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

<ol>
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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>for cleaning tail prior to injection</td>
		</tr>
		<tr>
			<td>dissection scissors</td>
			<td>Fisher Scientific</td>
			<td>08-951-5</td>
			<td>for mouse dissection and lung tissue inflation</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&#39;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&#39;s fur during dissection; use caution - flammable</td>
		</tr>
		<tr>
			<td>Evan&#39;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&#39;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&#39;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 Video at JoVE.com

Pathological Analysis of Lung Metastasis Following Lateral Tail-Vein Injection of Tumor Cells Video (Video) | JoVE

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。

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

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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

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