JP2020510414A - 免疫細胞および薬物ホーミング、移動ならびに腫瘍細胞毒性についての高スループット3dアッセイ - Google Patents
免疫細胞および薬物ホーミング、移動ならびに腫瘍細胞毒性についての高スループット3dアッセイ Download PDFInfo
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Abstract
Description
本明細書で使用する場合、文脈でそうでないことが明示されていない限り、単数形「a」、「an」、および「the」は複数の指示対象を包含する。したがって、例えば「〜の構造の底面」に対する言及は、文脈でそうでないことが明示されていない限り、2つ以上のそのような「構造の底面」を有する例を包含する。
セノン;オリゴヌクレオチド;オナプリストン;オンダンセトロン;オンダンセトロン;オラシン(oracin);口腔サイトカインインデューサー;オルマプラチン;オサテロン;オキサリプラチン;オキサウノマイシン;パクリタキセル;パクリタキセルアナログ;パクリタキセル誘導体;パラウアミン;パルミトイルリゾキシン;パミドロン酸;パナキシトリオール;パノミフェン;パラバクチン;パゼリプチン(pazelliptine);ペガスパルカーゼ;ペルデシン;ペントサンポリスルフェートナトリウム;ペントスタチン;ペントロゾール;パーフルブロン;ペルフォスファミド;ペリリルアルコール;フェナジノマイシン;フェニルアセテート;ホスファターゼ阻害剤;ピシバニール;塩酸ピロカルピン;ピラルビシン;ピリトレキシム;プラセチンA;プラセチンB;プラスミノーゲンアクチベータ阻害剤;白金錯体;白金化合物;白金−トリアミン複合体;ポルフィマーナトリウム;ポルフィロマイシン;プレドニゾン;プロピルビス−アクリドン;プロスタグランジンJ2;プロテアソーム阻害剤;タンパク質A系免疫モジュレータ;タンパク質キナーゼC阻害剤;タンパク質キナーゼC阻害剤、微細藻類;タンパク質チロシンホスファターゼ阻害剤;プリンヌクレオシドホスホリラーゼ阻害剤;プルプリン;ピラゾロアクリジン;ピリドキシル化ヘモグロビンポリオキシエチレンコンジュゲート;rafアンタゴニスト;ラルチトレキセド;ラモセトロン;rasファルネシルタンパク質トランスフェラーゼ阻害剤;ras阻害剤;ras−GAP阻害剤;脱メチル化レテリプチン;レニウムRe186エチドロネート;リゾキシン;リボザイム;RIIレチナミド;ログレチミド;ロヒツキン(rohitukine);ロムルチド;ロキニメックス;ルビギノンB1;ルボキシル;サフィンゴール;サイントピン;SarCNU;サルコフィトール(sarcophytol)A;サルグラモスチム;Sdi1模倣物;セムスチン;老化由来(senescence derived)阻害剤1;センスオリゴヌクレオチド;シグナル変換阻害剤;シグナル変換モジュレータ;単鎖抗原結合タンパク質;シゾフィラン;ソブゾキサン;ボロカプチン酸ナトリウム(sodium borocaptate);フェニル酢酸ナトリウム;ソルベロール;ソマトメジン結合タンパク質;ソネルミン;スパルフォス酸;スピカマイシンD;スピロムスチン;スプレノペンチン;スポンジスタチン1;スクワラミン;幹細胞阻害剤;幹細胞分裂阻害剤;スチピアミド;ストロメリシン阻害剤;スルフィノシン;超活性血管作動性腸ペプチドアンタゴニスト;スラジスタ;スラミン;スワインソニン;合成グリコサミングリカン;タリムスチン;タモキシフェンメチオジド;タウロムスチン;タザロテン;テコガランナトリウム;テガフール;テルラピリリウム;テロメラーゼ阻害剤;テモポルフィン;テモゾロマイド;テニポシド;テトラクロロデカオキシド;テトラゾミン(tetrazomine);タリブラスチン(thaliblastine);チオコラリン;トロンボポエチン;トロンボポエチン模倣物;チマルファシン;チモポエチン受容体アゴニスト;チモトリナン;甲状腺刺激ホルモン;エチルエチオプルプリンスズ;チラパザミン;チタノセンジクロリド;トプセチン;トレミフェン;分化全能性幹細胞因子;翻訳阻害剤;トレチノイン;トリアセチルウリジン;トリシリビン;トリメトレキサート;トリプトレリン;トロピセトロン;ツロステリド;チロシンキナーゼ阻害剤;チルホスチン(tyrphostins);UBC阻害剤;ウベニメクス;尿生殖洞由来の成長阻害因子;ウロキナーゼ受容体アンタゴニスト;バプレオチド;バリオリン(variolin)B;ベクターシステム、赤血球遺伝子療法;ベラレソール(velaresol);べラミン(veramine);ベルディンス(verdins);ベルテポルフィン;ビノレルビン;ビンキサルチン;ビタキシン(vitaxin);ボロゾール;ザノテロン;ゼニプラチン;ジラスコルブ;およびジノスタチンスチマラマーが挙げられる。
細胞培養
A549/GFP細胞(Cell Biolabs,Inc.カタログ番号AKR−209)は、10%ウシ胎仔血清(FBS)(Corningカタログ番号35−010−CV)を追加したIscoveの修飾DMEM(IMDM)(Corningカタログ番号10−016−CM)中にウェルあたり2,000個の細胞で96ウェル楕円体マイクロプレート(Corningカタログ番号4515)に播種した。翌日、培地を、30ng/mLのヒトSDF−1α(SDF−1α)/CXCL12(Shenandoah Biotechnology Inc(商標)カタログ番号100−20)またはビヒクル対照を含む200μLのIMDM10%FBSと置換した。NK92−MI細胞を80μM細胞Tracker(商標)Blue CMHC Dye(Molecular Probes(商標)カタログ番号C2110)で1時間染色し、同時に血清を含まないIMDM中2μg/mLのプロスタグランジンE2(PGE2)(Tocrisカタログ番号2296)またはビヒクル対照で1時間処理した。HTS Transwell−96Well Permeable Supportを96ウェル楕円体プレートに入れた(概略図を図1Aおよび図4Cに示す)。NK−92MI細胞を次に無血清IMDM中に再懸濁させ、インサートのアピカルチャンバーに50,000細胞/インサートで播種した。24時間後、インサートを取り出し、楕円体マイクロプレートをフローサイトメトリーのために処理した。簡単に言うと、培地を除去し、150μLのTrypLE(商標)Select Enzyme(10X)(Gibco(商標)カタログ番号A1217701)で置換し、楕円体を最小のピペッティングで単細胞に分解することができるようになるまで37℃でインキュベートした。細胞を次にMiltenyi Biotec MacsQuant(登録商標)を利用してフローサイトメトリーにより分析した。
A549/GFP細胞のNK−92MI移動および殺腫瘍活性は、図4および図5で示すように、Corning 96ウェル楕円体マイクロプレートとともに市販のCorning HTS 96Transwellインサートを利用して評価した。悪性腫瘍構造中のある特定の免疫細胞の存在は、患者生存率の増加と相関することが示されている。免疫細胞毒性を研究するためにより一般的に用いられる2Dインビトロモデルとは異なり、腫瘍楕円体への免疫細胞浸透を観察するために3Dモデルを使用することができる。図6は、図5で示す実施形態にしたがって、3D中に培養されるA549/GFP腫瘍細胞へのNK細胞の移動を示すグラフである。データは2つの独立した研究の平均で示し、ボンフェローニポストテストを伴う一元配置分散分析でN=24。***=p<0.0001そして**=p<0.001。図6は、免疫細胞移動は、どのようにしてSDF−1αなどのケモカインの添加によって増強することができ、またPGE2などの阻害剤の添加によって抑制できるかを示す。NK移動は、化学誘引物質SDF−1αが楕円体マイクロプレート中に腫瘍楕円体とともに存在する場合、有意に増大した。図7は、図4A、図4B、および図5で示す実施形態にしたがって3Dに培養されたA549/GFP腫瘍細胞のNK誘発性細胞毒性を示すグラフである。反対に、免疫回避の形態としてがん細胞によって多くの場合分泌される免疫細胞機能の公知阻害剤であるPGE2にNK細胞が曝露された場合、移動が有意に減少した。殺腫瘍活性は、移動データとよく相関し、PGE2を含まないSDF−1αに曝露されたNK細胞で観察されたA549/GFP腫瘍細胞の生存率は最低である(図7)。
血液脳関門モデル
MDCKII/MDR1細胞をPiet Borst博士(Netherlands Cancer Institute、オランダ国アムステルダム)から入手し、10%ウシ胎仔血清(FBS)(Corningカタログ番号35−010−CV)を追加した100μLのDulbecco’s Modification of Eagle’s Medium(DMEM)(Corningカタログ番号10−013−CM)中で1cm2あたり100,000個の細胞でHTS96−ウェルTranswell(Corningカタログ番号3391または3977)中に播種した。アッセイの24時間前に培地を交換してそれらを5日間培養した。単層完全性をルシファーイエロー透過性(図10A)(シグマカタログ番号L0144)およびローダミン123輸送(図10B)(シグマカタログ番号R8004)により評価した。製造業者のプロトコル(データは不掲載)どおりに密着結合タンパク質ZO1(Thermo Fisherカタログ番号339188)およびオクルディン(Thermo Fisherカタログ番号331588)の存在を確認するために、MDCKII/MDR1単層の免疫染色を実施した。
LN229細胞(ATCC(登録商標)カタログ番号CRL−2611(商標))を、10%FBSを含むDMEM中でルーチン的に培養した。細胞をAccutase(登録商標)細胞分離溶液(Corningカタログ番号25−058−CI)で収集し、96ウェル楕円体マイクロプレートにウェルあたり1,000細胞でアッセイ前24時間播種した(図8B)。
図8Aから8Gに示すように、血液脳関門/神経膠腫球モデル試験を、血液脳関門をシミュレートするように構成された多孔質膜の使用を含むモデルにおいて治療薬の能動的移動および細胞毒性を検出するための本開示の方法の一実施形態で実施した。まず、図8Aに示すように、BBBをシミュレートするために使用するMDCKII/MDR1細胞を細胞培養インサート304の多孔質膜302上に播種した。LN229腫瘍細胞をCorning96ウェル楕円体プレート中で成長させた(図8Bに示すとおり)。楕円体に発生したLN229腫瘍細胞(図8Bに示すとおり)および細胞培養インサートの多孔質膜上のBBBをシミュレートするMDCKII/MDR1内皮細胞のコンフルエントな単層(図8Aに示すとおり)が形成されたら、細胞培養インサートをウェルに入れた。図8Cを参照して、薬物、シスプラチンまたはピペロングミンなどの薬物をインサートのキャビティに2時間添加した。薬物インキュベーション後、細胞培養インサートを取り出し、単層完全性について試験した(データは不掲載)。楕円体をさらに2日間培養し(図8Dおよび図8Eに示すとおり)、次いで、例えばCellTiter−Glo(登録商標)3Dなどの染色によって、腫瘍細胞溶解について分析した(図8Fに示すとおり。処理後の楕円体生理機能(図8Gに示すとおり)も試験した。図9は、化合物シスプラチンおよびピペロングミンとともに直接培養した48時間後のLN229楕円体の用量依存性細胞毒性を示すグラフである。濃度あたりN=12ウェルで2回の独立した研究を行う。図11は血液脳関門サロゲートの有無でのシスプラチンまたはピペロングミンのLN229 細胞毒性を示すグラフである。BBBの有無でのTranswellによる2時間の薬物曝露後48時間のLN229楕円体の生存百分率を示す。薬物なしの対照を100%生存率に標準化することによって生存率を評価した。データは3つの独立した研究の平均として示し、ボンフェローニポストテストを伴う一元配置分散分析でN=30。***=p<0.0001。図12Aおよび図12Bは、BBB有(図12B)およびBBB無(図12A)で見られるヒットを示すTocrisライブラリからの代表的なスクリーンを示すグラフである。上の点線は平均緩衝液対照であり、下の点線は緩衝液応答より低い3シグマを表す。図13Aおよび13Bは、BBBの有無で見られるヒットのコンピレーションを示す図12Aおよび図12Bのスクリーンの概要のグラフである。ヒットは、それらが3つの独立したスクリーンのうち少なくとも2つにおいて、緩衝液応答よりも低い3シグマであるか否かを考慮した。横縞のハッチングを施したボックス(緩衝液単独以外に)はBBBなしでのみ見られるヒットである。斜めのハッチングを施したボックスは、BBBの存在下および非存在下で見られるヒットであった。したがって、本明細書中で提示するデータは、Corning楕円体マイクロプレートとHTS Transwell96−ウェル透過性支持体との組み合わせを含み得る本開示の方法によって、結果として得られる神経膠腫球(または他の3D腫瘍楕円体)細胞毒性も調査しながら、BBBを通過することができる化合物と通過できない化合物とを区別することができる新規3Dモデルが可能になることを示す。
治療薬の能動的移動および細胞毒性を検出するためのアッセイ法であって:
a)腫瘍細胞を3D楕円体コンフォメーションに成長させるような構造のチャンバーを含む細胞培養製品中で腫瘍細胞を培養して楕円体を形成し、
b)多孔質膜を含むインサートを前記細胞培養製品に入れ、治療薬を当該インサート中に導入し、
c)前記インサートから前記細胞培養製品チャンバーへの前記治療薬の能動的移動を検出し、そして
d)腫瘍細胞応答を検出すること
を含む、アッセイ法。
前記チャンバーが:
側壁、
上部開口部、および
少なくとも1つの陥凹面を含む液体不透過性底部であって、当該底部の少なくとも一部が当該少なくとも1つの陥凹面中または上に低接着性または非接着性材料を含む、液体不透過性底部
を含む、実施形態1に記載のアッセイ法。
少なくとも1つの陥凹面を含む前記液体不透過性底部がガス透過性である、実施形態1または2に記載のアッセイ法。
前記側壁が不透明である、実施形態2に記載のアッセイ法。
前記底部の少なくとも一部が透明である、実施形態2から4のいずれか1つに記載のアッセイ法。
前記細胞培養製品が、1から約2000の前記チャンバーを含み、各チャンバーは互いに物理的に離間されている、実施形態2から5のいずれか1つに記載のアッセイ法。
前記少なくとも1つの陥凹面が同じチャンバー内で複数の陥凹面を含む、実施形態2から6のいずれか1つに記載のアッセイ法。
前記少なくとも1つの陥凹面が半球状面、前記側壁から前記底面へと30から約60度のテーパーを有する円錐面、またはそれらの組み合わせを含む、実施形態2から7のいずれか1つに記載のアッセイ法。
前記側壁面が、垂直円筒、前記チャンバー頂部から底面へと直径が減少する垂直円錐の一部、前記少なくとも1つの陥凹底面に対して、円錐遷移部を有する垂直な方形シャフト、またはそれらの組み合わせを含む、実施形態2に記載のアッセイ法。
前記製品が吸引のためにピペットチップを受容するためのチャンバー付属物をさらに含み、当該チャンバー付属物は前記チャンバーに隣接し、前記チャンバーと流体連通した表面を含み、当該チャンバー付属物は前記底面より上方に離間した第二底部を有し、当該第二底部はピペットから分配された流体を当該底面からそれさせる、実施形態2に記載のアッセイ法。
前記インサートがインサートプレートを含む、実施形態1から10のいずれか1つに記載のアッセイ法。
前記多孔質膜の少なくとも一部が血液脳関門をシミュレートするように構成される、実施形態1から11のいずれか1つに記載のアッセイ法。
前記多孔質膜の少なくとも一部が微小血管内皮細胞の本質的にコンフルエントな単層を含む、実施形態12に記載のアッセイ法。
前記治療薬の能動的移動および腫瘍細胞応答の両方がフローサイトメトリーによって検出される、実施形態1から13のいずれか1つに記載のアッセイ法。
腫瘍細胞応答の検出が、前記治療薬の前記腫瘍細胞楕円体への浸透を検出することを含む、実施形態1から14のいずれか1つに記載のアッセイ法。
腫瘍細胞応答の検出が、腫瘍細胞溶解を測定することを含む、実施形態1から15のいずれか一項に記載のアッセイ法。
前記治療薬が薬物または細胞治療薬を含む、実施形態1から16のいずれか1つに記載のアッセイ法。
前記治療薬が白血球またはリンパ球を含む、実施形態17に記載のアッセイ法。
前記治療薬が薬物である、実施形態10から18のいずれか一項に記載のアッセイ法。
11 楕円プレート
25 3D腫瘍細胞楕円体
30、310 治療薬
101 ウェル
105 中心軸
106 底面
110 空間
112 マイクロキャビティ
113 側壁
115 マイクロウェル
116 底面、最下点
118、418 上部開口部
119、419 底部
120 遷移ゾーン
121、421 側壁
130 フレーム、保護層
300、306、525 細胞
302 多孔質膜
304、400 インサート
312、キャビティ、インサートチェンバー
315 アッセイチャンバー
401 インサートプレート
420 キャビティ
500 培地
w 幅
d 深さ
Claims (19)
- 治療薬の能動的移動および細胞毒性を検出するためのアッセイ法であって:
a)腫瘍細胞を3D楕円体コンフォメーションに成長させるような構造のチャンバーを含む細胞培養製品中で腫瘍細胞を培養して楕円体を形成し、
b)多孔質膜を含むインサートを前記細胞培養製品に入れ、治療薬を該インサート中に導入し、
c)前記インサートから前記細胞培養製品チャンバーへの前記治療薬の能動的移動を検出し、そして
d)腫瘍細胞応答を検出すること
を含む、アッセイ法。 - 前記チャンバーが、
側壁、
上部開口部、および
少なくとも1つの陥凹面を含む液体不透過性底部であって、該底部の少なくとも一部が該少なくとも1つの陥凹面中または上に低接着性または非接着性材料を含む、液体不透過性底部
を含む、請求項1に記載のアッセイ法。 - 少なくとも1つの陥凹面を含む前記液体不透過性底部がガス透過性である、請求項1または2に記載のアッセイ法。
- 前記側壁が不透明である、請求項2に記載のアッセイ法。
- 前記底部の少なくとも一部が透明である、請求項2から4のいずれか一項に記載のアッセイ法。
- 前記細胞培養製品が、1から2000の前記チャンバーを含み、各チャンバーは互いに物理的に離間されている、請求項2から5のいずれか一項に記載のアッセイ法。
- 前記少なくとも1つの陥凹面が前記同じチャンバー内で複数の陥凹面を含む、請求項2から6のいずれか一項に記載のアッセイ法。
- 前記少なくとも1つの陥凹面が、半球状面と、前記側壁から前記底面へと30から60度のテーパーを有する円錐面、またはそれらの組み合わせを含む、請求項2から7のいずれか一項に記載のアッセイ法。
- 前記側壁面が垂直円筒と、前記チャンバーの頂部から底面へと直径が減少する垂直円錐の一部と、前記少なくとも1つの陥凹底面への円錐形遷移部を有する垂直方形シャフト、またはそれらの組み合わせとを含む、請求項2に記載のアッセイ法。
- 前記製品が吸引のためにピペットチップを受容するためのチャンバー付属物をさらに含み、該チャンバー付属物は、前記チャンバーに隣接し前記チャンバーと流体連通した表面を含み、該チャンバー付属物は前記底面より上方に離間した第二底部を有し、該第二底部はピペットから分配された流体を該底面からそれさせる、請求項2に記載のアッセイ法。
- 前記インサートがインサートプレートを含む、請求項1から10のいずれか一項に記載のアッセイ法。
- 前記多孔質膜の少なくとも一部が血液脳関門をシミュレートするように構成される、請求項1から11のいずれか一項に記載のアッセイ法。
- 前記多孔質膜の少なくとも一部が微小血管内皮細胞の本質的にコンフルエントな単層を含む、請求項12に記載のアッセイ法。
- 前記治療薬の能動的移動および腫瘍細胞応答の両方がフローサイトメトリーによって検出される、請求項1から13のいずれか一項に記載のアッセイ法。
- 腫瘍細胞応答の検出が、前記治療薬の前記腫瘍細胞楕円体への浸透を検出することを含む、請求項1から14のいずれか一項に記載のアッセイ法。
- 腫瘍細胞応答の検出が、腫瘍細胞溶解を測定することを含む、請求項1から15のいずれか一項に記載のアッセイ法。
- 前記治療薬が薬物または細胞治療薬を含む、請求項1から16のいずれか一項に記載のアッセイ法。
- 前記治療薬が白血球またはリンパ球を含む、請求項17に記載のアッセイ法。
- 前記治療薬が薬物である、請求項10から18のいずれか一項に記載のアッセイ法。
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