テクニカル サポートのため博士明分チャン ・ チェンさんは屋新を感謝致します。この作品は、台湾の科学技術省によって支えられた (ほとんど-103-2325-B-006-009、ほとんど-104-2325-B-006-001、ほとんど-105-2325-B-006-001、および MOST-106-2320-B-006-068-MY3) 厚生省と福祉 (MOHW106-TDU-B-211-144004)。また、国立成功大学、医学のコア研究室から多光子顕微鏡のサポートを感謝しております。

すべてのマウスの実験は、当研究所 (ケア、NCKU 医科大学実験動物の使用のためのガイド) のガイドラインに従って行われました。
1. 楽器、文化、メディア、料理の準備
<ol><li>実験的プロトコルを開始する前に取得 (尾静脈注射) を 26 G/0.5 cm の針を用いて 1 mL 注射器滅菌手術用縫合糸、3 mL シリンジ (肺灌流) の 24 G/3 cm 針ダルベッコ変更イーグル メディア (DMEM)、トリプシン EDTA 溶液 (5グラム/L のトリプシンと 0.53 mM)、ウシ胎児血清 (FBS)、1 x PBS (137 mM NaCl、KCl 2.7 mM、10 mM Na2フォー4と 1.8 mM KH2PO4)、6 cm 文化料理。</li>
</ol>2. 準備中と懸濁液中に腫瘍細胞の回復
<ol><li>滅菌ダルベッコ変更イーグル メディア (DMEM) 10% または 20% を含む準備 FBS たて 0.05%、1 x PBS 2 mM L グルタミンを追加およびトリプシン-EDTA。</li>
	<li>10 %dmem で細胞の培養 37 ° C で FBS、培養皿で 70-80% の合流があるまでそれらを育てます。</li>
	<li>2 mL の滅菌 1x PBS で 2 回洗濯に続いて、料理から文化メディア (DMEM) を削除します。</li>
	<li>0.05 %1 mL を加えてトリプシン-EDTA 料理に徹底的にすべてのセルをカバーするため。すぐに 800 μ L のソリューションを削除し、料理にのみ 200 μ L を残します。30 の 37 ° C で料理を孵化させなさい (携帯型) によって 1 分と細胞の大半がサスペンド状態になるまで待機します。</li>
	<li>新鮮な DMEM の 1 mL を追加 10% を含むと精力的にトリプシンの酵素活性を停止するお皿に直接 FB ピペットの細胞懸濁液を上下 1000 μ L ピペット単一細胞懸濁液を準備するとします。</li>
	<li>1.5 mL 遠心チューブにお皿からセルを転送、室温で 3 分間 162 x g 細胞をスピンします。</li>
	<li>上清を吸引し、37 ° C 2 時間の懸濁液で細胞を回転 1.5 mL 20 %fbs、エンド オーバー エンドを含む DMEM で腫瘍細胞を再懸濁します。 注: 媒体は回復中に黄色に変わったら場合、は、20% を含む新鮮な培地で古い培地を交換政府短期証券。</li>
</ol>3. 蛍光染色と Carboxyfluorescein サクシニミジルエステル (CFSE) と懸濁液中に腫瘍細胞の解析
<ol><li>懸濁液中の細胞回復後、用量を染色する蛍光を滴定の室を数えるノイバウアーのトリパン ブルーと適切な生菌数をカウントし、尾静脈注射。</li>
	<li>3 分の 1 mL にペレット細胞滅菌 1x PBS、162 x g 3 分で室温の遠心分離によって続いて再懸濁します常温 162 x g で細胞をスピンします。</li>
	<li>500 μ L の 10 を含む 1x PBS でペレットの細胞を再懸濁します %fbs と 0、5、10、20、または用量調節の 40 μ M CFSE 37 oC 暗闇の中で 10 分間の細胞懸濁液を孵化させなさいと。</li>
	<li>スピン ・ ダウン 3 分間室温で 162 x g でセルと 4 ml の 1% を含む DMEM の小球形にされた細胞を再懸濁します FBS。この洗浄工程を 3 回繰り返します。DMEM 最終的な洗浄のためだけの 4 ml 小球形にされた細胞を再懸濁します。</li>
	<li>尾静脈注射や CFSE 分類された細胞を蛍光活性化細胞のソーティング (FACS) による単一細胞の蛍光強度を定量化するために対象します。 注: は、FACS 解析や尾静脈注射を実行する前に氷の上細胞を保ちます。</li>
</ol>4. 肺植民地化アッセイ
<ol><li>20 μ m CFSE 分類されるセルを準備 (ステップ 3.1 3.4 を参照)。 注: 腫瘍細胞、FACS 解析から滴定結果に基づいてラベル付けのため CFSE の作業量を決定する (ステップ 3.3 を参照)。</li>
	<li>4-6 週のツインテールを暖かい-古い男性 c57bl/6 マウス (6 マウス) マウス尾静脈を拡張するためのマウスでごとに 5-10 分のハロゲン ランプを使用。</li>
	<li>徹底的にミックスし、(5 × 106セル/mL の最終的な細胞密度) の細胞懸濁液で任意の気泡を回避 (26Gx1/2) 1 mL 注射器 CFSE 分類された腫瘍細胞を吸引します。</li>
	<li>慎重に 1 を注入 x106 CFSE 分類された腫瘍細胞マウス落で提出する必要がありますマウスの尾静脈に DMEM を 200 μ l 添加します。 注: 注入中尾静脈の内腔に針を直接配置した場合腫瘍細胞必要があります簡単に配信循環に注射器にハードにプッシュすることがなく。腫瘍細胞をプッシュするは難しいが場合、彼らほとんど挿入されている尾静脈外の皮下組織に。</li>
	<li>マウス静脈内 CFSE 分類された腫瘍細胞を受信した 2 つのグループに分ける: 1 つのグループは共焦点顕微鏡に続いて肺灌流を受けるし、肺植民地化アッセイおよび他の 1 の ImageJ 定量化が残される 4 - 5 だけで長期肺植民地における週間の試金します。</li>
	<li>20、38、45 h 以上との時間コースと用量依存の滴定の間に静脈内注入 1 × 106腫瘍細胞はよく受け入れられ、適切な anesthetizer2350 75 mg/kg zoletil 50, 担癌マウスを麻酔します。 注: 麻酔中の乾燥を防ぐためのマウスの目に獣医軟膏を使用。</li>
	<li>縦外科はさみで皮膚と胸部に腹部から皮下組織を切る。外科はさみと心臓と肺を完全に公開するための鉗子で胸腔内を開きます。 注: 血管を切断しないように注意ください。</li>
	<li>上大静脈 (SVC) と下大静脈 (IVC) 肺灌灌流液の逆流を防ぐために滅菌手術用縫合糸とを結ぶ。</li>
	<li>肺から灌流液を排出する裂 (約 2-4 mm) を開く手術ハサミで左心室の壁をカットします。</li>
	<li>1x PBS を右心室に 3 mL 注射器で注入し、肺は完全に薄いに赤みを帯びた色からターンまで肺灌一定の吸引、排水ソリューションを削除します。</li>
</ol>5. 共焦点顕微鏡イメージングと肺植民地化腫瘍細胞の解析
<ol><li>胸腔内から今薄い肺を取り外し、外科はさみ鉗子24と気管と結合組織の靭帯の切除によって 5 つの葉。</li>
	<li>肺葉の配置の 2 つの棒の間にスペースを持つ網状テクスチャーを生成する担架のような肺ホルダーに示すよう図 3 bにナイロン縫合糸を巻き約二本の金属棒を用意します。6 cm 皿肺ホルダーをセットします。 注: 肺ホルダーは、共焦点顕微鏡の対物レンズの下に肺葉を安定させるために使用されます。</li>
	<li>ロッジ、肺ホルダーの網状組織、肺葉を修正します。1x PBS で肺葉を湿らすし、カバーします。</li>
	<li>件名記録肺植民地化腫瘍細胞蛍光画像をキャプチャする共焦点顕微鏡に肺のホルダーに肺葉をかくまっている 6 cm 培養皿です。</li>
	<li>イメージングのための対物レンズを使用して、(5 X、MPLN、NA: 0.1、WD: 20、空気)。</li>
	<li>NIBA (励起/蛍光; 470-495 nm/510-550 nm) にフィルター ホイールを回転、488 nm レーザーを使って、エキサイト CFSE 明らかに肺の葉に蛍光ドットのイメージを視覚化します。</li>
	<li>(マイタイ HP DeepSee レーザー) のフィルター ホイール R690 を回転 2.0 でスキャンする μ s/ピクセル スキャン速度し、HV、ゲインとオフセットを最適化レベル。</li>
	<li>10 で顕微鏡のソフトウェアを使用して蛍光画像 (512 × 512 ピクセル) をキャプチャ μ s/ピクセル スキャン速度。</li>
	<li>定量化し、前述21として ImageJ とグラフパッド ソフトウェアを用いた顕微鏡画像の統計的な分析します。</li>
</ol>6. 長期的な肺の植民地化の試金
メモ: 手順 4.1 4.5 から繰り返します。
<ol><li>4-5 週間の腫瘍細胞接種後マウスを犠牲にし、1 ~ 2 日間ブアン液25彼らの肺を修正します。</li>
	<li>定量化し、統計的に分析ソフトウェア21マウス肺から腫瘍結節。</li>
</ol>

著者が明らかに何もありません。

一緒に長期間肺植民地化アッセイ、短期的には遠く離れた臓器に Ctc によって肺菌を体内を用いてここで我々 の方法論は明確に発表し、植民地化で Ctc に組み立てられた polyFN の特定の役割を区別肺の血管外漏出と転移プロセス18,19,20,21につながった。直接確認できなかった長期の細胞追跡 ...

転移癌死1,2の主要な原因であります。主な組織由来腫瘍細胞懸濁液の循環を入力し、彼らの前に様々 な血行性課題、例えばアノイーキス、免疫の攻撃および血圧や幾何学的な制約からせん断応力に起因する損害を生き残る遠隔臓器転移3,4,5,6の成功を口述の重要なステップを植民地化することができます。そのため、積極的な努力は現在循環腫瘍細胞 (CTCs) を特徴付けると腫瘍悪性度、転移、がん患者7,8の生存率とこれらの特性を関連付けることで行われています。,9. がん転移のプロセスは特に体内イベントを示しています、動物モデルは転移10、11,12 の完全全身プロセスをキャプチャする唯一の方法.
CTCs 遠隔臓器1,2の植民地化を含む複数の携帯電話イベントを介して転移腫瘍組織になります。ただし、最も一般的に使用される転移アッセイ13,14,15は遠隔臓器の CTC を植民地化を観察する方法を行いません。したがって、CTC の植民地化の可視化のため体内の分析設計が急務します。いくつか体内と体外の短い用語肺植民地化の試金に設計されている、問題および不利な点のまま。例えば、緑色蛍光タンパク質 (GFP) をしながらの試金の過剰発現の腫瘍細胞は、これらに使用されている22,23, 安定 transfect し、十分な GFP の蛍光強度の下で腫瘍細胞クローンの作成にかかる時間顕微鏡。同様が長期細胞トレーサー CFSE による腫瘍細胞の一時的な染色は、gfp 発現する腫瘍細胞を置き換えるに雇われている、それは CFSE 分類された腫瘍細胞が接続されているかどうかを判断することは困難または内だけに存在します摘出臓器16,17の血管。
批判的に転移腫瘍組織18,19,20,21,22 の最終的な確立に貢献する高分子フィブロネクチン (polyFN) Ctc の表面上で組み立てが見つかりました.ここでは、短期的には肺の植民地化の試金が中断されたルイス肺癌細胞 (Llc) 安定 FN shRNA (shFN) またはスクランブル shRNA (shScr) を表現し、事前の付いた CFSE c57bl/6 マウスに静脈内接種を行った。2 〜 3 日後マウス肺最初灌リン酸緩衝生理食塩水 (PBS) 受ける前に血管内の未接続の Ctc を完全に削除する共焦点顕微鏡と肺をくわえて Llc の定量化します。明確に示した肺植民地化 shFN Llc の数と腫瘍結節はあった shScr Llc、実質的に植民地化の Ctc の成長を促進する上で Ctc で polyFN アセンブリの役割を裏付けると比較して大幅に削減、肺。我々 の研究は癌転移における polyFN の役割の検討をさらに保証します。

<table><tbody><tr><td>&lt;strong&gt;Material&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Bovine Serum Albumin&amp;nbsp;(BSA)</td><td>Cyrusbioscience (Taipei, Taiwan)</td><td>101-9048-46-8</td><td></td></tr><tr><td>Bouin&#039;s Fluid</td><td>MCC(medical chemical corporation)/POISON</td><td>456-A-1GL</td><td></td></tr><tr><td>CFSE Proliferation Dye</td><td>ebiosciences</td><td>65-0850-85</td><td>Full name: Carboxyfluorescein succinimidyl ester</td></tr><tr><td>Dulbecco&#039;s Modified Eagle Media (DMEM)&amp;nbsp;</td><td>(Gibco)ThermoFisher Scientific</td><td>12100-061</td><td></td></tr><tr><td>Ethylenediaminetetraacetic acid (EDTA)</td><td>Cyrusbioscience (Taipei, Taiwan)</td><td>101-6381-92-6</td><td>For prepared trypsin-EDTA solution( Final concentration: 0.53mM )&amp;nbsp;</td></tr><tr><td>Fetal bovine serum (FBS)</td><td>(Gibco)ThermoFisher Scientific</td><td>10437-028</td><td></td></tr><tr><td>Lewis lung carcinoma (LLC)</td><td>ATCC, Manassas, VA, USA</td><td>CRL-1642</td><td></td></tr><tr><td>L-Glutamine, USP&amp;nbsp;</td><td>(Gibco)ThermoFisher Scientific</td><td>21051-024</td><td></td></tr><tr><td>Potassium chloride (KCl)</td><td>Cyrusbioscience (Taipei, Taiwan)</td><td>101-7447-40-7</td><td>For prepared 1X PBS ( Final concentration: 2.7mM )</td></tr><tr><td>Potassium phosphate monobasic (KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;)</td><td>Cyrusbioscience (Taipei, Taiwan)</td><td>101-7778-77-0</td><td>For prepared 1X PBS ( Final concentration: 1.8mM )</td></tr><tr><td>Sodium chloride (NaCl)</td><td>Cyrusbioscience (Taipei, Taiwan)</td><td>101-7647-14-5</td><td>For prepared 1X PBS ( Final concentration: 137mM )</td></tr><tr><td>Sodium phosphate dibasic (Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;)</td><td>Cyrusbioscience (Taipei, Taiwan)</td><td>101-10039-32-4</td><td>For prepared 1X PBS ( Final concentration: 10mM )</td></tr><tr><td>Trypan Blue</td><td>Sigma Aldrich</td><td>T6146</td><td>0.5 g mix with 100 mL 1X PBS</td></tr><tr><td>Trypsin</td><td>Sigma Aldrich</td><td>T4799</td><td>For prepared trypsin-EDTA solution ( Final concentration: 5g/L )</td></tr><tr><td>Zoletil 50</td><td>Virbac</td><td></td><td>To dilute with 1X PBS&amp;nbsp;</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Compact Tabletop Centrifuge 2420</td><td>KUBOTA Co.</td><td>2420</td><td></td></tr><tr><td>Culture dish (6cm)</td><td>Wuxi NEST Biotechnology Co.</td><td>705001</td><td></td></tr><tr><td>Disposable syringe (with needle)</td><td>Perfect Medical Industry Co.</td><td></td><td>24G/3 cm;3 ml &amp;amp; 26G/0.5 cm;1 ml</td></tr><tr><td>End over end mixer</td><td>C.T.I YOUNG CHENN</td><td>TS-20</td><td>For suspended cells recovery&amp;nbsp;</td></tr><tr><td>FACSCalibur (FACS)</td><td>BD biosciences</td><td></td><td></td></tr><tr><td>Forceps</td><td>Dimeda</td><td>10.102.14</td><td></td></tr><tr><td>Forma Direct Heat CO&lt;sub&gt;2&lt;/sub&gt; incubator</td><td>Thermo Fisher Scientific Inc.</td><td>HEPA CLASS 100</td><td></td></tr><tr><td>Mouse restrainer (Cylindrical Restrainer 15-30 gm)</td><td>Stoelting</td><td>51338</td><td></td></tr><tr><td>Multiphoton Confocal Microscope BX61WI</td><td>Olympus</td><td>FV1000MPE</td><td></td></tr><tr><td>Neubauer counting chamber</td><td>Marienfeld-Superior</td><td>640010</td><td></td></tr><tr><td>Surgical scissor</td><td>Dimeda</td><td>08.370.11</td><td></td></tr><tr><td>Surgical sutures&amp;nbsp;</td><td>UNIK SURGICAL SUTURES MFG. CO.</td><td>NO. 0034</td><td>Black Braided silk; non-absorbable (25YD; U.S.P. 4/0)</td></tr><tr><td>1.5 mL microcentrifuge tube</td><td>Wuxi NEST Biotechnology Co.</td><td>615001</td><td></td></tr><tr><td>15 mL Greiner tube</td><td>Greiner bio-one</td><td>188271</td><td></td></tr></tbody></table>

the establishment of a lung colonization assay for circulating tumor cell visualization in lung tissues

転移は癌死の主要な原因です。血中循環がん細胞 (Ctc) Ctc によって肺を植民地化が非常に初期の肺転移過程に貢献、がん転移を促進する上での役割を積極的に検討されている.そのため、動物モデルが完全全身転移プロセスをキャプチャする唯一の方法です。体内の肺植民地化アッセイを設けて血管漏出する Ctc の貢献を調べるため実験的デザインの以前に問題が発生したことを考える長期的蛍光セル-トレーサー、carboxyfluoresceinサクシニミジルエステル (CFSE) が中断された腫瘍細胞をラベルに使用した、閉じ込められた Ctc 以外特に肺の除去、共焦点イメージングと定量前をクリアする肺灌流を行った。CTC 表面組み立てられた高分子フィブロネクチン (polyFN) は、転移性腫瘍組織の最終的な確立で肺を植民地化を仲介する発見されています。ここでは、特に肺の植民地化および血管外漏出の Ctc で polyFN アセンブリの要件をテストするため短期を行った肺植民地化アッセイを中断ルイス肺癌細胞 (Llc) (shFN) の FN-shRNA を安定に発現するかスクランブル shRNA (shScr) と 20 の前にラベルが付いた CFSE の μ M は c57bl/6 マウスに静脈内接種しました。マウス肺を植民地化する shFN LLC 細胞の能力は shScr LLC 細胞と比較すると大幅減少したことを実証します。したがって、この短期的な方法論が広く適用特に肺を植民地化する循環内で Ctc の能力を発揮する可能性があります。

体内の肺の植民地化分析を実行する前に肺の植民地化および/または転移を促進する管外中断された腫瘍細胞上の polyFN を仲介するかどうかをテストするのには、図 1 aに示されているように我々 まず滴定別CFSE は細胞透過性と共有抱合体細胞内アミン含有分子26,27中断された腫瘍細胞のと時間の?...

Watch this Scientific Journal Video about The Establishment of a Lung Colonization Assay for Circulating Tumor Cell Visualization in Lung Tissues at JoVE.com

The Establishment of a Lung Colonization Assay for Circulating Tumor Cell Visualization in Lung Tissues

血中循環がん細胞 (Ctc) がん転移中に肺を植民地化を促進する上での役割を解読する動物モデルです。ここでは、私たちが設立され、正常に実行体内法を具体的にテスト肺植民地化の Ctc の高分子フィブロネクチン (polyFN) アセンブリの要件。

肺組織に腫瘍のセルの視覚化を循環させるため肺植民地化アッセイの確立

Metastasis is the major cause of cancer death. The role of circulating tumor cells (CTCs) in promoting cancer metastasis, in which lung colonization by ...

the-establishment-lung-colonization-assay-for-circulating-tumor-cell

Universidad de Cartagena UDEC

Research

JoVE Journal

Cancer Research

9.2K ビュー.  National Cheng Kung University. 血中循環がん細胞 (Ctc) がん転移中に肺を植民地化を促進する上での役割を解読する動物モデルです。ここでは、私たちが設立され、正常に実行体内法を具体的にテスト肺植民地化の Ctc の高分子フィブロネクチン (polyFN) アセンブリの要件。

ビデオ: The Establishment of a Lung Colonization Assay for Circulating Tumor Cell Visualization in Lung Tissues

Lung Tumor Cell Recruitment Assay

To investigate the molecular mechanisms governing tumor metastasis, various assays using the mouse as a model animal have been proposed. Here, we demonstrate a simple assay to evaluate tumor cell extravasation or micrometastasis. In this assay, tumor cells were injected through the tail vein, and after a short period, the lungs were dissected and digested to count the accumulated labeled tumor cells. This assay skips the initial step of primary tumor invasion into the blood vessel and facilitates the study of events in the distant organ where tumor metastasis occurs. The number of cells injected into the blood vessel can be optimized to observe a limited number of metastases. It has been reported that stromal cells in the distant organ contribute to metastasis. Thus, this assay could be a useful tool to explore potential therapeutic drugs or devices for prevention of tumor metastasis.

This manuscript describes a method to quantify tumor cell accumulation in the lungs in an animal model of tumor metastasis.

肺腫瘍細胞募集試験

To investigate the molecular mechanisms governing tumor metastasis, various assays using the mouse as a model animal have been proposed. Here, we ...

がん研究

Isolation of Circulating Tumor Cells in an Orthotopic Mouse Model of Colorectal Cancer

Despite the advantages of easy applicability and cost-effectiveness, subcutaneous mouse models have severe limitations and do not accurately simulate tumor biology and tumor cell dissemination. Orthotopic mouse models have been introduced to overcome these limitations; however, such models are technically demanding, especially in hollow organs such as the large bowel. In order to produce uniform tumors which reliably grow and metastasize, standardized techniques of tumor cell preparation and injection are critical. 
We have developed an orthotopic mouse model of colorectal cancer (CRC) which develops highly uniform tumors and can be used for tumor biology studies as well as therapeutic trials. Tumor cells from either primary tumors, 2-dimensional (2D) cell lines or 3-dimensional (3D) organoids are injected into the cecum and, depending on the metastatic potential of the injected tumor cells, form highly metastatic tumors. In addition, CTCs can be found regularly. We here describe the technique of tumor cell preparation from both 2D cell lines and 3D organoids as well as primary tumor tissue, the surgical and injection techniques as well as the isolation of CTCs from the tumor-bearing mice, and present tips for troubleshooting.

We describe the establishment of orthotopic colorectal tumors via injection of tumor cells or organoids into the cecum of mice and the subsequent isolation of circulating tumor cells (CTCs) from this model.

結腸直腸癌モデルマウスにおける循環腫瘍細胞の単離

Despite the advantages of easy applicability and cost-effectiveness, subcutaneous mouse models have severe limitations and do not accurately simulate ...

Practical Considerations in Studying Metastatic Lung Colonization in Osteosarcoma Using the Pulmonary Metastasis Assay

The pulmonary metastasis assay (PuMA) is an ex vivo lung explant and closed cell culture system that permits researchers to study the biology of lung colonization in osteosarcoma (OS) by fluorescence microscopy. This article provides a detailed description of the protocol, and discusses examples of obtaining image data on metastatic growth using widefield or confocal fluorescence microscopy platforms. The flexibility of the PuMA model permits researchers to study not only the growth of OS cells in the lung microenvironment, but also to assess the effects of anti-metastatic therapeutics over time. Confocal microscopy allows for unprecedented, high-resolution imaging of OS cell interactions with the lung parenchyma. Moreover, when the PuMA model is combined with fluorescent dyes or fluorescent protein genetic reporters, researchers can study the lung microenvironment, cellular and subcellular structures, gene function, and promoter activity in metastatic OS cells. The PuMA model provides a new tool for osteosarcoma researchers to discover new metastasis biology and assess the activity of novel anti-metastatic, targeted therapies.

The goal of this article is to provide a detailed description of the protocol for the pulmonary metastasis assay (PuMA). This model permits researchers to study metastatic osteosarcoma (OS) cell growth in lung tissue using a widefield fluorescence or confocal laser-scanning microscope.

肺転移アッセイを用いた骨肉腫の肺転移を植民地化の勉強に実用的な考慮事項

The pulmonary metastasis assay (PuMA) is an ex vivo lung explant and closed cell culture system that permits researchers to study the biology of lung ...

Improved Visualization of Lung Metastases at Single Cell Resolution in Mice by Combined In-situ Perfusion of Lung Tissue and X-Gal Staining of lacZ-Tagged Tumor Cells

Metastasis is the main cause of death in the majority of cancer types and consequently a main focus in cancer research. However, the detection of micrometastases by radiologic imaging and the success in their therapeutic eradication remain limited.
While animal models have proven to be invaluable tools for cancer research1, the monitoring/visualization of micrometastases remains a challenge and inaccurate evaluation of metastatic spread in preclinical studies potentially leads to disappointing results in clinical trials2. Consequently, there is great interest in refining the methods to finally allow reproducible and reliable detection of metastases down to the single cell level in normal tissue. The main focus therefore is on techniques, which allow the detection of tumor cells in vivo, like micro-computer tomography (micro-CT), positron emission tomography (PET), bioluminescence or fluorescence imaging3,4. We are currently optimizing these techniques for in vivo monitoring of primary tumor growth and metastasis in different osteosarcoma models. Some of these techniques can also be used for ex vivo analysis of metastasis beside classical methods like qPCR5, FACS6 or different types of histological staining. As a benchmark, we have established in the present study the stable transfection or transduction of tumor cells with the lacZ gene encoding the bacterial enzyme &beta;-galactosidase that metabolizes the chromogenic substrate 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-Gal) to an insoluble indigo blue dye7 and allows highly sensitive and selective histochemical blue staining of tumor cells in mouse tissue ex vivo down to the single cell level as shown here. This is a low-cost and not equipment-intensive tool, which allows precise validation of metastasis8 in studies assessing new anticancer therapies9-11. A limiting factor of X-gal staining is the low contrast to e.g. blood-related red staining of well vascularized tissues. In lung tissue this problem can be solved by in-situ lung perfusion, a technique that was recently established by Borsig et al.12 who perfused the lungs of mice under anesthesia to clear them from blood and to fix and embed them in-situ under inflation through the trachea. This method prevents also the collapse of the lung and thereby maintains the morphology of functional lung alveoli, which improves the quality of the tissue for histological analysis. In the present study, we describe a new protocol, which takes advantage of a combination of X-gal staining of lacZ-expressing tumor cells and in-situ perfusion and fixation of lung tissue. This refined protocol allows high-sensitivity detection of single metastatic cells in the lung and enabled us in a recent study to detect "dormant" lung micrometastases in a mouse model13, which was originally described to be non-metastatic14.

Improved Visualization of Lung Metastases at Single Cell Resolution in Mice by Combined In-situ Perfusion of Lung Tissue and X-Gal Staining of lacZ-Tagged Tumor Cells

The novel protocol reported in the present study allows selective detection of lung metastases at single cell resolution in mice by combined in-situ lung perfusion and fixation and X-Gal staining of lacZ-tagged tumor cells.

組み合わせによるマウスのシングルセル解像度で肺転移の可視化の向上<em&gt;のin-situ</em肺組織とのX-gal染色の&gt;灌流<em&gt; lacZを</em&gt;タグ付き腫瘍細胞

Metastasis is the main cause of death in the majority of cancer types and consequently a main focus in cancer research. However, the detection of ...

医学

Micromanipulation of Circulating Tumor Cells for Downstream Molecular Analysis and Metastatic Potential Assessment

Blood-borne metastasis accounts for&#160;most cancer-related deaths and involves circulating tumor cells (CTCs) that are successful in establishing new tumors at distant sites. CTCs are found in the bloodstream of patients as single cells (single CTCs) or as multicellular aggregates (CTC clusters and CTC-white blood cell clusters), with the latter displaying a higher metastatic ability. Beyond enumeration, phenotypic and molecular analysis is extraordinarily important to dissect CTC biology and to identify actionable vulnerabilities. Here, we provide a detailed description of a workflow that includes CTC immunostaining and micromanipulation, ex vivo culture to assess&#160;proliferative and survival capabilities of individual cells, and in vivo metastasis-formation assays. Additionally, we provide a protocol to achieve the dissociation of CTC clusters into individual cells and the investigation of intra-cluster heterogeneity. With these approaches, for instance, we precisely quantify survival and proliferative potential of single CTCs and individual cells within CTC clusters, leading us to the observation that cells within clusters display better survival and proliferation in ex vivo cultures compared to single CTCs. Overall, our workflow offers a platform to dissect the characteristics of CTCs at the single cell level, aiming towards the identification of metastasis-relevant pathways and a better understanding of CTC biology.

Here, we present an integrated workflow to identify phenotypic and molecular features that characterize circulating tumor cells (CTCs). We combine live immunostaining and robotic micromanipulation of single and clustered CTCs with single cell-based techniques for downstream analysis and assessment of metastasis-seeding ability.

下流の分子分析と転移性電位評価のための循環腫瘍細胞のマイクロマニピュレーション

Blood-borne metastasis accounts for&#160;most cancer-related deaths and involves circulating tumor cells (CTCs) that are successful in establishing new ...

Semi-automatic PD-L1 Characterization and Enumeration of Circulating Tumor Cells from Non-small Cell Lung Cancer Patients by Immunofluorescence

Circulating tumor cells (CTCs) derived from the primary tumor are shed into the bloodstream or lymphatic system. These rare cells (1&#8722;10 cells per mL of blood) warrant a poor prognosis and are correlated with shorter overall survival in several cancers (e.g., breast, prostate and colorectal). Currently, the anti-EpCAM-coated magnetic bead-based CTC capturing system is the gold standard test approved by the U.S. Food and Drug Administration (FDA) for enumerating CTCs in the bloodstream. This test is based on the use of magnetic beads coated with anti-EpCAM markers, which specifically target epithelial cancer cells. Many studies have illustrated that EpCAM is not the optimal marker for CTC detection. Indeed, CTCs are a heterogeneous subpopulation of cancer cells and are able to undergo an epithelial-to-mesenchymal transition (EMT) associated with metastatic proliferation and invasion. These CTCs are able to reduce the expression of cell surface epithelial marker EpCAM, while increasing mesenchymal markers such as vimentin. To address this technical hurdle, other isolation methods based on physical properties of CTCs have been developed. Microfluidic technologies enable a label-free approach to CTC enrichment from whole blood samples. The spiral microfluidic technology uses the inertial and Dean drag forces with continuous flow in curved channels generated within a spiral microfluidic chip. The cells are separated based on the differences in size and plasticity between normal blood cells and tumoral cells. This protocol details the different steps to characterize the programmed death-ligand 1 (PD-L1) expression of CTCs, combining a spiral microfluidic device with customizable immunofluorescence (IF) marker set.

The characterization of circulating tumor cells (CTCs) is a popular topic in translational research. This protocol describes a semi-automatic immunofluorescence (IF) assay for PD-L1 characterization and enumeration of CTCs in non-small cell lung cancer (NSCLC) patient samples.

免疫蛍光による非小細胞肺癌患者からの循環腫瘍細胞の半自動PD-L1特性図と列挙

Circulating tumor cells (CTCs) derived from the primary tumor are shed into the bloodstream or lymphatic system. These rare cells (1&#8722;10 cells per mL ...

Multidimensional Coculture System to Model Lung Squamous Carcinoma Progression

Tumor-stroma interactions play a critical role in the development of lung squamous carcinoma (LUSC). However, understanding how these dynamic interactions contribute to tissue architectural changes observed during tumorigenesis remains challenging due to the lack of appropriate models. In this protocol, we describe the generation of a 3D coculture model using a LUSC primary cell culture known as TUM622. TUM622 cells were established from a LUSC patient-derived xenograft (PDX) and have the unique property to form acinar-like structures when seeded in a basement membrane matrix. We demonstrate that TUM622 acini in 3D coculture recapitulate key features of tissue architecture during LUSC progression as well as the dynamic interactions between LUSC cells and components of the tumor microenvironment (TME), including the extracellular matrix (ECM) and cancer-associated fibroblasts (CAFs). We further adapt our principal 3D culturing protocol to demonstrate how this system could be utilized for various downstream analyses. Overall, this organoid model creates a biologically rich and adaptable platform that enables one to gain insight into the cell-intrinsic and extrinsic mechanisms that promote the disruption of epithelial architectures during carcinoma progression and will aid the search for new therapeutic targets and diagnostic markers.

An in vitro model system was developed to capture tissue architectural changes during lung squamous carcinoma (LUSC) progression in a 3-dimensional (3D) co-culture with cancer-associated fibroblasts (CAFs). This organoid system provides a unique platform to investigate the roles of diverse tumor cell-intrinsic and extrinsic changes that modulate the tumor phenotype.

肺扁平上皮癌進行をモデル化する多次元共培養システム

Tumor-stroma interactions play a critical role in the development of lung squamous carcinoma (LUSC). However, understanding how these dynamic interactions ...

Combined Use of Tail Vein Metastasis Assays and Real-Time In Vivo Imaging to Quantify Breast Cancer Metastatic Colonization and Burden in the Lungs

Metastasis is the main cause of cancer-related deaths and there are limited therapeutic options for patients with metastatic disease. The identification and testing of novel therapeutic targets that will facilitate the development of better treatments for metastatic disease requires preclinical in vivo models. Demonstrated here is a syngeneic mouse model for assaying breast cancer metastatic colonization and subsequent growth. Metastatic cancer cells are stably transduced with viral vectors encoding firefly luciferase and ZsGreen proteins. Candidate genes are then stably manipulated in luciferase/ZsGreen-expressing cancer cells and then the cells are injected into mice via the lateral tail vein to assay metastatic colonization and growth. An in vivo imaging device is then used to measure the bioluminescence or fluorescence of the tumor cells in the living animals to quantify changes in metastatic burden over time. The expression of the fluorescent protein allows the number and size of metastases in the lungs to be quantified at the end of the experiment without the need for sectioning or histological staining. This approach offers a relatively quick and easy way to test the role of candidate genes in metastatic colonization and growth, and provides a great deal more information than traditional tail vein metastasis assays. Using this approach, we show that simultaneous knockdown of Yes associated protein (YAP) and transcriptional co-activator with a PDZ-binding motif (TAZ) in breast cancer cells leads to reduced metastatic burden in the lungs and that this reduced burden is the result of significantly impaired metastatic colonization and reduced growth of metastases.

The described approach combines experimental tail vein metastasis assays with in vivo live animal imaging to allow real-time monitoring of breast cancer metastasis formation and growth in addition to the quantification of metastasis number and size in the lungs.

尾静脈転移アッセイとリアルタイムインビボイメージングの併用による乳癌転移性コロニー形成と肺の負担の定量化

Metastasis is the main cause of cancer-related deaths and there are limited therapeutic options for patients with metastatic disease. The identification ...

Circulating Tumor Cell Lines: an Innovative Tool for Fundamental and Translational Research

Metastasis is a leading cause of cancer death. Despite improvements in treatment strategies, metastatic cancer has a poor prognosis. We thus face an urgent need to understand the mechanisms behind metastasis development, and thus to propose efficient treatments for advanced cancer. Metastatic cancers are hard to treat, as biopsies are invasive and inaccessible. Recently, there has been considerable interest in liquid biopsies including both cell-free circulating deoxyribonucleic acid (DNA) and circulating tumor cells from peripheral blood and we have established several circulating tumor cell lines from metastatic colorectal cancer patients to participate in their characterization. Indeed, to functionally characterize these rare and poorly described cells, the crucial step is to expand them. Once established, circulating tumor cell (CTC) lines can then be cultured in suspension or adherent conditions. At the molecular level, CTC lines can be further used to assess the expression of specific markers of interest (such as differentiation, epithelial or cancer stem cells) by immunofluorescence or cytometry analysis. In addition, CTC lines can be used to assess drug sensitivity to gold-standard chemotherapies as well as to targeted therapies. The ability of CTC lines to initiate tumors can also be tested by subcutaneous injection of CTCs in immunodeficient mice.
Finally, it is possible to test the role of specific genes of interest that might be involved in cancer dissemination by editing CTC genes, by short hairpin ribonucleic acid (shRNA) or Crispr/Cas9. Modified CTCs can thus be injected into immunodeficient mouse spleens, to experimentally mimic part of the metastatic development process in vivo.
In conclusion, CTC lines are a precious tool for future research and for personalized medicine, where they will allow prediction of treatment efficiency using the very cells that are originally responsible for metastasis.

Culturing CTCs allows a deeper functional characterization of cancer, through assaying specific marker expression, and assessing drug resistance and the ability to colonize the liver among other possibilities. Overall, CTC culture could be a promising clinical tool for personalized medicine to improve patient outcome.

循環腫瘍細胞株:基礎的・翻訳的研究のための革新的なツール

Metastasis is a leading cause of cancer death. Despite improvements in treatment strategies, metastatic cancer has a poor prognosis. We thus face an ...

CTC Immunofluorescence Assay : A Technique to  Characterize Circulating Tumor Cells

Source: Garcia, J., et al. <a href="https://jove.unicartagenaproxy.elogim.com/v/59873/semi-automatic-pd-l1-characterization-enumeration-circulating-tumor">Semi-automatic PD-L1 Characterization and Enumeration of Circulating Tumor Cells from Non-small Cell Lung Cancer Patients by Immunofluorescence.</a>&#160;J. Vis. Exp.&#160;(2019)
This video describes the Circulating Tumor Cells (CTC) Immunofluorescence assay, a technique to characterize the biomarkers on circulating tumor cells in non-small cell lung cancer patient samples. This assay could be used to monitor dynamic changes of the tumor and immune response, which may reflect the response to chemotherapy, radiation, and possible immunotherapy treatments.

CTC免疫蛍光アッセイ:循環腫瘍細胞の特性評価技術

Source: Garcia, J., et al. Semi-automatic PD-L1 Characterization and Enumeration of Circulating Tumor Cells from Non-small Cell Lung Cancer Patients by ...