私たちは、ハーバード大学医学部ニコンイメージングセンターは、特にジェニファー·ウォーターズ、ララペトラークとウェンディサーモン、訓練のため、それらのタイムラプス顕微鏡の使用を感謝したいと思います。また、貴重な議論のためにローザNgとアヒム·Besserに感謝します。この作品は、NIHグラント5695837（M. Iwanickiまで）とJSBにGM064346によってサポートされていました、博士ミリアムとシェルドンG.アデルソン医学研究財団（JSBまで）からの助成金によって。

1。卵巣癌細胞スフェロイドの形成<ol><li> RFP発現卵巣癌細胞は10％の基本培地（199 MCDB105の50:50混合物、10％不活化ウシ胎児血清および1％ペン連鎖球菌を含むカスタム細胞培養培地）で培養されています。ラベルが付いていない卵巣癌細胞にRFPを表現するために、プラスミドを含むRFPで細胞をトランスフェクトし、RFPを発現する細胞に対して選択されます。また、ウイルスベクターを一過的に蛍光タンパク質を発現するために使用されたり、細胞が赤色蛍光細胞トラッカー色素（Invitrogen）とプレインキュベートすることができます。 </li><li>前の卵巣癌細胞スフェロイドの形成には、低接着96ウェル丸底培養皿を準備する必要があります。低接着培養プレートを生成するために、30μlのポリHEMA（1ミリリットル95パーセントエタノールで6mgをpolyhydroxyethylmethacrylate）溶液を96ウェルコーニングの細胞培養皿の各ウェルに添加されています。 96ウェルプレートは、L、°C非加湿インキュベーターは、エタノールを蒸発させるために37でインキュベートされる各ウェルにポリHEMAのフィルムをeaving。このポリHEMA膜は、細胞は、懸濁液18に成長すると、強制的に井戸の底に付着するから細胞を防ぐことができます。 [別の方法として、超低添付培養プレート（Corning）は、ポリ-HEMAコートディッシュの代わりに使用できます。] </li><li>低接着培養プレートが用意された後、卵巣癌細胞、ペレット3分間900 RCFで卓上遠心機（ヘレウス）の細胞は、上清を吸引し、10％の基本培地に再懸濁のプレートをトリプシン処理。 </li><li>血球計算板を用いて細胞をカウントします。 </li><li> 10％の基本培地50μlの100細胞が存在するような細胞の濃度を調整します。 </li><li> 96ウェルのポリHEMAコーティングした培養皿の各ウェルに均一に懸濁希釈細胞懸濁液50μlのを追加します。 </li><li> 16時間37℃細胞培養インキュベーター中で96ウェルプレートをインキュベートします（この時間は、それがためにかかる時間の量に応じて増減されるべき各ウェル内の単一の多細胞スフェロイドを形成し、一緒にクラスタに卵巣癌細胞を可能にする多細胞スフェロイドまたは所望の実験条件）を形成する特定の細胞株。いくつかの腫瘍細胞は、この期間中にアポトーシスを受けることができるので、前のアポトーシスの誘導までの時間を選択することが重要です。 </li></ol> 2。中皮細胞単層の形成<ol><li>皿の各ウェルに5μgのフィブロネクチン/ mLのPBS溶液2mLのを追加し、室温で30分間インキュベートすることにより細胞培養フード、プレコートにフィブロネクチンで6ウェルガラスボトムマテック皿の井戸。マテック皿にガラス底の光学的品質は、高解像度顕微鏡イメージングを可能にします。 </li><li> GFPを発現する中皮細胞は、10％の基本培地中で培養されています。中皮細胞のプレートをトリプシン処理、3分間900RPMで卓上遠心機（ヘレウス）にスピンダウンし、上清を吸引し、10％の基本培地に再懸濁する。ザここで使用される中皮細胞は、それらが得られた時にすでにGFPを発現していましたが、ラベルが付いていない中皮細胞のcDNAを含むプラスミドでトランスフェクションGFP、緑色蛍光細胞トラッカー色素（Invitrogen）中で細胞をプレインキュベートすることによって製造することができる。 </li><li> 30分フィブロネクチンのインキュベーション（ステップ2.1）の後、2mLのPBSでマテック皿のウェルを洗浄する。 </li><li> 6ウェルマテック皿の各ウェルにウェルあたりPBSと板6×10 5中皮細胞を吸引除去する。中皮細胞がディッシュにアタッチし、単分子膜を形成できるようにするために一晩37℃のインキュベーター内でマテック皿をインキュベートします。 </li></ol> 3。中皮細胞クリアランスアッセイ<ol><li> 96ウェルポリ-HEMAコートプレートから卵巣癌のスフェロイドを収集するためのピペットを使用しています。 </li><li>中皮細胞単層を含む6ウェルマテック皿の1つのウェルから培地を吸引除去する。 2mLのPBSで1回洗浄します。 96ウェルpから回転楕円体のすべてを追加します。マテック皿（撮像することはできません料理の一部にスフェロイドの着陸を考慮してイメージを作成しようとしている回転楕円体の〜3倍の数）1つのウェルに遅い。 </li><li>少なくとも8時間の期間タイムラプスイメージングを行うことができる反転広視野蛍光顕微鏡のステージ上でマテック皿を置きます。回の実験では、複数の回転楕円体のインターカレーションイベントで、皿の中のイメージを複数の位置に電動ステージを使用しています。我々は統合されたパーフェクトフォーカスシステムとニコンの​​Ti-E反転電動広視野蛍光タイムラプス顕微鏡を使用して、低[20X-0.75の開口数（NA）]倍率/ NA微分干渉コントラスト（DIC）光学、0.52 NAニコンハロゲンネーター長い作動距離（LWD）コンデンサー、ニコンに高速（&lt;100ミリ秒の切り替え時間）は励起および発光フィルタ（GFP例40分の480、エム50分の525、RFP-mCherry例50分の575エム50分の640）、サッターは、高速送信と落射蛍光光路スマートシャットダウンTERS、ニコン線形でエンコードされた電動ステージ、浜松ORCA-AGは、電荷結合素子（CCD）カメラ、温度とCO 2制御と特注の顕微鏡のインキュベーション室、ニコンNIS-ElementsシリーズARソフトウェアバージョン3を冷却し、 TMCの防振台。 </li><li>卵巣癌細胞のスフェロイドは、皿の底に沈殿し、中皮細胞単層にアタッチされます。 8時間に20のGFP、RFPと位相画像+回転楕円体/単層相互作用、10分ごとに収集します。 </li><li> RFP発現卵巣癌細胞のスフェロイドは、単層の穴を作成するGFPを発現する中皮細胞単層に侵入します。 8時間後には、Elementsソフトウェア（またはそのようなイメージJなどの別の適切なソフトウェア）を使用して、GFP画像のブラックホールをトレースすることによって穴の大きさを測定します。時間ゼロで対応するRFPイメージの回転楕円体の大きさによって8時間で穴の大きさを分割することによって初期回転楕円体の大きさに穴の大きさを正規化します。この元の十分な、穴のサイズは一度しか測定されたが、それは優れたインターカレーションのダイナミクスを理解するために8時間の実験を通して複数回測定することができます。 </li></ol> 4。代表的な結果この例では、OVCA433スフェロイドを制御するために、タリン-1の減衰式を持っているOVCA433卵巣癌細胞スフェロイドの皮クリアランス能力を比較した。各グループからOVCA433スフェロイドは、ZT中皮細胞単層を含むマテック皿に追加されました。各グループから六スフェロイドは8時間（図4、映画3、ムービー4）は10分ごとに撮像した。拡散スフェロイドで単分子層で生産さの穴を測定し、各グループから6つの位置を平均した。タリン1ノックダウン細胞スフェロイドによって作成された平均隙間面積が制御スフェロイドで作成された平均面積よりも有意に小さかったことが図4に示すように、タリンはO皮クリアランスために必要であることが示唆されたVCA433卵巣癌細胞スフェロイド。 <img alt="図1" src="/files/ftp_upload/3888/3888fig1.jpg" /> 図1卵巣がんの転移。原発性卵巣腫瘍は卵巣表層上皮または卵管のいずれかから開発しています。腫瘍細胞/クラスタでは、原発腫瘍から離れると腹腔内に収集されます。腫瘍細胞は多細胞スフェロイドを形成するために集計してすることができます。スフェロイドは、腹腔の内側を覆う中皮細胞単層に接続します。中皮細胞は、スフェロイドは、基礎となる基底膜へのアクセス権を取得することができ、接続された卵巣癌の回転楕円体の下から除外されています。 ムービー1。卵巣がんの転移は<a href="http://www.jove.com/files/ftp_upload/3888/Movie_1_Lower_Quality.mp4">、ムービーを表示するには、ここをクリック</a> 。 <img alt="図2" src="/files/ftp_upload/3888/3888fig2.jpg" /> 図2。 </strオング&gt;中皮細胞は、ヒト腹膜組織の表面を整列および卵巣癌細胞のインプラントの下から除外されています。 <img alt="図3" src="/files/ftp_upload/3888/3888fig3.jpg" /> 図3中皮クリアランスアッセイ。卵巣癌のスフェロイドは、100をインキュベートすることによって形成されたRFPを発現している16時間37℃で、ポリ-HEMAコート96ウェル丸底培養皿に、ウェルあたり卵巣癌細胞を。ポリHEMAは、細胞懸濁液中に残り、ウェルあたり単一のクラスタを形成するためにお互いに付着することができ、培養皿に付着するから細胞を防ぐことができます。中皮細胞単層をフィブロネクチンコーティングした6ウェルマテック皿にウェルあたり6×10 5中皮細胞をプレーティングし、16時間37℃でプレートをインキュベートすることによって調製される。スフェロイドは、その後中皮単分子膜でマテック皿に移しており、2つの細胞集団は、ニコンのTi-E Iを用いて8時間、10分ごとに結像さnverted電動広視野蛍光タイムラプス顕微鏡および要素ソフトウェア。 映画（2）中皮クリアランスアッセイは<a href="http://www.jove.com/files/ftp_upload/3888/Movie_2_lower_quality.mp4">、ムービーを表示するには、ここをクリック</a> 。 <img alt="図4" src="/files/ftp_upload/3888/3888fig4.jpg" /> OVCA433スフェロイドにおけるタリン1発現の図4減衰は皮クリアランス能力を減少させます。タリン1の減衰発現の有無にかかわらずOVCA433スフェロイド（赤）に接続し、ZT中皮組織化単分子膜（緑色）に侵入することができました。 2つの細胞集団は、ニコンのTi-E反転電動広視野蛍光タイムラプス顕微鏡と要素ソフトウェアを使用して8時間10分ごとに撮像した。グラフは、タリン1減衰を示しています。大幅に中皮細胞のクリアランス（手段で緑色のバーが分位点プロット）を減少させます。 映画は、3。中皮組織化単分子膜（緑色）に侵入OVCA433スフェロイド（赤）を制御します。 <a href="http://www.jove.com/files/ftp_upload/3888/Movie_3.mov">ムービーを表示するには、ここをクリック</a> 。 OVCA433スフェロイドにおけるタリン1の発現（赤）の映画4。減衰は（緑）クリアランス能力を皮が減少します。 <a href="http://www.jove.com/files/ftp_upload/3888/Movie_4.mov">ムービーを表示するには、ここをクリック</a> 。

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Metastasis. 12, 305-314 (1994).</li><li>Zervantonakis, I. K., Kothapalli, C. R., Chung, S., Sudo, R., Kamm, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidic+devices+for+studying+heterotypic+cell-cell+interactions+and+tissue+specimen+cultures+under+controlled+microenvironments.">Microfluidic devices for studying heterotypic cell-cell interactions and tissue specimen cultures under controlled microenvironments.</a> Biomicrofluidics. 5, 13406-1310 (2011).</li><li>Brandt, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3D-extravasation+model+--+selection+of+highly+motile+and+metastatic+cancer+cells.">3D-extravasation model -- selection of highly motile and metastatic cancer cells.</a> Semin. Cancer Biol. 15, 387-395 (2005).</li><li>Condeelis, J., Segall, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravital+imaging+of+cell+movement+in+tumours.">Intravital imaging of cell movement in tumours.</a> Nat. Rev. Cancer. 3, 921-930 (2003).</li><li>Dai, J., Ting-Beall, H. P., Hochmuth, R. M., Sheetz, M. P., Titus, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myosin+I+contributes+to+the+generation+of+resting+cortical+tension.">Myosin I contributes to the generation of resting cortical tension.</a> Biophys. J. 77, 1168-1176 (1999).</li><li>Laferriere, J., Houle, F., Taher, M. M., Valerie, K., Huot, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transendothelial+migration+of+colon+carcinoma+cells+requires+expression+of+E-selectin+by+endothelial+cells+and+activation+of+stress-activated+protein+kinase-2+(SAPK2/p38)+in+the+tumor+cells.">Transendothelial migration of colon carcinoma cells requires expression of E-selectin by endothelial cells and activation of stress-activated protein kinase-2 (SAPK2/p38) in the tumor cells.</a> J. Biol. Chem. 276, 33762-33772 (2001).</li><li>Dong, C., Slattery, M. J., Rank, B. M., You, J. <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+characterization+and+micromechanics+of+tumor+cell+chemotactic+protrusion,+locomotion,+and+extravasation.">In vitro characterization and micromechanics of tumor cell chemotactic protrusion, locomotion, and extravasation.</a> Ann. Biomed. Eng. 30, 344-355 (2002).</li></ol>

我々は、開示することは何もありません。

ここで紹介する &quot;中皮クリアランスAssayは、&quot;偉大な空間的·時間的に詳細に、卵巣癌多細胞スフェロイドと中皮細胞単層の相互作用を監視するためのタイムラプス顕微鏡を使用しています。以前は、いくつかのグループ8-14卵巣癌細胞にアタッチし、中皮細胞単層に侵入することを示すためにエンドポイントアッセイを使用していた。このアッセイは、これら2つの細胞集団のダ?...

<table border="1"><tbody><tr><td> 試薬 </td><td> 会社 </td><td> カタログ番号 </td><td> コメント </td></tr><tr><td> OVCA433卵巣癌細胞</td><td></td><td></td><td>博士デニスSlamonからの贈り物</td></tr><tr><td> ZT中皮細胞</td><td></td><td></td><td>博士タンからの贈り物はインス</td></tr><tr><td>培地199 </td><td>ギブコ</td><td> 19950 </td><td></td></tr><tr><td> MCDB105 </td><td>セル·アプリケーション株式会社</td><td> 117から500 </td><td></td></tr><tr><td> FBS-熱不活性化</td><td>ギブコ</td><td> 10082 </td><td></td></tr><tr><td>ペン連鎖球菌</td><td>ギブコ</td><td> 15070 </td><td></td></tr><tr><td> 96ウェルプレート</td><td>コー​​ニングコースター</td><td> 3799 </td><td></td></tr><tR&gt; <td> Polyhydroxyethylmethacrylate（ポリHEMA） </td><td>シグマアルドリッチ</td><td> 192066-25G </td><td>ポリHEMAソリューションでは、95％エタノール1mlので6mgをポリHEMA粉末を溶解</td></tr><tr><td>エタノール</td><td> Pharmco-aaper </td><td> 111ACS200 </td><td>のdH 2 0で95％に希釈する</td></tr><tr><td>細胞培養フード</td><td> Nuaire </td><td> NU-425から300 </td><td></td></tr><tr><td>組織培養インキュベーター</td><td>サーモフィッシャーサイエンティフィック</td><td> 3110 </td><td></td></tr><tr><td>ポリHEMAプレートのインキュベーター</td><td> Lablineインスツルメンツ</td><td>帝国III 305 </td><td></td></tr><tr><td>卓上遠心機</td><td>ヘレウス</td><td> 75003429/01 </td><td></td></tr><tr><td> 6ウェルガラスボトムディッシュ</td><td>マテック株式会社。 </td><td> P06G-1.5-20-F </td><td></td></tr><tr><td>フィブロネクチン</td><td>シグマ</td><td> F1141-1mgの</td><td></td></tr><tr><td> PBS </td><td> Cellgro </td><td> 21から040-CV </td><td></td></tr><tr><td> タイムラプス顕微鏡： </td><td></td><td></td><td></td></tr><tr><td>顕微鏡</td><td>ニコン</td><td></td><td>統合されたパーフェクトフォーカスシステムとしたTi-E電動倒立蛍光タイムラプス顕微鏡</td></tr><tr><td>レンズ</td><td>ニコン</td><td></td><td> 20X-0.75の数値apeture </td></tr><tr><td>ハロゲンネーター</td><td>ニコン</td><td></td><td> 0.52 NA長作動距離コンデンサ</td></tr><tr><td>励起および発光フィルター</td><td>ニコンハウジング内のクロマ単一パス·フィルタ</td><td></td><td> GFP例40分の480、エム50分の525 RFP-mCherry例50分の575エム50分の640 </td></tr><tr><td>送信され、光路をEpifluoresce </td><td>サッター</tD&gt; <td></td><td>スマートシャッター</td></tr><tr><td>線形符号化された電動ステージ</td><td>ニコン</td><td></td><td></td></tr><tr><td>冷却電荷結合素子カメラ</td><td>浜松市</td><td> ORCA-AG </td><td></td></tr><tr><td>温度とCO 2制御と顕微鏡インキュベーションチャンバー</td><td>特注の</td><td></td><td></td></tr><tr><td>防振台</td><td> TMC </td><td></td><td></td></tr><tr><td> NIS-Elementsシリーズソフトウェア</td><td>ニコン</td><td></td><td>バージョン3 </td></tr></tbody></table>

in vitro mesothelial clearance assay that models the early steps of ovarian cancer metastasis

卵巣癌は、米国1の癌関連死の第五主要な原因である。治療への積極的な初期応答にもかかわらず、卵巣癌の女性の70〜90％が新たな転移を開発し、再発はしばしば2致命的である。それは、中間と後期段階の卵巣癌のためのより良い治療法を開発するために、二次転移が起こるかを理解するため、必要がある。悪性細胞が原発腫瘍部位から切り離し、腹腔内全体に普及させるときに卵巣がんの転移が発生します。播種細胞が腹腔3（図1、動画1）内の臓器に多クラスタ、または回転楕円体のいずれかのまま添付されていないか、またはインプラントを形成することができる。 腹腔内臓器のすべてが中皮細胞4-6（図2）のシングル、連続、層が並んでいます。しかし、中皮細胞は下から不在であるとして摘出したヒト腫瘍組織切片3,5-7（図2）の電子顕微鏡の研究によって明らかにされた腹膜腫瘍塊。これは中皮細胞が未知のプロセスによって、腫瘍塊の下から除外されていることを示唆している。 in vitro実験前の8中皮細胞に対するよりも原発性卵巣癌細胞は細胞外マトリックスへのより効率的に取り付けることが示され、さらに最近の研究では、原発性腹膜中皮細胞が実際に接着および浸潤に比べて卵巣癌細胞の接着および浸潤（に障壁を提供示した中皮細胞で覆われていない基板）9,10ます。これは中皮細胞は、卵巣癌の転移に対する障壁として作用することを示唆している。卵巣癌細胞は、この障壁を突破し、持っている皮を除外したことにより細胞および分子メカニズムは、最近まで不明であった。 ここでは、目を記述するin vitroアッセイのためのeの方法論をモデル生体内 （図3、映画2） の卵巣癌細胞のスフェロイドと中皮細胞との相互作用。我々のプロトコルは、中皮単層8月16日卵巣腫瘍細胞の相互作用を解析するための前述の方法から適応され、最初に卵巣腫瘍細胞はの排除を促進するためのミオシンとトラクション力のインテグリン依存性活性化を利用して表示するレポートに記載された腫瘍回転楕円体17の下から中皮細胞。このモデルは、相互作用の空間的および時間的な情報を提供し、リアルタイムで2つの細胞集団を監視するタイムラプス蛍光顕微鏡を利用しています。中皮細胞が緑色蛍光タンパク質（GFP）を発現しながら卵巣癌細胞は、赤色蛍光タンパク質（RFP）を発現する。 RFP発現卵巣癌細胞のスフェロイドは、GFPを発現する中皮単層に接続します。スフェロイドの広がり、侵入し、脇に単分子層に穴を作成中皮細胞を強制します。この穴は、GFPイメージの負の領域（黒）として可視化される。穴の面積は、次に定量的に制御および卵巣癌および/または中皮細胞の実験的集団の間のクリアランス活性の違いを分析するために測定することができます。このアッセイは、卵巣癌細胞（条件ごとに回転楕円体X 20から30回転楕円体当たり100細胞）のごく少数を必要とするので、貴重な原発腫瘍の細胞サンプルを使用して、このアッセイを行うことが可能である。さらに、このアッセイは、簡単にハイスループットスクリーニングに適応することができます。

Watch this Scientific Journal Video about In vitro Mesothelial Clearance Assay that Models the Early Steps of Ovarian Cancer Metastasis at JoVE.com

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

ここで説明した中皮クリアランスアッセイは、蛍光標識細胞とタイムラプスビデオ顕微鏡可視化と定量的に卵巣癌多細胞スフェロイドと中皮細胞単層の相互作用を測定するために利用しています。このアッセイは、卵巣癌転移の初期段階をモデル化します。

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 ...

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Research

JoVE Journal

Medicine

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

14.4K ビュー.  Harvard Medical School. ここで説明した中皮クリアランスアッセイは、蛍光標識細胞とタイムラプスビデオ顕微鏡可視化と定量的に卵巣癌多細胞スフェロイドと中皮細胞単層の相互作用を測定するために利用しています。このアッセイは、卵巣癌転移の初期段階をモデル化します。

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

Experimental Metastasis Assay

Metastasis is the leading cause of death in cancer patients. To understand the mechanism of metastasis, an experimental metastasis assay was established using immunodeficient mice. This article delineates the procedures involved in this assay, including sample preparation, intravenous injection, and culturing cells from lung metastases. Briefly, a pre-determined number of human cancer cells were prepared in vitro and directly injected into the circulation of immunodeficient mice through their tail veins. A small number of cells survive the turbulence in the circulation and grow as metastases in internal organs, such as lung. The injected mice are dissected after a certain period. The tissue distribution of metastases is determined under a dissecting microscope. The number of metastases in a specific tissue is counted and it directly correlates with the metastatic ability of the injected cancer cells. The arisen metastases are isolated and cultured in vitro as cell lines, which often show enhanced metastatic abilities than the parental line when injected again into immunodeficient mice. These highly metastatic derivatives become useful tools for identifying genes or molecular pathways that regulate metastatic progression.

This article describes the procedures of an experimental metastasis assay that is used to determine the metastatic potential of human cancer cell lines.

実験的転移アッセイ

Metastasis is the leading cause of death in cancer patients.  To understand the mechanism of metastasis, an experimental metastasis assay was established ...

医学

MAME Models for 4D Live-cell Imaging of Tumor: Microenvironment Interactions that Impact Malignant Progression

We have developed 3D coculture models, which we term MAME (mammary architecture and microenvironment engineering), and used them for live-cell imaging in real-time of cell:cell interactions. Our overall goal was to develop models that recapitulate the architecture of preinvasive breast lesions to study their progression to an invasive phenotype. Specifically, we developed models to analyze interactions among pre-malignant breast epithelial cell variants and other cell types of the tumor microenvironment that have been implicated in enhancing or reducing the progression of preinvasive breast epithelial cells to invasive ductal carcinomas. Other cell types studied to date are myoepithelial cells, fibroblasts, macrophages and blood and lymphatic microvascular endothelial cells. In addition to the MAME models, which are designed to recapitulate the cellular interactions within the breast during cancer progression, we have developed comparable models for the progression of prostate cancers. 
Here we illustrate the procedures for establishing the 3D cocultures along with the use of live-cell imaging and a functional proteolysis assay to follow the transition of cocultures of breast ductal carcinoma in situ (DCIS) cells and fibroblasts to an invasive phenotype over time, in this case over twenty-three days in culture. The MAME cocultures consist of multiple layers. Fibroblasts are embedded in the bottom layer of type I collagen. On that is placed a layer of reconstituted basement membrane (rBM) on which DCIS cells are seeded. A final top layer of 2% rBM is included and replenished with every change of media. To image proteolysis associated with the progression to an invasive phenotype, we use dye-quenched (DQ) fluorescent matrix proteins (DQ-collagen I mixed with the layer of collagen I and DQ-collagen IV mixed with the middle layer of rBM) and observe live cultures using confocal microscopy. Optical sections are captured, processed and reconstructed in 3D with Volocity visualization software. Over the course of 23 days in MAME cocultures, the DCIS cells proliferate and coalesce into large invasive structures. Fibroblasts migrate and become incorporated into these invasive structures. Fluorescent proteolytic fragments of the collagens are found in association with the surface of DCIS structures, intracellularly, and also dispersed throughout the surrounding matrix. Drugs that target proteolytic, chemokine/cytokine and kinase pathways or modifications in the cellular composition of the cocultures can reduce the invasiveness, suggesting that MAME models can be used as preclinical screens for novel therapeutic approaches.

We have developed 3D coculture models for live-cell imaging in real-time of interactions among breast tumor cells and other cells in their microenvironment that impact progression to an invasive phenotype. These models can serve as preclinical screens for drugs to target paracrine-induced proteolytic, chemokine/cytokine and kinase pathways implicated in invasiveness.

腫瘍の4DライブセルイメージングのためのMAMEモデル：微小環境の相互作用に影響を与える悪性進行

We have developed 3D coculture models, which we term MAME (mammary architecture and microenvironment engineering), and used them for live-cell imaging in ...

Multi-modal Imaging of Angiogenesis in a Nude Rat Model of Breast Cancer Bone Metastasis Using Magnetic Resonance Imaging, Volumetric Computed Tomography and Ultrasound

Angiogenesis is an essential feature of cancer growth and metastasis formation. In bone metastasis, angiogenic factors are pivotal for tumor cell proliferation in the bone marrow cavity as well as for interaction of tumor and bone cells resulting in local bone destruction. Our aim was to develop a model of experimental bone metastasis that allows in vivo assessment of angiogenesis in skeletal lesions using non-invasive imaging techniques.
For this purpose, we injected 105 MDA-MB-231 human breast cancer cells into the superficial epigastric artery, which precludes the growth of metastases in body areas other than the respective hind leg1. Following 25-30 days after tumor cell inoculation, site-specific bone metastases develop, restricted to the distal femur, proximal tibia and proximal fibula1. Morphological and functional aspects of angiogenesis can be investigated longitudinally in bone metastases using magnetic resonance imaging (MRI), volumetric computed tomography (VCT) and ultrasound (US).
MRI displays morphologic information on the soft tissue part of bone metastases that is initially confined to the bone marrow cavity and subsequently exceeds cortical bone while progressing. Using dynamic contrast-enhanced MRI (DCE-MRI) functional data including regional blood volume, perfusion and vessel permeability can be obtained and quantified2-4. Bone destruction is captured in high resolution using morphological VCT imaging. Complementary to MRI findings, osteolytic lesions can be located adjacent to sites of intramedullary tumor growth. After contrast agent application, VCT angiography reveals the macrovessel architecture in bone metastases in high resolution, and DCE-VCT enables insight in the microcirculation of these lesions5,6. US is applicable to assess morphological and functional features from skeletal lesions due to local osteolysis of cortical bone. Using B-mode and Doppler techniques, structure and perfusion of the soft tissue metastases can be evaluated, respectively. DCE-US allows for real-time imaging of vascularization in bone metastases after injection of microbubbles7.
In conclusion, in a model of site-specific breast cancer bone metastases multi-modal imaging techniques including MRI, VCT and US offer complementary information on morphology and functional parameters of angiogenesis in these skeletal lesions.

In the pathogenesis of bone metastasis, angiogenesis is a crucial process and therefore represents a target for imaging and therapy. Here, we present a rat model of site-specific breast cancer bone metastasis and describe strategies to non-invasively image angiogenesis in vivo using magnetic resonance imaging, volumetric computed tomography and ultrasound.

磁気共鳴イメージング、体積CT画像と超音波を用いた乳がんの骨転移のヌードラットモデルにおける血管新生のマルチモーダルイメージング

Angiogenesis is an essential feature of cancer growth and metastasis formation. In bone metastasis, angiogenic factors are pivotal for tumor cell ...

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 ...

Models of Bone Metastasis

Bone metastases are a common occurrence in several malignancies, including breast, prostate, and lung. Once established in bone, tumors are responsible for significant morbidity and mortality1. Thus, there is a significant need to understand the molecular mechanisms controlling the establishment, growth and activity of tumors in bone. Several in vivo models have been established to study these events and each has specific benefits and limitations. The most commonly used model utilizes intracardiac inoculation of tumor cells directly into the arterial blood supply of athymic (nude) BalbC mice. This procedure can be applied to many different tumor types (including PC-3 prostate cancer, lung carcinoma, and mouse mammary fat pad tumors); however, in this manuscript we will focus on the breast cancer model, MDA-MB-231. In this model we utilize a highly bone-selective clone, originally derived in Dr. Mundy's group in San Antonio2, that has since been transfected for GFP expression and re-cloned by our group3. This clone is a bone metastatic variant with a high rate of osteotropism and very little metastasis to lung, liver, or adrenal glands. While intracardiac injections are most commonly used for studies of bone metastasis2, in certain instances intratibial4 or mammary fat pad injections are more appropriate. Intracardiac injections are typically performed when using human tumor cells with the goal of monitoring later stages of metastasis, specifically the ability of cancer cells to arrest in bone, survive, proliferate, and establish tumors that develop into cancer-induced bone disease. Intratibial injections are performed if focusing on the relationship of cancer cells and bone after a tumor has metastasized to bone, which correlates roughly to established metastatic bone disease. Neither of these models recapitulates early steps in the metastatic process prior to embolism and entry of tumor cells into the circulation. If monitoring primary tumor growth or metastasis from the primary site to bone, then mammary fat pad inoculations are usually preferred; however, very few tumor cell lines will consistently metastasize to bone from the primary site, with 4T1 bone-preferential clones, a mouse mammary carcinoma, being the exception 5,6.
This manuscript details inoculation procedures and highlights key steps in post inoculation analyses. Specifically, it includes cell culture, tumor cell inoculation procedures for intracardiac and intratibial inoculations, as well as brief information regarding weekly monitoring by x-ray, fluorescence and histomorphometric analyses.

Animal models are frequently utilized to study cancer metastasis to bone. In this protocol we will describe two common methods of tumor inoculation for bone metastasis studies and briefly describe some of the analyses utilized to monitor and quantify these models.

骨転移モデル

Bone metastases are a common occurrence in several malignancies, including breast, prostate, and lung. Once established in bone, tumors are responsible ...

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 ...

In vitro Enrichment of Ovarian Cancer Tumor-initiating Cells

Evidence suggests that small subpopulations of tumor cells maintain a unique self-renewing and differentiation capacity and may be responsible for tumor initiation and/or relapse. Clarifying the mechanisms by which these tumor-initiating cells (TICs) support tumor formation and progression could lead to the development of clinically favorable therapies. Ovarian cancer is a heterogeneous and highly recurrent disease. Recent studies suggest TICs may play an important role in disease biology. We have identified culture conditions that enrich for TICs from ovarian cancer cell lines. Growing either adherent cells or non-adherent &#8216;floater&#8217; cells in a low attachment plate with serum free media in the presence of growth factors supports the propagation of ovarian cancer TICs with stem cell markers (CD133 and ALDH activity) and increased tumorigenicity without the need to physically separate the TICs from other cell types within the culture. Although the presence of floater cells is not common for all cell lines, this population of cells with innate low adherence may have high tumorigenic potential.Compared to adherent cells grown in the presence of serum, TICs readily form spheres, are significantly more tumorigenic in mice, and express putative stem cell markers. The conditions are easy to establish in a timely manner and can be used to study signaling pathways important for maintaining stem characteristics, and to identify drugs or combinations of drugs targeting TICs. The culture conditions described herein are applicable for a variety of ovarian cancer cells of epithelial origin and will be critical in providing new information about the role of TICs in tumor initiation, progression, and relapse.

In vitro Enrichment of Ovarian Cancer Tumor-initiating Cells

Tumor-initiating cells (TICs) may represent a viable therapeutic target for the treatment of ovarian cancer, a highly recurrent and fatal disease. We present a protocol for culture conditions that enrich for this highly tumorigenic population of cells.

卵巣がんの腫瘍開始細胞の体外富化

Evidence suggests that small subpopulations of tumor cells maintain a unique self-renewing and differentiation capacity and may be responsible for tumor ...

Three-Dimensional (3D) Tumor Spheroid Invasion Assay

Invasion of surrounding normal tissues is generally considered to be a key hallmark of malignant (as opposed to benign) tumors. For some cancers in particular (e.g., brain tumors such as glioblastoma multiforme and squamous cell carcinoma of the head and neck &#8211; SCCHN) it is a cause of severe morbidity and can be life-threatening even in the absence of distant metastases. In addition, cancers which have relapsed following treatment unfortunately often present with a more aggressive phenotype. Therefore, there is an opportunity to target the process of invasion to provide novel therapies that could be complementary to standard anti-proliferative agents. Until now, this strategy has been hampered by the lack of robust, reproducible assays suitable for a detailed analysis of invasion and for drug screening. Here we provide a simple micro-plate method (based on uniform, self-assembling 3D tumor spheroids) which has great potential for such studies. We exemplify the assay platform using a human glioblastoma cell line and also an SCCHN model where the development of resistance against targeted epidermal growth factor receptor (EGFR) inhibitors is associated with enhanced matrix-invasive potential. We also provide two alternative methods of semi-automated quantification: one using an imaging cytometer and a second which simply requires standard microscopy and image capture with digital image analysis.

Three-Dimensional 3D Tumor Spheroid Invasion Assay

Invasion of surrounding normal tissues is a defining characteristic of malignant tumors. We provide here a simple, semi-automated micro-plate assay of invasion into a natural 3D biomatrix that has been exemplified with a number of models of advanced human cancers.

三次元（3D）腫瘍スフェロイド浸潤アッセイ

Invasion of surrounding normal tissues is generally considered to be a key hallmark of malignant (as opposed to benign) tumors. For some cancers in ...

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 ...

Quantitation of Intra-peritoneal Ovarian Cancer Metastasis

Epithelial ovarian cancer (EOC) is the leading cause of death from gynecologic malignancy in the United States. Mortality is due to diagnosis of 75% of women with late stage disease, when metastasis is already present. EOC is characterized by diffuse and widely disseminated intra-peritoneal metastasis. Cells shed from the primary tumor anchor in the mesothelium that lines the peritoneal cavity as well as in the omentum, resulting in multi-focal metastasis, often in the presence of peritoneal ascites. Efforts in our laboratory are directed at a more detailed understanding of factors that regulate EOC metastatic success. However, quantifying metastatic tumor burden represents a significant technical challenge due to the large number, small size and broad distribution of lesions throughout the peritoneum. Herein we describe a method for analysis of EOC metastasis using cells labeled with red fluorescent protein (RFP) coupled with in vivo multispectral imaging. Following intra-peritoneal injection of RFP-labelled tumor cells, mice are imaged weekly until time of sacrifice. At this time, the peritoneal cavity is surgically exposed and organs are imaged in situ. Dissected organs are then placed on a labeled transparent template and imaged ex vivo. Removal of tissue auto-fluorescence during image processing using multispectral unmixing enables accurate quantitation of relative tumor burden. This method has utility in a variety of applications including therapeutic studies to evaluate compounds that may inhibit metastasis and thereby improve overall survival.

Ovarian cancer metastasis is characterized by numerous diffuse intra-peritoneal lesions, such that accurate visual quantitation of tumor burden is challenging. Herein we describe a method for in situ and ex vivo quantitation of metastatic tumor burden using red fluorescent protein (RFP)-labeled tumor cells and optical imaging.

腹腔内卵巣癌転移の定量

Epithelial ovarian cancer (EOC) is the leading cause of death from gynecologic malignancy in the United States.  Mortality is due to diagnosis of 75% of ...