Name: preclinical assessment of the bioactivity of the anticancer coumarin ot48 by spheroids, colony formation assays, and zebrafish xenografts
Uploaded: 2018-06-26T13:00:00.000+00:00
Duration: 9 min 20 s
Description: Watch this Scientific Journal Video about Preclinical Assessment of the Bioactivity of the Anticancer Coumarin OT48 by Spheroids, Colony Formation Assays, and Zebrafish Xenografts at JoVE.com

ソウル大学での研究、腫瘍微小環境グローバル コア研究センター (画する) 助成、[許可番号 2011 0030001]; 韓国の MEST 国立研究財団 (NRF) によってサポートされてソウル国立大学研究助成、頭脳韓国 (bk 21) プログラム プラス。

1. コロニー形成アッセイ
<ol><li>20,000 セル/cm2 RPMI1640 の 15 mL の濃度で培養 A549 細胞 10 添加培養液中の細胞 %fbs (ウシ胎児血清)、37 ° C、5% CO2で CO2インキュベーターで 75 cm2フラスコで 1% の抗生物質。</li>
	<li>実験の日、診断を使用して、セルの濃度を決定します。1.5 mL 遠心チューブに 7 分間 400 × gで遠心分離によって 50,000 の細胞を収集し、再 1 x 滅菌 PBS (リン酸緩衝生理食塩水) 1.5 mL チューブの 100 μ L のセルを中断します。</li>
	<li>15 mL チューブ、multipipette の半固体セルロース ベースの中型 (MCBM) の 1.1 mL を調剤します。条件ごとに 1 つの管を準備します。</li>
	<li>1.1 mL MCBM P200 ピペットを使用してに FBS の 110 μ L を追加します。</li>
	<li>種細胞 1,000 セル/mL。原液 (100 μ L の 1x PBS で 50,000 セル) から細胞懸濁液の 2.4 μ L を収集 P2 マイクロ ピペットを使用して、10% を添加した MCBM 1.1 mL を加える FBS。</li>
	<li>セルを追加した後渦 MCBM と細胞をよく混ぜるために最高速度で垂直方向にそれらを保持している 1 分の管。</li>
	<li>試験化合物 (A1210477 と OT48 単独または組み合わせ) を追加します。コントロールとしての別々 のチューブの準備使用される溶媒のため (ここでジメチルスルホキシド (DMSO))。化合物や条件ごとに追加ボリュームの濃度に応じて溶剤コントロール テスト混合物の希釈液の溶媒によって引き起こされる副作用を評価するために使用される溶剤の最高濃度が含まれます。</li>
	<li>1分間ボルテックス チューブ。</li>
	<li>それぞれの試金のため 12 ウェルの細胞培養プレートのセンターに P1000 ピペットとの混合物の 1 つの mL を追加します。</li>
	<li>空井戸 1 ml の滅菌水または湿度を安定させるために 1x PBS で塗りつぶします。</li>
	<li>37 ° C、5% CO2でインキュベーターで 10 日間の培養皿を孵化させなさい。</li>
	<li>培養 10 日後を最終濃度 1 mg/mL に到達する各ウェル MTT (methylthiazolyldiphenyl テトラゾリウム ブロマイド) 貯蔵液 (5 mg/mL) 200 μ L を追加します。</li>
	<li>37 ° C、5% CO2でインキュベーターで 10 分間インキュベートします。実行可能なコロニーになる紫。</li>
	<li>ホワイト バック グラウンド プレート (ラス白ダイヤ トレイ) を使用してデジタル カメラを使用してイメージをキャプチャします。次の設定を使用: 方法、デジタル化。露出型、精密;露出時間、マニュアル、1 秒。感度・解像度、高解像度。Tiff 形式で画像を保存します。</li>
	<li>ImageJ のソフトウェアを使用して実行可能なコロニーを定量化します。[選択] メニュー「画像 |種類 |8 ビット」。[選択] メニュー"調整 |しきい値"。コントロールに閾値を設定、すべての条件を一定に保つ[選択] メニュー"分析 |「粒子をを分析します。データを保存し、グラフィカルな表現の統計ソフトウェアで分析します。</li>
</ol>2. スフェロイド形成アッセイ
<ol><li>15 ml の 10 %fbs および 1% 抗生物質 RPMI 1640 培の 75 cm2フラスコで 20,000 セル/cm2の濃度で培養 A549 細胞。</li>
	<li>実験の日、準備、96 ウェル U 底プレート。</li>
	<li>すでに興味 (OT48 のここ 50 μ M) および/または A1210477 の 20 μ M の化合物が含まれている井戸に 10,000 のセルをプレートします。細胞培養液を 100 μ l 添加する最終的な音量を調整します。そのまま外側境界と均一な回転楕円体を形成する細胞の数は、フォーム回転楕円体に十分に考えられます。</li>
	<li>3 日間は、37 ° C、5% CO2インキュベーターで孵化させなさい。</li>
	<li>培養 3 日後回転楕円体の 4 倍の倍率を用いた光顕微鏡の画像をキャプチャします。</li>
</ol>3. ゼブラフィッシュ異種アッセイ
注: この技術的なアプローチは、概略図 (図 1) として視覚化です。
<ol><li>
ストック試薬の調製
	<ol>
<li>30 x Danieau の液 1 L を準備: 1740 mM の NaCl、21 mM KCl、12 mM MgSO4∙7H2O、18 mM Ca (3)2、および 150 mM HEPES。7.6 pH メーターを使用するバッファーの pH を調整します。</li>
		<li>1% (50 x) tricaine ソリューションを準備: エチル 3-アミノ安息香酸メタンスルホン酸 (tricaine) 2 mL の蒸留水で 20 mg を溶解します。</li>
		<li>1% フェノールレッド PBS 溶液を準備: 追加 2 μ L の滅菌 PBS 溶液 1.5 μ 管の 198 μ L にフェノールレッド ナトリウム塩溶液。</li>
		<li>3% セルロースを準備: 100 x Danieau の液 1 mL にセルロースの 3 g を追加。</li>
	</ol>
</li>
	<li>
ゼブラフィッシュの産卵と孵化
	<ol>
<li>別の一人の女性とパーティションのタンクの 1 つの男性ゼブラフィッシュ暗闇の中 28.5 ° C で UV 殺菌し、フィルター処理された水道水でいっぱい。分離の 24 h の後男女交尾を開始するための魚をミックスします。新鮮な Danieau の溶液 5 mL を含むペトリ皿に合うタンクから受精卵を転送します。</li>
		<li>Danieau の溶液 5 mL で 3 回卵を洗います。洗濯、ピペットと新鮮な溶液 5 mL に転送卵を吸い出しなさい慎重に。28.5 ° C で 8 時間インキュベートします。<ol>
<li>Danieau のソリューションのインキュベーションの 8 h 後 0.03 %1-フェニル-2-チオ色素沈着を抑制する (PTU) を含む Danieau のソリューションに変更します。汚染を防ぐために不透明な卵 (未受精または含む未開発胚) を整理します。</li>
		</ol>
</li>
	</ol>
</li>
	<li>
胚 dechorionation
	<ol>
<li>24 h はシャープな鉗子を用いた dechorionate ゼブラフィッシュ胚受精 (hpf) を投稿します。0.03% と Danieau のソリューションで孵化胚の孵化 48 まで 28.5 ° C で PTU hpf。</li>
	</ol>
</li>
	<li>
化合物処理セル準備
	<ol>
<li>20,000 セル/cm2で 25 cm2培養フラスコ種子 A549 細胞。播種の 24 h 後追加化合物 (50 μ M でここ OT48 と 20 μ M で A1210477 および/または) 37 ° C、5% CO2でインキュベーターで 24 時間。細胞死へそれらをコミットするが、細胞の生存を維持するために、各化合物の治療時間を設定します。今回はポイント個別に設定する必要がありますは、細胞生存率、形態と分子マーカーに基づいています。</li>
		<li>治療の終わりの前に 2 h は、染色細胞蛍光細胞の追跡の 4 μ m は、37 ° C、5% CO2で CM Dil を染めます。孵化の 2 h は後の 0.05% トリプシン-EDTA、細胞を trypsinize し、射出前にフェノールレッド PBS 溶液 50 μ L の 10 の6セルを希釈します。</li>
	</ol>
</li>
	<li>
異種移植腫瘍の形成のための癌細胞のマイクロ射出成形
	<ol>
<li>彼らは寒天プレートに落ち着くまで 5 分 0.02 %tricaine 溶液ゼブラフィッシュを麻酔します。</li>
		<li>マイクロ ピペットの引き手で 1.0 mm (外径 (OD) ガラス管) の外を引っ張る (番組の設定: p: 300、熱: 260、プル: 60、ヴェル: 50、および時間: 200)。シャープなエッジを生成する注射器、マイクロキャピ ラリーをカットします。セル/フェノール赤の 20 μ L で microcapillaries をロード-PBS ソリューション。射出圧力を設定し、2 を調剤する時注射あたり nL。</li>
		<li>100-200 セル卵黄嚢を注入注入 6 に 10 nL 3 に 5 注射を介して。注入後、魚の PTU ソリューションを含む新鮮な Danieau の溶液の 5 mL の 10 から 30 分を回復することができます。PTU ソリューションを含む Danieau の溶液の 1 mL を 24 ウェルプレートでディスパッチ魚と 28.5 ° C で 72 時間インキュベート</li>
		<li>培養 72 時間後 0.02 %tricaine 溶液で魚を麻酔し、3% セルロースのドロップでスライド ガラスに固定します。620 に 750 の波長の蛍光顕微鏡による 5 x 写真を撮る nm。</li>
		<li>ImageJ ソフトウェアによって蛍光腫瘍の大きさを定量化します。[選択] メニュー"分析 |メジャー"。蛍光信号に対応する値を保存し、グラフィカルな表現の統計ソフトウェアで分析します。</li>
	</ol>
</li>
</ol>

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著者が明らかに何もありません。

MCBM で得られたコロニー形成率はセルの種類によって異なります。通常、非付着細胞のコロニー数は付着細胞へと比較してはるかに高い。A549 細胞が 10 日後 30 に 40 コロニーを形成したことがわかった。以前、コロニーの数は 200 に 2509の間別の白血病細胞で報告しています。私たちの結果は、OT48 だけが単独でコロニー数の大幅な減少を作り出さなかった。ただし、BH3 と組?...

がんは細胞の突然変異によって引き起こされ、生化学的なシグナル伝達経路が中断された結果として制御されていない細胞分裂および細胞死への抵抗をトリガーします。腫瘍は、消化器系、神経系、循環系、その後隣接組織1の生理機能を妨げます。広範な研究努力にもかかわらず癌世界2の最も一般的な命にかかわる病気のままです。精密医学は、今後のがん治療の基盤として認識されています。新しい分子の実体は、臨床結果を改善するために既存の薬剤との組み合わせで定期的にテストされます。
ただし、新しい効率的なターゲットを絞った治療法の開発に関連する重要な制限の 1 つは薬物暴露3の正確な生物学的結果をシミュレートするために一般的に使用される試金の失敗です。ガン創はまだほとんど臨床試験4で検証することは困難である 2次元単層培養で培養したがん細胞株における治療薬の有効性のテストに依存しています。したがってより体内の腫瘍5のネイティブの機能を模倣するより良いがんモデルを開発する関心の高まりがあります。近年、3次元培養モデルに対する注目が高まって結果 3 D 腫瘍モデル5を生成する改善された方法論の開発。
深さ体内アッセイにより前に BH3 模倣との組み合わせで hydroxycoumarin OT486の力の理解を改善することができます 3 つの異なった 3 D 細胞培養の技術と手法を紹介します。本論文では、生体内で腫瘍形成テストでさらに肺癌細胞 OT48、BH3 オノマトペの併用の有効性を検証し、生活で癌の進行を監視するゼブラフィッシュ モデルに基づく植民地と SFAs を組み合わせた生物。
コロニー形成法は、日常的に両方の固体だけでなく、造血の癌に対する抗癌剤の有効性を評価するために使用されます。アッセイは、無限に増殖し、コロニー7を形成する細胞の能力を決定します。コロニー形成細胞の能力に及ぼす抗悪性腫瘍剤は、数の減少および/またはコロニーのサイズによって決定されます。
回転楕円体体外腫瘍モデルを表し、抗がん剤のスクリーニング低コストのプラットフォームとして。回転楕円体は、懸濁液中の成長の細胞の集合体をまたは 3 D マトリックスに埋め込まれています。このアプローチは、薬剤のスクリーニングおよび腫瘍の成長、増殖と免疫の相互作用のための8の研究に広く使用されます。
完全に新しい薬物の特性を理解して、齧歯動物の生体内で実験を行うことが不可欠です。しかし、この従来の方法は高価で時間がかかるです。近年、ゼブラフィッシュ (動脈分布) が安くかつ迅速に高めるために広く研究所生物になった。ゼブラフィッシュ表す 3 D セル文化アプローチ、9の脊椎動物の体内の設定の中で腫瘍を開発しました。
完全に、我々 はここで使用 Cfa、SFAs ゼブラフィッシュ生体内で腫瘍形成など 3 つの異なる 3 D 文化アプローチ BH3 模倣との組み合わせで肺がんがん A549 細胞モデルによる hydroxycoumarin OT48 の抗がん能力を発揮します。

<table><tbody><tr><td>&lt;strong&gt;Materials required for colony formation assays&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>A549</td><td>ATCC</td><td>CCL-185</td><td>37 C&amp;deg;</td></tr><tr><td>RPMI 1640</td><td>Lonza</td><td>30096</td><td>4 C&amp;deg;</td></tr><tr><td>FBS</td><td>Biowest</td><td>S1520-500</td><td>-20 C&amp;deg;</td></tr><tr><td>Penicillin-Streptomycin</td><td>Lonza</td><td>17-602E</td><td>-20 C&amp;deg;</td></tr><tr><td>Cell culture flask T75</td><td>SPL</td><td>70075</td><td>RT</td></tr><tr><td>PBS solution</td><td>Hyclone</td><td>SH30256.02</td><td>RT</td></tr><tr><td>1.5ml tube</td><td>Extragene</td><td>Tube-170-C</td><td>RT</td></tr><tr><td>15 ml tube</td><td>Hyundai Micro</td><td>H20015</td><td>RT</td></tr><tr><td>12 well plate</td><td>SPL</td><td>30012</td><td>RT</td></tr><tr><td>MethoCult</td><td>StemCell technologies</td><td>4230</td><td>-20 C&amp;deg;</td></tr><tr><td>Thiazolyl Blue Tetrazolium Bromide (MTT powder)</td><td>Sigma</td><td>M5622</td><td>4 C&amp;deg;</td></tr><tr><td>LAS4000</td><td>GE Healthcare Technologies</td><td></td><td>RT</td></tr><tr><td>&lt;strong&gt;Materials required for spheroid formation assay&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>A549</td><td>ATCC</td><td>CCL-185</td><td>37 C&amp;deg;</td></tr><tr><td>RPMI 1640</td><td>Lonza</td><td>30096</td><td>4 C&amp;deg;</td></tr><tr><td>FBS</td><td>Biowest</td><td>S1520-500</td><td>-20 C&amp;deg;</td></tr><tr><td>Penicillin-streptomycin</td><td>Lonza</td><td>17-602E</td><td>-20 C&amp;deg;</td></tr><tr><td>Cell culture flask T75</td><td>SPL</td><td>70075</td><td>RT</td></tr><tr><td>PBS solution</td><td>Hyclone</td><td>SH30256.02</td><td>RT</td></tr><tr><td>Trypsin-EDTA</td><td>Gibco</td><td>25-300-054</td><td>4 C&amp;deg;</td></tr><tr><td>Corning costar ultra low attachemnt 96 well plate</td><td>Corning</td><td>3474</td><td>RT</td></tr><tr><td>Microscopy</td><td>Nikon</td><td>Eclipse TS100</td><td>RT</td></tr><tr><td>&lt;strong&gt;Materials required for zebrafish xenografts&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>A549</td><td>ATCC</td><td>CCL-185</td><td>37 C&amp;deg;</td></tr><tr><td>RPMI 1640</td><td>Lonza</td><td>30096</td><td>4 C&amp;deg;</td></tr><tr><td>FBS</td><td>Biowest</td><td>S1520-500</td><td>-20 C&amp;deg;</td></tr><tr><td>Penicillin-streptomycin</td><td>Lonza</td><td>17-602E</td><td>-20 C&amp;deg;</td></tr><tr><td>Cell culture flask T25</td><td>SPL</td><td>70025</td><td>RT</td></tr><tr><td>Cell culture flask T75</td><td>SPL</td><td>70075</td><td>RT</td></tr><tr><td>Cell culture flask T175</td><td>SPL</td><td>71175</td><td>RT</td></tr><tr><td>1.5ml tube</td><td>Extragene</td><td>Tube-170-C</td><td>RT</td></tr><tr><td>24 well plate</td><td>SPL</td><td>30024</td><td>RT</td></tr><tr><td>Petridish</td><td>SPL</td><td>10100</td><td>RT</td></tr><tr><td>PBS solution</td><td>Hyclone</td><td>SH30256.02</td><td>RT</td></tr><tr><td>Trypsin-EDTA</td><td>Gibco</td><td>25-300-054</td><td>4 C&amp;deg;</td></tr><tr><td>Sodium Chloride</td><td>Sigma-Aldrich</td><td>71382</td><td>RT</td></tr><tr><td>Potassium chloride (KCL)</td><td>Sigma-Aldrich</td><td>P9541</td><td>RT</td></tr><tr><td>Magnesium sulfate heptahydrate (MgSO4.7H2O)</td><td>Sigma-Aldrich</td><td>M2773</td><td>RT</td></tr><tr><td>Calcium nitrate tetrahydrate (Ca(NO3)2)</td><td>Sigma-Aldrich</td><td>C1396</td><td>RT</td></tr><tr><td>HEPES solution</td><td>Sigma-Aldrich</td><td>H0887</td><td>RT</td></tr><tr><td>Ethyl 3-aminobenzoate methanesulfonate (Tricaine)</td><td>Sigma-Aldrich</td><td>E10521</td><td>RT</td></tr><tr><td>Phenol Red solution</td><td>Sigma-Aldrich</td><td>P0290</td><td>RT</td></tr><tr><td>Methylcellulose</td><td>Sigma-Aldrich</td><td>M0512</td><td>RT</td></tr><tr><td>1-phenyl-2-thiourea (PTU)</td><td>Sigma-Aldrich</td><td>P7629</td><td>RT</td></tr><tr><td>CM-Dil dye</td><td>Invitrogen</td><td>C7001</td><td>-20 C&amp;deg;</td></tr><tr><td>Glass capillary</td><td>World Precision Instruments</td><td>TW 100F-4</td><td>RT</td></tr><tr><td>Micropipette puller</td><td>Shutter instrument, USA</td><td>P-97</td><td>RT</td></tr><tr><td>Micro injector</td><td>World Precision Instruments</td><td>PV820</td><td>RT</td></tr><tr><td>Syringe</td><td>KOVAX</td><td>1ml</td><td>RT</td></tr><tr><td>Micro loader</td><td>Eppendorf</td><td>5242956003</td><td>RT</td></tr><tr><td>Glass slide</td><td>Marienfeld</td><td>HSU-1000612</td><td>RT</td></tr><tr><td>Fluorescence microscopy</td><td>Leica</td><td>DE/DM 5000B</td><td>RT</td></tr></tbody></table>

preclinical assessment of the bioactivity of the anticancer coumarin ot48 by spheroids, colony formation assays, and zebrafish xenografts

新規治療薬のin vitroとin vivoの前臨床スクリーニングは、がん創薬における重要なツールです。ひと癌細胞株は、2 D (二次元) 単層細胞培養治療化合物に対応がもっと生理学的に関連するモデルの薬剤の有効性を理解する 3 D 培養システムが開発されました。近年、パラダイム シフトは、3次元培養システム、腫瘍微小環境をより正確に模倣の新規分子の効力を検証するための前臨床研究で観察されました。これらのシステムはもっと生理学的に関連する方法で病気の状態を特徴付けるし、より良い機械論的洞察力と与えられた分子の薬理学的力の理解を得るを助けます。また、生体内で癌モデルの改良で現在の傾向とゼブラフィッシュは、生体内で腫瘍形成を評価し、治療薬の効果を研究する重要な脊椎動物モデルとして浮上しています。ここでは、調べた hydroxycoumarin OT48 の治療効果単独または肺がん細胞株 A549 BH3 オノマトペとの組み合わせでコロニー形成アッセイ (CFA)、スフェロイド形成アッセイ (SFA) を含む 3 つの異なる 3次元培養システムを用いた、ゼブラフィッシュ異種生体内で。

図 2肺癌細胞株 A549 でシードだった MCBM コロニーを形成する OT48 治療後単独で、または指定された濃度で綴じられた BH3 擬態 A1210477 との組み合わせでの。その結果、組み合わせを大幅に削減数、サイズおよび植民地の総面積 10 日間の培養後。
図 3、A549 細胞 BH3 擬態 A1210477 と OT4...

Watch this Scientific Journal Video about Preclinical Assessment of the Bioactivity of the Anticancer Coumarin OT48 by Spheroids, Colony Formation Assays, and Zebrafish Xenografts at JoVE.com

Preclinical Assessment of the Bioactivity of the Anticancer Coumarin OT48 by Spheroids, Colony Formation Assays, and Zebrafish Xenografts

回転楕円体、コロニー形成アッセイとゼブラフィッシュ異種移植片による抗癌性クマリン OT48 の生物活性の臨床評価

三次元培養とゼブラフィッシュを用いた抗癌剤のクマリンの前臨床スクリーニングを紹介します。

In vitro and in vivo pre-clinical screening of novel therapeutic agents are an essential tool in cancer drug discovery. Although human cancer cell lines ...

preclinical-assessment-bioactivity-anticancer-coumarin-ot48-spheroids

Universidad de Cartagena UDEC

Research

JoVE Journal

Cancer Research

8.3K ビュー.  Seoul National University. 三次元培養とゼブラフィッシュを用いた抗癌剤のクマリンの前臨床スクリーニングを紹介します。

ビデオ: Preclinical Assessment of the Bioactivity of the Anticancer Coumarin OT48 by Spheroids, Colony Formation Assays, and Zebrafish Xenografts

Testing the Vascular Invasive Ability of Cancer Cells in Zebrafish (Danio Rerio)

Cancer cell vascular invasion and extravasation is a hallmark of metastatic progression. Traditional in vitro models of cancer cell invasion of endothelia typically lack the fluid dynamics that invading cells are otherwise exposed to in vivo. However, in vivo systems such as mouse models, though more physiologically relevant, require longer experimental timescales and present unique challenges associated with monitoring and data analysis. Here we describe a zebrafish assay that seeks to bridge this technical gap by allowing for the rapid assessment of cancer cell vascular invasion and extravasation. The approach involves injecting fluorescent cancer cells into the precardiac sinus of transparent 2-day old zebrafish embryos whose vasculature is marked by a contrasting fluorescent reporter. Following injection, the cancer cells must survive in circulation and subsequently extravasate from vessels into tissues in the caudal region of the embryo. Extravasated cancer cells are efficiently identified and scored in live embryos via fluorescence imaging at a fixed timepoint. This technique can be modified to study intravasation and/or competition amongst a heterogeneous mixture of cancer cells by changing the injection site to the yolk sac. Together, these methods can evaluate a hallmark behavior of cancer cells and help uncover mechanisms indicative of malignant progression to the metastatic phenotype.

Testing the Vascular Invasive Ability of Cancer Cells in Zebrafish Danio Rerio

This method utilizes zebrafish embryos to efficiently test the vascular invasive ability of cancer cells. Fluorescent cancer cells are injected into the precardiac sinus or yolk sac of developing embryos. Cancer cell vascular invasion and extravasation is assessed via fluorescence microscopy of the tail region 24 to 96 hr later.

（ゼブラフィッシュにおける癌細胞の血管浸潤能をテストします<em&gt;ゼブラフィッシュ</em&gt;）

Cancer cell vascular invasion and extravasation is a hallmark of metastatic progression.  Traditional in vitro models of cancer cell invasion of ...

がん研究

Screening for Melanoma Modifiers using a Zebrafish Autochthonous Tumor Model

Genomic studies of human cancers have yielded a wealth of information about genes that are altered in tumors1,2,3. A challenge arising from these studies is that many genes are altered, and it can be difficult to distinguish genetic alterations that drove tumorigenesis from that those arose incidentally during transformation. To draw this distinction it is beneficial to have an assay that can quantitatively measure the effect of an altered gene on tumor initiation and other processes that enable tumors to persist and disseminate. Here we present a rapid means to screen large numbers of candidate melanoma modifiers in zebrafish using an autochthonous tumor model4 that encompasses steps required for melanoma initiation and maintenance. A key reagent in this assay is the miniCoopR vector, which couples a wild-type copy of the mitfa melanocyte specification factor to a Gateway recombination cassette into which candidate melanoma genes can be recombined5. The miniCoopR vector has a mitfa rescuing minigene which contains the promoter, open reading frame and 3'-untranslated region of the wild-type mitfa gene. It allows us to make constructs using full-length open reading frames of candidate melanoma modifiers. These individual clones can then be injected into single cell Tg(mitfa:BRAFV600E);p53(lf);mitfa(lf)zebrafish embryos. The miniCoopR vector gets integrated by Tol2-mediated transgenesis6 and rescues melanocytes. Because they are physically coupled to the mitfa rescuing minigene, candidate genes are expressed in rescued melanocytes, some of which will transform and develop into tumors. The effect of a candidate gene on melanoma initiation and melanoma cell properties can be measured using melanoma-free survival curves, invasion assays, antibody staining and transplantation assays.

A rapid way to screen for melanoma modifiers using a zebrafish autochthonous tumor model is presented. It takes advantage of the miniCoopR vector which allows for expression of candidate melanoma genes in melanocytes. A method to obtain melanoma-free survival curves, an invasion assay, a protocol for antibody staining of scale melanocytes and a melanoma transplantation assay are described.

ゼブラ土着腫瘍モデルを用いたメラノーマの修飾のためのスクリーニング

Genomic studies of  human cancers have yielded a wealth of information about genes that are altered  in tumors1,2,3. A challenge arising from these ...

医学

Drug Screening of Primary Patient Derived Tumor Xenografts in Zebrafish

Patient derived xenograft models are critical in defining how different cancers respond to drug treatment in an in vivo system. Mouse models are the standard in the field, but zebrafish have emerged as an alternative model with several advantages, including the ability for high-throughput and low-cost drug screening. Zebrafish also allow for in vivo drug screening with large replicate numbers that were previously only obtainable with in vitro systems. The ability to rapidly perform large scale drug screens may open up the possibility for personalized medicine with rapid translation of results back to clinic. Zebrafish xenograft models could also be used to rapidly screen for actionable mutations based on tumor response to targeted therapies or to identify new anti-cancer compounds from large libraries. The current major limitation in the field has been quantifying and automating the process so that drug screens can be done on a larger scale and be less labor-intensive. We have developed a workflow for xenografting primary patient samples into zebrafish larvae and performing large scale drug screens using a fluorescence microscope equipped imaging unit and automated sampler unit. This method allows for standardization and quantification of engrafted tumor area and response to drug treatment across large numbers of zebrafish larvae. Overall, this method is advantageous over traditional cell culture drug screening as it allows for growth of tumor cells in an in vivo environment throughout drug treatment, and is more practical and cost-effective than mice for large scale in vivo drug screens.

Zebrafish xenograft models allow for high-throughput drug screening and fluorescent imaging of human cancer cells in an in vivo microenvironment. We developed a workflow for large scale, automated drug screening on patient-derived leukemia samples in zebrafish using an automated fluorescence microscope equipped imaging unit.

ゼブラフィッシュにおける原発性腫瘍異種移植片の薬物スクリーニング

Patient derived xenograft models are critical in defining how different cancers respond to drug treatment in an in vivo system. Mouse models are the ...

Generation of Zebrafish Larval Xenografts and Tumor Behavior Analysis

Zebrafish larval xenografts are being widely used for cancer research to perform in vivo and real-time studies of human cancer. The possibility of rapidly visualizing the response to anti-cancer therapies (chemo, radiotherapy, and biologicals), angiogenesis and metastasis with single cell resolution, places the zebrafish xenograft model as a top choice to develop preclinical studies.

The zebrafish larval xenograft assay presents several experimental advantages compared to other models, but probably the most striking is the reduction of size scale and consequently time. This reduction of scale allows single cell imaging, the use of a relatively low number of human cells (compatible with biopsies), medium-high-throughput drug screenings, but most importantly enables a significant reduction of the time of the assay. All these advantages make the zebrafish xenograft assay extremely attractive for future personalized medicine applications.

Many zebrafish xenograft protocols have been developed with a wide diversity of human tumors; however, a general and standardized protocol to efficiently generate zebrafish larval xenografts is still lacking. Here we provide a step-by-step protocol, with tips to generate xenografts and guidelines for tumor behavior analysis, whole-mount immunofluorescence, and confocal imaging quantification.

Here, we provide a step-by-step protocol, with tips to generate xenografts and guidelines for tumor behavior analysis, whole-mount immunofluorescence, and confocal imaging quantification.

ゼブラフィッシュラーバル異種移植片の生成と腫瘍行動解析

Zebrafish larval xenografts are being widely used for cancer research to perform in vivo and real-time studies of human cancer. The possibility of rapidly ...

Establishing 3-Dimensional Spheroids from Patient-Derived Tumor Samples and Evaluating their Sensitivity to Drugs

Despite remarkable advances in understanding tumor biology, the vast majority of oncology drug candidates entering clinical trials fail, often due to a lack of clinical efficacy. This high failure rate illuminates the inability of the current preclinical models to predict clinical efficacy, mainly due to their inadequacy in reflecting tumor heterogeneity and the tumor microenvironment. These limitations can be addressed with 3-dimensional (3D) culture models (spheroids) established from human tumor samples derived from individual patients. These 3D cultures represent real-world biology better than established cell lines that do not reflect tumor heterogeneity. Furthermore, 3D cultures are better than 2-dimensional (2D) culture models (monolayer structures) since they replicate elements of the tumor environment, such as hypoxia, necrosis, and cell adhesion, and preserve the natural cell shape and growth. In the present study, a method was developed for preparing primary cultures of cancer cells from individual patients that are 3D and grow in multicellular spheroids. The cells can be derived directly from patient tumors or patient-derived xenografts. The method is widely applicable to solid tumors (e.g., colon, breast, and lung) and is also cost-effective, as it can be performed in its entirety in a typical cancer research/cell biology lab without relying on specialized equipment. Herein, a protocol is presented for generating 3D tumor culture models (multicellular spheroids) from primary cancer cells and evaluating their sensitivity to drugs using two complementary approaches: a cell-viability assay (MTT) and microscopic examinations. These multicellular spheroids can be used to assess potential drug candidates, identify potential biomarkers or therapeutic targets, and investigate the mechanisms of response and resistance.

The present protocol describes generating 3D tumor culture models from primary cancer cells and evaluating their sensitivity to drugs using cell-viability assays and microscopic examinations.

患者由来の腫瘍サンプルから3次元スフェロイドを樹立し、薬物に対する感受性を評価

Despite remarkable advances in understanding tumor biology, the vast majority of oncology drug candidates entering clinical trials fail, often due to a ...

Establishment of Zebrafish Patient-Derived Xenografts from Pancreatic Cancer for Chemosensitivity Testing

Cancer is one of the main causes of death worldwide, and the incidence of many types of cancer continues to increase. Much progress has been made in terms of screening, prevention, and treatment; however, preclinical models that predict the chemosensitivity profile of cancer patients are still lacking. To fill this gap, an in vivo patient-derived xenograft model was developed and validated. The model was based on zebrafish (Danio rerio) embryos at 2 days post fertilization, which were used as recipients of xenograft fragments of tumor tissue taken from a patient's surgical specimen. 
It is also worth noting that bioptic samples were not digested or disaggregated, in order to maintain the tumor microenvironment, which is crucial in terms of analyzing tumor behavior and the response to therapy. The protocol details a method for establishing zebrafish-based patient-derived xenografts (zPDXs) from primary solid tumor surgical resection. After screening by an anatomopathologist, the specimen is dissected using a scalpel blade. Necrotic tissue, vessels, or fatty tissue are removed and then chopped into 0.3 mm x 0.3 mm x 0.3 mm pieces. 
The pieces are then fluorescently labeled and xenotransplanted into the perivitelline space of zebrafish embryos. A large number of embryos can be processed at a low cost, enabling high-throughput in vivo analyses of the chemosensitivity of zPDXs to multiple anticancer drugs. Confocal images are routinely acquired to detect and quantify the apoptotic levels induced by chemotherapy treatment compared to the control group. The xenograft procedure has a significant time advantage, since it can be completed in a single day, providing a reasonable time window to carry out a therapeutic screening for co-clinical trials.

Preclinical models aim to advance the knowledge of cancer biology and predict treatment efficacy. This paper describes the generation of zebrafish-based patient-derived xenografts (zPDXs) with tumor tissue fragments. The zPDXs were treated with chemotherapy, the therapeutic effect of which was assessed in terms of cell apoptosis of the transplanted tissue.

化学感受性試験のためのゼブラフィッシュ患者由来膵臓癌異種移植片の樹立

Cancer is one of the main causes of death worldwide, and the incidence of many types of cancer continues to increase. Much progress has been made in terms ...

ゼブラフィッシュ幼生の効率的なIn vivo腫瘍研究への活用

A Simple, Rapid, and Effective Method for Tumor Xenotransplantation Analysis in Transparent Zebrafish Embryos

In vivo studies of tumor behavior are a staple of cancer research; however, the use of mice presents significant challenges in cost and time. Here, we present larval zebrafish as a transplant model that has numerous advantages over murine models, including ease of handling, low expense, and short experimental duration. Moreover, the absence of an adaptive immune system during larval stages obviates the need to generate and use immunodeficient strains. While established protocols for xenotransplantation in zebrafish embryos exist, we present here an improved method involving embryo staging for faster transfer, survival analysis, and the use of flow cytometry to assess disease burden. Embryos are staged to facilitate rapid cell injection into the yolk of the larvae and cell marking to monitor the consistency of the injected cell bolus. After injection, embryo survival analysis is assessed up to 7 days post injection (dpi). Finally, disease burden is also assessed by marking transferred cells with a fluorescent protein and analysis by flow cytometry. Flow cytometry is enabled by a standardized method of preparing cell suspensions from zebrafish embryos, which could also be used in establishing the primary culture of zebrafish cells. In summary, the procedure described here allows a more rapid assessment of the behavior of tumor cells in vivo with larger numbers of animals per study arm and in a more cost-effective manner.

著者スポットライト:ゼブラフィッシュにおける腫瘍異種移植を用いたがん治療の進歩

We describe a protocol for xenotransplantation into the yolk of transparent zebrafish embryos that is optimized by a simple, rapid staging method. Post-injection analyses include survival and assessing the disease burden of xenotransplanted cells by flow cytometry.

透明ゼブラフィッシュ胚における腫瘍異種移植解析のための簡便、迅速、効果的な方法

In vivo studies of tumor behavior are a staple of cancer research; however, the use of mice presents significant challenges in cost and time. Here, we ...

Rapid Evaluation of Toxicity of Chemical Compounds Using Zebrafish Embryos

The zebrafish is a widely used vertebrate model organism for the disease and phenotype-based drug discovery. The zebrafish generates many offspring, has transparent embryos and rapid external development. Zebrafish embryos can, therefore, also be used for the rapid evaluation of toxicity of the drugs that are precious and available in small quantities. In the present article, a method for the efficient screening of the toxicity of chemical compounds using 1-5-day post fertilization embryos is described. The embryos are monitored by stereomicroscope to investigate the phenotypic defects caused by the exposure to different concentrations of compounds. Half-maximal lethal concentrations (LC50) of the compounds are also determined. The present study required 3-6 mg of an inhibitor compound, and the whole experiment takes about 8-10 h to be completed by an individual in a laboratory having basic facilities. The current protocol is suitable for testing any compound to identify intolerable toxic or off-target effects of the compound in the early phase of drug discovery and to detect subtle toxic effects that may be missed in the cell culture or other animal models. The method reduces procedural delays and costs of drug development.

Zebrafish embryos are used for evaluating the toxicity of chemical compounds. They develop externally and are sensitive to chemicals, allowing detection of subtle phenotypic changes. The experiment only requires a small amount of compound, which is directly added to the plate containing embryos, making the testing system efficient and cost-effective.

ゼブラフィッシュ胚を用いる化学化合物の毒性の迅速評価

The zebrafish is a widely used vertebrate model organism for the disease and phenotype-based drug discovery. The zebrafish generates many offspring, has ...

回転楕円体、コロニー形成アッセイとゼブラフィッシュ異種移植片による抗癌性クマリン OT48 の生物活性の臨床評価

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（ゼブラフィッシュにおける癌細胞の血管浸潤能をテストします

ゼブラ土着腫瘍モデルを用いたメラノーマの修飾のためのスクリーニング

ゼブラフィッシュにおける原発性腫瘍異種移植片の薬物スクリーニング

ゼブラフィッシュラーバル異種移植片の生成と腫瘍行動解析

患者由来の腫瘍サンプルから3次元スフェロイドを樹立し、薬物に対する感受性を評価

化学感受性試験のためのゼブラフィッシュ患者由来膵臓癌異種移植片の樹立

透明ゼブラフィッシュ胚における腫瘍異種移植解析のための簡便、迅速、効果的な方法

透明ゼブラフィッシュ胚における腫瘍異種移植解析のための簡便、迅速、効果的な方法

透明ゼブラフィッシュ胚における腫瘍異種移植解析のための簡便、迅速、効果的な方法

ゼブラフィッシュ胚を用いる化学化合物の毒性の迅速評価