この作業は、寛大に子供がん財団（ボルティモア、MD）、ブランドン·キャリントンリー財団（シルバースプリング、MD）、DOD（W81XWH-10-1から0137）とNIH（R01CA108641）からの資金によってサポートされていました。 私たちは、博士アントンWellsteinと彼らの経験を共有し、提示された方法を最適化するために、私たちを助けるために彼の研究室のメンバーに感謝したいと思います。

1。準備<ol><li>すべてのステップは、組織培養フード内で無菌条件下で実施されるべきである。 </li><li>エクセリジェンスステーションは5％CO 2の存在下で、37℃のインキュベーター内に配置される。 </li><li>通路6より好ましくは低継HUVEC細胞を使用してください。また、細胞は収穫時にコンフルエント75％よりも多くされていないことを確認してください。 </li><li> HUVEC細胞は、増殖因子、サプリメント、5％ウシ胎児血清を含むEGM-2弾キット（ロンザバイオサイエンス社）を用いて再構成EGM-2培地中で増殖させる。 </li><li>トリプシンのすべての痕跡を、200xgで細胞をスピンPBSで1回洗浄し、そして示された細胞密度にそれらを再懸濁することにより細胞懸濁液から除去されるべきである。 </li><li> 37℃で1時間0.1％ゼラチンでコートエクセリジェンスE-プレート16°Cまたは一晩4℃でPBSでプレートを洗浄し、100μL再構成EGM-2メディアを追加。バックグラウンドを測定するためのエクセリジェンスシステム上の空白の読みを行う細胞の非存在下でのインピーダンス。 </li></ol> 2。 HUVEC単分子層の生成<ol><li>トリプシン処理によりHUVEC細胞のサブコンフルエントフラスコを収穫。 2.5×10 5細胞/ mlの最終濃度に再構成されたEGM-2培地で再懸濁した細胞。 </li><li> E-プレート含む100μLEGM-2メディア校正され、背景の各ウェルに、HUVEC細胞懸濁液（2.5×10 4 HUVEC細胞）の100μLを追加。 </li><li>直ちにエクセリジェンス機器にE-プレートを取り付けます。 </li><li> 10分間隔でインピーダンス測定値を実行するためのプログラムエクセリジェンスソフトウェア。 </li><li> （図2）セルインデックスの平坦化によって証明されるように、彼らは、単層を形成するまで、内皮細胞が18〜21時間増殖させて。 </li></ol> 3。細胞およびデータの正規化に侵入添加。 <ol><li>一度安定したHUVECの単層は、最終的な洞穴にトリプシン処理し、再懸濁により収穫腫瘍細胞を形成しています腫瘍細胞を成長させるために使用される培地中1×10 5細胞/ ml（例えば、10％FBSを含有するRPMI又はDMEM培地など）の性</li><li>ポーズエクセリジェンスソフトウェアのインピーダンス測定値を、駅からE-プレートを取り外します。 </li><li>吸引によりEGM-2メディアを取り出します。 1×10 4個の細胞を含む腫瘍細胞懸濁液100μLを追加します。一般的には、腫瘍細胞の比率1:2.5：内皮細胞播種は、アッセイに最適です。しかしながら、この比は、個々の細胞株に対して最適化することができる。 </li><li>インキュベーターでxCELLigenceシステム上のE-プレートを置き、10分間隔でインピーダンス測定値を継続する。 </li><li>侵略は、腫瘍細胞の侵入によって内皮接合と浸透の引き込みによる細胞指数の低下として次の6-12時間にわたってリアルタイムで監視することができます。 </li><li>腫瘍細胞に侵入の添加時に、結果を正規化します。エクセリジェンスソフトウェアは、任意の時点及び結果を正規化を可能にする直接ソフトウェアのウィンドウで表示することができます。 </li></ol> 4。代表的な結果： 転移性および非転移性細胞によるHUVEC単層浸潤の例を図3に示されている。 K7M2は、転移性骨肉腫細胞株である。これらの細胞は、この細胞系13,14の侵襲的な性質を占める細胞骨格リンカータンパク質エズリン、高レベルの発現。 HUVEC細胞はK7M2細胞でチャレンジされた場合、6時間以内に電気抵抗の低下がある。電気抵抗のこのドロップは、侵入腫瘍細胞によるHUVEC単層の侵略を表しています。 K12細胞株は、一方では、エズリンはるかに低いレベルで発現し、その結果、13 ​​以下で転移性である。細胞が付着またはHUVEC単層（図3）を通って侵入することができないように抵抗の少ない急なドロップK12細胞結果とHUVEC単層に挑戦。 <imgSRC = &quot;/ files/ftp_upload/2792/2792fig1.jpg&quot; ALT = &quot;図1&quot; /&gt; 図1。腫瘍細胞による内皮細胞の浸潤のためのワークフロープロセスの概略図 。 HUVEC細胞を、ゼラチンをコーティングしたE-プレート上に播種し、コンフルエントな単層を形成させる。単分子層を形成し、浸潤、内皮単層の侵入に起因するセル·インデックスの変化に応じてリアルタイムに監視されると、悪性細胞を添加する。 <img src="/files/ftp_upload/2792/2792fig2.jpg" alt="図2"/> 図2。 HUVEC単層の形成 。 HUVEC細胞はにアタッチし、ゼラチンコートした金電極上に広がるようにセルインデックスの推移。コンフルエントな単層の形成は、18時間後の細胞の安定化インデックスで表される。 <img src="/files/ftp_upload/2792/2792fig3.jpg" alt="図3"/> 図3。 K7M2とK12骨肉腫細胞によるHUVEC細胞の浸潤 。 CELの急激な減少L-インデックスは、高転移性K7M2細胞の導入の数時間以内に発生します。 K12細胞が、その一方で、より少ない転移であり、できるだけ効率的K7M2細胞などの内皮単層を貫通することができない。これは、HUVEC単層は、K12細胞でチャレンジされているセルインデックスで緩やかに減少によって表される。制御は、癌細胞の非存在下でHUVEC単層のインピーダンスを表す。実験はトリプリケートで実施された。

<ol>
	<li>Klein, C. 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.+The+metastasis+cascade.">Cancer. The metastasis cascade.</a> Science. 321, 1785-1787 (2008).</li><li>Chiang, A. C., Massague, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+basis+of+metastasis.">Molecular basis of metastasis.</a> N Engl J Med. 359, 2814-2823 (2008).</li><li>Orr, F. W., Wang, H. H., Lafrenie, R. M., Scherbarth, S., Nance, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interactions+between+cancer+cells+and+the+endothelium+in+metastasis.">Interactions between cancer cells and the endothelium in metastasis.</a> J Pathol. 190, 310-329 (2000).</li><li>Bacac, M., Stamenkovic, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metastatic+cancer+cell.">Metastatic cancer cell.</a> Annu Rev Pathol. 3, 221-247 (2008).</li><li>Christofori, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+signals+from+the+invasive+front.">New signals from the invasive front.</a> Nature. 441, 444-450 (2006).</li><li>Kramer, R. H., Nicolson, G. L. <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+tumor+cells+with+vascular+endothelial+cell+monolayers:+a+model+for+metastatic+invasion.">Interactions of tumor cells with vascular endothelial cell monolayers: a model for metastatic invasion.</a> Proc Natl Acad Sci U S A. 76, 5704-5708 (1979).</li><li>Giaever, I., Keese, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monitoring+fibroblast+behavior+in+tissue+culture+with+an+applied+electric+field.">Monitoring fibroblast behavior in tissue culture with an applied electric field.</a> Proc Natl Acad Sci U S A. 81, 3761-3764 (1984).</li><li>Giaever, I., Keese, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Micromotion+of+mammalian+cells+measured+electrically.">Micromotion of mammalian cells measured electrically.</a> Proc Natl Acad Sci U S A. 88, 7896-7900 (1991).</li><li>Tiruppathi, C., Malik, A. B., Del Vecchio, P. J., Keese, C. R., Giaever, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrical+method+for+detection+of+endothelial+cell+shape+change+in+real+time:+assessment+of+endothelial+barrier+function.">Electrical method for detection of endothelial cell shape change in real time: assessment of endothelial barrier function.</a> Proc Natl Acad Sci U S A. 89, 7919-7923 (1992).</li><li>Whipple, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epithelial-to-mesenchymal+transition+promotes+tubulin+Detyrosination+and+Microtentacles+that+enhance+endothelial+engagement.">Epithelial-to-mesenchymal transition promotes tubulin Detyrosination and Microtentacles that enhance endothelial engagement.</a> Cancer Res. 70, 8127-8137 (2010).</li><li>Boyd, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+cell-microelectronic+sensing+technique+for+profiling+cytotoxicity+of+chemicals.">A cell-microelectronic sensing technique for profiling cytotoxicity of chemicals.</a> Anal Chim Acta. 615, 80-87 (2008).</li><li>Khanna, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+membrane-cytoskeleton+linker+ezrin+is+necessary+for+osteosarcoma+metastasis.">The membrane-cytoskeleton linker ezrin is necessary for osteosarcoma metastasis.</a> Nat Med. 10, 182-186 (2004).</li><li>Ren, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+actin-cytoskeleton+linker+protein+ezrin+is+regulated+during+osteosarcoma+metastasis+by+PKC.">The actin-cytoskeleton linker protein ezrin is regulated during osteosarcoma metastasis by PKC.</a> Oncogene. 28, 792-802 (2009).</li></ol>

この原稿のための出版費用は親切にロシュ·ダイアグノスティックスによって提供されていました。

リアルタイムHUVEC浸潤アッセイは、監視侵略のため、シンプルでありながら強力な方法である。これは、エンドポイント解析に依存する他の伝統的な技術上の重要な利点である、リアルタイムで結果を提供します。アッセイは、転移の阻害剤または刺激剤として薬物、抗体、リガンドとタンパク質をテストするために変更することができる。癌/内皮細胞のクロストークに異なる薬剤の効果を同様に評価することができる。そのような培養培地中で増殖因子及び薬物として外因剤を導入する場合、それはHUVEC単層の完全性に影響を与えないようにすることが重要である。 HUVEC単層の破壊は、細胞に侵入し、細胞指数に変化がないことを保証するの不在下で外因剤を導入することによって試験することができる。したがって、適切なコントロールは、実験デザインの一部として含まれるべきである。 彼らはconfのを形成するように別の細胞株は、異なる細胞指数曲線を実証luent単層。曲線、ならびに最大セルインデックス達成の形状は、細胞型特異的である。 HUVEC細胞は16〜18時間後に、より高いセルインデックスで別の安定化、続いて4-6時間播種後セルインデックスの平坦化特性の過渡を示す。それゆえ、細胞が腫瘍細胞の添加前に少なくとも18時間コンフルエントな単層を形成させることが重要である。 セルインデックスは、電極/セル間期におけるイオン環境に依存しているので、そのような細胞形態および細胞 - 細胞接着の品質など様々な要因が覆われたウェル底の領域に加えて、セル·インデックスに影響を及ぼす。したがって、腫瘍細胞の浸潤により発生したセルインデックスの減少は、内皮接合部およびその後の腫瘍細胞浸潤の後退を含むいくつかの要因の関数である。 リアルタイム細胞分析装置：エクセリジェンス器具は、3つの異なる構成で製造される（RTCA）SP、RTCA MPとRTCA DP。 RTCA MP楽器が同時に6 96ウェルE-プレートに保持することができ、一方RTCA SPインストゥルメントは、一96ウェルE-プレートを保持しています。これは、薬物と化合物のハイスループットスクリーニングを可能にする。この記事の実験は3つの16ウェルE-プレートを保持できるRTCA DP機器を使用して実行されます。各E-プレート保持局は、複数のユーザが同時に実験を実行することを可能にする、独立して動作することができる。 RTCA DP器具はまた、CIMプレートを使用して移行を研究するために使用することができる。これらは8umのメジアン細孔径を有するポリエチレンテレフタレート（PET）膜によって分離され、上部及び下部チャンバーの16ウェルプレートである。電子センサーは、上部チャンバの多孔質膜の下側に配置されている。下部チャンバーは、上部チャンバー内の細胞のための化学誘引物質のための貯水池として機能します。しかし、CIMプレート上HUVEC浸潤アッセイの主な利点は、前者の腫瘍細胞浸潤I内皮細胞の浸潤が腫瘍および内皮細胞間のクロストークに依存するのは、細孔サイズによって制限されない。 HUVEC浸潤アッセイはまた、応用生物物理学（トロイ、NY）製、ECIS Z機​​器で行う​​ことができる。 ECIS Zインストゥルメントは、配列と電極構成の違いと、エクセリジェンス機器に同様の技術を利用しています。我々は成功した8ウェルECISアレイを用いたHUVEC浸潤アッセイを行った。それぞれ2.5×10 5〜1×10 5細胞：ウェル底面積が被めっき内皮細胞および腫瘍細胞の多くを必要とする、ECIS配列の方が大きいことに留意することが重要である。

<table border="1">
	<tbody>
 <tr>
 	<th>Name of the reagent</th>
 <th>Company</th>
 <th>Catalogue number</th>
 <th>Comments (optional)</th>
 </tr> 
 <tr>
 	<td>xCELLigence RTCA DP station</td>
 <td>Roche	</td>
 <td>05469759001</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>RTCA control unit</td>
 <td>Roche</td>
 <td>05454417001</td>
 <td>Notebook with preinstalled RTCA software</td>
 </tr>
 <tr>
 	<td>E-Plate 16</td>
 <td>Roche</td>
 <td>05469830001</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>HUVEC cells</td>
 <td>Lonza</td>
 <td>CC-2517</td>
 <td></td>
 </tr>
 <tr>
 	<td>EGM-2 media</td>
 <td>Lonza</td>
 <td>CC-3156</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>EGM-2 bullet kit</td>
 <td>Lonza</td>
 <td>CC-4176</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>0.1% Gelatin in 
sterile H2O
</td>
 <td>Millipore</td>
 <td>MCPROTO045</td>
 <td>&nbsp;</td>
 </tr>
 </tbody>
</table>

a real-time electrical impedance based technique to measure invasion of endothelial cell monolayer by cancer cells

悪性細胞の転移性播種は、ホスト内遠隔部位への輸送手段として血流に入るため内皮層を介して基底膜、血管内皮への腫瘍細胞の付着、内皮接合の後退及び腫瘍細胞の浸潤および最終的にマイグレーションの分解を必要とする1-3。循環系に一度、癌細胞壁をキャピラリーおよび転移性腫瘍が4,5形成するために周囲の組織に浸出する接着する。腫瘍細胞、内皮細胞との相互作用の様々な構成要素は、癌細胞とヒト臍帯静脈内皮細胞（HUVEC）を単層に挑戦することによってインビトロで複製することができる。電子と位相差顕微鏡を用いて行った研究では、イベントのin vitroでのシーケンスでかなり生体転移プロセス6 で表すことを示唆している。ここでは、リアルタイムinvasに監視し、定量化し、電気インピーダンスベースの手法を記述悪性腫瘍細胞による内皮細胞のイオンである。 イェーヴァーとキースは、最初の細胞集団、電極7,8の表面に成長したときにインピーダンスの変動を測定するための手法を説明した。ロシュ社製エクセリジェンス器具は、細胞はウェルの底部に面積の約80％を覆う金微小電極アレイで覆われた培養皿に添付して広がるように、電気インピーダンスの変化を測定するために同様の技術を利用する。細胞が付着して電極表面に広がるように、それは9-12電気インピーダンスの増加につながる。インピーダンスは、細胞によって覆われているウェル組織培養の全面積に正比例するセルインデックスと呼ばれる無次元パラメータとして表示される。したがって、セルインデックスは、細胞接着、拡散、形態および細胞密度をモニターするために使用することができる。 この資料に記載されている浸潤アッセイは、変化に基づいている電極/セル間期における電気インピーダンスで、悪性の細胞集団としてHUVEC単層（図1）を通って侵入する。腫瘍細胞による内皮接合の混乱、内皮単層および交換の引き込みは、インピーダンスの大きな変化につながる。これらの変更は、直接腫瘍細胞の浸潤能との相関、すなわち、極めて積極的な細胞による侵攻がセルインピーダンス、およびその逆の大きな変化につながる。この技術は、ボイデンチャンバーとマトリゲルアッセイなどの侵入を測定する既存の方法、上の二重の利点を提供する：1）内皮細胞、腫瘍細胞の相互作用がより密接にインビボプロセスにおいて模倣する、2）データは、リアルタイムで得られる時間および他の方法のエンドポイント解析とは対照的に、より容易に定量化である。

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A Real-time Electrical Impedance Based Technique to Measure Invasion of Endothelial Cell Monolayer by Cancer Cells

この記事では説明し<em&gt; in vitroで</em内皮細胞の単層を介して侵入する癌細胞を監視するため&gt;手法。データは、ウェル底の電極の表面にインピーダンスの変化の関数としてリアルタイムで取得される。

癌細胞による内皮細胞単層の侵入を測定するリアルタイム電気インピーダンス基づく手法

Metastatic dissemination of malignant cells requires degradation of basement membrane, attachment of tumor cells to vascular endothelium, retraction of ...

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27.2K ビュー.  Georgetown University. この記事では説明し in vitroで手法。データは、ウェル底の電極の表面にインピーダンスの変化の関数としてリアルタイムで取得される。

ビデオ: A Real-time Electrical Impedance Based Technique to Measure Invasion of Endothelial Cell Monolayer by Cancer Cells

Evaluation of Nanoparticle Uptake in Tumors in Real Time Using Intravital Imaging

Current technologies for tumor imaging, such as ultrasound, MRI, PET and CT, are unable to yield high-resolution images for the assessment of nanoparticle uptake in tumors at the microscopic level1,2,3, highlighting the utility of a suitable xenograft model in which to perform detailed uptake analyses. Here, we use high-resolution intravital imaging to evaluate nanoparticle uptake in human tumor xenografts in a modified, shell-less chicken embryo model. The chicken embryo model is particularly well-suited for these in vivo analyses because it supports the growth of human tumors, is relatively inexpensive and does not require anesthetization or surgery 4,5. Tumor cells form fully vascularized xenografts within 7 days when implanted into the chorioallantoic membrane (CAM) 6. The resulting tumors are visualized by non-invasive real-time, high-resolution imaging that can be maintained for up to 72 hours with little impact on either the host or tumor systems. Nanoparticles with a wide range of sizes and formulations administered distal to the tumor can be visualized and quantified as they flow through the bloodstream, extravasate from leaky tumor vasculature, and accumulate at the tumor site. We describe here the analysis of nanoparticles derived from Cowpea mosaic virus (CPMV) decorated with near-infrared fluorescent dyes and/or polyethylene glycol polymers (PEG) 7, 8, 9,10,11. Upon intravenous administration, these viral nanoparticles are rapidly internalized by endothelial cells, resulting in global labeling of the vasculature both outside and within the tumor7,12. PEGylation of the viral nanoparticles increases their plasma half-life, extends their time in the circulation, and ultimately enhances their accumulation in tumors via the enhanced permeability and retention (EPR) effect 7, 10,11. The rate and extent of accumulation of nanoparticles in a tumor is measured over time using image analysis software. This technique provides a method to both visualize and quantify nanoparticle dynamics in human tumors.

We present a novel approach to quantify nanoparticle localization in the vasculature of human xenografted tumors using dynamic, real-time intravital imaging in an avian embryo model.

生体内イメージングを使ってリアルタイムで腫瘍におけるナノ粒子の取り込みの評価

Current technologies for tumor imaging, such as ultrasound, MRI, PET and CT, are unable to yield high-resolution images for the assessment of nanoparticle ...

医学

Multi-photon Imaging of Tumor Cell Invasion in an Orthotopic Mouse Model of Oral Squamous Cell Carcinoma

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient management strategies. Orthotopic mouse models of oral cancer have been developed to facilitate the study of factors that impact invasion and serve as model system for evaluating anti-tumor therapeutics. In these systems, visualization of disseminated tumor cells within oral cavity tissues has typically been conducted by either conventional histology or with in vivo bioluminescent methods. A primary drawback of these techniques is the inherent inability to accurately visualize and quantify early tumor cell invasion arising from the primary site in three dimensions. Here we describe a protocol that combines an established model for squamous cell carcinoma of the tongue (SCOT) with two-photon imaging to allow multi-vectorial visualization of lingual tumor spread. The OSC-19 head and neck tumor cell line was stably engineered to express the F-actin binding peptide LifeAct fused to the mCherry fluorescent protein (LifeAct-mCherry). Fox1nu/nu mice injected with these cells reliably form tumors that allow the tongue to be visualized by ex-vivo application of two-photon microscopy. This technique allows for the orthotopic visualization of the tumor mass and locally invading cells in excised tongues without disruption of the regional tumor microenvironment. In addition, this system allows for the quantification of tumor cell invasion by calculating distances that invaded cells move from the primary tumor site. Overall this procedure provides an enhanced model system for analyzing factors that contribute to SCOT invasion and therapeutic treatments tailored to prevent local invasion and distant metastatic spread. This method also has the potential to be ultimately combined with other imaging modalities in an in vivo setting.

A comprehensive overview of the techniques involved in generating a mouse model of oral cancer and quantitative monitoring of tumor invasion within the tongue through multi-photon microscopy of labeled cells is presented. This system can serve as a useful platform for the molecular assessment and drug efficacy of anti-invasive compounds.

口腔扁平上皮癌の同所性マウスモデルにおける腫瘍細胞の浸潤の多光子イメージング

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient ...

Spheroid Assay to Measure TGF-&#946;-induced Invasion

TGF-&beta; has opposing roles in breast cancer progression by acting as a tumor suppressor in the initial phase, but stimulating invasion and metastasis at later stage1,2. Moreover, TGF-&beta; is frequently overexpressed in breast cancer and its expression correlates with poor prognosis and metastasis 3,4. The mechanisms by which TGF-&beta; induces invasion are not well understood.
TGF-&beta; elicits its cellular responses via TGF-&beta; type II (T&beta;RII) and type I (T&beta;RI) receptors. Upon TGF-&beta;-induced heteromeric complex formation, T&beta;RII phosphorylates the T&beta;RI. The activated T&beta;RI initiates its intracellular canonical signaling pathway by phosphorylating receptor Smads (R-Smads), i.e. Smad2 and Smad3. These activated R-Smads form heteromeric complexes with Smad4, which accumulate in the nucleus and regulate the transcription of target genes5. In addition to the previously described Smad pathway, receptor activation results in activation of several other non-Smad signaling pathways, for example Mitogen Activated Protein Kinase (MAPK) pathways6.
To study the role of TGF-&beta; in different stages of breast cancer, we made use of the MCF10A cell system. This system consists of spontaneously immortalized MCF10A1 (M1) breast epithelial cells7, the H-RAS transformed M1-derivative MCF10AneoT (M2), which produces premalignant lesions in mice8, and the M2-derivative MCF10CA1a (M4), which was established from M2 xenografts and forms high grade carcinomas with the ability to metastasize to the lung9. This MCF10A series offers the possibility to study the responses of cells with different grades of malignancy that are not biased by a different genetic background.
For the analysis of TGF-&beta;-induced invasion, we generated homotypic MCF10A spheroid cell cultures embedded in a 3D collagen matrix in vitro (Fig 1). Such models closely resemble human tumors in vivo by establishing a gradient of oxygen and nutrients, resulting in active and invasive cells on the outside and quiescent or even necrotic cells in the inside of the spheroid10. Spheroid based assays have also been shown to better recapitulate drug resistance than monolayer cultures11. This MCF10 3D model system allowed us to investigate the impact of TGF-&beta; signaling on the invasive properties of breast cells in different stages of malignancy.

Spheroid Assay to Measure TGF-&amp;#946;-induced Invasion

An assay to quantitatively measure Transforming Growth Factor (TGF)-&#x03B2;-induced invasion in 3-dimensional collagen gels is described. This assay takes advantage of the MCF10A series of cell lines, which represent different stages of breast cancer development. This method can be adopted to be used with other cell lines and might be used to investigate other potential activators or inhibitors of invasion.

TGF -β誘発性浸潤を測定する回転楕円体アッセイ

TGF-&beta; has opposing roles in breast cancer progression by acting as a tumor suppressor in the initial phase, but stimulating invasion and metastasis ...

Modeling and Imaging 3-Dimensional Collective Cell Invasion

A defining characteristic of cancer malignancy is invasion and metastasis 1. In some cancers (e.g. glioma 2), local invasion into surrounding healthy tissue is the root cause of disease and death. For other cancers (e.g. breast, lung, etc.), it is the process of metastasis, in which tumor cells move from a primary tumor mass, colonize distal sites and ultimately contribute to organ failure, that eventually leads to morbidity and mortality 3. It has been estimated that invasion and metastasis are responsible for 90&#37; of cancer deaths 4. As a result, there has been intense interest in identifying the molecular processes and critical protein mediators of invasion and metastasis for the purposes of improving diagnosis and treatment 5.
A challenge for cancer scientists is to develop invasion assays that sufficiently resemble the in vivo situation to enable accurate disease modeling 6. Two-dimensional cell motility assays are only informative about one aspect of invasion and do not take into account extracellular matrix (ECM) protein remodeling which is also a critical element. Recently, research has refined our understanding of tumor cell invasion and revealed that individual cells may move by elongated or rounded modes 7. In addition, there has been greater appreciation of the contribution of collective invasion, in which cells invade in strands, sheets and clusters, particularly in highly differentiated tumors that maintain epithelial characteristics, to the spread of cancer 8.
We present a refined method 9 for examining the contributions of candidate proteins to collective invasion 10. In particular, by engineering separate pools of cells to express different fluorescent proteins, it is possible to molecularly dissect the activities and proteins required in leading cells versus those required in following cells. The use of RNAi provides the molecular tool to experimentally disassemble the processes involved in individual cell invasion as well as in different positions of collective invasion. In this procedure, mixtures of fluorescently-labeled cells are plated on the bottom of a Transwell insert previously filled with Matrigel ECM protein, then allowed to invade "upwards" through the filter and into the Matrigel. Reconstruction of z-series image stacks, obtained by confocal imaging, into three-dimensional representations allows for visualization of collectively invading strands and analysis of the representation of fluorescently-labeled cells in leading versus following positions.

Models of tumor cell invasion into three-dimensional extracellular matrix better reflect the in vivo situation than two-dimensional motility assays. Using matrix invasion assays combined with confocal imaging of fluorescently-labeled cells, detailed information on invasion modes and the distinct contributions of leading versus following cells can be obtained.

モデリングとイメージング、3次元の集団細胞浸潤

A defining characteristic of cancer malignancy is invasion and metastasis 1. In some cancers (e.g. glioma 2), local invasion into surrounding healthy ...

A Multiplexed Luciferase-based Screening Platform for Interrogating Cancer-associated Signal Transduction in Cultured Cells

Genome-scale interrogation of gene function using RNA interference (RNAi) holds tremendous promise for the rapid identification of chemically tractable cancer cell vulnerabilities. Limiting the potential of this technology is the inability to rapidly delineate the mechanistic basis of phenotypic outcomes and thus inform the development of molecularly targeted therapeutic strategies. We outline here methods to deconstruct cellular phenotypes induced by RNAi-mediated gene targeting using multiplexed reporter systems that allow monitoring of key cancer cell-associated processes. This high-content screening methodology is versatile and can be readily adapted for the screening of other types of large molecular libraries.

Achieving a systems level understanding of cellular processes is a goal of modern-day cell biology. We describe here strategies for multiplexing luciferase reporters of various cellular function end-points to interrogate gene function using genome-scale RNAi libraries.

培養細胞における癌関連シグナル伝達を問い合わせるための多重ルシフェラーゼベースのスクリーニング·プラットフォーム

Genome-scale interrogation of gene function using RNA interference (RNAi)  holds tremendous promise for the rapid identification of chemically tractable  ...

Cerebrospinal Fluid MicroRNA Profiling Using Quantitative Real Time PCR

MicroRNAs (miRNAs) constitute a potent layer of gene regulation by guiding RISC to target sites located on mRNAs and, consequently, by modulating their translational repression. Changes in miRNA expression have been shown to be involved in the development of all major complex diseases. Furthermore, recent findings showed that miRNAs can be secreted to the extracellular environment and enter the bloodstream and other body fluids where they can circulate with high stability. The function of such circulating miRNAs remains largely elusive, but systematic high throughput approaches, such as miRNA profiling arrays, have lead to the identification of miRNA signatures in several pathological conditions, including neurodegenerative disorders and several types of cancers. In this context, the identification of miRNA expression profile in the cerebrospinal fluid, as reported in our recent study, makes miRNAs attractive candidates for biomarker analysis.
There are several tools available for profiling microRNAs, such as microarrays, quantitative real-time PCR (qPCR), and deep sequencing. Here, we describe a sensitive method to profile microRNAs in cerebrospinal fluids by quantitative real-time PCR. We used the Exiqon microRNA ready-to-use PCR human panels I and II V2.R, which allows detection of 742 unique human microRNAs. We performed the arrays in triplicate runs and we processed and analyzed data using the GenEx Professional 5 software.
Using this protocol, we have successfully profiled microRNAs in various types of cell lines and primary cells, CSF, plasma, and formalin-fixed paraffin-embedded tissues.

We describe a protocol of real time PCR to profile microRNAs in the cerebrospinal fluid (CSF). With the exception of RNA extraction protocols, the procedure can be extended to RNA extracted from other body fluids, cultured cells, or tissue specimens.

定量リアルタイムPCRを用いて脳脊髄液マイクロRNAプロファイリング

MicroRNAs (miRNAs) constitute a potent layer of gene regulation by guiding RISC to target sites located on mRNAs and, consequently, by modulating their ...

A Modified In vitro Invasion Assay to Determine the Potential Role of Hormones, Cytokines and/or Growth Factors in Mediating Cancer Cell Invasion

Blood serum serves as a chemoattractant towards which cancer cells migrate and invade, facilitating their intravasation into microvessels. However, the actual molecules towards which the cells migrate remain elusive. This modified invasion assay has been developed to identify targets which drive cell migration and invasion. This technique compares the invasion index under three conditions to determine whether a specific hormone, growth factor, or cytokine plays a role in mediating the invasive potential of a cancer cell. These conditions include i) normal fetal bovine serum (FBS), ii) charcoal-stripped FBS (CS-FBS), which removes hormones, growth factors, and cytokines and iii) CS-FBS + molecule (denoted &#8220;X&#8221;). A significant change in cell invasion with CS-FBS as compared to FBS, indicates the involvement of hormones, cytokines or growth factors in mediating the change. Individual molecules can then be added back to CS-FBS to assay their ability to reverse or rescue the invasion phenotype. Furthermore, two or more factors can be combined to evaluate the additive or synergistic effects of multiple molecules in driving or inhibiting invasion. Overall, this method enables the investigator to determine whether hormones, cytokines, and/or growth factors play a role in cell invasion by serving as chemoattractants or inhibitors of invasion for a particular type of cancer cell or a specific mutant. By identifying specific chemoattractants and inhibitors, this modified invasion assay may help to elucidate signaling pathways that direct cancer cell invasion.

A Modified In vitro Invasion Assay to Determine the Potential Role of Hormones, Cytokines and/or Growth Factors in Mediating Cancer Cell Invasion

This video article describes a modified in vitro method to examine the role of hormones, cytokines and/or growth factors in driving cancer cell invasion.

変更された<em&gt;インビトロ</em媒介する癌細胞浸潤におけるホルモン、サイトカインおよび/または成長因子の潜在的な役割を決定するために&gt;浸潤アッセイ

Blood serum serves as a chemoattractant towards which cancer cells migrate and invade, facilitating their intravasation into microvessels. However, the ...

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 ...

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 ...

Isolation of Human Lymphatic Endothelial Cells by Multi-parameter Fluorescence-activated Cell Sorting

Lymphatic system disorders such as primary lymphedema, lymphatic malformations and lymphatic tumors are rare conditions that cause significant morbidity but little is known about their biology. Isolating highly pure human lymphatic endothelial cells (LECs) from diseased and healthy tissue would facilitate studies of the lymphatic endothelium at genetic, molecular and cellular levels. It is anticipated that these investigations may reveal targets for new therapies that may change the clinical management of these conditions. A protocol describing the isolation of human foreskin LECs and lymphatic malformation lymphatic endothelial cells (LM LECs) is presented. To obtain a single cell suspension tissue was minced and enzymatically treated using dispase II and collagenase II. The resulting single cell suspension was then labelled with antibodies to cluster of differentiation (CD) markers CD34, CD31, Vascular Endothelial Growth Factor-3 (VEGFR-3) and PODOPLANIN. Stained viable cells were sorted on a fluorescently activated cell sorter (FACS) to separate the CD34LowCD31PosVEGFR-3PosPODOPLANINPos LM LEC population from other endothelial and non-endothelial cells. The sorted LM LECs were cultured and expanded on fibronectin-coated flasks for further experimental use.

The goal of this protocol is to isolate lymphatic endothelial cells lining human lymphatic malformation cyst-like vessels and foreskins using fluorescence-activated cell sorting (FACS). Subsequent cell culturing and expansion of these cells permits a new level of experimental sophistication for genetic, proteomic, functional and cell differentiation studies.

マルチパラメータ蛍光活性化細胞選別によるヒトリンパ内皮細胞の単離

Lymphatic system disorders such as primary lymphedema, lymphatic malformations and lymphatic tumors are rare conditions that cause significant morbidity ...