JP7464977B2 - Canine mesothelioma cell lines - Google Patents
Canine mesothelioma cell lines Download PDFInfo
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- JP7464977B2 JP7464977B2 JP2020101200A JP2020101200A JP7464977B2 JP 7464977 B2 JP7464977 B2 JP 7464977B2 JP 2020101200 A JP2020101200 A JP 2020101200A JP 2020101200 A JP2020101200 A JP 2020101200A JP 7464977 B2 JP7464977 B2 JP 7464977B2
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Description
特許法第３０条第２項適用 〔公開１〕 開催日 ：令和元年（２０１９年）６月１６日 集会名 ：第９８回日本獣医麻酔外科学会 開催場所 ：大宮ソニックシティ 公開者 ：玉田雄大、村上泰一、小畠利沙子、小林由佳、鍋田梨奈、打出毅 Article 30, paragraph 2 of the Patent Act applies [Publication 1] Date: June 16, 2019 Meeting name: 98th Japanese Society of Veterinary Anesthesia and Surgery Venue: Omiya Sonic City Publishers: Yudai Tamada, Taiichi Murakami, Risako Obata, Yuka Kobayashi, Rina Nabeta, Takeshi Uchide
特許法第３０条第２項適用 〔公開２Ａ〕 開催日 ：令和元年（２０１９年）１１月２０日 集会名 ：東京農工大学・岩手大学共同獣医学科卒業論文発表会 開催場所 ：国立大学法人東京農工大学 府中キャンパス 農学部本館 講堂 公開者 ：玉田雄大 Article 30, paragraph 2 of the Patent Act applies [Publication 2A] Date: November 20, 2019 Name of the meeting: Tokyo University of Agriculture and Technology and Iwate University Joint Veterinary Medicine Department Graduation Thesis Presentation Venue: Tokyo University of Agriculture and Technology, Fuchu Campus, Faculty of Agriculture Main Building, Auditorium Publicist: Yudai Tamada
特許法第３０条第２項適用 〔公開２Ｂ〕 配布日 ：令和元年（２０１９年）１１月 配布物 ：東京農工大学・岩手大学共同獣医学科卒業論文発表要旨集 公開者 ：玉田雄大Article 30, paragraph 2 of the Patent Act [Publication 2B] Distribution date: November 2019 Distribution material: Tokyo University of Agriculture and Technology / Iwate University Joint Veterinary Medicine Department Graduation Thesis Abstracts Publisher: Yudai Tamada
本発明は、イヌ中皮腫細胞株及び該細胞株が移植された非ヒト哺乳動物に関する。本発明はまた、該細胞株又は該非ヒト哺乳動物を用いた、中皮腫の治療又は予防に有効な物質のスクリーニング方法に関する。 The present invention relates to a canine mesothelioma cell line and a non-human mammal into which the cell line has been transplanted. The present invention also relates to a method for screening for a substance effective in treating or preventing mesothelioma, using the cell line or the non-human mammal.
中皮腫は、胸腔、腹腔、心膜腔、総鞘膜腔などの体腔を内張りする中皮細胞を起源とする悪性腫瘍である。ヒト中皮腫は希少がんの１つであるが、中皮腫患者数は増加傾向にあることが知られている。イヌ、ネコにおいても中皮腫はまれな腫瘍であり、イヌにおける罹患率は0.1%ほどで、雌よりも雄で多いとされ、好発犬種は知られていない。中皮腫の予後は一般に不良であり、例えばイヌにおける中央生存期間は標準的治療を用いたとしても1年程度である。ヒト中皮腫では胸膜中皮腫とアスベスト暴露との関連性が指摘されており、イヌにおいてもアスベスト暴露歴のある胸膜中皮腫が報告されているが、心膜や腹膜でみられる中皮腫の発生要因は不明である。 Mesothelioma is a malignant tumor originating from mesothelial cells that line body cavities such as the thoracic, abdominal, pericardial, and synovial cavities. Human mesothelioma is a rare cancer, but the number of mesothelioma patients is known to be on the rise. Mesothelioma is also a rare tumor in dogs and cats, with an incidence rate of about 0.1% in dogs, and is more common in males than in females. No particular breed is known to be more susceptible to mesothelioma. The prognosis for mesothelioma is generally poor, and the median survival time in dogs is about one year even with standard treatment. In human mesothelioma, a relationship between pleural mesothelioma and asbestos exposure has been pointed out, and pleural mesothelioma has been reported in dogs with a history of asbestos exposure, but the cause of mesothelioma seen in the pericardium and peritoneum is unknown.
中皮腫は広範囲に漿膜面を冒すため、積極的な外科的切除は行われず、また放射線に対して抵抗を示すことから放射線治療は適応されない（非特許文献１）。現在、ヒト中皮腫においては化学療法が中皮腫の標準治療となっており、シスプラチンとドキソルビシンの併用をはじめ、近年ではシスプラチンと代謝拮抗薬であるメトトレキセートの併用療法が良い成績を上げている（非特許文献２及び３）。一方、獣医学領域における化学療法に関する報告は著しく少なく、中皮腫罹患犬へシスプラチンの胸腔内投与を行ったところ胸水貯留が抑制されたとする報告や、イヌ悪性中皮腫2例に対しカルボプラチンとドキソルビシンを体腔内投与することによって体腔液の貯留が減少し生存期間の延長に繋がったとする報告があるのみで（非特許文献４及び５）、化学療法についての系統的な研究報告は存在しない。獣医学領域で行われている中皮腫の治療は、胸腔・腹腔穿刺による体腔液の抜去、心タンポナーデの回避処置としての心膜切除術等の対症療法が主軸となっている。 Because mesothelioma affects the serosal surface over a wide area, aggressive surgical resection is not performed, and radiation therapy is not applicable because the tumor is resistant to radiation (Non-Patent Document 1). Currently, chemotherapy is the standard treatment for human mesothelioma, and in recent years, combination therapy of cisplatin and methotrexate, an antimetabolite, has produced good results (Non-Patent Documents 2 and 3). On the other hand, there are very few reports on chemotherapy in the field of veterinary medicine, with only one report reporting that intrathoracic administration of cisplatin to dogs with mesothelioma suppressed pleural effusion, and another report reporting that intrathoracic administration of carboplatin and doxorubicin reduced pleural effusion and extended survival in two cases of canine malignant mesothelioma (Non-Patent Documents 4 and 5), and there are no systematic research reports on chemotherapy. In veterinary medicine, mesothelioma treatment is centered on symptomatic treatments such as pleural and abdominal paracentesis to remove fluid from the body cavities, and pericardiectomy to prevent cardiac tamponade.
イヌ中皮腫に対する有効な化学療法プロトコールの確立のためには、有効な化学療法薬のスクリーニングと選択が必須となるが、罹患動物を用いたこれらの研究には、本腫瘍の低発生率の問題や腫瘍性状の個体間における不均一性の問題が存在するため、in vitroにおける検討が必要である。しかしながら、現在、イヌの中皮腫由来の中皮腫モデル細胞株が存在しないことが、基礎的な研究を行う上での大きな障壁となっている。 To establish an effective chemotherapy protocol for canine mesothelioma, it is essential to screen and select effective chemotherapeutic drugs. However, in vitro studies are necessary for these studies using affected animals because of the low incidence of this tumor and the heterogeneity of tumor characteristics among individuals. However, the current lack of a mesothelioma model cell line derived from canine mesothelioma is a major barrier to conducting basic research.
ヒト中皮腫の治療法の開発を進める上でも、中皮腫モデル細胞株は有用である。しかし中皮腫由来の細胞株であっても中皮腫の性質を必ずしも保持しているわけではなく、中皮腫モデルとして好適な細胞株の作製がなおも望まれている。 Mesothelioma model cell lines are also useful in developing treatments for human mesothelioma. However, even cell lines derived from mesothelioma do not necessarily retain the properties of mesothelioma, and there is still a need to create cell lines suitable as mesothelioma models.
本発明は、イヌ中皮腫細胞株を提供することを課題とする。 The objective of the present invention is to provide a canine mesothelioma cell line.
本発明者らは、上記課題を解決するため鋭意検討を重ねた結果、中皮腫モデルとして好適なイヌ中皮腫細胞株を樹立することに成功し、本発明を完成するに至った。 As a result of extensive research to solve the above problems, the inventors have succeeded in establishing a canine mesothelioma cell line suitable as a mesothelioma model, thus completing the present invention.
すなわち、本発明は以下を包含する。
［１］ カルボプラチン、ドキソルビシン、及びビノレルビンのそれぞれに対して薬剤感受性を有するイヌ中皮腫細胞株。
［２］ 受託番号NITE P-03216を有するイヌ中皮腫細胞MC19009株である、［１］に記載のイヌ中皮腫細胞株。
［３］ 以下の工程：
(a) 中皮腫を有するイヌから胸水サンプルを採取する工程、
(b) 工程(a)で採取した胸水サンプル中の細胞を液体培地中で培養する工程、及び
(c) 培養した細胞の薬剤感受性を試験し、カルボプラチン、ドキソルビシン、及びビノレルビンのそれぞれに対して薬剤感受性を有する細胞を選抜する工程
を含む、イヌ中皮腫細胞株の作製方法。
［４］ ［３］に記載の方法により作製された、イヌ中皮腫細胞株。
［５］ ［１］、［２］、及び［４］のいずれかに記載のイヌ中皮腫細胞株が移植された、非ヒト哺乳動物。
［６］ 非ヒト哺乳動物が免疫不全である、［５］に記載の非ヒト哺乳動物。
［７］ 中皮腫モデル動物である、［５］又は［６］に記載の非ヒト哺乳動物。
［８］ 被験物質の存在下、［１］、［２］、及び［４］のいずれかに記載のイヌ中皮腫細胞株を培養し、該細胞株の細胞生存率を測定することを含む、中皮腫の治療又は予防に有効な物質のスクリーニング方法。
［９］ ［５］～［７］のいずれかに記載の非ヒト哺乳動物に被験物質を投与し、腫瘍に対する治療又は予防効果を評価することを含む、中皮腫の治療又は予防に有効な物質のスクリーニング方法。
That is, the present invention includes the following.
[1] A canine mesothelioma cell line sensitive to carboplatin, doxorubicin, and vinorelbine.
[2] The canine mesothelioma cell line described in [1], which is the canine mesothelioma cell line MC19009 having the accession number NITE P-03216 .
[3] The following steps:
(a) obtaining a pleural fluid sample from a dog with mesothelioma;
(b) culturing the cells in the pleural fluid sample collected in step (a) in a liquid culture medium; and
(c) A method for producing a canine mesothelioma cell line, comprising the steps of testing the drug sensitivity of the cultured cells and selecting cells having drug sensitivity to each of carboplatin, doxorubicin, and vinorelbine.
[4] A canine mesothelioma cell line prepared by the method described in [3].
[5] A non-human mammalian animal having transplanted therein the canine mesothelioma cell line according to any one of [1], [2], and [4].
[6] The non-human mammal according to [5], wherein the non-human mammal is immunodeficient.
[7] The non-human mammal according to [5] or [6], which is a mesothelioma model animal.
[8] A method for screening for a substance effective in treating or preventing mesothelioma, comprising culturing a canine mesothelioma cell line described in any one of [1], [2], and [4] in the presence of a test substance and measuring the cell viability of the cell line.
[9] A method for screening for a substance effective in treating or preventing mesothelioma, comprising administering a test substance to the non-human mammal according to any one of [5] to [7], and evaluating the therapeutic or preventive effect on a tumor.
以下、本発明を詳細に説明する。 The present invention is described in detail below.
本発明は、イヌ中皮腫細胞株、特に、中皮腫モデルとして好適なイヌ中皮腫細胞株に関する。 The present invention relates to a canine mesothelioma cell line, in particular, a canine mesothelioma cell line suitable as a mesothelioma model.
本発明において、「イヌ中皮腫細胞株」とは、イヌ中皮腫に由来する細胞株を意味する。本発明において「細胞株」は、生体内及び腫瘍から分離され、人為的に培養された細胞を指し、初代培養細胞であっても株化細胞であってもよい。 In the present invention, the term "canine mesothelioma cell line" refers to a cell line derived from canine mesothelioma. In the present invention, the term "cell line" refers to cells that have been isolated from a living body or a tumor and artificially cultured, and may be either primary cultured cells or established cell lines.
本発明において、「中皮腫」とは、胸腔、腹腔、心膜腔及び総鞘膜腔などの体腔を内張りする中皮細胞を起源とする悪性腫瘍を意味する。本発明における中皮腫は、発生部位による分類に従って胸膜中皮腫、腹膜中皮腫又は心膜中皮腫であってよく、組織型による分類に従って上皮型、肉腫型、繊維形成型又は二相型の中皮腫であってよい。 In the present invention, "mesothelioma" refers to a malignant tumor originating from mesothelial cells lining body cavities such as the thoracic, abdominal, pericardial and synovial cavities. Mesothelioma in the present invention may be pleural mesothelioma, peritoneal mesothelioma or pericardial mesothelioma according to the site of origin, and may be epithelial, sarcomatoid, desmoplastic or biphasic mesothelioma according to the histological type.
本発明に係るイヌ中皮腫細胞株は、中皮腫モデルとして好適な薬剤感受性を有することが好ましく、特に、中皮腫の治療に使用可能な抗癌剤（抗中皮腫薬）であるカルボプラチン、ドキソルビシン、及びビノレルビンのそれぞれに対して薬剤感受性を有することがさらに好ましい。カルボプラチンは白金製剤の１つであり、白金製剤は、DNA鎖内及びDNA鎖間架橋の形成によりDNA合成阻害を引き起こすことによって抗腫瘍効果を示す。ドキソルビシン及びビノレルビンは、カルボプラチンとは異なる作用機序を有する。ドキソルビシンは、腫瘍細胞のDNA塩基対間への挿入により核酸合成酵素の反応を阻害することによって抗腫瘍効果を示す。ビノレルビンは、有糸分裂微小管のチューブリンに選択的に作用し、その重合を阻害することによって抗腫瘍効果を示す。 The canine mesothelioma cell line of the present invention preferably has drug sensitivity suitable for use as a mesothelioma model, and more preferably has drug sensitivity to each of carboplatin, doxorubicin, and vinorelbine, which are anticancer drugs (antimesothelioma drugs) that can be used to treat mesothelioma. Carboplatin is a platinum drug, which exerts an antitumor effect by causing inhibition of DNA synthesis through the formation of intrastrand and interstrand crosslinks. Doxorubicin and vinorelbine have a different mechanism of action from carboplatin. Doxorubicin exerts an antitumor effect by inhibiting the reaction of nucleic acid synthesis enzymes through insertion between DNA base pairs in tumor cells. Vinorelbine exerts an antitumor effect by selectively acting on tubulin in mitotic microtubules and inhibiting their polymerization.
本発明に係るイヌ中皮腫細胞株は、さらに、他の抗中皮腫薬に対する薬剤感受性も有し得る。本発明に係るイヌ中皮腫細胞株は、例えば、カルボプラチンとは異なる作用機序を有するゲムシタビンに対する薬剤感受性を有していてもよい。本発明に係るイヌ中皮腫細胞株はまた、カルボプラチンとゲムシタビンの併用に対して強い感受性を有していてもよい。一実施形態では、カルボプラチンとゲムシタビンの併用は、本発明に係るイヌ中皮腫細胞株に対し、単剤と比較して増強された細胞増殖抑制効果（抗腫瘍効果）をもたらすものであり得る。本発明に係るイヌ中皮腫細胞株は、カルボプラチン、ドキソルビシン、ビノレルビン、及びゲムシタビンからなる群から選択される少なくとも１つ（例えば、１つ又は２つ以上）と他の抗癌剤（抗中皮腫薬）の併用療法に対する薬剤感受性を有していてもよい。 The canine mesothelioma cell line of the present invention may further have drug sensitivity to other anti-mesothelioma drugs. The canine mesothelioma cell line of the present invention may have drug sensitivity to, for example, gemcitabine, which has a mechanism of action different from that of carboplatin. The canine mesothelioma cell line of the present invention may also have strong sensitivity to a combination of carboplatin and gemcitabine. In one embodiment, the combination of carboplatin and gemcitabine may provide an enhanced cell proliferation inhibitory effect (antitumor effect) to the canine mesothelioma cell line of the present invention compared to a single agent. The canine mesothelioma cell line of the present invention may have drug sensitivity to a combination therapy of at least one (e.g., one or more) selected from the group consisting of carboplatin, doxorubicin, vinorelbine, and gemcitabine with another anticancer drug (anti-mesothelioma drug).
本発明に係るイヌ中皮腫細胞株は、作用機序の異なる抗中皮腫薬カルボプラチン、ドキソルビシン、及びビノレルビンのそれぞれに対して薬剤感受性を有し、その薬剤感受性は中皮腫患者に対するそれらの薬剤の奏効率の高さ（中皮腫患者での薬剤応答性）をよく反映していること、さらに、好ましくはカルボプラチンとゲムシタビンの併用による抗腫瘍効果の増強の点でも中皮腫患者における薬剤応答性をよく反映していることから、中皮腫モデルとして非常に有用である。 The canine mesothelioma cell line of the present invention is highly useful as a mesothelioma model because it is sensitive to the anti-mesothelioma drugs carboplatin, doxorubicin, and vinorelbine, which have different mechanisms of action, and its drug sensitivity closely reflects the high efficacy rate of these drugs in mesothelioma patients (drug responsiveness in mesothelioma patients).Furthermore, the enhanced antitumor effect of the combined use of carboplatin and gemcitabine also closely reflects the drug responsiveness in mesothelioma patients.
本発明に係るイヌ中皮腫細胞株は、以下の工程：
(a) 中皮腫を有するイヌから胸水サンプルを採取する工程、及び
(b) 胸水サンプル中の細胞を液体培地中で培養する工程
を含む、イヌ中皮腫細胞株の作製方法により作製することができる。本発明は、イヌ中皮腫細胞株の作製方法も提供する。
The canine mesothelioma cell line of the present invention can be prepared by the following steps:
(a) obtaining a pleural fluid sample from a dog having mesothelioma; and
(b) The canine mesothelioma cell line can be prepared by a method for preparing a canine mesothelioma cell line, the method comprising the step of culturing cells in a pleural effusion sample in a liquid medium. The present invention also provides a method for preparing a canine mesothelioma cell line.
上記イヌは、任意の中皮腫を有していてよいが、例えば、胸膜中皮腫、腹膜中皮腫又は心膜中皮腫であってよい。 The dog may have any type of mesothelioma, for example pleural, peritoneal or pericardial mesothelioma.
胸水サンプルは、常法により採取することができるが、例えば、胸腔穿刺により採取してもよい。胸腔穿刺は、例えば、超音波ガイド下で行ってもよいが、それに限定されない。 Pleural fluid samples can be collected by standard methods, including, but not limited to, thoracentesis. Thoracentesis may be performed under ultrasound guidance, for example.
一実施形態では、胸水サンプルをそのまま液体培地に添加し、それを培養することにより、胸水サンプル中の細胞を培養してもよい。別の実施形態では、胸水サンプルから回収した細胞を洗浄後に液体培地に添加し、それを培養することにより、胸水サンプル中の細胞を培養してもよい。 In one embodiment, the cells in the pleural fluid sample may be cultured by adding the pleural fluid sample directly to a liquid medium and culturing the liquid medium. In another embodiment, the cells collected from the pleural fluid sample may be cultured by adding the cells to a liquid medium after washing and culturing the liquid medium.
本発明のイヌ中皮腫細胞株の作製方法において、液体培地は、細胞培養、特に初代培養に適した任意の培地であってよく、例えばRPMI-1640培地などのRPMI（Roswell Park Memorial Institute）培地であってよい。液体培地は、非働化ウシ胎児血清（FBS）、イヌ血清などの血清を含有していてもよい。液体培地は、マイコプラズマ、細菌、真菌等の感染を予防するための薬剤、例えばPrimocinTM、PlasmosinTM、NormocinTM、ペニシリン、ストレプトマイシンなどの抗生物質を含有してもよい。 In the method for producing a canine mesothelioma cell line of the present invention, the liquid medium may be any medium suitable for cell culture, particularly primary culture, for example, RPMI (Roswell Park Memorial Institute) medium such as RPMI-1640 medium. The liquid medium may contain serum such as inactivated fetal bovine serum (FBS) or dog serum. The liquid medium may contain an antibiotic such as Primocin ™ , Plasmosin ™, Normocin™ , penicillin, streptomycin, etc., for preventing infection with mycoplasma, bacteria, fungi, etc.
このイヌ中皮腫細胞株の作製方法はまた、(c) 培養した細胞の薬剤感受性を試験し、カルボプラチン、ドキソルビシン、及びビノレルビンのそれぞれに対して薬剤感受性を有する細胞を選抜する工程を、さらに含んでもよい。一実施形態では、培養した細胞の薬剤感受性を試験し、ゲムシタビン（単剤）、又はカルボプラチンとゲムシタビンの併用に対して薬剤感受性を有する細胞をさらに選抜してもよい。 This method for producing a canine mesothelioma cell line may also further include the step of (c) testing the drug sensitivity of the cultured cells and selecting cells that are sensitive to each of carboplatin, doxorubicin, and vinorelbine. In one embodiment, the drug sensitivity of the cultured cells may be tested and cells that are sensitive to gemcitabine (single agent) or a combination of carboplatin and gemcitabine may be further selected.
本発明において薬剤感受性とは、生体投与可能な濃度に対応する濃度（例えば、臨床的薬用量に対応する細胞処理濃度）の特定の薬剤（特に、抗癌剤）で本発明のイヌ中皮腫細胞株を処理したときに、同じイヌ中皮腫細胞株の当該薬剤非処理群と比較して、当該イヌ中皮腫細胞株の薬剤処理群の細胞生存率が低下する（好ましくは、統計学的に有意に低下する）ことを意味する。一実施形態では、本発明における薬剤感受性は、その細胞生存率が薬剤用量依存的に低下する傾向によって示されるものであってもよい。 In the present invention, drug sensitivity means that when the canine mesothelioma cell line of the present invention is treated with a specific drug (particularly an anticancer drug) at a concentration corresponding to a concentration that can be administered to the body (e.g., a cell treatment concentration corresponding to a clinical drug dose), the cell viability of the drug-treated group of the canine mesothelioma cell line is reduced (preferably, statistically significantly reduced) compared to a group of the same canine mesothelioma cell line that is not treated with the drug. In one embodiment, drug sensitivity in the present invention may be indicated by a tendency for the cell viability to decrease in a drug dose-dependent manner.
本発明のイヌ中皮腫細胞株の作製方法はまた、(a)及び(b)の工程、又は(a)～(c)の工程に加えて、細胞における中皮腫マーカーの発現を試験する工程をさらに含んでもよい。任意の中皮腫マーカーの発現を試験することができ、例えば、AE1/AE3、CK5及びWT-1の発現を試験してもよい。１つ以上の中皮腫マーカー、例えばAE1/AE3、CK5及びWT-1の発現が陽性であれば、そのイヌ中皮腫細胞株が中皮細胞由来であること、すなわち中皮腫細胞であることを確認することができる。細胞における中皮腫マーカーの発現の有無は、常法により試験することができる。例えば、AE1/AE3、CK5及びWT-1の発現が陽性であることは、それぞれ、抗AE1/AE3抗体、抗CK5抗体及び抗WT-1抗体を用いた免疫蛍光染色により試験することができる。 The method for producing a canine mesothelioma cell line of the present invention may further include, in addition to steps (a) and (b) or steps (a) to (c), a step of testing the expression of a mesothelioma marker in the cells. The expression of any mesothelioma marker may be tested, for example, the expression of AE1/AE3, CK5 and WT-1 may be tested. If the expression of one or more mesothelioma markers, for example AE1/AE3, CK5 and WT-1, is positive, it can be confirmed that the canine mesothelioma cell line is derived from mesothelial cells, i.e., is a mesothelioma cell. The presence or absence of expression of a mesothelioma marker in the cells can be tested by a conventional method. For example, the positive expression of AE1/AE3, CK5 and WT-1 can be tested by immunofluorescence staining using anti-AE1/AE3 antibody, anti-CK5 antibody and anti-WT-1 antibody, respectively.
本発明は、本発明のイヌ中皮腫細胞株の作製方法により作製されたイヌ中皮腫細胞株も提供する。 The present invention also provides a canine mesothelioma cell line produced by the method for producing a canine mesothelioma cell line of the present invention.
好ましい実施形態では、本発明のイヌ中皮腫細胞株は、上皮様の細胞形態を示し、10継代以上継代することができ、かつ／又はAE1/AE3、CK5及びWT-1の発現について陽性である。 In a preferred embodiment, the canine mesothelioma cell line of the present invention exhibits epithelial-like cell morphology, can be passaged for 10 or more passages, and/or is positive for the expression of AE1/AE3, CK5, and WT-1.
本発明のイヌ中皮腫細胞株の好ましい例は、後述の実施例に示されたイヌ中皮腫細胞MC18001株、MC19001株、及びMC19009株である。 Preferred examples of the canine mesothelioma cell lines of the present invention are the canine mesothelioma cell lines MC18001, MC19001, and MC19009 shown in the Examples below.
イヌ（犬）中皮腫細胞MC19009株は、2020年5月14日付で、独立行政法人製品評価技術基盤機構 特許微生物寄託センター（NPMD）（日本国千葉県木更津市かずさ鎌足2-5-8 122号室）に、受託番号NITE P-03216で寄託されている。イヌ中皮腫細胞MC19009株は、上皮様の細胞形態を示し、好ましくは10継代以上継代することができ、AE1/AE3、CK5及びWT-1の発現について陽性である。さらに、イヌ中皮腫細胞MC19009株は、カルボプラチン、ドキソルビシン、及びビノレルビンのそれぞれに対して薬剤感受性を有し、カルボプラチンとゲムシタビンの併用に対して強い感受性を有しており、中皮腫患者における薬剤応答性をよく反映していることから、中皮腫モデルとして非常に有用である。 The canine mesothelioma cell MC19009 strain was deposited on May 14, 2020 at the National Institute of Technology and Evaluation, Patent Microorganism Depository Center (NPMD) (Room 122, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan) under the accession number NITE P-03216 . The canine mesothelioma cell MC19009 strain exhibits epithelial-like cell morphology, can be preferably passaged for 10 or more passages, and is positive for the expression of AE1/AE3, CK5, and WT-1. Furthermore, the canine mesothelioma cell MC19009 strain is drug sensitive to each of carboplatin, doxorubicin, and vinorelbine, and has strong sensitivity to the combination of carboplatin and gemcitabine, which well reflects the drug responsiveness in mesothelioma patients, making it very useful as a mesothelioma model.
本発明はまた、本発明のイヌ中皮腫細胞株が移植された、非ヒト哺乳動物に関する。本発明において、「非ヒト哺乳動物」とは、ヒトを除く哺乳動物を意味する。非ヒト哺乳動物は、免疫不全であってもよい。非ヒト哺乳動物は、非ヒトかつ非イヌ哺乳動物であってもよい。非ヒト哺乳動物としては、以下に限定されないが、マウス、ラット、ハムスター等のげっ歯類、チンパンジー、ゴリラ、アカゲザル等の霊長類、ブタ、イヌ、ウサギなどが挙げられる。免疫不全である非ヒト哺乳動物は、例えば、重症複合免疫不全（SCID）マウスであってよい。本発明の非ヒト哺乳動物において、イヌ中皮腫細胞株は、生体内の任意の部位に移植されてもよいが、例えば、皮下又は腹腔内に移植されていてもよい。本発明のイヌ中皮腫細胞株が移植された非ヒト哺乳動物は、移植箇所に腫瘍を有し得る。一実施形態では、本発明のイヌ中皮腫細胞株が移植された非ヒト哺乳動物において、その移植されたイヌ中皮腫細胞株から形成される腫瘍は、中皮腫であることが好ましく、例えば上皮型、肉腫型、繊維形成型、又は二相型の中皮腫であってよい。本発明のイヌ中皮腫細胞株が移植された非ヒト哺乳動物は、中皮腫モデル動物として有利に用いることができる。 The present invention also relates to a non-human mammal into which the canine mesothelioma cell line of the present invention has been transplanted. In the present invention, the term "non-human mammal" refers to a mammal other than humans. The non-human mammal may be immunodeficient. The non-human mammal may be a non-human and non-canine mammal. Non-human mammals include, but are not limited to, rodents such as mice, rats, and hamsters, primates such as chimpanzees, gorillas, and rhesus monkeys, pigs, dogs, and rabbits. The immunodeficient non-human mammal may be, for example, a severe combined immunodeficient (SCID) mouse. In the non-human mammal of the present invention, the canine mesothelioma cell line may be transplanted at any site in the body, for example, subcutaneously or intraperitoneally. The non-human mammal into which the canine mesothelioma cell line of the present invention has been transplanted may have a tumor at the site of transplantation. In one embodiment, in a non-human mammal into which the canine mesothelioma cell line of the present invention has been transplanted, the tumor formed from the transplanted canine mesothelioma cell line is preferably mesothelioma, and may be, for example, epithelial, sarcomatoid, desmoplastic, or biphasic mesothelioma. The non-human mammal into which the canine mesothelioma cell line of the present invention has been transplanted can be advantageously used as a mesothelioma model animal.
本発明のイヌ中皮腫細胞株及び非ヒト哺乳動物は、中皮腫の治療又は予防に有効な物質のスクリーニングに有用である。本発明は、本発明のイヌ中皮腫細胞株又は非ヒト哺乳動物を用いた、中皮腫の治療又は予防に有効な物質のスクリーニング方法を提供する。 The canine mesothelioma cell line and non-human mammal of the present invention are useful for screening for substances effective in treating or preventing mesothelioma. The present invention provides a method for screening for substances effective in treating or preventing mesothelioma using the canine mesothelioma cell line or non-human mammal of the present invention.
より具体的には、本発明は、被験物質の存在下、本発明のイヌ中皮腫細胞株を培養し、該細胞株の細胞生存率を測定することを含む、中皮腫の治療又は予防に有効な物質のスクリーニング方法を提供する。本発明のイヌ中皮腫細胞株の培養は、例えば上述の液体培地を用いて行うことができ、例えば35～38℃（典型的には37℃）で行うことができるが、この条件に限定されない。被験物質の細胞処理濃度は、生体投与可能な濃度に対応する濃度（例えば、臨床的薬用量に対応する細胞処理濃度）であることが好ましい。本発明のスクリーニング方法では、例えば、被験物質の存在下（例えば培地中に被験物質を添加した状態で）、本発明のイヌ中皮腫細胞株を所定時間（典型的には、48～96時間、例えば72時間）培養した後、細胞数を計数し、被験物質の非存在下で本発明のイヌ中皮腫細胞株を同時間培養した場合の細胞数（コントロール）と比較し、本発明のイヌ中皮腫細胞株の細胞生存率を決定する。細胞生存率(%)は以下の式に従って算出すればよい。
細胞生存率(%)＝（被験物質の存在下で本発明のイヌ中皮腫細胞株を所定時間培養後の細胞数）/（被験物質の非存在下で本発明のイヌ中皮腫細胞株を同じ時間培養後の細胞数）×100
More specifically, the present invention provides a method for screening for a substance effective in treating or preventing mesothelioma, comprising culturing the canine mesothelioma cell line of the present invention in the presence of a test substance and measuring the cell viability of the cell line. The canine mesothelioma cell line of the present invention can be cultured, for example, using the above-mentioned liquid medium, at, for example, 35 to 38°C (typically 37°C), but is not limited to these conditions. The cell treatment concentration of the test substance is preferably a concentration corresponding to a concentration that can be administered to a living body (for example, a cell treatment concentration corresponding to a clinical drug dose). In the screening method of the present invention, for example, the canine mesothelioma cell line of the present invention is cultured for a predetermined time (typically 48 to 96 hours, for example 72 hours) in the presence of the test substance (for example, in a state in which the test substance is added to the medium), and then the number of cells is counted and compared with the number of cells (control) obtained when the canine mesothelioma cell line of the present invention is cultured for the same time in the absence of the test substance to determine the cell viability of the canine mesothelioma cell line of the present invention. The cell viability (%) may be calculated according to the following formula:
Cell viability (%) = (cell number after culturing the canine mesothelioma cell line of the present invention for a given period of time in the presence of a test substance) / (cell number after culturing the canine mesothelioma cell line of the present invention for the same period of time in the absence of a test substance) x 100
細胞生存率（%）は、呈色アッセイに基づいて算出することもできる。具体的には、例えば、被験物質の存在下（例えば培地中に被験物質を添加した状態）又は被験物質の非存在下（例えば培地中に被験物質を添加しない状態；コントロール）で、本発明のイヌ中皮腫細胞株を所定時間（典型的には、48～96時間、例えば72時間）にわたり液体培地中で培養した後、生細胞計数キット（例えば、Cell Counting Kit-8（同仁化学研究所））などを用いて、細胞内脱水素酵素により還元されて水溶性ホルマザンを生成するテトラゾリウム塩WST-8を発色基質とする呈色反応を行い、波長450nmで吸光度を測定する。並行して、呈色アッセイ試薬を含む同じ液体培地を用いて、バックグラウンドの測定を行う。被験物質の存在下で細胞を培養した群（処理群）の吸光度（Asample）、被験物質の非存在下で細胞を培養した群（コントロール）の吸光度（Acontrol）、バックグラウンドの吸光度（Ablank）に基づき、処理群の細胞生存率（%）を以下の式に従って算出することができる。
細胞生存率（%）＝(Asample-Ablank)/(Acontrol-Ablank)×100
The cell viability (%) can also be calculated based on a color assay. Specifically, for example, the canine mesothelioma cell line of the present invention is cultured in a liquid medium for a predetermined time (typically 48 to 96 hours, for example 72 hours) in the presence of a test substance (for example, a state in which the test substance is added to the medium) or in the absence of a test substance (for example, a state in which the test substance is not added to the medium; control), and then a color reaction is carried out using a viable cell counting kit (for example, Cell Counting Kit-8 (Dojindo Laboratories)) or the like, using the tetrazolium salt WST-8, which is reduced by intracellular dehydrogenase to generate water-soluble formazan as a color-developing substrate, and the absorbance is measured at a wavelength of 450 nm. In parallel, background measurements are carried out using the same liquid medium containing a color assay reagent. Based on the absorbance (A sample ) of the group in which cells were cultured in the presence of the test substance (treatment group), the absorbance (A control ) of the group in which cells were cultured in the absence of the test substance (control), and the background absorbance (A blank ), the cell viability (%) of the treatment group can be calculated according to the following formula.
Cell viability (%) = (A sample - A blank )/(A control - A blank ) x 100
以上のようにして算出した細胞生存率に基づいて、被験物質が細胞生存率にどのような影響を及ぼしたかを評価することができる。被験物質の存在下で細胞生存率の低下（例えば、統計学的に有意な低下）が認められれば、被験物質が中皮腫に対する抗腫瘍活性を有すると判断することができ、すなわちその被験物質を中皮腫の治療又は予防に有効な物質として同定することができる。 Based on the cell viability calculated as described above, it is possible to evaluate the effect of the test substance on cell viability. If a decrease in cell viability (e.g., a statistically significant decrease) is observed in the presence of the test substance, it can be determined that the test substance has antitumor activity against mesothelioma, i.e., the test substance can be identified as a substance effective in treating or preventing mesothelioma.
本発明はまた、本発明のイヌ中皮腫細胞株が移植された非ヒト哺乳動物（本発明の非ヒト哺乳動物）に被験物質を投与し、腫瘍に対する治療又は予防効果を評価することを含む、中皮腫の治療又は予防に有効な物質のスクリーニング方法を提供する。本方法において、本発明の非ヒト哺乳動物への被験物質の投与は、経口投与であっても非経口投与であってもよい。非経口投与としては、以下に限定されないが、腫瘍内投与、静脈内投与、動脈内投与、筋肉内投与、腹腔内投与、経皮投与、皮下投与、皮内投与、舌下投与、経腸投与、経腟投与、硬膜外投与、鼻腔投与等が挙げられる。 The present invention also provides a method for screening for a substance effective in treating or preventing mesothelioma, comprising administering a test substance to a non-human mammal (non-human mammal of the present invention) into which a canine mesothelioma cell line of the present invention has been transplanted, and evaluating the therapeutic or preventive effect on a tumor. In this method, the test substance may be administered orally or parenterally to the non-human mammal of the present invention. Parenteral administration includes, but is not limited to, intratumoral administration, intravenous administration, intra-arterial administration, intramuscular administration, intraperitoneal administration, transdermal administration, subcutaneous administration, intradermal administration, sublingual administration, enteral administration, vaginal administration, epidural administration, nasal administration, etc.
本方法において、被験物質を投与する、中皮腫モデル動物としての本発明の非ヒト哺乳動物は、腫瘍、特に中皮腫を有しているか又はそれを形成する能力を有する。 In this method, the non-human mammal of the present invention as a mesothelioma model animal to which the test substance is administered has a tumor, particularly mesothelioma, or has the ability to form a tumor.
本方法において、腫瘍に対する治療効果は、被験物質によって腫瘍の増殖若しくは転移が抑制されるかどうか、又は非ヒト哺乳動物の生存期間が延長されるかどうかを調べることにより評価することができる。例えば、本発明のイヌ中皮腫細胞株が移植され腫瘍が形成された本発明の非ヒト哺乳動物に被験物質を投与し、所定期間後に腫瘍体積を測定し、被験物質を投与していない場合の腫瘍体積と比べ、腫瘍体積の減少が見られる場合には、被験物質は抗腫瘍活性を有すると判断することができ、すなわちその被験物質を中皮腫の治療に有効な物質として同定することができる。あるいは、本発明のイヌ中皮腫細胞株が移植され腫瘍が形成された本発明の非ヒト哺乳動物において、被験物質の投与の所定期間後に、被験物質を投与していない場合と比較して、腫瘍の転移発生頻度が低下するか、及び／又は平均生存期間が延長される場合には、被験物質は抗腫瘍活性を有すると判断することができ、すなわちその被験物質を中皮腫の治療に有効な物質として同定することができる。 In this method, the therapeutic effect on tumors can be evaluated by examining whether the test substance inhibits tumor growth or metastasis or extends the survival time of the non-human mammal. For example, a test substance is administered to a non-human mammal of the present invention in which a tumor has been formed by transplanting the canine mesothelioma cell line of the present invention, and the tumor volume is measured after a predetermined period of time. If a decrease in tumor volume is observed compared to the tumor volume in the absence of the test substance, the test substance can be determined to have antitumor activity, i.e., the test substance can be identified as a substance effective for treating mesothelioma. Alternatively, in a non-human mammal of the present invention in which a tumor has been formed by transplanting the canine mesothelioma cell line of the present invention, if the frequency of tumor metastasis is reduced and/or the average survival time is extended compared to the absence of the test substance after a predetermined period of administration of the test substance, the test substance can be determined to have antitumor activity, i.e., the test substance can be identified as a substance effective for treating mesothelioma.
本方法において、腫瘍に対する予防効果は、被験物質によって腫瘍の発生若しくは再発が抑制されるかどうか、又は非ヒト哺乳動物の生存期間が延長されるかどうかを調べることにより評価することができる。例えば、本発明のイヌ中皮腫細胞株が移植されたが腫瘍を未だ形成していないか、又は移植されたイヌ中皮腫細胞株から形成した腫瘍の切除又は完全退縮後の本発明の非ヒト哺乳動物に被験物質を投与し、所定期間後に腫瘍形成を観察し、腫瘍体積を測定し、被験物質を投与していない場合の腫瘍形成及び腫瘍体積と比べ、腫瘍形成が阻止若しくは遅延（例えば、統計学的に有意な遅延）しているか又は腫瘍体積の減少（例えば、統計学的に有意な減少）が見られる場合には、被験物質は抗腫瘍活性を有すると判断することができ、すなわちその被験物質を中皮腫の予防に有効な物質として同定することができる。あるいは、本発明のイヌ中皮腫細胞株が移植されたが腫瘍を未だ形成していないか、又は移植されたイヌ中皮腫細胞株から形成した腫瘍の切除又は完全退縮後の本発明の非ヒト哺乳動物において、被験物質の投与の所定期間後に、被験物質を投与していない場合と比較して、腫瘍の発生若しくは再発の頻度が低下するか、及び／又は平均生存期間が延長される場合には、被験物質は抗腫瘍活性を有すると判断することができ、すなわちその被験物質を中皮腫の予防に有効な物質として同定することができる。 In this method, the preventive effect against tumors can be evaluated by examining whether the test substance inhibits the onset or recurrence of tumors or extends the survival time of the non-human mammal. For example, a test substance is administered to a non-human mammal of the present invention in which the canine mesothelioma cell line of the present invention has been transplanted but no tumor has yet formed, or in which a tumor formed from the transplanted canine mesothelioma cell line has been resected or completely regressed, and tumor formation is observed after a predetermined period of time, and the tumor volume is measured. If tumor formation is prevented or delayed (e.g., statistically significant delay) or tumor volume is reduced (e.g., statistically significant reduction) compared to the tumor formation and tumor volume in the absence of the test substance, the test substance can be determined to have antitumor activity, i.e., the test substance can be identified as a substance effective in preventing mesothelioma. Alternatively, in a non-human mammal of the present invention into which the canine mesothelioma cell line of the present invention has been transplanted but which has not yet formed a tumor, or in which a tumor formed from the transplanted canine mesothelioma cell line has been resected or completely regressed, if the frequency of tumor development or recurrence is reduced and/or the average survival time is extended after a specified period of administration of the test substance compared to when the test substance is not administered, the test substance can be determined to have antitumor activity, i.e., the test substance can be identified as a substance effective in preventing mesothelioma.
本発明のスクリーニング方法に供する被験物質としては、以下に限定されないが、例えば、有機化合物、無機化合物、タンパク質、抗体、ペプチド、アミノ酸、核酸、化合物ライブラリー、遺伝子ライブラリーの発現産物、細胞抽出物、細胞培養上清、発酵微生物産生物、海洋生物抽出物、植物抽出物、原核細胞抽出物（細菌抽出物等）、真核単細胞抽出物（真菌抽出物、真核藻類抽出物等）若しくは動物細胞抽出物等を挙げることができる。これらは精製物であっても、また植物、動物又は微生物等の抽出物等のように粗精製物であってもよい。本発明のスクリーニング方法に供する被験物質は、抗癌剤、細胞増殖抑制剤、免疫チェックポイント阻害剤等の薬剤であってもよい。また被験物質の製造方法は特に制限されず、天然物から単離されたものであっても、化学的又は生化学的に合成されたものであっても、また遺伝子工学的に調製されたものであってもよい。 The test substances used in the screening method of the present invention include, but are not limited to, organic compounds, inorganic compounds, proteins, antibodies, peptides, amino acids, nucleic acids, compound libraries, expression products of gene libraries, cell extracts, cell culture supernatants, fermentation microbial products, marine organism extracts, plant extracts, prokaryotic cell extracts (bacterial extracts, etc.), eukaryotic single cell extracts (fungal extracts, eukaryotic algae extracts, etc.), and animal cell extracts. These may be purified products or crude products such as extracts of plants, animals, or microorganisms. The test substances used in the screening method of the present invention may be drugs such as anticancer drugs, cell growth inhibitors, and immune checkpoint inhibitors. The method for producing the test substances is not particularly limited, and the test substances may be isolated from natural products, chemically or biochemically synthesized, or genetically engineered.
以下、実施例を用いて本発明をさらに具体的に説明する。但し、本発明の技術的範囲はこれら実施例に限定されるものではない。 The present invention will be described in more detail below using examples. However, the technical scope of the present invention is not limited to these examples.
［実施例１］イヌ中皮腫細胞株の作製
外科的処置による心膜切除術を受け、病理組織検査にて心膜中皮腫と診断された3匹のイヌ（症例1～3）から、超音波ガイド下で胸腔穿刺により無菌的に胸水サンプルを採取した。採取した胸水サンプルを200gで3分間、遠心分離し、沈渣細胞をリン酸緩衝食塩水（PBS）で洗浄した。当該細胞を1×106細胞/mlの細胞数となるように液体培地（10重量%の割合で非働化ウシ胎児血清（FBS）を添加し、さらに500mg/mlの濃度でPrimocin（登録商標）（ナカライテスク株式会社）を添加したRPMI-1604培地）に浮遊させ、75cm2培養フラスコに10mlずつ播種し、CO2インキュベーター内で37℃で培養した。2日ごとに培地交換を行った。
Example 1: Preparation of a canine mesothelioma cell line Three dogs (cases 1-3) were surgically treated with pericardiectomy and diagnosed with pericardial mesothelioma by histopathological examination. Pleural fluid samples were collected aseptically by thoracocentesis under ultrasound guidance. The collected pleural fluid samples were centrifuged at 200 g for 3 minutes, and the sediment cells were washed with phosphate-buffered saline (PBS). The cells were suspended in a liquid medium (RPMI-1604 medium containing 10% by weight of inactivated fetal bovine serum (FBS) and 500 mg/ml of Primocin (registered trademark) (Nacalai Tesque, Inc.)) to a cell count of 1 x 106 cells/ml, and seeded in 10 ml portions in 75 cm2 culture flasks and cultured at 37°C in a CO2 incubator. The medium was replaced every two days.
培養フラスコ内の細胞密度が70～80%程度になった時点で継代を行った。アスピレーターを用いて培養フラスコ内の培地をすべて除去した後、PBS 4mlで細胞表面を洗浄した。アスピレーターを用いてPBSを除去し、0.25重量%トリプシン/エチレンジアミン四酢酸（EDTA）溶液 1 mlを培養フラスコに添加し、CO2インキュベーター内で10分間静置後、FBSを含むRPMI-1640培地 3 mlを添加しトリプシンを不活化させた。これにより得られた細胞浮遊液を15 mlコニカルチューブに移した後、200gで3分間、遠心分離した。得られた沈渣細胞の細胞数を血球計算盤で算出し、液体培地を用いて1×106 細胞/mlとなるように調整し、75cm2フラスコに10 ml播種し、培養フラスコ内の細胞密度が70～80%程度になるまで（4日間）、37℃で培養し、上記と同様の工程により継代を行った。同様にして、継代を繰り返した。 Subculture was performed when the cell density in the culture flask reached about 70-80%. After removing all the medium from the culture flask using an aspirator, the cell surface was washed with 4 ml of PBS. The PBS was removed using an aspirator, and 1 ml of 0.25% by weight trypsin/ethylenediaminetetraacetic acid (EDTA) solution was added to the culture flask. After standing in a CO2 incubator for 10 minutes, 3 ml of RPMI-1640 medium containing FBS was added to inactivate the trypsin. The cell suspension thus obtained was transferred to a 15 ml conical tube and centrifuged at 200 g for 3 minutes. The cell number of the obtained sediment cells was calculated using a hemocytometer, adjusted to 1 x 106 cells/ml using liquid medium, seeded in a 75 cm2 flask with 10 ml, and cultured at 37°C until the cell density in the culture flask reached about 70-80% (4 days), and subcultured by the same process as above. Subculture was repeated in the same manner.
その結果、イヌ初代培養中皮腫細胞として得られた、症例1由来の培養細胞（MC18001株）、症例2由来の培養細胞（MC19001株）及び症例3由来の培養細胞（MC19009株）はそれぞれ、上皮様の細胞形態を示し（図１Ａ～Ｃ）、対数的な増殖を示した。MC18001株、MC19001株及びMC19009株すべてについて、10継代以上の継代を達成することができた。MC19009株は、独立行政法人 製品評価技術基盤機構 特許微生物寄託センター（NPMD）（日本国千葉県木更津市かずさ鎌足2-5-8 122号室）に、受託番号NITE P-03216で寄託された。 As a result, the cultured cells from case 1 (MC18001 strain), case 2 (MC19001 strain), and case 3 (MC19009 strain) obtained as primary cultured canine mesothelioma cells each showed epithelial-like cell morphology (Fig. 1A-C) and logarithmic growth. All of the MC18001, MC19001, and MC19009 strains were successfully passaged for 10 or more passages. The MC19009 strain was deposited at the National Institute of Technology and Evaluation, Patent Microorganisms Depositary (NPMD) (Room 122, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan) under the accession number NITE P-03216 .
［実施例２］免疫蛍光染色による中皮腫細胞マーカー発現の確認
抗AE1/AE3抗体（Novus）、抗CK5抗体（GeneTex）、及び抗WT-1抗体（Dako）の3つの抗体を用い、イヌ中皮腫細胞における中皮腫細胞マーカーAE1/AE3、CK5及びWT-1の発現を蛍光抗体染色法によって以下のように確認した。
[Example 2] Confirmation of mesothelioma cell marker expression by immunofluorescence staining Using three antibodies, anti-AE1/AE3 antibody (Novus), anti-CK5 antibody (GeneTex), and anti-WT-1 antibody (Dako), the expression of mesothelioma cell markers AE1/AE3, CK5, and WT-1 in canine mesothelioma cells was confirmed by fluorescent antibody staining as follows.
8穴マイクロスライドガラスに1000個/ウェルとなるように、イヌ中皮腫細胞MC19009株を播種し、インキュベーター内で24時間培養した。アスピレーターで8穴マイクロスライドガラスの培地を除去し、PBSで5分間ウェルを洗浄した後、ウェルに4重量%パラホルムアルデヒド（PFA）を添加し、15分間細胞を固定し、PBSで3回、1回につき5分間、ウェルを洗浄した。次いで、ウェルに0.2重量%トリトンX（triton-X）を添加し、10分間透過処理をした後、再度PBSで3回、1回につき5分間、ウェルを洗浄した。その後、10重量%ウシ血清アルブミン（BSA）をウェルに添加し、30分間ブロッキングした後、PBSで3回、1回につき5分間、ウェルを洗浄した。次いで、PBSで希釈した抗AE1/AE3抗体（1:100）、抗CK5抗体（1:400）又は抗WT-1抗体（1:400）をウェルに添加し、室温で3時間静置した。その後、PBSで3回、1回につき5分間、ウェルを洗浄し、PBSで希釈した蛍光色素標識2次抗体（1:500）（Thermo Fisher Scientific）及びヘキスト（登録商標）（Thermo Fisher Scientific）（1:1000）をウェルに添加し、室温で1時間静置した。最後にPBSで3回、1回につき5分間、ウェルを洗浄した後、カバーガラスで封入し、蛍光顕微鏡で観察した。 MC19009 canine mesothelioma cells were seeded on an 8-well microslide glass at 1000 cells/well and cultured in an incubator for 24 hours. The medium was removed from the 8-well microslide glass using an aspirator, and the wells were washed with PBS for 5 minutes. After that, 4% by weight paraformaldehyde (PFA) was added to the wells to fix the cells for 15 minutes, and the wells were washed three times with PBS for 5 minutes each time. Next, 0.2% by weight triton-X was added to the wells for 10 minutes of permeabilization, and the wells were washed again three times with PBS for 5 minutes each time. Then, 10% by weight bovine serum albumin (BSA) was added to the wells, and the wells were blocked for 30 minutes, and then washed three times with PBS for 5 minutes each time. Next, anti-AE1/AE3 antibody (1:100), anti-CK5 antibody (1:400), or anti-WT-1 antibody (1:400) diluted with PBS was added to the wells and left to stand at room temperature for 3 hours. After that, the wells were washed three times with PBS for 5 minutes each time, and fluorescent dye-labeled secondary antibody (1:500) (Thermo Fisher Scientific) and Hoechst (registered trademark) (Thermo Fisher Scientific) (1:1000) diluted with PBS were added to the wells and left to stand at room temperature for 1 hour. Finally, the wells were washed three times with PBS for 5 minutes each time, and then the wells were sealed with a cover glass and observed under a fluorescent microscope.
イヌ中皮腫細胞MC19009株は、中皮腫細胞マーカーであるAE1/AE3、CK5及びWT-1の3つすべての発現を示した（図２Ａ～Ｃ）。同様の方法で、イヌ中皮腫細胞MC18001株及びMC19001株もAE1/AE3、CK5及びWT-1の3つの中皮腫マーカーすべての発現を示した。 The canine mesothelioma cell line MC19009 showed expression of all three mesothelioma cell markers, AE1/AE3, CK5, and WT-1 (Figures 2A-C). Using a similar method, the canine mesothelioma cell lines MC18001 and MC19001 also showed expression of all three mesothelioma markers, AE1/AE3, CK5, and WT-1.
［実施例３］イヌ中皮腫培養細胞を移植した免疫不全マウスの作製及び組織学的診断
イヌ中皮腫細胞を重症複合免疫不全（SCID）マウス（三共ラボ）の腹腔内又は皮下に移植し、腫瘍形成能の検証及び組織標本による中皮腫の診断を行った。
[Example 3] Creation of immunodeficient mice transplanted with cultured canine mesothelioma cells and histological diagnosis Canine mesothelioma cells were transplanted intraperitoneally or subcutaneously into severe combined immunodeficient (SCID) mice (Sankyo Lab), and tumor formation ability was verified and mesothelioma was diagnosed using tissue specimens.
PBSで希釈したイヌ中皮腫細胞MC18001株の浮遊液50μl（5×105細胞）をSCIDマウスの皮下に移植した。また、PBSで希釈したイヌ中皮腫細胞MC18001株の浮遊液200μl（5×105細胞）を別個体のSCIDマウスの腹腔内に移植した。 50 μl (5× 105 cells) of a suspension of canine mesothelioma cell line MC18001 diluted in PBS was subcutaneously transplanted into a SCID mouse, and 200 μl (5× 105 cells) of a suspension of canine mesothelioma cell line MC18001 diluted in PBS was intraperitoneally transplanted into another SCID mouse.
イヌ中皮腫細胞MC18001株を皮下に移植したSCIDマウスにおいて、移植後52日目に解剖に供したところ、12mm大の腫瘤が移植部位に観察された（図３Ａ）。また、同系統（MC18001株）のイヌ中皮腫培養細胞を腹腔内に移植したSCIDマウスにおいても、移植後52日目に解剖に供したところ、腸管膜の漿膜表面に白色の結節状病変が多数観察された（図３Ｂ）。 When SCID mice were subcutaneously transplanted with MC18001 canine mesothelioma cells and dissected 52 days after transplantation, a 12 mm tumor was observed at the site of transplantation (Fig. 3A). In addition, when SCID mice were intraperitoneally transplanted with cultured canine mesothelioma cells of the same strain (MC18001 strain) and dissected 52 days after transplantation, numerous white nodular lesions were observed on the serosal surface of the intestinal membrane (Fig. 3B).
解剖に供したSCIDマウスから皮下の腫瘤及び腹腔内臓器（腸管）を採材し、ホルマリン固定した後、ヘマトキシリン・エオジン（HE）染色を行い、組織標本スライドを作製した。得られた皮下腫瘤、腹腔内結節の組織標本を顕微鏡で観察すると、類円形でクロマチン凝集し核小体明瞭な大小不同の核を有し、好酸性に富む細胞質を持つ細胞が腫瘤表面方向へ乳頭状に増殖する像が見られた。また腫瘤及び結節中心部では比較的細胞密度が低く、紡錘形の大小不同の核を持ち、細胞質の広い肉腫様の細胞の増殖が見られた(図４)。また腫瘍細胞の一部は脂肪組織への浸潤を伴っていた。これらの所見から、移植されたイヌ中皮腫細胞MC18001株から形成された皮下腫瘤及び結節状病変は病理組織学的に二相型（上皮型と肉腫型の混合型）の中皮腫と考えられた。 Subcutaneous tumors and abdominal organs (intestines) were collected from the dissected SCID mice, fixed in formalin, stained with hematoxylin and eosin (HE), and prepared as tissue slides. Microscopic observation of the obtained tissue specimens of the subcutaneous tumors and abdominal nodules revealed images of cells with round, chromatin-condensed, nuclei of various sizes with distinct nucleoli, and eosinophilic cytoplasm growing in a papillary pattern toward the surface of the tumor. In addition, the cell density was relatively low in the center of the tumor and nodule, and proliferation of sarcoma-like cells with spindle-shaped nuclei of various sizes and wide cytoplasm was observed (Figure 4). Some of the tumor cells also infiltrated into the adipose tissue. Based on these findings, the subcutaneous tumors and nodular lesions formed from the transplanted canine mesothelioma cell line MC18001 were histopathologically considered to be biphasic (mixed epithelial and sarcoma) mesothelioma.
［実施例４］薬剤単剤でのイヌ中皮腫細胞株に対する効果
中皮腫治療に用いられている抗癌剤であるドキソルビシン、ビノレルビン、カルボプラチン又はゲムシタビンの単剤を用いてイヌ中皮腫細胞株に対する薬剤感受性試験を行った。
[Example 4] Effects of single drugs on canine mesothelioma cell lines. Drug sensitivity tests were carried out on canine mesothelioma cell lines using single drugs of doxorubicin, vinorelbine, carboplatin or gemcitabine, which are anticancer drugs used in the treatment of mesothelioma.
96穴マイクロプレートの各ウェルに5000細胞/ウェルとなるように、イヌ中皮腫培養細胞（MC18001株、MC19001株又はMC19009株）の懸濁液を100μl播種し、CO2インキュベーター内で24時間培養した。その後、培地をアスピレーターで吸引し、抗癌剤（ドキソルビシン、ビノレルビン、カルボプラチン又はゲムシタビン）を添加した液体培地（RPMI1640 10% FBS）を各ウェルに加え、再びCO2インキュベーター内で72時間培養した。ドキソルビシン、ビノレルビン、カルボプラチンについては0.1、1、10又は100μMの濃度で、ゲムシタビンについては、1、10、100又は1000μMの濃度で、ウェルに添加した。イヌ中皮腫培養細胞を含む別のウェルには、抗癌剤を添加した液体培地に代えて、抗癌剤を添加していない液体培地を加え、同様の処理を行った（コントロール）。 100μl of suspension of canine mesothelioma cultured cells (MC18001, MC19001 or MC19009) was seeded in each well of a 96-well microplate at 5000 cells/well, and cultured in a CO2 incubator for 24 hours. After that, the medium was aspirated with an aspirator, and liquid medium (RPMI1640 10% FBS) containing an anticancer drug (doxorubicin, vinorelbine, carboplatin or gemcitabine) was added to each well, and cultured again in a CO2 incubator for 72 hours. Doxorubicin, vinorelbine and carboplatin were added to the wells at concentrations of 0.1, 1, 10 or 100μM, and gemcitabine was added to the wells at concentrations of 1, 10, 100 or 1000μM. To another well containing cultured canine mesothelioma cells, a liquid medium containing no anticancer drug was added instead of the liquid medium containing the anticancer drug, and the same treatment was carried out (control).
次いで、各ウェルから培地をアスピレーターで吸引除去し、液体培地（RPMI1640 10% FBS）100μl及びCell Counting Kit-8 溶液（同仁化学）10μlを各ウェルに添加し、インキュベーター内で2時間、呈色反応させた後、マイクロプレートリーダーを用いて波長450nmで吸光度を測定した。同時に、バックグラウンド測定のため、別のウェルに上記液体培地100μl及びCell Counting Kit-8 溶液10μlを加え、同様に吸光度を測定した（バックグラウンド）。抗癌剤処理を施した群（処理群）の吸光度（Asample）、抗癌剤処理を施していない群（コントロール）の吸光度（Acontrol）、バックグラウンドの吸光度（Ablank）に基づき、各薬剤濃度における各処理群の細胞生存率（%）を以下の式に従って算出した。
細胞生存率（%）＝(Asample-Ablank)/(Acontrol-Ablank)×100
Next, the medium was removed from each well with an aspirator, and 100 μl of liquid medium (RPMI1640 10% FBS) and 10 μl of Cell Counting Kit-8 solution (Dojindo Chemical) were added to each well. After the color reaction was allowed to proceed in an incubator for 2 hours, the absorbance was measured at a wavelength of 450 nm using a microplate reader. At the same time, 100 μl of the above liquid medium and 10 μl of Cell Counting Kit-8 solution were added to another well for background measurement, and the absorbance was measured in the same manner (background). Based on the absorbance of the group treated with anticancer drugs (treated group) (A sample ), the absorbance of the group not treated with anticancer drugs (control) (A control ), and the background absorbance (A blank ), the cell viability (%) of each treatment group at each drug concentration was calculated according to the following formula.
Cell viability (%) = (A sample - A blank )/(A control - A blank ) x 100
3系統のイヌ中皮腫細胞株それぞれについて、上述の薬剤感受性試験を6回行い、平均細胞生存率と標準誤差を算出した。図５にその結果を示す。ドキソルビシン処理群及びビノレルビン処理群では、抗癌剤濃度が高いほど細胞生存率が低く、用量依存的な細胞増殖抑制効果が見られた。ドキソルビシンの典型的な臨床的薬用量に対応する細胞処理濃度は1～10μM、ビノレルビンの典型的な臨床的薬用量に対応する細胞処理濃度は0.1～1μMである。ここで「臨床的薬用量に対応する細胞処理濃度」は、臨床的薬用量の薬剤をヒトに投与した場合の最高血中濃度をもとに算出した細胞処理濃度である。カルボプラチン処理群では、典型的な臨床的薬用量に対応する細胞処理濃度である100μMで高い細胞毒性が見られた。一方、ゲムシタビン処理群では、細胞毒性は確認されたが、用量依存的な細胞増殖抑制効果は見られなかった。ゲムシタビンの典型的な臨床的薬用量に対応する細胞処理濃度は100μMである。 The above-mentioned drug sensitivity test was performed six times for each of the three canine mesothelioma cell lines, and the average cell viability and standard error were calculated. The results are shown in Figure 5. In the doxorubicin-treated group and the vinorelbine-treated group, the higher the concentration of the anticancer drug, the lower the cell viability, and a dose-dependent cell proliferation inhibitory effect was observed. The cell treatment concentration corresponding to the typical clinical dose of doxorubicin is 1 to 10 μM, and the cell treatment concentration corresponding to the typical clinical dose of vinorelbine is 0.1 to 1 μM. Here, the "cell treatment concentration corresponding to the clinical dose" is the cell treatment concentration calculated based on the maximum blood concentration when the clinical dose of the drug is administered to humans. In the carboplatin-treated group, high cytotoxicity was observed at a cell treatment concentration of 100 μM, which is the typical clinical dose. On the other hand, in the gemcitabine-treated group, cytotoxicity was confirmed, but no dose-dependent cell proliferation inhibitory effect was observed. The cell treatment concentration corresponding to the typical clinical dose of gemcitabine is 100 μM.
臨床におけるヒト中皮腫に対する奏効率は、単剤の場合、カルボプラチンが高く、ドキソルビシン、ビノレルビン、ゲムシタビンはより低いことが知られている。上記の結果から、本発明のイヌ中皮腫細胞株はヒト中皮腫患者とよく似た薬剤感受性を示すことが示された。 It is known that the clinical response rate for human mesothelioma as a single agent is high for carboplatin, and lower for doxorubicin, vinorelbine, and gemcitabine. The above results demonstrate that the canine mesothelioma cell line of the present invention exhibits drug sensitivity very similar to that of human mesothelioma patients.
［実施例５］カルボプラチンとゲムシタビンの併用によるイヌ中皮腫細胞株に対する効果
ヒト中皮腫の治療において、ゲムシタビンをカルボプラチンやシスプラチンなどの白金製剤と併用することにより、奏効率が向上することが知られている。そこで、カルボプラチンとゲムシタビンの併用によるイヌ中皮腫細胞株に対する効果を検証する薬剤感受性試験を行った。
[Example 5] Effect of combined use of carboplatin and gemcitabine on canine mesothelioma cell lines In the treatment of human mesothelioma, it is known that the response rate is improved by combining gemcitabine with platinum preparations such as carboplatin and cisplatin. Therefore, a drug sensitivity test was performed to verify the effect of combined use of carboplatin and gemcitabine on canine mesothelioma cell lines.
96穴マイクロプレートの各ウェルに5000細胞/ウェルとなるように、3系統のイヌ中皮腫培養細胞（MC18001株、MC19001株又はMC19009株）の懸濁液を100μlずつ播種し、CO2インキュベーター内で24時間培養した。その後、培地をアスピレーターで吸引し、ゲムシタビンを添加した液体培地100μlを各ウェルに加え、CO2インキュベーター内で4時間培養した。次いで、カルボプラチンを添加してさらに68時間CO2インキュベーター内で培養した。カルボプラチンは100μMの濃度で、ゲムシタビンは0、1、10、100又は1000μMの濃度で用いた。イヌ中皮腫培養細胞を含む別のウェルには、抗癌剤を添加した液体培地に代えて、抗癌剤を添加していない液体培地を加え、同様の処理を行った（コントロール）。 100μl of suspension of three strains of canine mesothelioma cultured cells (MC18001, MC19001 or MC19009) was seeded in each well of a 96-well microplate at 5000 cells/well, and cultured in a CO2 incubator for 24 hours. After that, the medium was aspirated with an aspirator, and 100μl of liquid medium containing gemcitabine was added to each well, and cultured in a CO2 incubator for 4 hours. Carboplatin was then added and cultured in a CO2 incubator for another 68 hours. Carboplatin was used at a concentration of 100μM, and gemcitabine was used at concentrations of 0, 1, 10, 100 or 1000μM. In another well containing canine mesothelioma cultured cells, liquid medium without anticancer drug was added instead of the liquid medium containing anticancer drug, and the same treatment was performed (control).
次いで、各ウェルから培地をアスピレーターで吸引除去し、液体培地（RPMI1640 10% FBS）100μl及びCell Counting Kit-8 溶液（同仁化学）10μlを各ウェルに添加し、インキュベーター内で2時間、呈色反応させた後、マイクロプレートリーダーを用いて波長450nmで吸光度を測定した。同時に、バックグラウンド測定のため、別のウェルに上記液体培地100μl及びCell Counting Kit-8 溶液10μlを加え、同様に吸光度を測定した（バックグラウンド）。測定した吸光度に基づき、実施例４と同様に、コントロールに対する処理群の細胞生存率（%）を算出した。 The medium was then removed from each well using an aspirator, and 100 μl of liquid medium (RPMI1640 10% FBS) and 10 μl of Cell Counting Kit-8 solution (Dojindo Chemicals) were added to each well. After allowing the color reaction to occur in an incubator for 2 hours, the absorbance was measured at a wavelength of 450 nm using a microplate reader. At the same time, 100 μl of the above liquid medium and 10 μl of Cell Counting Kit-8 solution were added to another well for background measurement, and the absorbance was measured in the same manner (background). Based on the measured absorbance, the cell survival rate (%) of the treatment group relative to the control was calculated in the same manner as in Example 4.
3系統のイヌ中皮腫培養細胞それぞれについて、上述の薬剤感受性試験を6回行い、平均細胞生存率と標準誤差を算出した（図６）。MC18001株では、カルボプラチン単剤での細胞増殖抑制効果が大きいため、カルボプラチンとゲムシタビンの併用による相加効果はあまり見られなかった。一方、MC19001株又はMC19009株では、カルボプラチン100μM単剤を用いた場合には細胞生存率が約26～27%、カルボプラチン100μMとゲムシタビン1μM以上を併用した場合には細胞生存率が約4～13%であり、カルボプラチン100μMとゲムシタビン1μM以上を組み合わせることによりカルボプラチン単剤よりも高い細胞増殖抑制効果が得られた。 The above-mentioned drug sensitivity test was performed six times for each of the three lines of cultured canine mesothelioma cells, and the average cell viability and standard error were calculated (Figure 6). In the MC18001 line, carboplatin alone had a large cell proliferation inhibitory effect, so the additive effect of the combination of carboplatin and gemcitabine was not observed. On the other hand, in the MC19001 or MC19009 line, the cell viability was approximately 26-27% when carboplatin was used alone at 100 μM, and approximately 4-13% when carboplatin was used in combination with 100 μM carboplatin and 1 μM or more gemcitabine, indicating that the combination of 100 μM carboplatin and 1 μM or more gemcitabine provided a higher cell proliferation inhibitory effect than carboplatin alone.
本発明のイヌ中皮腫細胞株は、中皮腫の治療又は予防のための薬剤の開発や試験に用いることができる。
The canine mesothelioma cell lines of the present invention can be used in the development and testing of drugs for the treatment or prevention of mesothelioma.
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