JP2015033384A - Screening method for substance acting on maintenance of epithelial property of cell - Google Patents
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Abstract
Description
本発明は、スフェロイドの形態の変化を指標として、細胞の上皮性維持に作用する物質をスクリーニングする方法に関する。 The present invention relates to a method for screening a substance that acts on the maintenance of epitheliality of a cell, using a change in spheroid morphology as an index.
上皮−間葉転換（Ｅｐｉｔｈｅｌｉａｌ−Ｍｅｓｅｎｃｈｙｍａｌ Ｔｒａｎｓｉｔｉｏｎ：ＥＭＴ）とは、細胞が上皮としての特性を維持することができず、間葉系としての特性を獲得する現象である。近年、上皮−間葉転換が、癌細胞の浸潤・転移や組織のリモデリング・線維化などに関与することが報告されている。そのため、細胞の上皮性維持に作用する物質、すなわち、上皮−間葉転換を阻害する物質や、上皮−間葉転換の逆の現象である間葉−上皮転換（Ｍｅｓｅｎｃｈｙｍａｌ−Ｅｐｉｔｈｅｌｉａｌ Ｔｒａｎｓｉｔｉｏｎ：ＭＥＴ）を誘導する物質は、癌や線維症などの治療薬につながることが期待されている。 Epithelial-mesenchymal transition (EMT) is a phenomenon in which cells cannot maintain their characteristics as epithelium and acquire their characteristics as mesenchymal system. In recent years, epithelial-mesenchymal transition has been reported to be involved in cancer cell invasion / metastasis, tissue remodeling / fibrosis, and the like. Therefore, substances that affect the epithelial maintenance of cells, that is, substances that inhibit epithelial-mesenchymal transition, and mesenchymal-epithelial transition (MET), which is the reverse phenomenon of epithelial-mesenchymal transition, are used. Inducing substances are expected to lead to therapeutic drugs such as cancer and fibrosis.
従来、細胞の上皮性維持に作用する物質をスクリーニングする方法として、上皮−間葉転換が誘導された細胞に被験物質を接触させ、上皮−間葉転換状態を示すバイオマーカー量の変化を指標として評価する方法がある（例えば、特許文献１参照）。 Conventionally, as a method for screening a substance that acts on the maintenance of epitheliality of cells, a test substance is brought into contact with a cell in which epithelial-mesenchymal transition is induced, and a change in the amount of biomarker indicating an epithelial-mesenchymal transition state is used as an index There is a method of evaluation (for example, see Patent Document 1).
しかしながら、バイオマーカー量を指標とするスクリーニング方法は、その検出に手間とコストがかかる。それゆえ当該方法は、創薬初期段階のスクリーニングのような、大量のサンプルをスピーディーに評価しなければならないスクリーニングへの適用が困難である。また、低濃度のバイオマーカーの検出は難しいため、信頼性に乏しいという問題がある。 However, the screening method using the amount of biomarker as an index takes time and effort to detect it. Therefore, this method is difficult to apply to screening that requires rapid evaluation of a large number of samples, such as screening at an early stage of drug discovery. Moreover, since it is difficult to detect a low-concentration biomarker, there is a problem that reliability is poor.
そこで本発明は、迅速かつ低コストで実施でき、信頼性の高いデータの取得を可能とする細胞の上皮性維持に作用する物質のスクリーニング方法を提供することを目的とする。 Therefore, an object of the present invention is to provide a screening method for a substance that can be carried out quickly and at low cost, and that can acquire highly reliable data and that acts on the maintenance of epitheliality of cells.
本発明者らは、上皮細胞が発現する細胞間接着因子であるＥ−ｃａｄｈｅｒｉｎの発現量と、所定の培養基材上で培養される細胞が示す挙動との間に相関関係があることを見出した。すなわち、上皮−間葉転換によりＥ−ｃａｄｈｅｒｉｎの発現が低下して細胞の上皮性が維持出来なくなると、当該細胞は高い遊走能を獲得して所定の培養基材上でスフェロイドを形成しやすい状態になると共に、形成されるスフェロイドはＥ−ｃａｄｈｅｒｉｎの発現量に応じて異なる形態を呈することを見出した。そして、スフェロイド形態の変化を指標としたスクリーニング方法として本発明を完成させた。 The present inventors have found that there is a correlation between the expression level of E-cadherin, which is an intercellular adhesion factor expressed by epithelial cells, and the behavior exhibited by cells cultured on a predetermined culture substrate. It was. That is, when the expression of E-cadherin decreases due to epithelial-mesenchymal transition and the epitheliality of the cells cannot be maintained, the cells acquire high migration ability and are likely to form spheroids on a predetermined culture substrate As a result, it was found that the formed spheroids have different forms depending on the expression level of E-cadherin. And this invention was completed as a screening method which used the change of the spheroid form as a parameter | index.
本発明の要旨は以下の通りである。 The gist of the present invention is as follows.
本発明の細胞の上皮性維持に作用する物質のスクリーニング方法は、（ａ）スフェロイド形成可能な培養基材上で細胞を培養する工程と、（ｂ）前記細胞と被験物質とを接触させる工程と、（ｃ）被験物質が前記細胞の上皮性維持に及ぼす効果をスフェロイドの形態の変化を指標として評価する工程と、を含むことを特徴とする。 The screening method for a substance that acts to maintain epitheliality of cells of the present invention includes (a) culturing cells on a culture substrate capable of forming spheroids, and (b) contacting the cells with a test substance. And (c) evaluating the effect of the test substance on the maintenance of the epithelial properties of the cells using the change in the spheroid morphology as an index.
この場合、前記工程（ｃ）は、所定の大きさのスフェロイド数の測定により評価することができる。 In this case, the step (c) can be evaluated by measuring the number of spheroids having a predetermined size.
また、前記工程（ｃ）は、スフェロイド内の低酸素領域を検出可能な試薬を用いて評価することができる。 Moreover, the said process (c) can be evaluated using the reagent which can detect the hypoxia area | region in a spheroid.
前記工程（ａ）は、細胞接着面として機能する所定の凹凸構造を有する培養基材上で行うことができる。 The step (a) can be performed on a culture substrate having a predetermined concavo-convex structure that functions as a cell adhesion surface.
この場合、前記凹凸構造は、所定の平面形状からなる単位構造を規則的に複数配列したものであることが好ましい。 In this case, the concavo-convex structure is preferably one in which a plurality of unit structures each having a predetermined planar shape are regularly arranged.
また、凹凸構造は、単位構造間の幅が３μｍ以下で、平面方向の形状が多角形であると共に、最小内径が３μｍ以下の単位構造を規則的に複数配列して形成されているものであると良い。 In addition, the concavo-convex structure is formed by regularly arranging a plurality of unit structures having a width between unit structures of 3 μm or less, a planar shape of a polygon, and a minimum inner diameter of 3 μm or less. And good.
本発明のスクリーニング方法は、スフェロイドの形態の変化を指標として評価を行うため、迅速かつ低コストで目的の物質をスクリーニングすることができる。また、単層細胞よりも生体内に近い状態を反映すると考えられているスフェロイドを利用した方法であることから、効率的にスクリーニングを行うことができる。さらに、複数の指標を同時に捉えて評価することが可能であるため、信頼性の高いスクリーニング結果を得ることができる。 Since the screening method of the present invention is evaluated using the change in the spheroid morphology as an index, the target substance can be screened quickly and at low cost. Moreover, since it is a method using a spheroid considered to reflect a state closer to the living body than a monolayer cell, screening can be performed efficiently. Furthermore, since a plurality of indices can be captured and evaluated simultaneously, a highly reliable screening result can be obtained.
本発明のスクリーニング方法は、（ａ）スフェロイド形成可能な培養基材上で細胞を培養する工程と、（ｂ）前記細胞と被験物質とを接触させる工程と、（ｃ）被験物質が前記細胞の上皮性維持に及ぼす効果をスフェロイドの形態の変化を指標として評価する工程とを含むものである。 The screening method of the present invention comprises (a) a step of culturing cells on a culture substrate capable of forming spheroids, (b) a step of contacting the cells with a test substance, and (c) a test substance of the cell. And evaluating the effect on epithelial maintenance using a change in the spheroid morphology as an index.
本発明におけるフェロイドとは、三次元的に細胞同士が集合・凝集化した細胞集合体を意味する。 The pheroid in the present invention means a cell aggregate in which cells are aggregated and aggregated three-dimensionally.
また、本発明における細胞の上皮性維持とは、上皮−間葉転換を抑制・阻害された状態、または、間葉−上皮転換を促進・誘導された状態を意味する。したがって、本発明のスクリーニング方法は、上皮−間葉転換の阻害物質のスクリーニング方法と、間葉−上皮転換の誘導物質のスクリーニング方法とに大別される。 Further, the maintenance of epitheliality of cells in the present invention means a state where epithelial-mesenchymal transition is suppressed or inhibited, or a state where mesenchymal-epithelial transition is promoted / induced. Therefore, the screening method of the present invention is roughly divided into a screening method for an inhibitor of epithelial-mesenchymal transition and a screening method for an inducer of mesenchymal-epithelial transition.
上皮−間葉転換の阻害物質のスクリーニング方法は、例えば、スフェロイド形成可能な培養基材上で細胞を培養する工程と、当該細胞と上皮−間葉転換誘導剤とを接触させる工程と、培養細胞と被験物質とを接触させる工程と、被験物質が細胞の上皮−間葉転換に及ぼす効果をスフェロイドの形態の変化を指標として評価する工程と、により行うことができる。上皮−間葉転換誘導剤としては、例えば、TGF-β、TNF-α、EGF、IL-4等を用いることができる。なお、培養細胞と上皮−間葉転換誘導剤とを接触させる工程は、培養細胞と被験物質とを接触させる工程以前であればいつの時点で行っても良く、例えば、細胞播種時に行っても良いし、細胞播種から数日経過後に行っても良いし、培養細胞と上皮−間葉転換誘導剤とを接触させる工程と同時に行っても良い。 The method for screening an inhibitor of epithelial-mesenchymal transition includes, for example, a step of culturing cells on a culture substrate capable of forming spheroids, a step of contacting the cells with an epithelial-mesenchymal transition inducer, and cultured cells. And the step of contacting the test substance with each other, and the step of evaluating the effect of the test substance on the epithelial-mesenchymal transition of cells using the change in the spheroid morphology as an index. As the epithelial-mesenchymal transition inducer, for example, TGF-β, TNF-α, EGF, IL-4 and the like can be used. The step of contacting the cultured cell with the epithelial-mesenchymal transition inducer may be performed at any time before the step of contacting the cultured cell with the test substance, for example, at the time of cell seeding. In addition, it may be performed several days after cell seeding, or may be performed simultaneously with the step of bringing the cultured cells into contact with the epithelial-mesenchymal transition inducer.
間葉−上皮転換の誘導物質のスクリーニング方法は、例えば、スフェロイド形成可能な培養基材上で細胞を培養する工程と、培養細胞と被験物質とを接触させる工程と、被験物質が細胞の間葉−上皮転換に及ぼす効果をスフェロイドの形態の変化を指標として評価する工程と、により行うことができる。当該方法においては、Ｅ−ｃａｄｈｅｒｉｎ発現量の低い細胞、例えば、ヒト膵癌由来細胞株であるＭＩＡＰａＣａ−２、ＰＡＮＣ−１、Ａ１１６５等を用いて行うことが好ましい。なお、培養細胞と上皮−間葉転換誘導剤とを接触させる工程を挿入して間葉系細胞へと誘導した後に、被験物質と培養細胞とを接触させても良い。特に、上皮癌（例えば、Ａ５４９）や線癌（例えば、Ｃａｐａｎ−２）などの細胞を用いる場合は、播種時に上皮−間葉転換誘導剤による処理を行うことが好ましい。 A screening method for an inducer of mesenchymal-epithelial transformation includes, for example, a step of culturing cells on a culture substrate capable of forming spheroids, a step of contacting cultured cells with a test substance, The step of evaluating the effect on epithelial transformation with the change of the spheroid morphology as an index. In this method, it is preferable to use cells with low E-cadherin expression level, for example, MIAPaCa-2, PANC-1, A1165, etc., which are human pancreatic cancer-derived cell lines. In addition, after inserting the process which contacts a cultured cell and an epithelial-mesenchymal transition inducer and inducing | guiding | deriving to a mesenchymal cell, you may make a test substance and a cultured cell contact. In particular, when cells such as epithelial cancer (for example, A549) and line cancer (for example, Capan-2) are used, it is preferable to perform treatment with an epithelial-mesenchymal transition inducer at the time of seeding.
本発明におけるスフェロイドの形態の変化を指標とする評価は、ネガティブコントロール群と被験サンプル群との、スフェロイドの外観及び／又は内部構造の違いを比較することにより行うことができる。 The evaluation using the change in spheroid morphology in the present invention as an index can be performed by comparing the difference in the appearance and / or internal structure of the spheroid between the negative control group and the test sample group.
外観の違いは、スフェロイドの形（円形度）、大きさ、数等の変化を指標とすることができる。例えば、ネガティブコントロール群と比べて、被験サンプル群においてスフェロイドのサイズが大きくなった場合や、所定のサイズ以上のスフェロイド数が増加した場合や、スフェロイドの円形度がより１に近づいた場合には、被験物質が細胞の上皮性を維持する効果がある物質であると評価することができる。また、所定のサイズ以上のスフェロイド数の変化を指標とする代わりに（またはそれと並行して）、スフェロイドを形成していない細胞数の変化を指標としても良い。スフェロイドの形態の変化の一態様として捉えられるからである。 Differences in appearance can be indicated by changes in spheroid shape (circularity), size, number, and the like. For example, compared to the negative control group, when the size of the spheroid is increased in the test sample group, when the number of spheroids of a predetermined size or more is increased, or when the circularity of the spheroid is closer to 1, It can be evaluated that the test substance is a substance having an effect of maintaining the epitheliality of cells. Further, instead of (or in parallel with) a change in the number of spheroids having a predetermined size or more as an index, a change in the number of cells not forming spheroids may be used as an index. It is because it is grasped as one mode of change of the form of spheroid.
一方、内部構造の違いは、スフェロイド内の低酸素領域や酸素濃度の変化を指標とすることができる。例えば、ネガティブコントロール群と比べて、被験サンプル群においてスフェロイド内の低酸素領域が大きい場合には、細胞間接着のタイトなスフェロイドが形成されており、被験物質が細胞の上皮性を維持する効果がある物質であると評価することができる。 On the other hand, the difference in the internal structure can be determined by using a hypoxic region in the spheroid or a change in oxygen concentration as an index. For example, when the hypoxic region in the spheroid is large in the test sample group compared to the negative control group, a tight spheroid with intercellular adhesion is formed, and the test substance has an effect of maintaining the epitheliality of the cell. It can be evaluated as a certain substance.
なお、本発明のスクリーニング方法は、スフェロイドの外観の違いと内部構造の違いを同時に捉えることができるため、信頼性の高いデータの取得が可能である。さらに、本発明の方法は、視覚的パラメータに基づいて評価を行うことができるため、容易に経時的変化を追うことが可能である。 In addition, since the screening method of the present invention can simultaneously capture the difference in the appearance of the spheroid and the difference in the internal structure, it is possible to acquire highly reliable data. Furthermore, since the method of the present invention can be evaluated based on visual parameters, it is possible to easily follow changes over time.
スフェロイド形態の外観の違いの測定は、位相差顕微鏡等の生物顕微鏡を用いた観察・計測にて行うことができるほか、プレートリーダー、３次元的形状測定可能な装置、画像データを対象とした解析アルゴリズム等を用いて行うことができる。 The measurement of the difference in appearance of spheroid forms can be performed by observation and measurement using a biological microscope such as a phase contrast microscope, a plate reader, a device capable of measuring a three-dimensional shape, and analysis for image data This can be done using an algorithm or the like.
一方、内部構造の違いの測定は、低酸素領域を検出可能なものであればどのような方法であっても良いが、例えば、燐光を発する化合物を用いて行うことができる。燐光を発する化合物を用いれば、酸素による燐光の消光現象を利用して低酸素状態を可視化でき、低酸素領域及び酸素濃度を把握できるためである。当該化合物は、燐光を発して酸素による消光現象を生じさせるものであれば特に制限されないが、細胞透過性の高い長波長の燐光を発し、燐光寿命が長く、燐光量子収率の高い化合物であることが好ましい。当該化合物の例としては、Ｉｒ（ＩＩＩ）を中心金属として芳香族系分子を配位子とするイリジウム錯体が挙げられ、具体的には、Ｂｉｓ（２−ｂｅｎｚｏ［ｂ］ｔｈｉｏｐｈｅｎ−２−ｙｌ−ｐｙｒｉｄｉｎｅ）（ａｃｅｔｙｌａｃｅｔｏｎａｔｅ）ｉｒｉｄｉｕｍ（ＩＩＩ）が挙げられる。なお、燐光シグナルは定量化できるので、被験物質の上皮−間葉転換における阻害効率又は間葉−上皮転換誘導効率を容易に求めることができる。 On the other hand, the measurement of the difference in internal structure may be performed by any method as long as it can detect the low oxygen region. For example, it can be performed using a phosphorescent compound. This is because if a phosphorescent compound is used, a low oxygen state can be visualized by utilizing a phosphorescence quenching phenomenon by oxygen, and a low oxygen region and an oxygen concentration can be grasped. The compound is not particularly limited as long as it emits phosphorescence and causes a quenching phenomenon due to oxygen, but emits long-wavelength phosphorescence having high cell permeability, a long phosphorescence lifetime, and a high phosphorescence quantum yield. It is preferable. Examples of the compound include an iridium complex having Ir (III) as a central metal and an aromatic molecule as a ligand. Specifically, Bis (2-benzo [b] thiophen-2-yl- pyridine) (acetylacetonate) iridium (III). In addition, since the phosphorescence signal can be quantified, the inhibition efficiency or the mesenchymal-epithelial transition induction efficiency of the test substance in the epithelial-mesenchymal transition can be easily determined.
本発明におけるスフェロイド形成可能な培養基材は、通常の単層培養に用いられる培養基材よりも、細胞との接着性が抑制されているものであればどのようなものであっても良く、例えば、培養基材表面の親水性又は疎水性を改質したもの等を用いることができる。 The culture substrate capable of forming spheroids in the present invention may be any material as long as the adhesion to the cells is suppressed rather than the culture substrate used for normal monolayer culture, For example, those having modified hydrophilicity or hydrophobicity on the surface of the culture substrate can be used.
培養基材の材質は、細胞に対し無毒性のものであればどのようなものでも良く、例えば、「ポリスチレン」、「ポリエチレン」、「ポリプロピレン」、「ポリイミド」、「ポリ乳酸やポリ乳酸−ポリグリコール酸共重合体、ポリカプロラクトン等の生分解性ポリマー」、「ポリメチルペンテン等のポリオレフィン樹脂」、「環状オレフィン共重合体（ＣＯＣ）や環状オレフィン重合体（ＣＯＰ）等の環状オレフィン系熱可塑性樹脂」、「アクリル樹脂」、「光硬化性樹脂や熱硬化性樹脂等のその他の樹脂」、「酸化アルミニウム等の金属」、「ガラス」、「石英ガラス」、「シリコン」等を用いることができる。また、シリコンやガラス等からなる基板本体の表面に、「樹脂」、「フォトレジスト」、「酸化アルミニウム等の金属」等の被覆層が形成されたものを用いることもできる。 The material of the culture substrate may be any material as long as it is non-toxic to cells. For example, “polystyrene”, “polyethylene”, “polypropylene”, “polyimide”, “polylactic acid or polylactic acid-polylactic acid”. Glycolic acid copolymers, biodegradable polymers such as polycaprolactone "," polyolefin resins such as polymethylpentene ", and cyclic olefin thermoplastics such as cyclic olefin copolymer (COC) and cyclic olefin polymer (COP) "Resin", "Acrylic resin", "Other resins such as photo-curing resin and thermosetting resin", "Metal such as aluminum oxide", "Glass", "Quartz glass", "Silicon", etc. it can. Further, it is also possible to use a substrate body made of silicon, glass or the like on which a coating layer such as “resin”, “photoresist”, “metal such as aluminum oxide” is formed.
培養基材の表面は、細胞接着面として機能し得る限り、紫外線照射、ガンマ線照射、プラズマ照射や、種々の物質のコーティング等といった、細胞の接着性を制御するための処理が施されていても良い。 As long as the surface of the culture substrate can function as a cell adhesion surface, treatment for controlling cell adhesion such as ultraviolet irradiation, gamma ray irradiation, plasma irradiation, and coating of various substances may be performed. good.
また、本発明のスクリーニング方法は、細胞接着面として機能する所定の凹凸構造を有する培養基材を用いて行っても良い。 The screening method of the present invention may be performed using a culture substrate having a predetermined concavo-convex structure that functions as a cell adhesion surface.
凹凸構造としては、培養する細胞の性質に応じて、線状（ラインアンドスペース）、ピラー状、ホール状等、種々の形状とすることができるが、好ましくは、所定の平面形状からなる単位構造１を規則的に複数配列した構造の方が良い。例えば、図１に示すように、平面形状が多角形である単位構造１を複数連続したものとすることができる。この時、等方的に均一な構造上で細胞を成長させることができるという点で、正三角形、正方形、正六角形等の正多角形や、円形のものがより好ましい。また、ピラー状やホール状の凹凸構造と、平面形状からなる単位構造１から形成される凹凸構造とを組み合わせることも可能である。なお、培養細胞を生体内での状態に近付けるという観点からは、線２の幅は、３μｍ以下、２μｍ以下、１μｍ以下、７００ｎｍ以下、５００ｎｍ以下、２５０ｎｍ以下というように、小さくなるほど好ましい。線２の幅が小さくなるほど、凹凸構造面に接着した細胞は、多くの仮足を成長させながらスフェロイドを形成することができると考えられるためである。 The concavo-convex structure can be various shapes such as a line shape (line and space), a pillar shape, and a hole shape, depending on the properties of the cells to be cultured, but preferably a unit structure having a predetermined planar shape. A structure in which a plurality of 1s are regularly arranged is better. For example, as shown in FIG. 1, a plurality of unit structures 1 having a polygonal planar shape can be continuous. At this time, regular polygons such as regular triangles, squares, regular hexagons, and circular ones are more preferable because cells can be grown on an isotropically uniform structure. Moreover, it is also possible to combine a pillar-shaped or hole-shaped uneven structure and an uneven structure formed from the unit structure 1 having a planar shape. From the viewpoint of bringing the cultured cells closer to the state in vivo, the width of the line 2 is preferably as small as possible, such as 3 μm or less, 2 μm or less, 1 μm or less, 700 nm or less, 500 nm or less, or 250 nm or less. This is because, as the width of the line 2 becomes smaller, the cells adhered to the concavo-convex structure surface are considered to be able to form spheroids while growing many pseudopods.
また、単位構造１の深さは、培養する細胞の性質に応じて、１ｎｍ以上、１０ｎｍ以上、１００ｎｍ以上、２００ｎｍ以上、５００ｎｍ以上、１μｍ以上、１０μｍ以上、１００μｍ以上等種々の大きさに形成される。また、この凹凸のアスペクト比としては、０．２以上、０．５以上、１以上、２以上等種々のものがある。 Further, the depth of the unit structure 1 is formed in various sizes such as 1 nm or more, 10 nm or more, 100 nm or more, 200 nm or more, 500 nm or more, 1 μm or more, 10 μm or more, 100 μm or more, depending on the properties of the cells to be cultured. The Further, the aspect ratio of the unevenness includes various ones such as 0.2 or more, 0.5 or more, 1 or more, 2 or more.
また、単位構造１の最小内径（好ましくは最大内径）は、３μｍ以下であることが好ましく、２μｍ以下、１μｍ以下、７００ｎｍ以下、５００ｎｍ以下、２５０ｎｍ以下というように、小さくなるほど好ましい。ここで、内径とは、単位構造１に外接する２本の平行線間の距離を意味する。したがって、最小内径とは、単位構造１に外接する二本の平行線間の距離のうち最も短いものを言い、最大内径とは、単位構造１に外接する二本の平行線間の距離のうち最も長いものを言う。例えば、単位構造１が正六角形の場合には、対向する平行な辺と辺との間の距離が最小内径となり、対向する頂点間の距離が最大内径となる。また、単位構造１が長方形の場合には、短辺の長さが最小内径となり、対角線の長さが最大内径となる。 Further, the minimum inner diameter (preferably the maximum inner diameter) of the unit structure 1 is preferably 3 μm or less, and is preferably as small as 2 μm or less, 1 μm or less, 700 nm or less, 500 nm or less, or 250 nm or less. Here, the inner diameter means a distance between two parallel lines circumscribing the unit structure 1. Therefore, the minimum inner diameter refers to the shortest distance between two parallel lines circumscribing the unit structure 1, and the maximum inner diameter refers to the distance between two parallel lines circumscribing the unit structure 1 Say the longest. For example, when the unit structure 1 is a regular hexagon, the distance between the parallel sides facing each other is the minimum inner diameter, and the distance between the opposite vertices is the maximum inner diameter. When the unit structure 1 is rectangular, the length of the short side is the minimum inner diameter, and the length of the diagonal line is the maximum inner diameter.
凹凸構造の形成方法は、いかなる方法であっても良いが、例えば、ナノインプリント技術、溶液キャスト法、エッチング、ブラスト、コロナ放電等を用いることができる。この時、より精密に形状等を制御できる点で、ナノインプリント技術による方法が好ましい。 Any method may be used for forming the concavo-convex structure. For example, nanoimprint technology, solution casting, etching, blasting, corona discharge, or the like can be used. At this time, a method using a nanoimprint technique is preferable in that the shape and the like can be controlled more precisely.
以下、実施例により本発明を具体的に説明する。ただし、本発明は以下の記述に限定されるものではない。 Hereinafter, the present invention will be described specifically by way of examples. However, the present invention is not limited to the following description.
［Ｅ−ｃａｄｈｅｒｉｎ発現量とスフェロイド形態の相関関係］
（１）細胞播種および観察
細胞培養容器ＮａｎｏＣｕｌｔｕｒｅ（登録商標）ディッシュ（ＳＣＩＶＡＸ社製、３５ｍｍディッシュ、凹凸構造面の材質＝ポリメチルペンテン（三井化学社製ＴＰＸ）、凹凸構造の平面形状＝正方形、単位構造間の幅（線幅）＝７００ｎｍ、単位構造の最小内径＝３μｍ、深さ＝１μｍ）に、細胞を２．５×１０５ｃｅｌｌｓで播種し、培養３日目に光学顕微鏡にて観察を行った。細胞としては、ヒト膵臓癌細胞株、ＢｘＰＣ−３，Ｃａｐａｎ−１，Ｃａｐａｎ−２，ＡｓＰＣ−１，ＰＡＮＣ−１，ＭＩＡＰａＣａ−２を用いた。培地は、ＢｘＰＣ−３、ＡｓＰＣ−１，ＰＡＮＣ−１については１０％ＦＢＳ含有ＲＰＭＩ１６４０培地、Ｃａｐａｎ−１については１０％ＦＢＳ含有ＭＥＭ培地、Ｃａｐａｎ−２については１０％ＦＢＳ含有ＭｃＣｏｙ’ｓ ５Ａ培地、ＭＩＡＰａＣａ−２については１０％ＦＢＳ含有ＤＭＥＭ培地を用いた。
[Correlation between E-cadherin expression level and spheroid morphology]
(1) Cell seeding and observation cell culture vessel NanoCulture (registered trademark) dish (manufactured by SCIVAX, 35 mm dish, material of uneven structure surface = polymethylpentene (TPI manufactured by Mitsui Chemicals), planar shape of uneven structure = square, unit Cells were seeded at 2.5 × 10 5 cells in a width between structures (line width) = 700 nm, minimum inner diameter of unit structure = 3 μm, depth = 1 μm, and observed with an optical microscope on the third day of culture. went. As cells, human pancreatic cancer cell lines, BxPC-3, Capan-1, Capan-2, AsPC-1, PANC-1, and MIAPaCa-2 were used. The medium is 10% FBS-containing RPMI1640 medium for BxPC-3, AsPC-1 and PANC-1, 10% FBS-containing MEM medium for Capan-1, and 10% FBS-containing McCoy's 5A medium for Capan-2. For MIAPaCa-2, a DMEM medium containing 10% FBS was used.
（２）ウエスタンブロッティング
（１）と同様の細胞培養容器ＮａｎｏＣｕｌｔｕｒｅ（登録商標）ディッシュ（ＳＣＩＶＡＸ社製、３５ｍｍディッシュ）に、（１）と同様の細胞を播種し、５日間培養した後、当該細胞をピペッティングにより回収した。回収した当該細胞をＰＢＳで洗浄し、溶解バッファーにて溶解した細胞溶解液をサンプルとした。次に、各種細胞株のサンプル（タンパク質２０μｇ分）をＳＤＳポリアクリルアミドゲル電気泳動法（ＳＤＳ−ＰＡＧＥ）により分離し、その後、タンパク質をＰＶＤＦ膜に転写した。タンパク質の検出は、一次抗体として抗Ｅ−ｃａｄｈｅｒｉｎマウスモノクローナル抗体（ＢＤ Ｂｉｏｓｃｉｅｎｃｅｓ: Ｃａｔ＃６１０１８２)）、二次抗体としてＨＲＰラベルされた抗マウス抗体（Ｓａｎｔａ Ｃｒｕｚ: ｓｃ-２００５）、検出試薬としてＥＣＬ Ｐｌｕｓ Ｗｅｓｔｅｒｎ Ｂｌｏｔｔｉｎｇ Ｄｅｔｅｃｔｉｏｎ Ｓｙｓｔｅｍ（ＧＥヘルスケア社製）を用いて行った。
(2) Cells similar to Western blotting (1) Nanoculture (registered trademark) dish (manufactured by SCIVAX, 35 mm dish) are seeded with the same cells as (1) and cultured for 5 days. Collected by pipetting. The collected cells were washed with PBS, and a cell lysate dissolved in a lysis buffer was used as a sample. Next, samples of various cell lines (20 μg of protein) were separated by SDS polyacrylamide gel electrophoresis (SDS-PAGE), and then the protein was transferred to a PVDF membrane. Protein detection was performed using anti-E-cadherin mouse monoclonal antibody (BD Biosciences: Cat # 610182) as a primary antibody, HRP-labeled anti-mouse antibody (Santa Cruz: sc-2005) as a secondary antibody, and ECL Plus as a detection reagent. Western blotting detection system (manufactured by GE Healthcare) was used.
（１）および（２）の結果を図２および図３に示す。Ｅ−ｃａｄｈｅｒｉｎの発現量が高いＢｘＰＣ−３やＣａｐａｎ−２では、スフェロイドの円形度が１に近く、細胞接着のタイトなスフェロイドが形成されていることがわかる。これに対し、Ｅ−ｃａｄｈｅｒｉｎの発現量が低いＰＡＮＣ−１やＭＩＡＰａＣａ−２では、前述の細胞株のスフェロイドよりも円形度が１から離れ、細胞接着のルーズなスフェロイドが形成されていることがわかった。 The results of (1) and (2) are shown in FIGS. It can be seen that in BxPC-3 and Capan-2 where the expression level of E-cadherin is high, the roundness of the spheroids is close to 1, and spheroids with tight cell adhesion are formed. In contrast, PANC-1 and MIAPaCa-2, which have low expression levels of E-cadherin, show that spheroids having a circularity farther from 1 than the spheroids of the aforementioned cell lines are formed and loose spheroids for cell adhesion are formed. It was.
［上皮−間葉転換阻害剤がスフェロイドの形態に及ぼす効果］
（１）プレインキュベーション
細胞培養容器ＮａｎｏＣｕｌｔｕｒｅ（登録商標）プレート（ＳＣＩＶＡＸ社製、３８４ウェルプレート、凹凸構造面の材質＝ポリメチルペンテン（三井化学社製ＴＰＸ）、凹凸構造の平面形状＝正方形、単位構造間の幅（線幅）＝７００ｎｍ、単位構造の最小内径＝３μｍ、深さ＝１μｍ）に、５％ＦＢＳ及び１．０％Ｍａｔｒｉｇｅｌ（登録商標）含有ＤＭＥＭ培地を各ウェルに２５μｌずつ添加し、１０００×ｇで５分間遠心した。
[Effect of epithelial-mesenchymal transition inhibitor on spheroid morphology]
(1) Pre-incubation cell culture vessel NanoCulture (registered trademark) plate (manufactured by SCIVAX, 384 well plate, material of uneven structure surface = polymethylpentene (TPI manufactured by Mitsui Chemicals), planar shape of uneven structure = square, unit structure 25% of the DMEM medium containing 5% FBS and 1.0% Matrigel (registered trademark) was added to each well to the width (line width) = 700 nm, the minimum inner diameter of the unit structure = 3 μm, and the depth = 1 μm. Centrifugation was performed at 1000 × g for 5 minutes.
（２）細胞播種
ヒト肺がん細胞由来株Ａ５４９を５％ＦＢＳ含有ＤＭＥＭ培地で１３．５×１０４ｃｅｌｌｓ／ｍｌに調整した細胞懸濁液を、２５μｌずつ前述したプレートの各ウェルに添加した（細胞数は３．４×１０３ｃｅｌｌｓ／ｗｅｌｌ、培地添加物の最終濃度は５％ＦＢＳ、０．５％Ｍａｔｒｉｇｅｌ）。
(2) Cell seeding A cell suspension prepared by adjusting human lung cancer cell-derived strain A549 to 13.5 × 10 4 cells / ml with 5% FBS-containing DMEM medium was added in an amount of 25 μl to each well of the plate (cells). The number is 3.4 × 10 3 cells / well and the final concentration of the media additive is 5% FBS, 0.5% Matrigel).
（３）上皮−間葉転換の誘導剤及び阻害剤の添加
培養３日目に、上皮−間葉転換誘導剤であるＴＧＦ−β２（Ｒ＆Ｄｓｙｓｔｅｍｓ社製；３０２−Ｂ２−００２）及びＴＮＦ−α（Ｒ＆Ｄｓｙｓｔｅｍｓ社製；２１０−ＴＡ−０１０）を、それぞれ最終濃度が５ｎｇ／ｍｌ及び１０ｎｇ／ｍｌになるように各ウェルに添加した。それと同時に、ＴＧＦ−β２の阻害剤であるＳＢ４３１５４２（ＭｉｌｔｅｎｙｉＢｉｏｔｅｃ社製；１３０−０９５−５６１）を最終濃度が１０μＭになるように添加した。ＳＢ４３１５４２を添加しないすべてのウェルには最終濃度０．１％となるようにジメチルスルホキシドを添加した。
(3) Addition of inducer and inhibitor of epithelial-mesenchymal transition On the third day of the culture, TGF-β2 (manufactured by R & D systems; 302-B2-002) and TNF-α (epithelial-mesenchymal transition inducers) R & D systems; 210-TA-010) was added to each well so that the final concentrations were 5 ng / ml and 10 ng / ml, respectively. At the same time, SB431542 (manufactured by MiltenyiBiotec; 130-095-561), an inhibitor of TGF-β2, was added to a final concentration of 10 μM. Dimethyl sulfoxide was added to all wells to which SB431542 was not added to a final concentration of 0.1%.
（４）低酸素領域検出試薬の添加
培養５日目に、低酸素領域検出用試薬ＬＯＸ−１（ＳＣＩＶＡＸ社製）を最終濃度が２μＭになるように各ウェルに添加した。ＬＯＸ−１は、赤色燐光を発するイリジウム錯体を含有する試薬である。
(4) Addition of low oxygen region detection reagent On the fifth day of culture, a low oxygen region detection reagent LOX-1 (manufactured by SCIVAX) was added to each well so that the final concentration was 2 μM. LOX-1 is a reagent containing an iridium complex that emits red phosphorescence.
（５）観察
培養６日目に、蛍光顕微鏡による観察を行った。
(5) Observation On the sixth day of culture, observation was performed with a fluorescence microscope.
蛍光顕微鏡にて観察し、撮像した結果を図４に示す。まず、各ウェルの明視野像を比較すると、上皮−間葉転換誘導剤を添加していないウェル（コントロール）では、円形度が１に近く、コンパクトなスフェロイドが形成されている。これに対し、上皮−間葉転換誘導剤を添加したウェルでは、スフェロイドの大半が崩壊し、残ったスフェロイドの輪郭も凹凸が大きくなっていることがわかった。そして、上皮−間葉転換の誘導剤と阻害剤を添加したウェルでは、阻害剤を添加していないウェルと比較して、スフェロイドの崩壊が抑制されるとともにスフェロイドの輪郭も凹凸が小さくなっていることがわかった。一方、各ウェルの蛍光視野像を比較すると、コントロールに比べて上皮−間葉転換の誘導剤を添加したウェルでは、赤色強度が弱くかつ赤色領域が小さくなっており、ここに阻害剤を添加したウェルでは赤色強度が強くかつ赤色領域が大きくなっていることがわかった。すなわち、低酸素領域が大きくなっていることがわかった。また、このときの赤色シグナルを定量化した結果を図５に示す。上皮−間葉転換阻害剤を添加したウェルでは、添加していないウェルの約２倍の赤色シグナルがあることがわかった。 FIG. 4 shows the results of observation and imaging with a fluorescence microscope. First, when comparing bright field images of each well, in a well (control) to which no epithelial-mesenchymal transition inducer was added, the circularity was close to 1 and a compact spheroid was formed. In contrast, it was found that in the wells to which the epithelial-mesenchymal transition inducer was added, most of the spheroids collapsed, and the contours of the remaining spheroids were also uneven. And in the well which added the inducer and inhibitor of epithelial-mesenchymal transition, collapse of the spheroid is suppressed and the contour of the spheroid is also smaller than the well where the inhibitor is not added. I understood it. On the other hand, when the fluorescence field images of each well were compared, in the well to which the inducer of epithelial-mesenchymal transition was added compared to the control, the red intensity was weak and the red region was small, and the inhibitor was added here. It was found that the well had a strong red intensity and a large red region. That is, it was found that the hypoxic region was increased. Moreover, the result of quantifying the red signal at this time is shown in FIG. It was found that in the wells to which the epithelial-mesenchymal transition inhibitor was added, there was a red signal approximately twice that of the wells to which the epithelium-mesenchymal transition inhibitor was not added.
［上皮−間葉転換の誘導剤及び阻害剤添加時における遺伝子発現変化の検証］
実施例２で確認されたスフェロイドの形態の変化が、上皮−間葉転換及び上皮−間葉転換阻害を反映しているのか否かを検証するために、上皮又は間葉系マーカーとして知られている遺伝子について、その発現レベルをｑＲＴ−ＰＣＲにより測定した結果を図６〜図９に示す。（１）プレインキュベーション〜（３）上皮−間葉転換の誘導剤及び阻害剤の添加までの工程は、実施例２に示したものと同様に行った。そして、培養６日目に、各サンプルからＲＮｅａｓｙ Ｐｌｕｓ Ｍｉｎｉ Ｋｉｔ（ＱＩＡＧＥＮ社製）を用いて全ＲＮＡを抽出し、そのＲＮＡをＰｒｉｍｅＳｃｒｉｐｔ ＲＴ ｒｅａｇｅｎｔ Ｋｉｔ（タカラバイオ社製）を用いて逆転写したものを、ｑＲＴ−ＰＣＲに用いた。ＰＣＲにはＳＹＢＥＲ Ｐｒｅｍｉｘ Ｅｘ ＴａｑＩＩ（タカラバイオ社製）を用いた。またその反応および検出にはＴｈｅｒｍａｌ Ｃｙｃｌｅｒ Ｄｉｃｅ（タカラバイオ社製）を用いた。ＰＣＲに用いたプライマーは下記表１に示す。なおデータは、内部標準遺伝子（ＴＢＰ）の発現量で補正したうえで、コントロールの発現量を１とした場合の相対値として示した。
[Verification of changes in gene expression upon addition of inducers and inhibitors of epithelial-mesenchymal transition]
Known as epithelial or mesenchymal markers to verify whether the spheroid morphology changes identified in Example 2 reflect epithelial-mesenchymal transition and epithelial-mesenchymal transition inhibition The result of having measured the expression level by qRT-PCR about the gene which exists is shown in FIGS. The steps from (1) preincubation to (3) addition of an inducer and inhibitor of epithelial-mesenchymal transition were performed in the same manner as shown in Example 2. On day 6 of culture, total RNA was extracted from each sample using RNeasy Plus Mini Kit (QIAGEN), and the RNA was reverse transcribed using PrimeScript RT reagent Kit (Takara Bio). , QRT-PCR. SYBER Premix Ex TaqII (manufactured by Takara Bio Inc.) was used for PCR. For the reaction and detection, Thermal Cycler Dice (manufactured by Takara Bio Inc.) was used. The primers used for PCR are shown in Table 1 below. The data was corrected by the expression level of the internal standard gene (TBP) and expressed as a relative value when the expression level of the control was 1.
上皮マーカーとして知られているＥ−ｃａｄｈｅｒｉｎの発現量は、上皮−間葉転換誘導剤の添加によりコントロールの１割以下に減少し、阻害剤の添加によりコントロールの６割程度にまで回復している（図６）。一方、間葉系マーカーとして知られているＮ−ｃａｄｈｅｒｉｎの発現量は、上皮−間葉転換誘導剤の添加によりコントロールの約９倍にまで増加し、阻害剤の添加によりコントロールと同程度にまで減少している（図７）。同様に、間葉系マーカーであるＶｉｍｅｎｔｉｎと、Ｅ−ｃａｄｈｅｒｉｎの発現を抑制する転写因子であるＺＥＢ１の発現も、Ｎ−ｃａｄｈｅｒｉｎと近似の発現レベル変化を示した（図８および図９）。これらの結果より、実施例２で確認されたスフェロイドの形態の変化は、上皮−間葉転換及び上皮−間葉転換阻害時に生じる細胞の性質の変化を反映するものであることが示唆された。 The expression level of E-cadherin, known as an epithelial marker, decreased to 10% or less of the control by the addition of the epithelial-mesenchymal transition inducer and recovered to about 60% of the control by the addition of the inhibitor. (FIG. 6). On the other hand, the expression level of N-cadherin known as a mesenchymal marker is increased to about 9 times that of the control by the addition of the epithelial-mesenchymal transition inducer, and to the same level as the control by the addition of the inhibitor. It has decreased (FIG. 7). Similarly, expression of Vimentin, a mesenchymal marker, and expression of ZEB1, a transcription factor that suppresses the expression of E-cadherin, also showed an expression level change similar to N-cadherin (FIGS. 8 and 9). From these results, it was suggested that the change in the spheroid morphology confirmed in Example 2 reflects changes in the properties of the cells that occur when epithelial-mesenchymal transition and epithelial-mesenchymal transition are inhibited.
本発明方法は、抗癌剤や線維症治療薬等の薬剤スクリーニングに使用することができる。 The method of the present invention can be used for screening for drugs such as anticancer agents and therapeutic agents for fibrosis.
１ 単位構造
２ 線
1 unit structure 2 lines
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