MBは、MRCの交わりから助成金によってサポートされています。 KPとAHMはウェセックス医学研究および癌研究イギリス/ RCS（イングランド）（C28503/A10013）からの助成金によってサポートされています。我々はサウサンプトン組織化学研究ユニットの大学の支援に感謝しています。

結腸外植片からの初代線維芽細胞培養の1。設立<ol><li>外科手術室から直接、正常なヒト結腸粘膜組織のサンプルを得るし、PBS 7-10中に一時停止し、100単位/ mlペニシリン、100μg/ mlのストレプトマイシン、および0.25μg/ mlのファンギゾンを補った。 </li><li>研究室では、10cmの組織培養皿の中央に試料を置き、PBS /ペンストレプトマイシン/ファンギゾンで3回洗浄する。最後の洗浄の後に吸引しないでください。 </li><li>滅菌ピンセットやメスで、（約2mm）組織を小片にカットし、12ウェルプレート中のメスの刃を使用して描画十字の中心にそれぞれの作品を配置します。それが接着することができるように組織の上そっとメスを揺する。 </li><li> 750μlの培養中の検体は、一次線維芽細胞増殖培地submeべき（DMEM、20％ウシ胎児血清（FCS）、100 U / mlペニシリン、100μg/ mlのストレプトマイシン、および292μg/ mlのL-グルタミンを補充）フロートするためにそれを可能にすることなく、組織をRGE。 2日ごとに再給紙を除き、次の5日間にわたりプレートを邪魔しないようにします。 </li><li>次の5日間に線維芽細胞が徐々に組織の端から外に成長を開始します。約7日後に、これはより見えるようになります。後約3〜4週間、内外の成長に十分な線維芽細胞が存在する場合、各ウェルに750μlのトリプシンを追加します。細胞を、T-25組織培養フラスコに、プール、転移（5〜10分かかることがあります）取り外された後。 </li></ol> 2。器官の準備線維芽細胞を含浸させ、器官ゲルの調製： <ol><li> 10％FCSおよび292μg/ mlのL-グルタミンを補充したDMEM中の器官当たり5×10 5個の細胞を含む線維芽細胞懸濁液を調製する。 </li><li>以下の比率で、氷上器官ゲルを構成している。 <ul><li>ラットテールコラーゲンの7ボリューム：マトリゲルミックス（1:1） </li><li>フィルタリングさ10倍のDMEM 1ボリューム</li><li> FCSの1の体積</li><li> 5×10 5個の線維芽細胞を含む線維芽細胞懸濁液1容量 9ゲル（ゲルあたり1ミリリットル）のために10ミリリットルを用意し、気泡を避けるために、穏やかに混合。 </li></ul></li><li> 24ウェルプレートの各ウェルに1 mlを添加し、37℃で1時間インキュベート ゲルが設定できるように加湿した雰囲気の中でCと°。 1時間後、ゲルの上に1ミリリットルのDMEM（10％FCS）/供給過剰を追加し、一晩培養器に戻る。 </li><li>次の日、ゲルおよび5×10 5のCRC上皮細胞を含む培地のプレート1ミリリットルの上から吸引媒体。 </li></ol>ゲルコーティングされたナイロンシートの製造： <ol start="5"><li>事前に1.5センチメートル×1.5センチメートルを測定無菌、オートクレーブナイロンシートを準備します。 </li><li>滅菌ピンセットを用いて、10cmの培養皿（通常は4ナイロンシートを皿ごとに収容することができる）ナイロンシートの必要な数を配置する。 </li><li>次の順序で氷上ゲルを構成する（250μlの総ボリュームナイロンシートごとに必要となる）。 <ul><li>ラットテールコラーゲンの7容積</li><li>フィルタリングさ10倍のDMEM 1ボリューム</li><li> FCSの1の体積</li><li> 1体積のDMEM、10％FCS /グラット</li></ul></li><li>溶液が黄色の場合は、溶液がピンク色に変わるまで50μlのアリコートで0.1MのNaOHを加えることによって中和する。 </li><li>各ナイロンシートに、この溶液の250μlを加え、37℃でインキュベート °Cゲルが設定できるように30分間、5％CO 2の加湿雰囲気中。 </li><li> PBS中の1％グルタルアルデヒド溶液を構成しています。ゲルがセットされた後、各10cmの培養皿に10ミリリットルを加え、1時間4℃でインキュベートする。 </li><li>ウォッシュナイロンシートはPBSおよびDMEM中で1X（10％FCS）/グルタミン酸で3倍、4℃で、DMEM / Gluの中で一晩インキュベート</li></ol>侵入用スチールグリッド上に器官ゲルを上げる。 <ol start="12"><li>三脚の形成にステンレス鋼板を折り曲げて足場グリッドを事前に準備する。使用前にオートクレーブ。</li><li> 6ウェルプレートの各ウェルにスチールグリッドを配置し、エタノールで滅菌ピンセットを使用してください。 </li><li>各グリッド上に、コラーゲン側最上でゲルコーティングされたナイロンシートを置きます。 </li><li>慎重にエタノールで滅菌スパチュラを用いて上げたナイロンシートを24ウェルプレートからの器官のゲルを移す。 </li><li>ナイロンシートが媒体に接触しているが、水没しないまで、10％FCSを補充したDMEM、10％FCS /ウェルで供給過剰を埋める。媒体は器官ゲルに接触していないことを確認してください。 </li><li> 2日毎に培地を交換し、5％CO 2の加湿雰囲気中、37℃で14日間インキュベートする。 </li></ol> 3。器官固定<ol><li>井戸からナイロンシートを含む全器官を取り出して、ラップフィルム上に置きます。 </li><li>器官ときれいな使い捨てメスを用いてナイロンシートを二等分し、室温で24時間、ホルムアルデヒド中で両方の半分を修正。 </li><li> 70％のイーサンとホルムアルデヒドを交換してくださいオール24時間後と前に、パラフィンに包埋切片、および染色を一晩のままにしておきます。 </li></ol>侵略的マージンの4。レーザーマイクロダイセクション<ol><li>膜上10μmの厚さにセクションでは、スライドをマウントした。 </li><li> 1分間のキシレンを適用することにより脱パラフィン切片、次いでキシレンを除去し、さらに1分間、75％エタノールで固定する。 </li><li> 1分間0.125％クレシルバイオレット溶液でエタノールと汚れを取り除きます。クレバイオレットは、上皮細胞が強調表示され、間質から簡単に区別することを可能にする。 </li><li>クレシルバイオレットを除去し、100％エタノールでリンスする前にさらに1分間100％エタノールを加える。 30分間空気乾燥スライドを可能にします。 </li><li>選択したプラットフォーム（ 例えばライカASシステム）を用いたレーザーマイクロダイセクションを実施しています。 </li><li>顕微鏡ステージ上に下向き顕微解剖するために染色部をガラススライドを置きます。 </li><li>回収カセットに0.5ミリリットルマイクロチューブをマウントし、50μlを加える顕微解剖組織収集先となるキャップの細胞溶解緩衝液（ 例えば、DNA、RNAまたはタンパク質溶解緩衝液）。 </li><li>直視下、関心のある組織を同定するためにジョイスティックを使用して、ソフトウェア·インタフェースを使用して、腫瘍の浸潤先端部での顕微解剖するセルを強調表示する、染色された有機部に注釈を付ける。 </li><li>強調表示されたセクションを切り出し、マイクロチューブのキャップ内に顕微解剖した組織を推進する必要があり、どちらも、発射するレーザーを指示します。 </li><li>一旦十分な材料が収集され、収集カセットを取り出すマイクロ遠心チューブを閉じ、チューブの底に溶解緩衝液を描画するために穏やかに回す。 </li><li>マイクロダイセクションが完了するまで氷上にサンプルを置きます。適切な方法による処理サンプルは、目的の分析物を抽出する。 </li></ol>

<ol>
	<li>Nystrom, M. L., Thomas, G. J., Stone, M., Mackenzie, I. C., Hart, I. R., Marshall, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+quantitative+method+to+analyse+tumour+cell+invasion+in+organotypic+culture.">Development of a quantitative method to analyse tumour cell invasion in organotypic culture.</a> J Pathol. 205 (4), 468-475 (2005).</li><li>Zhang, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=miR-153+supports+colorectal+cancer+progression+via+pleiotropic+effects+that+enhance+invasion+and+chemotherapeutic+resistance.">miR-153 supports colorectal cancer progression via pleiotropic effects that enhance invasion and chemotherapeutic resistance.</a> Cancer Res. , (2013).</li><li>Bullock, M. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pleiotropic+actions+of+miR-21+highlight+the+critical+role+of+deregulated+stromal+microRNAs+during+colorectal+cancer+progression.">Pleiotropic actions of miR-21 highlight the critical role of deregulated stromal microRNAs during colorectal cancer progression.</a> Cell Death Dis. 20 (4), (2013).</li><li>Jenei, V., Nystrom, M. L., Thomas, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+invasion+in+an+organotypic+model.">Measuring invasion in an organotypic model.</a> Methods Mol Biol. 769, 223-232 (2011).</li><li>De Wever, O., Demetter, P., Mareel, M., Bracke, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stromal+myofibroblasts+are+drivers+of+invasive+cancer+growth.">Stromal myofibroblasts are drivers of invasive cancer growth.</a> Int J Cancer. 123 (10), 2229-2238 (2008).</li><li>Fries, K. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+of+fibroblast+heterogeneity+and+the+role+of+fibroblast+subpopulations+in+fibrosis.">Evidence of fibroblast heterogeneity and the role of fibroblast subpopulations in fibrosis.</a> Clin Immunol Immunopathol. 72 (3), 283-292 (1994).</li><li>Moutasim, K. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Betel-derived+alkaloid+up-regulates+keratinocyte+alphavbeta6+integrin+expression+and+promotes+oral+submucous+fibrosis.">Betel-derived alkaloid up-regulates keratinocyte alphavbeta6 integrin expression and promotes oral submucous fibrosis.</a> J Pathol. 223 (3), 366-377 (2011).</li><li>Winter, J., Jung, S., Keller, S., Gregory, R. I., Diederichs, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Many+roads+to+maturity:+microRNA+biogenesis+pathways+and+their+regulation.">Many roads to maturity: microRNA biogenesis pathways and their regulation.</a> Nat Cell Biol. 11 (3), 228-234 (2009).</li><li>Li, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+miRNA+expression+patterns+using+total+RNA+extracted+from+matched+samples+of+formalin-fixed+paraffin-embedded+(FFPE)+cells+and+snap+frozen+cells.">Comparison of miRNA expression patterns using total RNA extracted from matched samples of formalin-fixed paraffin-embedded (FFPE) cells and snap frozen cells.</a> BMC Biotechnol. 7 (36), (2007).</li><li>Szafranska, A. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accurate+molecular+characterization+of+formalin-fixed,+paraffin-embedded+tissues+by+microRNA+expression+profiling.">Accurate molecular characterization of formalin-fixed, paraffin-embedded tissues by microRNA expression profiling.</a> J Mol Diagn. 10 (5), 415-423 (2008).</li></ol>

著者らは、利害の相反を開示していない。

ここでは、特に上皮細胞および間質細胞の3次元共培養構築物における転移性進行の最も初期の段階で癌関連間質に侵入する能力を獲得した腫瘍細胞を分離し、特性化する方法について説明します。 ex vivoでのヒト結腸繊維芽細胞を含有する合成基質と並置CRC上皮細胞からなる共培養モデルは3次元でCRC浸潤を研究するために使用した。 インビボ条件の...

器官型共培養物は、必須の細胞外マトリックス成 ​​分1〜3を含むコラーゲンゲルにおける悪性上皮細胞および間質細胞を並置することによって3次元での生体内腫瘍微小環境を再構築するのを助ける組織工学モデルである。主に腫瘍浸潤を測定する方法として考えられ、organotypics インビボ動物モデルへの依存を低減し、そのような浸潤細胞が単分散状態2-4に人為的に強制されたトランスウェルアッセイのような他のインビトロ技術の欠点を回避する。線維芽細胞などのこれらのモデルにおける間質細胞の封入は、悪性の浸潤および転移3-4の調節における腫瘍微小環境の重要な役割を反映し、しかし、間質の表現型の異質性は、例えば、ある器官の生理的関連性を最大にするためにモデル、臓器の特定、解剖学的に正確なex vivoで可能な限り3,6線維芽細胞が含まれるべきである。 Organotypicsは、腫瘍細胞および間質細胞の間の相互作用を研究するための汎用プラットフォームであり、ますます化学的阻害剤および標的化遺伝子改変7,8の腫瘍浸潤に及ぼす影響を調べるために新規な方法で使用されている。浸潤性腫瘍マージンを研究することは、特にエキサイティングな見通しである。器官型モデルでは、確立された結腸直腸癌（CRC）細胞株は、典型的には、井戸重層上皮層を生成し、断面において、細胞外マトリックス（ECM）を浸潤する能力を獲得した細胞が容易に識別可能である。 インビボでの腫瘍および腫瘍関連間質細胞の確立マイクロ地形異質、レーザーマイクロダイセクションを使用して、これらの細胞を抽出し、分離して、それらを研究に照らして、大腸癌転移の起源に関する重要な生物学的な洞察を明らかにすることができるとどのようにそれがより効率的になることがあり対象とした。私N以下の方法論、組織サンプルを寄付し、すべての患者が書面によるインフォームドコンセントを提供し、研究は教育機関、地域研究倫理委員会によって承認された。

<table border="0" cellpadding="0" cellspacing="0" width="634">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Colorectal cancer cell lines - example shown SW480</td>
			<td style="width:132px;">ATCC</td>
			<td style="width:119px;">ATCC CCL-228</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Collagen</td>
			<td style="width:132px;">BD Biosciences</td>
			<td style="width:119px;">354265</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Matrigel</td>
			<td style="width:132px;">BD Biosciences</td>
			<td style="width:119px;">354234</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Nylon membrane</td>
			<td style="width:132px;">Merck Millipore</td>
			<td style="width:119px;">VVLP01300</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Metal grid</td>
			<td style="width:132px;">The Mesh Company</td>
			<td>WSS20-A4</td>
			<td>themeshcompany.com</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Laser microdissection platform</td>
			<td style="width:132px;">Leica Microsystems</td>
			<td style="width:119px;">Leica AS LMD</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Membrane mounted slides</td>
			<td style="width:132px;">Molecular devices</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Cresyl Violet</td>
			<td style="width:132px;">Merck Millipore</td>
			<td style="width:119px;">1052350025</td>
			<td></td>
		</tr>
	</tbody>
</table>

molecular profiling of the invasive tumor microenvironment in a 3-dimensional model of colorectal cancer cells and ex vivo fibroblasts

大腸がんに侵入（CRC）細胞は、姉妹細胞から自由になる間質に浸透し、細胞外マトリックス（ECM）を改造する能力を獲得している。細胞のこの表現型が異なるグループの生物学を特徴づけることは、実質的に転移カスケード時に初期事象の我々の理解を向上させることができます。 腫瘍浸潤、悪性上皮と癌関連間質間の双方向の相互作用によって促進さ動的なプロセスである。腫瘍間質界面での細胞特異的応答を調べるために、我々は器官共培養し、レーザーマイクロダイセクション技術を組み合わせている。 線維芽細胞などのキーの間質成分が、がん上皮細胞と共培養3 - dimentioanallyである、器官のモデルは、近pHの検討が浸潤や癌間質の相互作用を可能にする、高度に操作可能な実験的なツールですysiological条件。 レーザーマイクロダイセクション（LMD）は、ミクロンレベルの精度で腫瘍組織内の様々な階層の外科解剖と抽出を伴う手法である。 ゲノム、トランスクリプトームおよびエピジェネティックなプロファイリングでこれらの技術を組み合わせることで、我々は、腫瘍細胞に侵入し、間質組織、周囲の分子特性をよく理解できるようにする、そうすることで、潜在的に、CRCにおける薬剤開発のための新規バイオマーカーと機会を明らかにすることを目指しています。

我々は、CRC細胞株及び間質細胞の複数の組み合わせに対して、上記の方法を適用した。ここで紹介する一つの例は、一次ヒトex vivoで結腸線維芽細胞と上皮SW480 CRC細胞株である。 3次元共培養物は、浸潤性腫瘍前線におけるセル間の差次的に発現されるmiRNAの比較のために顕微鏡およびレーザーキャプチャーマイクロダイセクション（ 図2）に供した（ 図1）は ?...

Watch this Scientific Journal Video about Molecular Profiling of the Invasive Tumor Microenvironment in a 3-Dimensional Model of Colorectal Cancer Cells and Ex vivo Fibroblasts at JoVE.com

Molecular Profiling of the Invasive Tumor Microenvironment in a 3-Dimensional Model of Colorectal Cancer Cells and Ex vivo Fibroblasts

合成からのレーザーマイクロダイセクション細胞や間質、組織工学大腸癌モデルの分子プロファイリングは、浸潤性前面に腫瘍細胞および癌関連の間質細胞との間の界面に特徴的な生物学を特徴付け、分析する新規かつ操作可能なアプローチを表しています。

大腸癌細胞の3次元モデルにおける侵襲性腫瘍微小環境の分子プロファイリングと<em&gt;生体外</em&gt;線維芽細胞

Invading colorectal cancer (CRC) cells have acquired the capacity to break free from their sister cells, infiltrate the stroma, and remodel the ...

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Molecular Profiling of the Invasive Tumor Microenvironment in a 3-Dimensional Model of Colorectal Cancer Cells and Ex vivo Fibroblasts

11.0K ビュー.  University of Southampton School of Medicine. 合成からのレーザーマイクロダイセクション細胞や間質、組織工学大腸癌モデルの分子プロファイリングは、浸潤性前面に腫瘍細胞および癌関連の間質細胞との間の界面に特徴的な生物学を特徴付け、分析する新規かつ操作可能なアプローチを表しています。 

ビデオ: Molecular Profiling of the Invasive Tumor Microenvironment in a 3-Dimensional Model of Colorectal Cancer Cells and Ex vivo Fibroblasts

In vivo Bioluminescence Imaging of Tumor Hypoxia Dynamics of Breast Cancer Brain Metastasis in a Mouse Model

It is well recognized that tumor hypoxia plays an important role in promoting malignant progression and affecting therapeutic response negatively. There is little knowledge about in situ, in vivo, tumor hypoxia during intracranial development of malignant brain tumors because of lack of efficient means to monitor it in these deep-seated orthotopic tumors. Bioluminescence imaging (BLI), based on the detection of light emitted by living cells expressing a luciferase gene, has been rapidly adopted for cancer research, in particular, to evaluate tumor growth or tumor size changes in response to treatment in preclinical animal studies. Moreover, by expressing a reporter gene under the control of a promoter sequence, the specific gene expression can be monitored non-invasively by BLI. Under hypoxic stress, signaling responses are mediated mainly via the hypoxia inducible factor-1&alpha; (HIF-1&alpha;) to drive transcription of various genes. Therefore, we have used a HIF-1&alpha; reporter construct, 5HRE-ODD-luc, stably transfected into human breast cancer MDA-MB231 cells (MDA-MB231/5HRE-ODD-luc). In vitro HIF-1&alpha; bioluminescence assay is performed by incubating the transfected cells in a hypoxic chamber (0.1% O2) for 24 hr before BLI, while the cells in normoxia (21% O2) serve as a control. Significantly higher photon flux observed for the cells under hypoxia suggests an increased HIF-1&alpha; binding to its promoter (HRE elements), as compared to those in normoxia. Cells are injected directly into the mouse brain to establish a breast cancer brain metastasis model. In vivo bioluminescence imaging of tumor hypoxia dynamics is initiated 2 wks after implantation and repeated once a week. BLI reveals increasing light signals from the brain as the tumor progresses, indicating increased intracranial tumor hypoxia. Histological and immunohistochemical studies are used to confirm the in vivo imaging results. Here, we will introduce approaches of in vitro HIF-1&alpha; bioluminescence assay, surgical establishment of a breast cancer brain metastasis in a nude mouse and application of in vivo bioluminescence imaging to monitor intracranial tumor hypoxia.

In vivo Bioluminescence Imaging of Tumor Hypoxia Dynamics of Breast Cancer Brain Metastasis in a Mouse Model

Bioluminescence imaging of hypoxia inducible factor-1&alpha; activity is applied to monitor intracranial tumor hypoxia development in a breast cancer brain metastasis mouse model.

マウスモデルにおける乳がんの脳転移の腫瘍の低酸素動態の in vivo生物発光イメージングに

It is well recognized that tumor hypoxia plays an important role in promoting malignant progression and affecting therapeutic response negatively. There ...

医学

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 ...

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 ...

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 ...

Initiation of Metastatic Breast Carcinoma by Targeting of the Ductal Epithelium with Adenovirus-Cre: A Novel Transgenic Mouse Model of Breast Cancer

Breast cancer is a heterogeneous disease involving complex cellular interactions between the developing tumor and immune system, eventually resulting in exponential tumor growth and metastasis to distal tissues and the collapse of anti-tumor immunity. Many useful animal models exist to study breast cancer, but none completely recapitulate the disease progression that occurs in humans. In order to gain a better understanding of the cellular interactions that result in the formation of latent metastasis and decreased survival, we have generated an inducible transgenic mouse model of YFP-expressing ductal carcinoma that develops after sexual maturity in immune-competent mice and is driven by consistent, endocrine-independent oncogene expression. Activation of YFP, ablation of p53, and expression of an oncogenic form of K-ras was achieved by the delivery of an adenovirus expressing Cre-recombinase into the mammary duct of sexually mature, virgin female mice. Tumors begin to appear 6 weeks after the initiation of oncogenic events. After tumors become apparent, they progress slowly for approximately two weeks before they begin to grow exponentially. After 7-8 weeks post-adenovirus injection, vasculature is observed connecting the tumor mass to distal lymph nodes, with eventual lymphovascular invasion of YFP+ tumor cells to the distal axillary lymph nodes. Infiltrating leukocyte populations are similar to those found in human breast carcinomas, including the presence of &#945;&#946; and &#947;&#948; T cells, macrophages and MDSCs. This unique model will facilitate the study of cellular and immunological mechanisms involved in latent metastasis and dormancy in addition to being useful for designing novel immunotherapeutic interventions to treat invasive breast cancer.

Activation of latent mutations with adenovirus-Cre into the mammary ductal system results in a clinically relevant metastatic breast cancer. Incorporation of a YFP promoter allows tracking of distal metastatic tumor cells. This model is useful to study latent metastasis, anti-tumor immunity, and for designing novel immunotherapies to treat breast cancer.

乳癌の新規トランスジェニックマウスモデル：アデノ-Creを持つ乳管上皮の標的とすることにより、転移性乳癌の開始

Breast cancer is a heterogeneous disease involving complex cellular interactions between the developing tumor and immune system, eventually resulting in ...

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 ...

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 ...

Studying Pancreatic Cancer Stem Cell Characteristics for Developing New Treatment Strategies

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor initiation, metastasis and resistance to radio- and chemotherapy. Here we describe a specific methodology for culturing primary human pancreatic CSCs as tumor spheres in anchorage-independent conditions. Cells are grown in serum-free, non-adherent conditions in order to enrich for CSCs while their more differentiated progenies do not survive and proliferate during the initial phase following seeding of single cells. This assay can be used to estimate the percentage of CSCs present in a population of tumor cells. Both size (which can range from 35 to 250 micrometers) and number of tumor spheres formed represents CSC activity harbored in either bulk populations of cultured cancer cells or freshly harvested and digested tumors 1,2. Using this assay, we recently found that metformin selectively ablates pancreatic CSCs; a finding that was subsequently further corroborated by demonstrating diminished expression of pluripotency-associated genes/surface markers and reduced in vivo tumorigenicity of metformin-treated cells. As the final step for preclinical development we treated mice bearing established tumors with metformin and found significantly prolonged survival. Clinical studies testing the use of metformin in patients with PDAC are currently underway (e.g., NCT01210911, NCT01167738, and NCT01488552). Mechanistically, we found that metformin induces a fatal energy crisis in CSCs by enhancing reactive oxygen species (ROS) production and reducing mitochondrial transmembrane potential. In contrast, non-CSCs were not eliminated by metformin treatment, but rather underwent reversible cell cycle arrest. Therefore, our study serves as a successful example for the potential of in vitro sphere formation as a screening tool to identify compounds that potentially target CSCs, but this technique will require further in vitro and in vivo validation to eliminate false discoveries.

Pancreatic cancer stem cells (CSCs) can be expanded in vitro using the anchorage-independent sphere culture technique, which represents a powerful tool to study CSC biology and can serve as the first step to develop novel CSC-targeting therapies. Here the methodology for expanding, analyzing and targeting of pancreatic CSCs is provided.

新しい治療戦略を開発するための膵臓癌幹細胞特性を研究

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor ...

Isolation and Propagation of Circulating Tumor Cells from a Mouse Cancer Model

Cancer metastasis is the foremost cause of cancer-associated deaths. Recent studies have shown that circulating tumor cells (CTCs) are important in cancer metastasis. Indeed, the number of CTCs correlates with tumor size. Here, a detailed description is provided of a methodology for isolation and propagation of CTCs from a syngeneic mouse model of hepatocellular carcinoma (HCC) which allows for downstream analysis of potentially important molecular mechanisms of solid organ tumor metastasis. This method is efficient and reproducible. It is a non-invasive technique and, therefore, has potential to replace the invasive biopsy of tissues from humans which may be associated with complications. Therefore, the method discussed here allows for the isolation and propagation of CTCs from whole blood samples such that they can be examined and characterized. This has potential for future adaptation for clinical applications such as diagnosis, and personalized targeted therapy.

Circulating tumor cells (CTCs) have been shown to play an important role in tumor metastasis. Here, a method for the isolation and propagation of CTCs from the whole blood of a syngeneic mouse tumor model of hepatocellular carcinoma (HCC) metastasis is described.

マウス癌モデルからの循環腫瘍細胞の単離と伝播

Cancer metastasis is the foremost cause of cancer-associated deaths. Recent studies have shown that circulating tumor cells (CTCs) are important in cancer ...

A Brain Tumor/Organotypic Slice Co-culture System for Studying Tumor Microenvironment and Targeted Drug Therapies

Brain tumors are a major cause of cancer-related morbidity and mortality. Developing new therapeutics for these cancers is difficult, as many of these tumors are not easily grown in standard culture conditions. Neurosphere cultures under serum-free conditions and orthotopic xenografts have expanded the range of tumors that can be maintained. However, many types of brain tumors remain difficult to propagate or study. This is particularly true for pediatric brain tumors such as pilocytic astrocytomas and medulloblastomas. This protocol describes a system that allows primary human brain tumors to be grown in culture. This quantitative assay can be used to investigate the effect of microenvironment on tumor growth, and to test new drug therapies. This protocol describes a system where fluorescently labeled brain tumor cells are grown on an organotypic brain slice from a juvenile mouse. The response of tumor cells to drug treatments can be studied in this assay, by analyzing changes in the number of cells on the slice over time. In addition, this system can address the nature of the microenvironment that normally fosters growth of brain tumors. This brain tumor organotypic slice co-culture assay provides a propitious system for testing new drugs on human tumor cells within a brain microenvironment.

Many types of human brain tumors are localized to specific regions within the brain and are difficult to grow in culture. This protocol addresses the role of tumor microenvironment and investigates new drug treatments by analyzing fluorescent primary brain tumor cells growing in an organotypic mouse brain slice. &#160;

腫瘍微小環境と標的薬物療法研究のための脳の腫瘍/器官スライス共培養系

Brain tumors are a major cause of cancer-related morbidity and mortality. Developing new therapeutics for these cancers is difficult, as many of these ...