Name: evaluating the effectiveness of cancer drug sensitization in vitro and in vivo
Uploaded: 2015-02-06T14:00:00.000+00:00
Duration: 9 min 19 s
Description: Watch this Scientific Journal Video about Evaluating the Effectiveness of Cancer Drug Sensitization In Vitro and In Vivo at JoVE.com

この作品は、優秀オンタリオセンターとオンタリオ研究基金からJKへの補助金によって可能となった。 私たちは、撮影時の技術支援のためSiddika Pardhanとピーター·ファーガソンに感謝したいと思います。

注：以下のプロトコルは、接着細胞で使用するために設計されました。修飾は、懸濁液中で増殖させた非接着細胞に方法を適用することが必要である。プロトコールに記載されてCAM実験は、蛍光マーカー（ 例えば 、GFP、RFP など ） を発現する細胞で使用するために設計されている。ナイン日齢のニワトリ胚は、CAM実験のための実験プロトコルの2日目に必要とされる。 1.準備およびアンチセンスオリゴヌクレオチドによる腫瘍細胞のトランスフェクション（ASOは） <ol><li>興味のある細胞を含むT75フラスコ（S）から吸引する媒体は、1×PBSで洗浄し、0.25％トリプシン/ EDTAの2ミリリットルを追加します。各フラスコに完全増殖培地10mlを加え、50mlチューブに溶液中に細胞を移す。細胞をカウントし、最終濃度が1ml当たり5個の細胞1.0×10になるように音量を調整。 </li><li> T25フラスコのラベルつのグループ：1グループは1-8ラベル8フラスコを、含まれています、とuになりますリアルタイム代謝測定実験のためのsed。第2のグループはまた、8フラスコを含有し、 ニワトリ胚のCAM実験のために使用される。インキュベータ内の各フラスコ場所（37℃、5％CO 2 O / N）に、溶液中の細胞を2ml加える。 </li><li>翌朝（0日、時間0）、ラベル3 5ミリリットルのチューブを次のようにコントロール（非標的ASOまたはsiRNA）、「目的の標的」（すなわち 、標的とASOまたはsiRNA）、およびリポフェクタミン2000（または類似のトランスフェクション試薬）。適切なチューブで、無血清培地中で適切な濃度に株式ASOソリューションを希釈する。 <ol><li>適切なチューブにトランスフェクション試薬の必要量を添加し、5分間、無血清培地中でインキュベートする。 ASOチューブにトランスフェクション試薬溶液を加え、20分間インキュベートする。 </li></ol></li><li>各グループ内でのフラスコ1-4のそれぞれに制御ASOトランスフェクション試薬溶液の250μlのを追加し、250各群のフラスコ5-8のそれぞれに「目的の標的」ASOトランスフェクション試薬溶液μlの。 4時間（37ᵒC、5％CO 2）インキュベートする。 <ol><li>インキュベーションの間、2.1節で説明する手順を実行する。インキュベーション後、CAM実験群で各フラスコに、完全培地3mlを追加し、インキュベータO / N（37ᵒC、5％CO 2）に戻る。 セクション2.2の記載に従って代謝の実験グループを処理します。 セクション4の記述に従って設定CAM実験を処理します。 </li></ol></li></ol> 2.バイオセンサーチップの調製と細胞播種<ol><li>無菌環境では、慎重にPBS400μlの6バイオセンサーチップを洗う。 20分間の70％エタノール400μlのチップを滅菌する。 PBS400μlの3回洗浄する。 <ol><li>滅菌組織cultur内部のチップを置きますeは皿汚染を防止する。 「コントロール」ラベル皿に3つのチップを置き、「関心の対象」というラベル皿の中の3。 </li></ol></li><li> 「コントロール」と「目的の標的」として2 50mlチューブにラベルを付けます。 PBSで細胞を洗浄し、0.25％トリプシン/ EDTAの適切なボリュームを追加。単層が外れるまで待ってから、各フラスコから細胞を収集する。適切なチューブに溶液を堆積させる。 <ol><li>細胞を計数し、最終濃度6.0x10 5細胞/ mlとなるように音量を調整する。初期濃度が低すぎる場合、細胞を遠心分離し、再懸濁する。 </li></ol></li><li> （チップ当たり1.25×10 5細胞の合計）適切なグループ内の各バイオセンサーチップに細胞溶液の250μlのを追加します。チップの混乱のリスクを最小限に抑えるために、随時、このタスクを1グループを実行します。蒸発を防ぐために、湿度室内にチップを含む滅菌皿を置き、湿度茶を配置mberインキュベーター中でO / N（37ᵒC、5％CO 2）。 </li><li>翌朝（1日目）は、慎重に各チップから培地を吸引し、0.2％ウシ胎児血清（FBS）および1×ペニシリン及びストレプトマイシン（P / S）を補充した増殖培地を250μlと交換してください。 4時間（37ᵒC、5％CO 2）インキュベートする。 </li></ol> 3.代謝測定システム準備及び溶液の調製<ol><li>インキュベーションの間、必要な薬剤溶液と今後の分析のための代謝測定システムを準備する。 注：代謝測定システムは、6つのモジュールの合計が含まれています。各モジュールは、実験ごとに1つのチップに対応しています。 <ol><li>アッセイの間、車両を受け取るために「コントロール」とグループ「目的の標的」のそれぞれから1チップと、次の実験群に検定を細分化A中に定義された期間のための薬物治療を受けるために、各群から2チップssay、ウォッシュアウト期間中に車両が続く。 </li><li>薬物感研究のために、問題の特定の薬剤に合わせて薬物治療の長さ、タイミング、および濃度を変更する。 NOTE：シスプラチンについては、以下のステップがA549細胞を経験的に決定し、細胞株に依存して変化し得る。 4μL/分のデフォルトの流量ですべての段階を実行します。 増殖培地+ 0.2％FBS及び1×P / Sの6時間 培地中の6μMシスプラチンの24時間+ 0.2％FBS、1X P / S 増殖培地+ 0.2％FBS及び1×P / Sの24時間 増殖培地+ 0.2％FBS及び1×P / Sの18時間 0.1％のTriton-Xの4時間 注：これらの手順からの逸脱は、所望の流量と実験の期間に基づいて、メディアと薬物ソリューションの必要量を決定するための計算が必要になります。以下の節では、上記の工程に基づいている。 </li></ol></li><li>ラベル増殖培地+ 0.2％FBSのAN 50mlで14 50mlチューブに充填dは1×P / S。また、シスプラチンとのチューブが含まれている第三のチューブラックに1代謝測定システム管ラックに6、別の6、および残りの2を配置します。蒸発を防ぐために、空気透過性膜との最初の二つのラックをシール。 </li><li> 1X P / Sを含む培地+ 0.2％FBS中で（適切なボリュームの）6100μMのシスプラチンの溶液を調製する。 4ラベル5​​0ミリリットルチューブにシスプラチン液の必要量を分注する。 <ol><li>第三の代謝測定管ラックに残りの4つのスロットにチューブを置きます。チューブは希望biomodulesに対応し、適切なスロットに配置されていることを確認。空気透過性膜を有する管をシール。 </li></ol></li><li>準備し、標識6 50mlチューブを20 mlの培地中0.1％のTriton-Xの溶液のそれぞれに、それらを埋める。トリトンX溶液が残った細胞を溶解し、各チップ用の「ゼロ」値を提供する。第四の代謝測定単位管ラックにチューブを配置し、ウィットをシールH過度の蒸発を防ぐために空気permable膜。 </li><li> biomodulesにチップをロードし、実験を開始する。 </li></ol> 4. CAM実験薬物治療、注射注：CAM実験にニワトリ胚を調製するための手順は、手順は他の場所19に記載されている<ol><li> CAM実験 48時間のトランスフェクション後（2日目）の処理を開始します。 「コントロール」と車両のための2つのフラスコにグループ「目的の標的」、及びシスプラチンのための2つのサブ分割する。吸引し、フラスコから培地を新鮮な培地の3ミリリットル、または6μMシスプラチンのいずれかで補充する。 6時間（37ᵒC、5％CO 2）インキュベートする。 </li><li>洗った後、培地を吸引し、細胞をトリプシン処理。 4適切に標識15ミリリットルチューブに溶液中のトリプシン処理した細胞を収集します。 <ol><li> PBSで細胞を3回遠心機および洗浄。 5mlのPBSで細胞ペレットを再懸濁する細胞を数えるdは。 </li><li>最終細胞濃度が1.0×10 6細胞/ mlとなるように音量を調整する。注射のためにラベル5ミリリットルチューブに細胞溶液を移し、氷上に置きます。細胞が十分に混合されることを保証するために、注射の前に静かにチューブを反転。 </li></ol></li><li>注射のための（9日齢）24ニワトリ胚（グループあたり6）の合計を脇に置きます。 1mlシリンジに接続されたフレキシブルチューブに取り付けたガラス針を用いて注射を行う。噴射を行う個人は、治療群を知らされるべきである。 <ol><li>実体顕微鏡を用いて、CAMの静脈循環に100μlの容量で1.0×10 5個の細胞を注入する。細胞のフルボリュームを注入するために遅く、脈動技術を使用してください。柔らかい血液の流れを止めると、任意のこぼれを吸収するためにワイプで注射部位を軽くたたく。適切に各注射後の各胚コンテナにラベルを付けます。 </li></ol></li><li>存在感とdistriの確認蛍光顕微鏡を使用してそれらを視覚化することによって注射後のCAMの血管系における蛍光細胞のbution。腫瘍細胞の数が少ない表示され、または配布が悪いされる任意の胚に注意してください。 </li><li>湿度チャンバー内でニワトリ胚を置き、インキュベーター（37℃、湿度60％）に格納します。転移巣を列挙する前に7-9日間待ちます。 </li></ol> 5.転移巣の列挙<ol><li> 7-9日のインキュベーション期間の間、胚を監視し、死んだものを廃棄する。湿度室は、潜伏期間中、常に湿ったままであることを確認してください。 </li><li> 20Xの倍率で蛍光実体顕微鏡を用いて、規則的な格子状パターンで胚CAMの上面全体を走査する。 </li><li>その後胚当たりの最終合計数を集計、各視野における転移病巣の数を数える。各グループの6胚についてそうすることで平均数Oを提供します処置当たりF転移巣。 </li></ol>

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著者らは、開示することは何もない。

による臨床試験に関連する固有のコストとリスクを、十分に新規の抗癌治療レジメンを評価するために、より良く、より厳密な前臨床試験の方法論を開発する必要がある。現在一般的に使用される技術は、潜在的に有望な治療標的/薬剤とを識別する能力を制限し、あまり効果的であり得るものがあり、すべての展示弱点。我々は、従来の方法の欠点の多くに対処する新規な抗癌アプローチを...

標的および/ ​​または細胞傷害性癌治療に対する獲得 ​​耐性は治療の失敗、再発、および増加した患者の死亡率1をもたらすことができる重要な臨床的問題である。ほとんどの腫瘍における不均一性の高いレベルを考えると、十分に高い細胞数の腫瘍は、それらの細胞は生存のために2,3依存している分子経路を標的とする単一または併用療法に耐性の細胞のサブセットを含むこと数学的確実性である。このような腫瘍細胞は積極的疾患の再発につながる、治療中に選択することができる。同時に治療媒介選択の前または後のいずれかで、異なる癌細胞の生存メカニズムを標的とする新規治療法の開発は、このように臨床的に重要である。 腫瘍ゲノムにおけるゲノム不安定性および突然変異の高レベルは、非腫瘍宿主細胞4から癌細胞を区別する基本的な特性である。その結果、usefuDNA損傷化学療法の有効性を高め、抵抗性の発達を防止するために、Lの戦略は、積極的に腫瘍細胞5にDNA修復を阻害することである。これは、調査のアクティブなフィールドであり、新規なDNA修復対象の様々な前臨床設定において探索されている。これらの標的の小分子またはアンチセンスベースの阻害剤が開発されているとテスト6-8を受けている。目的は、最も有望な前臨床候補を特定し、臨床試験におけるそれらの安全性および有効性を評価することである。 臨床試験のコストが高く、（次善の前臨床評価を含む様々な理由のために）失敗のリスクは、新しい治療法9の開発に進行する手ごわい障害となっている。十分新しい治療標的および候補薬物を評価するための適切な厳密な前臨床モデルの使用は、臨床試験の10の高い故障率を減少させることができる</suP&gt;。 新規な抗癌療法の有効性を評価するためのいくつかの一般的に使用される前臨床の方法がある： インビトロ （細胞増殖アッセイ） 腫瘍細胞増殖を減少させる能力は、a）測定に対する腫瘍細胞の能力のa、b）療法誘発性の減少フォームの組織培養コロニー（コロニー形成アッセイ） インビトロ （レドックス染料変換） における腫瘍細胞の代謝活性におけるc）治療誘発性の減少、関連するインビトロの腫瘍細胞死（アポトーシス、壊死、オートファジーのd）の治療によって誘導される誘導インビボにおける有糸分裂等）11、及びe）ヒトおよびマウス異種移植片12-14の成長またはアブレーションの減少を治療によって誘発される。 記載されているインビトロの方法の主な弱点は、それらのどれも、候補治療法の効果の連続リアルタイム評価を提供しないことである。むしろ、それらは、選択された、広く分離された時点での情報を提供治療の過程。このような測定は、正確に腫瘍細胞応答の大きさおよびタイミングを反映する能力が減少している。 インビボのマウス異種移植モデルは、高コスト、完了するまでの時間の長さは、サブ最適な投与および治療 ​​のタイミング（スケジューリング）の危険性によって制限される。また、齧歯類異種移植モデルは、候補治療的介入への一次ヒト腫瘍細胞および樹立されたヒト腫瘍細胞株の応答15,16 のインビトロ評価に比べて、ヒトにおける臨床的有効性の限られた予測因子であるという証拠がある。 我々は、上記の一般的な手順の弱点に対処するようにして、前臨床の新しい薬物の組み合わせを評価するための新規な組合せのプロトコルを考案した。増殖、コロニー形成、またはレドックス色素変換アッセイの代わりに、我々は、リアルタイムで、細胞接着、呼吸、および酸性化を分析するために、代謝測定部を利用全体治療期間17の間に。同時に、我々は、浸潤および転移18,19のニワトリ胚漿尿膜（CAM）モデルを用いてインビボでの治療の組み合わせの効果を調べた。我々は、一般的に使用される化学療法剤シスプラチンの有効性を増強するBRCA2を標的とするアンチセンスオリゴヌクレオチド（ASO）の能力を評価するためにこれらの方法を使用する。

<table><tbody><tr><td>A549 cells</td><td>ATCC</td><td>CCL-185</td><td>http://www.atcc.org/products/all/CCL-185.aspx</td></tr><tr><td>AeraSeal film</td><td>Carl Roth GmbH</td><td></td><td></td></tr><tr><td>AMEM</td><td>Wisent Bioproducts</td><td>210-011-QK</td><td></td></tr><tr><td>Antisense oligodeoxynucleotides</td><td>Avecia</td><td></td><td>BRCA2 target sequence: 5&#039; - UAAGGAACGUCAAGAGAUAC - 3&#039; (bases 7241-7259 )</td></tr><tr><td>Axio Zoom V16 Microscope</td><td>Carl Zeiss</td><td></td><td>http://www.zeiss.ca/microscopy/en_ca/products/stereo-zoom-microscopes/axio-zoom-v16.html</td></tr><tr><td>Bionas Biosensor Chips</td><td>Bionas GmbH and Micronas GmbH</td><td>BIS8001D</td><td></td></tr><tr><td>Bionas Discovery 2500 System&amp;nbsp;</td><td>Bionas GmbH</td><td></td><td>http://www.bionas-discovery.com/prodservices/instruments/system2500/</td></tr><tr><td>Cisplatin</td><td>Sigma Aldrich</td><td>479306</td><td></td></tr><tr><td>Fertilized chicken eggs</td><td>Sourced locally</td><td></td><td></td></tr><tr><td>Fetal bovine serum&amp;nbsp;</td><td>Gibco - Life Technologies</td><td></td><td></td></tr><tr><td>Lipofectamine 2000&amp;nbsp;</td><td>Invitrogen - Life Technologies&amp;nbsp;</td><td>12566014</td><td>http://www.lifetechnologies.com/ca/en/home/life-science/protein-expression-and-analysis/transfection-selection/lipofectamine-2000.html</td></tr><tr><td>PBS</td><td>Wisent Bioproducts</td><td>311-010-CL</td><td></td></tr><tr><td>Trypsin (0.25%)/EDTA</td><td>Wisent Bioproducts</td><td>325-043-CL</td><td></td></tr></tbody></table>

evaluating the effectiveness of cancer drug sensitization in vitro and in vivo

による異質と同じ経路を標的ヒト癌、単剤療法、または併用レジメンに固有の変異のハイレベルに、失敗する可能性がある。重点は、治療への内因性および/または適応耐性に関与している経路の阻害の際に配置する必要があります。研究の活発な分野での作用を促進するDNA修復阻害剤の開発および試験され、そして、一般的に使用される化学療法および放射線療法に対する耐性を防ぐ。我々はDNA損傷薬剤シスプラチンに対して腫瘍細胞を感作するための手段としてBRCA2阻害の有効性を評価するための新規なプロトコルを使用する。腫瘍細胞の代謝（酸性および呼吸）分毎分で治療効果を描写するために72時間にわたりリアルタイムでモニターした。組み合わせでは、血管外遊出および浸潤のニワトリ胚漿尿膜（CAM）モデルを使用して、転移頻度の評価を行った。このプロトコルは、一般的に、新規な癌治療レジメンを評価するために、インビトロおよびインビボの方法を使用するの短所のいくつかに対処する。これは、より密接に候補標的及び薬剤間を区別し、さらなる発展のためには、最も有望な候補を選択するために、そのような細胞増殖アッセイ、細胞死アッセイ、およびin vivoのマウス異種移植研究におけるような一般的な方法に加えて使用することができる。

結果と数字は発表された研究22からの許可を得て適応さ。 BRCA2阻害は、シスプラチン処理した腫瘍細胞における呼吸の減少を誘導する BRCA2ターゲティングASOおよびシスプラチンで処理したヒトA549肺癌細胞は、対照ASOとシスプラチン単独を受けた細胞と比較して、呼吸における初期不可逆減少を示した。シスプラチン処理の24時間後にBR...

Watch this Scientific Journal Video about Evaluating the Effectiveness of Cancer Drug Sensitization In Vitro and In Vivo at JoVE.com

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 ...

evaluating-effectiveness-cancer-drug-sensitization-vitro

Research

JoVE Journal

Medicine

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

8.3K ビュー.  Western University. 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. 

ビデオ: Evaluating the Effectiveness of Cancer Drug Sensitization In Vitro and In Vivo

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 ...

がん研究

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

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 respond to therapeutic compounds in 2D (dimensional) monolayer cell cultures, 3D culture systems were developed to understand the efficacy of drugs in more physiologically relevant models. In recent years, a paradigm shift was observed in pre-clinical research to validate the potency of new molecules in 3D culture systems, more precisely mimicking the tumor microenvironment. These systems characterize the disease state in a more physiologically relevant manner and help to gain better mechanistic insight and understanding of the pharmacological potency of a given molecule. Moreover, with the current trend in improving in vivo cancer models, zebrafish has emerged as an important vertebrate model to assess in vivo tumor formation and study the effect of therapeutic agents. Here, we investigated the therapeutic efficacy of hydroxycoumarin OT48 alone or in combination with BH3 mimetics in lung cancer cell line A549 by using three different 3D culture systems including colony formation assays (CFA), spheroid formation assay (SFA) and in vivo zebrafish xenografts.

Here, we present the preclinical screening of anticancer coumarins using 3D culture and zebrafish.

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

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 ...

Potentiation of Anticancer Antibody Efficacy by Antineoplastic Drugs: Detection of Antibody-drug Synergism Using the Combination Index Equation

Potentiation of hostile monoclonal antibodies (mAb) by chemotherapeutic agents constitutes a valuable strategy for designing effective and safer therapy against cancer. Here we provide a protocol to identify a rational combination at the preclinical step. First, we describe a cell-based assay to assess the synergism between anticancer mAb and cytotoxic drugs, that uses the combination index equation of Chou and Talalay1. This includes the measurement of tumor cell drug- and antibody-sensitivity using an MTT assay, followed by an automated computer analysis to calculate the combination index (CI) values. CI values of &lt;1 indicate synergism between tested mAbs and cytotoxic agents1. To corroborate the in vitro findings in vivo, we further describe a method to assess the combination regimen efficacy in a xenograft tumor model. In this model, the combined regimen significantly delays tumor growth, which results in a significant extended survival in comparison to single-agent controls. Importantly, the in vivo experimentation reveals that the combination regimen is well tolerated. This protocol allows the effective evaluation of anticancer drug combinations in preclinical models and the identification of rational combination to evaluate in clinical trials.

This protocol describes how to assess synergism between an anticancer antibody and antineoplastic drugs in preclinical models by using the combination index equation of Chou and Talalay.

抗がん剤抗体抗悪性腫瘍薬による効果の増強: 組み合わせインデックス式を用いた抗体医薬の相乗作用の検出

Potentiation of hostile monoclonal antibodies (mAb) by chemotherapeutic agents constitutes a valuable strategy for designing effective and safer therapy ...

In Vitro Tumor Cell Rechallenge For Predictive Evaluation of Chimeric Antigen Receptor T Cell Antitumor Function

The field of chimeric antigen receptor (CAR) T cell therapy is rapidly advancing with improvements in CAR design, gene-engineering approaches and manufacturing optimizations. One challenge for these development efforts, however, has been the establishment of in vitro assays that can robustly inform selection of the optimal CAR T cell products for in vivo therapeutic success. Standard in vitro tumor-lysis assays often fail to reflect the true antitumor potential of the CAR T cells due to the relatively short co-culture time and high T cell to tumor ratio. Here, we describe an in vitro co-culture method to evaluate CAR T cell recursive killing potential at high tumor cell loads. In this assay, long-term cytotoxic function and proliferative capacity of CAR T cells is examined in vitro over 7 days with additional tumor targets administered to the co-culture every other day. This assay can be coupled with profiling T cell activation, exhaustion and memory phenotypes. Using this assay, we have successfully distinguished the functional and phenotypic differences between CD4+ and CD8+ CAR T cells against glioblastoma (GBM) cells, reflecting their differential in vivo antitumor activity in orthotopic xenograft models. This method provides a facile approach to assess CAR T cell potency and to elucidate the functional variations across different CAR T cell products.

Here, we describe an in vitro co-culture method to recursively challenge tumor-targeted T cells, which allows for phenotypic and functional analysis of antitumor T cell activity.

キメラ抗原受容体の T 細胞の抗腫瘍機能の予測評価のため培養腫瘍細胞の再投与に

The field of chimeric antigen receptor (CAR) T cell therapy is rapidly advancing with improvements in CAR design, gene-engineering approaches and ...

Drug-induced Sensitization of Adenylyl Cyclase: Assay Streamlining and Miniaturization for Small Molecule and siRNA Screening Applications

Sensitization of adenylyl cyclase (AC) signaling has been implicated in a variety of neuropsychiatric and neurologic disorders including substance abuse and Parkinson&#39;s disease. Acute activation of G&#945;i/o-linked receptors inhibits AC activity, whereas persistent activation of these receptors results in heterologous sensitization of AC and increased levels of intracellular cAMP. Previous studies have demonstrated that this enhancement of AC responsiveness is observed both in vitro and in vivo following the chronic activation of several types of G&#945;i/o-linked receptors including D2 dopamine and &#956; opioid receptors. Although heterologous sensitization of AC was first reported four decades ago, the mechanism(s) that underlie this phenomenon remain largely unknown. The lack of mechanistic data presumably reflects the complexity involved with this adaptive response, suggesting that nonbiased approaches could aid in identifying the molecular pathways involved in heterologous sensitization of AC. Previous studies have implicated kinase and Gb&#947; signaling as overlapping components that regulate the heterologous sensitization of AC. To identify unique and additional overlapping targets associated with sensitization of AC, the development and validation of a scalable cAMP sensitization assay is required for greater throughput. Previous approaches to study sensitization are generally cumbersome involving continuous cell culture maintenance as well as a complex methodology for measuring cAMP accumulation that involves multiple wash steps. Thus, the development of a robust cell-based assay that can be used for high throughput screening (HTS) in a 384&#160;well format would facilitate future studies. Using two D2 dopamine receptor cellular models (i.e.&#160;CHO-D2L and HEK-AC6/D2L), we have converted our 48-well sensitization assay (&#62;20 steps 4-5 days) to a five-step, single day assay in 384-well format. This new format is amenable to small molecule screening, and we demonstrate that this assay design can also be readily used for reverse transfection of siRNA in anticipation of targeted siRNA library screening.

Persistent activation of inhibitory G protein-coupled receptors results in sensitization of adenylyl cyclase signaling. To identify the essential molecular pathways, nonbiased approaches are necessary; however, this strategy requires the development of a scalable cell-based cAMP sensitization assay. Herein, we describe a sensitization assay for small molecule and siRNA screening.

アデニリルシクラーゼの薬物誘発性感作：アッセイは、小分子およびsiRNAスクリーニング用途のために合理化·小型化

Sensitization of adenylyl cyclase (AC) signaling has been implicated in a variety of neuropsychiatric and neurologic disorders including substance abuse ...

生物工学

Patient-Derived Tumor Explants As a "Live" Preclinical Platform for Predicting Drug Resistance in Patients

An understanding of drug resistance and the development of novel strategies to sensitize highly resistant cancers rely on the availability of suitable preclinical models that can accurately predict patient responses. One of the disadvantages of existing preclinical models is the inability to contextually preserve the human tumor microenvironment (TME) and accurately represent intratumoral heterogeneity, thus limiting the clinical translation of data. By contrast, by representing the culture of live fragments of human tumors, the patient-derived explant (PDE) platform allows drug responses to be examined in a three-dimensional (3D) context that mirrors the pathological and architectural features of the original tumors as closely as possible. Previous reports with PDEs have documented the ability of the platform to distinguish chemosensitive from chemoresistant tumors, and it has been shown that this segregation is predictive of patient responses to the same chemotherapies. Simultaneously, PDEs allow the opportunity to interrogate molecular, genetic, and histological features of tumors that predict drug responses, thereby identifying biomarkers for patient stratification as well as novel interventional approaches to sensitize resistant tumors. This paper reports PDE methodology in detail, from collection of patient samples through to endpoint analysis. It provides a detailed description of explant derivation and culture methods, highlighting bespoke conditions for particular tumors, where appropriate. For endpoint analysis, there is a focus on multiplexed immunofluorescence and multispectral imaging for the spatial profiling of key biomarkers within both tumoral and stromal regions. By combining these methods, it is possible to generate quantitative and qualitative drug response data that can be related to various clinicopathological parameters and thus potentially be used for biomarker identification.

This paper describes methods for the generation, drug treatment, and analysis of patient-derived explants for assessing tumor drug responses in a live, patient-relevant, preclinical model system.

患者の薬剤耐性を予測するための「生きた」前臨床プラットフォームとしての患者由来腫瘍エクスプラント

An understanding of drug resistance and the development of novel strategies to sensitize highly resistant cancers rely on the availability of suitable ...

Profiling Sensitivity to Targeted Therapies in EGFR-Mutant NSCLC Patient-Derived Organoids

Novel 3D cancer organoid cultures derived from clinical patient specimens represent an important model system to evaluate intratumor heterogeneity and treatment response to targeted inhibitors in cancer. Pioneering work in gastrointestinal and pancreatic cancers has highlighted the promise of patient-derived organoids (PDOs) as a patient-proximate culture system, with an increasing number of models emerging. Similarly, work in other cancer types has focused on establishing organoid models and optimizing culture protocols. Notably, 3D cancer organoid models maintain the genetic complexity of original tumor specimens and thus translate tumor-derived sequencing data into treatment with genetically informed targeted therapies in an experimental setting. Further, PDOs might foster the evaluation of rational combination treatments to overcome resistance-associated adaptation of tumors in the future. The latter focuses on intense research efforts in non-small-cell lung cancer (NSCLC), as resistance development ultimately limits the treatment success of targeted inhibitors. An early assessment of therapeutically targetable mechanisms using NSCLC PDOs could help inform rational combination treatments. This manuscript describes a standardized protocol for the cell culture plate-based assessment of drug sensitivities to targeted inhibitors in NSCLC-derived 3D PDOs, with potential adaptability to combinational treatments and other treatment modalities.

This protocol describes a standardized evaluation of drug sensitivities to targeted signaling inhibitors in NSCLC patient-derived organoid models.

EGFR変異型NSCLC患者由来オルガノイドにおける標的療法に対する感受性のプロファイリング

Novel 3D cancer organoid cultures derived from clinical patient specimens represent an important model system to evaluate intratumor heterogeneity and ...

A Combined 3D Tissue Engineered In Vitro/In Silico Lung Tumor Model for Predicting Drug Effectiveness in Specific Mutational Backgrounds

In the present study, we combined an in vitro 3D lung tumor model with an in silico model to optimize predictions of drug response based on a specific mutational background. The model is generated on a decellularized porcine scaffold that reproduces tissue-specific characteristics regarding extracellular matrix composition and architecture including the basement membrane. We standardized a protocol that allows artificial tumor tissue generation within 14 days including three days of drug treatment. Our article provides several detailed descriptions of 3D read-out screening techniques like the determination of the proliferation index Ki67 staining&#39;s, apoptosis from supernatants by M30-ELISA and assessment of epithelial to mesenchymal transition (EMT), which are helpful tools for evaluating the effectiveness of therapeutic compounds. We could show compared to 2D culture a reduction of proliferation in our 3D tumor model that is related to the clinical situation. Despite of this lower proliferation, the model predicted EGFR-targeted drug responses correctly according to the biomarker status as shown by comparison of the lung carcinoma cell lines HCC827 (EGFR -mutated, KRAS wild-type) and A549 (EGFR wild-type, KRAS-mutated) treated with the tyrosine-kinase inhibitor (TKI) gefitinib. To investigate drug responses of more advanced tumor cells, we induced EMT by long-term treatment with TGF-beta-1 as assessed by vimentin/pan-cytokeratin immunofluorescence staining. A flow-bioreactor was employed to adjust culture to physiological conditions, which improved tissue generation. Furthermore, we show the integration of drug responses upon gefitinib treatment or TGF-beta-1 stimulation - apoptosis, proliferation index and EMT - into a Boolean in silico model. Additionally, we explain how drug responses of tumor cells with a specific mutational background and counterstrategies against resistance can be predicted. We are confident that our 3D in vitro approach especially with its in silico expansion provides an additional value for preclinical drug testing in more realistic conditions than in 2D cell culture.

A Combined 3D Tissue Engineered In Vitro/In Silico Lung Tumor Model for Predicting Drug Effectiveness in Specific Mutational Backgrounds

We present a three-dimensional (3D) lung cancer model based on a biological collagen scaffold to study sensitivity towards non-small-cell-lung-cancer-(NSCLC)-targeted therapies. We demonstrate different read-out techniques to determine the proliferation index, apoptosis and epithelial-mesenchymal transition (EMT) status. Collected data are integrated into an in silico model for prediction of drug sensitivity.

操作された複合三次元組織<em&gt;インビトロ</em&gt; /<em&gt;インシリコ</em具体的な突然変異背景に薬物の有効性を予測するための&gt;肺腫瘍モデル

In the present study, we combined an in vitro 3D lung tumor model with an in silico model to optimize predictions of drug response based on a specific ...

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 ...

化学

An Organotypic High Throughput System for Characterization of Drug Sensitivity of Primary Multiple Myeloma Cells

In this work we describe a novel approach that combines ex vivo drug sensitivity assays and digital image analysis to estimate chemosensitivity and heterogeneity of patient-derived multiple myeloma (MM) cells. This approach consists in seeding primary MM cells freshly extracted from bone marrow aspirates into microfluidic chambers implemented in multi-well plates, each consisting of a reconstruction of the bone marrow microenvironment, including extracellular matrix (collagen or basement membrane matrix) and stroma (patient-derived mesenchymal stem cells) or human-derived endothelial cells (HUVECs). The chambers are drugged with different agents and concentrations, and are imaged sequentially for 96 hr through bright field microscopy, in a motorized microscope equipped with a digital camera. Digital image analysis software detects live and dead cells from presence or absence of membrane motion, and generates curves of change in viability as a function of drug concentration and exposure time. We use a computational model to determine the parameters of chemosensitivity of the tumor population to each drug, as well as the number of sub-populations present as a measure of tumor heterogeneity. These patient-tailored models can then be used to simulate therapeutic regimens and estimate clinical response.

We describe a high-throughput drug sensitivity assay for primary multiple myeloma cells. It consists of a reconstruction of the bone marrow microenvironment (including extracellular matrix and stroma) in multi-well plates, and a non-invasive method for longitudinal quantification of cell viability.

一次多発性骨髄腫細胞の薬剤感受性の特徴付けのための器官のハイスループットシステム

In this work we describe a novel approach that combines ex vivo drug sensitivity assays and digital image analysis to estimate chemosensitivity and ...

がん薬感の有効性を評価

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