Name: pathological analysis of lung metastasis following lateral tail-vein injection of tumor cells
Uploaded: 2020-05-20T14:45:00.000+00:00
Duration: 8 min 54 s
Description: Watch this Scientific Journal Video about Pathological Analysis of Lung Metastasis Following Lateral Tail-Vein Injection of Tumor Cells at JoVE.com

代表的なデータは、国立がん研究所(K22CA218549からS.T.S.)を通じて資金提供されました。ここに報告された包括的な分析方法の開発に協力することに加えて、我々は、オハイオ州立大学総合癌センター比較病理学およびマウスフェノタイピング共有リソース(ディレクター - クリスタ・ラ・ペルル、DVM、博士号)の組織学および免疫組織化学サービス、およびアルゴリズムの開発および分析のための病理イメージングコアに感謝します。

動物の使用は、OSU機関動物の世話と使用委員会(IACUC)承認プロトコル2007A0120-R4(PI:ジーナ・サイズモア博士)の下で大学実験動物資源(ULAR)規制に従いました。
1. 乳がん細胞の尾静脈注射
<ol><li>注射用細胞および注射器の調製<ol>
<li>使用するマウスの数と細胞濃度に基づいて適切な数の細胞をプレートします。 注: 注入される細胞の数と転移の発生に要する時間は、使用される細胞のラインによって異なっており、最適化する必要があります。本デモでは、1 x 106個のMDA-MB-231細胞を NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ(NSG)マウスに静脈内注射し、注射後24日以上で巨視的な肺病変が観察される。MVT1マウス乳腺腫瘍細胞株17に対して、3 x 106細胞が免疫に優れたFVB/Nマウスに注入され、14日22,23で多数の肺転移が観察される。</li>
		<li>吸気培地と1x PBSで細胞プレートをすすいだ。細胞を最小量でトリプシン化し、適切な量の培地を加え、造馬計または別の好ましい方法を用いて細胞を数える。トリパンブルー(0.4%)または他の生きている/死んだ細胞色素を使用して、生存可能な細胞数を決定することができます。</li>
		<li>ペレット細胞は180xgで5分間回転する。</li>
		<li>1回のマウスに100 μLの体積が注入されるよう、滅菌1x PBSの細胞の適切な数を再中断します。生存率を維持するために、細胞懸濁液を氷の上に保管してください。</li>
		<li>注入する前に、凝集を避けるために200 μLまたは1 mLピペットで細胞を十分に再懸濁させる。28Gインシュリンシリンジで100μLを引き上げる( 材料表参照)。</li>
		<li>シリンジを垂直に保ち、シリンジをタップし、プランジャーをゆっくりと調整して、気泡を除去します。静脈への気泡の注入は、致命的な可能性がある空気/ガス塞栓症を引き起こす可能性があります。</li>
	</ol></li>
	<li>横尾静脈注射 注:実験的な乳癌転移アッセイの場合、注射は生後6週齢の雌マウス>に対して行われる。<ol>
<li>尾でマウスを処理し、適切なサイズのスロットチューブ/拘束装置に動物をスライドさせます(使用する拘束装置 については、材料表 を参照)。</li>
		<li>拘束装置のプラグ部分を挿入し、横方向の尾静脈が見えやすいような側にマウスを置きます。マウスは生殖器、後部静脈、および2つの側尾静脈に沿った腹側動脈を有する。</li>
		<li>無菌ワイプでマウスの尾の表面をきれいにします。人差し指と親指の間の尾を非支配的な手で握り、わずかな緊張を加えます。</li>
		<li>尾の遠位部分から始まり、ベベルが終わる静脈に平行に針を挿入します。</li>
		<li>許可されている場合は、 慎重に 針を要約し、20〜30°の角度に曲げ。片手でアプローチまたは針のリタッピング装置を強くお勧めします。 注:これは静脈が崩壊する可能性があるため、吸引する必要はありません。しかし、最初に置かれたときに血の小さなフラッシュが見られるかもしれません。針は適切な配置で静脈にスムーズに進みます。</li>
		<li>ゆっくりと静脈に完全なボリュームを分配します。プランジャーを押しても抵抗があってはならない。</li>
		<li>抵抗が感じられる場合は、速やかに注射針を取り外します。必要に応じて、尾部の近位端または反対側の側面静脈に向かって移動する針(理想的には3回以下)を再挿入します。</li>
		<li>少量の血液は、注射後に置き換えられる可能性が高い。滅菌ガーゼで穏やかな圧力をかけ、無菌ワイプで拭きます。</li>
		<li>適切なシャープ容器に注射器を速やかに処分する。</li>
		<li>清潔で換気の良いケージにマウスを戻し、苦痛の兆候を監視します。</li>
		<li>マウスの転移形成(呼吸困難、姿勢のハンチ、体重減少)および一般的な苦痛の徴候を、週2~3倍に監視する。転移の発生までの時間は、細胞株とマウス株に依存します。</li>
		<li>in vivo生きた動物イメージング装置を用いる場合、尾静脈注射直後の画像マウスは、細胞の正常な注入を確認し、時間「ゼロ」データを得る(インビボ生物発光イメージングに関する具体的な詳細はここに含まれていないが、Yangらららによって記載されている)。</li>
	</ol></li>
</ol>2. 肺組織の固定と転移性肺腫瘍の負担の解析
<ol><li>組織病理学のための肺の構造様式を維持するための肺組織の膨張<ol>
<li>承認された安楽死手順(例えば、チャンバー容積/分の30〜70%の変位で二酸化炭素)が続いた後、マウスの死骸をピンで解剖板に固定します。スプレーまたは70%エタノールを適用して、解剖中にマウスの毛皮を邪魔さないようにします。 注:二酸化炭素窒息は、特に遅い流速で、期待される背景病変として肺出血を引き起こす可能性があります。</li>
		<li>正中線切開で胸郭を開き、腹膜を通して切開を頸部/尾様に伸ばし、xyphoidプロセスをつかんで横隔膜を切り取る。</li>
		<li>刃を鈍らせないように別のハサミのセットを使用して、胸骨の両側に沿って肋骨を切断し、慎重に肺が拡大するための余地を残す肋骨ケージを取り除きます。</li>
		<li>下顎下唾液腺とインフラヒロイド筋肉を除去することによって気管を分離します。気管の両側にピンを置くと、針の挿入中に不要な動きを防ぐことができます。</li>
		<li>26 G シリンジに 2~3 mL の 10% 中性緩衝ホルマリンを充填し、気管に挿入します。</li>
		<li>ゆっくりとホルマリンを注入し、肺が拡大するのを見てください(通常、ホルマリンの約1.5 mLが必要です)。</li>
		<li>ホルマリンが肺から漏れ始めたら(インフレを避ける)、鉗子で気管をつまみ、注射針を取り外し、呼吸器装置全体を取り外します。組織の追加のトリミングが後に行うことができるように、肺、心臓などをホルマリンに直接入れる。</li>
		<li>完全な処理、埋め込み、組織の切片、および標準的な方法を使用してヘマトキシリンおよびエオシン(H&E)染色。</li>
	</ol></li>
	<li>転移性肺腫瘍の負担の解析<ol>
<li>H&E染色された肺のセクションを、40倍の倍率で高解像度のスライドスキャナーでスキャンします(図3)。</li>
		<li>肺転移の定量化のための画像解析ソフトウェア(例えば、Visiopharm画像解析)に画像をインポートします。 注: 新しいユーザーは、画像解析ソフトウェアを使用するためにオンサイトまたはオンラインのトレーニングを受けることをお勧めします。市販のウェブページからも多数のウェビナーが利用できます。</li>
		<li>ソフトウェアのアプリライブラリから、Visiopharm 10118 H&E肺転移アプリを選択します。 注:このアプリの目的は、H&E染色されたスライドに肺転移をラベル付けし、定量化することです。10118 H&E肺転移アプリの一部として、最初の画像処理ステップは、組織検出アプリで肺組織をセグメント化します。第二の画像処理ステップは、肺組織内の転移を識別する転移検出アプリを使用しています。転移は、形状が間違っている領域、赤すぎる領域、または転移として識別するにはまばらすぎる領域と共に識別されます。</li>
		<li>形状とスパースを定義するパラメータを調整して、代表的な画像に最適にフィットします。腫瘍転移および正常肺組織のセグメント化領域は、組織タイプごとに異なる色ラベルを使用して表示することができる。 注: アプリが正常な肺組織から正確に転移を分離できない場合、Visiopharm Decision Forest プログラムを使用するカスタム アプリは、実験で行われたように記述する必要があります ( 図 2 および 図 3 を参照)。カスタム アルゴリズムを記述する詳細は以下のとおりです。それ以外の場合は、ステップ 2.2.9 に進みます。</li>
		<li>所望の画像上の複数の クラス (すなわち、肺組織(非腫瘍性)、転移、赤血球、上皮、および/または白いスペースを訓練することによって動作するDecision Forestプログラムを開きます。 図2では、腫瘍転移は青色、正常組織は緑色、気管支上皮は黄色である。また、赤血球は赤と空気空間にピンク色です。</li>
		<li>プロンプトされた一連のイエスまたはノーの質問に従って、各 クラス を適切に画像に合わせて訓練します。アルゴリズムの精度は、イエス/ノーの質問の数を決定します。分析のために、カスタム アルゴリズム/アプリは 50 (範囲 0 から 100) に設定された精度で書き込まれました。</li>
		<li>アルゴリズム/App. Visiopharm は、機能と呼ばれる 1 つまたは複数のレンズを通じて各クラスを表示する精度を高めるために、シャープ、ぼかし、形状による並べ替えなどを行うフィルターを適用して、各クラスの機能を調整します。特徴は、特定の色や強度を引き出すために、クラスが画像を見る方法を変更します。 注: カスタムアルゴリズムでは、8500 μm2 以上のメタスタは、メタスタとしてラベル付けされ、測定されます。これは、サイズの差異と転移が小さすぎて検出できない場合を示します。小さな誤った形状の領域と8500 μm2 の下の小さな転移領域は、正常な組織定量に含まれていた。</li>
		<li>アプリまたはカスタムアルゴリズムのいずれかから変更された設定を保存し、その後、全体のセットまたは一連のH&E染色組織にアルゴリズム/アプリを適用します。</li>
		<li>最後に、 表 1 に示した出力変数を含むすべての出力変数をエクスポートします。ミクロン二乗(μm2)の面積は、組織タイプごとに定量化することができ、パーセンテージは標本の総正味組織面積(すなわち、総組織から空気空間を差し引いた)に由来する。</li>
		<li>カスタムアルゴリズムを作成する場合は、米国獣医病理学者の米国大学によって認定された獣医病理学者の理事会と協議して組織マークアップをレビューし、正確な測定を保証し、組織タイプを区別します。</li>
	</ol></li>
</ol>

<ol>
	<li>Chambers, A. F., Groom, A. C., MacDonald, I. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissemination+and+growth+of+cancer+cells+in+metastatic+sites.">Dissemination and growth of cancer cells in metastatic sites.</a> Nature Reviews: Cancer. 2 (8), 563-572 (2002).</li><li>Steeg, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+metastasis.">Targeting metastasis.</a> Nature Reviews: Cancer. 16 (4), 201-218 (2016).</li><li>Gupta, G. P., Massague, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+metastasis:+building+a+framework.">Cancer metastasis: building a framework.</a> Cell. 127 (4), 679-695 (2006).</li><li>Steeg, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+metastasis:+mechanistic+insights+and+clinical+challenges.">Tumor metastasis: mechanistic insights and clinical challenges.</a> Nature Medicine. 12 (8), 895-904 (2006).</li><li>Chaffer, C. L., Weinberg, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+perspective+on+cancer+cell+metastasis.">A perspective on cancer cell metastasis.</a> Science. 331 (6024), 1559-1564 (2011).</li><li>Eckhardt, B. L., Francis, P. A., Parker, B. S., Anderson, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Strategies+for+the+discovery+and+development+of+therapies+for+metastatic+breast+cancer.">Strategies for the discovery and development of therapies for metastatic breast cancer.</a> Nature Reviews Drug Discovery. 11 (6), 479-497 (2012).</li><li>Gomez-Cuadrado, L., Tracey, N., Ma, R., Qian, B., Brunton, V. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+models+of+metastasis:+progress+and+prospects.">Mouse models of metastasis: progress and prospects.</a> Disease Models &amp; Mechanisms. 10 (9), 1061-1074 (2017).</li><li>Fantozzi, A., Christofori, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+models+of+breast+cancer+metastasis.">Mouse models of breast cancer metastasis.</a> Breast Cancer Research. 8 (4), 212 (2006).</li><li>Schoenenberger, C. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+c-myc+gene+expression+in+mammary+glands+of+transgenic+mice+induces+mammary+tumours+with+constitutive+milk+protein+gene+transcription.">Targeted c-myc gene expression in mammary glands of transgenic mice induces mammary tumours with constitutive milk protein gene transcription.</a> EMBO Journal. 7 (1), 169-175 (1988).</li><li>Nusse, R., Varmus, H. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Many+tumors+induced+by+the+mouse+mammary+tumor+virus+contain+a+provirus+integrated+in+the+same+region+of+the+host+genome.">Many tumors induced by the mouse mammary tumor virus contain a provirus integrated in the same region of the host genome.</a> Cell. 31 (1), 99-109 (1982).</li><li>Muller, W. J., Sinn, E., Pattengale, P. K., Wallace, R., Leder, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-step+induction+of+mammary+adenocarcinoma+in+transgenic+mice+bearing+the+activated+c-neu+oncogene.">Single-step induction of mammary adenocarcinoma in transgenic mice bearing the activated c-neu oncogene.</a> Cell. 54 (1), 105-115 (1988).</li><li>Lin, E. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progression+to+malignancy+in+the+polyoma+middle+T+oncoprotein+mouse+breast+cancer+model+provides+a+reliable+model+for+human+diseases.">Progression to malignancy in the polyoma middle T oncoprotein mouse breast cancer model provides a reliable model for human diseases.</a> American Journal of Pathology. 163 (5), 2113-2126 (2003).</li><li>Green, J. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+C3(1)/SV40+T-antigen+transgenic+mouse+model+of+mammary+cancer:+ductal+epithelial+cell+targeting+with+multistage+progression+to+carcinoma.">The C3(1)/SV40 T-antigen transgenic mouse model of mammary cancer: ductal epithelial cell targeting with multistage progression to carcinoma.</a> Oncogene. 19 (1), 1020-1027 (2000).</li><li>Iorns, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+mouse+model+for+the+study+of+human+breast+cancer+metastasis.">A new mouse model for the study of human breast cancer metastasis.</a> PloS One. 7 (10), 47995 (2012).</li><li>Kim, I. S., Baek, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+models+for+breast+cancer+metastasis.">Mouse models for breast cancer metastasis.</a> Biochemical and Biophysical Research Communications. 394 (3), 443-447 (2010).</li><li>Mariotto, A. B., Etzioni, R., Hurlbert, M., Penberthy, L., Mayer, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Estimation+of+the+Number+of+Women+Living+with+Metastatic+Breast+Cancer+in+the+United+States.">Estimation of the Number of Women Living with Metastatic Breast Cancer in the United States.</a> Cancer Epidemiology, Biomarkers and Prevention. 26 (6), 809-815 (2017).</li><li>Xiao, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Risk+factors+and+survival+outcomes+in+patients+with+breast+cancer+and+lung+metastasis:+a+population-based+study.">Risk factors and survival outcomes in patients with breast cancer and lung metastasis: a population-based study.</a> Cancer Medicine. 7 (3), 922-930 (2018).</li><li>Smid, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subtypes+of+breast+cancer+show+preferential+site+of+relapse.">Subtypes of breast cancer show preferential site of relapse.</a> Cancer Research. 68 (9), 3108-3114 (2008).</li><li>Kennecke, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metastatic+behavior+of+breast+cancer+subtypes.">Metastatic behavior of breast cancer subtypes.</a> Journal of Clinical Oncology. 28 (20), 3271-3277 (2010).</li><li>Soni, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Breast+cancer+subtypes+predispose+the+site+of+distant+metastases.">Breast cancer subtypes predispose the site of distant metastases.</a> American Journal of Clinical Pathology. 143 (4), 471-478 (2015).</li><li>Leone, B. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prognostic+impact+of+metastatic+pattern+in+stage+IV+breast+cancer+at+initial+diagnosis.">Prognostic impact of metastatic pattern in stage IV breast cancer at initial diagnosis.</a> Breast Cancer Research and Treatment. 161 (3), 537-548 (2017).</li><li>Pei, X. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Explant-cell+culture+of+primary+mammary+tumors+from+MMTV-c-Myc+transgenic+mice.">Explant-cell culture of primary mammary tumors from MMTV-c-Myc transgenic mice.</a> In Vitro Cellular and Developmental Biology: Animal. 40 (1-2), 14-21 (2004).</li><li>Mathsyaraja, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CSF1-ETS2-induced+microRNA+in+myeloid+cells+promote+metastatic+tumor+growth.">CSF1-ETS2-induced microRNA in myeloid cells promote metastatic tumor growth.</a> Oncogene. 34 (28), 3651-3661 (2015).</li><li>Yang, S., Zhang, J. J., Huang, X. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+models+for+tumor+metastasis.">Mouse models for tumor metastasis.</a> Methods in Molecular Biology. 928, 221-228 (2012).</li><li>La Perle, K. M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+Pathologists:+Ultimate+Control+Freaks+Seeking+Validation.">Comparative Pathologists: Ultimate Control Freaks Seeking Validation.</a> Veterinary Pathology. 56 (1), 19-23 (2019).</li><li>Blomberg, O. S., Spagnuolo, L., de Visser, K. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immune+regulation+of+metastasis:+mechanistic+insights+and+therapeutic+opportunities.">Immune regulation of metastasis: mechanistic insights and therapeutic opportunities.</a> Disease Models &amp; Mechanisms. 11 (10), (2018).</li><li>Gonzalez, H., Hagerling, C., Werb, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Roles+of+the+immune+system+in+cancer:+from+tumor+initiation+to+metastatic+progression.">Roles of the immune system in cancer: from tumor initiation to metastatic progression.</a> Genes and Development. 32 (19-20), 1267-1284 (2018).</li><li>Borowsky, A. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Syngeneic+mouse+mammary+carcinoma+cell+lines:+two+closely+related+cell+lines+with+divergent+metastatic+behavior.">Syngeneic mouse mammary carcinoma cell lines: two closely related cell lines with divergent metastatic behavior.</a> Clinical and Experimental Metastasis. 22 (1), 47-59 (2005).</li><li>Yang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunocompetent+mouse+allograft+models+for+development+of+therapies+to+target+breast+cancer+metastasis.">Immunocompetent mouse allograft models for development of therapies to target breast cancer metastasis.</a> Oncotarget. 8 (19), 30621-30643 (2017).</li><li>Resch, M., Neels, T., Tichy, A., Palme, R., Rulicke, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+assessment+of+tail-vein+injection+in+mice+using+a+modified+anaesthesia+induction+chamber+versus+a+common+restrainer+without+anaesthesia.">Impact assessment of tail-vein injection in mice using a modified anaesthesia induction chamber versus a common restrainer without anaesthesia.</a> Laboratory Animals. 53 (2), 190-201 (2019).</li><li>Rashid, O. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Is+tail+vein+injection+a+relevant+breast+cancer+lung+metastasis+model.">Is tail vein injection a relevant breast cancer lung metastasis model.</a> Journal of Thoracic Disease. 5 (4), 385-392 (2013).</li><li>Goodale, D., Phay, C., Postenka, C. O., Keeney, M., Allan, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+tumor+cell+dissemination+patterns+in+preclinical+models+of+cancer+metastasis+using+flow+cytometry+and+laser+scanning+cytometry.">Characterization of tumor cell dissemination patterns in preclinical models of cancer metastasis using flow cytometry and laser scanning cytometry.</a> Cytometry Part A. 75 (4), 344-355 (2009).</li><li>Goddard, E. T., Fischer, J., Schedin, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Portal+Vein+Injection+Model+to+Study+Liver+Metastasis+of+Breast+Cancer.">A Portal Vein Injection Model to Study Liver Metastasis of Breast Cancer.</a> Journal of Visualized Experiments. (118), (2016).</li><li>Wright, L. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Murine+models+of+breast+cancer+bone+metastasis.">Murine models of breast cancer bone metastasis.</a> BoneKEy Reports. 5, 804 (2016).</li><li>Simmons, J. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+Models+of+Bone+Metastasis.">Animal Models of Bone Metastasis.</a> Veterinary Pathology. 52 (5), 827-841 (2015).</li><li>Liu, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improving+orthotopic+mouse+models+of+patient-derived+breast+cancer+brain+metastases+by+a+modified+intracarotid+injection+method.">Improving orthotopic mouse models of patient-derived breast cancer brain metastases by a modified intracarotid injection method.</a> Scientific Reports. 9 (1), 622 (2019).</li><li>Kodack, D. P., Askoxylakis, V., Ferraro, G. B., Fukumura, D., Jain, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Emerging+strategies+for+treating+brain+metastases+from+breast+cancer.">Emerging strategies for treating brain metastases from breast cancer.</a> Cancer Cell. 27 (2), 163-175 (2015).</li><li>Brown, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Practical+Stereology+Applications+for+the+Pathologist.">Practical Stereology Applications for the Pathologist.</a> Veterinary Pathology. 54 (3), 358-368 (2017).</li><li>Aeffner, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Digital+Microscopy,+Image+Analysis,+and+Virtual+Slide+Repository.">Digital Microscopy, Image Analysis, and Virtual Slide Repository.</a> Institute for Laboratory Animal Research Journal. 59 (1), 66-79 (2018).</li></ol>

著者らは開示するものは何もない。

研究者が転移の実験モデルとして腫瘍細胞の静脈内注射を使用し続ける中で、得られた転移性腫瘍の負担を分析するための標準的な慣行は欠けている。場合によっては、特定の細胞株の操作や化学化合物の使用時に転移性腫瘍の負担に有意な差が認められる。しかし、他の例では、転移性播種と成長の微妙な違いは、徹底的な病理学的分析なしに見落とされたり、誤って解釈されたりする可?...

An erratum was issued for:&#160;<a href="https://www.jove.com/t/61270/pathological-analysis-lung-metastasis-following-lateral-tail-vein">Pathological Analysis of Lung Metastasis Following Lateral Tail-Vein Injection of Tumor Cells</a>. The author list was updated.
The author list was updated from:
Katie A. Thies1,&#160;Sue E. Knoblaugh2, and Steven T. Sizemore1 
1Department of Radiation Oncology, Arthur G. James Comprehensive Cancer Center and Richard L. Solove Research Institute, The Ohio State University Medical Center 
2Department of Veterinary Biosciences, Comparative Pathology and Digital Imaging Shared Resource, The Ohio State University
to:
Katie A. Thies1, Sarah Steck1, Sue E. Knoblaugh2, and Steven T. Sizemore1 
1Department of Radiation Oncology, Arthur G. James Comprehensive Cancer Center and Richard L. Solove Research Institute, The Ohio State University Medical Center 
2Department of Veterinary Biosciences, Comparative Pathology and Digital Imaging Shared Resource, The Ohio State University

An erratum was issued for:&#160;<a href="https://www.jove.com/t/61270/pathological-analysis-lung-metastasis-following-lateral-tail-vein">Pathological Analysis of Lung Metastasis Following Lateral Tail-Vein Injection of Tumor Cells</a>. The author list was updated.

Erratum: Pathological Analysis of Lung Metastasis Following Lateral Tail-Vein Injection of Tumor Cells

非常に複雑で非効率的なプロセス1であるにもかかわらず、転移は癌患者の罹患率と死亡率に大きく寄与する2である。実際、ほとんどの癌関連死は、疾患の転移性広がりに起因する3,4である。腫瘍細胞が正常に転移するためには、一次部位から切り離し、隣接する間質を通って侵入し、血液循環またはリンパ管に浸潤し、二次部位の毛細血管床に移動し、二次組織に飛び出し、増殖または成長して転移病変を形成する必要がある。マウスモデルの使用は、転移性播種および成長を担う分子機構の理解を深める上で重要であった6,7。ここでは、遺伝子組み換えマウスモデルと移植方法の両方がしばしば使用される乳がん転移に焦点を当てています。
遺伝子組み換え乳腺腫瘍モデルは、MMTV-LTR(マウス乳腺腫瘍ウイルス長末反復)およびWAP(乳清酸性タンパク質)を含む乳腺特異的プロモーターを利用して、乳腺上皮8の遺伝子導入の発現を促進する。ポリオーマ中期T抗原(PyMT)、ErbB2/Neu、c-Myc、Wnt-1、シミアンウイルス40(SV40)を含む腫瘍遺伝子は、この方法で発現されており、これらの遺伝モデルは原発性腫瘍の開始および進行を研究するのに有用であるが、遠くの器官に容易に転移する。さらに、これらの遺伝的マウスモデルは、多くの場合、自発的または実験的な転移モデルよりも時間とコストが非常に高いです。転移を研究する最も遺伝子組み換え乳腺腫瘍モデルの限界を考えると、移植技術はこの複雑なプロセスを研究する魅力的な方法となっています。これには、適切な細胞株の正交性、尾静脈、心臓内注射、頭蓋内注射が含まれる。
いくつかの乳癌細胞株は、乳腺脂肪パッド14,15への交所注入後に容易に転移するが、転移性腫瘍負担の一貫性および再現性は困難であり、そのような研究の持続期間は数ヶ月のオーダーで可能である。肺転移を評価するために、特に、尾静脈への静脈内注射は、典型的には数週間のスパンで起こる転移性広がりを伴うより再現性が高く、時間効果の高い方法である。しかし、静脈内注入モデルは転移性カスケードの初期ステップをバイパスするので、これらの研究の結果を解釈する際に注意が必要である。本デモでは、乳腺腫瘍細胞の尾静脈注射と、正確かつ包括的な分析方法を示す。
研究コミュニティは乳がん転移の複雑なプロセスを理解する上で大きな進歩を遂げましたが、現在150,000人以上の女性が転移性乳癌を持っていると推定されています16。ステージIV乳癌の患者のうち、>36%の患者が肺転移17を有する。しかし、部位特異的パターンおよび転移の発生率は、分子サブタイプ18、19、20、21に基づいて変化する可能性がある。乳癌関連肺転移を有する患者は、この疾患に対する有効な治療法および新規バイオマーカーを同定する必要性を強調するわずか21ヶ月の生存期間の中央値を有する17。腫瘍細胞の静脈内注射を含む実験的転移モデルの使用は、この重要な臨床的課題に関する我々の知識を引き続き進める。デジタルイメージング病理と、このプロトコルに記載されている転移性肺腫瘍負担分析の方法と組み合わせると、尾静脈注射は乳癌転移研究にとって貴重なツールです。

<table><tbody><tr><td>alcohol prep pads</td><td>Fisher Scientific</td><td>22-363-750</td><td>&lt;strong&gt;for cleaning tail prior to injection&lt;/strong&gt;</td></tr><tr><td>dissection scissors</td><td>Fisher Scientific</td><td>08-951-5</td><td>&lt;strong&gt;for mouse dissection and lung tissue inflation&lt;/strong&gt;</td></tr><tr><td>DMEM with L-Glutamine, 4.5g/L Glucose and Sodium Pyruvate</td><td>Fisher Scientific</td><td>MT10013CV</td><td>cell culture media base for MDA-MB-231 and MVT1 cell lines</td></tr><tr><td>Dulbecco&#039;s Phosphate-Buffered Salt Solution 1x</td><td>Fisher Scientific</td><td>MT21030CV</td><td>used for resuspending tumor cells for injection</td></tr><tr><td>ethanol (70 % solution)</td><td>OSU</td><td></td><td>used to minimize mouse&#039;s fur during dissection; use caution - flammable</td></tr><tr><td>Evan&#039;s blue dye</td><td>Millipore Sigma</td><td>E2129</td><td>used at 1 % in sterile PBS for practice with tail-vein injection method; use caution - dangerous reagent</td></tr><tr><td>Fetal Bovine Serum</td><td>Millipore Sigma</td><td>F4135</td><td>cell culture media additive; used at 10% in DMEM</td></tr><tr><td>forceps</td><td>Fisher Scientific</td><td>10-270</td><td>for dissection and lung tissue inflation</td></tr><tr><td>FVB/NJ mice</td><td>The Jackson Laboratory</td><td>001800</td><td>syngeneic mouse strain for MVT1 cells</td></tr><tr><td>hemacytometer (Bright-Line)</td><td>Millipore Sigma</td><td>Z359629</td><td>for use in cell culture to obtain cell counts</td></tr><tr><td>insulin syringe (28 G)</td><td>Fisher Scientific</td><td>14-829-1B</td><td>for tail-vein injections (BD 329424)</td></tr><tr><td>MDA-MB-231 cells</td><td>ATCC</td><td></td><td>human breast cancer cell line</td></tr><tr><td>MVT1 cells</td><td></td><td></td><td>mouse mammary tumor cells</td></tr><tr><td>needles (26 G)</td><td>Fisher Scientific</td><td>14-826-15</td><td>used to inflate the mouse&#039;s lungs</td></tr><tr><td>neutral buffered formalin (10%)</td><td>Fisher Scientific</td><td>245685</td><td>used as a tissue fixative and to inflate lung tissue; use caution - dangerous reagent</td></tr><tr><td>NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice</td><td>The Jackson Laboratory</td><td>005557</td><td>maintained by OSUCCC Target Validation Shared Resource</td></tr><tr><td>Penicillin Streptomycin 100x</td><td>ThermoFisher</td><td>15140163</td><td>cell culture media additive</td></tr><tr><td>sterile gauze</td><td>Fisher Scientific</td><td>NC9379092</td><td>for applying pressue to mouse&#039;s tail if bleeding occurs</td></tr><tr><td>syringe (5 mL)</td><td>Fisher Scientific</td><td>14-955-458</td><td>used to inflate mouse lung tissue</td></tr><tr><td>tail-vein restrainer</td><td>Braintree Scientific, Inc.</td><td>TV-150 STD</td><td>used to restrain mouse for tail-vein injections</td></tr><tr><td>Trypan blue (0.4 %)</td><td>ThermoFisher</td><td>15250061</td><td>used in cell culture to assess viability</td></tr><tr><td>Trypsin-EDTA 0.25 %</td><td>ThermoFisher</td><td>25200-114</td><td>used in cell culture to detach tumor cells from plate</td></tr></tbody></table>

pathological analysis of lung metastasis following lateral tail-vein injection of tumor cells

ほとんどのがん患者の罹患率と死亡率の主な原因である転移は、マウスの前臨床的にモデル化するのが難しい場合があります。自然転移モデルはごくわずかです。したがって、適切な細胞株の尾静脈注入を伴う実験転移モデルは転移研究の主力である。癌細胞が横尾静脈に注入されるとき、肺は植民地化の彼らの好ましい部位である。この技術の潜在的な制限は、転移性肺腫瘍の負担の正確な定量化である。一部の研究者は、事前に定義されたサイズのマクロ転移を数える、または/または組織の切除後の微小転移を含むが、他の研究者は正常な組織領域に対する転移性病変の領域を決定する。これらの定量方法はいずれも転移負担が高い場合に非常に困難な場合があります。本明細書において、画像解析ソフトを用いて転移性腫瘍負担を定量化するための高度な方法を用いて、肺転移の静脈内注射モデルを示す。このプロセスにより、平均転移サイズ、転移の総数、総転移領域など、複数のエンドポイントパラメータを調査して包括的な分析を行うことができます。さらに、この方法は、正確性を確保するために、米国獣医病理学者会(SEK)によって認定された獣医病理学者委員会によって見直されました。

尾静脈注射に標識されていない細胞を用いる場合、(1)マクロメタスターゼが観察される場合は壊死時まで肺の植民地化を確認することが困難であり、顕微鏡転移が存在する場合は(2)組織学的分析に従う。広範な転移性肺腫瘍の負担で、マウスは呼吸を働かされるであろう。他の腫瘍研究と同様に、マウスは研究期間中を通して注意深く監視されるべきである。標識された細胞の使用は、成功?...

Watch this Scientific Journal Video about Pathological Analysis of Lung Metastasis Following Lateral Tail-Vein Injection of Tumor Cells at JoVE.com

Pathological Analysis of Lung Metastasis Following Lateral Tail-Vein Injection of Tumor Cells

癌細胞の静脈内注射は転移研究でよく用いられるが、転移性腫瘍の負担は分析が難しい。本明細書において、転移の尾静脈注入モデルを示し、生じる転移性肺腫瘍の負担を分析する新しいアプローチを含む。

腫瘍細胞の横尾静脈注入後の肺転移の病理学的解析

Metastasis, the primary cause of morbidity and mortality for most cancer patients, can be challenging to model preclinically in mice. Few spontaneous ...

pathological-analysis-lung-metastasis-following-lateral-tail-vein

Research

JoVE Journal

Cancer Research

8.8K ビュー.  The Ohio State University Medical Center. 癌細胞の静脈内注射は転移研究でよく用いられるが、転移性腫瘍の負担は分析が難しい。本明細書において、転移の尾静脈注入モデルを示し、生じる転移性肺腫瘍の負担を分析する新しいアプローチを含む。

ビデオ: Pathological Analysis of Lung Metastasis Following Lateral Tail-Vein Injection of Tumor Cells

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

Experimental Metastasis Assay

Metastasis is the leading cause of death in cancer patients. To understand the mechanism of metastasis, an experimental metastasis assay was established using immunodeficient mice. This article delineates the procedures involved in this assay, including sample preparation, intravenous injection, and culturing cells from lung metastases. Briefly, a pre-determined number of human cancer cells were prepared in vitro and directly injected into the circulation of immunodeficient mice through their tail veins. A small number of cells survive the turbulence in the circulation and grow as metastases in internal organs, such as lung. The injected mice are dissected after a certain period. The tissue distribution of metastases is determined under a dissecting microscope. The number of metastases in a specific tissue is counted and it directly correlates with the metastatic ability of the injected cancer cells. The arisen metastases are isolated and cultured in vitro as cell lines, which often show enhanced metastatic abilities than the parental line when injected again into immunodeficient mice. These highly metastatic derivatives become useful tools for identifying genes or molecular pathways that regulate metastatic progression.

This article describes the procedures of an experimental metastasis assay that is used to determine the metastatic potential of human cancer cell lines.

実験的転移アッセイ

Metastasis is the leading cause of death in cancer patients.  To understand the mechanism of metastasis, an experimental metastasis assay was established ...

医学

A Portal Vein Injection Model to Study Liver Metastasis of Breast Cancer

Breast cancer is the leading cause of cancer-related mortality in women worldwide. Liver metastasis is involved in upwards of 30% of cases with breast cancer metastasis, and results in poor outcomes with median survival rates of only 4.8 - 15 months. Current rodent models of breast cancer metastasis, including primary tumor cell xenograft and spontaneous tumor models, rarely metastasize to the liver. Intracardiac and intrasplenic injection models do result in liver metastases, however these models can be confounded by concomitant secondary-site metastasis, or by compromised immunity due to removal of the spleen to avoid tumor growth at the injection site. To address the need for improved liver metastasis models, a murine portal vein injection method that delivers tumor cells firstly and directly to the liver was developed. This model delivers tumor cells to the liver without complications of concurrent metastases in other organs or removal of the spleen. The optimized portal vein protocol employs small injection volumes of 5 - 10 &#956;l, &#8805; 32 gauge needles, and hemostatic gauze at the injection site to control for blood loss. The portal vein injection approach in Balb/c female mice using three syngeneic mammary tumor lines of varying metastatic potential was tested; high-metastatic 4T1 cells, moderate-metastatic D2A1 cells, and low-metastatic D2.OR cells. Concentrations of &#8804; 10,000 cells/injection results in a latency of ~ 20 - 40 days for development of liver metastases with the higher metastatic 4T1 and D2A1 lines, and &#62; 55 days for the less aggressive D2.OR line. This model represents an important tool to study breast cancer metastasis to the liver, and may be applicable to other cancers that frequently metastasize to the liver including colorectal and pancreatic adenocarcinomas.

A surgical procedure was developed to deliver mammary tumor cells to the murine liver via portal vein injection. This model permits investigation of late stages of liver metastasis in a fully immune competent host, including tumor cell extravasation, seeding, survival, and metastatic outgrowth in the liver.

乳癌の肝転移を研究するためのポータル静脈注射モデル

Breast cancer is the leading cause of cancer-related mortality in women worldwide. Liver metastasis is involved in upwards of 30% of cases with breast ...

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 ...

Orthotopic Injection of Breast Cancer Cells into the Mice Mammary Fat Pad

A proper animal model is crucial for a better understanding of diseases. Animal models established by different methods (subcutaneous injections, xenografts, genetic manipulation, chemical reagents induction, etc.) have various pathological characters and play important roles in investigating certain aspects of diseases. Although no single model can totally mimic the whole human disease progression, orthotopic organs disease models with a proper stromal environment play an irreplaceable role in understanding diseases and screening for potential drugs. In this article, we describe how to implant breast cancer cells into the mammary fat pad in a simple, less invasive, and easy-to-handle way, and follow the metastasis to distant organs. With the proper features of primary tumor growth, breast and nipple pathological changes, and a high occurrence of other organs&#39; metastasis, this model maximumly mimics human breast cancer progression. Primary tumor growth in situ, long-distant metastasis, and the tumor microenvironment of breast cancer can be investigated by using this model.

Here, we present a protocol to implant breast cancer cells into the mammary fat pad in a simple, less invasive, and easy-to-handle way, and this mouse orthotopic breast cancer model with a proper mammary fat pad environment can be used to investigate various aspects of cancer.

マウス乳腺の脂肪パッドに乳癌細胞の同所性同種注入

A proper animal model is crucial for a better understanding of diseases. Animal models established by different methods (subcutaneous injections, ...

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 ...

Modeling Brain Metastasis Via Tail-Vein Injection of Inflammatory Breast Cancer Cells

Metastatic spread to the brain is a common and devastating manifestation of many types of cancer. In the United States alone, about 200,000 patients are diagnosed with brain metastases each year. Significant progress has been made in improving survival outcomes for patients with primary breast cancer and systemic malignancies; however, the dismal prognosis for patients with clinical brain metastases highlights the urgent need to develop novel therapeutic agents and strategies against this deadly disease. The lack of suitable experimental models has been one of the major hurdles impeding advancement of our understanding of brain metastasis biology and treatment. Herein, we describe a xenograft mouse model of brain metastasis generated via tail-vein injection of an endogenously HER2-amplified cell line derived from inflammatory breast cancer (IBC), a rare and aggressive form of breast cancer. Cells were labeled with firefly luciferase and green fluorescence protein to monitor brain metastasis, and quantified metastatic burden by bioluminescence imaging, fluorescent stereomicroscopy, and histologic evaluation. Mice robustly and consistently develop brain metastases, allowing investigation of key mediators in the metastatic process and the development of preclinical testing of new treatment strategies.

We describe a xenograft mouse model of breast cancer brain metastasis generated via tail-vein injection of an endogenously HER2-amplified inflammatory breast cancer cell line.

炎症性乳癌細胞の尾静脈注射による脳転移のモデル化

Metastatic spread to the brain is a common and devastating manifestation of many types of cancer. In the United States alone, about 200,000 patients are ...

Orthotopic Injection of Breast Cancer Cells into the Mammary Fat Pad of Mice to Study Tumor Growth.

Breast cancer growth can be studied in mice using a plethora of models. Genetic manipulation of breast cancer cells may provide insights into the functions of proteins involved in oncogenic progression or help to discover new tumor suppressors. In addition, injecting cancer cells into mice with different genotypes might provide a better understanding of the importance of the stromal compartment. Many models may be useful to investigate certain aspects of disease progression but do not recapitulate the entire cancerous process. In contrast, breast cancer cells engraftment to the mammary fat pad of mice better recapitulates the location of the disease and presence of the proper stromal compartment and therefore better mimics human cancerous disease. In this article, we describe how to implant breast cancer cells into mice orthotopically and explain how to collect tissues to analyse the tumor milieu and metastasis to distant organs. Using this model, many aspects (growth, angiogenesis, and metastasis) of cancer can be investigated simply by providing a proper environment for tumor cells to grow.

Cancer is a complex disease that is influenced by the tissue surrounding the tumor as well as local pro- and anti-inflammatory mediators. Therefore, orthotropic injection models, rather than subcutaneous models may be useful to study cancer progression in a manner that better mimics human pathology.

腫瘍増殖を研究するためにマウスの乳房脂肪パッド中に乳癌細胞の同所注入。

Breast cancer growth can be studied in mice using a plethora of models. Genetic manipulation of breast cancer cells may provide insights into the ...

Injection of Syngeneic Murine Melanoma Cells to Determine Their Metastatic Potential in the Lungs

Approximately 90% of human cancer deaths are linked to metastasis. Despite the prevalence and relative harm of metastasis, therapeutics for treatment or prevention are lacking. We report a method for the establishment of pulmonary metastases in mice, useful for the study of this phenomenon. Tail vein injection of B57BL/6J mice with B16-BL6 is among the most used models for melanoma metastases. Some of the circulating tumor cells establish themselves in the lungs of the mouse, creating "experimental" metastatic foci. With this model it is possible to measure the relative effects of therapeutic agents on the development of cancer metastasis. The difference in enumerated lung foci between treated and untreated mice indicates the efficacy of metastases neutralization. However, prior to the investigation of a therapeutic agent, it is necessary to determine an optimal number of injected B16-BL6 cells for the quantitative analysis of metastatic foci. Injection of too many cells may result in an overabundance of metastatic foci, impairing proper quantification and overwhelming the effects of anti-cancer therapies, while injection of too few cells will hinder the comparison between treated and controls.

Metastasis plays a profound role in the virulence of cancer, accounting for an estimated 90% of deaths. We report a protocol for a metastatic melanoma model in mice that is useful for determining the efficacy of therapeutic agents against this clinical phenomenon.

同系マウスメラノーマ細胞の注入は、肺におけるそれらの転移能を決定するために、

Approximately 90% of human cancer deaths are linked to metastasis.  Despite the prevalence and relative harm of metastasis, therapeutics for treatment or ...

An Orthotopic Mouse Model of Spontaneous Breast Cancer Metastasis

Metastasis is the primary cause of mortality of breast cancer patients. The mechanism underlying cancer cell metastasis, including breast cancer metastasis, is largely unknown and is a focus in cancer research. Various breast cancer spontaneous metastasis mouse models have been established. Here, we report a simplified procedure to establish orthotopic transplanted breast cancer primary tumor and resultant spontaneous metastasis that mimic human breast cancer metastasis. Combined with the bioluminescence live tumor imaging, this mouse model allows tumor growth and progression kinetics to be monitored and quantified. In this model, a low dose (1 x 104 cells) of 4T1-Luc breast cancer cells was injected into BALB/c mouse mammary fat pad using a tuberculin syringe. Mice were injected with luciferin and imaged at various time points using a bioluminescent imaging system. When the primary tumors grew to the size limit as in the IACUC-approved protocol (approximately 30 days), mice were anesthetized under constant flow of 2% isoflurane and oxygen. The tumor area was sterilized with 70% ethanol. The mouse skin around the tumor was excised to expose the tumor which was removed with a pair of sterile scissors. Removal of the primary tumor extends the survival of the 4T-1 tumor-bearing mice for one month. The mice were then repeatedly imaged for metastatic tumor spreading to distant organs. Therapeutic agents can be administered to suppress tumor metastasis at this point. This model is simple and yet sensitive in quantifying breast cancer cell growth in the primary site and progression kinetics to distant organs, and thus is an excellent model for studying breast cancer growth and progression, and for testing anti-metastasis therapeutic and immunotherapeutic agents in vivo.

An orthotopic breast cancer primary tumor model and surgical removal of primary tumor to extend mouse life to generate spontaneous metastasis are described. The tumor growth and progression are monitored and quantified by luciferase fluorescence imaging.