JP2008237098A - Animal model for analyzing metastasis of breast cancer to lung and/or liver - Google Patents
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Abstract
Description
本発明は、乳癌の肺及び／または肝臓転移解析用動物モデル、該モデル作成用の高転移性マウス乳癌細胞、並びに該細胞からなる肺及び／または肝臓転移抑制薬のスクリーニング用細胞材料及び同スクリーニング法に関する。 The present invention relates to an animal model for analyzing lung and / or liver metastasis of breast cancer, a highly metastatic mouse breast cancer cell for preparing the model, a cell material for screening a lung and / or liver metastasis inhibitor comprising the cell, and the same screening Regarding the law.
近年日本人女性に増えて来た乳癌は高率で骨髄、肺、肝臓等に転移を起こし、これらは患者に著しい苦痛を与え、患者の生活の質(QOL)を低下させ大きな社会問題となっているが、それにも関わらず、乳癌の転移のメカニズムの解明が進まないのは、これら組織への転移の動物モデルが存在しない事にその一因がある。また、このような転移の動物モデルは、新規な乳癌骨髄転移抑制薬のスクリーニング開発にとっても不可欠である。
これまでに存在する乳癌の転移モデルとしては、ヒトの乳癌(MDA-231)をヌードマウスに移植するモデルが広く使われて来た (非特許文献１参照)。
しかし、ヌードマウスでは免疫機構が低いため、最近注目されている癌細胞と宿主の免疫担当細胞の相互作用(非特許文献２参照)を解析する事ができないため、同系宿主のモデルの方が望ましい。また、このモデルは広く使われているが、左心室投与には熟練を要するという問題がある。一方、マウスモデルでよく用いられる4T1乳癌細胞は、ヒト乳癌によく類似した性質を有する事が知られている(非特許文献３参照)が、マウスの他の組織への転移能は必ずしも高くない（非特許文献３参照）。
As a metastasis model of breast cancer that has existed so far, a model in which human breast cancer (MDA-231) is transplanted into nude mice has been widely used (see Non-Patent Document 1).
However, since nude mice have a low immune mechanism, it is not possible to analyze the interaction between cancer cells that have recently been attracting attention and host immunocompetent cells (see Non-Patent Document 2). . Moreover, although this model is widely used, there is a problem that skill is required for left ventricular administration. On the other hand, 4T1 breast cancer cells often used in mouse models are known to have properties similar to human breast cancer (see Non-Patent Document 3), but their ability to metastasize to other tissues of mice is not necessarily high. (Refer nonpatent literature 3).
そこで、本発明の課題は、ヒト乳癌に類似した性質を有するとともに、肺、肝臓等の他の組織に対し高い転移能を有するマウスの乳癌細胞を用いて、新規な、乳癌の肺及び／または肝臓転移モデルを作成するとともに、該細胞を用いて乳癌の肺及び／または肝臓転移抑制薬のスクリーニング手段を新たに提供することにある。 Thus, an object of the present invention is to use a mouse breast cancer cell having a property similar to that of human breast cancer and having a high metastatic potential for other tissues such as lung and liver, to provide a novel breast cancer lung and / or In addition to creating a liver metastasis model, another object is to provide a screening means for a lung and / or liver metastasis inhibitor of breast cancer using the cells.
本発明者は、上記課題を解決するため、マウス乳癌細胞4T1を高転移性に改良することを着想し、薬剤耐性遺伝子を導入したマウス乳癌細胞4T1をマウスに静注した後、骨髄細胞を採取し、該細胞から薬剤耐性株を取り出して該薬剤耐性株をマウスに投与する手段を繰り返し、ついにマウス乳癌細胞由来であって骨髄高転移性の細胞を得ることに成功した。そして、さらに種々の実験を繰り返した結果、意外にも、該細胞が骨髄転移能以外に、肺あるいは肝臓に対して高い転移能を有するという知見を得た。そして該細胞を投与した動物モデルは、乳癌の肺または肝臓の解析用モデルとして有用であり、また、該細胞は乳癌の肺および／または肝臓転移抑制薬のスクリーニング手段として極めて有用であると確信し、本発明を完成するに至ったものである。 In order to solve the above problems, the present inventor conceived of improving mouse breast cancer cell 4T1 to be highly metastatic, and after intravenously injecting mouse breast cancer cell 4T1 introduced with a drug resistance gene into a mouse, bone marrow cells were collected. Then, the means for taking out the drug-resistant strain from the cells and administering the drug-resistant strain to the mice was repeated, and finally a mouse bone marrow cell-derived bone marrow highly metastatic cell was successfully obtained. As a result of repeating various experiments, the inventors have surprisingly found that the cells have high metastatic potential for lung or liver in addition to bone marrow metastasis. The animal model to which the cells are administered is useful as a model for analysis of lung or liver of breast cancer, and it is believed that the cells are extremely useful as a screening tool for lung and / or liver metastasis inhibitors of breast cancer. The present invention has been completed.
すなわち、本発明は以下のとおりである。
（１）マウス乳癌細胞４Ｔ１由来の細胞であって、肺及び／または肝臓転移能がマウス乳癌細胞４Ｔ１に比較し亢進されたマウス乳癌細胞をマウスに投与して得られた、乳癌の肺及び／または肝臓転移解析用動物モデル。
（２）以下のａ）からｄ）のプロセスから得られた高転移性マウス乳癌細胞をマウスに投与して得られた、乳癌の肺及び／または肝臓転移解析用動物モデル。
ａ）薬剤耐性遺伝子が導入されたマウス乳癌細胞４Ｔ１を、マウスに投与し、少なくとも１０日経過後、マウス骨髄から細胞を採取するプロセス、
ｂ）得られた細胞を、上記薬剤耐性遺伝子による耐性付与の対象薬剤を含有する培地で培養し、薬剤耐性細胞を採取するプロセス、
ｃ）得られた薬剤耐性細胞をマウスに投与するプロセス、
ｄ）得られた薬剤耐性細胞を用いて上記ａ）のマウスへの投与、細胞の採取及びｂ）の薬剤耐性細胞の採取の各プロセスを順次複数回繰り返し、高転移性マウス乳癌細胞を得るプロセス
（３）マウス乳癌細胞４ＴＩ由来の細胞であって、肺及び肝臓転移能がマウス乳癌細胞４Ｔ１に比較し亢進された高転移性マウス乳癌細胞からなることを特徴とする、乳癌の肺及び／または肝臓転移解析用動物モデル作成用細胞材料
（４）以下のａ）からｄ）のプロセスから得られた高転移性マウス乳癌細胞からなることを特徴とする乳癌の肺及び／または肝臓転移解析用動物モデル作成用細胞材料
ａ）薬剤耐性遺伝子が導入されたマウス乳癌細胞４Ｔ１を、マウスに投与し、少なくとも１０日経過後、マウス骨髄から細胞を採取するプロセス、
ｂ）得られた細胞を、上記薬剤耐性遺伝子による耐性付与の対象薬剤を含有する培地で培養し、薬剤耐性細胞を採取するプロセス、
ｃ）得られた薬剤耐性細胞をマウスに投与するプロセス、
ｄ）得られた薬剤耐性細胞を用いて上記ａ）のマウスへの投与、細胞の採取及びｂ）の薬剤耐性細胞の採取の各プロセスを順次複数回繰り返し、高転移性マウス乳癌細胞を得るプロセス
（５）上記（３）または（４）に記載の高転移性マウス乳癌細胞からなることを特徴とする、乳癌の肺お及び／または肝臓転移抑制薬のスクリーニング用細胞材料。
（６）マウスに、上記（３）または（４）に記載の高転移性マウス乳癌細胞、及び乳癌の肺及び／または肝臓転移抑制薬の候補物質を投与し、乳癌の肺及び／または肝臓転移抑の有無、程度を指標として、上記候補物質をスクリーニングすることを特徴とする、乳癌の肺及び／または肝臓転移移抑制薬のスクリーニング方法。
That is, the present invention is as follows.
(1) A mouse breast cancer cell 4T1-derived cell, which is obtained by administering to a mouse a mouse breast cancer cell whose lung and / or liver metastasis ability is enhanced compared to the mouse breast cancer cell 4T1, and Or an animal model for liver metastasis analysis.
(2) An animal model for analyzing lung and / or liver metastasis of breast cancer, obtained by administering to a mouse highly metastatic mouse breast cancer cells obtained from the following processes a) to d).
a) a process of administering mouse breast cancer cells 4T1 into which a drug resistance gene has been introduced to mice, and collecting the cells from mouse bone marrow after at least 10 days;
b) a process of culturing the obtained cells in a medium containing a target drug to be given resistance by the drug resistance gene, and collecting the drug resistant cells;
c) a process of administering the resulting drug resistant cells to mice,
d) A process for obtaining highly metastatic mouse breast cancer cells by sequentially repeating the processes of a) administration to the mouse, collection of cells, and collection of the drug resistant cells in b) a plurality of times using the obtained drug resistant cells. (3) A mouse breast cancer cell 4TI-derived cell, comprising a highly metastatic mouse breast cancer cell having enhanced lung and liver metastatic ability compared to mouse breast cancer cell 4T1, and / or a breast cancer lung and / or Animal material for preparing animal model for liver metastasis analysis (4) Breast cancer lung and / or liver metastasis analysis animal characterized by comprising highly metastatic mouse breast cancer cells obtained from the following processes a) to d) Cell material for modeling a) A process of administering mouse breast cancer cells 4T1 into which a drug resistance gene has been introduced to mice, and collecting cells from mouse bone marrow after at least 10 days;
b) a process of culturing the obtained cells in a medium containing a target drug to be given resistance by the drug resistance gene, and collecting the drug resistant cells;
c) a process of administering the resulting drug resistant cells to mice,
d) A process for obtaining highly metastatic mouse breast cancer cells by sequentially repeating the processes of a) administration to the mouse, collection of cells, and collection of the drug resistant cells in b) a plurality of times using the obtained drug resistant cells. (5) A cell material for screening for an agent for suppressing lung and / or liver metastasis of breast cancer, comprising the highly metastatic mouse breast cancer cell described in (3) or (4) above.
(6) A highly metastatic mouse breast cancer cell described in (3) or (4) above and a candidate substance for a lung and / or liver metastasis inhibitor of breast cancer are administered to a mouse, and the lung and / or liver metastasis of breast cancer is administered. A screening method for a lung and / or liver metastasis inhibitor for breast cancer, wherein the candidate substance is screened using the presence or absence and degree of suppression as an index.
本発明の転移性マウス乳癌細胞は、その親株であるマウス乳癌細胞４Ｔ１に比べて極めて肺、肝臓に対する転移性が高く、また、上記親株が保有するヒト乳癌に類似する性質をそのまま保有しているため、本発明の高転移性マウス乳癌細胞を投与したマウスは、ヒト乳癌細胞を投与した従来のマウスと比べ、ヒト乳癌細胞にたいするマウス免疫系の関与がないことと相俟って、極めて優れた乳癌の肺及び／または肝臓転移の動物モデルとなり、乳癌の及び／または肝臓転移の解析にとって有力な手段となる。 The metastatic mouse breast cancer cell of the present invention is extremely high in metastasis to the lung and liver as compared with the mouse breast cancer cell 4T1 which is the parent strain, and also has the property similar to the human breast cancer possessed by the parent strain. Therefore, the mice administered with the highly metastatic mouse breast cancer cells of the present invention were extremely superior in comparison with the conventional mice administered with human breast cancer cells, in combination with the absence of the mouse immune system for human breast cancer cells. It becomes an animal model of breast and / or liver metastasis of breast cancer, and is a powerful tool for analysis of breast cancer and / or liver metastasis.
すなわち、本発明の高転移性マウス乳癌細胞を投与したマウスは、該細胞投与から死亡するまでの間、その生体産生タンパク質の変化を解析したり、あるいは各段階のマウスから高転移性マウス乳癌細胞を採取し、その親株あるいは投与前の高転移性マウス乳癌細胞との遺伝子の発現状態等の比較を通じて、肺あるいは肝臓組織への転移メカニズム、その原因等の解明にとっても有効な手段となりうる。 That is, a mouse administered with the highly metastatic mouse breast cancer cell of the present invention can analyze changes in the biologically produced protein from the administration of the cell to death, or can be analyzed from each stage of the mouse to a highly metastatic mouse breast cancer cell. It can be an effective means for elucidating the mechanism of the metastasis to the lung or liver tissue, its cause, etc. through comparison of the gene expression state with the parental strain or highly metastatic mouse breast cancer cells before administration.
一方、本発明の高転移性マウス乳癌細胞は、マウスに対して静注あるいは皮下投与のみで肺及び／または肝臓組織に転移癌を形成させることが可能であり、特に肺に対する転移能は極めて高く、本発明の高転移性マウス乳癌細胞を使用することにより、容易かつ短期間に乳癌の肺及び／または肝臓転移状態を作りだすことが可能であるので、該高転移性マウス乳癌細胞と乳癌の肺及び／または肝臓転移抑制薬の候補物質とをマウスに投与して、高転移性マウス乳癌細胞の肺、肝臓への転移状況をみることにより、乳癌の肺及び／または肝臓への転移抑制薬のスクリーニングを効率的に行うことが可能となる。 On the other hand, the highly metastatic mouse breast cancer cell of the present invention can form metastatic cancer in the lung and / or liver tissue only by intravenous injection or subcutaneous administration to the mouse, and has particularly high metastatic potential for the lung. By using the highly metastatic mouse breast cancer cell of the present invention, it is possible to easily create a lung and / or liver metastasis state of breast cancer in a short period of time. And / or a candidate for a liver metastasis inhibitor is administered to a mouse, and the metastasis of highly metastatic mouse breast cancer cells to the lung and liver is observed. Screening can be performed efficiently.
本発明において使用する高転移性マウス乳癌細胞（以下、単に4T1E/M3細胞あるいはＦＭ３という場合がある。）は、マウス乳癌細胞４Ｔ１（以下、単に４Ｔ１細胞という場合がある。）由来の細胞であって、親株である４Ｔ１細胞（ＡＴＣＣ：CRL-2539）はATCC(American Type Culture Collection)から入手可能である。
４Ｔ１細胞は、ヒト乳癌細胞に転移能，増殖能，免疫学的特徴等の点で類似し、このような性質は本発明の高転移性マウス乳癌細胞も保有しているが、本発明の高転移性乳癌細胞は、肺、肝臓組織への転移能が高い点で特徴的である。本発明の高転移性マウス乳癌細胞の肺に対する転移能は、静注及び皮下移植の場合、親株であるマウス乳癌細胞４Ｔ１に比べ、１．５倍以上である。一方、本発明の高転移性マウス乳癌細胞の肝臓に対する転移能は、特にマウスに皮下投与した場合に顕著であり、この場合の転移能は、親株であるマウス乳癌細胞４Ｔ１に比べ、２倍以上である。
The highly metastatic mouse breast cancer cells used in the present invention (hereinafter sometimes simply referred to as 4T1E / M3 cells or FM3) are cells derived from mouse breast cancer cells 4T1 (hereinafter sometimes simply referred to as 4T1 cells). The parental 4T1 cells (ATCC: CRL-2539) are available from ATCC (American Type Culture Collection).
4T1 cells are similar to human breast cancer cells in terms of metastatic ability, proliferative ability, immunological characteristics, etc., and such properties also possess the highly metastatic mouse breast cancer cells of the present invention. Metastatic breast cancer cells are characteristic in that they have a high ability to metastasize to lung and liver tissues. In the case of intravenous injection and subcutaneous transplantation, the metastatic ability of the highly metastatic mouse breast cancer cells of the present invention to the lung is 1.5 times or more than that of the parental mouse breast cancer cell 4T1. On the other hand, the metastatic ability of the highly metastatic mouse breast cancer cells of the present invention to the liver is particularly remarkable when administered subcutaneously to mice. In this case, the metastatic ability is more than double that of the mouse breast cancer cell 4T1, which is the parent strain. It is.
なお、この静注した場合の転移能倍率は、薬剤耐性遺伝子を導入した、高転移性マウス乳癌細胞とその親株である４Ｔ１細胞（各5×105個/マウス)をそれぞれマウス尾静脈に投与し、10日後に投与マウスを犠牲死させて、マウスの肺、肝臓組織を採取して、マウスの肺、肝臓組織における各被験細胞由来の薬剤耐性遺伝子の発現量（ＲＮＡ量）に基づき算出されたものであり、親株である４Ｔ１細胞を試験した場合における肺、肝臓組織中の４Ｔ１細胞の同遺伝子発現量に対する、4T1E/M3細胞を試験した場合における肺、肝臓組織中の4T1E/M3細胞の遺伝子発現量の倍率である。
なお、この場合の転移能の倍率は、高転移性マウス乳癌細胞とその親株である４Ｔ１細胞（各2 X10５/マウス）をそれぞれマウスに皮下投与（各2 X10５/マウス）し、30日後に肺、肝臓組織を採取し、以下、上記と同様にして算出された倍率である。
In addition, the metastatic ability magnification in the case of intravenous injection was determined by administering highly metastatic mouse breast cancer cells into which a drug resistance gene was introduced and 4T1 cells (5 × 10 5 cells / mouse), each of which is a parent strain, to the mouse tail vein. 10 days later, the mice were sacrificed, and the lungs and liver tissues of the mice were collected, and calculated based on the expression level (RNA amount) of the drug resistance gene derived from each test cell in the lungs and liver tissues of the mice. 4T1E / M3 cells in 4T1E / M3 cells when 4T1E / M3 cells were tested against the expression level of 4T1 cells in the lungs and liver tissues when the parent strain 4T1 cells were tested. It is the magnification of gene expression level.
In this case, the ratio of metastatic potential is as follows: highly metastatic mouse breast cancer cells and their parent strains 4T1 cells (each 2 × 10 5 / mouse) were subcutaneously administered to each mouse (each 2 × 10 5 / mouse), 30 days Later, lungs and liver tissues are collected, and the magnifications are calculated in the same manner as described above.
本発明で使用する高転移性乳癌細胞は以下ａ）〜ｂ）の方法で得られる。
ａ）まず、マウス乳癌細胞４Ｔ１に薬剤耐性遺伝子を導入し、得られた薬剤耐性遺伝子導入細胞をマウスに投与し、一定時間経過後、マウスを殺して骨髄を採取し、骸骨髄から細胞を採取する。このプロセスにおいて使用する薬剤耐性遺伝子としては、ネオマイシン耐性遺伝子(ネオマイシンフォスフォトランスフェラーゼ)、ゼオシン耐性遺伝子等の抗生物質耐性遺伝子が挙げられるが、これら耐性遺伝子の種類につては特に制限がなく、市販されているこれら遺伝子を担持したプラスミド等を適宜用いることができ、また耐性遺伝子導入手法も、常法に従えばよく、例えば、市販品に添付されるプロトコルに従えばよい。また、上記薬剤耐性遺伝子導入細胞をマウス投与後の経過時間は、該細胞がマウス骨髄に転移させるための時間で、特に制限はないが、少なくとも１０日以上、好ましくは１０〜１２日である。
ｂ）次いで、上記骨髄ら採取された細胞を、上記薬剤耐性遺伝子による耐性付与の対象となる薬剤を含有する培地で培養し、該培地で生残する薬剤耐性株を採取する。薬剤耐性遺伝子としてネオマイシン耐性遺伝子（ネオマイシンフォスフォトランスフェラーゼ）を使用した場合には、例えばＧ４１８が挙げられ、ゼオシン(Zeocin)耐性遺伝子、ブラストサイジン(Blasticidin) 耐性遺伝子を使用した場合にはゼオシン(Zeocin)、ブラストサイジン(Blasticidin)等が挙げられる。
ｃ）上記ｂ）プロセスで得られた薬剤耐性細胞はマウスに投与され、再び、骨髄から細胞を採取し、該細胞中の上記薬剤含有培地で培養し、生残した細胞を採取する。以後、この採集された細胞を用いて上記ａ）マウス投与、細胞採取及びｂ）の薬剤による細胞選択操作を複数回、例えば３回以上繰り返して、４Ｔ１細胞が変異した高転移性マウス乳癌細胞を得る。
The highly metastatic breast cancer cells used in the present invention are obtained by the following methods a) to b).
a) First, a drug resistance gene is introduced into mouse breast cancer cell 4T1, and the obtained drug resistance gene-introduced cell is administered to the mouse. After a certain period of time, the mouse is killed and the bone marrow is collected, and the cell is collected from the bone marrow. To do. Drug resistance genes used in this process include antibiotic resistance genes such as neomycin resistance gene (neomycin phosphotransferase) and zeocin resistance gene, but there are no particular restrictions on the types of these resistance genes, which are commercially available. A plasmid carrying these genes can be used as appropriate, and the resistance gene introduction method may be in accordance with a conventional method, for example, in accordance with a protocol attached to a commercially available product. The elapsed time after administration of the drug-resistant gene-introduced cell to the mouse is a time for the cell to transfer to the mouse bone marrow, and is not particularly limited, but is at least 10 days or more, preferably 10 to 12 days.
b) Next, the cells collected from the bone marrow are cultured in a medium containing a drug to be given resistance by the drug resistance gene, and drug-resistant strains that survive in the medium are collected. When a neomycin resistance gene (neomycin phosphotransferase) is used as a drug resistance gene, for example, G418 is exemplified. When a zeocin resistance gene or a blasticidin resistance gene is used, zeocin (Zeocin) ), Blastcidin and the like.
c) The drug resistant cells obtained in the above b) process are administered to mice, and the cells are again collected from the bone marrow, cultured in the drug-containing medium in the cells, and the surviving cells are collected. Thereafter, using these collected cells, the above-mentioned a) mouse administration, cell collection and cell selection operation with the agent of b) are repeated a plurality of times, for example, 3 times or more, to obtain highly metastatic mouse breast cancer cells in which 4T1 cells are mutated. obtain.
このようにして得られた高転移性マウス乳癌細胞は、マウスに投与することにより、乳癌が肺あるいは肝臓に転移するあるいは転移した状態のマウスが作成できる。このマウスは、ヒト乳癌細胞をマウスに接種した場合とは異なり、ヒト乳癌細胞に対するマウス免疫系の関与がないため、より自然に近い状態の乳癌の肺及び／または肝臓転移を解析するための動物モデルとして有用である。 By administering the thus obtained highly metastatic mouse breast cancer cells to a mouse, a mouse in which the breast cancer has metastasized or metastasized to the lung or liver can be prepared. Unlike mice inoculated with human breast cancer cells, this mouse has no involvement of the mouse immune system against human breast cancer cells, so it is an animal for analyzing lung and / or liver metastasis of breast cancer in a more natural state Useful as a model.
高転移性マウス乳癌細胞をマウスに投与形態は、静注あるいは皮下移植が好ましい。静注の場合には、例えばマウス尾静脈、皮下移植の場合は、例えば背部または腹部皮下移植である。
高転移性マウス乳癌細胞５×１０５個／マウスをマウス尾静脈投与の場合には、通常５〜１０日で肺あるいは肝臓に転移し、高転移性マウス乳癌細胞２×１０５個／マウスをマウスの背部に移植する場合する場合、通常１５〜２０日で肺あるいは肝臓に転移するが、本発明はこれらの転移までの期間、あるいは投与する細胞数等には制限がなく、マウスが癌により自然死するまでの間、様々な状態の動物モデルを使用して、肺あるいは肝臓転移を解析することができる。
The administration form of highly metastatic mouse breast cancer cells to mice is preferably intravenous or subcutaneous transplantation. In the case of intravenous injection, for example, mouse tail vein, and in the case of subcutaneous transplantation, for example, dorsal or abdominal subcutaneous transplantation.
When 5 × 10 5 highly metastatic mouse breast cancer cells / mouse are administered to the tail vein of mice, they usually metastasize to the lung or liver in 5 to 10 days, and 2 × 10 5 highly metastatic mouse breast cancer cells / mouse are transferred. When transplanted to the back of a mouse, it usually metastasizes to the lung or liver in 15 to 20 days. However, the present invention has no limitation on the period until such metastasis or the number of cells to be administered. Until natural death, animal models of various states can be used to analyze lung or liver metastases.
例えば、転移直前と直後の、マウスにおける各種生理活性物質、免疫系物質の産生状況、または肺あるいは肝細胞における各種遺伝子の発現状況を解析したり、あるいは投与してから死亡する間の様々な状態のマウスから高転移性マウス乳癌細胞を採取して、その親株である４Ｔ１細胞あるいは投与前の高転移性マウス乳癌細胞とにおける各種遺伝子の発現状況等を比較解析し、乳癌の肺あるいは肝臓転移のメカニズム、その原因を解析することが可能となる。また、投与する細胞数を様々に変化させて、同様な解析を行うことも可能である。 For example, just before and after metastasis, various physiologically active substances, production status of immune system substances in mice, or various gene expression status in lungs or hepatocytes, or various states during death after administration High-metastatic mouse breast cancer cells were collected from these mice, and the expression status of various genes in the parental strain 4T1 cells or the highly metastatic mouse breast cancer cells before administration were compared and analyzed, and breast cancer lung or liver metastasis It becomes possible to analyze the mechanism and its cause. It is also possible to perform the same analysis by changing the number of cells to be administered.
さらに、上記高転移性マウス乳癌細胞を投与したマウスに、様々な生理活性物質あるいはその阻害物質を投与して、転移の状況変化あるいはマウスにおける各種遺伝子の発現状態の変化、あるいは採取された高転移性マウス乳癌細胞における遺伝子発現状態況の変化を観測して、これら生理活性物質が与える影響をとおして、乳癌の肺あるいは肝臓転移のメカニズムあるいはその抑制手法を探ることも可能となる。 Furthermore, by administering various physiologically active substances or inhibitors thereof to mice administered with the above highly metastatic mouse breast cancer cells, changes in metastasis status or changes in the expression status of various genes in mice, or collected high metastases It is also possible to investigate the mechanism of lung or liver metastasis of breast cancer or its suppression method through the influence of these physiologically active substances by observing changes in the state of gene expression in sex mouse breast cancer cells.
一方、より直接的には、本発明の高転移性マウス乳癌細胞は、乳癌の肺及び／または肝臓転移抑制薬のスクリーニング手段として有用である。これには、本発明の高転移性マウス乳癌細胞と乳癌の肺及び／または肝臓転移抑制薬の候補物質とマウスに投与し、一定時間経過後マウスを殺し、肺または肝臓転移の状況を観察し、転移の有無、程度から、使用した候補物質の転移抑制効果を判別することにより、使用した候補物質の中から乳癌の肺及び／または肝臓転移抑制薬をスクリーニングすることができる。また、以下に示す実施例３（５）および（６）に示される、本発明の高転移性マウス乳癌細胞は足場非依存的増殖能や傷修復能を利用して、in vitroにおいて、肺及び／または肝臓髄転移抑制薬の候補化合物がこれら能力を抑制するかどうかを指標にスクリーニングすることも可能であり、このような手法は、乳癌の肺及び／または肝臓転移抑制薬のスクリーニングの予備実験系として簡便で効率的手法となる。 On the other hand, more directly, the highly metastatic mouse breast cancer cells of the present invention are useful as a screening means for a breast and lung metastasis inhibitor. For this purpose, the highly metastatic mouse breast cancer cell of the present invention, a breast cancer lung and / or liver metastasis candidate drug and a mouse are administered to the mouse, and after a certain period of time, the mouse is killed and the state of lung or liver metastasis is observed. By discriminating the metastasis-suppressing effect of the used candidate substance from the presence / absence and degree of metastasis, a lung and / or liver metastasis inhibitor for breast cancer can be screened from the used candidate substances. In addition, the highly metastatic mouse breast cancer cells of the present invention shown in Examples 3 (5) and (6) shown below can be used in vitro for lung and It is also possible to perform screening based on whether or not a candidate compound of a liver metastasis inhibitor can suppress these abilities, and such a method is used as a preliminary experiment for screening lung cancer and / or liver metastasis inhibitors. It becomes a simple and efficient method as a system.
さらに、このようなスクリーニングあるいは、上記高転移性マウス乳癌細胞を使用する肺または肝臓転移メカニズムの解析に際しては、高転移性マウス乳癌細胞の作製において、薬剤耐性遺伝子の導入に加えて、ルシフェラーゼ遺伝子等の発光遺伝子を導入すれば、転移の状態は、発光強度を測定することにより把握できより効率的である。
以下に、本発明の実施例を示すが、本発明はこれら実施例により限定されるものではない。
Furthermore, in the case of such screening or analysis of lung or liver metastasis mechanisms using the above-mentioned highly metastatic mouse breast cancer cells, in addition to the introduction of drug resistance genes, in addition to the introduction of drug resistance genes, etc. If the luminescence gene is introduced, the state of metastasis can be grasped by measuring the luminescence intensity, which is more efficient.
Examples of the present invention are shown below, but the present invention is not limited to these examples.
高転移性マウス乳癌細胞の作製
マウス乳癌細胞4T1（ＡＴＣＣ：CRL-2539）をATCC(American Type Culture Collection)から入手し、この細胞に，BD Biosciences Clontech社製のネオマイシン耐性遺伝子を含むpEGFP-F ベクターを，QIAGEN 社製のトランスフェクション試薬Effectenを使用して導入した。
Preparation of highly metastatic mouse breast cancer cells Mouse breast cancer cell 4T1 (ATCC: CRL-2539) was obtained from ATCC (American Type Culture Collection), and pEGFP-F vector containing neomycin resistance gene produced by BD Biosciences Clontech. Was introduced using the transfection reagent Effecten manufactured by QIAGEN.
4T1E/M3細胞の転移能の測定
（１）BALB/cマウスをそれぞれ５匹づつ２群に分け、一方の群に4T1E/M3細胞をそれぞれ静脈内投与（5 X10５/マウス）し、他方の群に4T1E/M3細胞をそれぞれ静脈内投与（5 X10５/マウス）し、投与後の時間経過に対する生残率を調べた。
結果を図１に示す。図１によれば、親株４T1を投与した場合に比べて明らかに生存率が低下し、悪性度が更新していることが明らかである。
（２）上記（１）と同様にして、4T1E 細胞と4T1E/M3細胞をそれぞれBALB/cマウスの各群に静脈内投与（5 X106/マウス）し、10日後に肺、肝臓、骨髄（大腿骨、脛骨）からそれぞれRNAを抽出し、各1μgのtotalRNAを用いて逆転写(Reverse Transcription)反応を行わせcDNAを得た。ついで、これを用い、ネオマイシン耐性遺伝子(ネオマイシンフォスフォトランスフェラーゼ)をプライマーとしてリアルタイムPCRを行なって、外から投与した癌細胞がどの程度、上記組織に存在するかを調べた。定量的RT-PCRを行なって、外から投与した4T1E/M3細胞がどの程度これら組織に存在するかを調べた。結果を図２に示す。これによれば4T1E/M3細胞は、特に肺に対する転移能が高いことが明らかとなった。
（３）上記（１）と同様にして、BALB/cマウスを２群に分け、4T1細胞と4T1E/M3細胞をそれぞれに皮下投与（2 X10５/マウス）し、投与後の時間経過に対する生残率を調べた。結果を図３に示す。図３によれば、4T1E/M3細胞をBALB/cマウスに皮下投与（2 X10５/マウス）した場合の生存率は、親株の生存率と変化がなかった。
（４）上記（１）と同様にして、BALB/cマウスを２群に分け、4T1細胞と4T1E/M3細胞をそれぞれに皮下投与（2 X10５/マウス）し、30日後に肺、肝臓、骨髄（大腿骨、脛骨）からRNAを抽出し、上記（２）と同様にして、定量的RT-PCRを行なって、外から投与した癌細胞が各組織にどの程度存在するかを調べた。結果を図４に示す。これによれば、4T1E/M3細胞は親株に比べて、有意に各組織に対する転移能が亢進していることが明らかとなった。また、上記（２）の結果と対比すれば明らかなように、特に皮下投与の場合、4T1E/M3細胞は、その親株に比べ肝臓に対し転移能が増大する点が特徴的である。
Measurement of metastatic potential of 4T1E / M3 cells (1) Divide BALB / c mice into 2 groups of 5 mice, 4T1E / M3 cells were administered intravenously (5 X10 5 / mouse) to one group, and the other Each group was intravenously administered with 4T1E / M3 cells (5 × 10 5 / mouse), and the survival rate over time after administration was examined.
The results are shown in FIG. According to FIG. 1, it is clear that the survival rate is clearly reduced and the malignancy is updated as compared with the case where the parent strain 4T1 is administered.
(2) In the same manner as in (1) above, 4T1E cells and 4T1E / M3 cells were intravenously administered to each group of BALB / c mice (5 × 10 6 / mouse), and 10 days later, lung, liver, bone marrow ( RNA was extracted from each of the femur and tibia, and a reverse transcription reaction was performed using 1 μg of total RNA to obtain cDNA. Next, using this, real-time PCR was carried out using a neomycin resistance gene (neomycin phosphotransferase) as a primer, and the extent of cancer cells administered from the outside was examined in the tissue. Quantitative RT-PCR was performed to determine the extent of exogenously administered 4T1E / M3 cells in these tissues. The results are shown in FIG. This revealed that 4T1E / M3 cells have a particularly high ability to metastasize to the lung.
(3) In the same manner as in (1) above, BALB / c mice were divided into 2 groups, and 4T1 cells and 4T1E / M3 cells were administered subcutaneously (2 X10 5 / mouse) respectively. The remaining rate was examined. The results are shown in FIG. According to FIG. 3, the survival rate when 4T1E / M3 cells were subcutaneously administered to BALB / c mice (2 × 10 5 / mouse) was not different from the survival rate of the parent strain.
(4) In the same manner as in (1) above, BALB / c mice were divided into two groups, 4T1 cells and 4T1E / M3 cells were administered subcutaneously (2 × 10 5 / mouse), and 30 days later, lungs, liver, RNA was extracted from bone marrow (femur and tibia), and quantitative RT-PCR was performed in the same manner as in (2) above to examine the extent to which cancer cells administered from outside were present in each tissue. The results are shown in FIG. According to this, it became clear that 4T1E / M3 cells have significantly enhanced metastatic potential for each tissue compared to the parental strain. In addition, as is clear from the results of (2) above, 4T1E / M3 cells are characterized by an increased ability to metastasize to the liver, particularly when administered subcutaneously, compared to their parental strain.
4T1E/M3細胞のin vitroにおけるその他の性質についての検定
（１）高転移性乳癌細胞 4T1E/M3通常の増殖能
4T1E/M3細胞と親株4TIをそれぞれ９６ウェルプレートに 2 X103個/well まいて、０〜４日間培養し、培養後の細胞数の増加を cell counting kit8(同人化学社製)を用いた改変MTT Assayにより測定した。結果を図５に示す。図５に明らかなように、4TIE/M3細胞は親株4TIと通常の増殖能にほとんど差はない。
（２）高転移性乳癌細胞4T1E/M3のプラスチック表面への接着能
4T1E/M3細胞と親株4TIをそれぞれをプラスチック製96 well plateにまいて細胞単層を形成し、次いで0.017% EDTA-PDSを加えて、５〜6０分間培養後洗浄して剥がれた細胞を除去し、各培養時間後に接着している細胞数を cell counting kit8 を用いて測定した。結果を図６に示す。4TIE/M3細胞は親株4TIに比べ剥がれにくく、接着能が亢進していることが明らかである。
（３）高転移性乳癌細胞
4T1E/M3の骨髄由来内皮細胞への接着能
24 well plateに単層培養した内皮細胞の上にBCECF-AMで蛍光標識した4T1E/M3細胞及び親株4T1をそれぞれ加えて1hr 37℃ 5%CO2で培養後3回洗浄し、接着した細胞を蛍光プレートリーダー(励起波長480nm,蛍光波長：530nm)により測定した。AM で標識した各細胞を骨髄由来内皮細胞上にまき、１５〜７５分培養した。結果を図７に示す4T1E/M3細胞の骨髄由来内皮細胞への接着能は、親株より亢進していることが明らかである。
（４）高転移性乳癌細胞 4T1E/M3の接着分子遺伝子の発現
4T1E/M3細胞と4T1細胞とにおける発現遺伝子の比較を・2インテグリンと ICAM-1について、DNAマイクロアレイ解析と定量的RT-PCRにより行った。結果を図８に示す。
これによれば、これら遺伝子の発現は、ともに4T1E/M3細胞において亢進していることが明らかである。また、4T1細胞及び4T1E/M3細胞の各表面を・2インテグリンと ICAM-1 に対する抗体で染色し、フローサイトメトリーで解析した。結果を図９に示す。4TIE/M3細胞は親株に比べてβ2インテグリン及びICAM-1の細胞表面での発現が亢進していることが分かる。
（５）高転移性乳癌細胞 4T1E/M3の足場非依存的増殖能
4T1E/M3細胞と4T1細胞をそれぞれ６ｃｍ シャーレに０．３％軟寒天とともにまき１２日間培養した。使用した細胞数は各シャーレにつきそれぞれ１ｘ１０4個と２ｘ１０4個である。培養後、各シャーレ内に見られるコロニー数を計数し、コロニーの形状・大きさを顕微鏡撮影した。コロニー数及び観察された顕微鏡写真は、図１０および１１に示す。これらによれば4TIE/M3細胞は親株に比べて足場非依存的増殖能が亢進していることが明らかである。
（６）高転移性乳癌細胞 4T1E/M3の運動能
Assay for other properties of 4T1E / M3 cells in vitro (1) Highly metastatic breast cancer cells 4T1E / M3 normal proliferative capacity
4T1E / M3 cells and the parent strain 4TI are each in a 96-well plate and cultured at 2 × 10 3 cells / well for 0 to 4 days. Measured by MTT Assay. The results are shown in FIG. As is apparent from FIG. 5, 4TIE / M3 cells have almost no difference in normal growth ability from the parent strain 4TI.
(2) Adhesive ability of highly metastatic breast cancer cells 4T1E / M3 to plastic surfaces
4T1E / M3 cells and parent strain 4TI are spread on a plastic 96-well plate to form a cell monolayer, and then 0.017% EDTA-PDS is added. After incubation for 5 to 60 minutes, the detached cells are removed by washing. The number of cells adhering after each culture time was measured using a cell counting kit8. The results are shown in FIG. It is clear that 4TIE / M3 cells are less likely to peel off than the parent strain 4TI, and the adhesion ability is enhanced.
(3) Highly metastatic breast cancer cells
Adhesive ability of 4T1E / M3 to bone marrow-derived endothelial cells
4T1E / M3 cells fluorescently labeled with BCECF-AM and the parent strain 4T1 were added to the endothelial cells cultured in a single layer on a 24-well plate, cultured for 1 hr at 37 ° C, 5% CO 2 and washed three times. Measurement was performed with a fluorescence plate reader (excitation wavelength: 480 nm, fluorescence wavelength: 530 nm). Each cell labeled with AM was plated on bone marrow-derived endothelial cells and cultured for 15 to 75 minutes. It is clear that the adhesion ability of 4T1E / M3 cells shown in FIG. 7 to bone marrow-derived endothelial cells is higher than that of the parent strain.
(4) Highly metastatic breast cancer cells 4T1E / M3 adhesion molecule gene expression
Comparison of expressed genes between 4T1E / M3 cells and 4T1 cells was performed for 2 integrins and ICAM-1 by DNA microarray analysis and quantitative RT-PCR. The results are shown in FIG.
According to this, it is clear that the expression of these genes is enhanced in 4T1E / M3 cells. In addition, the surfaces of 4T1 cells and 4T1E / M3 cells were stained with antibodies against 2 integrin and ICAM-1 and analyzed by flow cytometry. The results are shown in FIG. It can be seen that the expression of β2 integrin and ICAM-1 on the cell surface is increased in 4TIE / M3 cells compared to the parent strain.
(5) Highly metastatic breast cancer cells 4T1E / M3 anchorage-independent growth ability
4T1E / M3 cells and 4T1 cells were each seeded in a 6 cm dish with 0.3% soft agar and cultured for 12 days. Number of cells used is four and 2x10 4 1x10 respectively per dish. After culturing, the number of colonies found in each petri dish was counted, and the shape and size of the colonies were photographed with a microscope. The number of colonies and the observed photomicrographs are shown in FIGS. According to these results, it is clear that 4TIE / M3 cells have enhanced anchorage-independent growth ability compared to the parent strain.
(6) Motility of highly metastatic breast cancer cells 4T1E / M3
Claims (6)
ａ）薬剤耐性遺伝子が導入されたマウス乳癌細胞４Ｔ１を、マウスに投与し、少なくとも１０日経過後、マウス骨髄から細胞を採取するプロセス、
ｂ）得られた細胞を、上記薬剤耐性遺伝子による耐性付与の対象薬剤を含有する培地で培養し、薬剤耐性細胞を採取するプロセス、
ｃ）得られた薬剤耐性細胞をマウスに投与するプロセス、
ｄ）得られた薬剤耐性細胞を用いて上記ａ）のマウスへの投与、細胞の採取及びｂ）の薬剤耐性細胞の採取の各プロセスを順次複数回繰り返し、高転移性マウス乳癌細胞を得るプロセス An animal model for analyzing lung and / or liver metastasis of breast cancer obtained by administering to a mouse highly metastatic mouse breast cancer cells obtained from the following processes a) to d).
a) a process of administering mouse breast cancer cells 4T1 into which a drug resistance gene has been introduced to mice, and collecting the cells from mouse bone marrow after at least 10 days;
b) a process of culturing the obtained cells in a medium containing a target drug to be given resistance by the drug resistance gene, and collecting the drug resistant cells;
c) a process of administering the resulting drug resistant cells to mice,
d) A process for obtaining highly metastatic mouse breast cancer cells by sequentially repeating the processes of a) administration to the mouse, collection of cells, and collection of the drug resistant cells in b) a plurality of times using the obtained drug resistant cells.
ａ）薬剤耐性遺伝子が導入されたマウス乳癌細胞４Ｔ１を、マウスに投与し、少なくとも１０日経過後、マウス骨髄から細胞を採取するプロセス、
ｂ）得られた細胞を、上記薬剤耐性遺伝子による耐性付与の対象薬剤を含有する培地で培養し、薬剤耐性細胞を採取するプロセス、
ｃ）得られた薬剤耐性細胞をマウスに投与するプロセス、
ｄ）得られた薬剤耐性細胞を用いて上記ａ）のマウスへの投与、細胞の採取及びｂ）の薬剤耐性細胞の採取の各プロセスを順次複数回繰り返し、高転移性マウス乳癌細胞を得るプロセス A cell material for creating an animal model for analyzing lung and / or liver metastasis of breast cancer, characterized by comprising highly metastatic mouse breast cancer cells obtained from the following processes a) to d): a) a drug resistance gene is introduced A process of administering the mouse breast cancer cell 4T1 to the mouse and collecting the cell from the mouse bone marrow after at least 10 days;
b) a process of culturing the obtained cells in a medium containing a target drug to be given resistance by the drug resistance gene, and collecting the drug resistant cells;
c) a process of administering the resulting drug resistant cells to mice,
d) A process for obtaining highly metastatic mouse breast cancer cells by sequentially repeating the processes of a) administration to the mouse, collection of cells, and collection of the drug resistant cells in b) a plurality of times using the obtained drug resistant cells.
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| WO2012015023A1 (en) * | 2010-07-29 | 2012-02-02 | 国立大学法人京都大学 | Method for screening anticancer agents |
| JP2015167521A (en) * | 2014-03-07 | 2015-09-28 | 国立研究開発法人産業技術総合研究所 | Establishing method of marrow highly metastatic mouse breast cancer cell strain of breast cancer |
| CN114196617A (en) * | 2021-12-22 | 2022-03-18 | 中国医学科学院基础医学研究所 | Organ-specific breast cancer metastasis model and establishment method and application thereof |
| CN114634913A (en) * | 2022-02-27 | 2022-06-17 | 复旦大学 | Method for constructing tumor dura mater metastasis model |
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| WO2005097832A2 (en) * | 2004-03-31 | 2005-10-20 | Genentech, Inc. | Humanized anti-tgf-beta antibodies |
| WO2007002373A2 (en) * | 2005-06-23 | 2007-01-04 | Baylor College Of Medicine | Use of mutant herpes simplex virus-2 for cancer therapy |
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| WO2005097832A2 (en) * | 2004-03-31 | 2005-10-20 | Genentech, Inc. | Humanized anti-tgf-beta antibodies |
| WO2007002373A2 (en) * | 2005-06-23 | 2007-01-04 | Baylor College Of Medicine | Use of mutant herpes simplex virus-2 for cancer therapy |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2012015023A1 (en) * | 2010-07-29 | 2012-02-02 | 国立大学法人京都大学 | Method for screening anticancer agents |
| US9018438B2 (en) | 2010-07-29 | 2015-04-28 | Kyoto University | Screening method for anticancer drugs |
| JP2015167521A (en) * | 2014-03-07 | 2015-09-28 | 国立研究開発法人産業技術総合研究所 | Establishing method of marrow highly metastatic mouse breast cancer cell strain of breast cancer |
| CN114196617A (en) * | 2021-12-22 | 2022-03-18 | 中国医学科学院基础医学研究所 | Organ-specific breast cancer metastasis model and establishment method and application thereof |
| CN114196617B (en) * | 2021-12-22 | 2023-12-22 | 中国医学科学院基础医学研究所 | Organ-specific breast cancer metastasis model, and establishment method and application thereof |
| CN114634913A (en) * | 2022-02-27 | 2022-06-17 | 复旦大学 | Method for constructing tumor dura mater metastasis model |
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