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1.クローン原性アッセイ<ol><li>以下の手順を使用して、エストロゲン依存性乳癌細胞株MCF-7およびT47D細胞の単一細胞懸濁液を調製<ol><li> MCF-7またはT-47D細胞との50％を超えるコンフルエントでない10cmの組織培養皿から培地（DMEM / 10％熱不活性化ウシ胎児血清/グルタミンおよびペニシリン/ストレプトマイシン）を吸引除去します。 PBSですすいでください。 1-4分間37℃で、DMEM高グルコースに溶解し、トリプシン0.25％/ 2.21 mMのEDTAでインキュベートします。 </li><li>単一細胞分布を確実にするために位相差顕微鏡下で1分間隔で細胞をチェック。ほぼ不変の、単一細胞の状態を達成するために、細胞 - 細胞接触を破壊するには、下に数回ピペッティングにより2mLのピペットで細胞を再懸濁します。 </li><li>あなたは、インキュベーションのわずか2分後の細胞の塊を観察場合、最大4分間37℃でトリプシンで細胞をインキュベートし続けています。彼らは電子に付着し残っている場合は、クローン原性研究のためにこれらの細胞を使用しないでくださいエラーがコロニー数の収率で導入されるため、トリプシン処理の4分後に他のACH。 注：細胞が凝集している場合は、形成されたコロニーの数をインキュベート数よりも少ない数の細胞の産物を反映します。細胞が過剰にトリプシン処理された場合、それらのクローン原性ポテンシャルが減少することができます。 </li><li>のために必要なボリューム全体を含むものマスターチューブ中連続希釈により、（細胞型または継代数に応じて、+ 500細胞/ ml）を24ウェルプレート、以下/ mlの培養培地1,500細胞の単一細胞懸濁液を調製します実験におけるすべての変数。 注：目標は800セル/ cm 2（約500〜1100個/ cm 2の範囲）の最終細胞密度です。目標は、比較的容易に計数を可能にする約100 + 50個のコロニーを得るためにある混雑を防止し、コロニーを実験perturbaによって増減したときに有意な統計的差異をもたらすのに十分なコロニーを可能にしますン。 </li></ol></li><li>以下の手順を使用してクローン形成密度で細胞をインキュベート<ol><li> 1,500細胞/ mlのマスター単一細胞懸濁液管から/ウェル1,500細胞のクローン原性密度で24ウェルのフィブロネクチンコーティングプレート上の四重のウェル中の細胞をインキュベートします。 3ミリリットルを描画し、2つのウェルのそれぞれ1 mlの培地を分配することによって、5ミリリットルピペットで細胞を含む培地を粉砕します。 注：フィブロネクチンでコーティングされたプレートは、商用ベンダーから予備被覆を購入する必要があります。制御品質の外側コーティングプレートは、凹凸面での自動化されたプロセスの結果は、このアッセイのために適さありません。 </li><li>細胞懸濁液の3ミリリットルを策定し、各1mlで2井戸を埋める、ピペッティングにより、5mlのピペットでダウンサスを混ぜます。 1ピペットから任意の時点でわずか2井戸を埋めます。上下にピペッティングして、再度、マスターチューブに細胞懸濁液にピペットで再懸濁細胞を、残りのボリュームを戻し、別の2 WELを埋めるために、別の3ミリリットルを描きます各1mlとLS。 注：細胞が連続的に沈殿しますので、マスター·チューブの連続的な混合が必要です。唯一の2つのウェルを満たすのに十分な量を描画すると、細胞は同様に、ピペットに沈降するので各ウェルに類似した細胞数を追加する必要があります。 </li><li>可能細胞は、それらのクローン原性の可能性（未発表の観察）を変調する室温とCO 2濃度の懸濁液中に座っているため、細胞を大量に配布するために、急速に働きます。 </li><li>コロニーアッセイのためのウェルにそれらをピペッティングの行為の間に細胞の空間分布を最適化します。ゆっくりウェルの中央に最終細胞濃度を含有する懸濁液をピペットで行ってください。細胞が底に沈殿する前に運動を促進するために、プレートを与えないでください。円形の混合が効果的によく6日目に高い細胞密度と無数のコンフルエントコロニーを作るの周囲に細胞を遠心分離しますので、プレートを旋回しないでください。 注：これは、懸濁液中の細胞でボリュームを導入した後、全く混合よりも少ないことが望ましいので、必要な場合以外は混ぜないでください。それは、細胞を混合する必要がある場合、それは平らな表面上に静止しながら垂直方向に前後にプレートを移動させることによってこれを行います。 </li><li> 6日間のメディアを変更せずに、37℃、5％CO 2で細胞をインキュベートします。 6日後のウェル中の細胞の小さな数を大幅に栄養またはサイトカイン組成も元の培地のpHには影響しません。 </li><li>以下のようにアッセイの時間経過を設計します。1ミリリットル新鮮な培地、または新鮮な培地を含有してメディアを既存の置き換え1日目フィブロネクチンコーティングされた基層上で細胞をインキュベートし、FGF-2、以下のように6日目に0日ステイン細胞に10 ng / mlで1.4で）。 ）1.3で、以下のように、3日目に任意の実験的摂動を行っています。 </li></ol></li><li>分子シグナリングまたは接着分子の実験的摂動を設定<ol><li> 3日目に、100を追加します56;ウェルに1mLの培地に摂動剤の意図された最終濃度の10倍を含有する溶液のリットル。混在させないでください。さらに3日間37℃、5％CO 2で細胞をインキュベートし続けます。 注：摂動剤は休止状態を支える役割を果たし得る阻害剤および接着分子、受容体または他の表面タンパク質、細胞内シグナル伝達経路の阻害剤、分子、因子、補因子または構造タンパク質のブロッキング剤の多様を含むことができます。 </li><li>以下のように、6日目のコロニーを染色します。 </li></ol></li><li>ステインコロニー<ol><li> 2％のエタノール/ 10mMのホウ酸ナトリウム（pHは9.0）溶液中で新たに作製した0.1％クリスタルバイオレットで培養中の6日後に染色細胞。吸引メディア、20分間各ウェルに1つmLのクリスタルバイオレット溶液を加えます。 </li><li>ウェル開口部が連続してタップを実行あふれるアイスバケツに鋭角に下にして、それらを浸漬することにより、プレートを洗浄シンク内の水。 、一度水中水平角にプレートを傾けよくダウン開いて、[1穏やかな流れる動きで除去する際に鋭角に倒します。 </li><li>ウェルの底に水がもはや青色でなくなるまで浸漬を2〜3回繰り返します。激しい洗浄はカウントとデータの誤りを大幅追加して、原因の実験的介入にあまり付着している細胞またはコロニーを除去することができます。 </li><li>それらに対応するラベルされたカバーに隣接する作業台の上にタオルの上に逆さにそれらを配置することで、一晩乾燥したプレート。 </li></ol></li><li>コロニーをカウント<ol><li>インキュベーション、染色、乾燥の6日後に各ウェルの成長と休眠コロニーの数を数えます。倒立位相差顕微鏡で40倍の倍率で最適にコロニーを数えます。増殖する細胞に比べて非常に大きいサイズの形態学的外観で、成長している12以下の細胞のコロニーとして&gt; 30細胞のコロニーをカウントし、大EXPA図1 7,15に示す割合を、核に大きな細胞質と細胞質nded。 注：彼らは簡単休眠アッセイにおいて非常に頻繁ではないとして13-29細胞のクラスターが正常にカウントされません。摂動が成長しているか、休止状態の細胞のいずれかの成長の可能性をシフトなら、彼らをカウントすることができます。これらの結果は、生物学的に有意に相関することが必要になります。 </li></ol></li></ol>免疫蛍光研究のための2クローン原性インキュベーション<ol><li>細胞数/ 24ウェル実験に類似した表面積に対応するために、6ウェルプレート中/ウェル約7,500-8,000細胞に細胞数を調整します。 </li><li>細胞の添加の前にイメージング研究のための6ウェルプレートの各ウェルベースにラウンド、滅菌、フィブロネクチンでコーティングされたカバースリップを置きます。 1.2で上記のクローン原性実験のために説明したように、各ウェルに3ミリリットルの容量でピペットで細胞を概説濃度で一度に2つのウェル）。 </li><li>細胞をインキュベート上記のように1.2.4と1.2.5で6日間。 1.3にコロニーアッセイ手順で説明したように）、300μlの量の10倍の濃度で、3日目に摂動要因を追加します。 </li><li>例えば、インテグリンα4、α5、α6、β1、β3、例えば、焦点接着複雑な分子FAK、パキシリンおよびビンキュリンのように接着分子を細胞に対する抗体で6日目の染色細胞は、このようなαチューブリンなどの運動性に関与するタンパク質、例えば、リン酸化Akt、ホスホERK、ホスホ-p38、ホスホJNK、例えば、または休眠におけるその役割のための調査の対象は、直接的または間接的のための標準的な技術を用いて、任意の他のタンパク質などのシグナル伝達経路のメンバー免疫蛍光染色。 <ol><li> -20℃で1：アセトン/メタノール1鉗子6日修正でスライドを削除します 20分、空気乾燥のためにCを°。代替の固定剤、必要に応じてパラホルムアルデヒドのような、使用されてもよいです。 2分間fの0.1％トリトンX-100、0.1％クエン酸ナトリウムと透過化細胞又は細胞内抗原の検出。 PBSで細胞を洗浄します。 </li><li>間接immunofluorecence染色、5％BSAを用いて室温で1時間または二次抗体が生成された種からの10％の免疫前血清でブロックスライドについて。 </li><li>経路メンバーまたはPBSにメーカーが推奨する特定の希釈液に希釈した休眠におけるその役割のための調査の対象である任意の他のタンパク質のシグナル伝達、接着分子、焦点複雑な分子をセルに一次抗体と4℃で一晩インキュベートし、0.1％TRITON X -100。 </li><li> PBSで3回洗浄します。 2時間室温で蛍光体結合抗体でカバースリップをインキュベートします。一例として、アレクサフルオロ488ロバ抗マウスIgG抗体は、マウスモノクローナル一次抗体を検出することができます。マウントにはDAPIで退色防止剤を用いてガラススライド上に細胞側を下カバースリップ。マニキュアで周囲をシール。 </li><li>直接免疫蛍光のために、BSAを行います）2.4.2に上記のようにブロックし、4℃で一晩蛍光体共役一次抗体とインキュベートします。 PBSで3回洗浄します。退色防止剤およびDAPIでスライドをインキュベートし、マニキュア液で密封します。 </li><li> 4でのアルミ箔とストアとカバースライドトレイ イメージングおよび写真撮影のためのC°いつまで数週間後に。 1,000倍の倍率でカメラを搭載した任意の蛍光顕微鏡イメージングシステムを使用して表示および写真の細胞。 </li><li>繊維状アクチン染色のために、1％ウシ血清アルブミン（BSA）でブロックスライド30分間、20分間、室温でBODIPY FL-ファラシジン（緑色）又はローダミンファロイジン（赤）でインキュベートします。退色防止剤を添加し、上記のように密封します。 </li></ol></li></ol>分子研究のための3クローン原性インキュベーション<ol><li>ウェスタンブロット<ol><li> fibronecに20,000細胞/ 60ミリメートルプレートおよび50,000細胞/ 100ミリメートルプレートのクローン原性密度でER +乳癌細胞MCF-7またはT47Dをインキュベート錫被覆された5％CO 2中37℃でプレート。 </li><li>溶解物からのタンパク質のためのmgの量を必要とする分子研究のために75,000細胞/ 100ミリメートルプレートのわずかに高い密度で細胞をインキュベートします。ウェスタンブロットを用いた分子の研究のために、またはノーザンブロットのためのRNA単離のために十分なタンパク質を収集するために実験点につき10プレートまで使用。 </li><li>ゲルローディングベースの細胞数は、非常に異なるサイズの成長と休眠細胞におけるタンパク質の発現を比較するために必要な付属物です。トリプシン処理により細胞を回収し、代わりに単一のエッジのブレードを用いて細胞を掻きのウェスタンブロットのための溶解液を調製するために、0.2％トリパンブルーで、血球計数器で一定量をカウントします。 </li><li> 2分間10,000×gで遠心分離し細胞を、吸引により培地を除去し、200μlの溶解緩衝液を追加し、溶解緩衝液中で細胞を超音波処理し、タンパク質濃度を決定します。 </li><li>細胞数によるタンパク質収量を分割することにより、細胞あたりのタンパク質の量を計算しますそれは、その発生量。タンパク質のa）の当量と同等の細胞数を表すb）のタンパク質の量の両方を表す別々のポリアクリルアミドゲルの各ウェルに溶解液をロードします。少なくとも25μgタンパク質を各ウェルにロードする必要があります。 注：これは、サイズおよびタンパク質含有量（ 図1）において著しく異なる成長および休眠細胞間の比較を可能にします。 </li></ol></li><li>フローサイトメトリー<ol><li> 3.1のように、トリプシン処理により100ミリメートルプレートからの細胞を収集し、標準的な蛍光活性化セルソーティング（FACS）によって分析すると、2.4で概説されるように、細胞内または細胞外のいずれかの抗原に対する一次または二次免疫蛍光染色を使用してプロトコル。 注：抗体標識によって決定されるようにトリプシン処理により組織培養プレートから細胞を剥離すると、膜タンパク質の濃度に影響を与えません。 </li></ol></li></ol>

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がん研究02から1140-CCR-E0および癌研究のためのルースエストロンゴールドバーグ記念（RW）に国防総省助成のDAMD17-01-C-0343とDAMD17-03-1-0524、ニュージャージー州委員会でサポートされています

我々のモデルは、骨髄中の休眠のいくつかの重要な要素で構成されています。それは、フィブロネクチン、骨髄の主要構成要素、FGF-2、成長因子が豊富に骨髄間質によって合成から成り、エストロゲンの長期間10に対する骨髄における休眠残る可能性が高いタイプである敏感な細胞を、構成されてい重く骨髄31,32とそれらの相互作用は、基層と主にクローン原性密度で細胞のイ...

疾患とすぐ小さな腫瘍が血管2,3を開発する、検出可能になる前に1乳癌細胞は、骨髄に転移します。転移プロセスは迅速であるが、非効率的です。細胞を、4日目あたり数百万人で、急速に新しい血管を入力しますが、いくつかは、遠隔臓器5への旅行を生き残ります。それにもかかわらず、いくつかの微小転移は、骨に生き残るためには、新たに診断された患者1からの骨髄吸引中の単一細胞または小細胞塊として求めることができます。これらの細胞は、それら6を除去する非常に目的のために投与されるアジュバント化学療法を、抵抗します。この抵抗は、骨髄微小環境7,8との相互作用によって開始される生存シグナル伝達によって、実質的に恵まれています。微小転移は、ローカライズされた乳がんの女性の約3分の1に見られ、単変量解析9により分析すると生存の独立した指標を表現することができます。一部micrometastasesは、成長を開始しているが、再発パターンは、細胞の種類によって異なります。トリプルネガティブ乳がんの患者は、休止状態の上に不十分な制御を示唆し、1〜4年の間に再発する傾向があります。 ER / PR +細胞を含む他の細胞型は、再発10の安定した、連続的な速度で、最大20年間の休眠残ることができます。 ER +とER-乳癌細胞株および腫瘍間休眠遺伝子発現シグネチャの差が異なる休眠電位11を反映しているが、骨髄間質との相互作用は、おそらく休止状態への重要な貢献を表します。 微小転移はまれであり、10以上の6倍によって造血細胞によって劣勢されているため、生体内で休眠の研究は非常に困難です。したがって、関連するモデルはメカニズムを示唆し、in vivoで検証可能な仮説を生成することができるインビトロのデータで提供する生成する必要があります。 mathe含む休眠モデルの数、maticalモデル12,13、in vitroモデル7,8,14,15、in vivoでの異種移植モデル16、in vitroでの組み合わせと異種移植モデル17,18と自発的な腫瘍および転移モデル19は 、癌細胞の休眠20にいくつかの洞察が得られています。これらの各モデルは、独自の制限があり、それ自体の分子シグナル伝達、より生物学的に関連するモデルで試験することが休眠を支配する相互作用に関する仮説を生成するために、主に有用です。 休眠の分子機構、ER +細胞中の再発をもたらす周期停止、再分化および治療 ​​抵抗性とメカニズムをもたらす微小環境との相互作用を定義する全体的な目標では、我々は、関連する要素を選択し提供するin vitroモデルを開発しました間質微小環境7。そのコンポにいる間、比較的まばらなこのモデルは、ネントは、休眠の重要な機能に影響を与える特定の分子メカニズムを導出するための研究を可能にするのに十分に頑強です。これらの実験は、 インビボで直接試験することができる仮説を生成します。モデルは、我々は休眠中の関連であることが実証いくつかの重要な要素に依存しています。これらは、エストロゲン依存性乳癌細胞の使用は、それらの相互作用は、基体と培地の可溶性成分、フィブロネクチン基質と塩基性線維芽細胞増殖因子（FGF-2）の存在下で主にクローン原性密度で細胞の培養を含みます培地中に。 私たちは、TGFβ22を介して媒介FGF-2 21による細胞周期停止の誘導を含む、in vitroでシステムを管理するメカニズムを、特徴に依存し、上皮表現型にPI3キナーゼ7,8およびERK 8と形態形成分化を介したシグナル伝達生存RhoAの不活性化、インテグリン45;生存7,15のための間質フィブロネクチンの5β1アップレギュレーションおよびライゲーション（ 図1）。 MCF-7細胞におけるFGF-2のin vitroでの細胞周期効果は濃度では10ng / mlの21,23下記少なくとも一つのログを開始します。理論的根拠は、乳管の形態形成、哺乳類系24-27の数の巡回拡大と景気後退を支配するFGF-2の発現の時間的制御に基づいていました。我々は、FGF-2は、3D培養28における乳管形態形成を含む、分化を誘導することを実証し、そのFGF-2の発現は、一般的に、ヒト腫瘍29の悪性形質転換と共に失われています。 FGR1の発現は29を調査し、MCF-7細胞は、すべての4 FGF受容体30を発現し続ける乳癌で無傷のままでした。休眠との関連では、FGF-2は、によってエクスポートされ、頻繁にそれは造血幹細胞33の保存に機能する骨髄間質31,32上に堆積します。我々DEMFGF-2はまた、それが豊富な形態形成分化を誘導する骨髄7において、フィブロネクチン基質上で培養されたER +乳癌細胞において休眠状態を誘発することonstrated。モデルでは、乳癌細胞は、増殖阻害され、RhoGap GRAF、上皮表現型および悪性進行による再急行インテグリンα5β1lostに再分化を介してのRho Aを不活性化します。彼らは、インテグリンα5β1を介してフィブロネクチンと結合し、それが細胞毒性療法7,8,15（ 図1）に、それらが耐性をレンダリング生存シグナル伝達を活性化します。ロークラスGTPアーゼの阻害は、休止状態の表現型34を誘導することが以前に実証されています。 ここでは、モデルを確立し、ER +乳癌細胞の休眠を支配する特定の分子·細胞メカニズムを研究する研究者を可能にする具体的な手順を概説します。モデルの使用方法を説明するためにここに提示された実験において、我々は、Akt阻害剤およびPI3K阻害剤とパンのRhoとRhoファミリー（ 図1B）阻害剤とRhoキナーゼ（ROCK）阻害剤の全てのメンバーとPI3K経路（ 図1B）を対象としました。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>MCF-7 cells</td>
			<td>ATCC</td>
			<td>HTB-22</td>
			<td></td>
		</tr>
		<tr>
			<td>T47D cells&#160;</td>
			<td>ATCC</td>
			<td>HTB-123</td>
			<td></td>
		</tr>
		<tr>
			<td>BD BioCoat Fibronectin 24 Well Clear Flat Bottom TC-Treated Multiwell Plate</td>
			<td>Corning</td>
			<td>354411</td>
			<td></td>
		</tr>
		<tr>
			<td>BD BioCoat Fibronectin 60 mm Culture Dishes</td>
			<td>Corning</td>
			<td>354403</td>
			<td></td>
		</tr>
		<tr>
			<td>BD BioCoat Fibronectin 100 mm Culture Dishes</td>
			<td>Corning</td>
			<td>354451</td>
			<td></td>
		</tr>
		<tr>
			<td>BD BioCoat 22x22mm #1 Glass Coverslip with a uniform application of human fibronectin</td>
			<td>Corning</td>
			<td>354088</td>
			<td></td>
		</tr>
		<tr>
			<td>6 Well tissue culture plate</td>
			<td>CellTreat</td>
			<td>229106</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco Modified Eagle Medium High Glucose 10X Powder</td>
			<td>Corning Life Sciences</td>
			<td>50-013-PB</td>
			<td></td>
		</tr>
		<tr>
			<td>Heat Inactivated, Fetal Bovine Serum</td>
			<td>Serum Source International</td>
			<td>FB02-500HI</td>
			<td></td>
		</tr>
		<tr>
			<td>0.25% Trypsin/2.21 mM EDTA</td>
			<td>Corning</td>
			<td>25-053-CI</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin Solution, 100X</td>
			<td>Corning</td>
			<td>30-002-CI</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutamine, 100x, Liquid</td>
			<td>Corning</td>
			<td>25-005-CI</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Human FGF&#160;basic</td>
			<td>R&#38;D Systems</td>
			<td>234-FSE-025</td>
			<td></td>
		</tr>
		<tr>
			<td>Akt Inhibitor (1L6-Hydroxymethyl-chiro-inositol-2-(R)-2-O-methyl-3-O-octadecyl-sn-glycerocarbonate)</td>
			<td>CalBiochem</td>
			<td>124005</td>
			<td></td>
		</tr>
		<tr>
			<td>LY294002&#160;</td>
			<td>CalBiochem</td>
			<td>19-142</td>
			<td>Chenical PI3K inhibitor</td>
		</tr>
		<tr>
			<td>C3 transferase&#160;</td>
			<td>Cytoskeleton</td>
			<td>CTO3</td>
			<td>Inhibits RhoA, RhoB, and RhoC, but not related GTPases such as Cdc42 or Rac1</td>
		</tr>
		<tr>
			<td>Y-27632 dihydrochloride&#160;</td>
			<td>Santa Cruz Biotechnology</td>
			<td>129830-28-2</td>
			<td></td>
		</tr>
		<tr>
			<td>BODIPY FL-Phallacidin (green)&#160;</td>
			<td>Molecular Probes</td>
			<td>B607</td>
			<td>Fluorochrome for fibrillar actin staining</td>
		</tr>
		<tr>
			<td>BODIPY FL-Rhodamine phalloidin (red)&#160;</td>
			<td>Molecular Probes</td>
			<td>R415</td>
			<td>Fluorochrome for fibrillar actin staining</td>
		</tr>
		<tr>
			<td>Alexa Fluor 488 Donkey anti-Mouse IgG Antibody, ReadyProbes Reagent&#160;</td>
			<td>Molecular Probes</td>
			<td>R37114</td>
			<td></td>
		</tr>
		<tr>
			<td>ProLong Gold Antifade Mountant with DAPI&#160;</td>
			<td>Molecular Probes</td>
			<td>P-36931</td>
			<td></td>
		</tr>
	</tbody>
</table>

an in vitro dormancy model of estrogen-sensitive breast cancer in the bone marrow: a tool for molecular mechanism studies and hypothesis generation

The study of breast cancer dormancy in the bone marrow is an exceptionally difficult undertaking due to the complexity of the interactions of dormant cells with their microenvironment, their rarity and the overwhelming excess of hematopoietic cells. Towards this end, we developed an in vitro 2D clonogenic model of dormancy of estrogen-sensitive breast cancer cells in the bone marrow. The model consists of a few key elements necessary for dormancy. These include 1) the use of estrogen sensitive breast cancer cells, which are the type likely to remain dormant for extended periods, 2) incubation of cells at clonogenic density, where the structural interaction of each cell is primarily with the substratum, 3) fibronectin, a key structural element of the marrow and 4) FGF-2, a growth factor abundantly synthesized by bone marrow stromal cells and heavily deposited in the extracellular matrix. Cells incubated with FGF-2 form dormant clones after 6 days, which consist of 12 or less cells that have a distinct flat appearance, are significantly larger and more spread out than growing cells and have large cytoplasm to nucleus ratios. In contrast, cells incubated without FGF-2 form primarily growing colonies consisting of >30 relatively small cells. Perturbations of the system with antibodies, inhibitors, peptides or nucleic acids on day 3 after incubation can significantly affect various phenotypic and molecular aspects of the dormant cells at 6 days and can be used to assess the roles of membrane-localized or intracellular molecules, factors or signaling pathways on the dormant state or survival of dormant cells. While recognizing the in vitro nature of the assay, it can function as a highly useful tool to glean significant information about the molecular mechanisms necessary for establishment and survival of dormant cells. This data can be used to generate hypotheses to be tested in vivo models.

<html>実験は、アッセイを再現するために行われました。実験の時間経過は、 図2Aに示されています。細胞を1日目にクローン原性密度でインキュベートし、FGF-2、新鮮な培地中では0日目に添加し、それらを染色し、コロニーを計数したときの細胞は6日目まで培養されます。システムに何らかの摂動が所望の10倍の最終濃度で100μlの容量で3日目に投与される。 図2Bは、?...

Watch this Scientific Journal Video about An In Vitro Dormancy Model of Estrogen-sensitive Breast Cancer in the Bone Marrow: A Tool for Molecular Mechanism Studies and Hypothesis Generation at JoVE.co

An In Vitro Dormancy Model of Estrogen-sensitive Breast Cancer in the Bone Marrow: A Tool for Molecular Mechanism Studies and Hypothesis Generation

We developed an in vitro model of dormancy in the bone marrow for estrogen-sensitive breast cancer cells. The goal of this protocol is to demonstrate use of the model for the study of the molecular and cellular biology of dormancy and for generation of hypotheses for subsequent testing in vivo.

アン<em&gt;インビトロ</em&gt;骨髄中のエストロゲン感受性乳癌の休眠モデル：分子機構の研究と仮説生成のためのツール

The study of breast cancer dormancy in the bone marrow is an exceptionally difficult undertaking due to the complexity of the interactions of dormant ...

an-vitro-dormancy-model-estrogen-sensitive-breast-cancer-bone-marrow

Research

JoVE Journal

Medicine

An In Vitro Dormancy Model of Estrogen-sensitive Breast Cancer in the Bone Marrow: A Tool for Molecular Mechanism Studies and Hypothesis Generation

8.2K ビュー.  Rutgers New Jersey Medical School. We developed an in vitro model of dormancy in the bone marrow for estrogen-sensitive breast cancer cells. The goal of this protocol is to demonstrate use of the model for the study of the molecular and cellular biology of dormancy and for generation of hypotheses for subsequent testing in vivo. 

ビデオ: An In Vitro Dormancy Model of Estrogen-sensitive Breast Cancer in the Bone Marrow: A Tool for Molecular Mechanism Studies and Hypothesis Generation

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 ...

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 ...

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 ...

Screening for Melanoma Modifiers using a Zebrafish Autochthonous Tumor Model

Genomic studies of human cancers have yielded a wealth of information about genes that are altered in tumors1,2,3. A challenge arising from these studies is that many genes are altered, and it can be difficult to distinguish genetic alterations that drove tumorigenesis from that those arose incidentally during transformation. To draw this distinction it is beneficial to have an assay that can quantitatively measure the effect of an altered gene on tumor initiation and other processes that enable tumors to persist and disseminate. Here we present a rapid means to screen large numbers of candidate melanoma modifiers in zebrafish using an autochthonous tumor model4 that encompasses steps required for melanoma initiation and maintenance. A key reagent in this assay is the miniCoopR vector, which couples a wild-type copy of the mitfa melanocyte specification factor to a Gateway recombination cassette into which candidate melanoma genes can be recombined5. The miniCoopR vector has a mitfa rescuing minigene which contains the promoter, open reading frame and 3'-untranslated region of the wild-type mitfa gene. It allows us to make constructs using full-length open reading frames of candidate melanoma modifiers. These individual clones can then be injected into single cell Tg(mitfa:BRAFV600E);p53(lf);mitfa(lf)zebrafish embryos. The miniCoopR vector gets integrated by Tol2-mediated transgenesis6 and rescues melanocytes. Because they are physically coupled to the mitfa rescuing minigene, candidate genes are expressed in rescued melanocytes, some of which will transform and develop into tumors. The effect of a candidate gene on melanoma initiation and melanoma cell properties can be measured using melanoma-free survival curves, invasion assays, antibody staining and transplantation assays.

A rapid way to screen for melanoma modifiers using a zebrafish autochthonous tumor model is presented. It takes advantage of the miniCoopR vector which allows for expression of candidate melanoma genes in melanocytes. A method to obtain melanoma-free survival curves, an invasion assay, a protocol for antibody staining of scale melanocytes and a melanoma transplantation assay are described.

ゼブラ土着腫瘍モデルを用いたメラノーマの修飾のためのスクリーニング

Genomic studies of  human cancers have yielded a wealth of information about genes that are altered  in tumors1,2,3. A challenge arising from these ...

Using Quantitative Real-time PCR to Determine Donor Cell Engraftment in a Competitive Murine Bone Marrow Transplantation Model

Murine bone marrow transplantation models provide an important tool in measuring hematopoietic stem cell (HSC) functions and determining genes/molecules that regulate HSCs. In these transplant model systems, the function of HSCs is determined by the ability of these cells to engraft and reconstitute lethally irradiated recipient mice. Commonly, the donor cell contribution/engraftment is measured by antibodies to donor- specific cell surface proteins using flow cytometry. However, this method heavily depends on the specificity and the ability of the cell surface marker to differentiate donor-derived cells from recipient-originated cells, which may not be available for all mouse strains. Considering the various backgrounds of genetically modified mouse strains in the market, this cell surface/ flow cytometry-based method has significant limitations especially in mouse strains that lack well-defined surface markers to separate donor cells from congenic recipient cells. Here, we reported a PCR-based technique to determine donor cell engraftment/contribution in transplant recipient mice. We transplanted male donor bone marrow HSCs to lethally irradiated congenic female mice. Peripheral blood samples were collected at different time points post transplantation. Bone marrow samples were obtained at the end of the experiments. Genomic DNA was isolated and the Y chromosome specific gene, Zfy1, was amplified using quantitative Real time PCR. The engraftment of male donor-derived cells in the female recipient mice was calculated against standard curve with known percentage of male vs. female DNAs. Bcl2 was used as a reference gene to normalize the total DNA amount. Our data suggested that this approach reliably determines donor cell engraftment and provides a useful, yet simple method in measuring hematopoietic cell reconstitution in murine bone marrow transplantation models. Our method can be routinely performed in most laboratories because no costly equipment such as flow cytometry is required.

Determining donor cell engraftment presents a challenge in mouse bone marrow transplant models that lack well-defined phenotypical markers. We described a methodology to quantify male donor cell engraftment in female transplant recipient mice. This method can be used in all mouse strains for the study of HSC functions.

競争力のあるマウス骨髄移植モデルにおけるドナー細胞の生着を​​決定するために、定量的リアルタイムPCRを用いて

Murine bone marrow transplantation models provide an important tool in measuring hematopoietic stem cell (HSC) functions and determining genes/molecules ...

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As such, primary or &#39;neuronal-like&#39; cell culture systems are commonly utilized. While&#160;these systems are relatively easy to work with, and are useful model systems in which various functional outcomes (such as cell death) can be readily quantified, the examined outcomes and pathways in cultured immature neurons (such as excitotoxicity-mediated cell death pathways) are not necessarily the same as those observed in mature brain, or in intact tissue. Therefore, there is the need to develop models in which cellular mechanisms in mature neural tissue can be examined. We have developed an in vitro technique that can be used to investigate a variety of molecular pathways in intact nervous tissue. The technique described herein utilizes rat cortical tissue, but this technique can be adapted to use tissue from a variety of species (such as mouse, rabbit, guinea pig, and chicken) or brain regions (for example, hippocampus, striatum, etc.). Additionally, a variety of stimulations/treatments can be used (for example, excitotoxic, administration of inhibitors, etc.). In conclusion, the brain slice model described herein can be used to examine a variety of molecular mechanisms involved in excitotoxicity-mediated brain injury.

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

We have developed a brain slice model which can be used to examine molecular mechanisms involved in excitotoxicity-mediated brain injury.&#160;This technique generates viable mature brain tissue and reduces animal numbers required for experimentation, whilst keeping the neuronal circuitry, cellular interactions, and postsynaptic compartments partly intact.

脳Microslicesの興奮毒性刺激<em&gt;インビトロ</em&gt;脳卒中のモデル

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As ...

Femoral Bone Marrow Aspiration in Live Mice

Serial sampling of the cellular composition of bone marrow (BM) is a routine procedure critical to clinical hematology. This protocol describes a detailed step-by-step technical procedure for an analogous procedure in live mice which allows for serial characterization of cells present in the BM. This procedure facilitates studies aimed to detect the presence of exogenously administered cells within the BM of mice as would be done in xenograft studies for instance. Moreover, this procedure allows for the retrieval and characterization of cells enriched in the BM such as hematopoietic stem and progenitor cells (HSPCs) without sacrifice of mice. Given that the cellular composition of peripheral blood is not necessarily reflective of proportions and types of stem and progenitor cells present in the marrow, procedures which provide access to this compartment without requiring termination of the mice are very helpful. The use of femoral bone marrow aspiration is illustrated here for cytological analysis of marrow cells, flow cytometric characterization of the hematopoietic stem/progenitor compartment, and culture of sorted HSPCs obtained by femoral BM aspiration compared with conventional marrow harvest.

This protocol describes a procedure for serial sampling of femoral bone marrow (BM) without requiring the sacrifice of mice. This procedure facilitates longitudinal studies of the BM composition of mice over time and provides serial access to cells within the BM for ex vivo and transplantation studies.

生きたマウスにおける大腿骨髄吸引

Serial sampling of the cellular composition of bone marrow (BM) is a routine procedure critical to clinical hematology. This protocol describes a detailed ...

An Ex vivo Model to Study Hormone Action in the Human Breast

The study of hormone action in the human breast has been hampered by lack of adequate model systems. Upon in vitro culture, primary mammary epithelial cells tend to lose hormone receptor expression. Widely used hormone receptor positive breast cancer cell lines are of limited relevance to the in vivo situation. Here, we describe an ex vivo model to study hormone action in the human breast. Fresh human breast tissue specimens from surgical discard material such as reduction mammoplasties or mammectomies are mechanically and enzymatically digested to obtain tissue fragments containing ducts and lobules and multiple stromal cell types. These tissue microstructures kept in basal medium without growth factors preserve their intercellular contacts, the tissue architecture, and remain hormone responsive for several days. They are readily processed for RNA and protein extraction, histological analysis or stored in freezing medium. Fluorescence activated cell sorting (FACS) can be used to enrich for specific cell populations. This protocol provides a straightforward, standard approach for translational studies with highly complex, varied human specimens.

An Ex vivo Model to Study Hormone Action in the Human Breast

We have developed a novel ex vivo model to study hormone action in the human breast. It is based on tissue microstructures isolated from surgical breast tissue specimens which preserve tissue architecture, intercellular interactions, and paracrine signaling.

アン<em&gt;エクスビボ</emヒト乳癌でホルモンの作用を研究するために&gt;モデル

The study of hormone action in the human breast has been hampered by lack of adequate model systems. Upon in vitro culture, primary mammary epithelial ...

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 ...

Utilization of the Soft Agar Colony Formation Assay to Identify Inhibitors of Tumorigenicity in Breast Cancer Cells

Given the inherent difficulties in investigating the mechanisms of tumor progression in vivo, cell-based assays such as the soft agar colony formation assay (hereafter called soft agar assay), which measures the ability of cells to proliferate in semi-solid matrices, remain a hallmark of cancer research. A key advantage of this technique over conventional 2D monolayer or 3D spheroid cell culture assays is the close mimicry of the 3D cellular environment to that seen in vivo. Importantly, the soft agar assay also provides an ideal tool to rigorously test the effects of novel compounds or treatment conditions on cell proliferation and migration. Additionally, this assay enables the quantitative assessment of cell transformation potential within the context of genetic perturbations. We recently identified peptidylarginine deiminase 2 (PADI2) as a potential breast cancer biomarker and therapeutic target. Here we highlight the utility of the soft agar assay for preclinical anti-cancer studies by testing the effects of the PADI inhibitor, BB-Cl-amidine (BB-CLA), on the tumorigenicity of human ductal carcinoma in situ (MCF10DCIS) cells.

Here, we document the use of the soft agar colony formation assay to test the effects of a peptidylarginine deiminase (PADI) enzyme inhibitor, BB-Cl-amidine, on breast cancer tumorigenicity in vitro.

乳癌細胞における腫瘍形成の阻害剤を同定するための軟寒天コロニー形成アッセイの活用

Given the inherent difficulties in investigating the mechanisms of tumor progression in vivo, cell-based assays such as the soft agar colony formation ...