Name: live imaging to study microtubule dynamic instability in taxane-resistant breast cancers
Uploaded: 2017-02-20T13:00:00.000+00:00
Duration: 6 min 2 s
Description: Watch this Scientific Journal Video about Live Imaging to Study Microtubule Dynamic Instability in Taxane-resistant Breast Cancers at JoVE.com

This research is supported by funding from CBCF (to ZW).

1.ライブイメージング用の細胞を準備<ol><li> 細胞培養および播種 <ol><li>ドセタキセル（MCF-7 TXT）及びその非耐性の親細胞株（MCF-7 CC）に対する耐性について選択されたMCF-7乳癌細胞を使用します。詳細な選択プロセスおよびこれらの選択された細胞株の特徴づけは、以前24に記載しました。 </li><li>ダルベッコ改変イーグル培地（DMEM）及びウシ胎児血清（FBS）10％90％からなる非必須アミノ酸を補充した培地中で、37℃で10cmの培養皿内のすべての細胞を増殖させます。 5％CO 2雰囲気中で培地を維持します。 5 nMのドセタキセルでMCF-7 TXT細胞を維持します。 </li><li>ライブイメージングのためのカバースリップ上にシード細胞。 70％アルコールでそれらをすすぎ、次いで一晩UVへの露光24によってmmのポリ-L-リジンでコーティングしたカバーガラスを滅菌します。 35ミリメートルの6ウェル培養プレートにカバースリップを置きます。 PL各ウェル中のエース1カバーガラス。 </li><li> 10 cmの培養皿から培地を除去し、PBSで細胞を洗浄。皿から細胞を剥離するために皿にトリプシンEDTAの0.5 mLを加え。細胞の剥離、次のトリプシンを中和するために皿に培地の5ミリリットルを追加します。 </li><li>単位体積あたりの細胞の数を決定するために、血球計数器で細胞を数えます。 </li><li>各ウェルの単位体積当たりの細胞数に基づいて、およそ10 5個の細胞を加えます。静かに振とうした後、細胞をカバーグラスに付着することを可能にするためにインキュベーターにバックプレートを置きます。 </li></ol></li><li> GFPタグ付きαチューブリンの発現 <ol><li>微小管ダイナミクスをアッセイするために、製造業者のプロトコルに従って、商業GFP伝達制御（ 例えば、BacMamによって）でGFPタグ付きαチューブリンで細胞を形質導入します。 </li><li> 37℃の細胞培養インキュベーター中で36時間、ウェル中の細胞は、COM可能にする培養plete接着および60％のコンフルエンス。 </li><li>次式に従って、各ウェルに追加するには、GFPタグαチューブリンの適切な量を計算します。 <img alt="方程式" src="/files/ftp_upload/55027/55027eq1.jpg" /> 細胞数は、標識の際にウェルの細胞の推定総数であり、所望のPPCは、セル当たりの粒子数です。 注：MCF-7細胞を播種は20の所望のPPCと10 5〜2×10から細胞数を倍増している必要があり、各カバースリップに必要な量は、したがって、40μLです。異なる細胞については、上記の式に基づいて算出ボリュームが最善ではないかもしれません。正確な量は、GFP-チューブリンの実際の発現レベルに応じて調整する必要があります。 </li><li>均一な溶液を確保するために反転させることによって、GFPタグ付きαチューブリンを数回混ぜます。ボルテックスしないでください。 注：コントロールのために、ヌル（コントロール）試薬は、任意の哺乳動物の遺伝子を欠いています要素とは、潜在的なバキュロウイルス媒介性効果とバックグラウンド蛍光を決定するのを助けるために使用することができます。非標的GFP伝達制御もセル全体を点灯しており、使用することができます。 </li><li>新鮮な培地で培養液を交換してください。各ウェルに新しいメディアの2 mLおよびGFPタグ付きαチューブリンの40μLを追加します。 </li><li>培養培地でGFPタグαチューブリンの完全な混合を可能にするために、静かにプレートを振ります。培養プレートをインキュベーターに戻し、GFPチューブリンの発現を可能にするために、24時間インキュベートします。 </li></ol></li><li> ドセタキセルで細胞を扱います 注：この実験は、微小管動的不安定性に対するドセタキセルの効果をテストすることです。したがって、細胞は、ドセタキセルの所望の濃度で処理しました。 <ol><li> DMEM 90％FBSおよび10％を構成し、非必須アミノ酸を補充した培地を調製。フェノールレッドはfluorescenに干渉する可能性があるため、フェノールレッドを含まないDMEMを使用しますGFPチューブリンのCE。 （10 nMの〜10μMの範囲）ドセタキセルの所望の濃度を追加します。ドセタキセルの原液をジメチルスルホキシド（DMSO）中10mMです。 </li><li>組織培養インキュベーター中で37°Cにドセタキセル含有培地を予め温めます。 </li><li>ドセタキセル含有培地での通常の培養培地を交換し、組織培養インキュベーター中で30分間インキュベートします。各ドセタキセル濃度についてウェルあたり少なくとも3カバースリップがあるはずです。 </li></ol></li></ol> 2.ライブイメージングは​​、微小管の動的不安定性を検査します<ol><li> システムのセットアップ <ol><li> 37℃、5％CO 2に維持し、チャンバ内の実験を行います。倒立蛍光顕微鏡にCCDカメラで60X、1.42 NA油の対物レンズを搭載した顕微鏡システムを用いて、時間経過により蛍光画像を取得します。 </li><li>ライブイメージングのための細胞を設定します。プリ暖かい両方の試料ホルダーと37°Cまでの媒体。調べるために、カバースリップを選択してください。試料ホルダーに関心のカバースリップをマウントして、ステップ1.3.1で製造したドセタキセル含有培地1mLでそれをインキュベートします。 </li></ol></li><li> 微小管動的不安定性のライブイメージング <ol><li>イメージングのために使用される細胞を同定します。彼らは、平坦で発現したGFP-チューブリンの高い蛍光強度を持ち、明確な微小管構造を有していなければなりません。 </li><li>まず調べるために、ステップ2.2.1から同定された細胞の群からの1つのセルを選択します。同定された細胞の周辺領域に焦点を当てています。露光時間と画像取得の頻度のためのプログラムを設定します。露光時間は、通常0.5秒であるように、画像は、あらゆる2つのSを取得します。 </li><li> 1.33から2.2.3へのステップのための追加の20分を許可します。このように、正確に50分ドセタキセルを加えた後、ステップ1.3.3で説明したように、（これは標準化のためである）イメージングを開始します。 </li><li>微小管dを記録しynamics 2分間、写真ごとに2秒を取ります。合計では、同定された細胞のための60の画像を取得します。 </li><li>次の識別されたセルに移動し、手順を繰り返し2.2.4に2.2.2。 5個の細胞が撮像されるまでの手順を繰り返します。時間は、ドセタキセルの添加後70分に到達する前に、ステップ1.3.3に記載のように、これを行うことが不可欠です。 </li><li>各処理条件については、繰り返しは3つの異なるカバースリップのために2.2.5に2.2.1を繰り返します。合計では、画像15細胞、十分なデータが微小管の動的不安定性を測定することです。 </li></ol></li></ol> 3.画像処理およびデータ解析<ol><li> 画像のデコンボリューション <ol><li>高品質を達成するために取得した画像をデコンボリューション。顕微鏡システムを装備したデコンボリューションプログラムを開きます。 </li><li> 「プロセス」をクリックして選択し、 &quot;逆畳み込みを。」 「入力」ウィンドウに画像ファイルをドラッグし、クリックして &quot;Doを」画像ファイルは、デコンボリューション、およびれますデコンボリューションファイルは自動的に新しいファイルとして保存されます。 </li></ol></li><li> 動画の生成 <ol><li>装備ソフトウェアで開くように復元画像ファイルをクリックします。 </li><li> 「ファイル」をクリックして選択し、「ムービーとして保存します。」 「AV」と「アニメーションスタイル」と「ムービーのフォーマット」を選択し「進みます。」 「100％」から「圧縮品質」と「フレームレート」を設定します &quot;5 /秒。」 </li><li> 「時間を通してアニメイト」のボックスをチェックして、クリックして &quot;それをしなさい。」ビデオが生成されます。 </li></ol></li><li> 微小管の短縮と成長率の測定 <ol><li>測定に使用される微小管を同定します。各セルについて、ほんの数微小管は、その短縮や成長がはっきりと完全に従っていることができるようになります。 </li><li>微小管Aの開始長さを示すフレームを識別するために、ビデオの各フレームを調べますndはほとんどの延長または短縮微小管とフレーム。 2微小管と2フレーム間の経過時間と長さの変化を測定します。 </li><li>時間によって長さの変化を分割することによって増殖または短縮の速度を計算する（単位はミクロン/ sです）。各処理条件については、少なくとも10個の細胞に従うと、少なくとも20微小管を測定します。 </li><li>各処理条件に対する平均と標準誤差を計算します。学生の試験を使用して条件間の統計的差異を分析します。表中および/またはグラフ内のデータを報告します。 </li></ol></li></ol>

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著者は、彼らが競合する金融利害関係を持たないことを宣言します。

in vitroおよびin vivoで ：微小管動的不安定性を測定するための2つの主要な方法があります。 インビトロの方法では、精製されたチューブリンは、コンピュータ増強経時微分干渉コントラスト顕微鏡法を用いて微小管の動的不安定性を測定するために使用されます。 インビボ法では、蛍光チューブリンのマイクロインジェクション、またはGFPチューブ?...

<html>乳癌死亡率の主要な原因は、転移1,2を介してです。例えば、ドセタキセルおよびパクリタキセルのようなタキサンは、現在、転移性乳癌2、3、4、5、6の処置における第一選択レジメンとして使用されます。彼らは、微小管ダイナミクスを混乱させる微小管標的とするエージェント（MTA）のグループの一部です。しかし、治癒的療法にタキサンを使用する最大の課題の一つは、疾患再発7につながる、癌細胞におけるタキサン耐性の発生です。薬剤耐性は、転移性乳癌7患者における全死亡の90％以上を占めています。 微小管は、α-およびβチューブリンヘテロ二量体の重合により形成されていますクラス= &quot;外部参照&quot;&gt; 8、9。微小管動態の正確な調節は、細胞の分極、細胞周期の進行、細胞内輸送、および細胞シグナル伝達を含む多くの細胞機能にとって重要です。微小管およびそれらの力学の調節不全は、細胞機能を破壊し、細胞死10、11をもたらします。彼らは、この調節不全を引き起こす方法に応じて、MTAの薬物は、微小管安定化剤（ 例えば、タキサン）または微小管destabalizing剤（ すなわち、ビンカアルカロイドまたはコルヒチン部位結合剤）20のいずれかに分類することができます。微小管の質量に対するその反対の効果にもかかわらず、十分な用量で、両方のクラスは、微小管ダイナミクス21への影響を介してがん細胞を殺すことができます。 主につながる、微小管スピンドル12を安定化させることにより、タキサン機能染色体不整列。紡錘体チェックポイント（SAC）のその後の永遠の活性化は、有糸分裂中の細胞を逮捕します。長時間の有糸分裂の停止は、その後アポトーシス13、14が発生します 。タキサンは、組み立てられたチューブリン16にのみ存在するβチューブリン8、15、上タキサン結合部位を介して微小管と相互作用します。 タキサン耐性のための複数のメカニズムは、 図9に示すように 、17が提案されています。これらのメカニズムは、原因薬剤排出タンパク質の過剰発現およびタキサン固有抵抗5への一般的な多剤耐性、9、18、19の両方が含まれます。例えば、タキサン耐性癌細胞は、特定のβ-浴槽の発現および機能が変化していてもよいですグロブリンは、5、9、19、20、21、22、23をアイソタイプ。微小管の動的不安定性を測定するためのin vivo法を用いて、我々は、非耐性、親MCF-7 CCセル 17と比較した場合、ドセタキセル耐性MCF-7 TXT細胞の微小管の動態は、ドセタキセル治療に非感受性であることを示しています。 より良いのMTAの機能と癌細胞におけるタキサン耐性の正確なメカニズムを理解するためには、微小管動態を測定することが不可欠です。ここで、我々はそうすることのin vivo法を報告しています。細胞中のGFPタグ付きチューブリンの発現と組み合わせてライブイメージングを用いて、我々は、MCF-7 TXTおよびMCF-7 CC細胞ととWiの微小管動態を測定することができますドセタキセル治療thout。結果は、私たちは、タキサン抵抗性を克服することができ、より効果的な薬物を設計するのに役立つことができます。

<table><tbody><tr><td>Dulbecco&#039;s Modified Eagle&#039;s Medium (DMEM)</td><td>Sigma-Aldrich</td><td>D5796</td><td></td></tr><tr><td>Non-essential amino acids</td><td>Life Technologies, Invitrogen</td><td>11140-050</td><td></td></tr><tr><td>FBS</td><td>Gibco, Invitrogen</td><td>12483</td><td></td></tr><tr><td>Anti-Anti (100x)</td><td>Life Technologies, Invitrogen</td><td>15240-062</td><td></td></tr><tr><td>docetaxel</td><td>Sigma-Aldrich</td><td>01885-5mg-F</td><td></td></tr><tr><td>DMEM phenol red-free</td><td>Gibco, Invitrogen</td><td>21063</td><td></td></tr><tr><td>CellLight Reagent *BacMam 2.0* GFP-tubulin</td><td>ThermoFisher Scientific</td><td>C10613</td><td>Key reagent for expressing GFP tubulin in cells</td></tr><tr><td>CellLight Reagent *BacMam 2.0* GFP</td><td>ThermoFisher Scientific</td><td>B10383</td><td>Control</td></tr><tr><td>Dimethyl Sulfoxide (DMSO)</td><td>Sigma-Aldrich+B9:AA9</td><td>472301</td><td>for dissoving decetaxel</td></tr><tr><td>22-mm glass coveslip</td><td>Fisher Scientifics</td><td>12-545-101</td><td></td></tr><tr><td>6-well culture plate</td><td>Greiner Bio-One International</td><td>6 Well Celi Culture Plate</td><td></td></tr><tr><td>DeltaVision Microscopy Imaging Systems</td><td>GE Health</td><td></td><td>This system is equipped with weather station for controlling temperature and CO2. It also equipped with Worx Software for deconvolution and time lapse control.</td></tr><tr><td>Trypsin-EDTA (0.25%), phenol red</td><td>ThermoFisher Scientific</td><td>25200056</td><td></td></tr><tr><td>Bright-Line Hemacytometer Set, Hausser Scientific</td><td>Hausser Scientific, Distributed by VWR</td><td>Supplier No.: 1492 VWR No.:15170-172</td><td></td></tr></tbody></table>

live imaging to study microtubule dynamic instability in taxane-resistant breast cancers

Taxanes such as docetaxel belong to a group of microtubule-targeting agents (MTAs) that are commonly relied upon to treat cancer. However, taxane resistance in cancerous cells drastically reduces the effectiveness of the drugs' long-term usage. Accumulated evidence suggests that the mechanisms underlying taxane resistance include both general mechanisms, such as the development of multidrug resistance due to the overexpression of drug-efflux proteins, and taxane-specific mechanisms, such as those that involve microtubule dynamics. 
Because taxanes target cell microtubules, measuring microtubule dynamic instability is an important step in determining the mechanisms of taxane resistance and provides insight into how to overcome this resistance. In the experiment, an in vivo method was used to measure microtubule dynamic instability. GFP-tagged &#945;-tubulin was expressed and incorporated into microtubules in MCF-7 cells, allowing for the recording of the microtubule dynamics by time lapse using a sensitive camera. The results showed that, as opposed to the non-resistant parental MCF-7CC cells, the microtubule dynamics of docetaxel-resistant MCF-7TXT cells are insensitive to docetaxel treatment, which causes the resistance to docetaxel-induced mitotic arrest and apoptosis. This paper will outline this in vivo method of measuring microtubule dynamic instability.

ここに提示されたプロトコルを使用して、我々は、通常の微小管動態（MCF-7 CC）とドセタキセル耐性（MCF-7 TXT）乳癌細胞に対するドセタキセルの効果を研究しました。画像の二組は、MCF-7 CC及びMCF-7 TXT細胞 （ 図1A）における微小管の成長と短縮にドセタキセル（0.5μM）の効果を示します。 <p class="jove_content" fo:keep-together....

Watch this Scientific Journal Video about Live Imaging to Study Microtubule Dynamic Instability in Taxane-resistant Breast Cancers at JoVE.com

Live Imaging to Study Microtubule Dynamic Instability in Taxane-resistant Breast Cancers

In this paper, we report a protocol describing an in vivo method to measure microtubule dynamic instability in docetaxel-resistant breast cancer cells (MCF-7TXT). In this method, a deconvolution microscopy imaging system is used to detect the expression of GFP-tubulin in target cells.

ライブイメージングは​​、タキサン耐性乳癌における微小管の動的不安定性を研究するために

Taxanes such as docetaxel belong to a group of microtubule-targeting agents (MTAs) that are commonly relied upon to treat cancer. However, taxane ...

live-imaging-to-study-microtubule-dynamic-instability-taxane

Universidad de Cartagena UDEC

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JoVE Core

JoVE Journal

Cell Biology

Molecular Biology

Cancer Research

Unit 2

Unit 1

Cell Division

The Cytoskeleton II: Microtubules and Intermediate Filaments

SCIENTISTS IN ACTION

7.5K ビュー.  University of Alberta. In this paper, we report a protocol describing an in vivo method to measure microtubule dynamic instability in docetaxel-resistant breast cancer cells (MCF-7TXT). In this method, a deconvolution microscopy imaging system is used to detect the expression of GFP-tubulin in target cells. 

ビデオ: Live Imaging to Study Microtubule Dynamic Instability in Taxane-resistant Breast Cancers

Live-imaging of Breast Epithelial Cell Migration After the Transient Depletion of TIP60

The wound-healing assay is efficient and one of the most economical ways to study cell migration in vitro. Conventionally, images are taken at the beginning and end of an experiment using a phase-contrast microscope, and the migration abilities of cells are evaluated by the closure of wounds. However, cell movement is a dynamic phenomenon, and a conventional method does not allow for tracking single-cell movement. To improve current wound-healing assays, we use live-cell imaging techniques to monitor cell migration in real time. This method allows us to determine the cell migration rate based on a cell tracking system and provides a clearer distinction between cell migration and cell proliferation. Here, we demonstrate the use of live-cell imaging in wound-healing assays to study the different migration abilities of breast epithelial cells influenced by the presence of TIP60. As cell motility is highly dynamic, our method provides more insights into the processes of wound healing than a snapshot of wound closure taken with the traditional imaging techniques used for wound-healing assays.

Here, we present the real-time monitoring of cell migration in a wound-healing assay using TIP60-depleted MCF10A breast epithelial cells. The implementation of live-cell imaging techniques in our protocol allows us to analyze and visualize single-cell movement in real time and across time.

乳腺上皮細胞移行 TIP60 の一時的な枯渇後のライブ イメージング

The wound-healing assay is efficient and one of the most economical ways to study cell migration in vitro. Conventionally, images are taken at the ...

がん研究

Monitoring Tumor Metastases and Osteolytic Lesions with Bioluminescence and Micro CT Imaging

Following intracardiac delivery of MDA-MB-231-luc-D3H2LN cells to Nu/Nu mice, systemic metastases developed in the injected animals. Bioluminescence imaging using IVIS Spectrum was employed to monitor the distribution and development of the tumor cells following the delivery procedure including DLIT reconstruction to measure the tumor signal and its location.
Development of metastatic lesions to the bone tissues triggers osteolytic activity and lesions to tibia and femur were evaluated longitudinally using micro CT. Imaging was performed using a Quantum FX micro CT system with fast imaging and low X-ray dose. The low radiation dose allows multiple imaging sessions to be performed with a cumulative X-ray dosage far below LD50. A mouse imaging shuttle device was used to sequentially image the mice with both IVIS Spectrum and Quantum FX achieving accurate animal positioning in both the bioluminescence and CT images. The optical and CT data sets were co-registered in 3-dimentions using the Living Image 4.1 software. This multi-mode approach allows close monitoring of tumor growth and development simultaneously with osteolytic activity.

An experimental mouse model of bone metastasis was established following intracardiac delivery of luciferase expressing mammary tumor cells. Tumor development and resulted osteolytic lesion were monitored longitudinally with bioluminescence and micro CT imaging.

生物発光とマイクロCTイメージングで腫瘍転移と溶骨性病変の監視

Following intracardiac delivery of MDA-MB-231-luc-D3H2LN cells to Nu/Nu mice, systemic metastases developed in the injected animals. Bioluminescence ...

医学

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 ...

Long-term Live-cell Imaging to Assess Cell Fate in Response to Paclitaxel

Live-cell imaging is a powerful technique that can be used to directly visualize biological phenomena in single cells over extended periods of time. Over the past decade, new and innovative technologies have greatly enhanced the practicality of live-cell imaging. Cells can now be kept in focus and continuously imaged over several days while maintained under 37 &#176;C and 5% CO2 cell culture conditions. Moreover, multiple fields of view representing different experimental conditions can be acquired simultaneously, thus providing high-throughput experimental data. Live-cell imaging provides a significant advantage over fixed-cell imaging by allowing for the direct visualization and temporal quantitation of dynamic cellular events. Live-cell imaging can also identify variation in the behavior of single cells that would otherwise have been missed using population-based assays. Here, we describe live-cell imaging protocols to assess cell fate decisions following treatment with the anti-mitotic drug paclitaxel. We demonstrate methods to visualize whether mitotically arrested cells die directly from mitosis or slip back into interphase. We also describe how the fluorescent ubiquitination-based cell cycle indicator (FUCCI) system can be used to assess the fraction of interphase cells born from mitotic slippage that are capable of re-entering the cell cycle. Finally, we describe a live-cell imaging method to identify nuclear envelope rupture events.

Live-cell imaging provides a wealth of information on single cells or whole populations that is unattainable by fixed cell imaging alone. Here, live-cell imaging protocols to assess cell fate decisions following treatment with the anti-mitotic drug paclitaxel are described.

長期的な住セルイメージ投射細胞の運命をパクリタ キセルに応答を評価するために

Live-cell imaging is a powerful technique that can be used to directly visualize biological phenomena in single cells over extended periods of time. Over ...

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 ...

Time-lapse Imaging of Primary Preneoplastic Mammary Epithelial Cells Derived from Genetically Engineered Mouse Models of Breast Cancer

Time-lapse imaging can be used to compare behavior of cultured primary preneoplastic mammary epithelial cells derived from different genetically engineered mouse models of breast cancer. For example, time between cell divisions (cell lifetimes), apoptotic cell numbers, evolution of morphological changes, and mechanism of colony formation can be quantified and compared in cells carrying specific genetic lesions. Primary mammary epithelial cell cultures are generated from mammary glands without palpable tumor. Glands are carefully resected with clear separation from adjacent muscle, lymph nodes are removed, and single-cell suspensions of enriched mammary epithelial cells are generated by mincing mammary tissue followed by enzymatic dissociation and filtration. Single-cell suspensions are plated and placed directly under a microscope within an incubator chamber for live-cell imaging. Sixteen 650 &mu;m x 700 &mu;m fields in a 4x4 configuration from each well of a 6-well plate are imaged every 15 min for 5 days. Time-lapse images are examined directly to measure cellular behaviors that can include mechanism and frequency of cell colony formation within the first 24 hr of plating the cells (aggregation versus cell proliferation), incidence of apoptosis, and phasing of morphological changes. Single-cell tracking is used to generate cell fate maps for measurement of individual cell lifetimes and investigation of cell division patterns. Quantitative data are statistically analyzed to assess for significant differences in behavior correlated with specific genetic lesions.

Time-lapse imaging is used to assess behavior of primary preneoplastic mammary epithelial cells derived from genetically engineered mouse models of breast cancer risk to determine if there are correlations between specific behavioral parameters and distinct genetic lesions.

乳がんの遺伝子改変マウスモデル由来の初代前癌乳腺上皮細胞のタイムラプスイメージング

Time-lapse  imaging can be used to compare behavior of cultured primary preneoplastic  mammary epithelial cells derived from different genetically ...

Real-time Imaging of Myeloid Cells Dynamics in ApcMin/+ Intestinal Tumors by Spinning Disk Confocal Microscopy

Myeloid cells are the most abundant immune cells within tumors and have been shown to promote tumor progression. Modern intravital imaging techniques enable the observation of live cellular behavior inside the organ but can be challenging in some types of cancer due to organ and tumor accessibility such as intestine. Direct observation of intestinal tumors has not been previously reported. A surgical procedure described here allows direct observation of myeloid cell dynamics within the intestinal tumors in live mice by using transgenic fluorescent reporter mice and injectable tracers or antibodies. For this purpose, a four-color, multi-region, micro-lensed spinning disk confocal microscope that allows long-term continuous imaging with rapid image acquisition has been used. ApcMin/+ mice that develop multiple adenomas in the small intestine are crossed with c-fms-EGFP mice to visualize myeloid cells and with ACTB-ECFP mice to visualize intestinal epithelial cells of the crypts. Procedures for labeling different tumor components, such as blood vessels and neutrophils, and the procedure for positioning the tumor for imaging through the serosal surface are also described. Time-lapse movies compiled from several hours of imaging allow the analysis of myeloid cell behavior in situ in the intestinal microenvironment.

Real-time Imaging of Myeloid Cells Dynamics in ApcMin/+ Intestinal Tumors by Spinning Disk Confocal Microscopy

By using transgenic reporter mice and injectable fluorescent labels, long-term intravital spinning disk confocal microscopy enables direct visualization of myeloid cell behavior into intestinal adenoma in the ApcMin/+ colorectal cancer model.

における骨髄細胞ダイナミクスのリアルタイムイメージング<em&gt;のApc<sup&gt;最小/ +</sup</em&gt;腸腫瘍ディスク共焦点顕微鏡スピニングすることにより

Myeloid cells are the most abundant immune cells within tumors and have been shown to promote tumor progression. Modern intravital imaging techniques ...

免疫・感染学

Simultaneous Visualization of the Dynamics of Crosslinked and Single Microtubules In Vitro by TIRF Microscopy

Microtubules are polymers of &#945;&#946;-tubulin heterodimers that organize into distinct structures in cells. Microtubule-based architectures and networks often contain subsets of microtubule arrays that differ in their dynamic properties. For example, in dividing cells, stable bundles of crosslinked microtubules coexist in close proximity to dynamic non-crosslinked microtubules. TIRF-microscopy-based in vitro reconstitution studies enable the simultaneous visualization of the dynamics of these different microtubule arrays. In this assay, an imaging chamber is assembled with surface-immobilized microtubules, which are either present as single filaments or organized into crosslinked&#160;bundles. Introduction of tubulin, nucleotides, and protein regulators allows direct visualization of associated proteins and of dynamic properties of single and crosslinked microtubules. Furthermore, changes that occur as dynamic single microtubules organize into bundles can be monitored in real-time. The method described here allows for a systematic evaluation of the activity and localization of individual proteins, as well as synergistic effects of protein regulators on two different microtubule subsets under identical experimental conditions, thereby providing mechanistic insights that are inaccessible by other methods.

Simultaneous Visualization of the Dynamics of Crosslinked and Single Microtubules In Vitro by TIRF Microscopy

Here, a TIRF microscopy-based in vitro reconstitution assay is presented to simultaneously quantify and compare the dynamics of two microtubule populations. A method is described to simultaneously view the collective activity of multiple microtubule-associated proteins on crosslinked microtubule bundles and single microtubules.

TIRF顕微鏡による インビトロでの 架橋小管と単一微小管のダイナミクスの同時可視化

Microtubules are polymers of &#945;&#946;-tubulin heterodimers that organize into distinct structures in cells. Microtubule-based architectures and ...

生化学

Through the Looking Glass: Time-lapse Microscopy and Longitudinal Tracking of Single Cells to Study Anti-cancer Therapeutics

The response of single cells to anti-cancer drugs contributes significantly in determining the population response, and therefore is a major contributing factor in the overall outcome. Immunoblotting, flow cytometry and fixed cell experiments are often used to study how cells respond to anti-cancer drugs. These methods are important, but they have several shortcomings. Variability in drug responses between cancer and normal cells, and between cells of different cancer origin, and transient and rare responses are difficult to understand using population averaging assays and without being able to directly track and analyze them longitudinally. The microscope is particularly well suited to image live cells. Advancements in technology enable us to routinely image cells at a resolution that enables not only cell tracking, but also the observation of a variety of cellular responses. We describe an approach in detail that allows for the continuous time-lapse imaging of cells during the drug response for essentially as long as desired, typically up to 96 hr. Using variations of the approach, cells can be monitored for weeks. With the employment of genetically encoded fluorescent biosensors numerous processes, pathways and responses can be followed. We show examples that include tracking and quantification of cell growth and cell cycle progression, chromosome dynamics, DNA damage, and cell death. We also discuss variations of the technique and its flexibility, and highlight some common pitfalls.

Here, we describe a method of long-term time-lapse microscopy to longitudinally track single cells in response to anti-cancer therapeutics.

鏡の国のアリス：単一細胞のタイムラプス顕微鏡と縦トラッキング抗がん治療薬を研究します

The response of single cells to anti-cancer drugs contributes significantly in determining the population response, and therefore is a major contributing ...

Extended Time-lapse Intravital Imaging of Real-time Multicellular Dynamics in the Tumor Microenvironment

In the tumor microenvironment, host stromal cells interact with tumor cells to promote tumor progression, angiogenesis, tumor cell dissemination and metastasis. Multicellular interactions in the tumor microenvironment can lead to transient events including directional tumor cell motility and vascular permeability. Quantification of tumor vascular permeability has frequently used end-point experiments to measure extravasation of vascular dyes. However, due to the transient nature of multicellular interactions and vascular permeability, the kinetics of these dynamic events cannot be discerned. By labeling cells and vasculature with injectable dyes or fluorescent proteins, high-resolution time-lapse intravital microscopy has allowed the direct, real-time visualization of transient events in the tumor microenvironment. Here we describe a method for using multiphoton microscopy to perform extended intravital imaging in live mice to directly visualize multicellular dynamics in the tumor microenvironment. This method details cellular labeling strategies, the surgical preparation of a mammary skin flap, the administration of injectable dyes or proteins by tail vein catheter and the acquisition of time-lapse images. The time-lapse sequences obtained from this method facilitate the visualization and quantitation of the kinetics of cellular events of motility and vascular permeability in the tumor microenvironment.

This protocol describes the use of multiphoton microscopy to perform extended time-lapse imaging of multicellular interactions in real time, in vivo at single cell resolution.

腫瘍微小環境におけるリアルタイム多細胞ダイナミクスの拡張タイムラプス生体内イメージング

In the tumor microenvironment, host stromal cells interact with tumor cells to promote tumor progression, angiogenesis, tumor cell dissemination and ...

ライブイメージングは、タキサン耐性乳癌における微小管の動的不安定性を研究するために

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乳腺上皮細胞移行 TIP60 の一時的な枯渇後のライブイメージング

生物発光とマイクロCTイメージングで腫瘍転移と溶骨性病変の監視

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

長期的な住セルイメージ投射細胞の運命をパクリタキセルに応答を評価するために

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

乳がんの遺伝子改変マウスモデル由来の初代前癌乳腺上皮細胞のタイムラプスイメージング

における骨髄細胞ダイナミクスのリアルタイムイメージング

TIRF顕微鏡によるインビトロでの架橋小管と単一微小管のダイナミクスの同時可視化

鏡の国のアリス：単一細胞のタイムラプス顕微鏡と縦トラッキング抗がん治療薬を研究します

腫瘍微小環境におけるリアルタイム多細胞ダイナミクスの拡張タイムラプス生体内イメージング

ライブイメージングは​​、タキサン耐性乳癌における微小管の動的不安定性を研究するために

要約

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乳腺上皮細胞移行 TIP60 の一時的な枯渇後のライブ イメージング

生物発光とマイクロCTイメージングで腫瘍転移と溶骨性病変の監視

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

長期的な住セルイメージ投射細胞の運命をパクリタ キセルに応答を評価するために

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

乳がんの遺伝子改変マウスモデル由来の初代前癌乳腺上皮細胞のタイムラプスイメージング

における骨髄細胞ダイナミクスのリアルタイムイメージング

TIRF顕微鏡による インビトロでの 架橋小管と単一微小管のダイナミクスの同時可視化

鏡の国のアリス：単一細胞のタイムラプス顕微鏡と縦トラッキング抗がん治療薬を研究します

腫瘍微小環境におけるリアルタイム多細胞ダイナミクスの拡張タイムラプス生体内イメージング

ライブイメージングは、タキサン耐性乳癌における微小管の動的不安定性を研究するために

乳腺上皮細胞移行 TIP60 の一時的な枯渇後のライブイメージング

長期的な住セルイメージ投射細胞の運命をパクリタキセルに応答を評価するために

TIRF顕微鏡によるインビトロでの架橋小管と単一微小管のダイナミクスの同時可視化