本研究の資金源：テルアビブ大学の技術移転会社、运动とシェバ·メディカルセンター（M. CA及びSI）、ICRFの共同ファンド - イスラエルのがん研究財団（M. CA。）とイスラエル科学財団（ SI）。

1。細胞培養の準備 MDA-MB-231細胞は、ATCC（ アメリカン·タイプ·カルチャー·コレクション ）から購入し、液体窒素中で保存した。 <ol><li>シード10 6 MDA-MB-231 Dulbeco改変イーグル培地（DMEM）、血清、1％L-グルタミン、及び1％Penstrep-アンホテリシンBのセルを許可し、10％の馬を含む10ミリリットル完全培地で92ミリメートル径シャーレ内の細胞約80-100％コンフルエントに増殖する。 </li><li>ディッシュから培地を取り外し、廃棄します。 </li><li> 0.25％血清のすべてのトレースを削除する（w / v）のトリプシン-EDTA溶液を用いて簡潔に細胞層を洗ってください。 </li><li>細胞層（通常は5〜15分以内）に分散するまで倒立顕微鏡により細胞を観察すると、皿トリプシン-EDTA溶液2.0 mlを加える。 </li><li> 18ミリリットル完全増殖培地を加え、穏やかにピペットで細胞を吸引。チューブに移します。 </li><li> 1,200 rpmで細胞懸濁液を遠心。 </li><li> CUの24ミリリットルで細胞ペレットを再懸濁メディアをlture。 </li><li> 35ミリガラスボトムディッシュ（25％コンフルエント程度）とインキュベーター内の場所（5％CO 2、37℃）に細胞懸濁液2 mlを加える。 </li><li> ソリューション： <ol><li> 10％FBS、1％抗生物質（100単位/ mlペニシリンG、100μg/mlの/ mlのストレプトマイシン、ペン連鎖球菌 - 両性溶液）及び2mMのL-グルタミンを含むDMEM：細胞増殖のための完全培地。 </li><li> 0.25％トリプシン-EDTAを含有するトリプシン-EDTA溶液。 </li></ol></li><li> 料理： 92ミリメートル直径ペトリ皿。 直径35mmのポリ-D-リジン被覆ガラス底培養皿。 </li></ol> 2。ライブ共焦点イメージングのための細胞の調製<ol><li>シード2×10部1で述べたように2ミリリットル完全培地中のガラス底培養皿に5 MDA-MB-231細胞。細胞培養は、60〜70％の合流を（シャーレ当たり3-4×10 5細胞程度）に達すると、トランスフェクションに進みます。 </li><li> 2つのプラスミドは、エンコーディングで細胞をトランスフェクト製造プロトコルに従って、リポソームトランスフェクション試薬ジェット-PIを使用して融合タンパク質γ-チューブリン-GFP（中心体の蛍光検出用）およびヒストン-RED（H2B-RED、染色体の蛍光検出用）をING。簡単に言えば、100μlの塩化ナトリウム（150mMの）とチューブに各プラスミドから2μgのを混在させる。第2のチューブに100μlの塩化ナトリウム（150mMの）とトランスフェクション試薬（100μL）を混合し、室温（RT）で5分間インキュベートする。その後、二つの溶液を組み合わせたミックス（軽度の渦を使用）とダウンスピン。室温で30分間インキュベートする。 </li><li>トランスフェクション混合物のインキュベーション中、PBSで1回細胞を洗浄しないサプリメントと暖かいDMEM 2mlの（37°C）で細胞培地を交換してください。 </li><li>穏やかにDMEMで細胞にトランスフェクション混合物を追加し、8時間（37℃、5％CO 2）インキュベーターにセルを返す。 </li><li> 24のためのインキュベーションの8時間後、2ミリリットル完全培地を含むDMEMを交換し、インキュベーターで細胞をインキュベートHR。 </li><li>トランスフェクション後24時間では、20μMPJ-34を含む2ミリリットル完全培地で細胞の培地を交換してください。 </li><li>さらに18時間（37℃、5％CO 2）、細胞をインキュベートする。 </li><li> 5％CO 2、37℃で細胞を維持するイメージングチャンバー内に少なくとも16時間の共焦点イメージングを生きるために件名のセル</li><li>並行して、以下のように蛍光顕微鏡を用いてトランスフェクション効率36時間後にトランスフェクションを調べます<ol><li>シード2 2ミリリットルの完全培地でウェル当たり1カバースリップを含む6ウェルプレートにおいて5×10 MDA-MB-231細胞。 </li><li>セクション2.2から2.5で述べたように細胞をトランスフェクトする。 </li><li> 36時間後にトランスフェクションは、冷メタノールでのインキュベーションによりカバースリップ上に取り付け、トランスフェクトされた細胞を固定：アセトン（1:1）ソリューション、7分、-20℃を</li><li>固定液を吸引除去し、化学フードで乾燥するように取り付け細胞とカバースリップしてみましょう。 </li><li> DAPIでゴールド退色試薬を延長し、目を聞かせて適用するeはカバースリップで6時間暗所で乾燥させる。 </li><li>蛍光顕微鏡下でスライドを調べ、細胞の総人口からトランスフェクションされた細胞（赤と緑の信号）（DAPIによるDNA染色）の割合を計算します。 100-200細胞がカウントされたとき、所望のトランスフェクション率は20〜40％程度である。 </li></ol></li></ol> 3。ライブ共焦点イメージングスキャナ設定の技術的パラメータ<ol><li> SCANMODE XYZT;ホール[エアリー] 1.00;ズーム3.5、解像度8ビット;レーザーDPSS 561 nmで、アルゴン、可視レーザー488nmの;レーザー彼/ Neの見える633 nmで、客観HCX PL APO CS 63X 1.40 OIL UV、開口数1.4;速度700 Hzのをスキャンし、屈折率1.52。 </li><li>画像3次元プレゼンテーションはIMARISイメージングソフトウェア7.0で調製した。 </li></ol> 4。固定した細胞における有糸分裂の共焦点イメージング<ol><li>シード2×10 5個のMDA-MB-231乳癌細胞（ATCC）、正常マウス胚線維芽細胞（MEF）、またはPARP1欠損ME2ミリリットル完全培地で6ウェルプレートにカバーガラス上のF（博士フランソワーズDantzerにより調製しPARP - / - ）。カバーガラスを2時間乾燥させ、6 - ウェルディッシュの各ウェルに入れ、滅菌DD水で洗浄することにより、以下の、96％エタノールで洗浄した。 </li><li>培地にPJ-34（10-30μM）を追加し、必要な期間（通常は最大96時間）のために、細胞をインキュベート。 </li><li> PBS（リン酸緩衝生理食塩水）で1回カバースリップを洗って、氷冷メタノール中でインキュベーションを用いて細胞を固定：アセトン（1:1）ソリューション、7分、-20℃ </li><li>固定液を吸引し、カバースリップ（この段階では、カバーガラスが数週間のために-20℃に保つことができる）化学フード中で乾燥させてください。 </li><li>細胞膜を透過性およびRTで1時間PBST中の10％NDS（通常ロバ血清）（ &#39;ブロッキング溶液&#39;）で細胞をブロックするPBST（PBS、0.1％のTween-20を補充）で1回カバースリップを洗浄する。 </li><li>プライマリーantibodiで透過固定された細胞をインキュベート室温で2時間、ES（スピンドルと中心体染色のため）。抗αチューブリン（1:250希釈）および抗-γチューブリン（1:200希釈）：抗体は、以下のようなブロッキング溶液で希釈する。一次抗体は、次のように適用されています：6ウェルプレートカバー（カバーは逆さまである）上の各カバースリップのためのブロッキング溶液中の抗体の混合物を100μl（ドロップ）を適用。優しく抗体ドロップは、ドロップに直面して播種した細胞上にカバースリップを置く。室温で2時間撹拌抗体と対向するカバースリップをインキュベートする。 </li><li>ウェルに戻ってカバーガラスを置き、PBSTで細胞を3回洗浄する。その後、蛍光二次抗体を用いてカバーガラス上の細胞を標識するために4.6で説明したのと同じ手順を使用します。暗闇の中で、1時間、RT用の二次抗体を用いてカバーガラス上の細胞をインキュベートする。抗体は、以下のようブロッキング溶液で希釈する。;とアレクサフルーア568（1:1,000希釈アレクサフルーア488（緑1:1,000希釈）;赤）。 </li><li>使用してカバースリップはDAPI（染色体染色用）とGold退色防止試薬を延長し、乾燥させる暗闇の中で室温で一晩インキュベートマウント。 </li><li>共焦点顕微鏡によるカバースリップを調べます。 </li></ol> 5。 ATP生産により測定し、細胞の生存率 ATP産生は、発光ATP検出アッセイキットにより測定される。 <ol><li>各ウェルに800μlの培地中の種子96ウェルプレート中の細胞、約20,000のセルを。三ブランクウェルは、媒体のバックグラウンド発光を決定するために使用されるべきである。 </li><li> 100μMに約10μMからATP標準希釈系列を用意し、氷上に保つ。 </li><li>各ウェルに洗剤液50μlを加え、オービタルシェーカー、700回転で5分間プレートを振る。 </li><li>キット内の &#39;基質緩衝&#39; 5mlの &#39;凍結乾燥基質&#39;の再構成する各バイアル。 </li><li>ウェルに再構成された基質溶液50μlを加え、振とうオービタルシェーカー、700回転で5分間プレート。 </li><li> 10分間、暗闇の中でプレートを保管してください。 </li><li> ELISAマイクロプレートリーダーで各ウェルのluminiscenceを測定します。 </li></ol>

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著者は、彼らが競合する経済的利益を持っていないことを宣言します。

ライブ共焦点イメージングは、有糸分裂（ 図3と補足情報）の間にライブマルチ中心体細胞ではPJ-34の細胞毒性効果をリアルタイムに資料を提供した。これは、PJ-34 5-9で細胞分裂異常細胞死の誘導を示唆し、余分な中心体デクラスタリングと細胞死にヒト癌細胞におけるPJ-34の細胞毒性を帰初のライブドキュメントだった。対照的に、超正規の中心体の双方向の焦点クラス...

PJ-34を含むフェナントレン由来PARP1阻害剤は、ストレス条件下でのエネルギ消費PARP1媒介DNA修復（脳卒中または心筋梗塞）1によって誘導されるアポトーシス細胞死からの静止細胞を保護するように設計された。しかし、最近我々はPJ-34のことを発見し、その誘導PARP1阻害よりも倍高い濃度で、独占的にヒト癌細胞2,3における細胞死を引き起こす可能性があります。細胞の増殖であったより迅速に、細胞をより効率的に根絶であった。 PJ-34の細胞傷害活性は、有糸分裂2余分中心体デクラスタリングに起因するものであった。多くのヒト癌細胞の港4,5 multicentrosomes。静止細胞を損なうか、またはいくつかの良性の増殖細胞が有糸分裂で2中心体を保有することなく、20μMPJ-34と港過剰中心体は、効率的に72〜96時間以内にこれらの細胞を根絶ヒト乳癌細胞MDA-MB-231、のインキュベーション<suP&gt; 2,3。良性細胞は、ヒト胸腺から調製されたMCF-10ヒト乳腺上皮細胞、ヒト内皮細胞（HUVEC）とプライマリ間葉細胞を含んでいた。これらの細胞は、PJ-34の細胞傷害活性に耐性であった。 PJ-34は、96時間のインキュベーションの間に彼らの細胞周期に干渉したり、中心体と双方向の焦点紡錘体形成2,3に影響を及ぼさなかった。 バイポーラ中心体アセンブリは、有糸分裂4,5における双極紡錘体形成のために重要です。したがって、以上の2中心体を持つ細胞は、2つの極4-9で彼らの余分中心体をクラスタリング、ほとんど理解していない分子機構を開発しました。彼らの中心体の双極アセンブリの失敗は逮捕はG2 / Mの逮捕で、細胞周期、細胞死に至るが4,5分裂の失敗に起因することが多歪んスピンドルと異常な染色体の分離を引き起こす可能性があります。余分中心体デクラスタリングの基礎となる分子メカニズムは集中的に調査している&lt;&gt; 10（商標）。健康な組織の5,10を温存しながら、この死のメカニズムを理解することは、癌細胞の排他的な根絶が可能になります。 従って、細胞分裂異常細胞死を活性化する化合物は、共焦点画像は、超中心体のクラスタリングに影響する分子を同定するために使用することができることを示唆しているヒト固形cancers.Our結果の広い範囲で効率的であり得る選択的癌療法の新たなモードを提供有糸分裂2,3、薬剤候補をターゲットに、これらの化合物の癌をレンダリング。 我々は、固定やライブヒト癌細胞（有糸分裂で余分中心体の高い発生で）対正常細胞をスキャンすることによってナントPJ-34の細胞傷害活性を文書化している。ヒト癌細胞においてPJ-34の細胞傷害活性を同定するために用いられる撮像手順のステップバイステップの説明が以下に含まれる。

<table border="1">
	<tbody>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>REAGENTS </td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Invitrogen (GIBCO)</td>
			<td>41965</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>FBS (Fetal bovine Serum)</td>
			<td>Invitrogen (GIBCO)</td>
			<td>12657</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Pen-Strep-Ampho solution</td>
			<td>Biological Industries, Israel</td>
			<td>03-033-1B</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>L-glutamine</td>
			<td>Invitrogen (GIBCO)</td>
			<td>25030-024</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>0.25% Tripsin-EDTA</td>
			<td>Invitrogen (GIBCO)</td>
			<td>25200</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>92 mm Petri dishes</td>
			<td>Nunc,Thermo scientific</td>
			<td>150350</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>35 mm poly-D-lysine coated glass bottom culture dishes</td>
			<td>MatTek Corporation, USA</td>
			<td>P35GC-0-14-C</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Luminescent ATP detection assay kit</td>
			<td>Abcam</td>
			<td>ab113849</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>NDS (Normal Donkey Serum)</td>
			<td>Jackson ImmunoResearch</td>
			<td>017-000-121</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Anti &alpha;-tubulin antibody</td>
			<td>Sigma</td>
			<td>T9026</td>
			<td>1:250 dilution (IF)</td>
		</tr>
		<tr>
			<td>Anti &gamma;-tubulin antibody</td>
			<td>Sigma</td>
			<td>T5192</td>
			<td>1:200 dilution (IF)</td>
		</tr>
		<tr>
			<td>Alexa Fluor 488 Goat Anti-Mouse IgG</td>
			<td>Invitrogen</td>
			<td>A-11017</td>
			<td>1:1,000 dilution (IF)</td>
		</tr>
		<tr>
			<td>Alexa Fluor 568 Donkey Anti-Rabbit IgG</td>
			<td>Invitrogen</td>
			<td>A-10042</td>
			<td>1:1,000 dilution (IF)</td>
		</tr>
		<tr>
			<td>ProLong Gold antifade reagent with DAPI (mounting)</td>
			<td>Invitrogen</td>
			<td>P36935</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>JetPEI (liposomal transfection reagent )</td>
			<td>Polyplus</td>
			<td>101-10</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>EQUIPMENT</td>
		</tr>
		<tr>
			<td>Confocal microscope</td>
			<td>Leica (Mannheim, Germany)</td>
			<td>TCS SP5II</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

cell death associated with abnormal mitosis observed by confocal imaging in live cancer cells

強力PARP1阻害剤として作用しフェナントレン誘導体は有糸分裂におけるマルチ中心体ヒト癌細胞における過剰中心体の双方向の焦点クラスタリングを防いだ。ナントPJ-34は、最も強力な分子であった。余分中心体のデクラスタリングは、マルチ中心体細胞では分裂障害と細胞死を引き起こす。ほとんどの固体ヒトの癌は超中心体の高い発生を持っている。 PJ-34の活性はライブヒト乳癌MDA-MB-231中心体に高度に豊富である蛍光γ-チューブリン、および、蛍光ヒストンH2Bのプレゼントにコードするベクターでトランスフェクトされた細胞の共焦点イメージングによってリアルタイムで文書化されました染色。異常な染色体の手配やdeclustered中心体を表すデクラスタ化γ-チューブリン病巣はPJ-34で処理した後、トランスフェクトMDA-MB-231細胞で検出された。 2紡錘体極でアンクラスタ余分中心体は、細胞死に先行。これらの結果は、foのリンクさR初めて余分中心体細胞分裂におけるデクラスタリング、および細胞死に至る分裂不全のヒト癌細胞におけるPJ-34の最近検出された排他的な細胞傷害活性。 2中心体と双方向の焦点スピンドルと有糸分裂を受けて正常な細胞を損なうことなく、多中心体ともっぱら根絶癌細胞PJ-34、固定した細胞の共焦点イメージングにより観察前の調査結果による。 PJ-34のこの細胞傷害活性は、他の強力なPARP1阻害剤によって共有されていなかった、とPARP1阻害のその独立性を示唆し、PARP1欠損MEF宿すextracentrosomesで観察された。共焦点イメージングを生きるには、有糸分裂中の細胞を根絶する新しい分子を同定するための有用なツールを提供した。

PJ-34安定した水溶性のフェナント1（ 図1）である。我々の以前の結果は、PJ-34で処理した固定マルチ中心体癌細胞のいくつかのタイプに細胞死および非クラスタ化された余分中心体を明らかにした。対照的に、正常な増殖細胞を2,3損なわれていなかった。中心体は、2つの固定余分中心体細胞でcentrine1とγ-チューブリンに対する抗体で二重標識により同定?...

Watch this Scientific Journal Video about Cell Death Associated with Abnormal Mitosis Observed by Confocal Imaging in Live Cancer Cells at JoVE.com

Cell Death Associated with Abnormal Mitosis Observed by Confocal Imaging in Live Cancer Cells

有糸分裂を受けている癌細胞でナントPJ-34の細胞傷害活性は、ライブ共焦点イメージングによりリアルタイムで文書化されました。 PJ-34根絶ヒト乳癌MDA-MB-231細胞分裂に余分中心体を保有する細胞。通常の双方向の焦点有糸分裂とは異なり、余分中心体は、PJ-34の存在下で2紡錘体極にクラスタ化されていなかった。

ライブ癌細胞で共焦点イメージングで観測異常有糸分裂と関連付けられる細胞死

Phenanthrene derivatives acting as potent PARP1 inhibitors prevented the bi-focal clustering of supernumerary centrosomes in multi-centrosomal human ...

cell-death-associated-with-abnormal-mitosis-observed-confocal-imaging

Research

JoVE Journal

Medicine

14.9K ビュー.  Sheba Medical Center. 有糸分裂を受けている癌細胞でナントPJ-34の細胞傷害活性は、ライブ共焦点イメージングによりリアルタイムで文書化されました。 PJ-34根絶ヒト乳癌MDA-MB-231細胞分裂に余分中心体を保有する細胞。通常の双方向の焦点有糸分裂とは異なり、余分中心体は、PJ-34の存在下で2紡錘体極にクラスタ化されていなかった。

ビデオ: Cell Death Associated with Abnormal Mitosis Observed by Confocal Imaging in Live Cancer Cells

High-Efficiency Transduction of Liver Cancer Cells by Recombinant Adeno-Associated Virus Serotype 3 Vectors

Recombinant vectors based on a non-pathogenic human parvovirus, the adeno-associated virus 2 (AAV2) have been developed, and are currently in use in a number of gene therapy clinical trials. More recently, a number of additional AAV serotypes have also been isolated, which have been shown to exhibit selective tissue-tropism in various small and large animal models1. Of the 10 most commonly used AAV serotypes, AAV3 is by far the least efficient in transducing cells and tissues in vitro as well as in vivo.
However, in our recently published studies, we have documented that AAV3 vectors transduce human liver cancer - hepatoblastoma (HB) and hepatocellular carcinoma (HCC) - cell lines extremely efficiently because AAV3 utilizes human hepatocyte growth factor receptor as a cellular co-receptor for binding and entry in these cells2,3.
In this article, we describe the steps required to achieve high-efficiency transduction of human liver cancer cells by recombinant AAV3 vectors carrying a reporter gene. The use of recombinant AAV3 vectors carrying a therapeutic gene may eventually lead to the potential gene therapy of liver cancers in humans.

In this article, we describe the identification of the adeno-associated virus serotype 3 (AAV3) as the most efficient vector for targeting human liver cancer cells.

組換えアデノ随伴ウイルスの血清型3つのベクトルによる肝癌細胞の高効率形質導入

Recombinant vectors based on a non-pathogenic human parvovirus, the adeno-associated virus 2 (AAV2) have been developed, and are currently in use in a ...

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 ...

Evaluation of Cancer Stem Cell Migration Using Compartmentalizing Microfluidic Devices and Live Cell Imaging

In the last 40 years, the United States invested over 200 billion dollars on cancer research, resulting in only a 5% decrease in death rate. A major obstacle for improving patient outcomes is the poor understanding of mechanisms underlying cellular migration associated with aggressive cancer cell invasion, metastasis and therapeutic resistance1. Glioblastoma Multiforme (GBM), the most prevalent primary malignant adult brain tumor2, exemplifies this difficulty. Despite standard surgery, radiation and chemotherapies, patient median survival is only fifteen months, due to aggressive GBM infiltration into adjacent brain and rapid cancer recurrence2. The interactions of aberrant cell migratory mechanisms and the tumor microenvironment likely differentiate cancer from normal cells3. Therefore, improving therapeutic approaches for GBM require a better understanding of cancer cell migration mechanisms. Recent work suggests that a small subpopulation of cells within GBM, the brain tumor stem cell (BTSC), may be responsible for therapeutic resistance and recurrence. Mechanisms underlying BTSC migratory capacity are only starting to be characterized1,4.
Due to a limitation in visual inspection and geometrical manipulation, conventional migration assays5 are restricted to quantifying overall cell populations. In contrast, microfluidic devices permit single cell analysis because of compatibility with modern microscopy and control over micro-environment6-9.
We present a method for detailed characterization of BTSC migration using compartmentalizing microfluidic devices. These PDMS-made devices cast the tissue culture environment into three connected compartments: seeding chamber, receiving chamber and bridging microchannels. We tailored the device such that both chambers hold sufficient media to support viable BTSC for 4-5 days without media exchange. Highly mobile BTSCs initially introduced into the seeding chamber are isolated after migration though bridging microchannels to the parallel receiving chamber. This migration simulates cancer cellular spread through the interstitial spaces of the brain. The phase live images of cell morphology during migration are recorded over several days. Highly migratory BTSC can therefore be isolated, recultured, and analyzed further. 
Compartmentalizing microfluidics can be a versatile platform to study the migratory behavior of BTSCs and other cancer stem cells. By combining gradient generators, fluid handling, micro-electrodes and other microfluidic modules, these devices can also be used for drug screening and disease diagnosis6. Isolation of an aggressive subpopulation of migratory cells will enable studies of underlying molecular mechanisms.

A compartmentalizing microfluidic device for investigating cancer stem cell migration is described. This novel platform creates a viable cellular microenvironment and enables microscopic visualization of live cell locomotion. Highly motile cancer cells are isolated to study molecular mechanisms of aggressive infiltration, potentially leading to more effective future therapies.

区分するマイクロ流体デバイスとライブセルイメージングを使用した癌幹細胞の移行の評価

In the last 40 years, the United States invested over 200 billion dollars on cancer research, resulting in only a 5% decrease in death rate. A major ...

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

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

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

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

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

Modeling Colitis-Associated Cancer with Azoxymethane (AOM) and Dextran Sulfate Sodium (DSS)

Individuals with inflammatory bowel disease (IBD), such as Crohn's disease (CD) or ulcerative colitis (UC) are at increased risk of developing colorectal cancer (CRC) over healthy individuals. This risk is proportional to the duration and extent of disease, with a cumulative incidence as high as 30% in individuals with longstanding UC with widespread colonic involvement.1 Colonic dysplasia in IBD and colitis associated cancer (CAC) are believed to develop as a result of repeated cycles of epithelial cell injury and repair while these cells are bathed in a chronic inflammatory cytokine milieu.2 While spontaneous and colitis-associated cancers share the quality of being adenocarcinomas, the sequence of underlying molecular events is believed to be different.3 This distinction argues the need for specific animal models of CAC.
Several mouse models currently exist for the study of CAC. Dextran sulfate sodium (DSS), an agent with direct toxic effects on the colonic epithelium, can be administered in drinking water to mice in multiple cycles to create a chronic inflammatory state. With sufficient duration, some of these mice will develop tumors.4 Tumor development is hastened in this model if administered in a pro-carcinogenic setting. These include mice with genetic mutations in tumorigenesis pathways (APC, p53, Msh2), as well as mice pre-treated with genotoxic agents (azoxymethane [AOM], 1,2-dimethylhydrazine [DMH]).5 
The combination of DSS with AOM as a model for colitis associated cancer has gained popularity for its reproducibility, potency, low price, and ease of use. Though they have a shared mechanism, AOM has been found to be more potent and stable in solution than DMH. While tumor development in other models generally requires several months, mice injected with AOM and subsequently treated with DSS develop adequate tumors in as little as 7-10 weeks.6, 7 Finally, AOM and DSS can be administered to mice of any genetic background (knock out, transgenic, etc.) without cross-breeding to a specific tumorigenic strain. Here, we demonstrate a protocol for inflammation-driven colonic tumorigenesis in mice utilizing a single injection of AOM followed by three seven-day cycles of DSS over a 10 week period. This model induces tumors with histological and molecular changes closely resembling those occurring in human CAC and provides a highly valuable model for the study of oncogenesis and chemoprevention in this disease.8

Modeling Colitis-Associated Cancer with Azoxymethane AOM and Dextran Sulfate Sodium DSS

We demonstrate a protocol in which administration of the genotoxic agent azoxymethane (AOM) followed by three cycles of the pro-inflammatory agent dextran sulfate sodium (DSS) rapidly and consistently generates colon tumors in mice with morphologic and molecular similarities to those seen in human colitis-associated cancer.

アゾキシメタン（AOM）とデキストラン硫酸ナトリウム（DSS）によるモデリング大腸炎関連癌

Individuals with inflammatory bowel disease (IBD), such as Crohn's disease (CD) or ulcerative colitis (UC) are at increased risk of developing colorectal ...

Isolation, Characterization and Comparative Differentiation of Human Dental Pulp Stem Cells Derived from Permanent Teeth by Using Two Different Methods

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human pulp-derived stem cells (hDPSCs) possess high proliferation potential with multi-lineage differentiation capacity compare to the ordinary source of adult stem cells1-8; therefore, hDPSCs could be the good candidates for autologous transplantation in tissue engineering and regenerative medicine. Along with these benefits, possessing the mesenchymal stem cells (MSC) features, such as immunolodulatory effect, make hDPSCs more valuable, even in the case of allograft transplantation6,9,10. Therefore, the primary step for using this source of stem cells is to select the best protocol for isolating hDPSCs from pulp tissue. In order to achieve this goal, it is crucial to investigate the effect of various isolation conditions on different cellular behaviors, such as their common surface markers &amp; also their differentiation capacity.
Thus, here we separate human pulp tissue from impacted third molar teeth, and then used both existing protocols based on literature, for isolating hDPSCs,11-13 i.e. enzymatic dissociation of pulp tissue (DPSC-ED) or outgrowth from tissue explants (DPSC-OG). In this regards, we tried to facilitate the isolation methods by using dental diamond disk. Then, these cells characterized in terms of stromal-associated Markers (CD73, CD90, CD105 &amp; CD44), hematopoietic/endothelial Markers (CD34, CD45 &amp; CD11b), perivascular marker, like CD146 and also STRO-1. Afterwards, these two protocols were compared based on the differentiation potency into odontoblasts by both quantitative polymerase chain reaction (QPCR) &amp; Alizarin Red Staining. QPCR were used for the assessment of the expression of the mineralization-related genes (alkaline phosphatase; ALP, matrix extracellular phosphoglycoprotein; MEPE &amp; dentin sialophosphoprotein; DSPP).14

The method described isolation and characterization of human Dental Pulp Stem Cells (hDPSCs) by using either enzymatic dissociation of pulp (DPSC-ED) or direct outgrowth of stem cells from pulp tissue explants (DPSC-OG). Then followed by in vitro comparative differentiation of both types of hDPSCs into odontoblasts.

単離、特徴付けと二つの異なる方法を使用して永久歯に由来するヒト歯髄幹細胞の分化の比較

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human ...

Live Imaging of Drug Responses in the Tumor Microenvironment in Mouse Models of Breast Cancer

The tumor microenvironment plays a pivotal role in tumor initiation, progression, metastasis, and the response to anti-cancer therapies. Three-dimensional co-culture systems are frequently used to explicate tumor-stroma interactions, including their role in drug responses. However, many of the interactions that occur in vivo in the intact microenvironment cannot be completely replicated in these in vitro settings. Thus, direct visualization of these processes in real-time has become an important tool in understanding tumor responses to therapies and identifying the interactions between cancer cells and the stroma that can influence these responses. Here we provide a method for using spinning disk confocal microscopy of live, anesthetized mice to directly observe drug distribution, cancer cell responses and changes in tumor-stroma interactions following administration of systemic therapy in breast cancer models. We describe procedures for labeling different tumor components, treatment of animals for observing therapeutic responses, and the surgical procedure for exposing tumor tissues for imaging up to 40 hours. The results obtained from this protocol are time-lapse movies, in which such processes as drug infiltration, cancer cell death and stromal cell migration can be evaluated using image analysis software.

We describe a method for imaging response to anti-cancer treatment in vivo and at single cell resolution.

乳癌のマウスモデルにおいて腫瘍微小環境における薬剤応答のイメージングを生きる

The tumor microenvironment plays a pivotal role in tumor initiation, progression, metastasis, and the response to anti-cancer therapies. Three-dimensional ...

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  ...

Assessing Phagocytic Clearance of Cell Death in Experimental Stroke by Ligatable Fluorescent Probes

We describe a new histochemical approach for visualization of phagocytic clearance in focal brain ischemia. The approach permits the study of elimination of dead cells in stroke by waste-management phagocytes of any cellular lineage. Although numerous cells of different origins that are capable of phagocytosis are present in ischemic brain, only part of them actively engulf and digest cell corpses. The selective visualization, quantification and analysis of such active phagocytic waste-management are helpful in assessing brain response to ischemia. Efficient cell death clearance is important for brain recovery from ischemic injury, as it opens the way for the subsequent regenerative processes. The failure to clean the corpses would result in a toxic reaction caused by non-degraded DNA and proteins. The described procedure uses fluorescent probes selectively ligated by a viral topoisomerase to characteristic DNA breaks produced in all phagocytes during engulfment and digestion of cells irreversibly damaged by ischemia. The method is a new tool for the investigation of brain reaction to ischemic injury.

We present a new fluorescence technique for selective in situ labeling of active phagocytic cells, which clear off cell corpses in stroke. The approach is important for assessing brain reaction to ischemia because only a small proportion of phagocytes present in ischemic brain participate in clearance of cell death.

連結可能な蛍光プローブによる実験脳卒中における細胞死の貪食クリアランスを評価する

We describe a new histochemical approach for visualization of phagocytic clearance in focal brain ischemia. The approach permits the study of elimination ...

Profiling of Estrogen-regulated MicroRNAs in Breast Cancer Cells

Estrogen plays vital roles in mammary gland development and breast cancer progression. It mediates its function by binding to and activating the estrogen receptors (ERs), ER&#945;, and ER&#946;. ER&#945; is frequently upregulated in breast cancer and drives the proliferation of breast cancer cells. The ERs function as transcription factors and regulate gene expression. Whereas ER&#945;&#39;s regulation of protein-coding genes is well established, its regulation of noncoding microRNA (miRNA) is less explored. miRNAs play a major role in the post-transcriptional regulation of genes, inhibiting their translation or degrading their mRNA. miRNAs can function as oncogenes or tumor suppressors&#160;and are also promising biomarkers. Among the miRNA assays available, microarray and quantitative real-time polymerase chain reaction (qPCR) have been extensively used to detect and quantify miRNA levels. To identify miRNAs regulated by estrogen signaling in breast cancer, their expression in ER&#945;-positive breast cancer cell lines were compared before and after estrogen-activation using both the &#181;Paraflo-microfluidic microarrays and Dual Labeled Probes-low density arrays. Results were validated using specific qPCR assays, applying both Cyanine dye-based and Dual Labeled Probes-based chemistry. Furthermore, a time-point assay was used to identify regulations over time. Advantages of the miRNA assay approach used in this study is that it enables a fast screening of mature miRNA regulations in numerous samples, even with limited sample amounts. The layout, including the specific conditions for cell culture and estrogen treatment, biological and technical replicates, and large-scale screening followed by in-depth confirmations using separate techniques, ensures a robust detection of miRNA regulations,&#160;and eliminates false positives and other artifacts. However, mutated or unknown miRNAs, or regulations at the primary and precursor transcript level, will not be detected. The method presented here represents a thorough investigation of estrogen-mediated miRNA regulation.

Molecular signaling through both estrogen and microRNAs are critical in breast cancer development and growth. Estrogen activates the estrogen receptors, which are transcription factors. Many transcription factors can regulate the expression of microRNAs, and estrogen-regulated microRNAs can be profiled using different large-scale techniques.

乳癌細胞におけるエストロゲン調節マイクロRNAのプロファイリング

Estrogen plays vital roles in mammary gland development and breast cancer progression. It mediates its function by binding to and activating the estrogen ...