この作品は、厚生R01 CA131990（BFSとRRM）の国立研究所によって、部分的には、サポートされていました。ライブセルイメージングは​​、コアがカルマノスがん研究所、ウェイン州立大学にNIHセンターの助成金によって、部分的には、P30 CA22453をサポートして顕微鏡、イメージングサイトメトリーリソースで実行され、母子保健と開発の国立研究所の出産期医学研究支部されました、ウェイン州立大学。

1。 DQ-基板を準備します。 <ol><li> 4で50μlのアリコートを、店舗に分割℃、DQ-基質は脱イオン水でDQ基板の1 mg / mlのストック溶液を調製し、バイアルを開く前に室温まで昇温を凍結乾燥することができ</li></ol> それが〜50から5分と熱が°Cの分散を容易にするための超音波水浴中でDQ基板を攪拌する必要があるかもしれません 。 <ol start="2"><li> 4℃一晩氷上で融解RBM°C; RBMは、すべての回で氷の上で処理する必要があります。 </li><li> 10Xリン酸塩を作るために生理食塩水（PBS）、80グラムのNaClを溶解させる（1.37 M）、2グラムのKCl（0.027 M）、14.4グラムのNa 2 HPO 4（1 M）と、2.4グラムのKH 2 PO 4（0.02 M）でバッファリング800ミリリットルの超純水。 1.0 M HClで7.4にPBS緩衝溶液のpHを調整します。体積1リットルに持って、濾過によって滅菌する。 </li><li>冷やしたコラーゲンIチルド10X PBSの1部に8部：氷上のコラーゲンI溶液を調製します。 pHを調整する混合物を滅菌した0.1 M NaOHを用いて7.2から7.6へ。 pHのストリップでpHをチェックし、滅菌水で10重量部に最終的な音量を調整します。 </li><li> 25μg/ mlで、2.4 mg / mlの最終濃度に予冷したチューブは、それぞれのコラーゲンでDQ-I型コラーゲンI溶液を希釈することによりI型コラーゲンマトリックス：DQ-I型コラーゲンを準備します。 </li><li>それぞれ、25μg/ mlに12から15 mg / mlの最終濃度に予冷したチューブ内のRBMにおけるDQ-IV型コラーゲンを（Cultrexまたはマトリゲル）で希釈して再構成基底膜（RBM）マトリックス：DQ-IV型コラーゲンを準備します。気泡を作成するのを避けるために穏やかにピペッティングを使用して氷の上で混ぜる。 </li></ol> 2。 MAME共培養を準備します。 共培養の模式図、図1を参照してください。 <ol><li>場所35 mmの培養皿またはIbiTreatμ-ディッシュ35 mmを使用する2つの長方形のプラスチックのカバーガラス（10分間、空気乾燥させ、70％アルコールで滅菌）。ここで方向性は、我々が学生のために使用され、カバーガラスおよびμ-料理の両方のためのものです正立顕微鏡で死ぬ。倒立顕微鏡の研究のために、私たちは唯一のμ-食器を使用しています。 </li><li> I型コラーゲンマトリックス：DQ-I型コラーゲンと線維芽細胞の希望の番号を混ぜる。 I型コラーゲンマトリックス：図2と図3に示すように、長期的な共培養のために、私たちはメディアに加え、DQ-I型コラーゲンの60μlを10μlの500線維芽細胞を使用しています。 </li><li>慎重にピペットは、100μlのピペットを使用して、線維芽細胞70μlの広がり：DQ-コラーゲンI：I型コラーゲンマトリックスの各カバースリップの表面全体に固化するために30分間CO 2なしで加湿インキュベーター中で37℃のままにします。 </li><li>転送35mmの平衡を10分間、5％CO 2で37℃のインキュベーターにカバースリップを含む料理をお楽しみいただけます。 </li><li>インキュベーターから取り出し、それらが室温になるまで、ボンネットの下に35-mmディッシュにしておきます。 </li><li>固化したDQ-I型コラーゲンの上にRBMマトリックス：埋め込まれた線維芽細胞のI型コラーゲンマトリックスDQ-IV型コラーゲンの60μlを添加します。ピペットチップ、CAとDQ-I型コラーゲン：コラーゲンIマトリックス線維芽細胞を傷つけ避けて、均等にRBMマトリックス：refully DQ-IV型コラーゲンを広げた。 </li><li>転送35ミリ固めるために10分間、5％CO 2で37℃のインキュベーターにカバースリップを含む料理をお楽しみいただけます。 DQ-IV型コラーゲン中：RBM行列が固化され、上皮細胞をトリプシン処理し、カウントされます。 </li><li> RBMマトリックス：DQ-IV型コラーゲンでコーティングされたカバースリップ上腫瘍/上皮細胞懸濁液50μlを置きます。 37℃インキュベーターにカバースリップを含む場所35-mmディッシュ。 RBM行列：セル40〜60分は、DQ-IV型コラーゲンにアタッチすることができます。図2と図3に示すように、長期的な共培養のために、私たちは2500年上皮または腫瘍細胞を使用しています。 </li><li>それぞれの35-mmディッシュに2％のRBMを含む培養液2 mlを追加します。任意の薬物療法を行う場合には、薬物は、この時点で追加する必要があります。 </li><li>イメージングする前に、所望の期間インキュベートします。長期培養のために、メディアは3〜4日ごとに変更する必要があります。細胞を維持することができる彼らの通常の培地インチ乳癌細胞株のMAME共培養のために、私たちはMEGM SingleQuotsを添加した上皮細胞基礎培地（MEBM-PRF）を使用します。 </li></ol> 3。共焦点顕微鏡によるライブMAME共培養の4D（3D +時間）イメージングを実行します。 <ol><li>示す証明の原理研究のために、MAME共培養は、当社の公開手順1に記載の蛍光細胞追跡色素で標識した。イメージングする前に、5％CO 2インキュベーターで37°CでPBSで洗浄した後、共培養は、37℃インキュベーター内で45分間MEGM培地中の5μMのCellTrackerオレンジとインキュベートし、PBSで再度洗浄し、30分間温めMEGMmediumと共にインキュベートした。 </li><li>画像MAME共培養では、ツァイスLSM-510 META共焦点顕微鏡で低倍率（10倍、水浸レンズ）に住んでいます。 </li><li>光のセクションは、構造体の全体の深さを通して間隔でキャプチャされます。我々の研究のために、私たちは10時の光学切片をキャプチャするμmの間隔で。 </li><li> 3Dで画像を再構成する光学切片を使用しています。我々は、3D再構成を生成するためにVolocityソフトウェアを使用します。 </li><li>細胞間相互作用とその周辺のマトリックスとの相互作用を可視化することができる：構造は、細胞ができるように3D再構成の映画を作る。我々はQuikTime Player 7でムービーを作りました。 </li></ol> 4。代表的な結果我々が開発したMAME共培養では、乳房の腫瘍とその微小環境を構成するさまざまな細胞成分間の相互作用のリアルタイムでの生細胞イメージングのための扱いやすい実験モデルである。ここでは、細胞を再現するためにMAMEモデルの能力を実証：乳がんの進行中および生細胞イメージングの細胞間相互作用は、時間の経過とともにそれらの相互作用に従うこと。我々は上の、上皮内MCF10.DCISヒト乳癌細胞株とヒト乳癌に関連した線維芽細胞ライン、WS-12Tiとの間の画像の相互作用する能力を示しています文化の中で3週間以上の期間。共培養は、3、16、23日に撮像した。共培養のボリューム全体を介して光のセクションは、図2の3つの時点の各々について示されている3Dと代表的な例に再建されました。図2の赤色蛍光はMCF10.DCISおよびWS-12Tiのセルを表します。 2つの細胞型の間で長期の共培養のために区別するためには、文化を確立する前に蛍光蛋白質と個々の細胞型をトランスフェクトまたは形質導入することができます。差動識別のための蛍光タンパク質で標識されMCF10.DCISおよびWS-12Ti細胞のMAMEの共培養は、図3に示されています。 MAME共培養に、この場合、DQ-コラーゲンで、基板を組み込むことによって、我々は侵襲的な表現型への移行に関連付けられているタンパク質分解の経時変化を評価し、定量化することができます。我々は、その結果蛍光開裂生成物を定量するための方法を確立していますDQ-コラーゲン分解2,3から。我々は、細胞核を標識し、カウントすることにより、細胞当たりベースでMAME共培養の全体の3Dボリューム全体の分解産物を正規化します。さらに、我々は細胞外および細胞内コンパートメントに劣化をローカライズし、我々は以前に2で説明したように、全体の細胞内と細胞外の分解産物を定量化することができます。図2および図3の緑色蛍光は、2つのDQ-コラーゲン基質の切断産物を表しています。この時点では、差動標識コラーゲンは、私たちはI型コラーゲンのIV型コラーゲンと分解の分解を区別できるようになることはできません。 MAME共培養における相互作用は動的であり、時間の経過とともに変更することができます。時間的および動的な情報を入手するには、ビューの同じフィールドには、週に数分から一定の期間にわたって繰り返してボリューム全体の標本を通じて取得されたイメージングと光学セクションすることができますしたがって、4-Dのデータを収集します。細胞数の変化、細胞遊走および細胞の相互作用、構造ボリューム、構造の形状、球形から構造の偏差、侵襲outgrowthsとタンパク質分解：図2に、我々は23日間で発生したという劇的な変化を示しています。さらに、我々はまた、MAME共培養は、低下させる彼らの、この期間に細胞外マトリックスを囲むためにボリュームの全体的な変化を示しています。図2Aに示すように、三日間で、ボリュームは110,000μmの3です。図2BとCに示すように対照的に、16でMAME共培養の量と23日は、唯一の46250μmの3 <img alt="図1" src="/files/ftp_upload/3661/3661fig1.jpg" /> 図1。 MAME共培養の模式図。プラスチック製カバースリップは、DQ-I型コラーゲンと線維芽細胞（マゼンタ）を含む、I型コラーゲンでコーティングされています。 DQ-colを含む再構成基底膜の2層目（RBM）ラゲンIVが追加されます。腫瘍細胞（赤）の上に播種し、培養培地に2％のRBMは、オーバーレイ（ホワイトドット）メディアのすべての変化であるされています。グリーンはDQ-コラーゲンIとIVの蛍光切断産物を表しています。 <img alt="図2" src="/files/ftp_upload/3661/3661fig2.jpg" /> 図2。 MCF10.DCISヒト乳癌細胞およびWS-12Tiヒト乳房線維芽細胞の共培養MAME。線維芽細胞がコラーゲンDQ-IV型コラーゲンを含むRBMとI / DQ-I型コラーゲンとオーバーレイに埋め込まれていた。 DCISの細胞はその後2％RBMを含むメディアとのRBMとオーバーレイに播種した。共培養の体積の減少によって示されるようにここに示す共培養23日間で、細胞増殖、DQ-IV型コラーゲンおよびI（緑）とコラーゲンマトリックスの劣化があった。細胞は、イメージング（赤）の前にin situで CellTrackerオレンジで標識することによって局在していた。同じライブ共培養は、Cで撮影した画像で23日間にわたって観察された共培養の3、16、23日でonfocal顕微鏡。得られた光学スライスはVolocityソフトウェアを使用して3Dで再構築されました。倍率10倍。 <img alt="図3" src="/files/ftp_upload/3661/3661fig3.jpg" /> 図3。 MAME 16日MCF10.DCISヒト乳癌細胞およびWS-12Tiヒト乳房線維芽細胞の共培養そのそれぞれ急行RFP（赤）とYFP（白）、。共培養が確立され、図2のように分析した。ここに示されている2つの細胞型は、しかし、差彼らは互いに区別することができるように標識されていました。この図では、高倍率の画像は、図2のものに比べ、DCIS細胞と線維芽細胞に近い地域でDQ-I型コラーゲンのびまん性タンパク質分解の表面でDQ-IV型コラーゲンの分解を示しています。倍率20倍。

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我々は、開示することは何もありません。

示したように、MAME共培養は、乳房の腫瘍とその微小環境を構成するさまざまな細胞成分間の相互作用のリアルタイムでライブセルイメージングに使用することができます。我々の研究室で進行中の研究では、我々は、タンパク質分解DCISから浸潤性乳管癌への移行に関連付けられた経路と同様に、このようなケモカイン/サイトカイン/増殖因子経路として、この移行に関与するタンパク質分解...

<table border="1"><tbody><tr><td> 試薬/機器 </td><td> 会社 </td><td> カタログ番号 </td><td> コメント </td></tr><tr><td>再構成基底膜（Cultrex） </td><td> Trevigen </td><td> 3445-005-01 </td><td>マトリゲル（BD Biosciences社）に匹敵する</td></tr><tr><td>コラーゲンI </td><td>凝集研究所</td><td> 5005-B </td><td></td></tr><tr><td> DQ-基質 （コラーゲンI、IV） </td><td>インビトロジェン</td><td> DQ-COL I-D12060 DQ-COL IV D12052 </td><td></td></tr><tr><td>を22 mmプラスティックカバースリップ</td><td>フィッシャー·サイエンティフィック株式会社</td><td> 12から547 </td><td>半分にカットし、使用前に10分間空気乾燥のために70％エタノールにしておきます。 </td></tr><tr><td>細胞培養培地</td><td>ロンザ</td><td> MEBM-PRF CC-3153 MEGM CC-4136 </td><td>赤遊離フェノール</td></tr><tr><td> μ-皿ibiTreat </td><td> Ibidi </td><td> 80136 </td><td></td></tr><tr><td> Volocityソフトウェア</td><td>パーキンエルマー</td><td>バージョン5.5 </td><td>イメージングソフトウェアの他のブランドは、3D再構成やムービーを生成するために使用することができます。 </td></tr></tbody></table>

mame models for 4d live-cell imaging of tumor: microenvironment interactions that impact malignant progression

我々は長期的MAME（M ammary rchitecture と m icroenvironment eの ngineering）3D共培養モデルを開発し、細胞のリアルタイムで生細胞イメージングのためにそれらを使用している：細胞の相互作用。私たちの全体的な目標は、侵襲的な表現型への進行を勉強するために侵入前の乳房病変のアーキテクチャを要約するモデルを開発することでした。具体的には、浸潤性乳管癌の上皮内乳房上皮細胞の進行を増強または低減に関与している腫瘍微小環境のプレ悪性の乳房上皮細胞の変異体および他の細胞型の間の相互作用を分析するモデルを開発しました。現在までに研究された他の細胞型は、上皮細胞、線維芽細胞、マクロファージ、血液とリンパの微小血管内皮細胞である。中に乳房内の細胞の相互作用を再現するように設計されているMAMEのモデルに加えて、癌の進行は、我々は前立腺癌の進行に匹敵するモデルを開発しました。 ここでは、ライブセルイメージングやin situで乳管癌（DCIS）時間の経過とともに浸潤表現型の細胞と線維芽細胞の共培養の推移をフォローするための機能タンパク質分解アッセイを使用するとともに、3次元共培養を確立するための手順を説明する文化の中で23日間以上このような場合。 MAME共培養では、複数のレイヤーで構成されています。線維芽細胞をI型コラーゲンの一番下の層に埋め込まれています。その上DCIS細胞が播種されている再構成基底膜（RBM）の層を配置されます。 2％のRBMの最後の最上位層が含まれており、メディアの変更があるたびに補充されています。侵襲的な表現型への進行に関連付けられたイメージのタンパク質分解には、私がRBの中間層と混合し、コラーゲンIとDQ-IV型コラーゲンの層と混合染料急冷（DQ）蛍光マトリックスタンパク質（DQ-コラーゲンを使用しM）と共焦点顕微鏡を使用して、ライブの文化を守ってください。光のセクションは、キャプチャされ処理され、Volocity可視化ソフトウェアと3Dで再構築されます。 MAME共培養23日間で、DCIS細胞が増殖し、大規模な侵襲的な構造に合体する。線維芽細胞が移行し、これらの侵襲的な構造に組み込まれになります。コラーゲンの蛍光タンパク質分解断片が細胞内に、DCISの構造の表面に関連して発見され、また周囲のマトリックス全体に分散されています。ターゲットタンパク分解、ケモカイン/サイトカインとキナーゼ経路または共培養の細胞組成の変更はMAMEモデルが新たな治療アプローチの臨床画面として使用することができることを示唆し、侵襲性を減らすことができる薬。

Watch this Scientific Journal Video about MAME Models for 4D Live-cell Imaging of Tumor: Microenvironment Interactions that Impact Malignant Progression at JoVE.com

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

我々は侵襲的な表現型への微小環境に影響を与える進行中の乳房の腫瘍細胞と他の細胞間の相互作用をリアルタイムでライブセルイメージングのための3D共培養モデルを開発しました。これらのモデルは、パラクリン誘起分解、ケモカイン/サイトカインと侵襲性に関与するキナーゼ経路を標的とする薬剤の臨床画面として使用できます。

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 ...

mame-models-for-4d-live-cell-imaging-tumor-microenvironment

Research

JoVE Journal

Medicine

14.3K ビュー.  Wayne State University. 我々は侵襲的な表現型への微小環境に影響を与える進行中の乳房の腫瘍細胞と他の細胞間の相互作用をリアルタイムでライブセルイメージングのための3D共培養モデルを開発しました。これらのモデルは、パラクリン誘起分解、ケモカイン/サイトカインと侵襲性に関与するキナーゼ経路を標的とする薬剤の臨床画面として使用できます。

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

Orthotopic Xenografting of Human Luciferase-Tagged Malignant Peripheral Nerve Sheath Tumor Cells for in vivo Testing of Candidate Therapeutic Agents

Although in vitro screens are essential for the initial identification of candidate therapeutic agents, a rigorous assessment of the drug's ability to inhibit tumor growth must be performed in a suitable animal model. The type of animal model that is best for this purpose is a topic of intense discussion. Some evidence indicates that preclinical trials examining drug effects on tumors arising in transgenic mice are more predictive of clinical outcome1and so candidate therapeutic agents are often tested in these models. Unfortunately, transgenic models are not available for many tumor types. Further, transgenic models often have other limitations such as concerns as to how well the mouse tumor models its human counterpart, incomplete penetrance of the tumor phenotype and an inability to predict when tumors will develop.
Consequently, many investigators use xenograft models (human tumor cells grafted into immunodeficient mice) for preclinical trials if appropriate transgenic tumor models are not available. Even if transgenic models are available, they are often partnered with xenograft models as the latter facilitate rapid determination of therapeutic ranges. Further, this partnership allows a comparison of the effectiveness of the agent in transgenic tumors and genuine human tumor cells. Historically, xenografting has often been performed by injecting tumor cells subcutaneously (ectopic xenografts). This technique is rapid and reproducible, relatively inexpensive and allows continuous quantitation of tumor growth during the therapeutic period2. However, the subcutaneous space is not the normal microenvironment for most neoplasms and so results obtained with ectopic xenografting can be misleading due to factors such as an absence of organ-specific expression of host tissue and tumor genes. It has thus been strongly recommended that ectopic grafting studies be replaced or complemented by studies in which human tumor cells are grafted into their tissue of origin (orthotopic xenografting)2. Unfortunately, implementation of this recommendation is often thwarted by the fact that orthotopic xenografting methodologies have not yet been developed for many tumor types.
Malignant peripheral nerve sheath tumors (MPNSTs) are highly aggressive sarcomas that occur sporadically or in association with neurofibromatosis type 13and most commonly arise in the sciatic nerve4. Here we describe a technically straightforward method in which firefly luciferase-tagged human MPNST cells are orthopically xenografted into the sciatic nerve of immunodeficient mice. Our approach to assessing the success of the grafting procedure in individual animals and subsequent non-biased randomization into study groups is also discussed.

Orthotopic Xenografting of Human Luciferase-Tagged Malignant Peripheral Nerve Sheath Tumor Cells for in vivo Testing of Candidate Therapeutic Agents

A method for reliably grafting luciferase-tagged human malignant peripheral nerve sheath tumor cells into the sciatic nerve of immunodeficient mice is described. The use of bioluminescence imaging to demonstrate proper establishment of tumor grafts and criteria for random segregation of animals into study groups are also discussed.

のために人間のルシフェラーゼ-タグ付き悪性末梢神経鞘腫瘍細胞の同所Xenografting in vivoで</em候補治療薬の>テスト

Although in vitro screens are essential for the initial identification of candidate therapeutic agents, a rigorous assessment of the drug's ability to ...

医学

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 ...

In vitro Mesothelial Clearance Assay that Models the Early Steps of Ovarian Cancer Metastasis

Ovarian cancer is the fifth leading cause of cancer related deaths in the United States1. Despite a positive initial response to therapies, 70 to 90 percent of women with ovarian cancer develop new metastases, and the recurrence is often fatal2. It is, therefore, necessary to understand how secondary metastases arise in order to develop better treatments for intermediate and late stage ovarian cancer. Ovarian cancer metastasis occurs when malignant cells detach from the primary tumor site and disseminate throughout the peritoneal cavity. The disseminated cells can form multicellular clusters, or spheroids, that will either remain unattached, or implant onto organs within the peritoneal cavity3 (Figure 1, Movie 1). 
All of the organs within the peritoneal cavity are lined with a single, continuous, layer of mesothelial cells4-6 (Figure 2). However, mesothelial cells are absent from underneath peritoneal tumor masses, as revealed by electron micrograph studies of excised human tumor tissue sections3,5-7 (Figure 2). This suggests that mesothelial cells are excluded from underneath the tumor mass by an unknown process. 
Previous in vitro experiments demonstrated that primary ovarian cancer cells attach more efficiently to extracellular matrix than to mesothelial cells8, and more recent studies showed that primary peritoneal mesothelial cells actually provide a barrier to ovarian cancer cell adhesion and invasion (as compared to adhesion and invasion on substrates that were not covered with mesothelial cells)9,10. This would suggest that mesothelial cells act as a barrier against ovarian cancer metastasis. The cellular and molecular mechanisms by which ovarian cancer cells breach this barrier, and exclude the mesothelium have, until recently, remained unknown. 
Here we describe the methodology for an in vitro assay that models the interaction between ovarian cancer cell spheroids and mesothelial cells in vivo (Figure 3, Movie 2). Our protocol was adapted from previously described methods for analyzing ovarian tumor cell interactions with mesothelial monolayers8-16, and was first described in a report showing that ovarian tumor cells utilize an integrin &#x2013;dependent activation of myosin and traction force to promote the exclusion of the mesothelial cells from under a tumor spheroid17. This model takes advantage of time-lapse fluorescence microscopy to monitor the two cell populations in real time, providing spatial and temporal information on the interaction. The ovarian cancer cells express red fluorescent protein (RFP) while the mesothelial cells express green fluorescent protein (GFP). RFP-expressing ovarian cancer cell spheroids attach to the GFP-expressing mesothelial monolayer. The spheroids spread, invade, and force the mesothelial cells aside creating a hole in the monolayer. This hole is visualized as the negative space (black) in the GFP image. The area of the hole can then be measured to quantitatively analyze differences in clearance activity between control and experimental populations of ovarian cancer and/ or mesothelial cells. This assay requires only a small number of ovarian cancer cells (100 cells per spheroid X 20-30 spheroids per condition), so it is feasible to perform this assay using precious primary tumor cell samples. Furthermore, this assay can be easily adapted for high throughput screening.

In vitro Mesothelial Clearance Assay that Models the Early Steps of Ovarian Cancer Metastasis

The mesothelial clearance assay described here takes advantage of fluorescently labeled cells and time-lapse video microscopy to visualize and quantitatively measure the interactions of ovarian cancer multicellular spheroids and mesothelial cell monolayers. This assay models the early steps of ovarian cancer metastasis.

in vitroで中皮クリアランスアッセイでそのモデルの卵巣がんの転移の初期段階を

Ovarian cancer is the fifth leading cause of cancer related deaths in the United States1. Despite a positive initial response to therapies, 70 to 90 ...

Heterotypic Three-dimensional In Vitro Modeling of Stromal-Epithelial Interactions During Ovarian Cancer Initiation and Progression

Epithelial ovarian cancers (EOCs) are the leading cause of death from gynecological malignancy in Western societies. Despite advances in surgical treatments and improved platinum-based chemotherapies, there has been little improvement in EOC survival rates for more than four decades 1,2. Whilst stage I tumors have 5-year survival rates &gt;85%, survival rates for stage III/IV disease are &lt;40%. Thus, the high rates of mortality for EOC could be significantly decreased if tumors were detected at earlier, more treatable, stages 3-5. At present, the molecular genetic and biological basis of early stage disease development is poorly understood. More specifically, little is known about the role of the microenvironment during tumor initiation; but known risk factors for EOCs (e.g. age and parity) suggest that the microenvironment plays a key role in the early genesis of EOCs. We therefore developed three-dimensional heterotypic models of both the normal ovary and of early stage ovarian cancers. For the normal ovary, we co-cultured normal ovarian surface epithelial (IOSE) and normal stromal fibroblast (INOF)&nbsp;cells, immortalized by retrovrial transduction of the catalytic subunit of human telomerase holoenzyme (hTERT) to extend the lifespan of these cells in culture. To model the earliest stages of ovarian epithelial cell transformation, overexpression of the CMYC oncogene in IOSE cells, again co-cultured with INOF cells. These heterotypic models were used to investigate the effects of aging and senescence on the transformation and invasion of epithelial cells. Here we describe the methodological steps in development of these three-dimensional model; these methodologies aren't specific to the development of normal ovary and ovarian cancer tissues, and could be used to study other tissue types where stromal and epithelial cell interactions are a fundamental aspect of the tissue maintenance and disease development.

Heterotypic Three-dimensional In Vitro Modeling of Stromal-Epithelial Interactions During Ovarian Cancer Initiation and Progression

We describe methodologies for establishing in vitro heterotypic three-dimensional models comprising ovarian fibroblasts and normal ovarian surface or ovarian cancer epithelial cells. We discuss the use of these models to study stromal-epithelial interactions that occur during ovarian cancer development.

三次元異<em&gt; in vitroで</em卵巣がんの発生と進行中に間質相互作用の上皮&gt;モデリング

Epithelial ovarian cancers (EOCs) are the leading cause of death from gynecological malignancy in Western societies. Despite advances in surgical ...

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 Novel High-resolution In vivo Imaging Technique to Study the Dynamic Response of Intracranial Structures to Tumor Growth and Therapeutics

We have successfully integrated previously established Intracranial window (ICW) technology 1-4 with intravital 2-photon confocal microscopy to develop a novel platform that allows for direct long-term visualization of tissue structure changes intracranially. Imaging at a single cell resolution in a real-time fashion provides supplementary dynamic information beyond that provided by standard end-point histological analysis, which looks solely at 'snap-shot' cross sections of tissue.
Establishing this intravital imaging technique in fluorescent chimeric mice, we are able to image four fluorescent channels simultaneously. By incorporating fluorescently labeled cells, such as GFP+ bone marrow, it is possible to track the fate of these cells studying their long-term migration, integration and differentiation within tissue. Further integration of a secondary reporter cell, such as an mCherry glioma tumor line, allows for characterization of cell:cell interactions. Structural changes in the tissue microenvironment can be highlighted through the addition of intra-vital dyes and antibodies, for example CD31 tagged antibodies and Dextran molecules.
Moreover, we describe the combination of our ICW imaging model with a small animal micro-irradiator that provides stereotactic irradiation, creating a platform through which the dynamic tissue changes that occur following the administration of ionizing irradiation can be assessed.
Current limitations of our model include penetrance of the microscope, which is limited to a depth of up to 900 &mu;m from the sub cortical surface, limiting imaging to the dorsal axis of the brain. The presence of the skull bone makes the ICW a more challenging technical procedure, compared to the more established and utilized chamber models currently used to study mammary tissue and fat pads 5-7. In addition, the ICW provides many challenges when optimizing the imaging.

A Novel High-resolution In vivo Imaging Technique to Study the Dynamic Response of Intracranial Structures to Tumor Growth and Therapeutics

We describe a novel in vivo imaging technique that couples fluorescent chimeric mice with intracranial windows and high-resolution 2-photon microscopy. This imaging platform aids studies of dynamic changes in brain tissue and microvasculature, at a single-cell level, following pathological insults and is adaptable to assess intracranial drug delivery and distribution.

新規高分解能<em生体内で&gt;</em腫瘍増殖と治療に頭蓋内構造物の動的応答を研究する&gt;イメージング技術

We have successfully integrated previously established Intracranial window (ICW) technology 1-4 with intravital 2-photon confocal microscopy to develop a ...

High-frequency Ultrasound Imaging of Mouse Cervical Lymph Nodes

High-frequency ultrasound (HFUS) is widely employed as a non-invasive method for imaging internal anatomic structures in experimental small animal systems. HFUS has the ability to detect structures as small as 30 &#181;m, a property that has been utilized for visualizing superficial lymph nodes in rodents in brightness (B)-mode. Combining power Doppler with B-mode imaging allows for measuring circulatory blood flow within lymph nodes and other organs. While HFUS has been utilized for lymph node imaging in a number of mouse&#160; model systems, a detailed protocol describing HFUS imaging and characterization of the cervical lymph nodes in mice has not been reported. Here, we show that HFUS can be adapted to detect and characterize cervical lymph nodes in mice. Combined B-mode and power Doppler imaging can be used to detect increases in blood flow in immunologically-enlarged cervical nodes. We also describe the use of B-mode imaging to conduct fine needle biopsies of cervical lymph nodes to retrieve lymph tissue for histological&#160; analysis. Finally, software-aided steps are described to calculate changes in lymph node volume and to visualize changes in lymph node morphology following image reconstruction. The ability to visually monitor changes in cervical lymph node biology over time provides a simple and powerful technique for the non-invasive monitoring of cervical lymph node alterations in preclinical mouse models of oral cavity disease.

This protocol describes the application of high-frequency ultrasound (HFUS) for imaging mouse cervical lymph nodes. This technique optimizes visualization and quantification of cervical lymph node morphology, volume and blood flow. Image-guided biopsy of cervical lymph nodes and processing of lymph tissue for histological evaluation is also demonstrated.

マウス子宮頸部リンパ節の高周波超音波イメージング

High-frequency ultrasound (HFUS) is widely employed as a non-invasive method for imaging internal anatomic structures in experimental small animal ...

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 ...

Bioluminescence-Based Tumor Quantification Method for Monitoring Tumor Progression and Treatment Effects in Mouse Lymphoma Models

Although bioluminescence imaging (BLI) shows promise for monitoring tumor burden in animal models of cancer, these analyses remain mostly qualitative. Here we describe a method for bioluminescence imaging to obtain a semi-quantitative analysis of tumor burden and treatment response. This method is based on the calculation of a luminoscore, a value that allows comparisons of two animals from the same or different experiments. Current BLI instruments enable the calculation of this luminoscore, which relies mainly on the acquisition conditions (back and front acquisitions) and the drawing of the region of interest (manual markup around the mouse). Using two previously described mouse lymphoma models based on cell engraftment, we show that the luminoscore method can serve as a noninvasive way to verify successful tumor cell inoculation, monitor tumor burden, and evaluate the effects of in situ cancer treatment (CpG-DNA). Finally, we show that this method suits different experimental designs. We suggest that this method be used for early estimates of treatment response in preclinical small-animal studies.

Bioluminescence imaging is a well-known tool for localizing tumors and metastases, but quantification of these images often requires complex calculations and particular instruments. We describe the easy-to-use luminoscore method, based on precise acquisition conditions, requiring no calculations, and enabling tumor burden and treatment response to be monitored in mouse models.

マウスリンパ腫モデルで腫瘍の進行と治療効果を監視するための生物発光に基づく腫瘍の定量法

Although bioluminescence imaging (BLI) shows promise for monitoring tumor burden in animal models of cancer, these analyses remain mostly qualitative. ...

Ex vivo Live Imaging of Lung Metastasis and Their Microenvironment

Metastasis is a major cause for cancer-related morbidity and mortality. Metastasis is a multistep process and due to its complexity, the exact cellular and molecular processes that govern metastatic dissemination and growth are still elusive. Live imaging allows visualization of the dynamic and spatial interactions of cells and their microenvironment. Solid tumors commonly metastasize to the lungs. However, the anatomical location of the lungs poses a challenge to intravital imaging. This protocol provides a relatively simple and quick method for ex vivo live imaging of the dynamic interactions between tumor cells and their surrounding stroma within lung metastasis. Using this method, the motility of cancer cells as well as interactions between cancer cells and stromal cells in their microenvironment can be visualized in real time for several hours. By using transgenic fluorescent reporter mice, a fluorescent cell line, injectable fluorescently labeled molecules and/or antibodies, multiple components of the lung microenvironment can be visualized, such as blood vessels and immune cells. To image the different cell types, a spinning disk confocal microscope that allows long-term continuous imaging with rapid, four-color image acquisition has been used. Time-lapse movies compiled from images collected over multiple positions and focal planes show interactions between live metastatic and immune cells for at least 4 hr. This technique can be further used to test chemotherapy or targeted therapy. Moreover, this method could be adapted for the study of other lung-related pathologies that may affect the lung microenvironment.

Ex vivo Live Imaging of Lung Metastasis and Their Microenvironment

We describe a relatively simple method for ex vivo live imaging of the tumor cell-stroma interactions within lung metastasis, utilizing fluorescent reporters in mice. Using spinning-disk confocal microscopy, this technique enables visualization of live cells for at least 4 hr and could be adapted to study other inflammatory lung conditions.

ex vivoでのライブ肺転移のイメージングとその微小環境

Metastasis is a major cause for cancer-related morbidity and mortality. Metastasis is a multistep process and due to its complexity, the exact cellular ...