SCREENING METHOD FOR SUBSTANCE ACTING ON MAINTENANCE OF EPITHELIAL PROPERTIES…

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1. A screening method for a substance on a maintenance of epithelial properties of comprising: (a) a step for culturing on a cell culture substrate can form a spheroid; (b) a step for the cells to contact a test and (c) a step for evaluating an effect of the substance acting on a maintenance of the properties of the cells with a in a morphology of the spheroid being as an

2. The screening method according to 1, wherein the step (c) carries out an through a measurement of a number of in a predetermined size.

3. The screening according to claim 1, wherein the (c) carries out an evaluation using a that can detect a hypoxic in a spheroid.

4. The screening method to claim 1, wherein the step (a) is out on a cell culture substrate a predetermined concavo-convex structure functions as a cell adhesion

5. The screening method according to 4, wherein the concavo-convex structure a plurality of unit structures with regularity, the unit being formed in a predetermined shape.

6. The screening method to claim 4, wherein the concavo-convex comprises a plurality of unit disposed with regularity, a between the unit structures equal to or smaller than 3 μm, the structure being formed in a shape in a plane direction, and a internal diameter of the unit being equal to or smaller 3 μm.

7. The screening method according to 2, wherein the step (c) carries out an using a reagent that can a hypoxic area in a spheroid.

8. The method according to claim 2, the step (a) is carried out on a cell substrate comprising a predetermined structure that functions as a adhesion surface.

9. The screening according to claim 3, wherein the (a) is carried out on a cell culture comprising a predetermined concavo-convex that functions as a cell surface.

Description:

TECHNICAL

The present invention relates to a of screening a substance that on a maintenance of the epithelial properties of with a change in a morphology of being as an indicator.

BACKGROUND ART

Transition (EMT) is a phenomenon in a cell cannot maintain as an epithelium, and obtains properties as a In recent years, it is reported epithelial-mesenchymal transition relates to and metastasis of a … cell, and and fibrosis of a tissue, etc. it is expected that substances acts on a maintenance of the epithelial of cells, i.e. substances inhibits the epithelial-mesenchymal transition, and inducing Mesenchymal-Epithelial Transition that is a reverse phenomenon of the transition result in a remedy of and fibrosis, etc.

Conventionally, as a of screening substances that act on a of the epithelial properties of cells, a is known which causes a substance to contact a cell to epithelial-mesenchymal transition is induced, and evaluates a change in a biomarker indicating an epithelial-mesenchymal transition as an indicator (see, for example, Literature 1).

PRIOR ART LITERATURE

Literature

Patent Literature 1: Publication No. WO 2009/111067

DISCLOSURE OF

Problem to be Solved by the Invention

A method using a biomarker as an indicator needs, however, a amount of preparation and costs for Accordingly, it is difficult to apply a method to a screening that a speedy evaluation for a plurality of like a screening in the early of a drug development. Moreover, it is to detect a biomarker of a low concentration, and the reliability is poor.

Hence, it is an of the present invention to provide a method which can be carried out and at a low cost, and which can obtain data for a substance that on a maintenance of epithelial properties of

Means for Solving the Problem

The of the present invention found a between the expression level of that is an cell adhesion expressed in an epithelial cell and a indicated by a cell cultured on a cell culture substrate. is, when the expression level of decreases due to the epithelial-mesenchymal transition and a becomes unable to maintain the properties, such a cell a high migration capability, and a condition to be likely to form a on the predetermined cell culture and the formed spheroid indicates a morphology in accordance with the level of E-cadherin. The inventors of the invention have accomplished the invention that is a screening with a change in the morphology of a being as an indicator.

The subject of the present invention are follows.

A method for a substance acting on a of an epithelial properties of cells to the present invention includes: (a) a for culturing cells on a cell substrate that can form a (b) a step for causing the cells to a test substance; and (c) a step for an effect of the test substance on a maintenance of the epithelial properties of the with a change in the morphology of the being as an indicator.

In this the step (c) may carry out an evaluation a measurement of a number of spheroids in a size.

In this case, the step (c) may carry out an evaluation a reagent that can detect a area in a spheroid.

The step (a) may be out on a cell culture substrate a predetermined concavo-convex structure functions as a cell adhesion

In this case, the concavo-convex may include a plurality of unit disposed with regularity, the structure being formed in a planar shape.

The concavo-convex may include a plurality of unit disposed with regularity, a between the unit structures equal to or smaller than 3 μm, the structure being formed in a shape in a plane direction, and a internal diameter of the unit being equal to or smaller 3 μm.

Effect of the Invention

The screening of the present invention can perform of a target substance quickly and at a low since such a method evaluation with a change in a of a spheroid as an indicator. Moreover, the utilizes a spheroid that is to reflect a condition further to condition in a biological object mono layer cells, enabling screening efficiently. since it is possible to perform while capturing a plurality of simultaneously, the method can obtain a screening result.

BRIEF OF DRAWINGS

FIG. 1 is a plan illustrating a concavo-convex structure of a culture substrate to be used in a of the present invention;

FIG. 2 is a illustrating morphologies of spheroids;

3 is a photograph and a graph illustrating expression levels;

FIG. 4 is a photograph illustrating an effect of an transition inhibitor acting on of spheroids;

FIG. 5 is a graph signal intensities of a hypoxic detection reagent;

FIG. 6 is a illustrating an expression level of an gene;

FIG. 7 is a graph an expression level of an N-cadherin

FIG. 8 is a graph illustrating an level of a Vimentin gene; and

FIG. 9 is a graph illustrating an level of a ZEB1 gene.

MODE FOR CARRYING OUT THE INVENTION

A method of the present invention (a) a process of culturing cells a cell culture substrate can form a spheroid, (b) a process of such cells to contact a substance, (c) a process of evaluating an of the test substance acting on a of the epithelial properties of the cells a change in the morphologies of the spheroid as an indicator.

A spheroid according to the invention means an aggregation of which have the cells gathered and aggregated.

A maintenance of the properties of cells according to the invention means a condition in an epithelial-mesenchymal transition is suppressed or or a condition in which a mesenchymal-epithelial is promoted or induced. Hence, the method of the present invention can be divided into a screening for an epithelial-mesenchymal transition inhibitor, and a method for a mesenchymal-epithelial transition

A screening method for an epithelial-mesenchymal inhibitor can be carried out through, for a process of culturing cells on a culture substrate that can a spheroid, a process of causing cells to contact an epithelial-mesenchymal inducer, a process of causing the cells to contact a test and a process of evaluating an effect of the substance acting on an epithelial-mesenchymal of the cells with a change in the of the spheroid being as an indicator. An transition inducer available is, for TGF-β, TNF-α, EGF, or The process of causing the cultured to contact the epithelial-mesenchymal transition may be executed in any instant before the of causing the cultured cells to the test substance is executed, and for may be executed at the time of cell may be executed when several have elapsed after the seeding, or may be executed simultaneously the process of causing the cultured to contact the epithelial-mesenchymal transition

A screening method for a mesenchymal-epithelial inducer can be carried out through, for a process of culturing cells on a culture substrate that can a spheroid, a process of causing the cells to contact a test and a process of evaluating an effect of the substance acting on a mesenchymal-epithelial of the cells with a change in the of the spheroid being as an indicator. to such a method, it is preferable to out such a method while a cell having a low E-cadherin level, such as MIAPaCa-2, or A1165 which is a cell derived from a human ….

The test substance may be with the cultured cells the process of causing the cultured to contact the epithelial-mesenchymal transition to induce the cultured cells to be cells. When, in particular, of an epithelial … (e.g. or a linear … (e.g.

etc. are used, it is preferable to a process by the epithelial-mesenchymal inducer at the of cell seeding.

An evaluation a change in a morphology of a spheroid as an according to the present invention can be out by comparing differences in external and/or internal structures of between a negative control and a test sample group.

The in an appearance can have an indicator is a change in a shape (a circularity), and number, etc. of the spheroid. for example, the size of the spheroid in the sample group becomes in comparison with that of the in the negative control group, the number of the spheroids having a equal to or larger than a size increases, or when the of the spheroid becomes further to 1, it can be evaluated that the test is a substance having an effect of the epithelial properties of cells.

Moreover, instead of (or together the indicator that is a change in the of spheroids having a size to or larger than the predetermined a change in the number of cells do not form a spheroid can be used as an This is because such an can be regarded as a form of a change in the of the spheroid.

Conversely, the difference in an structure can have an indicator is a change in a hypoxic area or an concentration in the spheroid. When, for the hypoxic area in the spheroid in the sample group is large in with that of the spheroid in the control group, a spheroid a tight cell-to-cell adhesion cells is formed, and thus it can be that the test substance an effect of maintaining the epithelial of cells.

Since the screening of the present invention can capture the in the external shape and the difference in the structure thereof at the same data having a high can be obtained. Moreover, since the of the present invention can perform based on a visual parameter, it possible to easily trace a over time.

A measurement of the in the external shape of the spheroid can be carried out through an observation and using a biological microscope a phase-contrast microscope, and can be also out through a plate reader, a that can measure a three-dimensional and an analysis algorithm for image etc.

Conversely, a measurement of the in the internal structure is not limited to any technique as long as a hypoxic is detectable, and for example, can be carried out a compound emitting phosphorescence. the phosphorescent compound is used, a condition can be made visible a quenching phenomenon of phosphorescence by and the hypoxic area and an oxygen can be grasped.

Such a compound is not to any particular one as long as it emits and causes the quenching phenomenon by and it is preferable that such a should emit phosphorescence of a wavelength having a high permeability, have a long life and have a high quantum yield. An example is an iridium complex having as a major metal and having of aromatic series as ligands, and specifically, is Bis(2-benzo[b]thiophen-2-yl-pyridine)(acetylacetonate)iridium(III). Since a signal can be quantified, an inhibitory in an epithelial-mesenchymal transition of the test or an induction efficiency in a mesenchymal-epithelial of the test substance can be easily

A cell culture substrate can form a spheroid according to the invention is not limited to any particular one as as an adherence property with a is suppressed in comparison with a culture substrate used in a monolayer culturing, and for example, a culture substrate having a property or a hydrophobic property can be used.

A material of a cell substrate is not limited to any particular one as as it is nontoxic to cells, and for example, “polyethylene”, “polypropylene”, “polyimide”, a polymer, such as polyactic polyactic-acid-polyglycolic-acid copolymer, or polycaprolactone”, a olefin resin like a “cyclic-olefin-based thermoplastic resin, as a cyclic olefin copolymer or a cyclic olefin polymer an “acrylic resin”, “other such as a photo-curable resin or a resin”, a “metal like oxide”, a “glass”, a “quartz or a “silicone” can be used. Moreover, one a coating layer, such as a a “photoresist”, or a “metal like oxide”, on the surface of a basal main body formed of a or glass, etc. can be used.

A for controlling the adherence of cells, as ultraviolet irradiation, gamma-ray plasma irradiation, or coating various kinds of substances, may be on the surface of a cell culture as long as it may function as a cell surface.

The screening method of the present may be carried out using a cell substrate having a predetermined structure that functions as a adhesion surface.

A concavo-convex can be formed in various shapes, as a linear shape (line and a pillar shape, or a hole in accordance with the properties of to be cultured, but a structure having a of unit structures 1 each in a predetermined planar shape and regularly is preferable. As shown in 1, for example, the plurality of unit 1 each formed in a polygonal shape can be disposed sequentially.

In case, from the standpoint of a growth on an isotropically uniform a regular polygon, such as a triangle, a square, or a regular or a circle is preferable. Moreover, it is to combine a pillar or hole concavo-convex structure with a structure formed of the unit 1 in a planar shape.

From the of causing cultured cells to be in a similar to a condition in a biological it is preferable that a line 2 have a width, such as to or smaller than 3 μm, equal to or than 2 μm, equal to or smaller 1 μm, equal to or smaller than 700 nm, to or smaller than 500 nm, or equal to or than 250 nm, and the smaller width is This is because that it is that the smaller the width of the 2 is, the more cells adhered to a structure plane can grow while forming a spheroid.

The structure 1 is formed so as to have a in various sizes, such as to or larger than 1 nm, equal to or than 10 nm, equal to or larger 100 nm, equal to or larger than 200 nm, to or larger than 500 nm, equal to or than 1 μm, equal to or larger 10 μm, or equal to or larger than 100 μm, in with a properties of cells to be Moreover, various aspect of such a concavity and convexity can be such as equal to or larger 0.2, equal to or larger 0.5, equal to or larger 1, equal to or larger than 2.

It is that the unit structure 1 have the minimum internal (preferably, the maximum internal of equal to or smaller than 3 μm, and equal to or smaller than 2 μm, to or smaller than 1 μm, equal to or than 700 nm, equal to or smaller 500 nm, or equal to or smaller than 250 nm, the diameter is preferable. In this an internal diameter means a between two parallel lines the external sides of the unit 1. Accordingly, the minimum internal means the shortest distance distances between the two parallel contacting the external sides of the structure 1, and the maximum internal means the longest distance the distances between the two parallel contacting the external sides of the structure 1. When, for example, the structure 1 is a regular hexagon, a between parallel sides with each other is the internal diameter, and a distance vertices facing with other is the maximum internal Moreover, when the unit 1 is a rectangle, the length of a short is the minimum diameter, and the length of a line is the maximum diameter.

A technique of the concavo-convex structure is not to any particular one, but for example, solution-casting, etching, blasting, or discharging can be applied. In this from the standpoint of controlling a etc. further highly a technique through the nanoimprinting is

Hereinafter, the present invention be explained in detail with to examples. The present invention is, not limited to the following description.

Example

Correlation Between Expression Level and Spheroid

(1) Cell Seeding and Observation

were seeded in a cell vessel NanoCulture (registered dish (made by SCIVAX 35 mm dish, material of concavo-convex surface=polymethylpentene (TPX made by Chemical Co. Ltd.), planar of concavo-convex structure=square, width unit structures (line nm, minimum internal diameter of structure=3 μM, and depth=1 μm) at 2.5×10 5 and were observed through an microscope at the third day of culturing.

The used were a human … cell strain, Capan-1, Capan-2, AsPC-1, and MIAPaCa-2. The culture media were a 10-%-FBS-containing RPMI1640 medium for BxPC-3, AsPC-1, and a 10-%-FBS-containing MEM culture medium for a 10-%-FBS-containing McCoy’s 5A culture for Capan-2, and a 10-%-FBS-containing DMEM medium for MIAPaCa-2.

(2) Western

The same cells as those of (1) also seeded in the same culture vessel NanoCulture trademark) dish (made by corporation, 35 mm dish) as that of and cultured for five days, and cells were collected by a The collected cells were by PBS, dissolved in a dissolving and cell dissolved solutions taken as samples.

Next, a of each cell strain to 20 μg protein) was separated through SDS gel electrophoresis (SDS-PAGE), and protein was to a PVDF film. A detection of was carried out using mouse monoclonal antibody (BD Biosciences: Cat as a primary antibody, an anti-mouse having undergone HRP labeling Cruz: sc-2005) as a secondary and an ECL Plus Western Blotting System (made by GE healthcare as a detection reagent.

The results of (1) and (2) are in FIGS. 2 and 3. According to BxPC-3 and having a high expression of E-cadherin, the circularity of spheroid was to 1, and it becomes clear that a having a tight cell-to-cell was formed. In contrast, according to and MIAPaCa-2 having a low expression of E-cadherin, the circularity was apart 1 more than the spheroid of the cell strains, and it becomes that a spheroid having cell adhesion was formed.

Example

Effect of Epithelial-mesenchymal Inhibitor Acting on Morphology of

(1) Pre-Incubation

5-%-FBS-containing DMEM medium and 1.0-%-Matrigel (registered containing DMEM culture were added in respective of a cell culture vessels (registered trademark) plate by SCIVAX corporation, 384-well material of concavity-convexity structure (TPX made by MITSUI Co. Ltd.), planar shape of structure=square, width between structures (line width)=700 nm, internal diameter of unit μm, and depth=1 μm) by 25 μl, and were subjected to force for five minutes at

(2) Cell Seeding

A cell having human-lung-…-cells derived A549 adjusted to 13.5×10 4 by a 5-%-FBS-containing DMEM culture was added to each well of the plate by 25 μl (the number of was 3.4×10 3 cells/well, and the final of the culture medium additive was 5% FBS and Matrigel).

(3) Addition of Epithelial-Mesenchymal Inducer and Inhibitor

At the third day of the TGF-β2 (made by RD systems 302-B2-002) and TNF-α (made by RD corporation, 210-TA-010) which epithelial-mesenchymal transition inducers added to each well in a way that a final concentration 5 ng/ml and 10 ng/ml, respectively. at the same time, SB431542 by Miltenyi Biotec corporation, that was an inhibitor of TGF-β2 was in such a way that a final became 10 μM.

Dimethyl-sulfoxide was added to all wells no SB431542 was added in such a way a final concentration became

(4) Addition of Hypoxic Area Reagent

At the fifth day of the culturing, a area detection reagent (made by SCIVAX corporation) was to each well in such a way a final concentration became 2 μM. is a reagent that contains complex which emits red

(5) Observation

An observation through a microscope was carried out at the sixth day of the

A result obtained through an and image-pickup by the fluorescent microscope is in FIG. 4. First of all, bright field images of wells are compared with other, in the case of the well (a where no epithelial-mesenchymal transition was added, compact spheroids a circularity close to 1 were

In contrast, in the case of the well the epithelial-mesenchymal transition inducers added, it becomes clear the majority of spheroids were and the contours of the remaining spheroids a large concavity and convexity. In the of the well where both transition inducer and inhibitor added, it becomes clear in comparison with the well no inhibitor was added, the collapse of were suppressed and the contours of the had a small concavity and convexity.

when fluorescent field of respective wells are compared each other, it becomes that in the case of the well the epithelial-mesenchymal transition inducer was in comparison with the control, the red was small and the red area was small, and in the of the well where the inhibitor was added, the red intensity was high and the red was large. That is, it becomes that a hypoxic area was

A result of quantifying the red signal at time is shown in FIG. 5. It clear that in the case of the where the epithelial-mesenchymal transition was added, the red signal was about as much as that of the well no inhibitor was added.

Third

Verification of Change in Gene at the time of Adding Epithelial-Mesenchymal Inducer and Inhibitor

In order to whether or not a change in the morphology of the confirmed in the second example an epithelial-mesenchymal transition and an epithelial-mesenchymal inhibition, a result of measuring an level of a gene that is as an epithelial or mesenchymal marker qRT-PCR is shown in FIGS. 6 to 9. The from (1) pre-incubation to (3) addition of the transition inducer and inhibitor carried out likewise the second

Next, at the sixth day of the culturing, all were extracted from sample using RNeasy Mini Kit (made by QIAGEN and such RNAs having reverse transcription using RT reagent Kit (made by TAKARABIO were used for qRT-PCR. Premix Ex Tagll (made by Inc.) was used for PCR. Thermal Cycler Dice by TAKARABIO Inc.) was used for a and a detection. The primers used for PCR are in the following table 1. Note data is corrected by the expression of internal standard gene and is shown as a relative value the expression level of the control is as 1.

Kawasaki GPZ 500 S (reduced effect)
Kawasaki GPZ 500 S (reduced effect)
Kawasaki GPZ 500 S (reduced effect)


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