The authors have no acknowledgements.

倫理の声明：すべての動物実験は、テキサス大学MDアンダーソンがんセンターの施設内動物管理使用委員会（IACUC）によって承認されました。 1.注射用癌細胞を準備します<ol><li> 1日か2日の注入前に癌細胞をシード。特殊媒体は、特定の細胞株について、文献に示されない限り、10％FBSを補充したDMEM / F12培地は、使用します。 </li><li> 37℃で2分 - 1のためのトリプシン処理（0.25％）の前に一回無血清培地との最初の洗浄により80％コンフルエンス - 手術の日に、収穫細胞は、彼らが70に到達したとき。 DMEM / F12培地をFBSを含有する10％が0.25％トリプシンをクエンチするために細胞に加えます。 </li><li> 3分間80×gで細胞を遠心分離します。細胞株に応じて、5×10 6細胞/ ml -無血清培地で洗浄細胞を2回1にハンクス平衡塩溶液（HBSS）で、残存血清を除去し、血球計で細胞を計数し、再懸濁します。氷の上に保管してください注射時まで。 注：MDA-MB-231乳癌細胞およびK1735メラノーマ細胞を本研究に使用した、両方の細胞株は、/ mlの濃度が2×10 6個の細胞で使用しました。過剰治療は転移アッセイの成果に影響を与える可能性があるとして、できるだけ短いトリプシン処理時間をおいてください。 </li></ol> 2.腫瘍細胞注射のためにマウスを準備<ol><li>腹腔内ケタミン/キシラジンのカクテルの（IP）注射（ケタミンを100mg / kgを、キシラジン10mg / kgの）によってマウスを麻酔。 </li><li>動物の足をつまんで完全に麻酔を確認し、応答の欠如を観察します。 </li><li> （タンパク質ゲルを鋳造するために使用される）ガラス板上にマウスを置き、ゴムバンドで固定します。 </li><li>必要に応じて、シェービングや脱毛製品の少量を適用し、紙タオルで髪を拭き取ることにより、首の毛を取り除きます。注：彼らは体毛を持っていないとして、ヌードマウスを使用している場合は、このステップをスキップします。 </li><li>適用することにより、首の皮膚をきれいにしてくださいポビドンヨードおよび70％のアルコール。 </li><li>解剖顕微鏡のステージ上にマウスを置き、 </li><li>手術用メスで約1センチの皮膚を切開してください。 </li><li>ぶっきらぼう下に頸動脈を露出させるために歯付き鉗子で筋肉を解剖。 </li><li>手術用鉗子で隣接する迷走神経から頸動脈を分離します。 </li><li>手術用鉗子を使用して、より小さなコットンボールに、綿球からいくつかの綿を分離湿らせ、そしてファッション。注射の意図された部位の頸動脈の下にこのバッファ湿コットンボールを置きます。 </li><li>コットンボールに縫合糸の遠位端と近位を置き、緩い結び目を作ります。注射部位への血流を遮断するために、近位結び目を締めます。 </li><li>コットンボールに頚動脈が新鮮な赤い血と完全に加圧された表示された場合は、癌細胞の注射に進みます。 </li></ol>頚動脈に3癌細胞注入<ol><li>ボルテックスし、100μlのOを描きますシリンジにF癌細胞。 </li><li>解剖顕微鏡下で、ゆっくりとコットンボールの上に座って頸動脈の内腔に（ベベルを上にして）31 G針を挿入します。 </li><li>ゆっくりと頸動脈に注射器から細胞を注入します。緩衝癌細胞が循環中に押し込まれたときに成功した注射は、近くの血管や筋肉組織の変色によって、顕微鏡下で観察することができます。 </li><li>ボリューム全体の注入が完了すると、逆流を防止するために穏やかに遠位頚動脈を持ち上げます。 </li><li>素早く注入プロセスを完了するために、遠位の結び目を締めます。 </li><li>注入が完了した後、合字を締めし、解剖顕微鏡下でマイクロはさみで余分な絹縫合糸をトリミング。創傷部位をカバーするために分離された筋肉を戻します。 2つのステープルで皮膚を閉じます。 </li></ol> 4.外科回復<ol><li>加熱パッド上に操作動物を移動します回復のため。動物が意識を回復するために60分 - それは30を取ることができます。一方で、術後の鎮痛などの皮下注射を介して、ブプレノルフィン（0.05ミリグラム/キログラム）を管理。注：鎮痛はIACUCプロトコルを承認し、ローカルに応じて提供されるべきです。 </li><li>動物が目覚めると、自分の通常の動作を再開したら、彼らの住宅の場所にマウスを返します。数日後に手術のためのゲル食品でマウスを提供します。外科的な記録を維持し、部屋に保管してください。 </li></ol> 5. in vivoイメージング非侵襲的<ol><li>ルシフェラーゼレポーター構築物で標識された細胞は、in vivoイメージングシステム14 に用いた癌細胞の注入効率を測定します。 注：イメージングは​​、研究動物に過度なストレスを回避するために、注射の日に実行されません。 </li></ol>

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The authors have nothing to disclose.

癌細胞の成功した頸動脈注射のための最も重要な手順は次のとおりです。手術の準備のために、マウスの1）深い麻酔。 2）綿のサポートの上に頸動脈の安定した配置。成功した注入後の頸動脈の3）タイト結紮。 30分〜の深い麻酔を解剖顕微鏡下での安定した外科的なパフォーマンスのために通常必要です。我々は、マウスの麻酔のために商業的にすぐに使用できるケタ?...

中枢神経系（CNS）への癌の転移は壊滅的な疾患であり、脳実質または軟膜（「脳転移が「この資料の両方を意味する）のいずれかを含むことができます。 1 1、2：それは&gt; 10で一次神経膠腫をより多かっ、支配的な頭蓋内悪性腫瘍です。肺癌、乳癌、および黒色腫は脳転移3,4の高い発生率を生成トップ三大腫瘍性疾患です。近年では、新たな癌治療中の印象的な進歩は劇的に生存を延長し、多くの癌患者の生活の質を改善しています。しかし、再発時に、脳転移の発生率が急速に上昇しています。例えば、抗HER2抗体トラスツズマブ（ハーセプチン）は、HER2 +乳癌患者で有意な臨床効果を示しました。まだ不穏な傾向が出てきましたこれらの患者における：その頭蓋外全身性疾患最初に、後に脳転移5、6、7を開発トラスツズマブ治療の恩恵を受けたものの1/3まで。悲しいことに、脳転移を有する患者は、通常、生活の質の外傷性劣化を経験し、ほとんどすべての現在の治療に対して難治性であり、診断後の彼らの1年生存率はわずか〜20％8です。 （ステロイド、頭蓋放射線療法、および選択された患者における外科的切除を含む）脳転移のための現在の治療は、単に9治癒的ではなく、姑息されています。そのため、脳転移は、新規癌治療のこの時代の次の印象的な課題として浮上しています。患者が診療所に日々直面している満たされていない課題を解決するために、我々は早急に優れた脳転移の根底にあるメカニズムを理解し、新しい治療法を開発するためにこの知識を使用する必要があります。 <pクラス= &quot;jove_content&quot;&gt;転移性癌細胞による脳の成功したコロニー形成は、血液脳関門（BBB）のバイパスのような複数の生物学的な選手の複雑な相互作用によって媒介される一連の機能の性能を必要とし、脳の固有から逃れますex vivoでまたはin vitroシステムで再現されているいずれも免疫防御的なメカニズム10、。したがって、in vivoモデル適切かつ忠実な脳転移の研究のために重要です。従来のインビボ転移アッセイは、肺から取り出すときにひっかかるする細胞の大部分をリードし、尾静脈注射を介してがん細胞を紹介します。脳転移病巣はほとんど肺11内の腫瘍負荷によって引き起こされる、動物の死を前に、これらのモデルで生産されていません。癌細胞の直接脳内注射は、CNSにおける一貫した腫瘍の成長を生じ、一次神経膠腫の研究で広く用いられています。しかし、SUCH注射は、BBBを損なうし、このモデルの生理的関連性についての懸念の両方の主要なポイント、注射部位の外傷性損傷を引き起こします。別の頻繁に使用される癌細胞の導入路、心臓内注射、管理し、CNSへの実験的転移を生み出すん簡単です。しかし、CNS以外の臓器サイトへの同時転移は常に生成され、動物の死亡率11を引き起こす可能性があります 。従って、このモデルの可変性の高い、動物の限られた数の生物学的機構又は治療薬の定量的評価のために、それは不適切になります。 ここでは、総頸動脈への癌細胞の注射を介して実験的脳転移を生成する手順を説明します。我々は、脳転移の転移カスケードに個々の遺伝子の貢献を分析し、治療的介入の有効性を評価するために、このアプローチを使用していますS 12、13。このアプローチの主な利点は、再現性の変動の低度の高度です。主な欠点は、顕微手術を実行するために必要な精巧さと器用さです。

<table border="0" cellpadding="0" cellspacing="0" width="1156">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:447px;">Ketamine hydrochloride/xylazine hydrochloride solution</td>
			<td style="width:151px;">Sigma-Aldrich</td>
			<td style="width:151px;">K113</td>
			<td style="width:411px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:447px;"></td>
			<td style="width:151px;"></td>
			<td style="width:151px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:447px;">Routine Stereomicroscope Leica M50</td>
			<td style="width:151px;">Leica</td>
			<td style="width:151px;">M50</td>
			<td>The microscope is modular and highly configurable to fit particular space requirements.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:447px;"></td>
			<td style="width:151px;"></td>
			<td style="width:151px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:447px;">Surgical disposable scapel</td>
			<td style="width:151px;">Integra Miltex</td>
			<td style="width:151px;">4-410</td>
			<td>to make skin incisions</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:447px;">Tissue Forceps - 1x2 Teeth</td>
			<td style="width:151px;">Fine Science Tools</td>
			<td style="width:151px;">11021-12</td>
			<td>to bluntly dissect mucles</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:447px;">Dumont #5 - Mirror Finish Forceps</td>
			<td style="width:151px;">Fine Science Tools</td>
			<td style="width:151px;">11252-23</td>
			<td>to separate and prepare the carotid artery for injection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:447px;">Spring Scissors</td>
			<td style="width:151px;">Fine Science Tools</td>
			<td style="width:151px;">15025-10</td>
			<td>to cut sutures</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:447px;">EZ Clip Kit</td>
			<td style="width:151px;">Stoelting</td>
			<td style="width:151px;">59020</td>
			<td>for wound cloure</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:447px;">BD Insulin Syringe</td>
			<td style="width:151px;">Becton Dickinson</td>
			<td style="width:151px;">328438</td>
			<td>for cell injection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:447px;"></td>
			<td style="width:151px;"></td>
			<td style="width:151px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:447px;">IVIS Spectrum In Vivo Imaging System</td>
			<td style="width:151px;">PerkinElmer</td>
			<td style="width:151px;">124262</td>
			<td></td>
		</tr>
	</tbody>
</table>

intracarotid cancer cell injection to produce mouse models of brain metastasis

転移は、患者の体内の二次部位での広がりと悪性細胞の増殖は、癌関連死亡率の&gt; 90％を占めます。最近、新しい治療法で印象的な進歩は飛躍的に多くの癌患者の生存および生活の質の向上を延長しています。悲しいことに、脳転移再発の発生率が急上昇して、すべての現在の治療は、単なる姑息です。したがって、良好な実験動物モデルが緊急疾患生物学の詳細な研究を容易にし、前臨床評価のための新規な治療法を評価するために必要とされます。しかし、癌細胞の尾静脈注射を介して、 インビボ転移アッセイにおける標準は、主に肺転移病変を生成します。動物は通常、脳転移のいずれかの意味の伸長前に肺腫瘍負荷に屈します。腫瘍細胞の心臓内注射は、脳を含む複数の器官部位への転移病変を生成します。しかし、variabiこのモデルを用いて製造腫瘍増殖のリティは、治療効果を評価する際に、その有用性を減衰大きいです。脳転移の研究のための信頼性と一貫性の動物モデルを生成するには、ここでは、腫瘍細胞の頸動脈内注射を介して家のマウス（ ハツカネズミ ）における実験的脳転移を製造するための手順について説明します。このアプローチは、このように基本的な生物学的メカニズムを研究し、新規治療薬を評価するために研究努力を促進する、いずれかの同様の増殖率および死亡率の特性を有する脳転移担持マウスの大量生産を可能にします。

注射の質を評価することができる2つのポイントがあります。最初の可能性は、注入の間、血管の色を変化させる操作者の観察によるものです。貧しい注射からの漏れが簡単に解剖顕微鏡下で観察することができます。サポートコットンボールにマウス本体（ 図1A）とその頸動脈（ 図1B）の安定的かつ安全な配置は、頸動脈への円滑かつ成功?...

Watch this Scientific Journal Video about Intracarotid Cancer Cell Injection to Produce Mouse Models of Brain Metastasis at JoVE.com

Intracarotid Cancer Cell Injection to Produce Mouse Models of Brain Metastasis

脳転移は、治療の選択肢が緩和的に残っているが、その発生率が増加しているとして緊急に満たされていない医療ニーズとなっています。癌細胞の頸動脈内動脈注射を介して脳転移の実験動物モデルを作成することは、疾患の生物学および新規介入計画の評価の機構研究を容易にします。

脳転移のマウスモデルを生成するために頸動脈内癌細胞注入

Metastasis, the spread and growth of malignant cells at secondary sites within a patient&#39;s body, accounts for &#62; 90% of cancer-related mortality. ...

intracarotid-cancer-cell-injection-to-produce-mouse-models-brain

Research

JoVE Journal

Cancer Research

26.2K ビュー.  The University of Texas MD Anderson Cancer Center. 脳転移は、治療の選択肢が緩和的に残っているが、その発生率が増加しているとして緊急に満たされていない医療ニーズとなっています。癌細胞の頸動脈内動脈注射を介して脳転移の実験動物モデルを作成することは、疾患の生物学および新規介入計画の評価の機構研究を容易にします。 

ビデオ: Intracarotid Cancer Cell Injection to Produce Mouse Models of Brain Metastasis

Intra-iliac Artery Injection for Efficient and Selective Modeling of Microscopic Bone Metastasis

Intra-iliac artery (IIA) injection is an efficient approach to introduce metastatic lesions of various cancer cells in animals. Compared to the widely used intra-cardiac and intra-tibial injections, IIA injection brings several advantages. First, it can deliver a large quantity of cancer cells specifically to hind limb bones, thereby providing spatiotemporally synchronized early-stage colonization events and allowing robust quantification and swift detection of disseminated tumor cells. Second, it injects cancer cells into the circulation without damaging the local tissues, thereby avoiding inflammatory and wound-healing processes that confound the bone colonization process. Third, IIA injection causes very little metastatic growth in non-bone organs, thereby preventing animals from succumbing to other vital metastases, and allowing continuous monitoring of indolent bone lesions. These advantages are especially useful for the inspection of progression from single cancer cells to multi-cell micrometastases, which has largely been elusive in the past. When combined with cutting-edge approaches of biological imaging and bone histology, IIA injection can be applied to various research purposes related to bone metastases.

This manuscript provides the detailed procedure of intra-iliac artery (IIA) injection, a technique to deliver cancer cells specifically to hind limb tissues including bones to establish experimental bone metastases. Although initially established with breast tumor models, this protocol can be easily extended to other cancer types.

顕微鏡骨転移の効率的かつ選択的モデリングのためのイントラ腸骨動脈注入

Intra-iliac artery (IIA) injection is an efficient approach to introduce metastatic lesions of various cancer cells in animals. Compared to the widely ...

がん研究

Surgical Procedures and Methodology for a Preclinical Murine Model of De Novo Mammary Cancer Metastasis

A rate-limiting aspect of transgenic mouse models of mammary adenocarcinoma is that primary tumor burden in mammary tissue typically defines study end-points. Thus, studies focused on elucidating mechanisms of late-stage de novo metastasis are compromised, as are studies examining efficacy of anti-cancer therapies targeting mediators of metastasis in the adjuvant setting. Numerous murine mammary cancer models have been developed via targeted expression of dominant oncoproteins to mammary epithelial cells yielding models variably mimicking histopathologic and transcriptome-defined breast cancer subtypes common in women1. While much has been learned regarding the biology of mammary carcinogenesis with these models, their utility in identifying molecules regulating growth of late-stage metastasis are compromised as mice are typically euthanized at earlier time points due to significant primary tumor burden. Moreover, since a significant percentage of women diagnosed with breast cancer receive adjuvant therapy after surgical resection of primary tumors and prior to presence of detectable metastatic disease, preclinical models of de novo metastasis are urgently needed as platforms to evaluate new therapies aimed at targeting metastatic foci. To address these deficiencies, we developed a murine model of de novo mammary cancer metastasis, wherein primary mammary tumors are surgically resected, and metastatic foci subsequently develop over a 115 day post-surgical period. This long latency provides a tractable model to identify functionally significant regulators of metastatic progression in mice lacking primary tumor, as well as a model to evaluate preclinical therapeutic efficacy of agents aimed at blocking functionally significant molecules aiding metastatic tumor survival and growth.

Surgical Procedures and Methodology for a Preclinical Murine Model of De Novo Mammary Cancer Metastasis

Pre-clinical models evaluating adjuvant therapy targeting breast cancer metastasis are lacking. To address this, we developed a murine model of de novo pulmonary mammary adenocarcinoma metastasis, wherein therapies administered in the adjuvant setting (post surgical resection of primary tumors) can be evaluated for efficacy in impacting previously seeded pulmonary metastases.

前臨床マウスモデルのための外科手術手順および方法論<em&gt;デ・ノヴォ</em&gt;乳がん転移

A rate-limiting aspect of transgenic mouse models of mammary adenocarcinoma is that primary tumor burden in mammary tissue typically defines study ...

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 ...

Generation of Prostate Cancer Cell Models of Resistance to the Anti-mitotic Agent Docetaxel

Microtubule targeting agents (MTAs) are a mainstay in the treatment of a wide range of tumors. However, acquired resistance to chemotherapeutic drugs is a common mechanism of disease progression and a prognostic-determinant feature of malignant tumors. In prostate cancer (PC), resistance to MTAs such as the taxane Docetaxel dictates treatment failure as well as progression towards lethal stages of disease that are defined by a poor prognosis and high mortality rates. Though studied for decades, the array of mechanisms contributing to acquired resistance are not completely understood, and thus pose a significant limitation to the development of new therapeutic strategies that could benefit patients in these advanced stages of disease. In this protocol, we describe the generation of Docetaxel-resistant prostate cancer cell lines that mimic lethal features of late-stage prostate cancer, and therefore can be used to study the mechanisms by which acquired chemoresistance arises. Despite potential limitations intrinsic to a cell based model, such as the loss of resistance properties over time, the Docetaxel-resistant cell lines produced by this method have been successfully used in recent studies and offer&#160;the opportunity to advance our molecular understanding of acquired chemoresistance in lethal prostate cancer.

Resistance to cancer therapies contributes to disease progression and death. Determining the mechanistic underpinnings of resistance is crucial for improving therapeutic response. This manuscript details the protocol to generate taxane-resistant cell models of prostate cancer (PC) to help dissecting the pathways involved in progression to Docetaxel resistance in PC patients.

抗有糸分裂エージェント ドセタキセルに対する抵抗性の前立腺癌細胞モデルの生成

Microtubule targeting agents (MTAs) are a mainstay in the treatment of a wide range of tumors. However, acquired resistance to chemotherapeutic drugs is a ...

Live-Cell Imaging Assays to Study Glioblastoma Brain Tumor Stem Cell Migration and Invasion

Glioblastoma (GBM) is an aggressive brain tumor that is poorly controlled with the currently available treatment options. Key features of GBMs include rapid proliferation and pervasive invasion into the normal brain. Recurrence is thought to result from the presence of radio- and chemo-resistant brain tumor stem cells (BTSCs) that invade away from the initial cancerous mass and, thus, evade surgical resection. Hence, therapies that target BTSCs and their invasive abilities may improve the otherwise poor prognosis of this disease. Our group and others have successfully established and characterized BTSC cultures from GBM patient samples. These BTSC cultures demonstrate fundamental cancer stem cell properties such as clonogenic self-renewal, multi-lineage differentiation, and tumor initiation in immune-deficient mice. In order to improve on the current therapeutic approaches for GBM, a better understanding of the mechanisms of BTSC migration and invasion is necessary. In GBM, the study of migration and invasion is restricted, in part, due to the limitations of existing techniques which do not fully account for the in vitro growth characteristics of BTSCs grown as neurospheres. Here, we describe rapid and quantitative live-cell imaging assays to study both the migration and invasion properties of BTSCs. The first method described is the BTSC migration assay which measures the migration toward a chemoattractant gradient. The second method described is the BTSC invasion assay which images and quantifies a cellular invasion from neurospheres into a matrix. The assays described here are used for the quantification of BTSC migration and invasion over time and under different treatment conditions.

Here, we describe live-cell imaging techniques to quantitatively measure the migration and invasion of glioblastoma brain tumor stem cells over time and under multiple treatment conditions.

住セルイメージ投射アッセイ研究神経膠腫脳腫瘍幹細胞の遊走と浸潤に

Glioblastoma (GBM) is an aggressive brain tumor that is poorly controlled with the currently available treatment options. Key features of GBMs include ...

Patient-derived Orthotopic Xenograft Models for Human Urothelial Cell Carcinoma and Colorectal Cancer Tumor Growth and Spontaneous Metastasis

Cancer patients have poor prognoses when lymph node (LN) involvement is present in both high-grade urothelial cell carcinoma (HG-UCC) of the bladder and colorectal cancer (CRC). More than 50% of patients with muscle-invasive UCC, despite curative therapy for clinically-localized disease, will develop metastases and die within 5 years, and metastatic CRC is a leading cause of cancer-related deaths in the US. Xenograft models that consistently mimic UCC and CRC metastasis seen in patients are needed. This study aims to generate patient-derived orthotopic xenograft (PDOX) models of UCC and CRC for primary tumor growth and spontaneous metastases under the influence of LN stromal cells mimicking the progression of human metastatic diseases for drug screening. Fresh UCC and CRC tumors were obtained from consented patients undergoing resection for HG-UCC and colorectal adenocarcinoma, respectively. Co-inoculated with LN stromal cell (LNSC) analog HK cells, luciferase-tagged UCC cells were intra-vesically (IB) instilled into female non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice, and CRC cells were intra-rectally (IR) injected into male NOD/SCID mice. Tumor growth and metastasis were monitored weekly using bioluminescence imaging (BLI). Upon sacrifice, primary tumors and mouse organs were harvested, weighed, and formalin-fixed for Hematoxylin and Eosin and immunohistochemistry staining. In our unique PDOX models, xenograft tumors resemble patient pre-implantation tumors. In the presence of HK cells, both models have high tumor implantation rates measured by BLI and tumor weights, 83.3% for UCC and 96.9% for CRC, and high distant organ metastasis rates (33.3% detected liver or lung metastasis for UCC and 53.1% for CRC). In addition, both models have zero mortality from the procedure. We have established unique, reproducible PDOX models for human HG-UCC and CRC, which allow for tumor formation, growth, and metastasis studies. With these models, testing of novel therapeutic drugs can be performed efficiently and in a clinically-mimetic manner.

This protocol describes the generation of patient-derived orthotopic xenograft models by intra-vesically instilling high-grade urothelial cell carcinoma cells or intra-rectally injecting colorectal cancer cells into non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice for primary tumor growth and spontaneous metastases under the influence of lymph node stromal cells, which mimics the progression of human metastatic diseases.

ヒト尿路上皮細胞癌および大腸癌腫瘍増殖および自発性転移に対する患者由来の眼科医異所性Xenograft移植片モデル

Cancer patients have poor prognoses when lymph node (LN) involvement is present in both high-grade urothelial cell carcinoma (HG-UCC) of the bladder and ...

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

Metastasis, the primary cause of morbidity and mortality for most cancer patients, can be challenging to model preclinically in mice. Few spontaneous metastasis models are available. Thus, the experimental metastasis model involving tail-vein injection of suitable cell lines is a mainstay of metastasis research. When cancer cells are injected into the lateral tail-vein, the lung is their preferred site of colonization. A potential limitation of this technique is the accurate quantification of the metastatic lung tumor burden. While some investigators count macrometastases of a pre-defined size and/or include micrometastases following sectioning of tissue, others determine the area of metastatic lesions relative to normal tissue area. Both of these quantification methods can be exceedingly difficult when the metastatic burden is high. Herein, we demonstrate an intravenous injection model of lung metastasis followed by an advanced method for quantifying metastatic tumor burden using image analysis software. This process allows for investigation of multiple end-point parameters, including average metastasis size, total number of metastases, and total metastasis area, to provide a comprehensive analysis. Furthermore, this method has been reviewed by a veterinary pathologist board-certified by the American College of Veterinary Pathologists (SEK) to ensure accuracy.

Intravenous injection of cancer cells is often used in metastasis research, but the metastatic tumor burden can be difficult to analyze. Herein, we demonstrate a tail-vein injection model of metastasis and include a novel approach to analyze the resulting metastatic lung tumor burden.

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 ...

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 ...

The Establishment and Utilization of Patient Derived Xenograft Models of Central Nervous System Metastasis

The development of novel therapies for central nervous system (CNS) metastasis has been hindered by the lack of preclinical models that accurately represent the disease. Patient derived xenograft (PDX) models of CNS metastasis have been shown to better represent the phenotypic and molecular characteristics of the human disease, as well as better reflect the heterogeneity and clonal dynamics of human patient tumors compared to historic cell line models. There are multiple sites that can be used to implant patient-derived tissue when setting up preclinical trials, each with their own advantages and disadvantages, and each suited for studying different aspects of the metastatic cascade. Here, the protocol describes how to establish PDX models and present three different approaches for utilizing CNS metastasis PDX models in pre-clinical studies, discussing each of their applications and limitations. These include flank implantation, orthotopic injection in the brain, and intracardiac injection. Subcutaneous flank implantation is the easiest to monitor and, therefore, most convenient for pre-clinical studies. In addition, metastases to brain and other tissues from flank implantation were observed, indicating that the tumor has undergone multiple steps of metastasis, including intravasation, extravasation, and colonization. Orthotopic injection in the brain is the best option for recapitulating the brain tumor microenvironment and is useful for determining the efficacy of biologics to cross the blood-brain barrier (BBB) but bypasses most steps of the metastatic cascade. Intracardiac injection facilitates metastasis to the brain and is also useful for studying organ tropism. While this method forgoes earlier steps of the metastatic cascade, these cells will still have to survive circulation, extravasate, and colonize. The utility of a PDX model, is therefore, impacted by the route of tumor inoculation and the choice of which one to utilize should be dictated by the scientific question and overall goals of the experiment.

Central nervous system metastasis PDX models represent the phenotypic and molecular characteristics of human metastasis, making them excellent models for preclinical studies. Described here is how to establish PDX models and the inoculation routes that are best used for preclinical studies.

中枢神経系転移の患者由来異種移植モデルの確立と利用

The development of novel therapies for central nervous system (CNS) metastasis has been hindered by the lack of preclinical models that accurately ...

Portal Vein Injection of Colorectal Cancer Organoids to Study the Liver Metastasis Stroma

Hepatic metastasis of colorectal cancer (CRC) is a leading cause of cancer-related death. Cancer-associated fibroblasts (CAFs), a major component of the tumor microenvironment, play a crucial role in metastatic CRC progression and predict poor patient prognosis. However, there is a lack of satisfactory mouse models to study the crosstalk between metastatic cancer cells and CAFs. Here, we present a method to investigate how liver metastasis progression is regulated by the metastatic niche and possibly could be restrained by stroma-directed therapy. Portal vein injection of CRC organoids generated a desmoplastic reaction, which faithfully recapitulated the fibroblast-rich histology of human CRC liver metastases. This model was tissue-specific with a higher tumor burden in the liver when compared to an intra-splenic injection model, simplifying mouse survival analyses. By injecting luciferase-expressing tumor organoids, tumor growth kinetics could be monitored by in vivo imaging. Moreover, this preclinical model provides a useful platform to assess the efficacy of therapeutics targeting the tumor mesenchyme. We describe methods to examine whether adeno-associated virus-mediated delivery of a tumor-inhibiting stromal gene to hepatocytes could remodel the tumor microenvironment and improve mouse survival. This approach enables the development and assessment of novel therapeutic strategies to inhibit hepatic metastasis of CRC.

Portal vein injection of colorectal cancer (CRC) organoids generates stroma-rich liver metastasis. This mouse model of CRC hepatic metastasis represents a useful tool to study tumor-stroma interactions and develop novel stroma-directed therapeutics such as adeno-associated virus-mediated gene therapies.

肝転移間質を研究するための結腸直腸癌オルガノイドの門脈注入

Hepatic metastasis of colorectal cancer (CRC) is a leading cause of cancer-related death. Cancer-associated fibroblasts (CAFs), a major component of the ...