KR20180009889A - Method for culturing breast cancer cell and method for screening drugs related to breast cancer cell using the same - Google Patents

Abstract

The present invention provides a method for culturing an epithelial-mesenchymal transition (EMT) induced breast cancer cell which comprises a step of co-culturing a fat cell and the breast cancer cell physically separated from the fat cell, and a method for screening a drug related to a breast cell using the same. The method for screening a drug related to a breast cancer suppresses activity and expression of an EMT-related factor of the EMT induced breast cancer cell or selects a substance to be detected which has a negative influence on an expression position.

Description

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for culturing breast cancer cells and a method for screening breast cancer cells using the same,

The present invention relates to a method for culturing breast cancer cells, and more particularly, to a method for culturing breast cancer cells using co-culture with adipocytes and a method for screening breast cancer-related drugs using the same.

Breast cancer is a mass of breast cancer cells. Various factors such as female hormones, family history, history, fertility and dietary habits have been raised as causes of breast cancer, but nothing has been clarified yet. The incidence of breast cancer is rapidly increasing due to increased sensitivity of wired tissues, westernization of dietary life, and pollution of living environment. Treatment of breast cancer includes surgical treatment, radiation therapy, chemotherapy, and antihormonal therapy, but more effective methods are needed to prevent or inhibit breast cancer cells.

BACKGROUND OF THE INVENTION [0002] Techniques as a background of the invention have been made in order to facilitate understanding of the present invention. And should not be construed as an admission that the matters described in the technical background of the invention are present in the prior art.

Conversion of tumors to metastatic malignant tumors in the early stages of breast cancer may be associated with epithelial-mesenchymal transition (EMT) activity. In addition, the cell phenotype may be transferred from the epithelial stage to the mesodermal stage, which may be a hallmark of tumor formation.

EMT is basically the transformation of epithelial cells morphologically and functionally into mesenchymal cells. Typical EMT features may be that the cell shape changes from a circular shape to a spindle shape and that the intercellular polarity is lost and separated into individual cells. EMT-mediated mesenchymal cells promote the detachment of early tumors and subsequent metastatic cancer cells, which may lead to progression of metastasis.

In breast cancer, EMT is associated with cancer stem cell induction, tumor recurrence and tumor metastasis in breast cancer of basal or basal-like type, and is closely related to tumor aggressiveness and poor prognosis It is relevant. EMT is known to be associated with mesenchymal formation, neural tube involvement, fibrosis, and cancer progression, which is accompanied by embryogenesis. During the EMT process, the epithelial cells lose their polarity and cell-cell binding ability, resulting in morphological and functional transformations like mesenchymal stem cells. EMT can enhance tumor metastasis by enhancing cell migration and migration to other sites of cancer cells. As a result, expression of epithelial cell phenotypic factors such as fusion membrane, desmosome and cytoskeletal elements is reduced. The inventors of the present invention studied cell interactions as one of many factors causing EMT, and found that EMT was induced using a co-culture technique, which is a method of raising two cells together to cause metabolic interaction We were able to cultivate breast cancer cells.

In particular, the inventors of the present invention have proposed an indirect co-culturing method that permits mass transfer for interactions between cells by separating two intercellular spaces. As a result, cell division was facilitated, the effect of co-culture of each cell was rapidly analyzed, and EMT-induced breast cancer cells were cultured using an indirect co-culture method. Accordingly, a problem to be solved by the present invention is to provide a method for culturing EMT-induced breast cancer cells.

Another object of the present invention is to provide a method for screening breast cancer-related drugs by inhibiting the activity and expression of EMT-related factors of EMT-induced breast cancer cells or selecting substances to be detected that have a negative effect on the expression site .

The problems of the present invention are not limited to the above-mentioned problems, and other problems not mentioned can be clearly understood by those skilled in the art from the following description.

According to one embodiment of the present invention, there is provided a method for culturing EMT-induced breast cancer cells comprising co-culturing adipocytes and breast cancer cells physically separated from the adipocytes.

As used herein, the term "cultivation" means the cultivation of organs, tissues and cells of an organism in an artificial condition.

As used herein, the term "fat cells" refers to cells that contain lipids. For example, the adipocyte may be selected from the group consisting of monocytes, marsupials, vaginas, blood vessels, suckers, meat foods, insects, equipments, equipment, utensils, Adipose and adipocyte-derived adipocytes can be used.

The adipocyte is most preferably a human-derived cell, but is not limited thereto. Specifically, the adipocyte may regulate the expression of an EMT-related gene when co-cultured with breast cancer cells, and the regulation may be upregulation of expression or downregulation of expression.

As used herein, the term "breast cancer cell" means a cell derived from cancer tissue of a breast cancer patient.

As used herein, the term "co-cultivation" means mixed culture of two or more cells. For example, the co-culture may be a direct co-culture and an indirect co-culture. Specifically, in the direct co-culture, two cells can be separated using a fluorescence activated cell sorter (FACS), but the present invention is not limited thereto.

The co-culture of the present invention may preferably be an indirect co-culture amount. Specifically, it can indirectly co-localize between breast cancer cells and adipocytes, allowing the movement of the substances required for interaction. More specifically, the Transwell system may be used as the co-culture method, but the present invention is not limited thereto.

As used herein, the term "EMT" is an epithelial-mesenchymal transition in which epithelial cells lose cell polarity and cell-cell adhesion and acquire metastatic and invasive properties, Which is a multipotent stromal cell capable of differentiating into cells.

The EMT may be required in many developmental processes, including mesoderm formation and neural tube formation. EMT can also be involved in the initiation of metastasis in wound healing, organ fibrosis and cancer progression.

One of the important phenomena in EMT is the loss of E-cadherin. In the present invention, the loss of E-carderine in EMT-induced breast cancer cells could be confirmed. SNAI1 / Snail1, SNAI2 / Snail2, ZEB1, ZEB2, E47 and KLF8, which directly bind to the E-carderine promoter and inhibit its transcription, and indirectly E - Twist, Goosecoid, E2.2 (aka TCF4), homeobox proteins SIX1 and FOXC2, which inhibit carderine.

Furthermore, EMT induction of breast cancer cells may be dependent on the type of breast cancer cells and the type of adipocyte. In addition, EMT induction can reduce the polarity between epithelial cells, allowing easy separation into single cells and facilitating cell dispersion.

The method for culturing EMT-induced breast cancer cells according to an embodiment of the present invention includes arranging the breast cancer cells disposed in a container having a plurality of pores and adipocytes arranged separately from the breast cancer cells to interact with each other through a solubility factor Lt; / RTI >

As used herein, the term "pore" means a fine pore and the diameter of the pore may be from 0.1 [mu] m to 1 [mu] m, preferably from 0.2 [mu] m to 0.5 [mu] m through which the solubility factor can pass. In the range of diameter, the soluble factors secreted from the adipocytes pass through but do not pass the macromolecules of the medium composition, so that mutual contamination of the medium can be minimized.

As used herein, the term "container" means a container in which the cells are cultured. For example, a container in which the breast cancer cells and fat cells are cultured. The container may be made of glass or plastic. In particular, a part of the frame made of impermeable plastic may have at least one window made of a semitransparent film, which has the advantages of structural stability and cost reduction.

The semi-permeable membrane can be any material that prevents the passage of microalgae while allowing passage of gas, water, and ions. However, the membrane can be made of any material such as cellulose, cellulose acetate (CA), cellulose 2.5 acetate, cellulosetriacetate (CTA), modified cellulose, curophan, polyvinylalcohol (PVA), poly (methyl methacrylate) Polyacrylonitrile (PAN), poly (lactic-co-glycolic acid), polylactic acid (PLA), and polylactic acid copolymers ), Polypropylene (PP), polyacrylamide, polysulfone (PSF), and polyethylene-vinylacetate can be used.

As the impermeable film, conventionally used polyethylene, polystyrene, polypropylene, polyethylene terephthalate (PET), hidensity polyethylene (HDPE), polycarbonate may be used. However, various materials can be used without being limited thereto.

As used herein, the term "solubility factor" refers to a substance that has solubility in breast cancer cells and adipocytes in factors causing or causing the biological phenomenon during culture. For example, among the factors that induce EMT, E-carderine, integrin, N-cadherin, cadherin-11, vimentin, related protein 1), HMGA2 (high mobility group proteins characterized by AT-hook 2), TGF-ß (Transforming Growth Factor-β) and S100A4 (S100 calcium-binding protein A4) may be solubility factors, Do not.

As used herein, the term "interaction" refers to interrelationships between organisms, including species such as other species in a biological community. The interaction may be an interaction of breast cancer cells and adipocytes.

The breast cancer cells of the method for culturing EMT-induced breast cancer cells according to an embodiment of the present invention may be selected from the group consisting of a luminal A type cell, a luminal type B type cell, And a luminal-basal mixing type.

The cell types of the breast cancer can be classified into two groups depending on the expression of the estrogen, progesterone, Her-2, Her-1 and Cytokeratin 5/3 selectable factors, such as a luminal A type, a luminal B type, a vesicular type, Type and ERBB2 type. Luminal A type is able to express estrogen and progesterone. Luminal B type can express estrogen and Her-2 among the selectivity factors. Bethle type is Her-1 and Cytokeratin 5 / 6 can be expressed. The normal-like type may not result in the expression of a screening factor, and the ERBB2 type may be distinguished by the overexpression of ERBB2. For example, in the present invention, breast cancer cells of the vesicular type, the luminal type and the mixed type of luminal-vesicle were used, but not limited thereto, various types of breast cancer cells can be used.

Methods for culturing breast cancer cells of EMT induced vesicular type, luminal A type, luminal B type, normal-like type, ERBB2 type and luminal-vesicular mixed type can be used for screening methods for breast cancer- have.

On the other hand, the beryllium type inhibits extracellular growth factors such as epidermal growth factor (HGF), hepatocyte growth factor (HGF), TGF-ß, fibroblast growth factors (FGF) and insulin- Hedgehog) and Wnt signaling molecules (Wnt signaling molecules). Specifically, the EMT induction process may be associated with an increase in transcription factor expression of Snail1, Snail2 (Slug), SIP-1 (ZEB-3), ZEB-1 (DEF1) and TWIST1 have.

In the culture of the EMT-induced breast cancer cells according to an embodiment of the present invention, the culture medium in which the breast cancer cells are cultured is DMEM (Dulbecco's modified Eagle's medium), EMEM (Eagle's minimum essential medium), Ham's F-12 medium and Leibovitz's L -15 medium, but not limited thereto, a medium containing various antibacterial substances or additives can be used. For example, in one embodiment of the present invention, DMEM medium containing FBS, penicillin-streptomycin, and insulin was used.

The method for culturing EMT-induced breast cancer cells according to an embodiment of the present invention may be a method for culturing breast cancer cells different from cancer-associated adipocytes (CAAs).

As used herein, the term "cancer-associated adipocyte" refers to an adipocyte surrounding a tumor, which is characterized by having an inactive phenotype.

The method of co-culturing adipocytes and breast cancer cells according to an embodiment of the present invention is distinguished from a method of co-culturing breast cancer cells with the CAAs which are adipocytes associated with cancer.

According to one embodiment of the present invention, EMT-induced breast cancer cells cultured by co-culturing adipocytes and breast cancer cells physically separated from the adipocytes are cultured in the EMT- A method for screening a breast cancer-related drug which comprises inhibiting the activity and expression of an EMT-related factor or selecting a substance to be detected which has a negative effect on the expression site.

As used herein, the term "control group" refers to a group that does not have any manipulations or conditions to determine whether the results of the experiment are properly derived, . For example, in order to analyze EMT induction of breast cancer cells in a coculture group in which breast cancer cells are co-cultured with adipocytes, a group in which only adipocytes are not cultured and breast cancer cells are cultured can be set as a control group.

As used herein, the term "EMT-associated factor" is not to be construed as indicating a factor associated with induction of EMT and expression after EMT induction.

For example, the EMT-associated factor may be, but is not limited to, E-carderine, integrin, N-carderine, carderine-11, visentin, TWIST1, HMGA2, TGF-ß and S100A4 as described above.

As used herein, the term "substance to be detected" may mean, but is not limited to, the activity of an EMT-associated factor and a substance that acts to inhibit expression or negatively affect the expression site.

As used herein, the term "breast cancer-related drug" means a substance that acts to suppress the expression of breast cancer cells and further the transfer-related response. For example, the drug may act to inhibit the activity and expression of EMT-related factors or to adversely affect the expression site.

As used herein, the term "screening " refers to drug screening, which may be the activity of EMT-associated factors in breast cancer and the search for candidate substances that inhibit expression or have negative effects on expression sites, It does not.

The drug may be a breast cancer-related drug, a therapeutic agent and a preventive substance.

When EMT induced breast cancer cells cultured through co-culture of the aforementioned adipocytes and breast cancer cells are found to be inhibited in induction of EMT through various methods after treatment of the substance, the substance can be used as a candidate substance of an EMT inhibitory drug You can decide.

Methods for determining whether EMT induction is inhibited include, but are not limited to, for example, methods for measuring whether the expression of EMT-related factors decreases, whether the degree of cell migration and invasion decreases, or the shape of cells changes can do.

Accordingly, the screening method for a breast cancer-related drug according to an embodiment of the present invention may further include the step of measuring the expression of an EMT-related factor in the breast cancer cell.

The EMT-associated factor according to one embodiment of the present invention may be E-carderine, integrin, N-carderine, carderine-11, visentin, TWIST1, HMGA2, TGF- Do not.

As used herein, the term "cardinal" means a group of molecules essential for animal cells to adhere to each other, and may be expressed in different shapes depending on the cells.

The cardinalin may additionally include at least one of R-carderine, P-carderine, M-carderine, EP-carderine, B-carderine, T-carderine, L-CAM, carderine-5, carderine-6, carderine-7, carderine-8, carderine-9 and carderine-10.

As used herein, the term " visentin "means an intermediate light filament protein specific for mesenchymal cells such as fibroblasts, leukocyte cells, and the like. Molecular weight Molecular weight is about 54,000 and may be expressed in malignant tumors such as breast cancer.

As used herein, the term "TWIST1" is a transcription factor of the basic helix-loop-helix and means a factor encoded in the human TWIST1 gene.

As used herein, the term "HMGA2" refers to a rapid grouping protein characterized by AT-hook 2, the DNA binding site.

As used herein, the term "TGF-β" refers to a transforming growth factor β.

As used herein, the term "S100A4" means the protein A4 binding to S100 calcium.

The measurement of E-carderine and vimentin expression according to an embodiment of the present invention may be performed by immunocytochemical analysis, western blotting and real-time PCR, Without being limited thereto, the E-carderine and the vimentin expression can be measured in various ways.

As used herein, the term "immunohistochemical analysis" refers to a method of studying the chemical composition of a cell using the properties of an antigen-antibody reaction.

The immunohistochemical analysis includes an enzyme-labeled antibody method in which an antibody is labeled with a fluorescent dye, an enzyme such as a peroxidase is labeled, an enzyme-labeled antibody method is used to obtain a chromogenic material from the enzyme-degraded substrate, and a radioactive isotope- and radioisotope labeling antibody method using autoradiography. However, various methods can be used for immunohistochemical analysis without limitation.

As used herein, the term " protein inhalation "means a method of immobilizing a protein separated by electrophoresis on a hydrophobic membrane and detecting a protein for the purpose of using a specific antibody, but there is a variety of inhalation methods . For example, the inhalation method may be, but not limited to, the Southern suction method and the Northern suction method.

As used herein, the term "real-time PCR" means a PCR method that quantitatively displays the product as a result of the chain-polymerization reaction in real time, but not limited thereto, various PCR methods may be used. For example, the PCR method may be SSCP (single strand conformation polymorphism), RACE (rapid amplification of cDNA ends), ISPCR (in situ PCR), or DDRT-PCR (differential display reverse transcriptase PCR) .

The method for screening a breast cancer-related drug according to an embodiment of the present invention may further include the step of measuring the expression of an EMT-related gene in the breast cancer cell.

As used herein, the term "EMT-associated gene" means a gene encoding an EMT-associated factor, but is not limited thereto.

The EMT-related gene according to an embodiment of the present invention may be, but is not limited to, the TWIST1 gene, the MMP9 gene, the zinc finger transcription repressor nail gene, the HMGA2 gene, and the TGF-ß gene.

The expression of the EMT-associated gene may be increased by adipocytes, and an increase in expression may mean upregulation of expression.

The method for screening a breast cancer-related drug according to an embodiment of the present invention may further include measuring a degree of movement and invasion of the breast cancer cell.

As used herein, the term "migration of cells" refers to the movement of cells, and the movement of the cells may be a central processing step in the development and maintenance of multicellular organisms. For example, the migration of the cells may, but is not limited to, the migration of EMT-induced breast cancer cells.

As used herein, the term "invasion of a cell" means that a cell can gain mobility and migrate to another tissue. For example, the tissue may be a cancer cell, specifically, but not exclusively, an EMT-induced breast cancer cell.

The degree of migration and invasion of the breast cancer cells can be induced by up-regulation of EMT-related factor expression. The step of measuring the degree of migration and invasion of the breast cancer cell can be used in a screening method of a breast cancer-related drug.

The method for screening a breast cancer-related drug according to an embodiment of the present invention may further include monitoring a shape change of the breast cancer cell.

The shape change may mean morphological variation of EMT induced breast cancer cells. For example, the morphological variation may be, but is not limited to, changing into a spindle shape.

As used herein, the term "monitoring" refers to the step of visualizing changes, shaping control, and verification. For example, the monitoring can monitor morphological variation of EMT-induced breast cancer cells.

The present invention provides a method for culturing EMT-induced breast cancer cells comprising co-culturing adipocytes and breast cancer cells physically separated from the adipocytes, and is capable of screening breast cancer-related drugs using the same .

In addition, co-culturing adipocytes and breast cancer cells according to one embodiment of the present invention may be used to determine whether the morphological changes of cultured breast cancer cells, the degree of migration and invasion of breast cancer cells are increased, and whether EMT- A method for screening candidates of breast cancer-related drugs can be provided by confirming the degree of induction of EMT through measurement of whether expression is increased.

The effects according to the present invention are not limited by the contents exemplified above, and more various effects are included in the specification.

1 shows a device for a method for culturing EMT-induced breast cancer cells according to an embodiment of the present invention.
FIG. 2A shows experimental results showing morphological changes of MCF-7, 453S and 231 compared to the control group in the method of culturing EMT-induced breast cancer cells according to an embodiment of the present invention.
FIG. 2b shows experimental results showing the morphological changes of 453 and 468 compared with the control in a method of providing a method of culturing EMT-induced breast cancer cells according to an embodiment of the present invention.
FIG. 3A shows experimental results showing mobility and invasiveness of coculture group and control group cultured according to the method of culturing EMT-induced breast cancer cells according to an embodiment of the present invention.
FIG. 3b is a graph illustrating the number of invasive breast cancer cells cultured according to the method of culturing EMT-induced breast cancer cells according to an embodiment of the present invention and the control group.
4A, 4B and 4C are graphs showing the expression levels of E-carderine and visemen in cultured coculture group and control group according to the method of providing EMT-induced breast cancer cells according to an embodiment of the present invention The results of one experiment.
FIG. 5A is a graph showing the results of the measurement of the relative expression levels of MMP9 mRNA in the coculture group and the control group cultured according to the method of culturing EMT-induced breast cancer cells according to an embodiment of the present invention.
FIG. 5B is a graph showing the results of measurement of the relative expression levels of TWIST1 mRNA in the coculture group and the control group cultured according to the method of culturing EMT-induced breast cancer cells according to an embodiment of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS The advantages and features of the present invention, and the manner of achieving them, will be apparent from and elucidated with reference to the embodiments described hereinafter in conjunction with the accompanying drawings. It should be understood, however, that the invention is not limited to the disclosed embodiments, but is capable of many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, To fully disclose the scope of the invention to those skilled in the art, and the invention is only defined by the scope of the claims.

Hereinafter, a method of culturing EMT-induced breast cancer cells and measuring whether EMT is induced by co-culturing breast cancer cells and adipocytes in a physically separated space will be described in detail. Whether or not EMT has been induced can be judged based on morphological changes of breast cancer cells, measurement of degree of movement and invasion of breast cancer cells, measurement of expression of EMT-related factors and genes, and the like. In addition, the methods described above can also be applied to screening for breast cancer-related drugs in a manner that inhibits the activity and expression of EMT-related factors or selects the substances to be detected that adversely affect the expression site, according to various embodiments of the invention .

Breast cancer cell culture

According to the method of culturing EMT-induced breast cancer cells according to an embodiment of the present invention, breast cancer cells were cultured as follows. MDA-MB-435S (hereinafter referred to as "435S"), MDA-MB-231 (hereinafter referred to as "231"), MDA- (Hereinafter referred to as '468') and MCF-7 (hereinafter referred to as 'MCF-7'). The cell types of MCF-7 and 453 are of the luminal type, the cell type of 468 is the luminal-vesicle mixed type, and the cell type of 435S, 231 is the vesicular type. The 453, 435S, 231 and 468 were cultured in DMEM medium supplemented with 10% FBS and 1% penicillin-streptomycin under 5% CO 2. MCF-7 was cultured in DMEM supplemented with 10% FBS, 1% penicillin-streptomycin and 0.1 mg / ml insulin.

Culture of adipocytes

According to the method for culturing EMT-induced breast cancer cells according to an embodiment of the present invention, adipocytes were cultured as follows. The adipocytes were cultured in DMEM medium supplemented with 10% FBS after differentiating adipocytes 3T3-L1 (preadipocytes 3T3-L1), which is different from CAAs, cancer-associated adipocytes, into adipocytes.

Co-culture of the cultured adipocytes and breast cancer cells can be more effectively performed in relation to the induction of EMT of breast cancer cells than co-culturing the cancer-associated adipocyte CAAs and breast cancer cells.

Example 1: Co-culture of breast cancer cells and adipocytes

In order to examine the effect of indirect co-culture between breast cancer cells and adipocytes, coculture group and control group were divided as follows.

The coculture group was divided into 453 and coculture groups of adipocytes, 435S and coculture groups of adipocytes, 231 and coculture groups of adipocytes, 468 and coculture groups of adipocytes, and coculture groups of MCF-7 and adipocytes.

In the control group, only cancer cells were cultured without adipocytes. Specifically, 453 control group, 435S control group, 231 control group, 468 control group and MCF-7 control group were divided.

The co-cultures were cultured for 8 days using a transwell culture plate.

FIG. 1 shows the arrangement of breast cancer cells and adipocytes by enlarging the Transwell well culture plate used for culturing the five coculture groups and the control group. Fig. 1 (a) is a layout diagram of each cell in a Transwell culture plate of both coculture groups, in which breast cancer cells were arranged in the upper layer and fat cells in the lower layer. Fig. 1 (b) shows an enlargement of the Transwell culture plate of both the coculture group of Fig. 1 (a). Specifically, it shows that pores are present in the upper layer and that the solubility factors in the upper and lower layers can be exchanged with the pores during the culture. Fig. 1 (c) shows the arrangement of cells in the Transwell culture plate of the control group, showing that breast cancer cells were placed only in the upper layer.

In all of the examples below, in order to identify EMT-induced breast cancer cells, the same co-culture method and cell arrangement in the Transwell culture plate were applied in all five co-fed groups and the control group in the same manner.

Example 2: Observation of morphological changes of breast cancer cells

The morphological changes of breast cancer cells in coculture group and control group were compared to identify EMT - induced breast cancer cells. Morphological changes in breast cancer cells of both coculture groups were confirmed. Specifically, morphological changes of the coculture group were analyzed using an optical microscope.

In Fig. 2A, it was confirmed that mammary cells of both MCF-7, 453S and 231 were morphologically changed to spindle-shaped cells and EMT was induced. However, in FIG. 2B, it can be seen that there is no morphological change in breast cancer cells of both coculture groups 453 and 468, unlike breast cancer cells of other coculture group in which EMT is induced.

As described above, the morphological changes in the EMT-induced breast cancer cells of MCF-7, 453S, and 231 in the coculture of MCF-7, 453S, and 231 were observed, and the morphological changes revealed whether EMT was induced in breast cancer cells in breast cancer- Can be used to confirm. For example, among screening candidates, a substance that inhibits morphological changes in breast cancer cells may be a substance that inhibits EMT induction of breast cancer cells.

Example 3: Movement and invasion of breast cancer cells

For breast cancer cell migration and invasion observation, breast cancer cells of 453, 435S, 231, 468 and MCF-7 were stained with 5% glutaraldehyde for 30 minutes and stained with 0.5% crystal violet for 30 minutes.

3A and 3B, the migration and invasion of stained breast cancer cells in both coculture and control groups are compared.

Figures 3 (b) and 3 (b) show counts of migrated and invaded cells in both coculture and control groups.

The migration and invasion observations of the breast cancer cells were performed with an optical microscope at an x200 magnification, and the number of cells in five random fields was counted, and all the above procedures were repeated three times.

3A and 3B show migration and invasion in breast cancer cells of both coculture groups of 453, 435S, 231, 468 and MCF-7.

Figures 3 (b) and 3 (b) show the migration and invasive cell counts in breast cancer cells of both coculture groups of 435S, 231 and 468.

As a result, migration and invasion were confirmed in all five breast cancer cell lines. Especially, the increase of the degree of migration and invasion in breast cancer cells of both coculture group of 435S, 231 and 468 among coculture group was remarkably increased.

In both coculture groups, breast cancer cell migration and invasion increased, indicating that breast cancer cells were induced by co - cultured adipocytes.

EMT-induced breast cancer cells showed a decrease in intercellular polarity, and as a result, they were separated into individual single cells. In addition, EMT-induced breast cancer cells can be confirmed to have improved cell mobility due to the enhanced mobility.

In Example 2 described above, there was no morphological variation in spindle shape specifically related to EMT induction in breast cancer cells of both coculture groups of 453 and 468. However, movement and invasion of both cooperatives 453 and 468 are confirmed. In other words, although there is no morphological variation, metastasis of cancer cells may be induced by EMT induction.

As described above, migration and invasion increases in both breast and cervical cancer cell lines co-cultured with adipocytes. The increase in migration and invasion of breast cancer cells indicates that EMT is induced in breast cancer cells in breast cancer-related drug screening . ≪ / RTI > For example, among screening candidates, substances that inhibit the increase in the degree of migration and invasion of breast cancer cells may be substances that inhibit EMT induction of breast cancer cells.

Example 4: Measurement of EMT-related factor expression in breast cancer cells

The expression of EMT-related factors, E-carderin and visentin, in five coculture groups of breast cancer cells after culture was determined using immunohistochemistry, protein inhalation and real-time PCR to identify EMT-induced breast cancer cells . Other EMT-related factors, N-carderine, TWIST1, HMGA2, TGF-ß and S100A4, were measured by immunochemical staining. By measuring the expression of the EMT-related factor, it was confirmed that adipocytes induce EMT of breast cancer cells.

The breast cancer cells used for immunohistochemical analysis, protein inhalation method, real time PCR and immunochemical staining included breast cancer cells in patients diagnosed with invasive ductal carcinoma, and the breast cancer cells were obtained through surgery. The patient did not include patients undergoing hormone therapy or previous chemotherapy. Hematoxylin and eosin (H & E) staining was performed on resected breast cancer cells. Specifically, the breast cancer cell tissue used a breast cancer cell tissue having a fibrous stroma and a breast cancer cell tissue having an adipose stroma. The breast cancer cell tissue having the above-mentioned fatty epilepsy was used in which 50% or more of the entire tumor stroma was the fatty epileptic. H & E stained breast cancer cell tissue was marked on the surface of a paraffin block. The indicated breast cancer cell tissue was obtained using a biopsy punch.

The breast cancer cell tissue thus obtained was placed in a microarray for analysis, and each well was linked to data containing clinical pathological data.

Immunohistochemical analysis

First, 5 coculture breast cancer cells were fixed in 4% paraformaldehyde for 20 minutes at room temperature, permeabilized with 0.2% Triton X-100 for 10 minutes, and then washed with PBS.

Next, the breast cancer cells were blocked for 1 hour in a blocking solution of PBS containing 5% goat serum and incubated with the primary antibody for 1 hour.

Next, the cells were washed with PBS-T (PBS containing 0.1% Tween-20), and then cultured with the primary antibody at room temperature. The cells were incubated with fluorescently tagged secondary antibody for 1 hour Lt; / RTI > at room temperature.

Finally, the cultured breast cancer cells were stained with Hoechst 333442 (Hoechst 33342) and fixed on a slide glass using an encapsulating agent. The results were visualized at ⅹ400 magnification using a confocal laser scanning microscope equipped with fluorescence and digital image analysis.

Immunochemical staining

First, immunochemical staining was performed using formalin fixed and paraffin-fixed breast cancer cell tissue. The sections of the breast cancer cell tissues were obtained using a microtome, and the tissues of the breast cancer cells were then transferred to a slide and dried at 62 ° C for 30 minutes. The dried slides were incubated with the primary antibody for 32 min. Immunodetection was performed with biotinylated antimouse immunoglobulin.

The primary antibody can be obtained by using the aforementioned EMT factors N-carderine, TWIST1, HMGA2, TGF-ß and S100A and ER (estrogen receptor), PR (progesterone receptor) and HER-2 (human epidermal growth facror-2) Respectively.

After incubation with the primary antibody, breast cancer cell lines were immunostained using streptavidin biotin kit and 3,3'-diaminobenzidine chromogen as substrates .

Finally, the immuno-detected products were contrast dyed with Harris hematoxylin for optical microscopy analysis and were analyzed by semi-quantitative method using EMT-related factors, N-carderine, TWIST1, HMGA2, TGF-ß And the degree of expression of S100A4 were evaluated. Table 1 shows the criteria for evaluating the degree of expression. The evaluation was carried out according to the following criteria.

The immunochemical staining scores of the EMT-related factors N-carderine, TWIST1, HMGA2, TGF-ß and S100A4 were evaluated as 2 + points on the tumor tissues of breast cancer cells of both cows.

Analysis using protein inhalation method

First, breast cancer cells from both coculture groups were dissolved in RIPA buffer containing protease inhibitor (RIPA buffer) and centrifuged at 13000 xg for 15 minutes at 4 ° C.

Next, the concentration of the protein was measured by BCA assay, and the same amount of protein was separated by SDS-PAGE, followed by protein inhalation.

Finally, the protein was detected and analyzed using a chemiluminescence kit.

Analysis using real-time PCR

453, 435S, 231, 468, and MCF-7 were isolated using RNeasy kit and real-time PCR was performed. In this case, the heat cycle condition is a cycle of 5 minutes at 42 ° C, 10 seconds at 95 ° C, and 5 seconds at 95 ° C and 33 seconds at 60 ° C for 40 times. The relative expression level of mRNA was normalized using a housekeeping gene (GAPDH).

Statistical analysis

All analyzed data were analyzed statistically by SPSS statistical program. Statistical differences between the control and coculture groups were analyzed by Student's t test (Stuedent's t test). If the p-value of all data is less than 0.05, the data is statistically significant.

FIG. 4A shows the comparison of the expression of E-carderin (green) and vimentin (red) around the nucleus (blue) of breast cancer cells of coculture group and control group using immunohistochemical analysis. MCF-7 coculture breast cancer cells showed a decreased expression of E-carderin (downregulation of expression) and a slight increase in viseme expression (upregulation of expression). FIG. 4B shows the expression of E-carderine and vimentin in the coculture group and the control group using immunoblotting. Expression of E-carderin decreased in both MCF-7 breast cancer cells and vimentin expression was increased in breast cancer cells of 435s and 231 coculture groups.

FIG. 4c shows the results of analysis of the relative expression levels of Vimentin mRNA in both coculture group and control group using real-time PCR. In the breast cancer cells of 435s and 231 coculture groups, expression of visenger mRNA was about twice as high as that of the control group.

Thus, the increase in the expression of EMT-related factors in the breast cancer cells of the 435s and 231 coculture groups can be recognized as the EMT induction of 435s and 231 breast cancer cells by adipocytes.

As described above, EMT-induced breast cancer cells were found in 5 groups of cocultured mammary gland cells co-cultured with adipocytes, showing a marked increase in the expression level of EMT-related factors as compared with that of control breast cancer cells. The increased expression of the breast cancer EMT-associated factor can be used to determine whether EMT is induced in breast cancer cells in screening for breast cancer-related drugs. For example, among screening candidates, a substance that inhibits the increase in the expression of EMT-related factors may be a substance that inhibits EMT induction of breast cancer cells.

Example 5: Measurement of EMT-associated gene expression in breast cancer cells

Expression of the EMT-related genes TWIST1 gene, MMP9 gene, zinc finger transcriptional repressuris nail gene, HMGA2 gene and TGF-ß gene was analyzed by using 453, 435S, 231, 468 and MCF-7 Were measured in breast cancer cells of both coculture groups. Expression of the gene was analyzed using real-time PCR.

5A, the amount of MMP9 mRNA expression of breast cancer cells in MCF-7, 435s, and 231 coculture groups was about twice as high as that of the control group.

In FIG. 5b, the TWIST1 mRNA expression level of breast cancer cells in the coculture group of 435s was about two times higher than that of the control group, and the amount of TWIST1 mRNA expression of breast cancer cells in the coculture group of 231 was about three times higher than that of the control group. In addition, the amount of TWIST1 mRNA expression of breast cancer cells in the 468 coculture group was about 1.2 times higher than that of the control group.

As described above, the expression levels of EMT-related genes were significantly increased in breast cancer cells of 5 coculture groups co-cultured with adipocytes compared with control breast cancer cells, and as a result, EMT-induced breast cancer cells could be identified. The increased expression of the breast cancer EMT-associated gene can be used to determine whether EMT is induced in breast cancer cells in breast cancer screening. For example, among screening candidates, substances that inhibit the increase in the expression of EMT-related genes may be substances that inhibit the induction of EMT of breast cancer cells.

In the five examples described above, adipocytes were found to cause morphological variation of breast cancer cells and to increase migration and invasion. In addition, we confirmed that adipocytes affect the expression of EMT-associated factors and EMT-associated factor genes in breast cancer cells. Therefore, indirect co-culture of the adipocytes and breast cancer cells induces EMT of the breast cancer cells, and further, EMT induction of breast cancer cells is dependent on the type of breast cancer cells and the type of adipocytes.

Although the embodiments of the present invention have been described in detail with reference to the accompanying drawings, it is to be understood that the present invention is not limited to those embodiments and various changes and modifications may be made without departing from the scope of the present invention. . Therefore, the embodiments disclosed in the present invention are intended to illustrate rather than limit the scope of the present invention, and the scope of the technical idea of the present invention is not limited by these embodiments. Therefore, it should be understood that the above-described embodiments are illustrative in all aspects and not restrictive. The scope of protection of the present invention should be construed according to the following claims, and all technical ideas within the scope of equivalents should be construed as falling within the scope of the present invention.

Claims (13)

(EMT) -induced breast cancer cells comprising co-culturing adipocytes and breast cancer cells physically separated from the adipocytes. The method according to claim 1,
Wherein the breast cancer cells disposed in a container having a plurality of pores in the co-culturing step and the fat cells disposed separately from the breast cancer cells are arranged to interact through a solubility factor. The method according to claim 1,
The breast cancer cells may be selected from the group consisting of a luminal A type cell, a luminal B type cell, a basal type cell, and a luminal-basal mixed type type cells of a breast cancer cell. The method according to claim 1,
The culture medium in which the breast cancer cells are cultured is a culture method of breast cancer cells comprising at least one selected from the group consisting of DMEM (Dulbecco's modified Eagle's medium), EMEM (Eagle's minimum essential medium), Ham's F-12 medium and Leibovitz's L- . The method according to claim 1,
Wherein said adipocyte is different from cancer-associated adipocytes (CAAs). Induced factors and EMT-induced factors in the EMT-induced breast cancer cells using the cultured EMT-induced breast cancer cells co-cultured with adipocytes and physically separated breast cells from the adipocytes, A step of screening for a substance to be detected which inhibits the expression or has a negative influence on the expression site. The method according to claim 6,
And measuring the expression of an EMT-related factor in the breast cancer cell. 8. The method of claim 7,
The EMT-associated factors include E-cadherin, integrin, N-cadherin, cadherin-11, vimentin, TWIST1 at least one selected from the group consisting of: -related protein 1), HMGA2 (high mobility group proteins characterized by an AT-hook 2), TGF-ß (Transforming Growth Factor-?) and S100A4 ≪ / RTI > 9. The method of claim 8,
Wherein the measurement of E-carderine and the vimentin expression is selected from the group consisting of immunocytochemical analysis, protein western blotting and real-time PCR. Screening method. The method according to claim 6,
And measuring the expression of the EMT-related gene in the breast cancer cell. 11. The method of claim 10,
The EMT-associated gene comprises at least one selected from the group consisting of TWIST1 gene, MMP9 (matrix metallopeptidase 9) gene, zinc finger transcriptional repressor Snail gene, HMGA2 gene and TGF-β gene Screening methods for breast cancer-related drugs. The method according to claim 6,
And measuring the degree of migration and invasion of the breast cancer cells. The method according to claim 6,
And monitoring the shape change of the breast cancer cell.

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