Name: looking for driver pathways of acquired resistance to targeted therapy: drug resistant subclone generation and sensitivity restoring by gene knock-down
Uploaded: 2017-12-11T14:00:00.000+00:00
Duration: 8 min 59 s
Description: Watch this Scientific Journal Video about Looking for Driver Pathways of Acquired Resistance to Targeted Therapy: Drug Resistant Subclone Generation and Sensitivity Restoring by Gene Knock-down at JoV

著者の原稿を編集のため博士ベロニカ ザノーニを感謝したいです。

注: このプロトコルは、具体的にはトラスツズマブ HER 陽性胃癌細胞株の抵抗性獲得機構の解析に調整されています。必要なすべての実験室の器械は、材料表で報告されます。
1. 対象となる治療に抵抗性サブクローンの世代
注: これは、最も重要かつ異なる細胞を表わすかもしれないない別の感度にターゲットを絞った治療阻害剤独立して腫瘍 histotype や薬物濃度からという事実のために、プロトコルの手順を標準化することは困難使用されます。
<ol><li>文化 NCI N87 セル RPMI 培牛胎児血清 (10%) とグルタミン (2 mM, 1%)、37 ° C で単分子膜としての線、25 cm2培養フラスコにシードします。</li>
	<li>先発の線量としてトラスツズマブの各のフラスコ 10 μ g/mL の培養液を追加します。彼らは成長を再開するまでは、先発の線量に細胞を公開します。 注: 標的治療阻害剤の先発の線量は 10 倍、血漿中のピーク濃度よりも低いはずです。血漿中のピーク濃度は、通常次の標準療法の複数用量患者血中薬物濃度の最高レベルです。この値は、薬物動態に関する研究から取得することができます。</li>
	<li>トリパン ブルー色素排除法と細胞の成長を監視します。(通常 2 〜 3 週間の期間) 後細胞の成長を再開すると、トラスツズマブの 10 倍高い線量集中する細胞を公開します。</li>
	<li>(約 2 週間) 後の高い薬物濃度 (少なくとも 2.5 - 4 倍プラズマ ピークよりも高い) の存在にもかかわらず、成長細胞維持までトラスツズマブ (から 400 μ g/mL に 100 μ g/mL) 量が徐々 に増加する細胞を公開します。</li>
	<li>
濃度の培養液でターゲット療法阻害のあらゆる変化で標的治療への抵抗のレベルを評価するクローン形成法を実行し、相対抵抗 IC50インデックス (RR IC50) を次のように定義します。
	<ol>
<li>24 ウェル培養皿 500 細胞を播きます。 注: 5 ウェルズは、条件セットごとに準備されなければなりません。</li>
		<li>100 μ g/mL 400 μ G/ml までターゲット療法 (トラスツズマブ) 阻害剤の濃度を上げるに細胞を公開します。</li>
		<li>15 日間の 37 ° C で CO2インキュベーター内のセルを孵化させなさい。</li>
		<li>インキュベーション後、修正し、次のようにサンプルを染色します。<ol>
<li>10 mL の 1x PBS の媒体とリンスのセルを削除します。1x PBS を削除し、固定液 (酢酸酸: メタノール、1:7、(巻/巻)) の 2-3 mL を追加します。固定の解決を削除し、0.5% クリスタル バイオレット溶液を追加します。2 時間室温で孵化させなさい</li>
			<li>クリスタル ・ バイオレット液をピペットで吸引によって慎重に削除し、クリスタル バイオレット ソリューション残渣を洗い流す水道水でプレートを浸します。2 日間室温で空気乾燥します。</li>
		</ol>
</li>
		<li>倒立顕微鏡 (4 倍) を使用して 50 以上の細胞のコロニーをカウントします。 注: この 2 つの独立した観測者が必要です。</li>
		<li>RR IC50をターゲット治療抵抗性 subclone セルの IC50値と元/ナイーブ細胞の値の比率として計算します。</li>
	</ol>
</li>
</ol>2. 検証プロトコル候補者に対象療法抵抗性遺伝子 (R 遺伝子)
注: 遺伝子発現の解析後、署名に関連付けられているターゲット療法と任意の候補遺伝子獲得抵抗性を通じて抵抗のドライバーを調査する:) RT-PCR および b) ウエスタン ブロット分析。
<ol><li>
リアルタイム RT-PCR
	<ol>
<li>細胞を用いた酸ためチオシアン酸-フェノール-クロロホルム混合物からの総 RNA の抽出 (材料の表を参照してください)。</li>
		<li>ランダム hexamer プライマーを使用して逆のトランスクリプション (RT) 反応を行い、熱 cycler を次のように設定: 46 ° C、20 分で RT、RT 不活化 1 分 95 ° C で 5 分間 25 ° C でプライミングします。</li>
		<li>20 μ L 反応の総 RNA の使用 400 ng。反応混合物を次のようにサンプルの準備: 7 μ L RNase フリー H2O、2 μ L cDNA (10 ng/μ L)、ターゲット固有の蛍光標識プローブ、x 1 μ 20 と 10 μ L 2 x ミックス含むバッファー、dNTPs、DNA ポリメラーゼ (材料の表を参照してください)。</li>
		<li>各ウェル プレートに反応混合物の 20 μ L を追加し、次のプログラムを実行: 95 ° C、10 分 (1 サイクル) で、2 分の 50 ° C でステージを開催40 サイクル 95 ° c、15 秒と 1 分の 60 ° c。</li>
	</ol>
</li>
	<li>
西部のしみ
	<ol>
<li>0.05%/トリプシン EDTA 溶液 2 mL を追加し、動作するようにトリプシンの 2 〜 3 分を許可するには、細胞懸濁液を回復します。</li>
		<li>中型含む 10% 牛胎児血清トリプシン (例えば、各 1 mL のトリプシンのための媒体の 2 mL) を中和し、1,200 x g で 5 分間遠心冷 1x PBS で破棄上清および洗浄細胞の 2 つのボリュームを追加します。</li>
		<li>5 分 1,200 × g で遠心分離し、上澄みを廃棄します。換散バッファーと細胞ペレットを再懸濁します、4 ° C、30 分でロッカーで孵化させなさい。<ol>
<li>換散バッファーの量によって異なります (例えば1 x 10 の6セルを 100 μ L); 細胞数毎回新鮮な換散バッファーを準備することを確認します。換散バッファーの準備 (氷の上を残す) の 1 ml: 975 μ M を使用して-10 μ L 100 μ M PMSF 阻害剤、15 μ L ホスファターゼとプロテアーゼ阻害剤は、哺乳類の溶解バッファーあたり。</li>
		</ol>
</li>
		<li>4 ° C 15 分で 13,000 × g で遠心沈降法による細胞のライセート上清タンパク質を回復します。</li>
		<li>BCA タンパク質アッセイを用いたタンパク質の濃度を決定します。</li>
		<li>蛋白質のサンプル (蛋白質の少なくとも 20-30 μ g) と 3 分間 100 ° C で熱に塩化物イオン サンプル バッファー x 4 を追加します。</li>
		<li>プレキャスト 4 20% ゲルを使用し、塩化物イオンとタンパク質溶液の 20-30 μ g、タンパク質標準サンプルごとの 5-10 μ L を読み込みます。</li>
		<li>トリス/GLICYNE/SDS 1 x バッファーと西部のしみリングを記入し、(時間停止染料はゲル、それ以外の場合、いくつかの追加分の実行の終わりに達したことを確認する) とき 110 V 30-60 分のためのゲルを実行</li>
		<li>PVDF 膜に蛋白質を転送する Electroblot (材料表参照) 半乾燥システムを使用しています。</li>
		<li>1 時間室温でシェーカーで適切なタンパク質溶液 (ミルク/BSA 5%) に浸漬して膜をブロックします。</li>
		<li>アンチ IQGAP1 一次抗体と 1: 400 ソリューションは、5% ミルク ソリューション (材料の表を参照してください)。</li>
		<li>一晩冷蔵室のシェーカーの一次抗体溶液中膜を配置します。</li>
		<li>次の日、抗体溶液を破棄し、T TBS 1 x 溶液中浸漬 (1 T TBS x: 1 x Tween20 で Tris バッファーは生理食塩水を追加 0.1%) は 7 分間室温でシェーカーで。</li>
		<li>ソリューションと置換 x T-tbs 系 1 を破棄します。3 回繰り返します。</li>
		<li>標準的な西部の抗体を追加することによってしみの検出 1: 10,000 のセカンダリ マウス抗体溶液を作成 (材料の表を参照してください); シェーカーに膜を置くと2 時間室温に置いたまま。</li>
		<li>抗体溶液を破棄し、7 分間室温でシェーカーで T TBS 1 x 溶液中浸漬します。</li>
		<li>ソリューションと置換 x T-tbs 系 1 を破棄します。3 回繰り返します。</li>
		<li>化学発光液 5 分間膜を浸漬し、化学発光のイメージャの膜をイメージします。</li>
	</ol>
</li>
</ol>3. 候補 R 遺伝子ノックダウンによる標的治療阻害剤感受性の復元
<ol><li>
セル準備
	<ol>
<li>Trypsinization 単一細胞懸濁液を準備します。細胞当日 transfection を実行します。<ol>
<li>洗って NCI N87 PBS のセルし、5 ~ 10 分追加 2 RPMI 培地含む 10% 牛胎児血清トリプシン (例えば、各 1 mL のトリプシンのための媒体の 2 mL) を中和する量の 0.05% トリプシン/EDTA 溶液で 37 ° C で孵化させなさい。</li>
			<li>トリパン ブルー染色を用いた検定とセルをカウントします。各条件の T25 cm2フラスコにシード 2.5 x 105セル (60% 合流)。 注: 播種のための細胞の適切な数は、細胞倍加時間と実験の期間によって決定されます。目的は、ターゲット遺伝子ノックダウン実験の全体の持続期間のために達成するためにです。9 T25 cm2フラスコは、トランスフェクション条件とクローン形成法 (コントロール、負トランスフェクション コントロール、遺伝子ターゲット transfection のため 3 フラスコ 3 フラスコ 3 フラスコ) に最適です。</li>
		</ol>
</li>
	</ol>
</li>
	<li>
トランスフェクション
	<ol>
<li>遺伝子ターゲットを準備し、次のように各 T25 cm2フラスコのコントロール トランスフェクション ミックスを負します。<ol>
<li>各標的遺伝子の siRNA オリゴヌクレオチドまたは最小限不可欠なトランスフェクション媒体 (比率 1:55; 参照テーブルの材料) のネガティブ コントロール siRNA オリゴヌクレオチドの 12.5 μ L を希釈します。</li>
			<li>カチオン性リポソームのトランスフェクション試薬 (siRNA オリゴヌクレオチドの標的遺伝子と 1:1 の比率; 参照材料テーブル) を追加します。20 分間室温でインキュベートします。 注: トランスフェクション ミックスの容量は 700 μ L です。</li>
			<li>各 T25 cm2フラスコにトランスフェクション ミックス 700 μ L を追加します。1 〜 3 日の 37 ° C でセルを孵化させなさい。</li>
		</ol>
</li>
		<li>RT-PCR および西部のしみの分析による遺伝子ノックダウンを確認します。推定 R 遺伝子ノック ダウン (繰り返し手順 1.5) コロニー形成実験を通じてターゲットを絞った治療エージェントと感度復原の影響を再確認してください。</li>
	</ol>
</li>
</ol>

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著者が明らかに何もありません。

パーソナライズし、対象と治療法は、成長している熱意を誘発しているが、これらのいわゆる 'スマート' 療法として従来の化学療法薬、すなわち、薬物抵抗性の急速かつ予測不可能な獲得同じ主要なハードルに直面します。標的療法の結果を改善するためにそれらを引き起こすメカニズムの理解を通じて薬剤耐性問題を解決されなければなりません。ここでは、私たちは癌細胞ライン...

分子細胞生物学の重要な発見をフォロー アップして、新規に合成された抗がん剤の分子の数は、選択的に腫瘍のタイプの広い配列で発癌性シグナル伝達経路をターゲットに開発されています。特に、腫瘍のユニークなプロパティを持つ対象の治療薬の 2 つのカテゴリ、すなわち合成化合物、遺伝子組換え抗体の臨床成功とで劇的に変化した癌治療を達成しているが知られています。ここ数十年1,2,3。
それにもかかわらず、その印象的な初期応答にもかかわらず治療に、癌患者の大半はすべての標的治療薬、モノクローナル抗体とキナーゼ阻害剤への薬剤耐性を開発しました。結果として、薬剤耐性、古典的な抗がん薬4の主要なハードルはまだ新たな標的治療5,6にとって大きな挑戦であります。
標的治療への抵抗は本質的なものがあります (つまり、プライマリ) または (すなわち、セカンダリ) 取得します。本質的な抵抗では、薬物治療7への応答の期間後に発生する二次抵抗に対しの療法への応答のde novoの欠如について説明します。後者は原始的な腫瘍の大部分の内で腫瘍細胞の小さなコホートの発生によって引き起こされるまたは遠位の不可解な解剖学的ニッチに囲まれた、90% までを展示抵抗 1 つまたは複数のターゲット治療薬。主に目標とされた療法に抵抗性の発現の基礎となる分子機構に起因ターゲット遺伝子変異とその理解はまだはるかに完全な8その他生存シグナル伝達経路の冗長活性化。
この作品は、体外サイレンシングの説明への薬剤耐性細胞のサブクローンの世代から始まって、ターゲット治療薬抵抗性獲得のメカニズムを調査するため時間と費用有効方法を提案阻害剤のテストと作業仮説を検証するために使用する重要なツールに感度を復元するために使用する手順。特に、我々 のアプローチは、胃癌トラスツズマブ (例えばハーセプチン)、HER2 タンパク質9の細胞外ドメインを対象とするヒト化モノクローナル抗体に抵抗機構の調査に使用されました。化学療法 + トラスツズマブは HER2 陽性転移性乳癌に対する標準のファーストライン治療として広く受け入れられます。治療が両方生体外で有意な活性を持つその抗 HER2 を示す最近の臨床研究のおかげで、生体内でHER2 陽性胃癌モデル10,11分子薬 HER2 をターゲットにされています。広範囲の臨床試験で検討した、いくつかはまだ進行中、胃食道がん12,13,14,15症例。
これらの研究は、トラスツズマブに16、他の標的治療阻害剤の観察されるものと同様に抵抗を発生している患者数は上昇を強調しています。特に、トラスツズマブの応答率は 47% で、トラスツズマブ + 化学療法を受けている患者の全生存中央値はわずか 2.7 ヶ月化学療法単独で17の患者よりも長くなります。これは、プライマリ トラスツズマブ抵抗は普及しているが、セカンダリのトラスツズマブ抵抗は避けられない示唆されました。このため、緊急にこの腫瘍18内腫瘍の遺伝の不均質を与えさらに問題は、胃癌 HER2 標的治療抵抗性の原因とメカニズムを解明する必要があります。
さらに、胃癌トラスツズマブへの抵抗のメカニズムはこの状態の信頼性の高い前臨床モデル代表の取得の難しさのため、部分的に説明するは困難証明しました。大島らは、トラスツズマブ19治療後小さな残留腹膜転移の繰り返された文化から成るトラスツズマブ抵抗性胃癌細胞株の生体内での選択法を報告しました。ただし、筐体の特定動物取り扱い専門、動物倫理委員会の承認の必要性は、時間とコストのかかるメソッドにすることに貢献しました。この作品で述べる方法は簡単で、広く適用できると異なる腫瘍 histotypes20の他の治療薬に抵抗性のメカニズム解明のために簡単に拡張可能性があります。

<table><tbody><tr><td>Trizol Reagent</td><td>Invitrogen</td><td>15596026</td><td>TRIzol Reagent is a reagent for isolating high-quality total RNA or simultaneously isolating RNA, DNA, and protein from a variety of biological samples</td></tr><tr><td>ISCRIPT CDNA SYNTHESIS KIT</td><td>Bio-Rad</td><td>1708891</td><td>The iScript cDNA synthesis kit is a sensitive and easy-to-use first-strand cDNA synthesis kit for gene expression analysis using real-time qPCR.</td></tr><tr><td>TaqMan gene expression assay</td><td>Life Technologies</td><td>4331182</td><td>Applied Biosystems TaqMan Gene Expression Assays consist of a pair of unlabeled PCR primers and a TaqMan probe with an Applied Biosystems FAM or VIC dye label on the 5&amp;rsquo; end and minor groove binder (MGB) and nonfluorescent quencher (NFQ) on the 3&amp;rsquo; end.</td></tr><tr><td>M-PER Mammalian Protein Extraction Reagent</td><td>Thermo Scientific</td><td>78501</td><td>Thermo Scientific M-PER Mammalian Protein Extraction Reagent is designed to provide highly efficient total soluble protein extraction from cultured mammalian cells.</td></tr><tr><td>Halt Protease and Phosphatase Inhibitor Cocktail (100X)</td><td>Thermo Scientific</td><td>78440</td><td>Thermo Scientific Halt Protease and Phosphatase Inhibitor Cocktail (100X) provides the convenience of a single solution with full protein sample protection for cell and tissue lysates.</td></tr><tr><td>Pierce BCA Protein Assay Kit</td><td>Thermo Scientific</td><td>23225</td><td>The Thermo Scientific Pierce BCA Protein Assay Kit is a two-component, high-precision, detergent-compatible assay reagent set to measure (A562nm) total protein concentration compared to a protein standard.</td></tr><tr><td>4x Laemmli Sample Buffer</td><td>Bio-Rad</td><td>1610747</td><td>Use 4x Laemmli Sample Buffer for preparation of samples for SDS PAGE. For reduction of samples, add a reducing agent such as 2-mercaptoethanol to the buffer prior to mixing with the sample.</td></tr><tr><td>4&amp;ndash;20% Mini-PROTEAN TGX Precast Protein Gels</td><td>Bio-Rad</td><td>456-1094</td><td>Long-life TGX (Tris-Glycine eXtended) Gels have a novel formulation and can be used for both standard denaturing protein separations as well as native electrophoresis.</td></tr><tr><td>Precision Plus Protein WesternC Blotting Standards</td><td>Bio-Rad</td><td>1610376</td><td>Precision Plus Protein Prestained Standards are available in Dual Color, All Blue, Kaleidoscope, and Dual Xtra formats. All four have the same gel migration patterns, with 3 high-intensity reference bands (25, 50, and 75 kD).</td></tr><tr><td>10x Tris/Glycine/SDS</td><td>Bio-Rad</td><td>1610732</td><td>10x Tris/glycine/SDS is a premixed running buffer for separating protein samples by SDS-PAGE.</td></tr><tr><td>Trans-Blot Turbo Mini PVDF Transfer Packs</td><td>Bio-Rad</td><td>1704156</td><td>Trans-Blot Turbo Mini PVDF Transfer Packs for fast, efficient transfer of proteins from mini gels using the Trans-Blot Turbo Transfer System. Each vacuum sealed, ready-to-use transfer pack contains two buffer-saturated ion reservoir stacks and a prewetted PVDF membrane.</td></tr><tr><td>Precision Protein StrepTactin-HRP Conjugate</td><td>Bio-Rad</td><td>1610381</td><td>StrepTactin-Horseradish Peroxidase (HRP) Conjugate for chemiluminescent or colorimetric detection of Precision Plus Protein Unstained Protein Standards or Precision Plus Protein WesternC Protein Standards on western blots.</td></tr><tr><td>Immun-Star WesternC solution</td><td>Bio-Rad</td><td>5572</td><td>Immun-Star WesternC solution, a method of protein detection , detects mid-femtogram amounts of protein.</td></tr><tr><td>Universal Negative Control</td><td>Invitrogen</td><td>12935300</td><td>Negative Control siRNA has no significant sequence similarity to mouse, rat, or human gene sequences. The control has also been tested in cell-based screens and proven to have no significant effect on cell proliferation, viability, or morphology.</td></tr><tr><td>opti-MEM Glutamax Medium</td><td>Thermo Fisher Scientific</td><td>51985026</td><td>Opti-MEM Medium is an improved Minimal Essential Medium (MEM) that allows for a reduction of Fetal Bovine Serum supplementation by at least 50% with no change in growth rate or morphology.</td></tr><tr><td>Silencer Select Pre-Designed &amp;amp; Validated siRNA</td><td>Ambion</td><td>4390824</td><td>RNA interference (RNAi) is the best way to effectively knock down gene expression to study protein function in a wide range of cell types.</td></tr><tr><td>Lipofectamine RNAiMAX Transfection Reagent</td><td>Invitrogen</td><td>13778075</td><td>Lipofectamine RNAiMAX Transfection Reagent offers an advanced, efficient solution for siRNA delivery. No other siRNA specific transfection reagent provides such easy and efficient siRNA delivery in a wide variety of cell lines including common cell types, stem cells and primary cells, as well as traditionally hard-to-transfect cell types.</td></tr><tr><td>Mouse anti-IQGAP1</td><td>Invitrigen</td><td>33-8900</td><td>working concentration: 1:400 in 5% milk solution</td></tr><tr><td>goat anti-mouse IgG-HRP: sc-2005</td><td>Santa Cruz</td><td>sc-2005</td><td>working concentration: 1:10000 in 5% milk solution</td></tr><tr><td>PMSF</td><td>Sigma Aldrich</td><td>P 7626</td><td>this is an inhibitor of serine proteases and acetylcholinesterase</td></tr><tr><td>Thermocycler for RT-PCR</td><td>MJ Research</td><td>MJ Research PTC-200 Thermal Cycler</td><td>it allows temperature homogeneity and a ramping speed of up to 3&amp;deg;C/sec. The protocol can be performed also with other thermalcyclers</td></tr><tr><td>Real Time RT-PCR instrument</td><td>Applied Biosystems</td><td>7500 RT-PCR system</td><td>This is a five-color platform that uses fluorescence-based polymerase chain reaction (PCR) reagents to provide a relative quantification using comparative C&lt;sub&gt;T&lt;/sub&gt; assay type. The protocol can be performed also with other thermalcyclers</td></tr><tr><td>Microvolume Spectrophotometers</td><td>Thermo Scientific</td><td>Nanodrop</td><td>It provides an accurate and fast acid nucleid measurement using little volume of sample</td></tr><tr><td>Blotting system</td><td>Bio-Rad</td><td>Trans-Blot Turbo system</td><td>It perfoms a semy-dry transfer in a few minutes</td></tr><tr><td>Image acquisition system and gel documentation</td><td>Bio-Rad</td><td>Chemidoc image system</td><td>It is easy to use for chemiluminescent detection</td></tr></tbody></table>

looking for driver pathways of acquired resistance to targeted therapy: drug resistant subclone generation and sensitivity restoring by gene knock-down

過去 20 年間には、細胞毒性薬から医療がんの標的治療へのシフトを見てきました。標的治療薬より印象的な臨床効果と従来の治療法よりも最小化された副作用に示されてが、薬剤耐性の利点の主な制限となっています。臨地実習における標的薬抵抗性のいくつかの/体外・体内の前臨床モデルは、主に 2 つの戦略を使用して開発されている: 獲得抵抗性とii) の in vitro 遺伝子型をモデル化するための i) 遺伝子操作/生体内の抵抗性モデルの選択。本研究では薬剤耐性細胞のサブクローンの世代からの説明から始まって、ターゲット治療薬抵抗性獲得に責任があるメカニズムを調査するため統一的枠組を提案します。阻害剤に感度を復元するために使用するプロシージャを黙らせます。この単純な時間と費用の有効アプローチは広く適用できる、異なる腫瘍 histotypes の他のターゲット治療薬に抵抗性の機構を調査するために簡単に拡張できます。

図 1では、実験の手順のフレームワークについて説明します。HER2 とトラスツズマブ感受性の高レベルを表現する 3 胃癌細胞株 (IC50 < 薬物、図 2 a、左のグラフのピーク血漿中濃度) ターゲットを絞った治療剤を含む培養液中で育った。用量には、トラスツズマブ (10 μ G/ml) のピーク血漿中濃度は開始用量として設定?...

Watch this Scientific Journal Video about Looking for Driver Pathways of Acquired Resistance to Targeted Therapy: Drug Resistant Subclone Generation and Sensitivity Restoring by Gene Knock-down at JoV

Looking for Driver Pathways of Acquired Resistance to Targeted Therapy: Drug Resistant Subclone Generation and Sensitivity Restoring by Gene Knock-down

これは癌管理の非常に満たされていない医療ニーズである標的治療薬抵抗性獲得のメカニズムを調査時間および費用効果の in vitroプロトコルです。

標的治療への抵抗を取得ドライバーの経路を探して: 薬剤耐性 Subclone 世代と感受性遺伝子ノックダウンによる復元

The past two decades have seen a shift from cytotoxic drugs to targeted therapy in medical oncology. Although targeted therapeutic agents have shown more ...

looking-for-driver-pathways-acquired-resistance-to-targeted-therapy

Universidad San Sebastian

Research

JoVE Journal

Cancer Research

7.1K ビュー.  Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori (IRST) IRCCS. これは癌管理の非常に満たされていない医療ニーズである標的治療薬抵抗性獲得のメカニズムを調査時間および費用効果の in vitroプロトコルです。

ビデオ: Looking for Driver Pathways of Acquired Resistance to Targeted Therapy: Drug Resistant Subclone Generation and Sensitivity Restoring by Gene Knock-down

Using RNA-sequencing to Detect Novel Splice Variants Related to Drug Resistance in In Vitro Cancer Models

Drug resistance remains a major problem in the treatment of cancer for both hematological malignancies and solid tumors. Intrinsic or acquired resistance can be caused by a range of mechanisms, including increased drug elimination, decreased drug uptake, drug inactivation and alterations of drug targets. Recent data showed that other than by well-known genetic (mutation, amplification) and epigenetic (DNA hypermethylation, histone post-translational modification) modifications, drug resistance mechanisms might also be regulated by splicing aberrations. This is a rapidly growing field of investigation that deserves future attention in order to plan more effective therapeutic approaches. The protocol described in this paper is aimed at investigating the impact of aberrant splicing on drug resistance in solid tumors and hematological malignancies. To this goal, we analyzed the transcriptomic profiles of several in vitro models through RNA-seq and established a qRT-PCR based method to validate candidate genes. In particular, we evaluated the differential splicing of DDX5 and PKM transcripts. The aberrant splicing detected by the computational tool MATS was validated in leukemic cells, showing that different DDX5 splice variants are expressed in the parental vs. resistant cells. In these cells, we also observed a higher PKM2/PKM1 ratio, which was not detected in the Panc-1 gemcitabine-resistant counterpart compared to parental Panc-1 cells, suggesting a different mechanism of drug-resistance induced by gemcitabine exposure.

Using RNA-sequencing to Detect Novel Splice Variants Related to Drug Resistance in In Vitro Cancer Models

Here we describe a protocol aimed at investigating the impact of aberrant splicing on drug resistance in solid tumors and hematological malignancies. To this goal, we analyzed the transcriptomic profiles of parental and resistant in vitro models through RNA-seq and established a qRT-PCR based method to validate candidate genes.

に薬剤耐性に関連する新規スプライス変異体を検出するために、RNA配列決定を使用して、<em&gt;インビトロ</em&gt;がんモデル

Drug resistance remains a major problem in the treatment of cancer for both hematological malignancies and solid tumors. Intrinsic or acquired resistance ...

がん研究

Establishing Dual Resistance to EGFR-TKI and MET-TKI in Lung Adenocarcinoma Cells In Vitro with a 2-step Dose-escalation Procedure

Drug resistance is a major challenge in cancer therapy. The generation of resistant sublines in vitro is necessary for discovering novel mechanisms to overcome this challenge. Here, a 2-step dose-escalation method for establishing dual-resistance to an epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitor (TKI), gefitinib, and a MET-TKI, PHA665752, is described. This method is based on simple stepwise dose-escalation of inhibitors for inducing acquired resistance in cell lines. The alternate method for generating resistant sublines involves exposing the cells to high concentrations of the inhibitor in one step. The stepwise dose-escalation method has a higher possibility of successfully inducing acquired resistance than this method. Activating EGFR mutations are biomarkers of a response to treatment with EGFR-TKI, which is an applied first-line treatment for non-small cell lung cancers (NSCLC) that harbor these mutations. However, despite reports of effective responses, the use of EGFR-TKI is limited because tumors inevitably acquire resistance. The major mechanisms behind EGFR-TKI resistance include a secondary mutation at the gatekeeper site, T790M in exon 20 of EGFR, and a bypass signal of MET. Thus, a potential solution for this issue would be a combination of EGFR-TKI and MET-TKI. This combined treatment has been shown to be effective in an in vitro study model. Acquired gefitinib-resistance was established through MET-amplification by stepwise dose-escalation of gefitinib for 12 months, and a cell line named PC-9MET1000 was generated in a previous study. To further investigate the mechanisms of acquired MET-TKI and EGFR-TKI resistance, a MET-TKI, PHA665752, was administered to these cells with stepwise dose-escalation in the presence of gefitinib for 12 months. This protocol has also been successfully applied for a number of combination therapies to establish acquired resistance to other inhibitor molecules.

Establishing Dual Resistance to EGFR-TKI and MET-TKI in Lung Adenocarcinoma Cells In Vitro with a 2-step Dose-escalation Procedure

An in vitro method for establishing dual resistance to an EGFR-TKI and a MET-TKI in cancer cells is described. This method is useful for developing treatments for patients with EGFR-mutations, who exhibit disease progression despite EGFR-TKI treatment with MET-amplification. It can also be modified for inhibitors targeting other molecules.

肺腺癌細胞の in Vitro 2 ステップ増量の手順でデュアル抵抗性 EGFR TKI と会った TKI の確立

Drug resistance is a major challenge in cancer therapy. The generation of resistant sublines in vitro is necessary for discovering novel mechanisms to ...

Assessment of Resistance to Tyrosine Kinase Inhibitors by an Interrogation of Signal Transduction Pathways by Antibody Arrays

Cancer patients with an aberrant regulation of the protein phosphorylation networks are often treated with the tyrosine kinase inhibitors. Response rates approaching 85% are common. Unfortunately, patients often become refractory to the treatment by altering their signal transduction pathways. An implementation of the expression profiling with microarrays can identify the overall mRNA-level changes, and proteomics can identify the overall changes in protein levels or can identify the proteins involved, but the activity of the signal transduction pathways can only be established by interrogating post-translational modifications of the proteins. As a result, the ability to identify whether a drug treatment is successful or whether resistance arose, or the ability to characterize any alterations in the signaling pathways, is an important clinical challenge. Here, we provide a detailed explanation of antibody arrays as a tool which can identify system-wide alterations in various post-translational modifications (e.g., phosphorylation). One of the advantages of using antibody arrays includes their accessibility (an array does not require either an expert in proteomics or costly equipment) and speed. The availability of arrays targeting a combination of post-translational modifications is the primary limitation. In addition, unbiased approaches (phosphoproteomics) may be more suitable for the novel discovery, whereas antibody arrays are ideal for the most widely characterized targets.

Here, we present a protocol for antibody arrays to identify alterations in signaling pathways in various cellular models. These changes, caused by drugs/hypoxia/ultra-violet light/radiation, or by overexpression/downregulation/knockouts, are important for various disease models and can indicate whether a therapy will be effective or can identify mechanisms of drugs resistance.

抗体のアレイによるシグナル伝達経路の尋問でチロシンキナーゼ阻害剤に対する抵抗性の評価

Cancer patients with an aberrant regulation of the protein phosphorylation networks are often treated with the tyrosine kinase inhibitors. Response rates ...

Methods for Evaluating the Role of c-Fos and Dusp1 in Oncogene Dependence

The demonstration of tyrosine kinase inhibitors (TKIs) in treating chronic myeloid leukemia (CML) has heralded a new era in cancer therapeutics. However, a small population of cells does not respond to TKI treatment, resulting in minimal residual disease (MRD); even the most potent TKIs fail to eradicate these cells. These MRD cells serve as a reservoir to develop resistance to therapy. Why TKI treatment is ineffective against MRD cells is not known. Growth factor signaling is implicated in supporting the survival of MRD cells during TKI treatment, but a mechanistic understanding is lacking. Recent studies demonstrated that an elevated c-Fos and Dusp1 expression as a result of convergent oncogenic and growth factor signaling in MRD cells mediate TKI resistance. The genetic and chemical inhibition of c-Fos and Dusp1 renders CML exquisitely sensitive to TKIs and cures CML in both genetic and humanized mouse models. We identified these target genes using multiple microarrays from TKI-sensitive and -resistant cells. Here, we provide methods for target validation using in vitro and in vivo mouse models. These methods can easily be applied to any target for genetic validation and therapeutic development.

Here, we describe protocols for the genetic and chemical validation of c-Fos and Dusp1 as a drug target in leukemia using in vitro and in vivo genetic and humanized mouse models. This method can be applied to any target for genetic validation and therapeutic development.

C-fos の役割と Dusp1 の遺伝子依存性の評価方法

The demonstration of tyrosine kinase inhibitors (TKIs) in treating chronic myeloid leukemia (CML) has heralded a new era in cancer therapeutics. However, ...

Establishing Cell Lines Overexpressing DR3 to Assess the Apoptotic Response to Anti-mitotic Therapeutics

Studying the function of a gene of interest can be achieved by manipulating its level of expression, such as decreasing its expression with knockdown cell lines or increasing its expression with overexpression cell lines. Transient and stable transfection are two methods that are often used for exogenous gene expression. Transient transfection is only useful for short-term expression, whereas stable transfection allows exogenous genes to be integrated into the host cell genome where it will be continuously expressed. As a result, stable transfection is usually employed for research into long-term genetic regulation. Here we describe a simple protocol to generate a stable cell line overexpressing tagged death receptor 3 (DR3) to explore DR3 function. We picked single clones after a retroviral infection in order to maintain the homogeneity and purity of the stable cell lines. The stable cell lines generated using this protocol render DR3-deficient HT29 cells sensitive to antimitotic drugs, thus reconstituting the apoptotic response in HT29 cells. Moreover, the FLAG tag on DR3 compensates for the unavailability of good DR3 antibody and facilitates the biochemical study of the molecular mechanism by which antimitotic agents induce apoptosis.

Establishing a stable cell line overexpressing a gene of interest to study gene function can be done by stable transfection-picking single clones after transfecting them via retroviral infection. Here we show that HT29-DR3 cell lines generated in this way elucidate the mechanisms by which death receptor 3 (DR3) contributes to antimitotics-induced apoptosis.

抗有糸分裂治療に対するアポトーシス応答を評価するために DR3 を過剰発現細胞株の確立

Studying the function of a gene of interest can be achieved by manipulating its level of expression, such as decreasing its expression with knockdown cell ...

Establishment and Characterization of Three Afatinib-resistant Lung Adenocarcinoma PC-9 Cell Lines Developed with Increasing Doses of Afatinib

Acquired resistance to molecular target inhibitors is a severe problem in cancer therapy. Lung cancer remains the leading cause of cancer-related death in most countries. The discovery of "oncogenic driver mutations," such as epidermal growth factor receptor (EGFR)-activating mutations, and subsequent development of molecular targeted agents of EGFR tyrosine kinase inhibitors (TKIs) (gefitinib, erlotinib, afatinib, dacomitinib, and osimertinib) have dramatically altered lung cancer treatment in recent decades. However, these drugs are still not effective in patients with non-small cell lung cancer (NSCLC) carrying EGFR-activating mutations. Following acquired resistance, the systemic progression of NSCLC remains a significant obstacle in treating patients with EGFR mutation-positive NSCLC. Here, we present a stepwise dose escalation method for establishing three independent acquired afatinib-resistant cell lines from NSCLC PC-9 cells harboring EGFR-activating mutations of 15-base pair deletions in EGFR exon 19. Methods for characterizing the three independent afatinib-resistance cell lines are briefly presented. The acquired resistance mechanisms to EGFR TKIs are heterogeneous. Therefore, multiple cell lines with acquired resistance to EGFR-TKIs must be examined. Ten to twelve months are required to obtain cell lines with acquired resistance using this stepwise dose escalation approach. The discovery of novel acquired resistance mechanisms will contribute to the development of more effective and safe therapeutic strategies.

A method for establishing afatinib-resistance cell lines from lung adenocarcinoma PC-9 cells was developed, and resistant cells were characterized. The resistant cells can be used to investigate epidermal growth factor receptor tyrosine kinase inhibitor-resistance mechanisms, applicable for patients with non-small cell lung cancer.

アファチニブ耐性肺腺癌PC-9細胞株の確立と特徴付けは、アファチニブの用量の増加とともに開発された

Acquired resistance to molecular target inhibitors is a severe problem in cancer therapy. Lung cancer remains the leading cause of cancer-related death in ...

Pooled shRNA Library Screening to Identify Factors that Modulate a Drug Resistance Phenotype

Understanding clinically relevant driver mechanisms of acquired chemo-resistance is crucial for elucidating ways to circumvent resistance and improve survival in patients with acute myeloid leukemia (AML). A small fraction of leukemic cells that survive chemotherapy have a poised epigenetic state to tolerate chemotherapeutic insult. Further exposure to chemotherapy allows these drug persister cells to attain a fixed epigenetic state, which leads to altered gene expression, resulting in the proliferation of these drug-resistant populations and eventually relapse or refractory disease. Therefore, identifying epigenetic modulations that necessitate the survival of drug-resistant leukemic cells is critical. We detail a protocol to identify epigenetic modulators that mediate resistance to the nucleoside analog cytarabine (AraC) using pooled shRNA library screening in an acquired cytarabine-resistant AML cell line. The library consists of 5,485 shRNA constructs targeting 407 human epigenetic factors, which allows high-throughput epigenetic factor screening.

High-throughput RNA interference (RNAi) screening using a pool of lentiviral shRNAs can be a tool to detect therapeutically relevant synthetic lethal targets in malignancies. We provide a pooled shRNA screening approach to investigate the epigenetic effectors in acute myeloid leukemia (AML).

薬物耐性表現型を調節する因子を同定するためのプールされたshRNAライブラリースクリーニング

Understanding clinically relevant driver mechanisms of acquired chemo-resistance is crucial for elucidating ways to circumvent resistance and improve ...

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

Implementation of In Vitro Drug Resistance Assays: Maximizing the Potential for Uncovering Clinically Relevant Resistance Mechanisms

Although targeted therapies are initially effective, resistance inevitably emerges. Several methods, such as genetic analysis of resistant clinical specimens, have been applied to uncover these resistance mechanisms to facilitate follow-up care. Although these approaches have led to clinically relevant discoveries, difficulties in attaining the relevant patient material or in deconvoluting the genomic data collected from these specimens have severely hampered the path towards a cure. To this end, we here describe a tool for expeditious discovery that may guide improvement in first-line therapies and alternative clinical management strategies. By coupling preclinical in vitro or in vivo drug selection with next-generation sequencing, it is possible to identify genomic structural variations and/or gene expression alterations that may serve as functional drivers of resistance. This approach facilitates the spontaneous emergence of alterations, enhancing the probability that these mechanisms may be observed in the patients. In this protocol we provide guidelines to maximize the potential for uncovering single nucleotide variants that drive resistance using adherent lines.

Implementation of In Vitro Drug Resistance Assays: Maximizing the Potential for Uncovering Clinically Relevant Resistance Mechanisms

Drug resistance to targeted therapeutics is widespread and the need to identify mechanisms of resistance--prior to or following clinical onset--is critical for guiding alternative clinical management strategies. Here, we present a protocol to couple derivation of drug-resistant lines in vitro with sequencing to expedite discovery of these mechanisms.

の実装<em&gt;インビトロ</em&gt;薬剤耐性アッセイ：臨床的に関連性のメカニズムを暴くための可能性を最大化

Although targeted therapies are initially effective, resistance inevitably emerges. Several methods, such as genetic analysis of resistant clinical ...

A Method for Screening and Validation of Resistant Mutations Against Kinase Inhibitors

The discovery of BCR/ABL as a driver oncogene in chronic myeloid leukemia (CML) resulted in the development of Imatinib, which, in fact, demonstrated the potential of targeting the kinase in cancers by effectively treating the CML patients. This observation revolutionized drug development to target the oncogenic kinases implicated in various other malignancies, such as, EGFR, B-RAF, KIT and PDGFRs. However, one major drawback of anti-kinase therapies is the emergence of drug resistance mutations rendering the target to have reduced or lost affinity for the drug. Understanding the mechanisms employed by resistant variants not only helps in developing the next generation inhibitors but also gives impetus to clinical management using personalized medicine. We reported a retroviral vector based screening strategy to identify the spectrum of resistance conferring mutations in BCR/ABL, which has helped in developing the next generation BCR/ABL inhibitors. Using Ruxolitinib and JAK2 as a drug target pair, here we describe in vitro screening methods that utilizes the mouse BAF3 cells expressing the random mutation library of JAK2 kinase.

Emergence of genetic resistance against kinase inhibitor therapy poses significant challenge for effective cancer therapy. Identification and characterization of resistant mutations against a newly developed drug helps in better clinical management and next generation drug design. Here, we describe our protocol for in vitro screening and validation of resistant mutations.

キナーゼ阻害剤に対して耐性変異のスクリーニングおよび検証のための方法

The discovery of BCR/ABL as a driver oncogene in chronic myeloid leukemia (CML) resulted in the development of Imatinib, which, in fact, demonstrated the ...

標的治療への抵抗を取得ドライバーの経路を探して: 薬剤耐性 Subclone 世代と感受性遺伝子ノックダウンによる復元

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に薬剤耐性に関連する新規スプライス変異体を検出するために、RNA配列決定を使用して、