to the mouse tail vein, the metastasis rate to the bone marrow (vertebral bone) was 77%, which was significantly higher than 14% of the parent cell line 4T1E. The metastasis rate when transplanted subcutaneously to mice was as low as 20% (Non-patent Document 4). When the former cancer is administered intravenously, the initial process of entering the nearby blood vessel from the site where the cancer originally occurred (primary focus) is also skipped, so the latter when transplanted subcutaneously, Since it is a model for analyzing metastasis to the bone marrow that is closer to actual metastasis, it is desirable that the bone marrow be highly metastatic even when transplanted subcutaneously. Therefore, in order to produce an excellent bone marrow metastasis analysis model animal, establishment of breast cancer cells having high bone marrow metastasis ability even when transplanted subcutaneously has been eagerly desired.

Japanese Patent No. 4936379

Bandyopadhyay A, Elkahloun A, Baysa SJ, Wang L, Sun LZ.Development and gene expression profiling of a metastatic variant of the human breast cancer MDA-MB-435 cells.Cancer Biol Ther 2005; 4: 168-74. Condeelis J, Pollard JW. Macrophages: obligate partners for tumor cell migration, invasion, and metastasis.Cell 2006; 124: 263-6. Aslakson CJ, Miller FR.Selective events in the metastatic process defined by analysis of the sequential dissemination of subpopulations of a mouse mammary tumor.Cancer Res 1992; 52: 1399-405. Clin Exp Metastasis. (2008) 25: 517-529 Clin Exp Metastasis. (2009) 26: 817-828 Clin Exp Metastasis. (2012) 29: 327-338

  An object of the present invention is to further improve the mouse 4T1E / M3 breast cancer cell line having a high metastatic ability to bone marrow and other tissues such as lung and liver, and to provide a breast cancer cell line having a high bone marrow metastatic ability even when subcutaneously transplanted. An object is to establish a method for establishing a mouse bone marrow high metastasis mouse breast cancer cell line for breast cancer, and to provide a model animal for bone marrow high metastasis of breast cancer using the established strain. It is another object of the present invention to provide a screening method for a bone marrow metastasis inhibitor for breast cancer transplanted with the established strain.

In order to solve the above-mentioned problems, the present inventor uses the 4T1E / M3 breast cancer cell line derived from mouse breast cancer cell 4T1 strain (Patent Document 1, Non-Patent Document 4) as a parent strain, and has a high bone marrow metastasis ability even when subcutaneously transplanted. An attempt was made to establish a breast cancer cell line having high malignancy. As a result of various trials and errors, first, it penetrates into the bottom surface of the inner well having the microwell of the double well, collects the cells that have passed through the hole and dropped into the lower well, and then repopulates the inner well after culturing. Repeat in vitro to select cells with high motility in the vertical direction in vitro, then subcutaneously administer to mice to collect bone marrow cells from vertebrae, and subculture the drug resistant strain in the cells Thus, it was possible to establish a breast cancer cell line having high bone marrow metastasis ability even by subcutaneous transplantation. When the obtained breast cancer cell line is subcutaneously transplanted into a mouse, it is confirmed that bone marrow metastasis occurs with a high probability of almost 100%, and the obtained bone marrow metastasis mouse becomes an excellent model mouse for bone marrow metastasis analysis of breast cancer. The breast cancer cells and the subcutaneously transplanted mice are also extremely useful as a screening tool for breast cancer bone marrow metastasis inhibitors.
Furthermore, the present inventors conducted a comprehensive analysis of the expressed genes for the breast cancer cell line, and found that the expression of the Cadherin 17 gene was significantly increased compared to the parent line (4T1E / M3 line). Cadherin17 has been reported to be a marker of cancer malignancy in various cancers, but there has been no report on breast cancer. Cadherin17 is the first marker of malignancy of breast cancer bone marrow metastasis. It became clear.
By obtaining the above knowledge, the present invention has been completed.

That is, the present invention is as follows.
[1] A method for establishing a bone marrow highly metastatic mouse breast cancer cell line, which comprises using the mouse breast cancer cell 4T1E / M3 cell line as a parent line and including the following steps (a) to (d):
(A) A mouse breast cancer cell 4T1E / M3 cell line into which a drug resistance gene has been introduced is sown onto the inner well of a double well having a plurality of microholes at the bottom of the inner well, and the lower well is passed through the hole. The cells that have fallen in are collected and plated on the inner well again after culturing. A process of collecting and culturing cells that have fallen into the lower well after repeating this series of operations 8 to 12 times,
(B) a step of administering the obtained cells subcutaneously to a mouse;
(C) collecting mouse vertebrae, collecting bone marrow cells, culturing in a medium containing a target drug to be given resistance by the drug resistance gene, and collecting drug resistant cells;
(D) A step of culturing the obtained drug-resistant cells in a normal medium, stabilizing the character, and establishing a bone marrow highly metastatic mouse breast cancer cell line.
[2] The method for establishing a bone marrow highly metastatic mouse breast cancer cell line according to [1], further comprising the following step (e):
(E) A step of repeating steps (b) to (d) using the established cell line obtained in step (d).
[3] A bone marrow highly metastatic mouse breast cancer cell line established by the establishment method of [1] or [2].
[4] Bone marrow highly metastatic mouse breast cancer cell line according to [3] above, wherein Cdhn17 (cadherin17) is expressed at least 10 times higher than the parent cell (4T1E / M3 cell line) .
[5] An animal model mouse for bone marrow metastasis analysis of breast cancer, obtained by transplanting the bone marrow highly metastatic mouse breast cancer cell line according to [3] or [4] to a mouse by subcutaneous administration or intravenous injection.
[6] A method for screening a test substance that is a candidate for a bone marrow metastasis inhibitor of breast cancer, comprising the following (a) to (c);
(A) administering a test substance to the bone marrow highly metastatic mouse breast cancer cell line according to claim 3 or 4, and culturing;
(B) One or more of the anchorage-independent growth ability, vertical motility, and intracellular cadherin17 expression level of the cell line after administration were measured, and the measured values were measured in advance. Comparing the measured value in the cell line before administration;
(C) A step of evaluating and selecting a test substance as a candidate substance for a bone marrow metastasis inhibitor for breast cancer when the measured value after administration is significantly reduced as compared with the measured value before administration.
[7] The test substance in the step (a) is a test peptide or a test nucleic acid molecule, and administration of the test substance to the cell line is performed by adding a nucleic acid encoding the test peptide or the test nucleic acid molecule. The method according to [6] above, wherein the method is by introduction into cells.
[8] A method for screening a test substance that is a candidate for a bone marrow metastasis suppressant for breast cancer, wherein the test substance is obtained from a target mouse with high bone marrow metastasis according to [3] or [4]. The mouse is administered together with or before or after administration of the sexual mouse breast cancer cell line, and the mouse after administration is sacrificed after breeding for a certain period of time, and its ability to metastasize to the bone marrow A screening method for a bone marrow metastasis inhibitor for breast cancer, characterized by selecting a test substance that has been evaluated with the presence or absence or degree as an index and that has significantly suppressed metastasis to the bone marrow as a candidate for a bone marrow metastasis inhibitor for breast cancer .
[9] A method for screening a test substance that is a candidate for a bone marrow metastasis inhibitor of breast cancer, the method comprising the following (a) to (d);
(A) A test substance is administered to the subject mouse together with the bone marrow highly metastatic mouse breast cancer cell line according to claim 3 or 4 or before or after administration of the cell line, and the mouse after administration is fixed. The process of rearing the period,
(B) sacrificing the mouse obtained in step (a) and measuring the metastasis rate to the bone marrow and / or the area of the metastasized cancer cell region in the bone marrow tissue,
(C) The bone marrow highly metastatic mouse breast cancer cell line is administered without administering the test substance, the mouse after administration is bred for the same period as in step (a), and the resulting mouse is sacrificed to the bone marrow. Measuring the metastasis rate and / or the area of the metastatic cancer cell region in the bone marrow tissue,
(D) The measured value obtained in step (b) is compared with the measured value obtained in step (c). If the measured value is significantly decreased, the test substance is a candidate for a bone marrow metastasis inhibitor for breast cancer. The process of evaluating a substance.
[10] A screening kit for a bone marrow metastasis inhibitor for breast cancer, comprising the bone marrow highly metastatic mouse breast cancer cell line described in [3] or [4].
[11] The screening kit according to [10], further comprising at least one of the following (1) to (4):
(1) a soft agar medium for measuring the growth-independent growth ability of the cell line,
(2) a well provided with a plate having a plurality of microholes at the top for measuring the motility in the vertical direction of the cell line;
(3) a device for measuring the expression level of Cadherin17 in the cell line,
(4) A mouse to be transplanted with the cell line and a transplantation device.

  The mouse breast cancer cells established according to the present invention have a very high bone marrow metastasis compared to the parental mouse breast cancer cell 4T1E / M3 strain, and a high bone marrow that causes bone marrow metastasis with a very high probability of 100% just by subcutaneous implantation. A metastatic mouse breast cancer cell line. In addition, since the above-mentioned parent strain possesses properties similar to those of human breast cancer, the mouse transplanted with the high bone marrow metastatic mouse breast cancer cell of the present invention is more human than the conventional mouse administered with human breast cancer cells. Combined with the lack of involvement of the mouse immune system against breast cancer cells, it becomes an extremely excellent animal model for bone marrow metastasis analysis of breast cancer and is an effective means for analysis of bone marrow metastasis of breast cancer.

  That is, a mouse administered with the highly metastatic mouse breast cancer cell of the present invention can analyze changes in the biologically produced protein from the administration of the cell to death, or can be analyzed from each stage of the mouse to a highly metastatic mouse breast cancer cell. It can be an effective means for elucidating the mechanism of the metastasis to the bone marrow and its cause through comparison of the gene expression state with the parental strain or highly metastatic mouse breast cancer cells before administration.

On the other hand, since the bone marrow highly metastatic mouse breast cancer cell of the present invention can form a metastatic cancer in the bone marrow with a very high probability only by subcutaneous administration to the mouse, the metastasis of the breast cancer can be easily and quickly performed in the mouse bone marrow. A state can be created. Inhibition of breast cancer metastasis by administering a candidate substance for a breast cancer metastasis suppressant to a mouse simultaneously with or before or after transplantation of bone marrow highly metastatic mouse breast cancer cells of the present invention. It becomes possible to perform screening of agents efficiently.
Further, the bone marrow highly metastatic mouse breast cancer cell of the present invention exhibits remarkable activity even in the anchorage-independent growth ability and the vertical motility, and therefore, using these growth ability and motility as an index, a breast cancer metastasis inhibitor candidate Can be used as a cell material or kit for in vitro screening.
Furthermore, it was revealed that the high bone marrow metastatic mouse breast cancer cell of the present invention has a significantly high expression of the Cadherin17 gene even when compared with the parent strain (4T1E / M3 strain), and Cdhn17 (Cadherin17) is a bone marrow metastasis of breast cancer. Since it has been found to be used as a (malignancy) marker, in vitro screening for a breast cancer metastasis inhibitor candidate can also use a change in the expression level of Cdhn17 as an index.

Phase contrast micrographs of parent cell 4T1E / M3 strain (EM3) and FP10SC1 and FP10SC2 cells of the present invention. Images were taken with an objective lens 20x and an eyepiece 10x. 4T1E / M3 cells, FP10SC1 cells, and FP10SC2 cells were seeded on a 96-well plate at 3 × 10 ³ cells / well, and MTT reagent 10 uL / well was added on that day and 1 to 4 days later, at 37 ° C., 5% CO ₂ for 4 hours. After culturing, normal proliferation ability was measured by measuring absorbance at 450 nm. There was almost no difference in normal proliferation ability among 4T1E / M3 cells, FP10SC1 cells, and FP10SC2 cells. After 5 mL / dish of 0.5% soft agar medium, 4T1E / M3 cells, FP10SC1 cells, and FP10SC2 cells are suspended in a medium containing 0.3% soft agar, and 2 × 10 ⁴ cells / It was sown in a 6 cm dish and the number of colonies formed after 12 days was counted under a microscope. A is the number of colonies and B is a picture of the colonies. Apparently, FP10SC1 cells and FP10SC2 cells had markedly enhanced anchorage-independent growth ability. A chamber (commercially available, chemotaxel) with a polycarbonate membrane with 8um holes on the bottom is placed in a 24-well plate containing 800uL / well of medium, and 2 x 10 ⁴ / well breast cancer cells from above. (4T1E / M3, FP10SC1 and FP10SC2) were sprinkled, and the number of cells falling below the chamber after 24 hours was counted under a microscope. A shows a case where bone marrow-derived endothelial cells are cultured in a monolayer on a polycarbonate membrane and then spreads breast cancer cells, and B shows a case where cells are seeded without endothelial cells. In both cases A and B, the motility of FP10SC1,2 cells in the vertical direction was greatly enhanced. 4T1E / M3 cells, FP10SC1 cells and FP10SC2 cells were each administered subcutaneously to mice (BALB / c female, 8 weeks old) at 1 × 10 ⁶ cells / mouse, and vertebrae were collected on day 25 and tissue sections were prepared. HE (hematoxylin and eosin) staining. The micrographs of objective lens 4x and 40x are shown. Cancer cells have a large cytoplasm and nucleus and can be distinguished from normal cells. Comparison of mRNA expression levels of Cadherin 17 analyzed by extracting RNA from 4T1E / M3 cells, FP10SC1 cells, and FP10SC2 cells and performing real-time RT-PCR. Cadherin17 gene expression was enhanced in FP10SC1,2 cells.

Hereinafter, the present invention will be described in more detail.
1. Bone marrow highly metastatic mouse breast cancer cell FP10SC strain of the present invention and its establishment method (1-1) Mouse breast cancer cell 4T1E / M3 used as parent strain in the present invention Mouse breast cancer cell 4T1E / M3 (hereinafter simply referred to as 4T1E / M3 cells) are derived from mouse breast cancer cells 4T1 (hereinafter also simply referred to as 4T1 cells), and 4T1 cells (ATCC: CRL-2539) are available from ATCC (American Type Culture Collection). is there. The 4T1 cell-derived 4T1E / M3 cells inherit the properties of the parental 4T1 cells, such as metastatic ability, proliferative ability, and immunological characteristics, and in addition to the ability to metastasize to the bone marrow, lung and liver tissues It is characterized by high metastatic potential.
4T1E / M3 cells can be established according to the method described in Patent Document 1 or Non-Patent Document 4. Specifically, it is obtained by the following methods (a) to (c).
(A) First, mouse breast cancer cells 4T1 into which a drug resistance gene has been introduced are intravenously injected from the tail vein of the mouse (1 × 10 ⁶ cells / mouse). After 10 to 12 days, the mouse is sacrificed and the femur is removed. Bone marrow is collected, and cells are collected from the bone marrow.
(B) Next, the cells collected from the bone marrow are cultured in a medium containing a drug to be given resistance by the drug resistance gene, and a drug-resistant strain that survives in the medium is collected.
(C) The drug resistant cells obtained in the above process (b) are again subjected to the intravenous injection process from the mouse tail vein, and the cell collection process from the bone marrow of the femur and the cell selection process using the drug in (b) are repeated. These series of operations are repeated a plurality of times, for example, 3 times or more to obtain highly metastatic mouse breast cancer cells 4T1E / M3 cells in which 4T1 cells are mutated.
Examples of the drug resistance gene used in the present invention include antibiotic resistance genes such as neomycin resistance gene, zeocin resistance gene, and blasticidin resistance gene, but there is no particular limitation. A supported plasmid or the like can be appropriately used, and the resistance gene introduction method may be in accordance with a conventional method, for example, in accordance with a protocol attached to a commercially available product. When the neomycin resistance gene is used as a drug resistance gene, the selection medium contains G418 or the like, and when a zeocin resistance gene or a blasticidin resistance gene is used, zeocin, blasticidin or the like is contained in the medium.
Compared with its parental 4T1 cell, 4T1E / M3 cells have dramatically increased bone marrow metastatic potential, and the bone marrow metastasis rate when administered to the mouse tail vein is as high as 77%. . However, the bone marrow metastasis rate when administered subcutaneously to mice is as low as 20%. Considering the metastasis process of cancer, cancer enters a nearby blood vessel from the site where it originally occurred (primary focus), goes around the blood vessel to other organs, and then enters the metastasis destination organ and proliferates. follow. Intravenous administration of cancer skips the first stage of metastasis, so subcutaneous administration is closer to actual metastasis. In other words, in order to be truly highly malignant highly metastatic cancer cells, even when administered subcutaneously, it is necessary to be highly metastatic cancer cells in the bone marrow, and in that sense, bone marrow metastasis activity of 4T1E / M3 cells ( Malignancy) was not enough.

(2-2) Method for Establishing Bone Marrow Metastatic Mouse Breast Cancer Cell FP10SC of the Present Invention The bone marrow highly metastatic mouse breast cancer cell FP10SC cell of the present invention uses the 4T1E / M3 cell as a parent cell, and the following steps (a) to Established by (c) or further by step (d).
(A) In vitro selection process of cells with high motility in the vertical direction
A 4T1E / M3 cell line is used as a parent cell line, and an in vitro selection process is provided to select cells with high motility in the vertical direction. Specifically, a plurality of micro-holes, for example, about 6-10 microns, preferably using a double wells with polycarbonate filters holes of 8 microns open about 5000-6000 cells / cm ^2, the inside of the filter surface The cells are introduced and the cells that have passed through the holes are collected and cultured. In this example, two types of wells were combined, and a commercially available double well (made by Kurabo Industries, Chemotaxel) provided with a plurality of micro holes (diameter of about 8 μm) on the inner well bottom (made of polycarbonate film) was used. However, the present invention is not limited to this, and it may be a culture well or plate in which a membrane having a plurality of microholes, preferably a polycarbonate membrane, is placed above the culture well or plate. The step of seeding the obtained cells again on the filter surface is performed 6 times or more, preferably 8 to 12 times. This process may be further repeated, but the increase in mobility reaches a peak at the stage where the process is repeated 10 times, and therefore 10 times is most preferable from the viewpoint of efficiency. The cells obtained at this stage are also referred to as “FP10 cells”.
Even in the FP10 cells at this stage, the bone marrow metastasis rate when subcutaneously administered to the back of the mouse was 60%, which was significantly higher than that of the parent strain 4T1E / M3 cells.
In the present invention, “bone marrow metastasis rate” refers to the ratio of the number of mice in which metastatic cancer is observed in vertebral bone marrow tissue after a lapse of a certain number of days to the total number of mice administered with cancer cells. Specifically, the target cancer cells were administered subcutaneously on the back of the mouse (1 × 10 ⁶ cells / mouse), and the treated mice were sacrificed 22-24 days later, and tissue sections of bone marrow tissue collected from the vertebrae of the mice were obtained. The metastatic state was observed visually with a microscope, and the ratio of bone marrow metastasized cancer mice to all administered mice was measured.

(B) Mouse subcutaneous administration step and bone marrow cell collection step
(a) The FP10 cells obtained in the step are subcutaneously administered to mice, and after 20 to 26 days have elapsed (depending on the type and age of the mice, for example, in the case of BALB / c mice (female 8 weeks old), 22 to 24 days later is preferred.) Mice are sacrificed, their vertebrae are collected and bone marrow cells are collected, and bone marrow cells are collected from the bone marrow.
(C) Cell selection step using a drug The recovered bone marrow cells are cultured for 8 to 14 days, preferably 10 to 12 days, in a medium containing a drug (for example, G418 in the case of a neomycin resistance gene) to which resistance is imparted by a drug resistance gene. Culture and collect drug-resistant strains that survive in the medium. The drug-resistant strain obtained here is a stable strain whose trait does not change even if culture is continued, and a bone marrow highly metastatic mouse breast cancer cell FP10SC1 cell line can be established.
(D) Further, the FP10SC1 cell line is again subjected to the subcutaneous administration step to the mouse in (b), the cell collection step from the vertebral bone marrow, and the cell selection step with the drug in (c). The drug-resistant strain obtained here is a stable strain whose trait does not change even if culture is continued, and a bone marrow highly metastatic mouse breast cancer cell FP10SC2 cell line can be established.
Although a series of these operations may be repeated, both the FP10SC1 cell line and the FP10SC2 cell line have a bone marrow metastasis rate of 100% (Table 1), and the vertical motility is higher than that of the FP10SC1 cell line. In view of the fact that cell lines have fallen, the in vivo selection process is preferably 1 to 2 times.
The FP10SC1 strain, FP10SC2 strain or the bone marrow highly metastatic mouse breast cancer cell line established by repeating the steps (b) and (c) is also referred to as “4T1E / M3 / FP10SC cell line” or simply “FP10SC cell”.

(2-3) Characteristics of the FP10SC cell line of the present invention The bone marrow highly metastatic mouse breast cancer cell FP10SC cell of the present invention has an extremely high bone marrow metastasis compared to the parental mouse breast cancer cell 4T1E / M3 line, and is subcutaneous. It is a highly malignant breast cancer cell line that causes bone marrow metastasis with a very high probability of 100% just by transplanting.
In addition, the normal proliferation ability is similar to that of the parent cell (4T1E / M3 strain), but the anchorage-independent proliferation ability is 7 to 20 times that of the parent cell (FIG. 3).
Here, as the anchorage-independent growth ability, the growth ability in a medium containing 0.3% soft agar was observed. Specifically, 2 × 10 ⁴ test cancer cells were seeded per dish in a medium containing 0.3% soft agar and cultured for 12 days, and the number of colonies found in each dish was counted under a microscope.
In addition, when the motility in the vertical direction was measured, it was 3 to 6 times that of the parent cell, both with and without endothelial cells (FIG. 4).
Here, the vertical motility measurement method is as follows: 4 × 10 ⁴ test cancer cells per well are inoculated from the top of the polycarbonate membrane with microholes inside the double well, and 24 hours later. The number of cells falling under the chamber was counted under a microscope. At that time, in order to measure the motility in the vertical direction through the endothelial cells, bone marrow-derived endothelial cells are cultured in a monolayer on a polycarbonate membrane, and test cancer cells are sprinkled from the monolayer.

(2-4) Highly expressed genes in the FP10SC cell line of the present invention By comprehensive analysis of the expressed genes for the FP10SC cell line of the present invention, the expression level of the gene is higher than that of the parent cell (mouse breast cancer cell 4T1E / M3 line). As a result of performing quantitative RT-PCR on the gene that was greatly changed, it was revealed that the expression of Cdhn17 (Cadherin17) was particularly greatly enhanced (FIG. 6). The FP10SC cell line of the present invention is a bone marrow highly metastatic mouse breast cancer cell in which Cdhn17 is expressed more than 10 times, preferably 10 to 20 times higher than the parent cell (mouse breast cancer cell 4T1E / M3 strain). It can also be expressed as being.
Cdhn17 (cadherin17) belongs to the cadherin superfamily, which is a glycoprotein that regulates cell adhesion, but has seven cadherin domains and the intracellular domain has only about 20 amino acid residues. The structure is different from cadherin. Cdhn17 is known to function as a peptide transporter and adhesion molecule, but recently, its involvement in cancer metastasis and cancer malignancy has been reported in liver cancer, stomach cancer, colon cancer, etc. (Biochimica et Biophysica Acta 1806, 138-145, 2010). Furthermore, in highly metastatic colon cancer, Cdhn17 increased cell adhesion and proliferation, and suppression of Cdhn17 expression suppressed colon cancer cell proliferation and liver metastasis (Oncogene 2013 Apr 22 on line ( doi: 10.1038 / onc.2013.117)) and suppression of Cdhn17 expression suppresses proliferation, adhesion, movement and invasion of gastric cancer cells (PLOS ONE 2013 8, 3, e56959), and anti-Cdhn17 antibody is Reports that cancer growth and metastasis to the lung were suppressed (PLOS ONE 2013, 8, 9. e72386), significantly increased Cdhn17 expression in cancer tissues of gastric cancer patients compared to non-expression A report of low survival (Ann Surg Oncol 2012, 19, 1529-1534) and a correlation between Cdhn17 expression and cancer malignancy in tissue sections of ovarian cancer patients (Int J Gynecol Cancer 2012, 22 1170-1176), but there is no report examining the role of Cdhn17 in breast cancer.
In the present invention, it has been found for the first time that Cdhn17 (cadherin17) is used as a marker for bone marrow metastasis (grade of malignancy) of breast cancer.

(2-5) Production of a model mouse for bone marrow metastasis analysis of breast cancer
Unlike mice inoculated with human breast cancer cells, mice with bone marrow metastasis transplanted with FP10SC cells do not activate the mouse immune system against heterologous cells. Thus, the mouse is also useful as an animal model for analyzing bone marrow metastasis of breast cancer.
When preparing a model mouse for bone marrow metastasis analysis of breast cancer, the administration form of FP10SC cells to the mouse may be intravenous injection or subcutaneous transplantation, but subcutaneous transplantation is preferred, and back or abdominal subcutaneous transplantation is particularly preferred. In the case of intravenous injection using a mouse tail vein or the like, intravenous injection from a mouse tail vein or the like is preferable.
Usually, in the case of mouse tail vein administration, if 5 × 10 ⁵ or more of FP10SC cells, preferably 1 × 10 ⁶ cells / mouse, are intravenously injected, it usually metastasizes to bone marrow in 5 to 10 days and is subcutaneously transplanted into mice. In this case, if 5 × 10 ⁵ or more, preferably 1 × 10 ⁶ FP10SC cells / mouse are transplanted subcutaneously on the back or abdomen of the mouse, the metastasis usually takes place in 15 to 20 days. However, in the present invention, there is no limitation on the period until these metastases or the number of cells to be administered, etc., and bone marrow metastasis is analyzed using animal models in various states until the mice spontaneously die from cancer. can do.

For example, mice in various states before and after death after analysis or administration of various physiologically active substances, immune system substances production status, or various gene expression status in bone marrow cells immediately before and after metastasis It is possible to collect mouse breast cancer cells that have metastasized to bone marrow cells, compare the expression status of various genes in bone marrow highly metastatic FP10SC cells before administration, and analyze the mechanism and cause of breast cancer bone marrow metastasis Become. It is also possible to perform the same analysis by changing the number of cells to be administered.
Furthermore, various physiologically active substances or inhibitors thereof are administered to mice administered with the above FP10SC cells to change the status of metastasis, changes in the expression of various genes in the mouse, particularly the Cadherin17 gene, or collected bone marrow By observing changes in the gene expression state in metastatic mouse breast cancer cells, it is possible to explore the mechanism of bone marrow metastasis of breast cancer or a method for its suppression through the influence of these physiologically active substances.

(2-6) Screening for bone marrow metastasis inhibitor for breast cancer The FP10SC cell of the present invention is useful as a screening means for a bone marrow metastasis inhibitor for breast cancer.
Specifically, FP10SC cells of the present invention and a bone marrow metastasis inhibitor candidate substance for breast cancer were administered to mice (1 × 10 ⁶ cells / mouse), and the mice were sacrificed after a certain period of time, Can be screened from the candidate substances used to screen for a bone marrow metastasis inhibitor of the breast cancer. In addition, FP10SC cells can be induced against FP10SC cells in vitro by utilizing the property of anchorage-independent growth ability described in (2-3) above, or the ability of vertical motility through or without endothelial cells. It is also possible to perform screening using an index as to whether a candidate substance for a bone marrow metastasis inhibitor suppresses these abilities. Such a technique is a simple and efficient technique, particularly as a preliminary experimental system for screening for a bone marrow metastasis inhibitor.
Here, when the candidate substance for the bone marrow metastasis inhibitor is a peptide, a low molecular weight compound or the like, it is preferable to add it to the medium of FP10SC cells. However, the gene encoding the candidate peptide may be a normal expression vector such as a mammalian expression vector. It may be a method of introducing into a cell and expressing it using a nucleic acid introduction method into a mammalian cell. When the candidate substance is a nucleic acid molecule such as siRNA, a method of introducing it into a cell together with a known carrier compound can be used.
Furthermore, using Cadherin17 or its gene as a marker and culturing a candidate substance for a bone marrow metastasis inhibitor of breast cancer with FP10SC cells, or introducing a candidate gene or RNAi nucleic acid molecule into FP10SC cells, the expression level of Cadherin17 gene in FP10SC cells Preliminary screening for a bone marrow metastasis inhibitor is also possible by observing whether the decrease is caused by RT-PCR or Western blotting.

  In addition, in such a screening or analysis of bone marrow metastasis mechanism using the above-mentioned highly metastatic mouse breast cancer cells, in addition to the introduction of drug resistance genes, in addition to the introduction of drug resistance genes, If a fluorescent gene such as a luminescent gene or GFP gene is introduced, the state of metastasis can be grasped by luminescence or fluorescence observation, and the accurate transfer rate can be quantitatively grasped by measuring the luminescence intensity or fluorescence intensity. Because it is more efficient.

Examples of the present invention are shown below, but the present invention is not limited to these examples.
Other terms and concepts in the present invention are based on the meanings of terms that are conventionally used in the field, and various techniques used to implement the present invention include those that clearly indicate the source. Except for this, it can be easily and reliably carried out by those skilled in the art based on known documents and the like. In addition, various analyzes were performed by applying the methods described in the analytical instruments or reagents used, kit instruction manuals, catalogs, and the like.
In addition, the description content in the technical literature, the patent publication, and the patent application specification cited in this specification shall be referred to as the description content of the present invention.

[Example 1]
(1-1) Establishment of 4T1E / M3 cell line Mouse breast cancer cell 4T1 line (ATCC: CRL-2539) was obtained from ATCC (American Type Culture Collection), and according to the method described in Patent Document 1 or Non-Patent Document 4, A 4T1E / M3 cell line was established.
Specifically, a pEGFP-F vector (BD Biosciences Clontech) containing a neomycin resistance gene was introduced into the 4T1 strain using the transfection reagent Effecten (QIAGEN), and the obtained 4T1E cells were transferred to BALB. / c mice (female 8 weeks old) were administered with tail vein (1 × 10 ⁶ mice / mouse). Ten to twelve days later, bone marrow cells were collected from bone marrow (femur and tibia), cultured in a medium containing G418 (neomycin derivative, 120ug / mL), and the proliferated cells were again administered to mice three times. 4T1E / M3 cell line was established.

(1-2) Selection of cells with high motility Using the 4T1E / M3 cell line obtained in Example (1-1) as a parent strain, cells with high motility in the vertical direction were selected.
Specifically, a polycarbonate filter with an 8 micron hole is used on the bottom (Chemotaxel, Kurabo Industries), and this is a slightly larger well (24-well plate, Corning Coaster, even another company's product). Make a double well.
Put 800 uL / well culture medium (10% FBS (fetal calf serum), 10 mM HEPES-containing RPMI-1640 medium) in the outer well, and inject the cells into the inner well (4 × 10 ⁴ cells per well) When 200uL / well is applied so that it becomes / ml), highly motile cells pass through the hole and fall under the membrane. After 24 hours, the cells that had fallen into the lower well were collected, cultured in the above RPMI-1640 medium for 3-4 days, and seeded again in the inner well. This series of operations was repeated 10 times to select only cells with high motility. These cells are also referred to as “FP10 cells”. FP10 cells were subcutaneously administered to mice at 1 × 10 ⁶ pieces, and the vertebrae were collected 25 days later, immersed in formalin solution, fixed, immersed in decalcified solution, decalcified, embedded in paraffin resin, and 5- A 6um tissue section was prepared, stained with HE (hematoxylin and eosin), and observed under a microscope. As a result of determining the presence or absence of bone marrow metastasis, metastasis was observed in 6 of 10 animals (metastasis rate 60%) compared to the parent strain It was found that the bone marrow metastasis rate was significantly increased. However, in order to establish more highly metastatic cells, the following operation was performed.

(1-3) Establishment of FP10SC1 cell line and FP10SC2 cell line 1 × 10 ⁶ FP10 cells obtained in Example (1-2) per 2 to 3 BALB / c mice (8 weeks old female) After subcutaneous administration, vertebrae were collected 24 days later and bone marrow cells were collected, and cultured in the aforementioned RPMI-1640 medium containing G418 (120 ug / mL) for 10-12 days to obtain FP10SC1 strain. This FP10SC1 strain was further subcutaneously administered to 2 to 3 mice at 1 × 10 ⁶ mice. After 24 days, the vertebrae were removed and bone marrow cells were collected, and the above RPMI-164 medium containing G418 (120 ug / mL) was used. After culturing for 10 to 12 days, FP10SC2 strain was established.
FIG. 1 shows micrographs of the parent strain (4T1E / M3 cell line, expressed as EM3), and the established FP10SC1 and FP10SC2 strains. Both FP10SC1 and FP10SC2 are 4T1E / M3 / FP10SC cell lines that are combined with the 4T1E / M3 strain as the parental strain. Also called “FP10SC cell line”.

[Example 2] Assay for in vitro properties of FP10SC cells (2-1) Photomicrograph of each cell Position of parental 4T1E / M3 cells (EM3) and FP10SC1 cells and FP10SC2 cells established in the present invention A phase contrast micrograph (20 × 10 times) is shown in FIG. FP10SC1 cells and FP10SC2 cells tended to be slightly rounder and less adherent than 4T1E / M3 cells.

(2-2) Measurement of normal proliferation ability of FP10SC cells
The normal growth ability of the 4T1E / M3 / FP10SC cell line and the parental line (4T1E / M3 cell line) was compared by a modified MTT assay.
Specifically, FP10SC1 and FP10SC2 strains adjusted to a concentration of 1.5 × 10 ⁴ cells / ml and the parent strain were seeded in a 96-well plate at 200 ul / well and cultured for 0 to 4 days. Measurement was performed by a modified MTT Assay using cell counting kit 8 (manufactured by Dojin Chemical Co., Ltd.). After adding 10uL / well of MTT reagent (Wako Pure Chemical Industries, Ltd.) and incubating for 4 hours at 37 ° C and 5% CO ₂ , the absorbance at 450nm (OD450) was measured with a microplate reader and compared, and FP10SC1, FP10SC2 The cells showed almost no difference in growth ability from the parental strain 4T1E / M3 cells (FIG. 2).

(2-3) Anchorage-independent growth ability of FP10SC cells
The above culture medium containing 0.5% agarose was seeded at 5 mL / dish in a 6 cm diameter dish, and the parent strain (4T1E / M3 cell line) prepared at 1 × 10 ⁴ / mL in the same medium containing 0.3% agarose. ), FP10SC1 cells and FP10SC2 cells were seeded at 2 mL / dish, and cultured at 37 ° C., 5% CO ₂ for 12 days. The number of cells used is 2 × 10 ^{4 for} each dish. After incubation, the number of colonies found in each dish was counted under a microscope, and the shape and size of the colonies were photographed under a microscope. The number of colonies and the observed micrographs are shown in FIGS. 3A and 3B. According to these, FP10SC1 cells and FP10SC2 cells are greatly enhanced compared to the parent strain `` anchorage-independent growth ability '' (ability to grow without adhering to the substrate) which is an index of cancer cell malignancy it is obvious.

(2-4) Vertical motility of FP10SC cells Next, the vertical motility was measured using the same double well as used in (1-2). Place a chamber with a polycarbonate membrane with a hole of 8um on the bottom (made by Kurabo Industries, Chemotaxel) in a 24-well plate (such as Corning Coaster) containing 800uL / well medium, and 2 × from above 4T1E / M3 cells and FP10SC1, FP10SC2 cells prepared at 10 ⁵ / mL were seeded at 200 uL / well, and the number of cells that had fallen under the chamber after 24 hours was counted under a microscope. The results are shown in FIGS. 4A and 4B.
FIG. 4A is a measurement of “vertical motility through bone marrow-derived endothelial cells”. Before inoculating 4T1E / M3 cells and FP10SC1, FP10SC2 cells, fibronectin (20 ug / mL, 60 ul / well). The bone marrow-derived endothelial cells prepared at 2 × 10 ⁵ / mL were spread in double wells at a rate of 2 × 10 ⁵ / mL on the polycarbonate membrane coated with), and a complete monolayer was formed after 2 days under the microscope. Confirmed with. When 4T1E / M3 cells and FP10SC1, FP10SC2 cells were sprinkled on the bone marrow-derived endothelial cell monolayer, and the number of cells that fell below the chamber after 24 hours was counted, the bone marrow-derived endothelial cells of FP10SC1, FP10SC2 cells were It was revealed that the motility in the vertical direction was greatly enhanced compared to the parent strain (4T1E / M3 cell line).
FIG. 4B is a measurement of “vertical motility without bone marrow-derived endothelial cells”. 4T1E / M3 cells and FP10SC1, FP10SC2 cells were directly placed on a polycarbonate membrane without a bone marrow-derived endothelial cell monolayer. The number of cells that fell under the chamber after 24 hours. As in the case of using bone marrow-derived endothelial cells (FIG. 4A), it was revealed that the motility in the vertical direction of FP10SC1 and FP10SC2 was greatly enhanced compared to the parent strain (4T1E / M3 cell strain).

[Example 3] Measurement of bone marrow metastatic potential in mice subcutaneously administered with FP10SC cells
FP10SC1 and FP10SC2 strains were subcutaneously administered to the back of BALB / c mice (8 weeks old female) (1 × 10 ⁶ / mouse), and vertebrae were collected 25 days later and tissue sections were made to confirm the presence or absence of metastasis . The test was performed twice for 6 mice and 9 mice each, and it was found that any cell line caused metastasis to bone marrow tissue of 100% vertebrae (Table 1). At the same time, the same experiment was conducted with 6 and 10 mice in the parent strain (4T1E / M3 cell line), but the metastasis rate remained at 20 to 33%. FIG. 5 shows a tissue section photograph of the vertebrae of mice administered with the FP10SC1 and FP10SC2 strains.
In this example, BALB / c mice (female 8 weeks old) were each divided into 3 groups of 6 or 9-10 mice, and 4T1E / M3 cells were subcutaneously administered to each group (1 × 10 ⁶ / mouse). Then, FP10SC1 and FP10SC2 cells were administered subcutaneously (1 × 10 ⁶ / mouse) to the remaining two groups, respectively, and lungs and vertebrae were collected after 25 days and fixed in 10% formalin solution. The vertebrae were further decalcified by dipping in decalcification fluid. These were embedded in paraffin resin, sliced into 5-6 um to prepare tissue sections, stained with HE (hematoxylin and eosin), and analyzed for the presence or absence of metastasis to the lung and spine. For the lung, metastasis was observed in all of the parental strain (4T1E / M3 cell line) and the FP10SC1,2 cell administration group.
On the other hand, for the bone marrow tissue of the spine, the metastasis rate of the parent strain was 20-30%, whereas the metastasis rate of FP10SC1 and FP10SC2 cells was 100% (metastasis to the spine of all mice administered) Observed) revealed that the ability to metastasize to spinal bone marrow tissue was significantly enhanced (Table 1). FIG. 5 shows a photograph of a tissue section of a vertebra of a mouse administered with each cell. The part where cancer cells have metastasized and proliferated is large in both nucleus and cytoplasm, and can be clearly distinguished from the part that has not metastasized under the microscope.
Test cancer cells are administered as shown in FIG. 5, bone marrow tissue sections of the spine are prepared, and the metastasis rate when the presence or absence of metastasis to the vertebra is analyzed is shown as (Table 1). For example, when metastasis is seen in 6 out of 6 animals, it is shown as 100%. EM3 cell administration had a metastasis rate of 20-30%, whereas FP10SC1,2 cells caused metastasis in all mice administered (metastasis rate 100%).

[Example 4] Analysis of gene expression in FP10SC cell line Total RNA was collected from each of the parent strain (4T1E / M3 cell line) and FP10SC2 cells, and the gene expression was compared by DNA microarray analysis. As a result of quantitative RT-PCR for the gene that greatly changed, it was revealed that the expression of Cdhn17 (cadherin17) was particularly greatly enhanced (FIG. 6). Cdhn17 (cadherin17) is a protein that has been reported to be associated with various cancer metastasis and cancer malignancy, but there has been no report examining the role of bone marrow metastasis and Cdhn17 in breast cancer. In this example, the correlation with the malignancy of bone marrow metastasis in breast cancer was revealed for the first time.

Claims (11)

A method of establishing a bone marrow highly metastatic mouse breast cancer cell line, which comprises using the mouse breast cancer cell 4T1E / M3 cell line as a parent line and comprising the following steps (a) to ( f ):
(A) a murine breast cancer cells 4T1E / M3 cell line drug resistance gene has been introduced, rather蒔onto the inside wells of the double well having a plurality of small holes inside the well bottom step,
(B) recovering cells that have passed through the hole on the inner well and have fallen into the lower well (c) repeating the steps of (a) and (b) a plurality of times;
( D ) a step of administering the obtained cells subcutaneously to a mouse;
(E) a mouse vertebrae were taken, the bone marrow cells were collected from the vertebrae, the collected bone marrow cells were cultured in a medium containing a target drug resistance-conferring by the drug resistance gene, harvesting drug-resistant cells step,
( F ) a step of culturing the obtained drug-resistant cells in a normal medium, stabilizing the trait, and establishing a bone marrow highly metastatic mouse breast cancer cell line (however, the establishment method is the same as that used in the step (a)). Heavy wells do not contain extracellular matrix or vascular endothelial cells at the bottom of the inner well) . The method for establishing a bone marrow highly metastatic mouse breast cancer cell line according to claim 1, further comprising the following step ( g ):
( G ) A step of repeating steps ( d ) to ( f ) using the established cell line obtained in step ( f ).   A bone marrow highly metastatic mouse breast cancer cell line established by the establishment method according to claim 1 or 2.   The bone marrow highly metastatic mouse breast cancer cell line according to claim 3, wherein Cdhn17 (cadherin17) is expressed at least 10 times higher than the parent cell (4T1E / M3 cell line).   An animal model mouse for bone marrow metastasis analysis of breast cancer obtained by subcutaneously administering or intravenously injecting the bone marrow highly metastatic mouse breast cancer cell line according to claim 3 or 4 to the mouse. A method for screening a test substance that is a candidate for a bone marrow metastasis inhibitor of breast cancer, comprising the following (a) to (c):
(A) administering a test substance to the bone marrow highly metastatic mouse breast cancer cell line according to claim 3 or 4, and culturing;
(B) One or more of the anchorage-independent growth ability, vertical motility, and intracellular cadherin17 expression level of the cell line after administration were measured, and the measured values were measured in advance. Comparing the measured value in the cell line before administration;
(C) A step of evaluating and selecting a test substance as a candidate substance for a bone marrow metastasis inhibitor for breast cancer when the measured value after administration is significantly reduced as compared with the measured value before administration. The screening method according to claim 6, comprising:
The test substance in the step (a) is a test peptide or a test nucleic acid molecule, and administration of the test substance to the cell line enters the cell of the nucleic acid encoding the test peptide or the test nucleic acid molecule. it is those of the introduction, mETHODS. A method for screening a test substance that is a candidate for a bone marrow metastasis inhibitor for breast cancer,
The test substance is administered to the subject mouse together with the bone marrow highly metastatic mouse breast cancer cell line according to claim 3 or 4 or before or after administration of the cell line, and the mouse after administration is bred for a certain period of time. Bone marrow metastasis to a test substance that was sacrificed and evaluated for its ability to metastasize to the bone marrow using the presence or degree of breast cancer cell metastasis to the bone marrow tissue of the spine as an index. A screening method for a bone marrow metastasis inhibitor for breast cancer, which comprises selecting as a drug candidate. A method for screening a test substance that is a candidate for a bone marrow metastasis inhibitor for breast cancer, comprising the following (a) to (d);
(A) A test substance is administered to the subject mouse together with the bone marrow highly metastatic mouse breast cancer cell line according to claim 3 or 4 or before or after administration of the cell line, and the mouse after administration is fixed. The process of rearing for a period,
(B) sacrificing the mouse obtained in step (a) and measuring the metastasis rate to the bone marrow and / or the area of the metastasized cancer cell region in the bone marrow tissue,
(C) The bone marrow highly metastatic mouse breast cancer cell line is administered without administering the test substance, the mouse after administration is bred for the same period as in step (a), and the resulting mouse is sacrificed to the bone marrow. Measuring the metastasis rate and / or the area of the metastatic cancer cell region in the bone marrow tissue,
(D) The measured value obtained in step (b) is compared with the measured value obtained in step (c). If the measured value is significantly decreased, the test substance is a candidate for a bone marrow metastasis inhibitor for breast cancer. The process of evaluating a substance.   A kit for screening for a bone marrow metastasis inhibitor for breast cancer, comprising the bone marrow highly metastatic mouse breast cancer cell line according to claim 3 or 4. The screening kit according to claim 10, further comprising at least one of the following (1) to (4):
(1) a soft agar medium for measuring the growth-independent growth ability of the cell line,
(2) a well provided with a plate having a plurality of microholes at the top for measuring the motility in the vertical direction of the cell line;
(3) a device for measuring the expression level of Cadherin17 in the cell line,
(4) A mouse to be transplanted with the cell line and a transplantation device.

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