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 border="0" cellpadding="0" cellspacing="0" >
	<tbody>
		<tr >
			<td >Name of Material/ Equipment</td>
			<td >Company</td>
			<td >Catalog Number</td>
			<td >Comments/Description</td>
		</tr>
		<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&#160;</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&#39; - UAAGGAACGUCAAGAGAUAC - 3&#39; (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&#160;</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&#160;</td>
			<td >Gibco - Life Technologies</td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >Lipofectamine 2000&#160;</td>
			<td >Invitrogen - Life Technologies&#160;</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

8.2K 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

An In Vitro System to Study Tumor Dormancy and the Switch to Metastatic Growth

Recurrence of breast cancer often follows a long latent period in which there are no signs of cancer, and metastases may not become clinically apparent until many years after removal of the primary tumor and adjuvant therapy. A likely explanation of this phenomenon is that tumor cells have seeded metastatic sites, are resistant to conventional therapies, and remain dormant for long periods of time 1-4.
The existence of dormant cancer cells at secondary sites has been described previously as quiescent solitary cells that neither proliferate nor undergo apoptosis 5-7. Moreover, these solitary cells has been shown to disseminate from the primary tumor at an early stage of disease progression 8-10 and reside growth-arrested in the patients' bone marrow, blood and lymph nodes 1,4,11. Therefore, understanding mechanisms that regulate dormancy or the switch to a proliferative state is critical for discovering novel targets and interventions to prevent disease recurrence. However, unraveling the mechanisms regulating the switch from tumor dormancy to metastatic growth has been hampered by the lack of available model systems.
in vivo and ex vivo model systems to study metastatic progression of tumor cells have been described previously 1,12-14. However these model systems have not provided in real time and in a high throughput manner mechanistic insights into what triggers the emergence of solitary dormant tumor cells to proliferate as metastatic disease. We have recently developed a 3D in vitro system to model the in vivo growth characteristics of cells that exhibit either dormant (D2.OR, MCF7, K7M2-AS.46) or proliferative (D2A1, MDA-MB-231, K7M2) metastatic behavior in vivo . We demonstrated that tumor cells that exhibit dormancy in vivo at a metastatic site remain quiescent when cultured in a 3-dimension (3D) basement membrane extract (BME), whereas cells highly metastatic in vivo readily proliferate in 3D culture after variable, but relatively short periods of quiescence. Importantly by utilizing the 3D in vitro model system we demonstrated for the first time that the ECM composition plays an important role in regulating whether dormant tumor cells will switch to a proliferative state and have confirmed this in in vivo studies15-17. Hence, the model system described in this report provides an in vitro method to model tumor dormancy and study the transition to proliferative growth induced by the microenvironment.

An In Vitro System to Study Tumor Dormancy and the Switch to Metastatic Growth

A modified 3-D in vitro system is presented in which growth characteristics of several tumor cell lines in reconstituted basement membrane correlate with the dormant or proliferative behavior of the tumor cells at a metastatic secondary site in vivo.

アンインビトロ</em転移性増殖に腫瘍の休眠とスイッチを研究する>システム

Recurrence of breast cancer often follows a long latent period in which there are no signs of cancer, and metastases may not become clinically apparent ...

医学

Live Imaging of Drug Responses in the Tumor Microenvironment in Mouse Models of Breast Cancer

The tumor microenvironment plays a pivotal role in tumor initiation, progression, metastasis, and the response to anti-cancer therapies. Three-dimensional co-culture systems are frequently used to explicate tumor-stroma interactions, including their role in drug responses. However, many of the interactions that occur in vivo in the intact microenvironment cannot be completely replicated in these in vitro settings. Thus, direct visualization of these processes in real-time has become an important tool in understanding tumor responses to therapies and identifying the interactions between cancer cells and the stroma that can influence these responses. Here we provide a method for using spinning disk confocal microscopy of live, anesthetized mice to directly observe drug distribution, cancer cell responses and changes in tumor-stroma interactions following administration of systemic therapy in breast cancer models. We describe procedures for labeling different tumor components, treatment of animals for observing therapeutic responses, and the surgical procedure for exposing tumor tissues for imaging up to 40 hours. The results obtained from this protocol are time-lapse movies, in which such processes as drug infiltration, cancer cell death and stromal cell migration can be evaluated using image analysis software.

We describe a method for imaging response to anti-cancer treatment in vivo and at single cell resolution.

乳癌のマウスモデルにおいて腫瘍微小環境における薬剤応答のイメージングを生きる

The tumor microenvironment plays a pivotal role in tumor initiation, progression, metastasis, and the response to anti-cancer therapies. Three-dimensional ...

Slow-release Drug Delivery through Elvax 40W to the Rat Retina: Implications for the Treatment of Chronic Conditions

Diseases of the retina are difficult to treat as the retina lies deep within the eye. Invasive methods of drug delivery are often needed to treat these diseases. Chronic retinal diseases such as retinal oedema or neovascularization usually require multiple intraocular injections to effectively treat the condition. However, the risks associated with these injections increase with repeated delivery of the drug. Therefore, alternative delivery methods need to be established in order to minimize the risks of reinjection. Several other investigations have developed methods to deliver drugs over extended time, through materials capable of releasing chemicals slowly into the eye. In this investigation, we outline the use of Elvax 40W, a copolymer resin, to act as a vehicle for drug delivery to the adult rat retina. The resin is made and loaded with the drug. The drug-resin complex is then implanted into the vitreous cavity, where it will slowly release the drug over time. This method was tested using 2-amino-4-phosphonobutyrate (APB), a glutamate analogue that blocks the light response of the retina. It was demonstrated that the APB was slowly released from the resin, and was able to block the retinal response by 7 days after implantation. This indicates that slow-release drug delivery using this copolymer resin is effective for treating the retina, and could be used therapeutically with further testing.

This paper details how Elvax 40W can be used as a slow-release method for drug delivery to the adult rat retina. The protocol for preparing, loading, and delivering the drug-resin complex to the eye is described.

ラット網膜へのエルバックス40Wを通じて徐放性ドラッグデリバリー：慢性症状の治療のための含意

Diseases of the retina are difficult to treat as the retina lies deep within the eye. Invasive methods of drug delivery are often needed to treat these ...

Carotid Artery Infusions for Pharmacokinetic and Pharmacodynamic Analysis of Taxanes in Mice

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study model. As the use of mouse models are often a vital step in preclinical drug discovery and drug development1-8, it is necessary to design a system to introduce drugs into mice in a uniform, reproducible manner. Ideally, the system should permit the collection of blood samples at regular intervals over a set time course. The ability to measure drug concentrations by mass-spectrometry, has allowed investigators to follow the changes in plasma drug levels over time in individual mice1, 9, 10. In this study, paclitaxel was introduced into transgenic mice as a continuous arterial infusion over three hours, while blood samples were simultaneously taken by retro-orbital bleeds at set time points. Carotid artery infusions are a potential alternative to jugular vein infusions, when factors such as mammary tumors or other obstructions make jugular infusions impractical. Using this technique, paclitaxel concentrations in plasma and tissue achieved similar levels as compared to jugular infusion. In this tutorial, we will demonstrate how to successfully catheterize the carotid artery by preparing an optimized catheter for the individual mouse model, then show how to insert and secure the catheter into the mouse carotid artery, thread the end of the catheter out through the back of the mouse&#8217;s neck, and hook the mouse to a pump to deliver a controlled rate of drug influx. Multiple low volume retro-orbital bleeds allow for analysis of plasma drug concentrations over time.

This method was developed with the goal of delivering a steady drug solution via the carotid artery, to assess the pharmacokinetics of novel drugs in mouse models.

マウスにおけるタキサンの薬物動態学的および薬力学的分析のための頸動脈煎茶

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study ...

In Vivo and Ex Vivo Approaches to Study Ovarian Cancer Metastatic Colonization of Milky Spot Structures in Peritoneal Adipose

High-grade serous ovarian cancer (HGSC), the cause of widespread peritoneal metastases, continues to have an extremely poor prognosis; fewer than 30% of women are alive 5 years after diagnosis. The omentum is a preferred site of HGSC metastasis formation. Despite the clinical importance of this microenvironment, the contribution of omental adipose tissue to ovarian cancer progression remains understudied. Omental adipose is unusual in that it contains structures known as milky spots, which are comprised of B, T, and NK cells, macrophages, and progenitor cells surrounding dense nests of vasculature. Milky spots play a key role in the physiologic functions of the omentum, which are required for peritoneal homeostasis. We have shown that milky spots also promote ovarian cancer metastatic colonization of peritoneal adipose, a key step in the development of peritoneal metastases. Here we describe the approaches we developed to evaluate and quantify milky spots in peritoneal adipose and study their functional contribution to ovarian cancer cell metastatic colonization of omental tissues both in vivo and ex vivo. These approaches are generalizable to additional mouse models and cell lines, thus enabling the study of ovarian cancer metastasis formation from initial localization of cells to milky spot structures to the development of widespread peritoneal metastases.

In Vivo and Ex Vivo Approaches to Study Ovarian Cancer Metastatic Colonization of Milky Spot Structures in Peritoneal Adipose

We outline a protocol that implements both in vivo and ex vivo approaches to study ovarian cancer colonization of peritoneal adipose tissues, particularly the omentum. Furthermore, we present a protocol to quantitate and analyze immune cell-structures in the omentum known as milky spots, which promote metastases of peritoneal adipose.

生体内および生体外では腹膜脂肪にミルキースポット構造の卵巣がん転移性コロニー形成を研究するためのアプローチで

High-grade serous ovarian cancer (HGSC), the cause of widespread peritoneal metastases, continues to have an extremely poor prognosis; fewer than 30% of ...

Studying Pancreatic Cancer Stem Cell Characteristics for Developing New Treatment Strategies

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor initiation, metastasis and resistance to radio- and chemotherapy. Here we describe a specific methodology for culturing primary human pancreatic CSCs as tumor spheres in anchorage-independent conditions. Cells are grown in serum-free, non-adherent conditions in order to enrich for CSCs while their more differentiated progenies do not survive and proliferate during the initial phase following seeding of single cells. This assay can be used to estimate the percentage of CSCs present in a population of tumor cells. Both size (which can range from 35 to 250 micrometers) and number of tumor spheres formed represents CSC activity harbored in either bulk populations of cultured cancer cells or freshly harvested and digested tumors 1,2. Using this assay, we recently found that metformin selectively ablates pancreatic CSCs; a finding that was subsequently further corroborated by demonstrating diminished expression of pluripotency-associated genes/surface markers and reduced in vivo tumorigenicity of metformin-treated cells. As the final step for preclinical development we treated mice bearing established tumors with metformin and found significantly prolonged survival. Clinical studies testing the use of metformin in patients with PDAC are currently underway (e.g., NCT01210911, NCT01167738, and NCT01488552). Mechanistically, we found that metformin induces a fatal energy crisis in CSCs by enhancing reactive oxygen species (ROS) production and reducing mitochondrial transmembrane potential. In contrast, non-CSCs were not eliminated by metformin treatment, but rather underwent reversible cell cycle arrest. Therefore, our study serves as a successful example for the potential of in vitro sphere formation as a screening tool to identify compounds that potentially target CSCs, but this technique will require further in vitro and in vivo validation to eliminate false discoveries.

Pancreatic cancer stem cells (CSCs) can be expanded in vitro using the anchorage-independent sphere culture technique, which represents a powerful tool to study CSC biology and can serve as the first step to develop novel CSC-targeting therapies. Here the methodology for expanding, analyzing and targeting of pancreatic CSCs is provided.

新しい治療戦略を開発するための膵臓癌幹細胞特性を研究

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor ...

Depletion of Mouse Cells from Human Tumor Xenografts Significantly Improves Downstream Analysis of Target Cells

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic patient tumors in different assays1. Human tumor xenografts represent the gold standard with respect to tumor biology, drug discovery, and metastasis research 2-4. Tumor xenografts can be derived from different types of material like tumor cell lines, tumor tissue from primary patient tumors4 or serially transplanted tumors. When propagated in vivo, xenografted tissue is infiltrated and vascularized by cells of mouse origin. Multiple factors such as the tumor entity, the origin of xenografted material, growth rate and region of transplantation influence the composition and the amount of mouse cells present in tumor xenografts. However, even when these factors are kept constant, the degree of mouse cell contamination is highly variable.
Contaminating mouse cells significantly impair downstream analyses of human tumor xenografts. As mouse fibroblasts show high plating efficacies and proliferation rates, they tend to overgrow cultures of human tumor cells, especially slowly proliferating subpopulations. Mouse cell derived DNA, mRNA, and protein components can bias downstream gene expression analysis, next-generation sequencing, as well as proteome analysis 5. To overcome these limitations, we have developed a fast and easy method to isolate untouched human tumor cells from xenografted tumor tissue. This procedure is based on the comprehensive depletion of cells of mouse origin by combining automated tissue dissociation with the benchtop tissue dissociator and magnetic cell sorting. Here, we demonstrate that human target cells can be can be obtained with purities higher than 96% within less than 20 min independent of the tumor type.

Human tumor xenografts are vascularized and infiltrated by cells of mouse origin during the growth phase in vivo. To circumvent the bias caused by these contaminating cells during downstream analysis, we developed a method allowing for comprehensive depletion of all mouse cells by magnetic cell sorting.

ヒト腫瘍異種移植片からマウス細胞の枯渇は大幅に標的細胞の下流分析を向上させます

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic ...

Forskolin-induced Swelling in Intestinal Organoids: An In Vitro Assay for Assessing Drug Response in Cystic Fibrosis Patients

Recently-developed cystic fibrosis transmembrane conductance regulator (CFTR)-modulating drugs correct surface expression and/or function of the mutant CFTR channel in subjects with cystic fibrosis (CF). Identification of subjects that may benefit from these drugs is challenging because of the extensive heterogeneity of CFTR mutations, as well as other unknown factors that contribute to individual drug efficacy. Here, we describe a simple and relatively rapid assay for measuring individual CFTR function and response to CFTR modulators in vitro. Three dimensional (3D) epithelial organoids are grown from rectal biopsies in standard organoid medium. Once established, the organoids can be bio-banked for future analysis. For the assay, 30-80 organoids are seeded in 96-well plates in basement membrane matrix and are then exposed to drugs. One day later, the organoids are stained with calcein green, and forskolin-induced swelling is monitored by confocal live cell microscopy at 37 &#176;C. Forskolin-induced swelling is fully CFTR-dependent and is sufficiently sensitive and precise to allow for discrimination between the drug responses of individuals with different and even identical CFTR mutations. In vitro swell responses correlate with the clinical response to therapy. This assay provides a cost-effective approach for the identification of drug-responsive individuals, independent of their CFTR mutations. It may also be instrumental in the development of future CFTR modulators.

Forskolin-induced Swelling in Intestinal Organoids: An In Vitro Assay for Assessing Drug Response in Cystic Fibrosis Patients

This protocol describes an assay for measuring CFTR function and CFTR modulator responses in cultured tissue from subjects with cystic fibrosis (CF). Biopsy-derived intestinal organoids swell in a cAMP-driven fashion, a response that is defective (or strongly reduced) in CF organoids and can be restored by exposure to CFTR modulators.

フォルスコリン誘発性腸オルガノイドに腫れ：<em&gt;インビトロ</em&gt;アッセイ嚢胞性線維症患者における薬物反応を評価するための

Recently-developed cystic fibrosis transmembrane conductance regulator (CFTR)-modulating drugs correct surface expression and/or function of the mutant ...

Optimized Analysis of In Vivo and In Vitro Hepatic Steatosis

Establishing a system of procedures to qualitatively and quantitatively characterize in vivo and in vitro hepatic steatosis is important for metabolic study in the liver. Here, numerous assays are described to comprehensively measure the phenotype and parameters of hepatic steatosis in mouse and hepatocyte models. 
Combining the physiological, histological, and biochemical methods, this system can be used to assess the progress of hepatic steatosis. In vivo, the measurements of body weight and nuclear magnetic resonance (NMR) provide a general understanding of mice in a non-invasive manner. Hematoxylin and Eosin (H&amp;E) and Oil Red O staining determine the histological morphology and lipid deposition of liver tissue under nutrient overload conditions, such as high-fat diet feeding. Next, the total lipid contents are isolated by chloroform/methanol extraction, which are followed by a biochemical analysis for triglyceride and cholesterol. Moreover, mouse primary hepatocytes are treated with high glucose plus insulin to stimulate lipid accumulation, an efficient in vitro model to mimic diet-induced hyperglycemia and hyperinsulinemia in vivo. Then, the lipid deposition is measured by Oil Red O staining and chloroform/methanol extraction. Oil Red O staining determines intuitive hepatic steatotic phenotypes, while the lipid extraction analysis determines the parameters that can be analyzed statistically. The present protocols are of interest to scientists in the fields of fatty liver diseases, insulin resistance, and type 2 diabetes.

Optimized Analysis of In Vivo and In Vitro Hepatic Steatosis

Here, optimized methods to generate in vivo and in vitro models of hepatic steatosis and to analyze the steatotic phenotypes and related physiological parameters are described.

の最適化分析<em&gt;インビボ</em&gt;と<em&gt;インビトロ</em&gt;脂肪肝

Establishing a system of procedures to qualitatively and quantitatively characterize in vivo and in vitro hepatic steatosis is important for metabolic ...

Preparation of Herbal Medicine: Er-Xian Decoction and Er-Xian-containing Serum for In Vivo and In Vitro Experiments

Traditional herbal medicine, an alternative medicine in the clinical setting, has received increased attention in recent years. Before delivery to the body, an additional extraction procedure is commonly required to release the active constituents from raw herbs. Water decoction is a classical extraction procedure that is still broadly used in the clinical settings. Here, we propose a detailed protocol for er-xian decoction (EXD) in order to apply herbal decoctions to experimental studies. The calculation of an animal-appropriate dose is described, as well as the four main steps of EXD: soaking, water decoction, filtration, and concentration. In addition, serum-containing EXD is introduced to rats as a means of in vitro validation. Here, rats were orally administered EXD for three days. Blood samples were then collected, inactivated, centrifuged, and filtered. The serum, diluted with the culture medium, can be utilized to treat cells or tissues in vitro. For example, EXD was applied to both in vivo and in vitro studies and demonstrated that EXD enhances osteogenesis. This protocol can be used as a reference for the preparation and application of herbal medicines.

Preparation of Herbal Medicine: Er-Xian Decoction and Er-Xian-containing Serum for In Vivo and In Vitro Experiments

Here, we present the preparation of an er-xian decoction (EXD) in four steps&#8212;soaking, decoction, filtration, and concentration&#8212;and demonstrate the administration of a prepared EXD-containing serum to rats. These methods are applicable to the in vivo and in vitro study of herbal decoctions such as traditional Chinese medicines.

漢方薬の準備：Er-Xian煎じ薬とEr-Xian含有血清<em&gt;インビボ</em&gt;と<em&gt;インビトロ</em&gt;実験

Traditional herbal medicine, an alternative medicine in the clinical setting, has received increased attention in recent years. Before delivery to the ...