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

Research

JoVE Journal

Cancer Research

8.4K 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

Anticancer Metal Complexes: Synthesis and Cytotoxicity Evaluation by the MTT Assay

Titanium (IV) and vanadium (V) complexes are highly potent anticancer agents. A challenge in their synthesis refers to their hydrolytic instability; therefore their preparation should be conducted under an inert atmosphere. Evaluation of the anticancer activity of these complexes can be achieved by the MTT assay.
The MTT assay is a colorimetric viability assay based on enzymatic reduction of the MTT molecule to formazan when it is exposed to viable cells. The outcome of the reduction is a color change of the MTT molecule. Absorbance measurements relative to a control determine the percentage of remaining viable cancer cells following their treatment with varying concentrations of a tested compound, which is translated to the compound anticancer activity and its IC50 values. The MTT assay is widely common in cytotoxicity studies due to its accuracy, rapidity, and relative simplicity.
Herein we present a detailed protocol for the synthesis of air sensitive metal based drugs and cell viability measurements, including preparation of the cell plates, incubation of the compounds with the cells, viability measurements using the MTT assay, and determination of IC50 values.

A method for synthesis of air-sensitive titanium and vanadium anticancer agents is described, along with the evaluation of their cytotoxic activity towards human cancer cell line by the MTT Assay.

MTTアッセイにより合成と細胞毒性の評価：抗がん金属錯体

Titanium (IV) and vanadium (V) complexes are highly potent anticancer agents. A challenge in their synthesis refers to their hydrolytic instability; ...

化学

Sample Extraction and Simultaneous Chromatographic Quantitation of Doxorubicin and Mitomycin C Following Drug Combination Delivery in Nanoparticles to Tumor-bearing Mice

Combination chemotherapy is frequently used in the clinic for cancer treatment; however, associated adverse effects to normal tissue may limit its therapeutic benefit. Nanoparticle-based drug combination has been shown to mitigate the problems encountered by free drug combination therapy. Our previous studies have shown that the combination of two anticancer drugs, doxorubicin (DOX) and mitomycin C (MMC), produced a synergistic effect against both murine and human breast cancer cells in vitro. DOX and MMC co-loaded polymer-lipid hybrid nanoparticles (DMPLN) bypassed various efflux transporter pumps that confer multidrug resistance and demonstrated enhanced efficacy in breast tumor models. Compared to conventional solution forms, such superior efficacy of DMPLN was attributed to the synchronized pharmacokinetics of DOX and MMC and increased intracellular drug bioavailability within tumor cells enabled by the nanocarrier PLN. 
To evaluate the pharmacokinetics and bio-distribution of co-administered DOX and MMC in both free solution and nanoparticle forms, a simple and efficient multi-drug analysis method using reverse-phase high performance liquid chromatography (HPLC) was developed. In contrast to previously reported methods that analyzed DOX or MMC individually in the plasma, this new HPLC method is able to simultaneously quantitate DOX, MMC and a major cardio-toxic DOX metabolite, doxorubicinol (DOXol), in various biological matrices (e.g., whole blood, breast tumor, and heart). A dual fluorescent and ultraviolet absorbent probe 4-methylumbelliferone (4-MU) was used as an internal standard (I.S.) for one-step detection of multiple drug analysis with different detection wavelengths. This method was successfully applied to determine the concentrations of DOX and MMC delivered by both nanoparticle and solution approaches in whole blood and various tissues in an orthotopic breast tumor murine model. The analytical method presented is a useful tool for pre-clinical analysis of nanoparticle-based delivery of drug combinations.

This protocol describes an efficient and convenient analytical process of sample extraction and simultaneous determination of multiple drugs, doxorubicin (DOX), mitomycin C (MMC) and a cardio-toxic DOX metabolite, doxorubicinol (DOXol), in the biological samples from a preclinical breast tumor model treated with nanoparticle formulations of synergistic drug combination.

サンプル抽出、ドキソルビシン、マイトマイシン C 以下の組み合わせへのドラッグデリバリー ナノ粒子の担癌マウスの同時のガスクロマトグラフィーによる定量

Combination chemotherapy is frequently used in the clinic for cancer treatment; however, associated adverse effects to normal tissue may limit its ...

がん研究

Mass Spectrometry and Luminogenic-based Approaches to Characterize Phase I Metabolic Competency of In Vitro Cell Cultures

Xenobiotic metabolizing enzymes play a key function in the biotransformation of medicines and toxicants by adding functional groups that increase solubility and facilitate excretion. On some occasions those structural modifications lead to the formation of new toxic products. In order to reduce animal testing, chemical risk can be assessed using metabolically competent cells. The expression of metabolic enzymes, however, is not stable over time in many in vitro primary culture systems and is often partial or absent in cell lines. Therefore, the study of medicines, additives, and environmental pollutants metabolism in vitro should ideally be conducted in cell systems where metabolic activity has been characterized. We explain here an approach to measure the activity of a class of metabolic enzymes (Human Phase I) in 2D cell lines and primary 3D cultures using chemical probes and their metabolic products quantifiable by UPLC mass spectrometry and luminometry. The method can be implemented to test the metabolic activity in cell lines and primary cells derived from a variety of tissues.

Mass Spectrometry and Luminogenic-based Approaches to Characterize Phase I Metabolic Competency of In Vitro Cell Cultures

Metabolic competency of in vitro systems is a key requirement for the biotransformation and disposition of drugs and toxicants. In this protocol, we describe the application of reference metabolic probes to assess phase I metabolism in cell cultures.

のフェーズI代謝コンピテンシーを特徴づけるために質量分析とルミネベースのアプローチ<em&gt;インビトロ</em&gt;細胞培養

Xenobiotic metabolizing enzymes play a key function in the biotransformation of medicines and toxicants by adding functional groups that increase ...

生化学

Formation of Covalent DNA Adducts by Enzymatically Activated Carcinogens and Drugs In Vitro and Their Determination by 32P-postlabeling

Covalent DNA adducts formed by chemicals or drugs with carcinogenic potency are judged as one of the most important factors in the initiation phase of carcinogenic processes. This covalent binding, which is considered the cause of tumorigenesis, is now evaluated as a central dogma of chemical carcinogenesis. Here, methods are described employing the reactions catalyzed by cytochrome P450 and additional biotransformation enzymes to investigate the potency of chemicals or drugs for their activation to metabolites forming these DNA adducts. Procedures are presented describing the isolation of cellular fractions possessing biotransformation enzymes (microsomal and cytosolic samples with cytochromes P450 or other biotransformation enzymes, i.e., peroxidases, NADPH:cytochrome P450 oxidoreductase, NAD(P)H:quinone oxidoreductase, or xanthine oxidase). Furthermore, methods are described that can be used for the metabolic activation of analyzed chemicals by these enzymes as well as those for isolation of DNA. Further, the appropriate methods capable of detecting and quantifying chemical/drug-derived DNA adducts, i.e., different modifications of the 32P-postlabeling technique and employment of radioactive-labeled analyzed chemicals, are shown in detail.

Formation of Covalent DNA Adducts by Enzymatically Activated Carcinogens and Drugs In Vitro and Their Determination by 32P-postlabeling

Evaluating the potency of environmental chemicals and drugs, to be enzymatically bioactivated to intermediates generating covalent DNA adducts, is an important field in the development of cancer and its treatment. Methods are described for compound activation to form DNA adducts, as well as techniques for their detection and quantification.

共有結合 DNA 付加体生成活性酵素によって発ガン性物質の in Vitro薬物と32p-ポストラベルで決意

Covalent DNA adducts formed by chemicals or drugs with carcinogenic potency are judged as one of the most important factors in the initiation phase of ...

High-Throughput In Vitro Assay using Patient-Derived Tumor Organoids

Patient-derived tumor organoids (PDOs) are expected to be a preclinical cancer model with better reproducibility of disease than traditional cell culture models. PDOs have been successfully generated from a variety of human tumors to recapitulate the architecture and function of tumor tissue accurately and efficiently. However, PDOs are unsuitable for an in vitro high-throughput assay system (HTS) or cell analysis using 96-well or 384-well plates when evaluating anticancer drugs because they are heterogeneous in size and form large clusters in culture. These cultures and assays use extracellular matrices, such as Matrigel, to create tumor tissue scaffolds. Therefore, PDOs have a low throughput and high cost, and it has been difficult to develop a suitable assay system. To address this issue, a simpler and more accurate HTS was established using PDOs to evaluate the potency of anticancer drugs and immunotherapy. An in vitro HTS was created that uses PDOs established from solid tumors cultured in 384-well plates. An HTS was also developed for assessment of antibody-dependent cellular cytotoxicity activity to represent the immune response using PDOs cultured in 96-well plates.

High-Throughput In Vitro Assay using Patient-Derived Tumor Organoids

A highly accurate in vitro high-throughput assay system was developed to evaluate anticancer drugs using patient-derived tumor organoids (PDOs), similar to cancer tissues but are unsuitable for in vitro high-throughput assay systems with 96-well and 384-well plates.

患者由来腫瘍オルガノイドを用いたイン ビトロ アッセイのハイスループット

Patient-derived tumor organoids (PDOs) are expected to be a preclinical cancer model with better reproducibility of disease than traditional cell culture ...

Evaluating the Effectiveness of Cancer Drug Sensitization In Vitro and In Vivo

Due to the high level of heterogeneity and mutations inherent in human cancers, single agent therapies, or combination regimens which target the same pathway, are likely to fail. Emphasis must be placed upon the inhibition of pathways that are responsible for intrinsic and/or adaptive resistance to therapy. An active field of investigation is the development and testing of DNA repair inhibitors that promote the action of, and prevent resistance to, commonly used chemotherapy and radiotherapy. We used a novel protocol to evaluate the effectiveness of BRCA2 inhibition as a means to sensitize tumor cells to the DNA damaging drug cisplatin. Tumor cell metabolism (acidification and respiration) was monitored in real-time for a period of 72 hr to delineate treatment effectiveness on a minute by minute basis. In combination, we performed an assessment of metastatic frequency using a chicken embryo chorioallantoic membrane (CAM) model of extravasation and invasion. This protocol addresses some of the weaknesses of commonly used in vitro and in vivo methods to evaluate novel cancer therapy regimens. It can be used in addition to common methods such as cell proliferation assays, cell death assays, and in vivo murine xenograft studies, to more closely discriminate amongst candidate targets and agents, and select only the most promising candidates for further development.

Evaluating the Effectiveness of Cancer Drug Sensitization In Vitro and In Vivo

Here, real-time monitoring of tumor cell metabolism, combined with an in vivo chicken embryo chorio-allantoic membrane (CAM) model of metastasis, are used to evaluate novel anti-cancer targets/agents for their ability to sensitize tumor cells to DNA damaging chemotherapeutics.

がん薬感の有効性を評価<em&gt;インビトロ</em&gt;と<em&gt;インビボ</em

Due to the high level of heterogeneity and mutations inherent in human cancers, single agent therapies, or combination regimens which target the same ...

医学

In Vitro and In Vivo Evaluation of Photocontrolled Biologically Active Compounds - Potential Drug Candidates for Cancer Photopharmacology

Photocontrolled, biologically active compounds are an emerging class of &#34;smart&#34; drug candidates. They provide additional safety in systemic chemotherapy due to their precise spatiotemporal activation by directing a benign, non-ionizable light to a specific location within the patient&#39;s body. This paper presents a set of methods to evaluate the in vitro potency and ex vivo efficiency of the photoactivation of photocontrolled, biologically active compounds as well as the in vivo efficacy at early stages of drug development. The methodology is applied to anticancer cytotoxic peptides, namely, the diarylethene-containing analogs of a known antibiotic, gramicidin S. The experiments are performed using 2D (adherent cells) and 3D (spheroids) cell cultures of a cancer cell line (Lewis lung carcinoma, LLC), live tissue surrogates (pork meat mince), and an allograft cancer model (subcutaneous LLC) in immunocompetent mice. The selection of the most effective compounds and estimation of realistic phototherapeutic windows are performed via automated fluorescence microscopy. The photoactivation efficiency at varying illumination regimens is determined at different depths in a model tissue, and the optimal light dosage is applied in the final therapeutic in vivo experiment.

In Vitro and In Vivo Evaluation of Photocontrolled Biologically Active Compounds - Potential Drug Candidates for Cancer Photopharmacology

This protocol presents a set of experiments adopted for the evaluation of photoswitchable anticancer peptides, that can be used in the preclinical screening of such compounds. This includes cytotoxicity assessment in 2D and 3D cell cultures, the evaluation of ex vivo (model tissue) photoisomerization efficiency, and in vivo efficacy.

光制御された生物学的に活性な化合物のin vitroおよびin vivo評価-癌光薬理学の潜在的な薬剤候補

Photocontrolled, biologically active compounds are an emerging class of &#34;smart&#34; drug candidates. They provide additional safety in systemic ...

Utilization of the Soft Agar Colony Formation Assay to Identify Inhibitors of Tumorigenicity in Breast Cancer Cells

Given the inherent difficulties in investigating the mechanisms of tumor progression in vivo, cell-based assays such as the soft agar colony formation assay (hereafter called soft agar assay), which measures the ability of cells to proliferate in semi-solid matrices, remain a hallmark of cancer research. A key advantage of this technique over conventional 2D monolayer or 3D spheroid cell culture assays is the close mimicry of the 3D cellular environment to that seen in vivo. Importantly, the soft agar assay also provides an ideal tool to rigorously test the effects of novel compounds or treatment conditions on cell proliferation and migration. Additionally, this assay enables the quantitative assessment of cell transformation potential within the context of genetic perturbations. We recently identified peptidylarginine deiminase 2 (PADI2) as a potential breast cancer biomarker and therapeutic target. Here we highlight the utility of the soft agar assay for preclinical anti-cancer studies by testing the effects of the PADI inhibitor, BB-Cl-amidine (BB-CLA), on the tumorigenicity of human ductal carcinoma in situ (MCF10DCIS) cells.

Here, we document the use of the soft agar colony formation assay to test the effects of a peptidylarginine deiminase (PADI) enzyme inhibitor, BB-Cl-amidine, on breast cancer tumorigenicity in vitro.

乳癌細胞における腫瘍形成の阻害剤を同定するための軟寒天コロニー形成アッセイの活用

Given the inherent difficulties in investigating the mechanisms of tumor progression in vivo, cell-based assays such as the soft agar colony formation ...

Chick Heart Invasion Assay for Testing the Invasiveness of Cancer Cells and the Activity of Potentially Anti-invasive Compounds

The goal of the chick heart assay is to offer a relevant organ culture method to study tumor invasion in three dimensions. The assay can distinguish between invasive and non-invasive cells, and enables study of the effects of test compounds on tumor invasion. Cancer cells - either as aggregates or single cells - are confronted with fragments of embryonic chick heart. After organ culture in suspension for a few days or weeks the confronting cultures are fixed and embedded in paraffin for histological analysis. The three-dimensional interaction between the cancer cells and the normal tissue is then reconstructed from serial sections stained with hematoxylin-eosin or after immunohistochemical staining for epitopes in the heart tissue or the confronting cancer cells. The assay is consistent with the recent concept that cancer invasion is the result of molecular interactions between the cancer cells and their neighbouring stromal host elements (myofibroblasts, endothelial cells, extracellular matrix components, etc.). Here, this stromal environment is offered to the cancer cells as a living tissue fragment. Supporting aspects to the relevance of the assay are multiple. Invasion in the assay is in accordance with the criteria of cancer invasion: progressive occupation and replacement in time and space of the host tissue, and invasiveness and non-invasiveness in vivo of the confronting cells generally correlates with the outcome of the assay. Furthermore, the invasion pattern of cells in vivo, as defined by pathologists, is reflected in the histological images in the assay. Quantitative structure-activity relation (QSAR) analysis of the results obtained with numerous potentially anti-invasive organic congener compounds allowed the study of structure-activity relations for flavonoids and chalcones, and known anti-metastatic drugs used in the clinic (e.g., microtubule inhibitors) inhibit invasion in the assay as well. However, the assay does not take into account immunological contributions to cancer invasion.

Here, we present a protocol to study the invasion of tumor cells into living normal tissue fragments in three dimensions. This organ culture technique is mainly applied to test potentially anti-invasive drugs in vitro.

テストのためのひよこ心臓浸潤アッセイ癌細胞の浸潤性および潜在的に抗浸潤化合物の活性

The goal of the chick heart assay is to offer a relevant organ culture method to study tumor invasion in three dimensions. The assay can distinguish ...

High Content Screening Analysis to Evaluate the Toxicological Effects of Harmful and Potentially Harmful Constituents (HPHC)

Cigarette smoke (CS) is a major risk factor for cardiovascular and lung diseases. Because CS is a complex aerosol containing more than 7,000 chemicals1 it is challenging to assess the contributions of individual constituents to its overall toxicity. Toxicological profiles of individual constituents as well as mixtures can be however established in vitro, by applying high through-put screening tools, which enable the profiling of Harmful and Potentially Harmful Constituents (HPHCs) of tobacco smoke, as defined by the U.S. Food and Drug Administration (FDA).2
For an initial assessment, an impedance-based instrument was used for a real-time, label-free assessment of the compound&#39;s toxicity. The instrument readout relies on cell adhesion, viability and morphology that all together provide an overview of the cell status. A dimensionless parameter, named cell index, is used for quantification. A set of different staining protocols was developed for a fluorescence imaging-based investigation and a HCS platform was used to gain more in-depth information on the kind of cytotoxicity elicited by each HPHC.
Of the 15 constituents tested, only five were selected for HCS-based analysis as they registered a computable LD50 (&#60; 20 mM). These included 1-aminonaphtalene, Arsenic (V), Chromium (VI), Crotonaldehyde and Phenol. Based on their effect in the HCS, 1-aminonaphtalene and Phenol could be identified to induce mitochondrial dysfunction, and, together with Chromium (VI) as genotoxic based on the increased histone H2AX phosphorylation. Crotonaldehyde was identified as an oxidative stress inducer and Arsenic as a stress kinase pathway activator.
This study demonstrates that a combination of impedance-based and HCS technologies provides a robust tool for in vitro assessment of CS constituents.

High Content Screening Analysis to Evaluate the Toxicological Effects of Harmful and Potentially Harmful Constituents HPHC

The objective of the study was to assess the biological impact of 15 cigarette smoke constituents using a combination of an impedance-based real time cell analyzer and a high-content screening (HCS)-based platform for toxicological assessment in vitro. This study provides information on effective doses, toxicity and modes of action of the tested compounds.

有害と潜在的に有害な成分の毒性効果を評価するためにハイコンテントスクリーニング分析（HPHC）

Cigarette smoke (CS) is a major risk factor for cardiovascular and lung diseases. Because CS is a complex aerosol containing more than 7,000 chemicals1 it ...

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

要約

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MTTアッセイにより合成と細胞毒性の評価：抗がん金属錯体

サンプル抽出、ドキソルビシン、マイトマイシン C 以下の組み合わせへのドラッグデリバリーナノ粒子の担癌マウスの同時のガスクロマトグラフィーによる定量

のフェーズI代謝コンピテンシーを特徴づけるために質量分析とルミネベースのアプローチ

共有結合 DNA 付加体生成活性酵素によって発ガン性物質の in Vitro薬物と³²p-ポストラベルで決意

患者由来腫瘍オルガノイドを用いたインビトロアッセイのハイスループット

がん薬感の有効性を評価

光制御された生物学的に活性な化合物のin vitroおよびin vivo評価-癌光薬理学の潜在的な薬剤候補

乳癌細胞における腫瘍形成の阻害剤を同定するための軟寒天コロニー形成アッセイの活用

テストのためのひよこ心臓浸潤アッセイ癌細胞の浸潤性および潜在的に抗浸潤化合物の活性

有害と潜在的に有害な成分の毒性効果を評価するためにハイコンテントスクリーニング分析（HPHC）

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

要約

さらに動画を探す

MTTアッセイにより合成と細胞毒性の評価：抗がん金属錯体

サンプル抽出、ドキソルビシン、マイトマイシン C 以下の組み合わせへのドラッグデリバリー ナノ粒子の担癌マウスの同時のガスクロマトグラフィーによる定量

のフェーズI代謝コンピテンシーを特徴づけるために質量分析とルミネベースのアプローチ

共有結合 DNA 付加体生成活性酵素によって発ガン性物質の in Vitro薬物と32p-ポストラベルで決意

患者由来腫瘍オルガノイドを用いたイン ビトロ アッセイのハイスループット

がん薬感の有効性を評価

光制御された生物学的に活性な化合物のin vitroおよびin vivo評価-癌光薬理学の潜在的な薬剤候補

乳癌細胞における腫瘍形成の阻害剤を同定するための軟寒天コロニー形成アッセイの活用

テストのためのひよこ心臓浸潤アッセイ癌細胞の浸潤性および潜在的に抗浸潤化合物の活性

有害と潜在的に有害な成分の毒性効果を評価するためにハイコンテントスクリーニング分析（HPHC）

サンプル抽出、ドキソルビシン、マイトマイシン C 以下の組み合わせへのドラッグデリバリーナノ粒子の担癌マウスの同時のガスクロマトグラフィーによる定量

共有結合 DNA 付加体生成活性酵素によって発ガン性物質の in Vitro薬物と³²p-ポストラベルで決意

患者由来腫瘍オルガノイドを用いたインビトロアッセイのハイスループット