The authors acknowledge funding sources from D&#8217;Youville College School of Pharmacy, Buffalo, NY.

1.様々なメディアやその他のコンポーネントを準備<ol><li>実験の前に、テストされるいずれかの通常のFBSを加え、チャコール処理FBS、またはチャコール処理FBSを加えた構成要素を含むDMEMまたは他の指​​定されたメディアからなるメディアを準備。複数のコンポーネントが、各実験で試験することができることに留意されたい。 </li><li>秤量し、ホルモン、成長因子、または適切な生理学的濃度でチャコール処理血清中に溶解させるサイトカインを希釈する。 </li></ol> 2.氷の上にコラーゲンマトリックスを準備します<ol><li> 200μlの10倍のPBS（pH7.4）を、5.4μlの1NのNaOH、二重蒸留H 2 O600μlの、および1.2ミリリットルのコラーゲンI：私は氷の上で、以下の滅菌濾過のコンポーネントを追加することによって、2.2 mg / mlの位置にマトリクス2ミリリットルコラーゲンを準備（3.63 mg / mlの時）。 </li><li>プレートの準備ができるまで氷上にコラーゲンI溶液にしてください。 </li></ol> 3. &lt;アッセイのための移行/浸潤プレートを準備します/ pの&gt; <ol><li>試験すべき各細胞株について、12ウェルインサートを含有したもので、24ウェルチャンバープレートが使用。化学誘引物質メディアを追加し、設定した実験用インサートを転送するためのインサートを含有していない追加の12の井戸を使用してください。 注：細胞株をここで使用するためのポリエチレンテレフタレート（PET）膜および8μmの孔サイズを有するプレート上のコラーゲンマトリックスが最適である。しかし、プレコーティングしたプレートにおけるマトリゲルマトリックスはまた、研究された細胞株に応じて減少する孔径を有する置換されていてもよい。 </li><li>明らかに、分析される条件あたり3ウェル（コントロールのFBSの移行、CS-FBS遊走、浸潤FBS、およびCS-FBS浸潤ならびにFBS移行、非侵襲的な細胞株）を使用して、プレートを標識する。コラーゲンまたはマトリゲルマトリックスを通ると、膜の細孔を通して移動によるPET膜の細孔を通して動き、およびアッセイ侵略によって、アッセイの移行。各セルの状態のために異なる色のマーカーを使用めっきプロセスのid。 </li></ol> 4.侵攻マトリックスを分注<ol><li>慎重に浸潤アッセイのために使用されるインサートにコラーゲンマトリックス溶液の75μlのピペット。泡を避けるためには十分注意してください。表面に反転したピペットチップを適用することにより、気泡を分散させる。 </li><li>固化するゲルを可能にするために30分間37℃、5％CO 2インキュベーターにコラーゲンコートインサートプレートに移す。 </li></ol> 5.プレート膜または侵入マトリックス上に細胞<ol><li>一方、細胞をトリプシン処理し、10％FBSでメディアを追加する。テーブルトップ遠心機で5分間200×gでスピン細胞と無血清培地中で3回すすいでください。 </li><li>無血清培地中で再懸濁します。血球計数器または自動スライドカウンターで細胞を数える。 5×10 4細胞/ mlの最終濃度になるように無血清培地を加える。 </li><li>コラーゲンマトリックスは、（30分後）固化した場合には、いずれかでメディアの700μlのを追加2％を各ウェルにFBSまたはチャコール処理FBSを定義した。プレート当たり12のインサートの： <ul><li> 3インサートは、メディアとのコラーゲンと井戸を持っている+ 2％FBS </li><li> 3インサートは、メディアとのコラーゲンと井戸を持っている+ 2％CS-FBS </li><li> 3インサートは、メディア+ 2％FBSとのコラーゲンや井戸がありません</li><li> 3インサートは、メディアとのコラーゲンや井戸を持っていない+ 2％CS-FBS </li></ul><ol><li>テストされている多くの要因に各条件に対応するコラーゲンコントロールで応じて追加のプレートを使用してください。 </li></ol></li><li>すべての遊走および浸潤インサートインサートあたり500μlの細胞を播種、5×10 4細胞/ mlでインサートに細胞懸濁液を加える。 </li><li> 37℃で22時間、細胞をインキュベートします。 </li></ol> 6.移行と浸潤細胞の数を定量<ol><li>別々のウェルに、メタノール固定液、エオシン、およびヘマトキシリン​​を使用して、ウェルの染色を設定します。 </li><li>各ウェルから細胞とマトリックスを削除するには綿棒を使用してください。各ウェルのための第二綿棒アプリケーションとRrepeat。 </li><li>鉗子を用いて、各々が連続して3の溶液のそれぞれに1秒間に5回挿入浸す。 </li><li>インサートは、O / Nを乾燥することができます。 </li><li>どちらのI）エッジの周りを慎重に切断、メスでフィルターを取り外し、カバーガラスとイマージョンオイルをスライド上にマウント、またはii）の挿入がO ​​/ Nが反転し、乾燥し、顕微鏡観察のために直接挿入を使用できるようにする。 </li><li>次の日は、20Xの目的で、顕微鏡下でスライドまたは挿入を表示し、フィルタの異なる領域から5の画像を取る。一貫性を向上させるために、4外側のフィールドと1センターを取る。 </li><li> ImageJソフトウェアを使用して、すべての条件のために細胞をカウントし、以下の式に適用されます。 </li><li>次のようにパーセントの侵略を決定します。 私は= Aを挿入コラーゲンを通じて浸潤細胞の＃の平均 コントロールインサートを通って移動する細胞の＃= Bの平均 ％浸潤=（a / bの）* 100 </li><li>次のように2％のFBSに侵攻インデックスを決定： （2％FBS）でアッセイされる細胞の％の侵略= C ％（2％FBS）での制御の非侵襲細胞の浸潤= D 浸潤指数（FBS）=の（c / d）の</li><li>次のように2％CS-FBSに侵攻インデックスを決定： ％の（2％CS-FBS）でアッセイされる細胞の浸潤= E ％の（2％CS-FBS）でコントロールの非侵襲性細胞の浸潤= fは 浸潤インデックス（CS-FBS）=の（e / f）と</li></ol>チャコール処理FBS + X nに合わせて複数の要因とチャコール処理FBSを比較7.実験プロトコール<ol><li> 「X」またはコンポーネントの組み合わせのためのさまざまなコンポーネントを使用し、必要に応じて手順を複数回繰り返す。 </li><li>遊走および浸潤効果に対する各要因の寄与「X」を決定するための計算を適用します。 </li></ol>

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The authors have nothing to disclose.

腫瘍転移は、多段階プロセスである。細胞は、それらが離れた部位に輸送されている基底膜、リンパ系または血液の微小血管のいずれかにintravasate、を突破しなければならない。腫瘍細胞は、その後滲出とmacrometastasis 12に植民地化する。間葉移行（EMT）、腫瘍の浸潤、および転移に対する上皮を通じて進行はステロイドホルモン13,14によって強化された、成長は15〜18、</sup...

新規な浸潤アッセイは、癌細胞浸潤における化学誘引物質として特定のホルモン、サイトカインおよび/または成長因子の関与を同定することを目的に開発された。細胞外マトリックス（ECM）を通って浸潤する腫瘍細胞の能力は、転移表現型1-3の特徴である。浸潤チャンバーは広範囲の癌細胞の浸潤および遊走を研究するためのin vitroツールとして使用されており、in vivoでの腫瘍浸潤および転移のメカニズムとして知識を提供してもよい。 チャンバは、細胞培養プレートのウェル内にネストされた円筒形の細胞培養インサートから成る。インサートの底部は、規定の孔サイズの半透過性ポリカーボネートまたはポリスチレン膜である。 標準浸潤アッセイでは、細胞は無血清培地でインサートメンブレン上に播種し、血清または血清のような化学誘引物質で充填されている細胞培養ウェルに入れ秒化学誘引力アップら。この力ドライブ細胞を、検出し、細胞数を定量化するために、従来の染料を用いて染色することができる細胞外マトリックス（浸潤）のコーティングを通して、あるいは半透膜（マイグレーション）を介して移動したりする。細胞数は、次に、非侵襲的細胞株における遊走および浸潤の程度に応じて正規化される。この方法では、研究者は、遺伝子操作を含む、種々の条件下で異なる細胞型の浸潤能を評価することを可能にする。 ホルモン、サイトカインおよび成長因子は、血清の重要な構成要素であり、ますます浸潤性の表現型4を駆動すると関連することが示されている。しかし、侵入を媒介するこれらの化学誘引血清分子の性質、役割、および特異性はまだとらえどころのないまま。課題は、血清または血清のような化学誘引物質の特定の要因が運転を担当するかを決定するために残っている癌細胞浸潤ならびにどの分子が侵入プロセスを阻害することができる。本稿では、癌細胞の化学誘引および浸潤を駆動する分子として、ホルモン、サイトカインおよび/または血清中に存在する成長因子の関与の可能性を評価するための方法論を説明します。 この提案された修正されたプロトコルでは、木炭ストリッピングは、2％チャコール処理FBS（CS-FBS）を生成するために、2％ウシ胎児血清（FBS）からホルモン、サイトカインおよび増殖因子を除去するために使用される。 2％FBSおよび2％CS-FBSの両方が癌細胞浸潤を駆動するために化学誘引力を設定するための薬剤として使用される。 FBSは、まず、最も顕著に、ホルモン、サイトカインおよび増殖因子の減少/相対的な非存在下での癌浸潤表現型を研究するための能力を含むいくつかの利点が得られる通常の2％と比較して、化学誘引剤として2％CS-FBSを用いた。また、ホルモン、成長因子の集団を除去するかどうかを評価するために研究者を可能にし、cyのtokinesは侵襲的な指標の増加または減少を引き起こす。アッセイは、その後（ 図2および3を参照）、「X」と称される個々の成分の添加は、減少し、増加を阻害するか、元の値に侵襲的なインデックスを復元することができるかどうかを決定するように設計される。この方法は、木炭ストリッピング時に血清から除去される成分の生理的レベルを達成するために特異的であるが、それはまた、木炭ストリッピングはまた、シグナル伝達経路に影響を与えることができることに留意すべきである。例えば、木炭ストリッピングは、骨前駆細胞のアルカリホスファターゼ活性を低下させ、MAPK活性化剤5の還元により脂肪生成を誘導することが報告されている。チャコール処理メディアは、市販されており、プロトコルに基づくことがデキストランで木炭を結合し、ウシ胎児血清O / N 6と共にインキュベート概説されている。 このアッセイは、RESPOで開発されたNSEはザッカーら 7によって報告された結果に著者は、通常の2％FBSをメディアに向けて移行するときのギャップ結合コミュニケーションに影響を与える変異体の表現型は侵略指数の増加を示したことを実証した。この増加は、チャコール処理血清の置換で除去した。この結果から、著者らは、この変異体は、親油性分子（具体的には、ホルモン、成長因子、またはサイトカイン）7に向かって遊走に影響することを結論付けた。これはよくホルモン、成長因子、サイトカイン、腫瘍促進および浸潤4に関与するシグナル伝達経路を媒介することが知られている。したがって、科学的な研究者がどのような要因（s）が検討され、それらの特定の癌細胞または変異体の腫瘍浸潤を駆動しているかを決定するために重要である。成分の濃度との差に応じて決定されるようなアッセイは、それらの生理学的濃度で、個々の成分の役割に対処するために設計されている＆＃8220; X &quot;正常FBSおよびCS-FBS中。このアッセイの開発を通じて、研究者らは、彼らのシステムを管理する特定の侵襲的な経路へのより多くの洞察を得ることができます。 ホルモン、成長因子、およびサイトカインは、多くの場合、腫瘍促進8として分類されている。 EGFのようなこれらの要因のいくつかは、浸潤チャンバー9における化学誘引物質の直接の供給源として使用される。したがって、これらは、直接腫瘍浸潤、血清の主要成分を表す可能性が高いようである。このプロトコルは、研究者は、癌細胞の浸潤能を媒介する、ホルモン、成長因子、およびサイトカインの関与を評価することができ、従来のインビトロ浸潤アッセイにシンプルであり、有意な変更を提案している。しかしながら、アッセイは、貴重な時間とrを使用しないように、手順は、分析の初期段階で彼らの研究のために有効であるかどうかについての回答と調査者を提供するように設計されesources場合は不要と考えられる。この方法では、CS-FBSを使用し、潜在的に癌細胞の浸潤を仲介リード候補として追求する分子れるように研究者の裁量に依存しています。これらの分析の結果は、血清成分が特定の細胞株または変異体の化学誘引物質または阻害剤が検討されているとして機能する特定するのに有用であることを証明する必要があります。さらに、このアプローチは、研究者が癌細胞浸潤を促進または阻害のいずれかの重要なシグナル伝達経路の特定に役立つことができる。したがって、将来の薬剤設計を演出。

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	<tbody>
		<tr height="22">
			<td height="22" style="height:22px;width:367px;">Item</td>
			<td style="width:195px;">Vendor</td>
			<td style="width:136px;">Catalog #</td>
			<td style="width:289px;">Comments/Description</td>
		</tr>
		<tr height="42">
			<td>BioCoat Control Inserts with 8.0&#181;m PET Membrane 
			(12 in each of two 24-Well Plates)</td>
			<td>Corning</td>
			<td>354578</td>
			<td>polyethylene teraphthalate (PET) membranes&#160;&#160;</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Collagen I, rat tail</td>
			<td>Corning</td>
			<td>354236</td>
			<td>Source = rat tail tendon, purity = 90%</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Fixative; Diff-Quick</td>
			<td>Thermo Fisher Scientific</td>
			<td>NC9844047</td>
			<td>Methanol-based fixative, Hmatoxylin/Eosin</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Fetal bovine Serum</td>
			<td>Life Technologies</td>
			<td>16000</td>
			<td>Designated as FBS</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Charcoal Stripped Fetal Bovine Serum</td>
			<td>Life Technologies</td>
			<td>12676</td>
			<td>Designated as CS-FBS</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Fetal bovine Serum</td>
			<td>Thermo Scientific Hyclone</td>
			<td>SH30070</td>
			<td>Designated as FBS</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Charcoal Stripped Fetal Bovine Serum</td>
			<td>Thermo Scientific Hyclone</td>
			<td>SH30068</td>
			<td>Designated as CS-FBS</td>
		</tr>
	</tbody>
</table>

a modified in vitro invasion assay to determine the potential role of hormones, cytokines and/or growth factors in mediating cancer cell invasion

血清は、癌細胞が微小血管に彼らの血管内を容易に移行し、侵入している方に化学誘引物質として機能します。しかし、細胞が移動した方に実際の分子が、とらえどころのないまま。この修​​正された浸潤アッセイは、細胞遊走および浸潤を駆動する標的を同定するために開発された。この技術は、特定のホルモン、成長因子、またはサイトカインが癌細胞の浸潤能を媒介する役割を果たしているかどうかを決定するための3つの条件の下で侵入度とを比較する。これらの条件は、ホルモン、成長因子、およびサイトカインおよびiii）CS-FBS +分子（示される「X」）を除去するのi）通常のウシ胎児血清（FBS）、ii）のチャコール処理FBS（CS-FBS）を含む。 FBSと比較して、CS-FBSと細胞浸潤の有意な変化は、変化を媒介するホルモン、サイトカインまたは成長因子の関与を示している。個々の分子は、再度それらの能力をアッセイするためにCS-FBSを追加し直すことができる侵入表現型を詩や救助。さらに、2つ以上の因子は、添加剤または駆動または浸潤を阻害するのに複数の分子の相乗効果を評価するために組み合わせることができる。全体として、この方法は、ホルモン、サイトカイン、および/または成長因子が癌細胞の特定のタイプまたは特定の変異体の化学誘引物質または浸潤の阻害剤として機能することにより、細胞浸潤において役割を果たしているかどうかを決定するために研究者を可能にする。特定の化学誘引物質および阻害剤を同定することによって、この変更された浸潤アッセイは、癌細胞の浸潤を導くシグナル伝達経路を解明するのに役立ち得る。

浸潤指数は非侵襲細胞株に対する標準化によれば、各条件について計算される。我々の実験のために、我々は1205Luメラノーマ細胞株確立バリアント安定細胞株我々の侵襲線などと同様1205Lu細胞論理対照として10を誘導された前悪性非侵襲的変異体、WM793を使用する。それは、真皮の主成分であるため、我々はまた、侵略マトリックスとしてコラーゲンIを利用する。これは、最適侵入マ...

Watch this Scientific Journal Video about A Modified In vitro Invasion Assay to Determine the Potential Role of Hormones, Cytokines and/or Growth Factors in Mediating Cancer Cell Invasion at JoVE.com

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 ...

a-modified-vitro-invasion-assay-to-determine-potential-role-hormones

Research

JoVE Journal

Medicine

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

10.6K ビュー.  Roswell Park Cancer Institute. 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.

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

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

Metastatic dissemination of malignant cells requires degradation of basement membrane, attachment of tumor cells to vascular endothelium, retraction of endothelial junctions and finally invasion and migration of tumor cells through the endothelial layer to enter the bloodstream as a means of transport to distant sites in the host1-3. Once in the circulatory system, cancer cells adhere to capillary walls and extravasate to the surrounding tissue to form metastatic tumors4,5. The various components of tumor cell-endothelial cell interaction can be replicated in vitro by challenging a monolayer of human umbilical vein endothelial cells (HUVEC) with cancer cells. Studies performed with electron and phase-contrast microscopy suggest that the in vitro sequence of events fairly represent the in vivo metastatic process6. Here, we describe an electrical-impedance based technique that monitors and quantifies in real-time the invasion of endothelial cells by malignant tumor cells.
Giaever and Keese first described a technique for measuring fluctuations in impedance when a population of cells grow on the surface of electrodes7,8. The xCELLigence instrument, manufactured by Roche, utilizes a similar technique to measure changes in electrical impedance as cells attach and spread in a culture dish covered with a gold microelectrode array that covers approximately 80% of the area on the bottom of a well. As cells attach and spread on the electrode surface, it leads to an increase in electrical impedance9-12. The impedance is displayed as a dimensionless parameter termed cell-index, which is directly proportional to the total area of tissue-culture well that is covered by cells. Hence, the cell-index can be used to monitor cell adhesion, spreading, morphology and cell density.
The invasion assay described in this article is based on changes in electrical impedance at the electrode/cell interphase, as a population of malignant cells invade through a HUVEC monolayer (Figure 1). The disruption of endothelial junctions, retraction of endothelial monolayer and replacement by tumor cells lead to large changes in impedance. These changes directly correlate with the invasive capacity of tumor cells, i.e., invasion by highly aggressive cells lead to large changes in cell impedance and vice versa. This technique provides a two-fold advantage over existing methods of measuring invasion, such as boyden chamber and matrigel assays: 1) the endothelial cell-tumor cell interaction more closely mimics the in vivo process, and 2) the data is obtained in real-time and is more easily quantifiable, as opposed to end-point analysis for other methods.

This article describes an in vitro technique for monitoring cancer cells invading through a monolayer of endothelial cells. The data is acquired in real-time as a function of changes in impedance on the surface of electrodes at the well bottom.

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 ...

医学

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

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

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

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

アンインビトロ</em転移性増殖に腫瘍の休眠とスイッチを研究する>システム

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

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.

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 ...

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 ...

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 ...

Focus Formation: A Cell-based Assay to Determine the Oncogenic Potential of a Gene

Malignant transformation of cells is typically associated with increased proliferation, loss of contact inhibition, acquisition of anchorage-independent growth potential, and the ability to form tumors in experimental animals1. In NIH 3T3 cells, the Ras signal transduction pathway is known to trigger many of these events, what is known as Ras transformation. The introduction of an overexpressed gene in NIH 3T3 cells may promote morphological transformation and loss of contact inhibition, which can help determine the oncogenic potential of that gene of interest. An assay that provides a straightforward method to assess one aspect of the transforming potential of an oncogene is the Focus Formation Assay (FFA)2. When NIH 3T3 cells divide normally in culture, they do so until they reach a confluent monolayer. However, in the presence of an overexpressed oncogene, these cells can begin to grow in dense, multilayered foci1 that can be visualized and quantified by crystal violet or Hema 3 staining. In this article we describe the FFA protocol with retroviral transduction of the gene of interest into NIH 3T3 cells, and how to quantify the number of foci through staining. Retroviral transduction offers a more efficient method of gene delivery than transfection, and the use of an ecotropic murine retrovirus provides a biosafety control when working with potential human oncogenes.

The Focus Formation Assay provides a straightforward method to assess the transforming potential of a candidate oncogene.

フォーカス形成：細胞ベースのアッセイ遺伝子の発癌性を決定するために、

Malignant transformation of cells is typically associated with increased proliferation, loss of contact inhibition, acquisition of anchorage-independent ...

Establishment of a Human Multiple Myeloma Xenograft Model in the Chicken to Study Tumor Growth, Invasion and Angiogenesis

Multiple myeloma (MM), a malignant plasma cell disease, remains incurable and novel drugs are required to improve the prognosis of patients. Due to the lack of the bone microenvironment and auto/paracrine growth factors human MM cells are difficult to cultivate. Therefore, there is an urgent need to establish proper in vitro and in vivo culture systems to study the action of novel therapeutics on human MM cells. Here we present a model to grow human multiple myeloma cells in a complex 3D environment in vitro and in vivo. MM cell lines OPM-2 and RPMI-8226 were transfected to express the transgene GFP and were cultivated in the presence of human mesenchymal cells and collagen type-I matrix as three-dimensional spheroids. In addition, spheroids were grafted on the chorioallantoic membrane (CAM) of chicken embryos and tumor growth was monitored by stereo fluorescence microscopy. Both models allow the study of novel therapeutic drugs in a complex 3D environment and the quantification of the tumor cell mass after homogenization of grafts in a transgene-specific GFP-ELISA. Moreover, angiogenic responses of the host and invasion of tumor cells into the subjacent host tissue can be monitored daily by a stereo microscope and analyzed by immunohistochemical staining against human tumor cells (Ki-67, CD138, Vimentin) or host mural cells covering blood vessels (desmin/ASMA).
In conclusion, the onplant system allows studying MM cell growth and angiogenesis in a complex 3D environment and enables screening for novel therapeutic compounds targeting survival and proliferation of MM cells.

Human multiple myeloma (MM) cells require the supportive microenvironment of mesenchymal cells and extracellular matrix components for survival and proliferation. We established an in vivo chicken embryo model with engrafted human myeloma and mesenchymal cells to study effects of cancer drugs on tumor growth, invasion and angiogenesis.

腫瘍の増殖、浸潤及び血管新生を研究するために、ニワトリでのヒト多発性骨髄腫異種移植モデルの確立

Multiple myeloma (MM), a malignant plasma cell disease, remains incurable and novel drugs are required to improve the prognosis of patients. Due to the ...

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 ...

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