The authors would like to thank the MSKCC Tissue Procurement Service Team (TPS), specifically, Maria Corazon Mariano, Priscilla McNeil, Anas Idelbi, Daniel Navarrete and Katrina Allen, in all of their efforts in the successful pursuit of this project and funding from the following sources: 5 R21 CA158609-02 and the Conquer Cancer Foundation and the Breast Cancer Research Foundation. In addition, the authors would like to thank Eric Cottington PhD, Vice President of the Office of Research and Project Administration, the Office of Technology Development, Research Outreach and Compliance and RTM Information Systems Support, in the support of the submission of this manuscript.

注：患者組織の調達を制度的に審査委員会を通じて認定されました（IRB）は、メモリアル·スローン·ケタリングがんセンターで生体試料と臨床プロトコル（それぞれのプロトコル番号09から121と11から041を）承認の。 1組織調達<ol><li>患者の原発腫瘍/転移の調達 注：現在まで、このプロトコルは外科的に除去膵臓癌、胃癌および乳癌の腫瘍タイプ、ならびに、リンパ腫転移に行われている。 <ol><li>無菌流出防止の容器内に密閉し、滅菌漏れ防止プラスチック片袋での病理部門に宅配便または気送管システムによって試料を提供するために外科チームを指揮。ホルマリン又は固定剤なしで新鮮な状態で試料を輸送する外科チームを指揮。 </li><li>無菌技術を使用して、新鮮な試料を採取する病理医や病理医のアシスタントに指示し、その株式会社層流フードで無菌手袋や楽器のludes使用。 </li><li>外科処置の完了から30分の下で厳密に維持され、調達検体の収穫時間を記録します。冷たい虚血の考慮の影響を考慮して、&gt; 30分18の冷たい虚血時間とともに切除標本からサ ​​ンプルを取ることはありません。 </li><li>無菌環境を維持するために、層流フード中で約0.5cm 3〜1.0センチメートル3の原発腫瘍標本を除去するために病理部門要員を導く。可能な場合には、潜在的な率直な中心壊死（細胞死）を回避するために、インデックス病変の周囲から腫瘍組織を選択します。 注：壊死組織は、次の基準のいずれかにより肉眼的に認識可能性があります。色や組織の蒼白の喪失;壊死組織が柔らかく、もろいある、強度の低下;壊死性と生存組織との間に明確な境界。 </li><li> pathologiを指示STまたは診断評価のための腫瘍の直接のサンプリング後過剰（外科的廃棄）である末梢組織を提供するために、病理学者の助手。約5mlの1％抗生物質（ペニシリンおよびストレプトマイシン）を含む最小必須培地（MEM）のを含む15ミリリットルの無菌コニカル遠心管に試料を置きます。 </li><li>利用できる（ 例えば、乳房切除標本の腋窩尾部のリンパ節）は、同じサイズ（0.5 cm 3のセンチ3〜1.0）の著しくポジティブに関連するリンパ節試料を調達し、原発腫瘍の薬物反応と比較した場合。原発腫瘍試料と同様に、約5mlの1％抗生物質（ペニシリンおよびストレプトマイシン）を含む最小必須培地（MEM）のを含む15ミリリットルの無菌の円錐遠心管に関連するリンパ節試料を置く。 </li><li>手術標本が許す（ 例えば、乳房切除標本）のサイズならば、 すなわち （（原発腫瘍から離れた）正常組織のサンプルを削除約5mlの転送のみを目的とし、1％の抗生物質を含有する最小必須培地（MEM）のを含む15ミリリットルの無菌コニカル遠心管において正常濃密/繊維状乳房実質）と場所。研究施設に移した後、将来の分子解析用のC冷凍庫-80℃で凍結バイアル、「スナップ」を凍結し、店に「正常な」試料を移す。 </li><li>濡れた氷の上ですべての試料を置き、すぐにex vivoで新鮮な組織切片のための実験室のスペースに輸送する。ていないセクション通常の検体の操作を行いますが、むしろ将来分子解析のためのCの冷凍庫-80℃で保管してください。将来の分子解析のために-80℃の冷凍庫で凍結バイアル、「スナップ」を凍結し、ストアへの原発腫瘍の一部及び関連する転移を転送します。 </li></ol></li><li>臨床研究からの組織の調達（ 例えば 、オプションのコア針生検（CNB）） 注：私たちは、患者から腫瘍組織を得る治療開始前にアクセス可能な腫瘍転移のCNBを受ける彼らの同意が臨床プロトコルに登録した。 CNBSは、今日まで、乳房、辺縁帯リンパ腫、マントル細胞リンパ腫、転移性傍神経節腫、子宮頸部（扁平上皮）及び卵巣癌を有する患者に対して実施されてきた。 <ol><li>インターベンショナルラジオロジー又は関連部門からの臨床担当者は、前処理コア針生検を実行し、約5mlの1％抗生物質（ペニシリンおよびストレプトマイシン）を含む最小必須培地（MEM）のを含む15ミリリットルの無菌の円錐遠心管に試料を配置しています。 </li><li>濡れた氷の上の生検標本を置き、すぐにex vivoで新鮮な組織切片のための実験室のスペースに輸送する。 </li></ol></li></ol>セクショニングとビブラトーム設定のための材料の調製<ol><li>セクショニングのための材料の作製<ol><li> AGA物4gを添加することにより、4％アガロース溶液を作る100mlのPBSに上昇した。オートクレーブ（サイクルの設定：液体、121℃、30分、平方インチ当たり15ポンド）最初の使用前の溶液。その後の使用のために、アガロースは室温で固化することを心に留め、各使用の前に（30秒間隔の高熱）を液化し、適切〜25℃（ すなわち 、暖かいタッチする）まで冷却し、マイクロ波中、4％アガロースを配置。 </li><li>メディアを埋め込む非永久組織として、4％アガロースを使用してください。とき4％アガロースを十分に冷却し、滅菌ピンセット埋め込み型内に組織標本を配置し、4％アガロースに注ぐと、液体状態のままである。 </li><li>ポジション埋め込む金型の切削面の壁に直接（〜2mmの距離）の近くではなく、組織標本。 4％のアガロースは、したがって、適切に迅速に組織標本を置き、迅速に固化し始めます。切片の間に、溶液中に保つために55℃の水浴中で4％アガロースを保つ。 </li><li>セクション分析のために24ウェルプレートに1mlのO追加各ウェルにfを完全培地（ すなわち 、MEM + 10％ウシ胎児血清+ 1％抗生物質）。切片の期間を通じて、RT（〜25℃）で24ウェルプレートを保管してください。 </li></ol></li><li>ビブラトーム設定の調製<ol><li> 0〜10（0ヘルツ）[振動ミクロトームを使用して、[（2.5ミリメートル/秒）0〜10（0.00ミリメートル/秒）]刃が試料に接近する速度を設定し、ブレードの周波数（100ヘルツ） ]。組織（ すなわち、密度/硬さ）の質感に合わせて設定を調整します。 </li><li>浸潤性乳管癌の原発性腫瘍の場合は、それぞれ6および図3に示すように、速度や周波数に設定を調整します。密度の低い/事務所腫瘍組織では、速度を低下させ、それに応じて周波数を上げる。 </li><li> （ - 999ミクロン1）計器限界内で切片厚を設定してください。これらの分析のために、200ミクロンで切片厚を設定してください。 </li><li>試料ディスクの股関節に液状の接着剤の薄層を用いてアガロース包埋標本をマウントtが濡れた氷に包まれているメディアのリザーバ内に浸されている。 </li></ol></li></ol> 3組織セクショニング、トリートメント＆分析<ol><li>完全培地1mlでマルチウェルプレートに滅菌ピンセットと場所を使用して切片化後すぐに精密スライスされたセクションを削除します。十分な切片応答と相関分析のために取得されると、標準的な組織培養条件に設定された組織培養インキュベーター中で治療反応のためのマルチウェルプレートに置く。 </li><li>培養された成果物とは対照的に、用量依存性の応答性を確認する目的で処理された部分をブックエンド、複数の制御部を取る。 （ - 4時間〜2）18だけでなく冷たい虚血時間の任意の有害な影響の中、 - （40分〜30）急速に切片組織を実行します。わずか1時間の順応期間を以下のセクションでの治療を行います。 </li><li>治療関連（1時間の順応期間の後、漸増用量を追加します。 <em&gt;例えば、0 -治療期間のための標準的な組織培養条件下でのセクションと家への薬物の10μMの濃度）。この研究では、24および/または48時間の評価期間を使用しています。この研究で試験したが、コントロール（車両のみ）の組織切片は、ピリオドまでと96時間評価期間を含むために、実行可能なままになります。 </li><li>処置後、RT Oで10％緩衝ホルマリンと場所のいずれかを含有するチューブ中に組織切片を置くことによって、無菌条件下およびホルマリン固定の下のすべてのセクションを削除/ Nまたは4％パラホルムアルデヒドを含むチューブに（バイアルから16％のパラホルムアルデヒドを希釈4％の最終濃度にPBSで）と室温で2時間置く。 <ol><li>大きな組織切片を修正しました。（ 例えば 、〜6ミリメートル×4 mm）の10％緩衝ホルマリンOにおける厚さ200μm/ RTでN。小さ ​​なセクションを修正しました。（ 例えば 、1ミリメートル×1 mm）で、室温で2時間、4％パラホルムアルデヒド中で厚さ200μm。 注：固定液Fの容量の効率的な浸透のためにixativeは組織の容積を20Xする必要があります。適切な固定利用の確保すべての組織の大きさのための固定液1ミリリットルのための非常に小さな組織のサイズでこのプロトコルは、結果（ 例えば、6ミリメートル3組織= 650μl容量）ことを考える。 </li><li>固定後の組織標本を病理部門担当者がパラフィン包埋持つ。ヘマトキシリンおよびエオシン染色し、免疫組織化学的に評価した（ 例えば、アポトーシス）に帯電スライド上節検体のパラフィンブロックを。 </li></ol></li></ol>

<ol>
	<li>Abercrombie, M., Ambrose, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interference+Microscope+Studies+of+Cell+Contacts+in+Tissue+Culture.">Interference Microscope Studies of Cell Contacts in Tissue Culture.</a> Experimental Cell Research. 15 (58), 332-345 (1958).</li><li>Coriell, L. L., McAllister, R. M., Wagner, B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Criteria+for+Determining+Malignancy+in+Tissue-Culture+Cell+Lines+in+the+Albino+Rat.">Criteria for Determining Malignancy in Tissue-Culture Cell Lines in the Albino Rat.</a> New York Academy of Science. 5, 351-355 (1957).</li><li>Paul, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Cancer+Cell+in+Vitro:+A+Review.">The Cancer Cell in Vitro: A Review.</a> Cancer Research. 22, 431-440 (1962).</li><li>Dean, J. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Therapeutic+response+to+CDK4/6+inhibition+in+breast+cancer+defined+by+ex+vivo+analysis+of+human+tumors.">Therapeutic response to CDK4/6 inhibition in breast cancer defined by ex vivo analysis of human tumors.</a> Cell Cycle. 11 (14), 2756-2761 (2012).</li><li>Bray, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bcl-2+Modulation+to+Activate+Apoptosis+in+Prostate+Cancer.">Bcl-2 Modulation to Activate Apoptosis in Prostate Cancer.</a> Molecular Cancer Research. 7 (9), 1487-1496 (2009).</li><li>Quail, D. F., Joyce, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microenvironmental+regulation+of+tumor+progression+and+metastasis.">Microenvironmental regulation of tumor progression and metastasis.</a> Nature Medicine. 19 (11), 1423-1437 (2013).</li><li>Nakasone, E. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+Tumor-Stroma+Interactions+during+Chemotherapy+Reveals+Contributions+of+the+Microenvironment+to+Resistance.">Imaging Tumor-Stroma Interactions during Chemotherapy Reveals Contributions of the Microenvironment to Resistance.</a> Cancer Cell. 21, 488-503 (1016).</li><li>Shi, Y., Hogue, J., Dixit, D., Koh, J., Olson, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+and+genetic+studies+of+isolated+cells+from+parathyroid+tumors+reveal+the+complex+pathogenesis+of+parathyroid+neoplasia.">Functional and genetic studies of isolated cells from parathyroid tumors reveal the complex pathogenesis of parathyroid neoplasia.</a> PNAS. 111 (8), 3092-3097 (2014).</li><li>Vidal, S. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+cancer+stem+cells+from+human+prostate+cancer+samples.">Isolation of cancer stem cells from human prostate cancer samples.</a> J. Vis. Exp. (85), e51332 (2014).</li><li>Wanping, X., Neckers, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+the+Molecular+Chaperone+Heat+Shock+Protein+90+Provides+a+Multifaceted+Effect+on+Diverse+Cell+Signaling+of+Cancer+Cells.">Targeting the Molecular Chaperone Heat Shock Protein 90 Provides a Multifaceted Effect on Diverse Cell Signaling of Cancer Cells.</a> Clinical Cancer Research. 13, 1625-1629 (2007).</li><li>Moulick, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Affinity-based+proteomics+reveal+cancer-specific+networks+coordinated+by+Hsp90.">Affinity-based proteomics reveal cancer-specific networks coordinated by Hsp90.</a> Nature Chemical Biology. 7 (11), 818-826 (1038).</li><li>Alpaugh, M. L., Tomlinson, J. S., Ye, Y., Barsky, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Relationship+of+sialyl-Lewis(x/a)+underexpression+and+E-cadherin+overexpression+in+the+lymphovascular+embolus+of+inflammatory+breast+cancer.">Relationship of sialyl-Lewis(x/a) underexpression and E-cadherin overexpression in the lymphovascular embolus of inflammatory breast cancer.</a> American Journal of Pathology. 161 (2), 619-628 (2002).</li><li>Chu, K., Boley, K. M., Moraes, R., Barsky, S. H., Robertson, F. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+paradox+of+E-cadherin:+role+in+response+to+hypoxia+in+the+tumor+microenvironment+and+regulation+of+energy+metabolism.">The paradox of E-cadherin: role in response to hypoxia in the tumor microenvironment and regulation of energy metabolism.</a> Oncotarget. 4 (3), 446-462 (2013).</li><li>Vaira, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preclinical+model+of+organotypic+culture+for+pharmacodynamic+profiling+of+human+tumors.">Preclinical model of organotypic culture for pharmacodynamic profiling of human tumors.</a> PNAS. 107 (18), 8352-8356 (2010).</li><li>Centenera, M. M., Raj, G. V., Knudsen, K. E., Tilley, W. D., Butler, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ex+vivo+culture+of+human+prostate+tissue+and+drug+development.">Ex vivo culture of human prostate tissue and drug development.</a> Nature Reviews Urology. 10, 483-487 (2013).</li><li>Jhaveri, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heat+shock+protein+90+inhibitors+in+the+treatment+of+cancer:+current+status+and+future+directions.">Heat shock protein 90 inhibitors in the treatment of cancer: current status and future directions.</a> Expert Opinion on Investigational Drugs. 5, 611-628 (2014).</li><li>Gerecitano, J. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+124-PU-H71PET+imaging+to+predict+intratumoral+concentration+in+patients+on+a+phase+I+trial+of+PU-H71.">Using 124-PU-H71PET imaging to predict intratumoral concentration in patients on a phase I trial of PU-H71.</a> Journal of Clinical Oncology. 31, 11076-11 (2013).</li><li>Yildiz-Aktas, I., Dabbs, D. J., Bhargava, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+cold+ischemic+time+on+the+immunohistochemical+evaluation+of+estrogen+receptor,+progesterone+receptor,+and+HER2+expression+in+invasive+breast+carcinoma.">The effect of cold ischemic time on the immunohistochemical evaluation of estrogen receptor, progesterone receptor, and HER2 expression in invasive breast carcinoma.</a> Modern Pathology. 25, 1098-1105 (2012).</li></ol>

No conflicts of interest disclosed.

有効な治療戦略を開発しようとすると、がんの生物学者は、重要な課題に直面しています。確立された癌細胞に発達中の薬剤のテストラインが正確にPDXモデルでインビボ応答およびin vivo実験に反映しないことができ、労働集約的で極めて高価である。上記の、ex vivoでの適用は、原発性腫瘍患者14の技術は、15は、現在確立された癌細胞株および患者由来の...

有効な癌治療を開発することが極めて困難であることが証明された。癌細胞株および腫瘍外植-ならびに異種移植片は、半世紀以上の1,2,3のための癌研究に使用されている。現在までに、両方の確立された癌細胞株および患者由来の異種移植片（PDX）における薬剤感受性と耐性の分子分析が不可欠である。しかし、確立された癌細胞株における化合物の試験は、多くの場合、 インビボでの有効性の予測ではなく、特にPDXモデルにおいて、動物においてin vivo研究対応する、非常に高価であり、時間がかかる。これらのモデル系、腫瘍の進行および治療戦略に応じて、ネイティブの微小環境の影響に通知することができない、すなわちの限界は、これらの分析を補完するための追加の方法を開発する研究分野をリードしてきました。最近の中でも、高まり注意が患者TUM のex vivo解析に向けて注目されている癌治療反応は、癌細胞の固有の分子組成に排他的ではなく、むしろ大幅に伝統的な培養法によって再現したことができない腫瘍細胞の微小環境6,7機能によって影響されることにより、より深く理解するか、外植片4、5 /またはPDX。上記の文脈におけるエクスビボ分析が（ すなわち 、隣接する周囲の腫瘍細胞の微小環境の影響が）、むしろ、携帯分離株8,9 のex vivo分析よりも、生存可能な原発腫瘍/転移切片の評価を意味する。 私たちは忠実に、応答（IC 50）に通知するオフターゲット効果と分子を可能にし、患者の原発腫瘍と関連する転移（ すなわち 、リンパ節）の両方のex vivo手法（ すなわち 、精密スライスされた新鮮な組織切片）でここに報告抵抗とフィードバックのメカニズムの解析。さらに、therapeの相関分析バイオマーカーおよび遺伝子発現プロファイルに対するUTIC感受性/抵抗性（ すなわち 、高い薬物応答が特定の生物学的プロフィールを有する患者と一致する）関心のある試験薬に応答する可能性が高い患者を同定するための努力を行うことができる。マルチパラメータ方式でex vivoでの手法や評価の適用は、患者の選択と臨床転帰の全体的な改善に向けた動きである。 エクスビボ治療応答解析は、癌治療薬の前臨床および臨床開発における標準的なツールになる可能性があり、治療上の開発戦略における個別化医療のアプローチに向けたステップとして想定される。

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			<td style="width:167px;">Prepare 4% and 6% before use</td>
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			<td style="width:167px;">The media is prepared at Memorial Sloan Kettering Cancer Center&#160;</td>
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			<td height="42" style="height:42px;width:216px;">16% Formaldehyde solution</td>
			<td style="width:96px;">Thermo Scientific</td>
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ex vivo treatment response of primary tumors and/or associated metastases for preclinical and clinical development of therapeutics

The molecular analysis of established cancer cell lines has been the mainstay of cancer research for the past several decades. Cell culture provides both direct and rapid analysis of therapeutic sensitivity and resistance. However, recent evidence suggests that therapeutic response is not exclusive to the inherent molecular composition of cancer cells but rather is greatly influenced by the tumor cell microenvironment, a feature that cannot be recapitulated by traditional culturing methods. Even implementation of tumor xenografts, though providing a wealth of information on drug delivery/efficacy, cannot capture the tumor cell/microenvironment crosstalk (i.e., soluble factors) that occurs within human tumors and greatly impacts tumor response. To this extent, we have developed an ex vivo (fresh tissue sectioning) technique which allows for the direct assessment of treatment response for preclinical and clinical therapeutics development. This technique maintains tissue integrity and cellular architecture within the tumor cell/microenvironment context throughout treatment response providing a more precise means to assess drug efficacy.

この研究のために、ex vivoでの技術は、熱ショックタンパク質90阻害剤（Hsp90i）の治療感受性/耐性の相関分析に用いた。このHsp90i、乳癌の原発腫瘍の前臨床評価において、ER +浸潤性乳管癌（IDC）、および関連するリンパ節転移を分析したex vivoでの治療応答を&#160; （ 図1）。複数200μmの連続切片をHsp90iの車両のみと漸増用量（0.25、0.50、1.0および2.5μM）で?...

Watch this Scientific Journal Video about Ex Vivo Treatment Response of Primary Tumors and/or Associated Metastases for Preclinical and Clinical Development of Therapeutics at JoVE.com

Ex Vivo Treatment Response of Primary Tumors and/or Associated Metastases for Preclinical and Clinical Development of Therapeutics

確立された癌細胞株および異種移植片は、過去数十年の間、癌研究の主流となっている。しかし、最近の証拠は治療応答を大幅に腫瘍細胞の微小環境によって影響されることを示唆している。そこで、薬剤開発目的のために原発腫瘍標本のex vivo分析を開発した。

エキソビボ処理前臨床のための原発腫瘍および/ ​​または関連した転移の応答と治療薬の臨床開発

The molecular analysis of established cancer cell lines has been the mainstay of cancer research for the past several decades. Cell culture provides both ...

ex-vivo-treatment-response-primary-tumors-andor-associated-metastases

Research

JoVE Journal

Medicine

Ex Vivo Treatment Response of Primary Tumors and/or Associated Metastases for Preclinical and Clinical Development of Therapeutics

14.6K ビュー.  Memorial Sloan Kettering Cancer Center. 確立された癌細胞株および異種移植片は、過去数十年の間、癌研究の主流となっている。しかし、最近の証拠は治療応答を大幅に腫瘍細胞の微小環境によって影響されることを示唆している。そこで、薬剤開発目的のために原発腫瘍標本のex vivo分析を開発した。 

ビデオ: Ex Vivo Treatment Response of Primary Tumors and/or Associated Metastases for Preclinical and Clinical Development of Therapeutics

Thermal Ablation for the Treatment of Abdominal Tumors

Percutaneous thermal ablation is an emerging treatment option for many tumors of the abdomen not amenable to conventional treatments. During a thermal ablation procedure, a thin applicator is guided into the target tumor under imaging guidance. Energy is then applied to the tissue until temperatures rise to cytotoxic levels (50-60 &deg;C). Various energy sources are available to heat biological tissues, including radiofrequency (RF) electrical current, microwaves, laser light and ultrasonic waves. Of these, RF and microwave ablation are most commonly used worldwide.
During RF ablation, alternating electrical current (~500 kHz) produces resistive heating around the interstitial electrode. Skin surface electrodes (ground pads) are used to complete the electrical circuit. RF ablation has been in use for nearly 20 years, with good results for local tumor control, extended survival and low complication rates1,2. Recent studies suggest RF ablation may be a first-line treatment option for small hepatocellular carcinoma and renal-cell carcinoma3-5. However, RF heating is hampered by local blood flow and high electrical impedance tissues (eg, lung, bone, desiccated or charred tissue)6,7. Microwaves may alleviate some of these problems by producing faster, volumetric heating8-10. To create larger or conformal ablations, multiple microwave antennas can be used simultaneously while RF electrodes require sequential operation, which limits their efficiency. Early experiences with microwave systems suggest efficacy and safety similar to, or better than RF devices11-13.
Alternatively, cryoablation freezes the target tissues to lethal levels (-20 to -40 &deg;C). Percutaneous cryoablation has been shown to be effective against RCC and many metastatic tumors, particularly colorectal cancer, in the liver14-16. Cryoablation may also be associated with less post-procedure pain and faster recovery for some indications17. Cryoablation is often contraindicated for primary liver cancer due to underlying coagulopathy and associated bleeding risks frequently seen in cirrhotic patients. In addition, sudden release of tumor cellular contents when the frozen tissue thaws can lead to a potentially serious condition known as cryoshock 16.
Thermal tumor ablation can be performed at open surgery, laparoscopy or using a percutaneous approach. When performed percutaneously, the ablation procedure relies on imaging for diagnosis, planning, applicator guidance, treatment monitoring and follow-up. Ultrasound is the most popular modality for guidance and treatment monitoring worldwide, but computed tomography (CT) and magnetic resonance imaging (MRI) are commonly used as well. Contrast-enhanced CT or MRI are typically employed for diagnosis and follow-up imaging.

A thermal tumor ablation procedure is described. The entire procedure is detailed, including pretreatment planning and imaging studies, anesthesia, adjuvant techniques to facilitate a percutaneous approach, imaging guidance of the ablation device to the tumor, thermal treatment, post-treatment care and follow-up.

腹部腫瘍の治療のための熱的アブレーション

Percutaneous thermal ablation is an emerging treatment option for many tumors of the abdomen not amenable to conventional treatments. During a thermal ...

医学

Monitoring Tumor Metastases and Osteolytic Lesions with Bioluminescence and Micro CT Imaging

Following intracardiac delivery of MDA-MB-231-luc-D3H2LN cells to Nu/Nu mice, systemic metastases developed in the injected animals. Bioluminescence imaging using IVIS Spectrum was employed to monitor the distribution and development of the tumor cells following the delivery procedure including DLIT reconstruction to measure the tumor signal and its location.
Development of metastatic lesions to the bone tissues triggers osteolytic activity and lesions to tibia and femur were evaluated longitudinally using micro CT. Imaging was performed using a Quantum FX micro CT system with fast imaging and low X-ray dose. The low radiation dose allows multiple imaging sessions to be performed with a cumulative X-ray dosage far below LD50. A mouse imaging shuttle device was used to sequentially image the mice with both IVIS Spectrum and Quantum FX achieving accurate animal positioning in both the bioluminescence and CT images. The optical and CT data sets were co-registered in 3-dimentions using the Living Image 4.1 software. This multi-mode approach allows close monitoring of tumor growth and development simultaneously with osteolytic activity.

An experimental mouse model of bone metastasis was established following intracardiac delivery of luciferase expressing mammary tumor cells. Tumor development and resulted osteolytic lesion were monitored longitudinally with bioluminescence and micro CT imaging.

生物発光とマイクロCTイメージングで腫瘍転移と溶骨性病変の監視

Following intracardiac delivery of MDA-MB-231-luc-D3H2LN cells to Nu/Nu mice, systemic metastases developed in the injected animals. Bioluminescence ...

A Novel High-resolution In vivo Imaging Technique to Study the Dynamic Response of Intracranial Structures to Tumor Growth and Therapeutics

We have successfully integrated previously established Intracranial window (ICW) technology 1-4 with intravital 2-photon confocal microscopy to develop a novel platform that allows for direct long-term visualization of tissue structure changes intracranially. Imaging at a single cell resolution in a real-time fashion provides supplementary dynamic information beyond that provided by standard end-point histological analysis, which looks solely at 'snap-shot' cross sections of tissue.
Establishing this intravital imaging technique in fluorescent chimeric mice, we are able to image four fluorescent channels simultaneously. By incorporating fluorescently labeled cells, such as GFP+ bone marrow, it is possible to track the fate of these cells studying their long-term migration, integration and differentiation within tissue. Further integration of a secondary reporter cell, such as an mCherry glioma tumor line, allows for characterization of cell:cell interactions. Structural changes in the tissue microenvironment can be highlighted through the addition of intra-vital dyes and antibodies, for example CD31 tagged antibodies and Dextran molecules.
Moreover, we describe the combination of our ICW imaging model with a small animal micro-irradiator that provides stereotactic irradiation, creating a platform through which the dynamic tissue changes that occur following the administration of ionizing irradiation can be assessed.
Current limitations of our model include penetrance of the microscope, which is limited to a depth of up to 900 &mu;m from the sub cortical surface, limiting imaging to the dorsal axis of the brain. The presence of the skull bone makes the ICW a more challenging technical procedure, compared to the more established and utilized chamber models currently used to study mammary tissue and fat pads 5-7. In addition, the ICW provides many challenges when optimizing the imaging.

A Novel High-resolution In vivo Imaging Technique to Study the Dynamic Response of Intracranial Structures to Tumor Growth and Therapeutics

We describe a novel in vivo imaging technique that couples fluorescent chimeric mice with intracranial windows and high-resolution 2-photon microscopy. This imaging platform aids studies of dynamic changes in brain tissue and microvasculature, at a single-cell level, following pathological insults and is adaptable to assess intracranial drug delivery and distribution.

新規高分解能<em生体内で&gt;</em腫瘍増殖と治療に頭蓋内構造物の動的応答を研究する&gt;イメージング技術

We have successfully integrated previously established Intracranial window (ICW) technology 1-4 with intravital 2-photon confocal microscopy to develop a ...

Adaptation of Semiautomated Circulating Tumor Cell (CTC) Assays for Clinical and Preclinical Research Applications

The majority of cancer-related deaths occur subsequent to the development of metastatic disease. This highly lethal disease stage is associated with the presence of circulating tumor cells (CTCs). These rare cells have been demonstrated to be of clinical significance in metastatic breast, prostate, and colorectal cancers. The current gold standard in clinical CTC detection and enumeration is the FDA-cleared CellSearch system (CSS). This manuscript outlines the standard protocol utilized by this platform as well as two&#160;additional adapted protocols that describe the detailed process of user-defined marker optimization for protein characterization of patient CTCs and a comparable protocol for CTC capture in very low volumes of blood, using standard CSS reagents, for studying in vivo preclinical mouse models of metastasis. In addition, differences in CTC quality between healthy donor blood spiked with cells from tissue culture versus patient blood samples are highlighted. Finally, several commonly discrepant items that can lead to CTC misclassification errors are outlined. Taken together, these protocols will provide a useful resource for users of this platform interested in preclinical and clinical research pertaining to metastasis and CTCs.

Adaptation of Semiautomated Circulating Tumor Cell CTC Assays for Clinical and Preclinical Research Applications

Circulating tumor cells (CTCs) are prognostic in several metastatic cancers. This manuscript describes the gold standard CellSearch system (CSS) CTC enumeration platform and highlights common misclassification errors. In addition, two&#160;adapted protocols are described for user-defined marker characterization of CTCs and CTC enumeration in preclinical mouse models of metastasis using this technology.

臨床および前臨床研究用途のための半自動循環腫瘍細胞（CTC）アッセイの適応

The majority of cancer-related deaths occur subsequent to the development of metastatic disease. This highly lethal disease stage is associated with the ...

A Preclinical Murine Model of Hepatic Metastases

Numerous murine models have been developed to study human cancers and advance the understanding of cancer treatment and development. Here, a preclinical, murine pancreatic tumor model of hepatic metastases via a hemispleen injection of syngeneic murine pancreatic tumor cells is described. This model mimics many of the clinical conditions in patients with metastatic disease to the liver. Mice consistently develop metastases in the liver allowing for investigation of the metastatic process, experimental therapy testing, and tumor immunology research.

A preclinical, murine model of hepatic metastases performed via a hemispleen injection technique.

肝転移の前臨床マウスモデル

Numerous murine models have been developed to study human cancers and advance the understanding of cancer treatment and development. Here, a preclinical, ...

Isolated Hepatic Perfusion as a Treatment for Liver Metastases of Uveal Melanoma

Isolated hepatic perfusion (IHP) is a procedure where the liver is surgically isolated and perfused with a high concentration of the chemotherapeutic agent melphalan. Briefly, the procedure starts with the setup of a percutaneous veno-venous bypass from the femoral vein to the external jugular vein. Via a laparotomy, catheters are then inserted into the proper hepatic artery and the caval vein. The portal vein and the caval vein, both supra- and infrahepatically, are then clamped. The arterial and venous catheters are connected to a heart lung machine and the liver is perfused with melphalan (1 mg/kg body weight) for 60 min. This way it is possible to locally perfuse the liver with a high dose of a chemotherapeutic agent, without leakage to the systemic circulation.
In previous studies including patients with isolated liver metastases of uveal melanoma, an overall response rate of 33-100% and a median survival between 9 and 13 months, have been reported. The aim of this protocol is to give a clear description of how to perform the procedure and to discuss IHP as a treatment option for liver metastases of uveal melanoma.

Here, we present a protocol how to perform an isolated liver perfusion (IHP) with melphalan and we also discuss IHP as a treatment option for liver metastases of uveal melanoma.

ブドウ膜黒色腫の肝転移の治療として単離した肝灌流

Isolated hepatic perfusion (IHP) is a procedure where the liver is surgically isolated and perfused with a high concentration of the chemotherapeutic ...

Generation of Microtumors Using 3D Human Biogel Culture System and Patient-derived Glioblastoma Cells for Kinomic Profiling and Drug Response Testing

The use of patient-derived xenografts for modeling cancers has provided important insight into cancer biology and drug responsiveness. However, they are time consuming, expensive, and labor intensive. To overcome these obstacles, many research groups have turned to spheroid cultures of cancer cells. While useful, tumor spheroids or aggregates do not replicate cell-matrix interactions as found in vivo. As such, three-dimensional (3D) culture approaches utilizing an extracellular matrix scaffold provide a more realistic model system for investigation. Starting from subcutaneous or intracranial xenografts, tumor tissue is dissociated into a single cell suspension akin to cancer stem cell neurospheres. These cells are then embedded into a human-derived extracellular matrix, 3D human biogel, to generate a large number of microtumors. Interestingly, microtumors can be cultured for about a month with high viability and can be used for drug response testing using standard cytotoxicity assays such as 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and live cell imaging using Calcein-AM. Moreover, they can be analyzed via immunohistochemistry or harvested for molecular profiling, such as array-based high-throughput kinomic profiling, which is detailed here as well. 3D microtumors, thus, represent a versatile high-throughput model system that can more closely replicate in vivo tumor biology than traditional approaches.

Patient-derived xenografts of glioblastoma multiforme can be miniaturized into living microtumors using 3D human biogel culture system. This in vivo-like 3D tumor assay is suitable for drug response testing and molecular profiling, including kinomic analysis.

Kinomicプロファイリングと薬物反応検査のための3D人間バイオゲル培養システムと患者由来の神経膠芽腫細胞を用いた微小腫瘍の発生

The use of patient-derived xenografts for modeling cancers has provided important insight into cancer biology and drug responsiveness. However, they are ...

Assessment of Spontaneous Alternation, Novel Object Recognition and Limb Clasping in Transgenic Mouse Models of Amyloid-&#946; and Tau Neuropathology

Here we describe a staged, behavioral testing approach that can be used to screen for compounds that exhibit in vivo efficacy on cognitive and functional motor behaviors in transgenic mouse models of &#946;-amyloidosis and tauopathy. The paradigm includes tests for spontaneous alternation in a Y-maze, novel object recognition, and limb clasping. These tests were selected because they: 1) interrogate function of cognitive or motor domains and the correlate neural circuitry relevant to the human disease state, 2) have clearly defined endpoints, 3) have easily implementable quality control checks, 4) can be run in a moderate throughput format, and 5) require little intervention by the investigator. These methods are designed for investigators looking to screen compounds for activity in short-term and working memory tasks, or functional motor behaviors associated with Alzheimer's disease mouse models. The methods described here use behavioral tests that engage a number of different brain regions including hippocampus and various cortical areas. Investigators that desire cognitive tests that specifically assess cognition mediated by a single brain region could use these techniques to supplement other behavioral tests.

Assessment of Spontaneous Alternation, Novel Object Recognition and Limb Clasping in Transgenic Mouse Models of Amyloid-&amp;#946; and Tau Neuropathology

Described here is a staged, behavioral screening approach that can be used to screen for compounds that exhibit in vivo efficacy on cognitive and functional motor behaviors in transgenic mouse models of &#946;-amyloidosis and tauopathy. These methods are optimized to screen compounds for activity in short-term and working memory tasks.

アミロイド-βおよびタウのトランスジェニックマウスモデルにおける自発的交互作用の評価、新規対象の認識および肢の把握Neuropathology

Here we describe a staged, behavioral testing approach that can be used to screen for compounds that exhibit in vivo efficacy on cognitive and functional ...

Treatment of Liver Metastases Using an Internal Target Volume Method for Stereotactic Body Radiotherapy

The prognosis of patients with metastatic cancers has improved in the past decades due to effective chemotherapy and oligometastatic surgery. For inoperable patients, local ablation therapies, such as stereotactic body radiotherapy (SBRT), can provide effective local tumor control with minimal toxicity. Because of its high precision and accuracy, SBRT delivers a higher radiation dose per fraction, is more effective, and targets smaller irradiation volumes than does conventional radiotherapy. In addition, steep dose gradients from target lesions to surrounding normal tissues are achieved using SBRT; thus, SBRT provides more effective tumor control and exhibits fewer side effects than conventional radiotherapy. The use of SBRT is prevalent for treating intracranial lesions (known as stereotactic radiosurgery); however, it is now also used for treating spinal and adrenal metastases. Because of advancements in image-guided assistance and respiratory motion management, several studies have investigated the use of SBRT for treating lung or liver tumors, which move as a patient breathes. The results of these studies have suggested that SBRT favorably controls tumors in the case of moving lesions.
Four-dimensional computed tomography (4D-CT) with an abdominal compressor (AC) is clinically convenient for effective respiratory motion management. Because this method is noninvasive and allows free breathing, its use reduces complications. Furthermore, patients consider this method convenient. Moreover, it is considered more efficient than other methods of respiratory motion management by physicians and therapists. The use of 4D-CT with an AC for treating pulmonary lesions has also been widely investigated, and the technique is gaining acceptance for treating hepatic lesions. However, the protocols for using 4D-CT with an AC for treating hepatic lesions are different from those used for treating pulmonary lesions. In this article, we describe a new protocol for SBRT with 4D-CT and an AC for treating liver metastases.

Stereotactic body radiotherapy (SBRT) requires rigorous accuracy and precision for delivering high radiation doses per fraction to small treatment volumes to improve tumor control and simultaneously reduce toxicity. Herein, we present a noninvasive and clinically convenient respiratory motion management protocol for SBRT for liver metastases.

体幹部定位放射線照射のため内部のターゲット ボリュームによる肝転移の治療

The prognosis of patients with metastatic cancers has improved in the past decades due to effective chemotherapy and oligometastatic surgery. For ...

Contractions of Human-iPSC-derived Cardiomyocyte Syncytia Measured with a Ca-sensitive Fluorescent Dye in Temperature-controlled 384-well Plates

Spontaneously contracting syncytia of cardiomyocytes derived from human-induced pluripotent stem cells (hiPSC-CM) are a useful model of human cardiac physiology and pharmacology. Various methods have been proposed to record this spontaneous activity and to evaluate drug effects, but many of these methods suffer from limited throughput and/or physiological relevance. We developed a high-throughput screening system to quantify the effects of exogenous compounds on hiPSC-CM's beating frequency, using a Ca-sensitive fluorescent dye and a temperature-controlled imaging multi-well plate reader. We describe how to prepare the cell plates and the compound plates and how to run the automated assay to achieve high sensitivity and reproducibility. We also describe how to transform and analyze the fluorescence data to provide reliable measures of drug effects on spontaneous rhythm. This assay can be used in drug discovery programs to guide chemical optimization away from, or toward, compounds affecting human cardiac function.

Spontaneously contracting syncytia of cardiomyocytes derived from human-induced pluripotent stem cells are useful models of human cardiac physiology and pharmacology. Here we present a high-throughput screening system to quantify the effects of exogenous compounds on beating frequency, using a Ca-sensitive fluorescent dye and a temperature-controlled imaging multi-well plate reader.

人間-iPSC 由来心筋細胞分離の収縮測定 384 ウェル プレートの温度制御の Ca 感受性蛍光色素

Spontaneously contracting syncytia of cardiomyocytes derived from human-induced pluripotent stem cells (hiPSC-CM) are a useful model of human cardiac ...

エキソビボ処理前臨床のための原発腫瘍および/ ​​または関連した転移の応答と治療薬の臨床開発

エキソビボ処理前臨床のための原発腫瘍および/ または関連した転移の応答と治療薬の臨床開発