Name: modeling the early steps of ovarian cancer dissemination in an organotypic culture of the human peritoneal cavity
Uploaded: 2015-12-31T15:00:00.000+00:00
Duration: 9 min
Description: Watch this Scientific Journal Video about Modeling the Early Steps of Ovarian Cancer Dissemination in an Organotypic Culture of the Human Peritoneal Cavity at JoVE.com

We thank all residents and attending physicians, notably Dr. A.F. Haney (the University of Chicago, Department of Obstetrics and Gynecology) for collecting omental biopsies. Also, we thank Stacey Tobin and Gail Isenberg for carefully editing this manuscript. This work was supported by Bears Care, the charitable beneficiary of the Chicago Bears Football Club, the National Institute of Neurological Disorders and Stroke (NINDS) R21 NS075702, and the National Cancer Institute grant R01 CA111882 to E.L.

記載されている全ての研究プロトコルは、シカゴ施設内倫理委員会の大学（IRB）によって検討されています。インフォームドコンセントは、手術前に各患者から得られたものであり、この研究は、シカゴ大学IRBによって承認されました。生物学的安全キャビネットタイプ2との手袋は、保護のためにヒト組織を取り扱う際に使用されるべきであり、細胞の汚染のリスクを低減します。 一次形質転換されていない間質細胞の単離および培養1。 <ol><li>ヒト組織収集と準備。 <ol><li>腹部手術の間に除去さヒト大網、2 cm 3での標本を取得し、直ちにRTリン酸緩衝生理食塩水（PBS）（ 図2A）の組織を浸します。 X 2センチメートル大網3センチメートルの作品は、一般的に100万初代ヒト中皮細胞（HPMC）20万の一次ヒト線維芽細胞または正常大網線維芽細胞（NOF）が得られます。 </li><li>組織が3用0.5×gで中に浸漬したPBSをスピンダウン分できるだけ早く採取後（PBS中&lt;2時間）、50mlのコニカルチューブに新鮮な20ミリリットルのPBSに組織を移します。直径15cm、無菌培養皿（ 図2B）にメスで切り刻むことによって5ミリメートル3個に組織をカット。 </li></ol></li><li>初代ヒト中皮細胞の単離8 <ol><li> HPMCを単離するために、50mlコニカルチューブにみじん切りの組織を転送し、固体片がトップ（ 図2C＆D）に浮動するために1分を待ちます。 HPMCおよび赤血球（RBC）は、底部に液体中に存在するであろう。チューブの底から、新しいコニカルチューブに液体を除去するために、ピペットを使用してください。 3分間0.5×gで液体をスピンダウンし、HPMC / RBCペレットから上清を吸引除去します。 <ol><li>プロセスを3回繰り返します。組織は、上昇ピペットでおよびHPMC / RBC管に底から液体を除去、スピンダウン、および削除することができ、みじん切り組織に20ミリリットルのPBSを追加上清。すべてのスピンが完了し、上清を吸引した後、ペレットは、HPMCおよびRBCを含みます。 </li></ol></li><li>プレート10％ウシ胎児血清[FBS]、1％MEMビタミン[93ミリグラム/ L]、1％MEM非必須アミノ酸[81.4との完全成長培地15mlの（DMEM中で75cm 2のフラスコ中に、ペレットから全セルMG / L]、1％のペニシリンストレプトマイシン[ペニシリン - ストレプトマイシン、100U / mlペニシリンおよび100μg/ mlストレプトマイシン]）。 </li><li>追加のHPMCを分離するために、二次PBS洗浄を行ってください。 <ol><li>組織はトップにフロートするために二PBS洗浄のため、200 rpmで、20mlのPBSで10分間、37℃で、残りの固形組織を振る、その後、1分待ちます。 、新しいチューブに、チューブの底部から液体を除去する3分間の0.5×gで遠心分離し、上清を吸引除去します。 </li><li>二PBSのプレートのすべてのHPMC / RBCが完全増殖培地15ml中の別々の75cm 2のフラスコ中で洗います。 </li></ol></li><li>任意のRを単離するために、ボリュームによってPBS溶液：1、0.25％トリプシンの25mM EDTA：HPMCをemaining、1 20mlに10分間、200 rpmで37℃の度を細分化した組織を横に振ります。組織が上昇することを許可する、新しい50mlチューブにチューブの底に液体を収集し、0.5 GX 3分でスピンダウン。 <ol><li>上清を吸引除去し、完全増殖培地15mlに別々の75cm 2のフラスコ内のすべてのHPMC / RBCをめっき。 </li></ol></li><li>培養メッキ37℃で細胞を加湿環境下で5％CO 2。フィードは、使用済み培地を除去せずに3日目および5に完全成長培地を15mlを加えることによって、細胞を播種しました。 HPMCは、分割前に5〜7日間培養することができます。 <ol><li>元の表現型の脱分化および変更を最小限に抑えるために、すべての実験で（継代2まで）の低継代HPMCを使用してください。位相コントラストcapabilitieプラスチック微分干渉コントラスト機能や4-20x目的とした20倍目的で、好ましくは広視野顕微鏡を使用して、S細胞のすべての画像を撮影する（顕微鏡の位相コントラストフィルタ）、および3,3&#39;-ジアミノベンジジン（DAB）染色を免疫組織化学的に分析する明視野で10倍対物レンズ（）。 </li></ol></li><li> HPMCは立方形であり、免疫組織化学8（ 図3A、C、E）を行うことにより 、サイトケラチン8およびビメンチンを発現することを確認します。 </li></ol></li><li> NOF分離8 <ol><li> 1：100Xペニシリン - ストレプトマイシンの1：1混合物をPBS中のヒアルロニダーゼの714単位の活動/ mlのコラゲナーゼタイプIIIの1500単位の活動/ mlの1を組み合わせることにより、コラゲナーゼタイプIII溶液（10倍）の株式の10ミリリットルを準備します。 </li><li> 1％MEMビタミン[93ミリグラム/ L]で無血清培地で希釈10倍コラゲナーゼタイプIII溶液10ml（DMEM中で6時間、200 rpmでHPMC分離後に残るみじん切り大網組織、37℃を振って、 1％MEM非必須アミノ酸[81.4 mg / Lで]、1％のペニシリンストレプトマイシン[ペニシリン - ストレプトマイシン、100U / mlペニシリンおよび100μg/ mlストレプトマイシン]）。消化された組織は、不透明になりますし、いくつかの繊維の破片（ 図2E）を有することができます 。 </li><li> NOF細胞を含有する溶液を遠心 室温で3分間0.5×gで懸濁液中で、上清を吸引し、完全増殖培地13ミ ​​リリットルで75cm 2のフラスコ中でペレットをプレート。 </li><li>古いメディアを削除し、24時間後に、新鮮な完全増殖培地15mlで交換してください。 NOFは、分割前に1-3日間培養することができます。細胞がコンフルエントに達したときにNOFの注意増殖が停止します。 </li><li>一次ヒト線維芽細胞は平坦であることを確認し、細長い細胞およびビメンチンを発現するが、免疫組織化学8（ 図3B、D、F）を行うことにより 8サイトケラチンありません。脱分化を最小限にするために（理想的に継代3前）の実験のための低通路NOFを使用します そして、元の表現型の修正。細胞の全ての位相画像を撮影するためのプラスチック製のDIC機能を搭載した20倍目的で広視野顕微鏡を使用して、と明視野で10倍の目的は、免疫組織化学的DAB染色を分析します。 </li></ol></li></ol> 2.メッキ器官培養8 <ol><li>わずか5分3ミリリットル0.25％のトリプシン/ 25mMのEDTAに続いて10mlのPBSですすいで75センチ培養フラスコからリリースNOF。完全増殖培地の少なくとも3倍のボリュームで、トリプシンを中和します。 </li><li> 50mlコニカルチューブにトリプシン処理した細胞を移し、0.5 GX 3分でスピンダウン。上清を除去し、完全増殖培地5mlにまで戻って細胞をもたらします。培養フラスコから回収した細胞をカウントするセルカウンタを使用してください。 </li><li>黒、透明底、96ウェルプレートに100μlのあたり2,000〜4,000 NOFをプレートに完全増殖培地の適切な量で細胞を希釈します。メッキ前に、細胞混合物をラット尾コラーゲンI0.5μgの/ 100μLを加えます。細胞は、少なくとも4時間、加湿環境において、または細胞になるまで5％CO 2中で37℃で放置プレート表面に付着します。 </li><li>ステップ2.1および2.2に記載したのと同じ方法を用いて培養フラスコからHPMCを解除してください。すでに96ウェルプレート中のI型コラーゲンでメッキNOFの上に50μlのあたり10,000〜20,000 HPMCをプレートに完全増殖培地の適切な量で細胞を希釈します。細胞は、実験を開始する前に、少なくとも18時間、加湿環境で、5％CO 2中、37℃で座ってみましょう。 </li><li>完全な増殖培地でHeyA8卵巣癌細胞を発現性培養緑色蛍光タンパク質（GFP）。 HeyA8卵巣癌細胞を蛍光カイアシ類CGFP（pCDH-CMV-MCS-EF1）を発現するレンチウイルスベクターの発現によって標識されています。 HeyA8卵巣癌細胞は、使用日の80〜90％コンフルエント（対数増殖期）でなければなりません。培養プレートから細胞を離し、初代培養細胞のためのステップ2.1から2.2に記載したのと同じ方法を使用してカウントします。 </li><li>卵巣癌細胞を、37℃で15〜20分間、完全増殖培地中で回復させます緩く密封された蓋付きの円錐管。 </li></ol> 3.接着アッセイ8 <ol><li>回収後、0.5×gで3分間回転させることにより卵巣癌細胞をペレット化し、上清を除去し、50,000細胞/ 100μlの濃度を達成するために、無血清培地の適切な量で細胞を希釈します。 </li><li>使用済み培地を除去し、各ウェルに無血清培地中の癌細胞懸濁液100μlを追加するHPMC / NOF器官培養物を96ウェルプレートを反転します。 4時間30分間、加湿環境中、5％CO 2中37℃でプレートをインキュベートします。 </li><li>培地および非接着細胞を除去するために、96ウェルプレートを反転させます。慎重に、ゆっくりと各ウェルに100μlのPBSをピペット、再度プレートを反転させるために、低電力でマルチチャンネルピペットを使用してください。 PBSで一回以上の洗浄を繰り返します。 </li><li> PBSを除去し、各ウェルに100μlの4％パラホルムアルデヒドを添加することが反転します。プレートは、20マイルのために座ってできるようにします細胞を固定する場合は、N。 20分後、プレートを反転し、100μlのPBSを追加します。 </li><li>ウェルの総蛍光を報告することができるか、細胞の数を定量することができることに留意されたいです。定量化のために、100万細胞/ mlの濃度でHeyA8細胞を用いて、重複の最初のプレートの標準曲線。 <ol><li> 、ダウン100万個の細胞をスピン培地を除去し、それらを20分間1ミリリットルの4％パラホルムアルデヒド中で座ってできるようにすることで、標準曲線を作成します。 </li><li>再び細胞をスピンし、1mlのPBS（再集計セルが100万/ mlの濃度を確認するために）で起動します。連でプレートそれぞれに50,000、40,000、30,000 20,000 10,000 5000 1000、および0細胞だけでなく、各ウェルに50μlの最終総容量をPBSを追加します。 </li></ol></li><li>ボトムは高いウェル（標準曲線上で50,000）をスケーリング、蛍光プレートリーダー（励起= 488nmで、放射= 528 nm）の上で培養プレートをお読みください。 organotypに付着何卵巣癌細胞を計算するために、標準曲線を使用して各ウェル（ 図4A-C）におけるIC文化。 </li></ol> 4.増殖アッセイ10 <ol><li>卵巣癌細胞を、0.5×gで3分間回転させることによって細胞をペレット化（工程2.5および上記2.6を参照）を回収した後、1％のFBS、1％MEMと1％FBS培地の適切な量（DMEMで細胞を希釈した後に4000細胞の濃度を達成するために、ビタミン[93ミリグラム/ L]、1％MEM非必須アミノ酸、[81.4ミリグラム/ L]、1％のペニシリンストレプトマイシン[ペニシリン - ストレプトマイシン、100 U / mlペニシリンおよび100μg/ mlストレプトマイシン]） / 150μL。 </li><li>使用済み培地を除去し、各ウェルに1％FBS培地中の癌細胞懸濁液の150μlを添加するHPMC / NOF器官培養物を96ウェルプレートを反転します。 72時間加湿環境で、5％CO 2中、37℃でプレートをインキュベートします。 </li><li>ボトムは、相対的な増殖を評価するために、一日一回（励起= 488nmで、= 528 nmの発光）蛍光プレートリーダー上で培養プレートを読みます細胞の。 48時間（ 図5A-C）でメディアを変え、96時間アッセイを続行します。 （プレートが望ましい反転）のメディアを変更する際の文化を乱さないように注意してください。 </li></ol> 5.浸潤アッセイ8 <ol><li> 3D培養をメッキする前に、24ウェル培養プレートインサート（8μmの孔径）に200μlのPBS中7.5μgのラット尾コラーゲンIを追加します。プレートをインキュベートするO / Nが、その後慎重にすぐにコラーゲン被覆インサート（上記のステップ2）にHPMC / NOFの器官培養液をメッキする前にピペットでPBSを削除してみましょう。 </li><li>ステップ3.1のように卵巣癌細胞を準備します。インサートの器官培養物に卵巣癌細胞をプレートに、慎重にインサートの上部から余分なメディアを削除するには、ピペットを使用しています。各ウェルに無血清培地中で卵巣癌細胞懸濁液100μlを加えます。 </li><li>で癌細胞を誘導するために十分に挿入下に完全成長培地750μlのを追加器官培養物にvasion。 24時間加湿環境で、5％CO 2中、37℃でプレートをインキュベートします。 注意：器官培養物が挿入を渡り、インサートの底面に残る侵入卵巣癌細胞を。 </li><li>空のウェルに挿入を移動した後、24時間後、トングと挿入を把握し、簡単に、PBSのビーカーでそれをすすいでください。 1分の合計のための綿棒の両側に各インサートの上部をスクラブ。すばやく各ウェル中の750μlの4％パラホルムアルデヒドを含むプレートにカバーを置きます。各インサートは750μlのPBSで満たされたウェルへの挿入を転送する前に、10分間パラホルムアルデヒド中で座ってできるようにします。 </li><li> 2.5倍の倍率で顕微鏡で浸潤した細胞（インサートの底部に接着固定）を表示します。ウェル全体が視野内にある場合、回避、10倍に倍率を調整し、5写真、ウェルの各象限における中央付近の1と1を取りますエッジに近すぎる画像を撮影します。各ウェルについての同じパターンに従ってください。 </li><li>そのプロトコルに従って、各ウェル内のフィールドごとに浸潤した細胞の平均数（ 図6A-C）を取得するには、各画像内のセルの数をカウントするために、製造元のプログラムを使用します。 </li></ol>

<ol>
	<li>Siegel, R., Ma, J., Zou, Z., Jemal, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+statistics.">Cancer statistics.</a> CA Cancer J. Clin. 65 (1), 5-29 (2015).</li><li>Lengyel, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ovarian+cancer+development+and+metastasis.">Ovarian cancer development and metastasis.</a> Am. J. Pathol. 177, 1053-1064 (2010).</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> Nat. Med. 19, 1423-1437 (2013).</li><li>Correia, A. L., Bissell, M. 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+tumor+microenvironment+is+a+dominant+force+in+multidrug+resistance.">The tumor microenvironment is a dominant force in multidrug resistance.</a> Drug Resist Updat. 15, 39-49 (2012).</li><li>Hanahan, D., Coussens, 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=Accessories+to+the+crime:+Functions+of+cells+recruited+to+the+tumor+microenvironment.">Accessories to the crime: Functions of cells recruited to the tumor microenvironment.</a> Cancer Cell. 21, 309-322 (2012).</li><li>Nieman, K. M., 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=Adipocytes+promote+ovarian+cancer+metastasis+and+provide+energy+for+rapid+tumor+growth.">Adipocytes promote ovarian cancer metastasis and provide energy for rapid tumor growth.</a> Nat. Med. 17, 1498-1503 (2011).</li><li>Daya, D., McCaughy, W. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathology+of+the+peritoneum:+A+review+of+selected+topics.">Pathology of the peritoneum: A review of selected topics.</a> Semin. Diagn. Pathol. 8, 277-289 (1991).</li><li>Kenny, H. A., Krausz, T., Yamada, S. D., Lengyel, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+a+novel+3D+culture+model+to+elucidate+the+role+of+mesothelial+cells,+fibroblasts+and+extra-cellular+matrices+on+adhesion+and+invasion+of+ovarian+cancer+cells.">Use of a novel 3D culture model to elucidate the role of mesothelial cells, fibroblasts and extra-cellular matrices on adhesion and invasion of ovarian cancer cells.</a> Int. J. Cancer. 121, 1463-1472 (2007).</li><li>Kenny, H. A., Kaur, S., Coussens, L. M., Lengyel, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+initial+steps+of+ovarian+cancer+cell+metastasis+are+mediated+by+MMP-2+cleavage+of+vitronectin+and+fibronectin.">The initial steps of ovarian cancer cell metastasis are mediated by MMP-2 cleavage of vitronectin and fibronectin.</a> J. Clin. Invest. 118, 1367-1379 (2008).</li><li>Kenny, H. A., 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=Mesothelial+cells+promote+early+ovarian+cancer+metastasis+through+fibronectin+secretion.">Mesothelial cells promote early ovarian cancer metastasis through fibronectin secretion.</a> J. Clin. Invest. 124, 4614-4628 (2014).</li><li>Strobel, T., Cannistra, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=&#946;1-integrins+partly+mediate+binding+of+ovarian+cancer+cells+to+peritoneal+mesothelium+in+vitro.">&#946;1-integrins partly mediate binding of ovarian cancer cells to peritoneal mesothelium in vitro.</a> Gynecol. Oncol. 73, 362-367 (1999).</li><li>Strobel, T., Swanson, L., Cannistra, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+inhibition+of+CD44+limits+intra-abdominal+spread+of+a+human+ovarian+cancer+xenograft+in+nude+mice:+A+novel+role+for+CD+44+in+the+process+of+peritoneal+implantation.">In vivo inhibition of CD44 limits intra-abdominal spread of a human ovarian cancer xenograft in nude mice: A novel role for CD 44 in the process of peritoneal implantation.</a> Cancer Res. 57, 1228-1232 (1997).</li><li>Iwanicki, M., 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=Ovarian+cancer+spheroids+use+myosin-generated+force+to+clear+the+mesothelium.">Ovarian cancer spheroids use myosin-generated force to clear the mesothelium.</a> Cancer Discov. 1, 144-157 (2011).</li><li>Lessan, K., Aguiar, D., Oegema, T. R., Siebenson, L., Skubitz, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD44+and+&#946;1+integrin+mediate+ovarian+carcinoma+cell+adhesion+to+peritoneal+mesothelial+cells.">CD44 and &#946;1 integrin mediate ovarian carcinoma cell adhesion to peritoneal mesothelial cells.</a> Am. J. Pathol. 154, 1525-1537 (1999).</li><li>Ahmed, N., Riley, C., Rice, G., Quinn, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+integrin+receptors+for+fibronectin,+collagen+and+laminin+in+the+regulation+of+ovarian+carcinoma+functions+in+response+to+a+matrix+microenvironment.">Role of integrin receptors for fibronectin, collagen and laminin in the regulation of ovarian carcinoma functions in response to a matrix microenvironment.</a> Clin. Exp. Metastasis. 22, 391-402 (2005).</li><li>Kaur, 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=&#946;3-integrin+expression+on+tumor+cells+inhibits+tumor+progression,+reduces+metastasis,+and+is+associated+with+a+favorable+prognosis+in+patients+with+ovarian+cancer.">&#946;3-integrin expression on tumor cells inhibits tumor progression, reduces metastasis, and is associated with a favorable prognosis in patients with ovarian cancer.</a> Am. J. Pathol. 175, 2184-2196 (2009).</li><li>Niedbala, M. J., Crickard, K., Bernacki, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+degradation+of+extracellular+matrix+by+human+ovarian+carcinoma+cells.">In vitro degradation of extracellular matrix by human ovarian carcinoma cells.</a> Clin. Exp. Metastasis. 5, 181-197 (1987).</li><li>Kanemoto, T., Martin, G. R., Hamilton, T. C., Fridman, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+synthetic+peptides+and+protease+inhibitors+on+the+interaction+of+a+human+ovarian+cancer+cell+line+(NIH:OVCAR-3)+with+a+reconstituted+basement+membrane+(matrigel).">Effects of synthetic peptides and protease inhibitors on the interaction of a human ovarian cancer cell line (NIH:OVCAR-3) with a reconstituted basement membrane (matrigel).</a> Invasion Metastasis. 11, 84-92 (1991).</li><li>Rieppi, M., 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=Mesothelial+cells+induce+the+motility+of+human+ovarian+carcinoma+cells.">Mesothelial cells induce the motility of human ovarian carcinoma cells.</a> Int. J. Cancer. 80, 303-307 (1999).</li><li>Barbolina, M. V., Adley, B. P., Ariztia, E. V., Liu, Y., Stack, M. S. <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+membrane+type+1+matrix+metalloproteinase+activity+in+ovarian+carcinoma+cells+via+collagen-induced+EGR1+expression.">Microenvironmental regulation of membrane type 1 matrix metalloproteinase activity in ovarian carcinoma cells via collagen-induced EGR1 expression.</a> J. Biol. Chem. 282, 4924-4931 (2007).</li><li>Burleson, K. M., 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=Ovarian+carcinoma+ascites+spheroids+adhere+to+extracellular+matrix+components+and+mesothelial+cell+monolayers.">Ovarian carcinoma ascites spheroids adhere to extracellular matrix components and mesothelial cell monolayers.</a> Gynecol. Oncol. 93, 170-181 (2004).</li><li>Suzuki, N., 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=HMOCC-1,+a+human+monoclonal+antibody+that+inhibits+adhesion+of+ovarian+cancer+cells+to+human+mesothelial+cells.">HMOCC-1, a human monoclonal antibody that inhibits adhesion of ovarian cancer cells to human mesothelial cells.</a> Gynecol. Oncol. 95, 290-298 (2004).</li><li>Kishikawa, T., 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=Two+distinct+pattern+of+peritoneal+involvement+shown+by+in+vitro+and+in+vivo+ovarian+cancer+dissemination+models.">Two distinct pattern of peritoneal involvement shown by in vitro and in vivo ovarian cancer dissemination models.</a> Invas. Metast. 15, 11-21 (1995).</li><li>Casey, R. C., 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=Establishment+of+an+in+vitro+assay+to+measure+the+invasion+of+ovarian+carcinoma+cells+through+mesothelial+cell+monolayers.">Establishment of an in vitro assay to measure the invasion of ovarian carcinoma cells through mesothelial cell monolayers.</a> Clin. Exp. Metastasis. 20, 343-356 (2003).</li><li>Niedbala, M. J., Crickard, K., Bernacki, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interactions+of+human+ovarian+tumor+cells+with+human+mesothelial+cells+grown+on+extracellular+matrix.">Interactions of human ovarian tumor cells with human mesothelial cells grown on extracellular matrix.</a> Exp. Cell Res. 160, 499-513 (1985).</li><li>White, E. A., Kenny, H. A., Lengyel, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-Dimensional+modeling+of+ovarian+cancer.">Three-Dimensional modeling of ovarian cancer.</a> Adv. Drug Deliv. Rev. 79-80, 184-192 (2014).</li><li>Kenny, H. A., 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=Quantitative+high+throughput+screening+using+a+primary+human+three-dimensional+organotypic+culture+predicts+in+vivo+efficacy.">Quantitative high throughput screening using a primary human three-dimensional organotypic culture predicts in vivo efficacy.</a> Nature Comm. , (2015).</li><li>Sawada, 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=c-Met+overexpression+is+a+prognostic+factor+in+ovarian+cancer+and+an+effective+target+for+inhibition+of+peritoneal+dissemination+and+invasion.">c-Met overexpression is a prognostic factor in ovarian cancer and an effective target for inhibition of peritoneal dissemination and invasion.</a> Cancer Res. 67, 1670-1680 (2007).</li><li>Kenny, H. A., Lengyel, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MMP-2+functions+as+an+early+response+protein+in+ovarian+cancer+metastasis.">MMP-2 functions as an early response protein in ovarian cancer metastasis.</a> Cell cycle. 8, 683-688 (2009).</li><li>Sawada, 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=Loss+of+E-cadherin+promotes+ovarian+cancer+metastasis+via+alpha+5-integrin,+which+is+a+therapeutic+target.">Loss of E-cadherin promotes ovarian cancer metastasis via alpha 5-integrin, which is a therapeutic target.</a> Cancer Res. 68, 2329-2339 (2008).</li><li>Mitra, A. 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=Ligand-independent+activation+of+c-Met+by+fibronectin+and+&#945;(5)&#946;(1)-integrin+regulates+ovarian+cancer+invasion+and+metastasis.">Ligand-independent activation of c-Met by fibronectin and &#945;(5)&#946;(1)-integrin regulates ovarian cancer invasion and metastasis.</a> Oncogene. 30, 1566-1576 (2011).</li></ol>

The authors have nothing to disclose.

腹膜微小環境の器官モデルは、卵巣癌の普及における微小環境の細胞と生来のコンポーネントの両方の個人や集団の機能（複数可）を評価するために設立されました。卵巣癌細胞の接着、増殖、および浸潤を調べるために、3次元器官型培養物をプレーティングし、カスタマイズするための特定のプロトコルが提供されます。初代ヒト大網中皮細胞及び線維芽細胞は、患者から単離し、正常な...

Ovarian cancer is the deadliest gynecologic malignancy1. The majority of patients are diagnosed after the cancer has disseminated throughout the peritoneal cavity. Once the cancer has spread throughout the peritoneal cavity, cytoreductive surgery and chemotherapy are often not sufficient treatment to prevent cancer recurrence and chemoresistance, resulting in a less than 30% 5-year survival rate. Ovarian cancer metastasis is predominantly limited to the peritoneal cavity, and several other cancer types, including gastric, pancreatic, and colon cancers, metastasize to the same anatomic sites in the peritoneal cavity. In general, ovarian cancer cells detach from the in situ carcinoma in the fallopian tube or the primary ovarian tumor, travel in peritoneal fluid as single cells or spheroids, and attach to mesothelium-lined surfaces of the omentum, bowel, and abdominal wall2. 
The tumor microenvironment plays an important role in disease progression and chemoresistance in many cancers3-6. The peritoneal cavity is a unique microenvironment, with a mesothelial cell monolayer covering the majority of surfaces (Figure 1A)7. The mesothelial lining acts as a barrier that creates a low-friction surface, which tends to be protective against cancer cell adhesion8. Immediately underneath this mesothelial-lined surface is a layer made predominantly of fibroblasts and extracellular matrix (ECM), which promote cancer cell adhesion and invasion8. Ovarian cancer cells secrete factors that induce changes in the mesothelial cell lining that enhance ovarian cancer cell adhesion, invasion, and metastasis9,10. Ovarian cancer cells adhere to the mesothelial surface via integrin and CD44-mediated mechanisms (Figure 1B)11-16. 
Historically, several 3D models have been developed to investigate ovarian cancer interactions with the microenvironment. Some of the first models studied ovarian cancer-ECM interface17-21, ovarian cancer-mesothelial cell communication13,14,21-24, or both25 (reviewed by us 26). Niedbala et al. discovered that ovarian cancer cells display a quicker and firmer adhesion to ECM than to mesothelial cells or to plastic alone25. However, these models did not histologically resemble the peritoneal microenvironment. Therefore, we established a 3D organotypic model to more thoroughly replicate the ovarian cancer microenvironment. In order to better understand the role of the microenvironment and the interaction between cancer and peritoneal cells in the peritoneal dissemination of ovarian cancer, we have developed a 3D organotypic in vitro culture model of the peritoneal cavity lining (schematic in Figure 1C). The proposed model is composed of primary human fibroblasts and ECM, covered with a layer of primary human mesothelial cells-each cell type is isolated from human omentum. Histologically, this model resembles the normal peritoneal or omental lining, and provides a surface on which we can study the tumor microenvironment, the interaction between cancer cells and normal tissue, and the processes of cancer cell adhesion, invasion, and proliferation8.

<table><tbody><tr><td>&lt;strong&gt;1. Isolation and culture of primary cells&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>PBS</td><td>Fisher Scientific</td><td>SH3001304</td><td></td></tr><tr><td>Single-edged razor blades</td><td>Fisher Scientific</td><td>12-640</td><td></td></tr><tr><td>15 cm culture dishes</td><td>BD Biosciences</td><td>353025</td><td></td></tr><tr><td>Glass flask</td><td>?</td><td>?</td><td></td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Life Technologies</td><td>16000044_3616914956</td><td></td></tr><tr><td>DMEM with L-Glutamine</td><td>Corning</td><td>10-013-CV</td><td></td></tr><tr><td>MEM Vitamins</td><td>Corning</td><td>25-020-Cl</td><td></td></tr><tr><td>MEM Nonessential amino acids</td><td>Corning</td><td>25-025-CI</td><td></td></tr><tr><td>Penicillin-Streptomycin</td><td>Corning</td><td>30-002-CI</td><td></td></tr><tr><td>Shaker&amp;nbsp;</td><td>Thermo-Fisher</td><td>MaxQ 4450</td><td></td></tr><tr><td>Centrifuge</td><td>Eppendorf</td><td>5702</td><td></td></tr><tr><td>Incubator</td><td>Thermo-Fisher</td><td>Forma Series II Water Jacketed CO2 Incubator Model 3100</td><td></td></tr><tr><td>Trypsin EDTA, 1x (0.25%)</td><td>Corning</td><td>25-053-CI</td><td></td></tr><tr><td>Hyaluronidase</td><td>Worthington Biochemical</td><td>LS002592</td><td></td></tr><tr><td>T-75 Flasks</td><td>BD Biosciences</td><td>353136</td><td></td></tr><tr><td>T-175 Flasks</td><td>BD Biosciences</td><td>353112</td><td></td></tr><tr><td>Pipet tips</td><td>Rainin</td><td>P2, P10, P20, P200 and P1000</td><td></td></tr><tr><td>Pipet tips</td><td>Corning</td><td>Filtered tips P2, P10, P20, P200 and P1000</td><td></td></tr><tr><td>&lt;strong&gt;Name of Reagent/ Equipment&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments/Description&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;2. Plating 3D culture&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Cell Counter</td><td>Invitrogen</td><td>Countess</td><td></td></tr><tr><td>Countess Cell Counting Chamber Slides</td><td>Invitrogen</td><td>C10313</td><td></td></tr><tr><td>Trypan Blue Stain (0.4%)</td><td>Gibco</td><td>15250-061</td><td></td></tr><tr><td>Collagen Type I (Rat Tail)</td><td>BD Biosciences</td><td>354236</td><td></td></tr><tr><td>96 well plate, clear bottom, black</td><td>BD Biosciences</td><td>353219</td><td></td></tr><tr><td>&lt;strong&gt;Name of Reagent/ Equipment&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments/Description&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;3. Adhesion assay&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Multichannel pipet</td><td>Eppendorf</td><td>Xplorer 300</td><td></td></tr><tr><td>Paraformaldehyde solution 4% in PBS</td><td>Santa Cruz Biotechnology</td><td>sc-281692</td><td></td></tr><tr><td>Plate reader</td><td>Molecular Devices</td><td>Minimax</td><td></td></tr><tr><td>&lt;strong&gt;Name of Reagent/ Equipment&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments/Description&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;5. Invasion assay&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Cell Culture Inserts (8um, 24-well)</td><td>BD Biosciences</td><td>353097</td><td></td></tr><tr><td>Cotton swabs</td><td>Q-tips</td><td>cotton swabs</td><td></td></tr><tr><td>Microscope</td><td>Zeiss</td><td>Axiovert 200m</td><td></td></tr><tr><td>Cell Profiler</td><td>public domain</td><td></td><td></td></tr><tr><td>24 well plate</td><td>BD Biosciences</td><td>353047</td><td></td></tr><tr><td>&lt;strong&gt;Name of Reagent/ Equipment&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments/Description&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;6. Antibodies&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Anti-Integrin &amp;alpha;V&amp;beta;3 Antibody, clone LM609</td><td>EMD Millipore</td><td>MAB1976</td><td></td></tr><tr><td>Beta 1&amp;nbsp;</td><td>Oncosynergy</td><td>OS2966</td><td></td></tr><tr><td>Alpha 5 [CD49e]</td><td>ID Pharmingen</td><td>555615</td><td></td></tr><tr><td>Beta 4 [CD104]</td><td>EMD Millipore</td><td>MAB 2058</td><td></td></tr></tbody></table>

modeling the early steps of ovarian cancer dissemination in an organotypic culture of the human peritoneal cavity

The pattern of ovarian cancer metastasis is markedly different from that of most other epithelial tumors, because it rarely spreads hematogenously. Instead, ovarian cancer cells exfoliated from the primary tumor are carried by peritoneal fluid to metastatic sites within the peritoneal cavity. These sites, most notably the abdominal peritoneum and omentum, are organs covered by a mesothelium-lined surface. To investigate the processes of ovarian cancer dissemination, we assembled a complex three-dimensional culture system that reconstructs the lining of the peritoneal cavity in vitro. Primary human fibroblasts and mesothelial cells were isolated from human omentum. The fibroblasts were then mixed with extracellular matrix and covered with a layer of the primary human mesothelial cells to mimic the peritoneal and omental surfaces encountered by metastasizing ovarian cancer cells. The resulting organotypic model is, as shown, used to examine the early steps of ovarian cancer dissemination, including cancer cell adhesion, invasion, and proliferation. This model has been used in a number of studies to investigate the role of the microenvironment (cellular and acellular) in early ovarian cancer dissemination. It has also been successfully adapted to high throughput screening and used to identify and test inhibitors of ovarian cancer metastasis.

器官培養物を、最初にI型コラーゲンと初代ヒト線維芽細胞を混合した後、中皮細胞の5倍の数でこの文化を重ねることによって組み立てました。卵巣癌細胞の接着、浸潤または増殖を研究するために追加された前培養物を少なくとも18時間インキュベートしました。各アッセイは、複数を繰り返した（N = 5）及び浸潤アッセイ（N = 3）（N = 3~5）の異なる患者及び多数のウェルから得られた三次?...

Watch this Scientific Journal Video about Modeling the Early Steps of Ovarian Cancer Dissemination in an Organotypic Culture of the Human Peritoneal Cavity at JoVE.com

Modeling the Early Steps of Ovarian Cancer Dissemination in an Organotypic Culture of the Human Peritoneal Cavity

Here, we present a protocol to construct a three-dimensional in vitro model of the lining of the peritoneal cavity, composed of primary human mesothelial cells and fibroblasts layered with extracellular matrix, as a tool to investigate ovarian cancer cell adhesion, invasion, and proliferation.

ヒト腹腔器官培養における卵巣癌普及の初期段階のモデル化

The pattern of ovarian cancer metastasis is markedly different from that of most other epithelial tumors, because it rarely spreads hematogenously. ...

modeling-early-steps-ovarian-cancer-dissemination-an-organotypic

Research

JoVE Journal

Medicine

26.5K ビュー.  University of Chicago. Here, we present a protocol to construct a three-dimensional in vitro model of the lining of the peritoneal cavity, composed of primary human mesothelial cells and fibroblasts layered with extracellular matrix, as a tool to investigate ovarian cancer cell adhesion, invasion, and proliferation. 

ビデオ: Modeling the Early Steps of Ovarian Cancer Dissemination in an Organotypic Culture of the Human Peritoneal Cavity

Murine Experimental Model of Original Tumor Development and Peritoneal Metastasis via Orthotopic Inoculation with Ovarian Carcinoma Cells

Epithelial ovarian carcinoma (EOC) is associated with a poor prognosis because it shows peritoneal dissemination. To improve the prognosis, it is important to control peritoneal dissemination. However, it is still unclear how tumor cells detach from primary lesions and attach to the mesothelium. The establishment of an appropriate animal model is needed to gain an understanding of the mechanism of peritoneal dissemination in vivo. In the current study, we introduce the process from the local injection of EOC cells into the murine ovarian surface to the development of metastasis, including the peritoneum and distant organs. Female nude mice (BALB/c nu/nu) at 8 weeks of age were used. Under a microscopic field of view, EOC cells (1 x 105 cells/&#181;l of medium-extracellular matrix (ECM)-based hydrogel/unilateral ovary/mouse) were injected into murine ovaries through a retroperitoneal approach from the dorsal flank. This proposed method is a less invasive procedure for the mouse and minimizes damage to the ovary. Here, we describe the methodological steps in the development of the original and metastatic tumor formation of EOC.

Murine Experimental Model of Original Tumor Development and Peritoneal Metastasis via Orthotopic Inoculation with Ovarian Carcinoma Cells

To clarify the multistep process of peritoneal dissemination of ovarian carcinoma, the current study presents a murine experimental model of original tumor development and peritoneal metastasis via orthotopic inoculation with these tumor cells.

オリジナル腫瘍の開発のマウス実験モデルと腹膜転移<em&gt;経由で</em卵巣癌細胞で&gt;同所接種

Epithelial ovarian carcinoma (EOC) is associated with a poor prognosis because it shows peritoneal dissemination. To improve the prognosis, it is ...

がん研究

In vitro Mesothelial Clearance Assay that Models the Early Steps of Ovarian Cancer Metastasis

Ovarian cancer is the fifth leading cause of cancer related deaths in the United States1. Despite a positive initial response to therapies, 70 to 90 percent of women with ovarian cancer develop new metastases, and the recurrence is often fatal2. It is, therefore, necessary to understand how secondary metastases arise in order to develop better treatments for intermediate and late stage ovarian cancer. Ovarian cancer metastasis occurs when malignant cells detach from the primary tumor site and disseminate throughout the peritoneal cavity. The disseminated cells can form multicellular clusters, or spheroids, that will either remain unattached, or implant onto organs within the peritoneal cavity3 (Figure 1, Movie 1). 
All of the organs within the peritoneal cavity are lined with a single, continuous, layer of mesothelial cells4-6 (Figure 2). However, mesothelial cells are absent from underneath peritoneal tumor masses, as revealed by electron micrograph studies of excised human tumor tissue sections3,5-7 (Figure 2). This suggests that mesothelial cells are excluded from underneath the tumor mass by an unknown process. 
Previous in vitro experiments demonstrated that primary ovarian cancer cells attach more efficiently to extracellular matrix than to mesothelial cells8, and more recent studies showed that primary peritoneal mesothelial cells actually provide a barrier to ovarian cancer cell adhesion and invasion (as compared to adhesion and invasion on substrates that were not covered with mesothelial cells)9,10. This would suggest that mesothelial cells act as a barrier against ovarian cancer metastasis. The cellular and molecular mechanisms by which ovarian cancer cells breach this barrier, and exclude the mesothelium have, until recently, remained unknown. 
Here we describe the methodology for an in vitro assay that models the interaction between ovarian cancer cell spheroids and mesothelial cells in vivo (Figure 3, Movie 2). Our protocol was adapted from previously described methods for analyzing ovarian tumor cell interactions with mesothelial monolayers8-16, and was first described in a report showing that ovarian tumor cells utilize an integrin &#x2013;dependent activation of myosin and traction force to promote the exclusion of the mesothelial cells from under a tumor spheroid17. This model takes advantage of time-lapse fluorescence microscopy to monitor the two cell populations in real time, providing spatial and temporal information on the interaction. The ovarian cancer cells express red fluorescent protein (RFP) while the mesothelial cells express green fluorescent protein (GFP). RFP-expressing ovarian cancer cell spheroids attach to the GFP-expressing mesothelial monolayer. The spheroids spread, invade, and force the mesothelial cells aside creating a hole in the monolayer. This hole is visualized as the negative space (black) in the GFP image. The area of the hole can then be measured to quantitatively analyze differences in clearance activity between control and experimental populations of ovarian cancer and/ or mesothelial cells. This assay requires only a small number of ovarian cancer cells (100 cells per spheroid X 20-30 spheroids per condition), so it is feasible to perform this assay using precious primary tumor cell samples. Furthermore, this assay can be easily adapted for high throughput screening.

In vitro Mesothelial Clearance Assay that Models the Early Steps of Ovarian Cancer Metastasis

The mesothelial clearance assay described here takes advantage of fluorescently labeled cells and time-lapse video microscopy to visualize and quantitatively measure the interactions of ovarian cancer multicellular spheroids and mesothelial cell monolayers. This assay models the early steps of ovarian cancer metastasis.

in vitroで中皮クリアランスアッセイでそのモデルの卵巣がんの転移の初期段階を

Ovarian cancer is the fifth leading cause of cancer related deaths in the United States1. Despite a positive initial response to therapies, 70 to 90 ...

医学

Heterotypic Three-dimensional In Vitro Modeling of Stromal-Epithelial Interactions During Ovarian Cancer Initiation and Progression

Epithelial ovarian cancers (EOCs) are the leading cause of death from gynecological malignancy in Western societies. Despite advances in surgical treatments and improved platinum-based chemotherapies, there has been little improvement in EOC survival rates for more than four decades 1,2. Whilst stage I tumors have 5-year survival rates &gt;85%, survival rates for stage III/IV disease are &lt;40%. Thus, the high rates of mortality for EOC could be significantly decreased if tumors were detected at earlier, more treatable, stages 3-5. At present, the molecular genetic and biological basis of early stage disease development is poorly understood. More specifically, little is known about the role of the microenvironment during tumor initiation; but known risk factors for EOCs (e.g. age and parity) suggest that the microenvironment plays a key role in the early genesis of EOCs. We therefore developed three-dimensional heterotypic models of both the normal ovary and of early stage ovarian cancers. For the normal ovary, we co-cultured normal ovarian surface epithelial (IOSE) and normal stromal fibroblast (INOF)&nbsp;cells, immortalized by retrovrial transduction of the catalytic subunit of human telomerase holoenzyme (hTERT) to extend the lifespan of these cells in culture. To model the earliest stages of ovarian epithelial cell transformation, overexpression of the CMYC oncogene in IOSE cells, again co-cultured with INOF cells. These heterotypic models were used to investigate the effects of aging and senescence on the transformation and invasion of epithelial cells. Here we describe the methodological steps in development of these three-dimensional model; these methodologies aren't specific to the development of normal ovary and ovarian cancer tissues, and could be used to study other tissue types where stromal and epithelial cell interactions are a fundamental aspect of the tissue maintenance and disease development.

Heterotypic Three-dimensional In Vitro Modeling of Stromal-Epithelial Interactions During Ovarian Cancer Initiation and Progression

We describe methodologies for establishing in vitro heterotypic three-dimensional models comprising ovarian fibroblasts and normal ovarian surface or ovarian cancer epithelial cells. We discuss the use of these models to study stromal-epithelial interactions that occur during ovarian cancer development.

三次元異<em&gt; in vitroで</em卵巣がんの発生と進行中に間質相互作用の上皮&gt;モデリング

Epithelial ovarian cancers (EOCs) are the leading cause of death from gynecological malignancy in Western societies. Despite advances in surgical ...

Assessment of Ovarian Cancer Spheroid Attachment and Invasion of Mesothelial Cells in Real Time

Ovarian cancers metastasize by shedding into the peritoneal fluid and dispersing to distal sites within the peritoneum. Monolayer cultures do not accurately model the behaviors of cancer cells within a nonadherent environment, as cancer cells inherently aggregate into multicellular structures which contribute to the metastatic process by attaching to and invading the peritoneal lining to form secondary tumors. To model this important stage of ovarian cancer metastasis, multicellular aggregates, or spheroids, can be generated from established ovarian cancer cell lines maintained under nonadherent conditions. To mimic the peritoneal microenvironment encountered by tumor cells in vivo, a spheroid-mesothelial co-culture model was established in which preformed spheroids are plated on top of a human mesothelial cell monolayer, formed over an extracellular matrix barrier. Methods were then developed using a real-time cell analyzer to conduct quantitative real time measurements of the invasive capacity of different ovarian cancer cell lines grown as spheroids. This approach allows for the continuous measurement of invasion over long periods of time, which has several advantages over traditional endpoint assays and more laborious real time microscopy image analyses. In short, this method enables a rapid, determination of factors which regulate the interactions between ovarian cancer spheroid cells invading through mesothelial and matrix barriers over time.

Ovarian cancer cell invasion into the mesothelial lining of the peritoneum is a dynamic process over time. Utilizing a real time analyzer, the invasive capacity of ovarian cancer cells in a spheroid-mesothelial cell co-culture model can be quantified over prolonged time periods, providing insights into factors regulating the metastatic process.

リアルタイムで中皮細胞の卵巣癌スフェロイドアタッチメントおよび浸潤の評価

Ovarian cancers metastasize by shedding into the peritoneal fluid and dispersing to distal sites within the peritoneum. Monolayer cultures do not ...

卵巣がん-大網相互作用のための3D ex vivoモデルの確立

An Ex Vivo Model of Ovarian Cancer Peritoneal Metastasis Using Human Omentum

Ovarian cancer is the deadliest gynecologic malignancy. The omentum plays a key role in providing a supportive microenvironment to metastatic ovarian cancer cells as well as immune modulatory signals that allow tumor tolerance. However, we have limited models that closely mimic the interaction between ovarian cancer cells and adipose-rich tissues. To further understand the cellular and molecular mechanisms by which the omentum provides a pro-tumoral microenvironment, we developed a unique 3D ex vivo model of cancer cell-omentum interaction. Using human omentum, we are able to grow ovarian cancer cells within this adipose-rich microenvironment and monitor the factors responsible for tumor growth and immune regulation. In addition to providing a platform for the study of this adipose-rich tumor microenvironment, the model provides an excellent platform for the development and evaluation of novel therapeutic approaches to target metastatic cancer cells in this niche. The proposed model is easy to generate, inexpensive, and applicable to translational investigations.

著者スポットライト:がんと脂肪微小環境の相互作用を調査するための高度なex vivoモデル 

This protocol describes the establishment of a three-dimensional (3D) ex vivo model of cancer cell-omentum interaction. The model provides a platform for elucidating pro-tumor mechanisms within the adipose niche and for testing novel therapies.

ヒト大網を用いた卵巣癌腹膜転移の Ex vivo モデル

Ovarian cancer is the deadliest gynecologic malignancy. The omentum plays a key role in providing a supportive microenvironment to metastatic ovarian ...

Modeling Ovarian Cancer Multicellular Spheroid Behavior in a Dynamic 3D Peritoneal Microdevice

Ovarian cancer is characterized by extensive peritoneal metastasis, with tumor spheres commonly found in the malignant ascites. This is associated with poor clinical outcomes and currently lacks effective treatment. Both the three-dimensional (3D) environment and the dynamic mechanical forces are very important factors in this metastatic cascade. However, traditional cell cultures fail to recapitulate this natural tumor microenvironment. Thus, in vivo-like models that can emulate the intraperitoneal environment are of obvious importance. In this study, a new microfluidic platform of the peritoneum was set up to mimic the situation of ovarian cancer spheroids in the peritoneal cavity during metastasis. Ovarian cancer spheroids generated under a non-adherent condition were cultured in microfluidic channels coated with peritoneal mesothelial cells subjected to physiologically relevant shear stress. In summary, this dynamic 3D ovarian cancer-mesothelium microfluidic platform can provide new knowledge on basic cancer biology and serve as a platform for potential drug screening and development.

To study ovarian tumor progression in a physiologically relevant model, multicellular spheroids were cultured in a microdevice under simulated fluid flow. This dynamic 3D model emulates the intraperitoneal environment with the cellular and mechanical components where ovarian cancer metastasis occurs.

ダイナミックな3D腹膜のマイクロデバイスに卵巣癌多細胞スフェロイドの挙動のモデル化

Ovarian cancer is characterized by extensive peritoneal metastasis, with tumor spheres commonly found in the malignant ascites. This is associated with ...

生物工学

Modeling Ovarian Cancer Communication for Imaging Mass Spectrometry Analysis: A Method to Identify Small Molecule Chemical Communicators in Metastatic Ovarian Tumor Development

Source: Zink, K. E. et. al. <a href="https://www.jove.com/t/59490">Capturing Small Molecule Communication Between Tissues and Cells Using Imaging Mass Spectrometry.</a> J. Vis. Exp. (2019)
This protocol describes a technique for sample preparation by co-culturing ovarian explants with the fallopian tube cancer cells. These samples are visualized using imaging mass spectroscopy, or IMS,&#160; to detect small molecule exchange between the cells and tissues leading to tumor development.

イメージング質量分析のための卵巣がんコミュニケーションのモデリング:転移性卵巣腫瘍の発生における低分子化学コミュニケーターを同定する方法

Source: Zink, K. E. et. al. Capturing Small Molecule Communication Between Tissues and Cells Using Imaging Mass Spectrometry. J. Vis. Exp. (2019)
This ...

JoVE Encyclopedia of Experiments

婦人科系がん

アッセイと技術

Modeling Ovarian Cancer Metastasis in Peritoneal Cavity Lining: A Method To Establish an In Vitro 3D Model of the Peritoneal Lining

Source: Peters, P. N. et al. <a href="https://www.jove.com/t/53541">Modeling the Early Steps of Ovarian Cancer Dissemination in an Organotypic Culture of the Human Peritoneal Cavity.</a> J. Vis. Exp. (2015)
In this video, we demonstrate the development of&#160; an in vitro &#160;three-dimensional model of the lining of the peritoneal cavity. This organotypic culture can help to investigate ovarian cancer cell adhesion, invasion, and proliferation.

腹腔内層における卵巣癌転移のモデリング:腹膜内層のin vitro 3Dモデルを確立する方法

Source: Peters, P. N. et al. Modeling the Early Steps of Ovarian Cancer Dissemination in an Organotypic Culture of the Human Peritoneal Cavity. J. Vis. ...

ヒト腹腔器官培養における卵巣癌普及の初期段階のモデル化

要約

さらに動画を探す

この動画の章

オリジナル腫瘍の開発のマウス実験モデルと腹膜転移

in vitroで中皮クリアランスアッセイでそのモデルの卵巣がんの転移の初期段階を

三次元異

リアルタイムで中皮細胞の卵巣癌スフェロイドアタッチメントおよび浸潤の評価

ヒト大網を用いた卵巣癌腹膜転移の Ex vivo モデル

ヒト大網を用いた卵巣癌腹膜転移の Ex vivo モデル

ヒト大網を用いた卵巣癌腹膜転移の Ex vivo モデル

ダイナミックな3D腹膜のマイクロデバイスに卵巣癌多細胞スフェロイドの挙動のモデル化

イメージング質量分析のための卵巣がんコミュニケーションのモデリング:転移性卵巣腫瘍の発生における低分子化学コミュニケーターを同定する方法

腹腔内層における卵巣癌転移のモデリング:腹膜内層のin vitro 3Dモデルを確立する方法