この研究のための資金は、癌研究英国からのものです。

1。蛍光タンパク質と細胞のレトロウイルスラベリング<ol><li> 0.25 × 10 6細胞におけるウェルあたり6ウェルの外プレートレトロウイルスパッケージング細胞（ 例えばフェニックス） 10％ウシ胎児血清（FBS）/ DMEMで料理。 </li><li>レトロウイルスDNAとトランスフェクション細胞を48時間後に細胞はメーカーの指示に従ってEffecteneを使用。 </li><li>後で培地で2回24時間の井戸を洗い流した後、ウェルあたり10％FBS / DMEM 1.5 mlを加える。 </li><li> 2 mlのマイクロチューブにピペッティングし、転送することにより、48時間後に組織培養の培地にパッケージ化ウイルスを収集する。 </li><li>ペレット任意のセ​​ルに5分間1600 rpmで遠心する。 </li><li> -80きれいなチューブに上清と格納、削除℃の</li><li>レトロウイルス形質導入される細胞を6ウェルディッシュのウェル当たり1.5 × 10 5細胞で行わ播種し、37℃で一晩加湿インキュベータ内に配置されます。 </li><li>翌日、細胞や広告からメディアを削除するウイルスストックのD 1mlを各ウェルに4μlのポリブレン（濃度2μg/ mL）で補った。インキュベーター内でプレートを交換してください。 </li><li> 5〜6時間以下のインキュベーション、各ウェルプレートに2 mlの10％FBS / DMEMを追加するには° C inbubatorは一晩37に置き換えられます。 </li><li>翌日、適切な選択培地を追加する前にメディアを交換し、細胞を24時間可能です。 </li><li>ときに（成長細胞の割合、およびレトロウイルス形質導入の効率性に依存するが、通常は4から14日から行われる）コンフルエントに、基準として非形質導入細胞を用いた蛍光用セルとソートをトリプシン処理、収集し、プールの細胞と蛍光-80℃、非形質導入細胞と凍結のアリコート℃を超えて将来の使用のために。 </li></ol> 2。逆マトリゲル浸潤アッセイ<ol><li>ゆっくりと氷（図1A）の完全なマトリゲル（すなわち、増殖因子を含む）のアリコートを解凍。 </li><li>氷冷PBSで一度解凍、希釈マトリゲル1:1（その他の追加と共に前の希釈にPBSで2倍の濃度でのトリートメント、図1B）。それは容易に重合する前にマトリゲルを処理するようにするには、すべてのプラスチック製品（例えばチップ、チューブなど）の氷冷する必要があります。 </li><li>コー​​ティングされていない多くの8ミクロンの細孔6.5ミリメートルの直径は、24ウェル組織培養プレートのウェルに必要なTranswellsとして挿入し、慎重に各ウエルに希釈したマトリゲルの100μlをピペットで37〜30分間インキュベートしておく℃で固化する（図1C）。 </li><li>この時間の間に、彼らの正常な成長に各前処理条件の細胞株に依存しミリリットルあたり4〜1の間× 10 5細胞、、（E. グラム 。siRNAは、薬物治療）で1つまたは複数の蛍光標識した細胞懸濁液を準備する培地（図1D）。 </li><li>マトリゲルが固化したときに、フィルタ（図1E）の上向き下側にTranswellsと細胞懸濁液の100μL添加して反転。 </li><li>慎重に24ウェル組織の基盤とTranswellsをカバー培養プレート、細胞懸濁液（図1F）の各液滴と接触させる。 </li><li>細胞接着性（図1G）を可能にするために4時間倒立状態でプレートをインキュベートする。 </li><li>この時間の後、プレートを右サイドアップにして、2 × 1 ml血清フリー培地（図2A）に順次浸漬して、各トランスウェルを洗う。 </li><li> （図2B）も必要な薬や治療法を含む3番目にTranswellsを残す。 </li><li>ピペット固化マトリゲル/ PBS混合物の上にトランスウェルに10％FBS / DMEMを加えた誘引物質（E. グラム 。25 ng / mlの時EGF）の100μlを、37℃で3-5日のために蓋とインキュベートを交換してください° Cで5％CO 2（図2C）。 </li></ol> 3。染色と可視化<ol><li>蛍光タンパク質を発現する細胞は、前の共焦点顕微鏡に以​​下の手順をスキップできます。 </li><li>新鮮な24ウェルディッシュと4μMのピペット1mlのマトリゲル、場所のTranswellsに侵入する画像非蛍光細胞へカルセインは、側面を波及し、上部と下部から染色するので、それぞれのマトリゲルプラグの上に染色液を午前。カルセインAM（カルセインのアセトキシメチルエステル）は、その染色細胞全体緑生細胞の色素であり、ない固定を必要としません。 </li><li> 37℃で1時間インキュベート·ポイントの細胞が完全に染色し共焦点顕微鏡によって撮像される準備ができている時、5％CO 2加湿雰囲気、でCを。 </li><li>また、マトリゲルに侵入する非蛍光細胞を固定し、3.9に3.5で後述するように染色されることがあります。 </li><li>新鮮な24穴プレートに各トランスウェルを転送する。オーバーレイ4パーセントpara-formaldehyde/0.2％トリトンX - 100を1ml。 </li><li> 0.5時間室温でインキュベートする。 </li><li>固定液を取り出して、1mlのPBSで3回洗浄する。 </li><li> 100μg/ mlのRNaseを使用して30分RNase処理で細胞質RNAを削除します。 PBSで2回洗浄する。 </li><li>可視化のためにPBSで希釈した0.01 mg / mlのヨウ化プロピジウム（PI）を追加し、DAで、室温で残す0.5時間RK。 PBSで3回洗浄する。この段階では、PIは、トランスウェルは、少なくとも1ヶ月、暗所で室温で保存することができる染色。 </li><li>共焦点顕微鏡による精密なイメージング法は、利用可能なリソースに依存しています。非浸漬20 X客観的および光学的切片をマトリゲルプラグの底から10〜15μmの捕捉以上の大規模なカバースリップ上に残存する少量のPBS（気泡が存在しないことを確認）で倒立共焦点顕微鏡、場所のトランスウェルを使用する。個々の光のスライスは、侵略の範囲（図3A）を定量化するかVolocity（図3B）などの適切なソフトウェアを使用して3次元のオブジェクトに組み込むために使用することができます。 </li></ol> 4。代表的な結果光学スライスのZシリーズの例を図3Aに示されています。このインスタンスでは、細胞は、カルセインAMで染色し、フィルタから浸潤細胞の数は、距離とともに減少する見ることができます。での定量化vasionは、それぞれの間隔で、または上記に詳述固定/染色法を用いて、各位置でのPI陽性核をカウントすることで負のピクセルに正のピクセル回答カルセインの比率を分析することによって行うことができます。カルセインの利点の一つである染色午前細胞の浸潤のその3次元再構成は、侵略のモード（図3B）の視覚的描写を与えて、そのようなVolocityとしてソフトウェアを使用して組み立てることができます。細胞を蛍光タンパク質の発現によってラベル付けされている場合は、各色のセルの位置はどちらの側（図3C）から、または再構築（図3D）を介してスライスを行うことで閲覧、3次元再構成で視覚化することができます。 <img alt="図1" src="/files/ftp_upload/3525/3525fig1.jpg" /> 図1逆浸潤アッセイのセットアップに必要な手順の模式図。 A）マトリゲルECMは、氷上で解凍。 B）マトリゲルをPBSで二回最終濃度で、任意の薬物治療を含むで1:1に希釈される。 C）<html>トランスウェルインサートは、マルチウェルプレートに入れ、およびマトリゲルは、それぞれにピペットされています。 D）細胞懸濁液は、所望の濃度でなさ。 E）マトリゲルを設定した後、プレートを反転して削除され、細胞はトランスウェルインサートの底面フィルター上にプレーティングされています。 F）倒立位置では、多層板を慎重に細胞懸濁液と接触すること、トランスウェルインサート上に置かれる。 G）細胞は4時間のためにフィルタに付着させています。 <img alt="図2" src="/files/ftp_upload/3525/3525fig2.jpg" /> 図2逆浸潤アッセイの模式図の継続。細胞が付着している）したら、二回バラセルを削除するには、無血清培地に各トランスウェルを浸す。 B）場所は、必要に応じて最終的なも含まれているメディアに加え、治療にトランスウェルを洗浄した。 C）必要に応じて治療法と走化性因子（例えば10％ウシ胎児血清）を含むメディアは、慎重にマトリゲル上に重層されています。 ENT&quot;&gt; <img alt="図3" src="/files/ftp_upload/3525/3525fig3.jpg" /> 図3。逆浸潤アッセイの結果の代表的な画像。 A）カルセインで染色した細胞の光学セクションでは、共焦点顕微鏡で15μmの間隔で撮影された、マトリゲルに侵入しています。側から見た焦点Zシリーズの画像のスタックからの細胞浸潤の3次元再構築のB）再建、。参考文献10から転載。側から見たマトリゲルに侵入GFPおよびRFP標識細胞のC）の三次元再構築。 GFPおよびRFP標識細胞の3次元再構成を介してD）光学スライス。参考文献10から転載。

<ol>
	<li>Olson, M. F., Sahai, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+actin+cytoskeleton+in+cancer+cell+motility.">The actin cytoskeleton in cancer cell motility.</a> Clin. Exp. Metastasis. 26, 273-287 (2008).</li><li>Hoelzinger, D. B., Demuth, T., Berens, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autocrine+factors+that+sustain+glioma+invasion+and+paracrine+biology+in+the+brain+microenvironment.">Autocrine factors that sustain glioma invasion and paracrine biology in the brain microenvironment.</a> J. Natl. Cancer. Inst. 99, 1583-1593 (2007).</li><li>Chaffer, C. L., Weinberg, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+perspective+on+cancer+cell+metastasis.">A perspective on cancer cell metastasis.</a> Science. 331, 1559-1564 (2011).</li><li>Hanahan, D., Weinberg, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Hallmarks+of+Cancer.">The Hallmarks of Cancer.</a> Cell. 100, 57-70 (2000).</li><li>Francia, G., Cruz-Munoz, W., Man, S., Xu, P., Kerbel, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+models+of+advanced+spontaneous+metastasis+for+experimental+therapeutics.">Mouse models of advanced spontaneous metastasis for experimental therapeutics.</a> Nat. Rev. Cancer. 11, 135-141 (2011).</li><li>Hooper, S., Marshall, J. F., Sahai, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+cell+migration+in+three+dimensions.">Tumor cell migration in three dimensions.</a> Methods. Enzymol. 406, 625-643 (2006).</li><li>Croft, D. R., Olson, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulating+the+conversion+between+rounded+and+elongated+modes+of+cancer+cell+movement.">Regulating the conversion between rounded and elongated modes of cancer cell movement.</a> Cancer Cell. 14, 349-351 (2008).</li><li>Wolf, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multi-step+pericellular+proteolysis+controls+the+transition+from+individual+to+collective+cancer+cell+invasion.">Multi-step pericellular proteolysis controls the transition from individual to collective cancer cell invasion.</a> Nat. Cell. Biol. 9, 893-904 (2007).</li><li>Hennigan, R. F., Hawker, K. L., Ozanne, B. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fos-transformation+activates+genes+associated+with+invasion.">Fos-transformation activates genes associated with invasion.</a> Oncogene. 9, 3591-3600 (1994).</li><li>Scott, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=LIM+kinases+are+required+for+invasive+path+generation+by+tumor+and+tumor-associated+stromal+cells.">LIM kinases are required for invasive path generation by tumor and tumor-associated stromal cells.</a> J. Cell. Biol. 191, 169-185 (2010).</li></ol>

著者らは、開示することは何もない。

マトリゲル浸潤アッセイは、従来、化学誘引物質誘発性運動性に向かってとし、下部のフィルタを通して細胞外マトリックス蛋白質の層の上に置かれた細胞を使用して設定されています。侵襲性がフィルターの下側にカウントできるセルの数の関数として採点した。上記の&quot;逆&quot;浸潤アッセイでほとんど差が実質的にあるが、彼らはマトリゲルを通って移動して浸潤細胞を可視化するこ?...

<table border="1"><tbody><tr><th>試薬の名前</th><th>会社</th><th>カタログ番号</th></tr><tr><td> DMEM（ダルベッコ変法イーグル培地） </td><td> GIBCO </td><td> 21969 </td></tr><tr><td>ウシ胎仔血清</td><td> PAA </td><td> A15 - 101 </td></tr><tr><td>ペニシリンストレプトマイシン</td><td> GIBCO </td><td> 15140 </td></tr><tr><td> 200mMのL -グルタミン（100倍） </td><td> GIBCO </td><td> 25050-032 </td></tr><tr><td>ピューロマイシン</td><td> シグマアルドリッチ </td><td> P8833 </td></tr><tr><td> 0.05％トリプシンEDTA </td><td> GIBCO </td><td> 25300 </td></tr><tr><td>ポリブレン</td><td> シグマアルドリッチ </td><td> AL - 118 </td></tr><tr><td>リポフェクトアミン2000試薬</td><td> インビトロジェン </td><td> 11668019 </td></tr><tr><td> 6.5ミリメートルTranswells、8.0μmの孔径</td><td> コーニング </td><td> 3422 </td></tr><tr><td>完全なマトリ</td><td> BDバイオサイエンス </td><td> 354234 </td></tr> &lt;TR&gt; <td>カルセインAM </td><td> インビトロジェン </td><td> C1430 </td></tr><tr><td> RNaseの</td><td> キアゲン </td><td> 19101 </td></tr><tr><td>ヨウ化プロピジウム</td><td> シグマアルドリッチ </td><td> P4864 </td></tr><tr><td>共焦点microcope </td><td> ライカ </td><td> SP2MP </td></tr></tbody></table>

modeling and imaging 3-dimensional collective cell invasion

癌の悪性腫瘍の特徴は、浸潤転移1です。いくつかの癌（E. グラム 。神経膠腫2）で、健康な組織を周囲に局所浸潤は、病気や死亡の根本的な原因です。他のがんの場合（E. グラム 。乳房、肺、 等 。）、それは腫瘍細胞は、原発腫瘍塊から移動遠位部位を植民地化し、最終的に臓器不全に関与する、転移のプロセスであり、それは最終的に罹患率につながると死亡3。それは、浸潤転移は、がんによる死亡4の90％に責任があると推定されている。その結果、改善の診断と治療5の目的のために浸潤転移の分子プロセスと重要なタンパク質のメディエーターを特定するのに強い関心が集まっている。 癌の科学者にとっての課題は、十分にin vivoの状況に似ている浸潤アッセイを開発することである正確な病気のモデリング6を有効にする。二次元の細胞の運動性アッセイは、侵略の一つの側面についてのみ有益であり、また重要な要素になっているアカウント細胞外マトリックス（ECM）タンパク質のリモデリングは考慮されていません。最近、研究では、腫瘍細胞の浸潤の我々の理解を精緻化し、個々の細胞は細長いまたは丸みのモード7で動く可能性があることを明らかにした。さらに、癌8の普及に、特に上皮特性を維持する高度に差別化された腫瘍の細胞はストランド、シートやサーバークラスタ内に侵入した集団的侵略の貢献のより深い理解を、、、があった。 私たちは、集団的侵略10に候補タンパク質の寄与を調べるための洗練された方法9を提示する。特に、異なる蛍光タンパク質を発現する細胞の工学的に個別のプールで、それは分子的に活動してproteiを分析することが可能です。NSは、以下の細胞で必要とされる対リードする細胞に必要。 RNAiの使用は、実験的に個々の細胞侵入だけでなく、集団の侵略の異なる位置に関連するプロセスを分解するための分子ツールを提供しています。この手順では、蛍光標識した細胞の混合物が以前にマトリゲルECM蛋白質で満たされたトランスウェルインサートの底に播種され、その後、フィルターを通過し、マトリゲルに&quot;上向き&quot;に侵入することができました。 3次元表現への共焦点イメージングで得られたZシリーズの画像スタック、、の再建は、総称してストランドとリードに対する次のような位置で蛍光標識した細胞の表現の分析を侵入の可視化が可能になります。

Watch this Scientific Journal Video about Modeling and Imaging 3-Dimensional Collective Cell Invasion at JoVE.com

Modeling and Imaging 3-Dimensional Collective Cell Invasion

三次元細胞外マトリックスへの腫瘍細胞の浸潤のモデルより反映<em&gt; in vivoで</em二次元の運動性アッセイより&gt;状況。蛍光標識細胞の共焦点イメージングと組み合わせたマトリックス浸潤アッセイを使用して、侵略モードと主要な対後の細胞の明確な寄与に関する詳細な情報を得ることができる。

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 ...

modeling-and-imaging-3-dimensional-collective-cell-invasion

Research

JoVE Journal

Medicine

16.6K ビュー.  University of Strathclyde. 三次元細胞外マトリックスへの腫瘍細胞の浸潤のモデルより反映 in vivoで状況。蛍光標識細胞の共焦点イメージングと組み合わせたマトリックス浸潤アッセイを使用して、侵略モードと主要な対後の細胞の明確な寄与に関する詳細な情報を得ることができる。

ビデオ: Modeling and Imaging 3-Dimensional Collective Cell Invasion

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 ...

医学

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

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

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

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

TGF -β誘発性浸潤を測定する回転楕円体アッセイ

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

3-Dimensional Resin Casting and Imaging of Mouse Portal Vein or Intrahepatic Bile Duct System

In organs, the correct architecture of vascular and ductal structures is indispensable for proper physiological function, and the formation and maintenance of these structures is a highly regulated process. The analysis of these complex, 3-dimensional structures has greatly depended on either 2-dimensional examination in section or on dye injection studies. These techniques, however, are not able to provide a complete and quantifiable representation of the ductal or vascular structures they are intended to elucidate. Alternatively, the nature of 3-dimensional plastic resin casts generates a permanent snapshot of the system and is a novel and widely useful technique for visualizing and quantifying 3-dimensional structures and networks.
A crucial advantage of the resin casting system is the ability to determine the intact and connected, or communicating, structure of a blood vessel or duct. The structure of vascular and ductal networks are crucial for organ function, and this technique has the potential to aid study of vascular and ductal networks in several ways. Resin casting may be used to analyze normal morphology and functional architecture of a luminal structure, identify developmental morphogenetic changes, and uncover morphological differences in tissue architecture between normal and disease states. Previous work has utilized resin casting to study, for example, architectural and functional defects within the mouse intrahepatic bile duct system that were not reflected in 2-dimensional analysis of the structure1,2, alterations in brain vasculature of a Alzheimer's disease mouse model3, portal vein abnormalities in portal hypertensive and cirrhotic mice4, developmental steps in rat lymphatic maturation between immature and adult lungs5, immediate microvascular changes in the rat liver, pancreas, and kidney in response in to chemical injury6.
Here we present a method of generating a 3-dimensional resin cast of a mouse vascular or ductal network, focusing specifically on the portal vein and intrahepatic bile duct. These casts can be visualized by clearing or macerating the tissue and can then be analyzed. This technique can be applied to virtually any vascular or ductal system and would be directly applicable to any study inquiring into the development, function, maintenance, or injury of a 3-dimensional ductal or vascular structure.

A method of visualizing and quantifying the 3-dimensional structure of mouse hepatic portal vein or intrahepatic bile duct is described. This resin cast technique can also be applied to other ductal or vascular systems and allows for in situ visualization or quantification of a system's intact communicating architecture.

3次元樹脂鋳造およびマウスの門脈や肝内胆管系の結

In  organs, the correct architecture of vascular and ductal structures is  indispensable for proper physiological function, and the formation and  ...

Molecular Imaging to Target Transplanted Muscle Progenitor Cells

Duchenne muscular dystrophy (DMD) is a severe genetic neuromuscular disorder that affects 1 in 3,500 boys, and is characterized by progressive muscle degeneration1, 2. In patients, the ability of resident muscle satellite cells (SCs) to regenerate damaged myofibers becomes increasingly inefficient4. Therefore, transplantation of muscle progenitor cells (MPCs)/myoblasts from healthy subjects is a promising therapeutic approach to DMD. A major limitation to the use of stem cell therapy, however, is a lack of reliable imaging technologies for long-term monitoring of implanted cells, and for evaluating its effectiveness. Here, we describe a non-invasive, real-time approach to evaluate the success of myoblast transplantation. This method takes advantage of a unified fusion reporter gene composed of genes (firefly luciferase [fluc], monomeric red fluorescent protein [mrfp] and sr39 thymidine kinase [sr39tk]) whose expression can be imaged with different imaging modalities9, 10. A variety of imaging modalities, including positron emission tomography (PET), single-photon emission computed tomography (SPECT), magnetic resonance imaging (MRI), optical imaging, and high frequency 3D-ultrasound are now available, each with unique advantages and limitations11. Bioluminescence imaging (BLI) studies, for example, have the advantage of being relatively low cost and high-throughput. It is for this reason that, in this study, we make use of the firefly luciferase (fluc) reporter gene sequence contained within the fusion gene and bioluminescence imaging (BLI) for the short-term localization of viable C2C12 myoblasts following implantation into a mouse model of DMD (muscular dystrophy on the X chromosome [mdx] mouse)12-14. Importantly, BLI provides us with a means to examine the kinetics of labeled MPCs post-implantation, and will be useful to track cells repeatedly over time and following migration. Our reporter gene approach further allows us to merge multiple imaging modalities in a single living subject; given the tomographic nature, fine spatial resolution and ability to scale up to larger animals and humans10,11, PET will form the basis of future work that we suggest may facilitate rapid translation of methods developed in cells to preclinical models and to clinical applications.

A non-invasive means to evaluate the success of myoblast transplantation is described. The method takes advantage of a unified fusion reporter gene composed of genes whose expression can be imaged with different imaging modalities. Here, we make use of a fluc reporter gene sequence to target cells via bioluminescence imaging.

移植筋前駆細胞を標的とする分子イメージング

Duchenne muscular dystrophy (DMD) is a severe genetic neuromuscular disorder that affects 1 in 3,500 boys, and is characterized by progressive muscle ...

Generation and 3-Dimensional Quantitation of Arterial Lesions in Mice Using Optical Projection Tomography

The generation and analysis of vascular lesions in appropriate animal models is a cornerstone of research into cardiovascular disease, generating important information on the pathogenesis of lesion formation and the action of novel therapies. Use of atherosclerosis-prone mice, surgical methods of lesion induction, and dietary modification has dramatically improved understanding of the mechanisms that contribute to disease development and the potential of new treatments.
Classically, analysis of lesions is performed ex vivo using 2-dimensional histological techniques. This article describes application of optical projection tomography (OPT) to 3-dimensional quantitation of arterial lesions. As this technique is non-destructive, it can be used as an adjunct to standard histological and immunohistochemical analyses.
Neointimal lesions were induced by wire-insertion or ligation of the mouse femoral artery whilst atherosclerotic lesions were generated by administration of an atherogenic diet to apoE-deficient mice.
Lesions were examined using OPT imaging of autofluorescent emission followed by complementary histological and immunohistochemical analysis. OPT clearly distinguished lesions from the underlying vascular wall. Lesion size was calculated in 2-dimensional sections using planimetry, enabling calculation of lesion volume and maximal cross-sectional area. Data generated using OPT were consistent with measurements obtained using histology, confirming the accuracy of the technique and its potential as a complement (rather than alternative) to traditional methods of analysis.
This work demonstrates the potential of OPT for imaging atherosclerotic and neointimal lesions. It provides a rapid, much needed ex vivo technique for the routine 3-dimensional quantification of vascular remodelling.

Ex vivo analysis of arterial lesions from animal models of cardiovascular disease classically relies on histological and immunohistochemical techniques. These provide 2-dimensional measurements in 3-dimensional lesions. This manuscript describes the generation of arterial lesions for quantitative analysis in 3-dimensions using optical projection tomography.

発生と光学投影トモグラフィを用いたマウスにおける動脈病変の3次元定量

The generation and analysis of vascular lesions in appropriate animal models is a cornerstone of research into cardiovascular disease, generating ...

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

Invading colorectal cancer (CRC) cells have acquired the capacity to break free from their sister cells, infiltrate the stroma, and remodel the extracellular matrix (ECM). Characterizing the biology of this phenotypically distinct group of cells could substantially improve our understanding of early events during the metastatic cascade.

Tumor invasion is a dynamic process facilitated by bidirectional interactions between malignant epithelium and the cancer associated stroma. In order to examine cell-specific responses at the tumor stroma-interface we have combined organotypic co-culture and laser micro-dissection techniques.
Organotypic models, in which key stromal constituents such as fibroblasts are 3-dimentioanally co-cultured with cancer epithelial cells, are highly manipulatable experimental tools which enable invasion and cancer-stroma interactions to be studied in near-physiological conditions.

Laser microdissection (LMD) is a technique which entails the surgical dissection and extraction of the various strata within tumor tissue, with micron level precision.
By combining these techniques with genomic, transcriptomic and epigenetic profiling we aim to develop a deeper understanding of the molecular characteristics of invading tumor cells and surrounding stromal tissue, and in doing so potentially reveal novel biomarkers and opportunities for drug development in CRC.&#160;&#160;&#160;

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

Molecular profiling of laser microdissected cells and stroma from synthetic, tissue-engineered colorectal cancer models represents a novel and manipulatable approach to characterize and dissect the distinctive biology at the interface between tumor cells at the invasive front and cancer associated stromal cells. &#160;

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 ...

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

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

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

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

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

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

Modeling Spontaneous Metastatic Renal Cell Carcinoma (mRCC) in Mice Following Nephrectomy

One of the key challenges to improved testing of new experimental therapeutics in renal cell carcinoma (RCC) is the development of models that faithfully recapitulate early- and late-stage metastatic disease progression. Typical tumor implantation models utilize ectopic or orthotopic primary tumor implantation, but few include systemic spontaneous metastatic disease that mimics the clinical setting. This protocol describes the key steps to develop RCC disease progression stages similar to patients. First, it uses a highly metastatic mouse tumor cell line in a syngeneic model to show orthotopic tumor cell implantation. Methods include superficial and internal implantation into the sub-capsular space with cells combined with matrigel to prevent leakage and early spread. Next it describes the procedures for excision of tumor-bearing kidney (nephrectomy), with critical pre- and post- surgical mouse care. Finally, it outlines the steps necessary to monitor and assess micro-and macro-metastatic disease progression, including bioluminescent imaging as well provides a detailed visual necropsy guide to score systemic disease distribution. The goal of this protocol description is to facilitate the widespread use of clinically relevant metastatic RCC models to improve the predictive value of future therapeutic testing.&#160;

Modeling Spontaneous Metastatic Renal Cell Carcinoma mRCC in Mice Following Nephrectomy

Models of spontaneous metastatic renal cell carcinoma (RCC) disease progression can be used for evaluating treatments in a clinically relevant setting. This protocol demonstrates different procedures for orthotopic kidney tumor cell implantation, proper nephrectomy, and finally outlines a necropsy guide for visual and bioluminescent scoring of metastatic burden and localization.

腎摘出後のマウスにおける自発転移性腎細胞癌（MRCC）をモデル化する

One of the key challenges to improved testing of new experimental therapeutics in renal cell carcinoma (RCC) is the development of models that faithfully ...

Isolation and Immortalization of Patient-derived Cell Lines from Muscle Biopsy for Disease Modeling

The generation of patient-specific cell lines represents an invaluable tool for diagnostic or translational research, and these cells can be collected from skin or muscle biopsy tissue available during the patient&#8217;s diagnostic workup. In this protocol, we describe a technique for live cell isolation from small amounts of muscle or skin tissue for primary cell culture. Additionally, we provide a technique for the immortalization of myogenic cell lines and fibroblast cell lines from primary cells. Once cell lines are immortalized, substantial expansion of patient-derived cells can be achieved. Immortalized cells are amenable to many downstream applications, including drug screening and in vitro correction of the genetic mutation. Altogether, these protocols provide a reliable tool to generate and preserve patient-derived cells for downstream applications.

This protocol describes techniques for live cell isolation and primary culture of myogenic and fibroblast cell lines from muscle or skin tissue. A technique for the immortalization of these cell lines is also described. Altogether, these protocols provide a reliable tool to generate and preserve patient-derived cells for downstream applications.

病気のモデリングのための筋生検からの患者由来の細胞株の単離と不死

The generation of patient-specific cell lines represents an invaluable tool for diagnostic or translational research, and these cells can be collected ...

A Cancer Cell Spheroid Assay to Assess Invasion in a 3D Setting

The invasive nature of cancer cell lines is thought to correlate with their metastatic potential. Most traditional assays, however, do not examine these invasive features in a three-dimensional environment and the resulting data suffer from reduced biological applicability. Here an approach is presented to visualize the invasive ability of cell lines in a physiologically relevant setting. The cancer cell spheroid invasion assay first utilizes gravity to generate spheroids within drops of media that hang from the lid of a cell culture dish. Next, these spheroids are embedded in a 3D matrix consisting of a mixture of basement membrane materials and type I collagen. Cancer cell egression from the spheroids into the surrounding matrix is then monitored over time. The method described here can be modified to examine invasion after coculture of different cell types, inclusion of drugs/inhibitors, or alterations in extracellular matrix (ECM) constituents.

This method evaluates cancer cell invasion from spheroids into a surrounding 3D matrix. Spheroids are generated via the hanging drop culture method and then embedded in a matrix comprised of basement membrane materials and type I collagen. Invasion out of the spheroids is subsequently monitored.

3Dの設定で侵略を評価するために、癌細胞スフェロイドアッセイ

The invasive nature of cancer cell lines is thought to correlate with their metastatic potential.  Most traditional assays, however, do not examine these ...