Research in Zhang lab was supported by X. H.-F. Z.'s NCI CA151293, CA183878, Breast Cancer Research Foundation, U.S. Department of Defense DAMD W81XWH-13-1-0195, a Pilot Award of CA149196-04, McNair Medical Institute and by H.W.'s U.S. Department of Defense DAMD W81XWH-13-1-0296.

全ての動物作業はベイラー医科大学の動物のケアのガイドラインに基づいて行われました。 1.細胞の調製注：異なる癌細胞株は、研究目的に応じIIA注入のために使用することができます。我々は、乳癌細胞株を使用しているMCF7、4T1、4T07、MDA-MB-361、MDA-MB-231、MDA-MB-436および研究における前立腺癌細胞株C4-2。我々は通常、我々の研究のために、GFPおよびホタルルシフェラーゼ標識癌細胞の両方を使用し、GFP-ルシフェラーゼ標識MCF7細胞株から、ここでいくつかのデータを示しています。 <ol><li> 5％CO 2中、37℃で、10％FBSおよび0.1mg / mlのペニシリン-ストレプトマイシンを含有するDMEM中で細胞を維持します。 </li><li> 0.25％トリプシン/ EDTA溶液を用いて90％のコンフルエンス - IIA注射前に、80℃で細胞をトリプシン処理。細胞計数のために10ミリリットルのPBSで細胞を細胞を2回洗浄し、再懸濁するために冷PBSを使用してください。 5分間、800×gでPBS、遠心セルを削除します。 </li><li>細胞を再懸濁氷冷PBS中で5×10 6細胞/ mlの濃度で。 </li><li> 1マウスのIIA注射のために100μlのGFP-およびホタルルシフェラーゼ標識癌細胞を使用してください。 注：したがって、各動物によって受信されたセルの数が5×10 5であり、この数は、細胞系の侵襲性に基づいて変更される必要があり得ます。 </li><li>氷上で細胞懸濁液を保管してください。 注：これは、注射のために準備が整うまで、振動は、細胞凝集を数分ごとに防止するために必要とされ得ます。 （例えば、MDA-MB-231のような）いくつかの細胞株について、（ 例えば 、45μmの細胞ストレーナーを通過する）、よりストリンジェントな手順は、凝集を防止するために必要とされてもよいです。血管の詰まりが組織の壊死を引き起こし、そのための実験を混乱させる場合がありますのでご了承ください。 </li></ol> 2.動物の準備<ol><li>動物の疼痛管理のために、手術前にマウスに栄養補助食品に劣らず24以上時間を5ミリグラム/ kg /日のカルプロフェン（または他の鎮痛剤）を得ました。管理します手術前に0.1mg / mlのブプレノルフィン皮下に60分の線量。注：オプションの手順余分エストラジオールを提供するために、動物の背面にエストラジオールパレットを注入することです。これは、ER +癌細胞（ 例えば 、MCF-7細胞）9のグローを加速していきます。 </li><li>腹腔内注射により - （100ミリグラム/キログラム80）とキシラジン（10 -12.5 mg / mlで）ケタミンで6週齢のマウス（約20グラム） - 4を麻酔。 </li><li>つま先のピンチと呼吸数とピンクの粘膜の50％減少（ 例えば、マウスの耳皮膚と足の皮膚）の観察による20グラムのマウスの鎮静の適切なレベルを確認してください。手術中にこれらのバイタルサイン15分毎に監視してください。注：動物のいかなる動きは、動物が十分に麻酔をかけ、手術の準備ができていることを示しています。 </li><li>乾燥を防ぐために、目に獣医軟膏を使用してください。 </li><li>下腹部、特に右鼠径部ここで手術や注射が意志の上でマウスを剃ります場所を取ります。 注：無胸腺ヌードマウスを使用している場合、これは不要です。 </li><li> 、その背中の上でマウスを置く彼らの自然な位置に足を広げ、テープ付き携帯解剖段ボールにつま先を貼り付けます。 </li><li> 70％イソプロピルエタノールに浸した滅菌綿棒で右鼠径部を拭き、ベタジン外科手術用スクラブで数回続きます。 </li></ol> 3.総腸骨静脈と動脈の場所と分離<ol><li>長い4 番目と第3ハンドルで開催された無菌の、＃10炭素鋼メスの刃を使用して、右下腹部にある5 番目の乳首は1.2センチメートル- 1.0の皮膚切開を行います。その後、切開部位を除いて動物の体をカバーするために、滅菌外科用ドレープを使用します。 </li><li>標準ベンチトップ解剖顕微鏡に動物を移動します。注射のために4倍の倍率を使用してください。 </li><li>解剖顕微鏡の4倍の倍率下では、脂肪組織とperitoneu間の鈍分離鉗子を挿入mおよび腸骨血管および神経を露出させるために両側に外側の組織を押し（ 図1参照 ）。 注：正中動脈枝の下部から大動脈の間の動脈は総腸骨動脈と呼ばれています。注入が行われる場所です。 </li><li>船に近い血管と神経の間（膜のような）結合組織、突破するためにストレートの微細鉗子のペアを1つ使用します。 </li><li>左手にまっすぐ細かい鉗子を切り替えます。右手を使用して、角度の付いた微細な鉗子のペアをつかみます。結合組織が破壊されたのと同じ位置に右手の鉗子を挿入します。そして船の下を通って右側の鉗子を貼り付けます。 </li><li>同時に、通過して右手の鉗子を導くために船の左側の結合組織に左手の鉗子を挿入します。 </li><li>右側の鉗子が船の下に到達したら、周囲のTIから血管を分離しようもう少しssue、その後、右側の鉗子の上にそれらを保ちます。 </li><li>右側の鉗子に4-0絹縫合糸の先端を提供するために左側の鉗子を使用し、その後、船の下を通って静かに、ゆっくりと縫合糸を引っ張ります。注意：今の両方の共通の静脈と動脈血管が縫合糸によって持ち上げられ（ 図2を参照）。 </li></ol> 4.インジェクションとポスト噴射ケア<ol><li> 31 Gインスリン注射器で注入のための細胞を準備します。注射あたりPBSに100μlの細胞懸濁液を使用してください。注入された細胞は、血液の流れを詰まらせ、ブロックしないように、細胞を分離し、凝集を避けるために、上下に数回をピペットとすることを確認します。 </li><li>左手に角度のついたピンセットで、縫合糸の指導に沿って船の下に鉗子を入れ、その後、鉗子の2つのアームを開き、鉗子を軽く開催された血管を持ちます。 注：鉗子の2つのアーム間の間隔がiになりますnjectionサイト。 </li><li>注射の準備ができて、上向きの針のベベルと、右手に100μlの細胞懸濁液と31 G針を保持します。 </li><li> （血液が針の先端を入力します）動脈内腔に針を挿入します。次に、細胞を注入することで、ゆっくりと細胞懸濁液を押してください。細胞懸濁液を容器に赤血球を離れてプッシュします。血管を壊したり、細胞懸濁液を漏れ出ないようにしてください。 </li><li>注入が完了すると、そっと針を引き戻すと、出血を止めるために、左側の鉗子で船を保持し続けます。その後、縫合糸を引き離します。 </li><li>左側の鉗子を引き離し、かつ迅速に出血を止めるために動脈切開領域を押して綿棒を使用しています。 注：通常5から10分間連続的圧力は、出血を止めるのに十分です。 </li><li>停止し、出血すると、手術領域の皮膚のエッジを閉じるには、皮膚の接着剤を使用しています。耳タグを作成し、注入された細胞の分布を調べるために、シグナリング生物発光を確認してください。&lt;BR /&gt;注：成功した注射は、 図3に類似した画像になります。 <ol><li>イントラ眼窩洞経由して75ミリグラム/ kgのマウスの濃度でPBS中100μlの15 mg / mlとルシフェリンの注射により生物発光シグナルを確認してください。 <ol><li>イントラ眼窩注射のために、マウスの目の両側に指を入れて眼球が若干、目のフレームから飛び出し眼球と鼻の間に28 Gインスリン注射針を挿入し、ゆっくりとルシフェリンを注入する注射器をプッシュするために圧力を使用。 注： - 硬組織（骨）を押す前に、3ミリメートル正しい位置で、針は2の深さに到達することができるはずです。 </li><li> （ 図3参照 ） を 5分以内に60秒- 10ためのインビボ動物全体のイメージング用のイメージング・システムにマウスを置きます。 注：オプションの手順余分エストラジオールを提供するために、動物の背面にエストラジオールペレットを移植することです。これは、ER +癌細胞（ 例えば 、MCF-7の成長を加速します細胞）15。 </li></ol></li></ol></li><li>それが目覚めるまで暖かい加熱パッド上でマウスを残します。 、手術後の期間中の裂開や痛みの兆候を腫れ、出血を監視します。痛みの兆候まで、疼痛管理のために与えられた鎮痛カバレッジ（carprofin栄養補助食品が提案）とのケージに戻ってマウスを置きます。手術後7日の間、動物に細心の注意とケアを払ってください。 </li></ol> 5.監視転移成長<ol><li>組織形態計測： <ol><li> 5日目、および以前14に記載されているようにパラフィン包埋それらを施す-収穫腫瘍はその後3 pH7.0の14％EDTA溶液でそれらを脱灰、24時間、4％パラホルムアルデヒド中で骨を固定する、骨組織を有します。 </li><li>セクションパラフィン埋め込む骨3μmの厚さで組織し、以前に14を説明したようにヘマトキシリン/エオシン染色の標準的な組織学的方法を実行します。 </li></ol></li><li> Immunohistochemis試してみてください。 <ol><li>被験者パラフィン包埋腫瘍担持骨は、先14に記載されているように様々な目的のために染色免疫組織化学するスライド。簡潔には、それぞれ、抗オステリックス抗体および抗アルカリホスファターゼ（ALP）抗体によるMCF7癌、抗GFP抗体による細胞、および免疫染色骨形成細胞（前骨芽細胞および骨芽細胞）免疫染色。 </li></ol></li></ol>

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The authors have nothing to disclose.

唯一の腸骨動脈は、癌細胞に注入の対象ではありますが、私たちは、周囲の組織からの両方の腸骨静脈と動脈の分離をお勧めします、とのバンドルとして一緒にそれらを持ち上げるために。静脈と動脈が広く互いに接触するためである、と静脈血管壁が薄く、壊れやすいです。がん細胞のみを動脈に注入されているがため、成功した注射のために、それは、一緒に2つの容器を保持するために...

転移は、固形腫瘍による死亡の90％以上を占めています。骨は、様々な癌の種類、特に乳癌および前立腺癌の転移によって影響を受ける最も一般的な臓器です。クリニックで診断すると、骨転移は通常、すでに多くの場合、神経学的症状を伴う、骨に溶骨性または骨芽細胞の変化のいずれかで進行した段階に入ってきました。 主に明白な溶骨性骨転移1-3に焦点を当てた以前の研究は、しかし、我々は現在、溶骨性プロセスの開始前に、骨の中に微小転移の理解が限られています。これは、少なくとも部分的には、適切な実験モデルとアプローチの欠如にあります。乳癌の遺伝子操作されたマウスモデルは、多くの場合、肺に転移するが、はるかに少ない効率的に骨4へ。同様に、同所移植された腫瘍はほとんどいくつかの骨の熱帯4T1乳腺carcinomで、自発的な骨転移を発症しませんサブクローンおよびMSPは、例外5-7としてPyMTトランスジェニックマウスモデルを過剰発現しました。イントラ脛骨掘削は骨8-10に癌細胞を提供することができますが、それはまた、局所組織への損傷および炎症を招きます。現在の乳癌細胞株の心臓内注射は、骨定着11-13を調査するための主要なアプローチとなっています。癌細胞は、左心室の中に導入された後、しかし、限られた割合は、最終的にそれが困難な定量化方法で微小転移を追跡すること、骨および骨髄に到達します。 本研究では、選択的に局所組織への損傷を引き起こすことなく、骨及び骨髄中の癌細胞を濃縮、後肢組織に癌細胞を送達するために、技術、すなわち内腸骨動脈（IIA）注入14を確立します 。なぜなら骨特異性のため、このアプローチは、無痛性癌細胞は最終的に、ANの前にコロニーを形成するのに十分な時間を可能にしますimalsは、他の重要な臓器における原発腫瘍または転移に屈します。このような生物発光イメージング、免疫蛍光染色および骨組織形態計測などの他の種々の技術と組み合わせると、IIA注入は、特にマルチセルに単一の癌細胞からの進行を追跡するために、骨転移に関連した研究目的の広い範囲のために有用である可能性があります微小転移。特に、我々はIIA注射は、癌細胞と骨微小環境における周囲のセルの様々なタイプの間の相互作用を可視化することを可能にすることを実証しました。

<table border="0" cellpadding="0" cellspacing="0" width="843">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Materials</td>
			<td style="width:303px;"></td>
			<td style="width:123px;"></td>
			<td style="width:203px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DMEM</td>
			<td style="width:303px;">HyClone</td>
			<td style="width:123px;">SH30022.01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">FBS</td>
			<td style="width:303px;">Gibco</td>
			<td style="width:123px;">16000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Pen/Strep Amphatericin B</td>
			<td style="width:303px;">Lonza Biowhittaker</td>
			<td style="width:123px;">17-745E</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">PBS</td>
			<td style="width:303px;">Lonza Biowhittaker</td>
			<td style="width:123px;">17-516F</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Trypsin/EDTA solution</td>
			<td style="width:303px;">HyClone</td>
			<td style="width:123px;">SH30042.01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">45uM cell strainer</td>
			<td style="width:303px;">VWR International Laboratory</td>
			<td style="width:123px;">195-2545</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MediGel CPF with carprofen&#160;</td>
			<td style="width:303px;">Controlled item from veterinary care in BCM</td>
			<td style="width:123px;"></td>
			<td>For pain management</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Buprenorphine&#160;</td>
			<td style="width:303px;">Controlled item from veterinary care in BCM</td>
			<td style="width:123px;"></td>
			<td>For pain management</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Estradiol pellet</td>
			<td style="width:303px;">Innovative Research of America</td>
			<td style="width:123px;">SE-121</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ketamine and xylazine</td>
			<td style="width:303px;">Controlled item from veterinary care in BCM</td>
			<td style="width:123px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Vet ointment</td>
			<td style="width:303px;">Controlled item from veterinary care in BCM</td>
			<td style="width:123px;"></td>
			<td>Avoid eye dryness</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Shaver</td>
			<td style="width:303px;">Oster</td>
			<td style="width:123px;">78005-050</td>
			<td>For furred mice</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Isopropyl ethanol</td>
			<td style="width:303px;">ACROS</td>
			<td style="width:123px;">67-63-0</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Betadine surgical scrub</td>
			<td style="width:303px;">Controlled item from veterinary care in BCM</td>
			<td style="width:123px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">#10 scalpel blades</td>
			<td style="width:303px;">Ted Pella, Inc</td>
			<td style="width:123px;">549-3CS-10</td>
			<td>Multiple</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">No. 3 handle</td>
			<td style="width:303px;">Ted Pella, Inc</td>
			<td style="width:123px;">541-31</td>
			<td>Need to be autoclaved</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sterile surgical drape</td>
			<td style="width:303px;">Sai Infusion Technology</td>
			<td style="width:123px;">PSS-SD1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Straight forceps&#160;</td>
			<td style="width:303px;">Roboz Surgical Instrument</td>
			<td style="width:123px;">RS-5132</td>
			<td>Need to be autoclaved</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Straight fine forceps</td>
			<td style="width:303px;">Fine Science Tools</td>
			<td style="width:123px;">11253-20</td>
			<td>Need to be autoclaved</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Edged fine forceps</td>
			<td style="width:303px;">Fine Science Tools</td>
			<td style="width:123px;">11253-25</td>
			<td>Need to be autoclaved</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">4-0 Vicryl silk suture</td>
			<td style="width:303px;">Johnson &#38; Johnson Health Care</td>
			<td style="width:123px;">J214H</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">31G insuline syringes</td>
			<td style="width:303px;">BD</td>
			<td style="width:123px;">328418</td>
			<td>Multiple</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Q-tips cotton swabs (Sterile)</td>
			<td style="width:303px;">VWR International Laboratory</td>
			<td style="width:123px;">89031-272</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Skin glue</td>
			<td style="width:303px;">Henry Schein Animal Health</td>
			<td style="width:123px;">31477</td>
			<td>For surgery site skin closure</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ear Tag Applicator</td>
			<td style="width:303px;">Fine Science Tools</td>
			<td style="width:123px;">24220-00</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ear tags</td>
			<td style="width:303px;">Fine Science Tools</td>
			<td style="width:123px;">24220-50</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">D-luciferin</td>
			<td style="width:303px;">Gold Biotechnology</td>
			<td style="width:123px;">LUCK</td>
			<td>Avoid light and put on ice</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">28G insulin syringes</td>
			<td style="width:303px;">BD</td>
			<td style="width:123px;">329410</td>
			<td>For intra-orbital injection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Paraformadehyde</td>
			<td style="width:303px;">Alfa Aesar</td>
			<td style="width:123px;">30525-89-4</td>
			<td>For tissue fixation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">EDTA</td>
			<td style="width:303px;">OmniPur</td>
			<td style="width:123px;">4050</td>
			<td>For bone tissue decalficication</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Equipment</td>
			<td style="width:303px;"></td>
			<td style="width:123px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Dissection microscope</td>
			<td style="width:303px;">Leica</td>
			<td style="width:123px;">Leica S6E stereo</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">IVIS Lumina II imaging system</td>
			<td>Advanced Molecular Vision</td>
			<td style="width:123px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Antibodies</td>
			<td></td>
			<td style="width:123px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Anti-GFP antibodies (JL-8)</td>
			<td>Clontech</td>
			<td>632381</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Anti-ALP antibodies</td>
			<td>Abcam</td>
			<td>ab108337</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Anti-Osterix antibodies</td>
			<td>Abcam</td>
			<td>ab22552</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

図1は、解剖学的位置及び総腸骨動脈（赤）および静脈（青）の関係を示す図です。 図2は、解剖顕微鏡下で腸骨血管や神経の相対的な位置を示しています。 図2Aに示されているように、血管と神経が腹腔壁の下に右であり、皮膚切開がなされ、腹膜が押しのけた後に明らかにするこ?...

Watch this Scientific Journal Video about Intra-iliac Artery Injection for Efficient and Selective Modeling of Microscopic Bone Metastasis at JoVE.com

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

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 ...

intra-iliac-artery-injection-for-efficient-selective-modeling

Research

JoVE Journal

Cancer Research

16.2K ビュー.  Baylor College of Medicine. 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. 

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

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 ...

がん研究

Intracarotid Cancer Cell Injection to Produce Mouse Models of Brain Metastasis

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. Recently, impressive advances in novel therapies have dramatically prolonged survival and improved quality of life for many cancer patients. Sadly, incidence of brain metastatic recurrences is fast rising, and all current therapies are merely palliative. Hence, good experimental animal models are urgently needed to facilitate in-depth studies of the disease biology and to assess novel therapeutic regimens for preclinical evaluation. However, the standard in vivo metastasis assay via tail vein injection of cancer cells produces predominantly lung metastatic lesions; animals usually succumb to the lung tumor burden before any meaningful outgrowth of brain metastasis. Intracardiac injection of tumor cells produces metastatic lesions to multiple organ sites including the brain; however, the variability of tumor growth produced with this model is large, dampening its utility in evaluating therapeutic efficacy. To generate reliable and consistent animal models for brain metastasis study, here we describe a procedure for producing experimental brain metastasis in the house mouse (Mus musculus) via intracarotid injection of tumor cells. This approach allows one to produce large number of brain metastasis-bearing mice with similar growth and mortality characteristics, thus facilitating research efforts to study basic biological mechanisms and to assess novel therapeutic agents.

Brain metastasis has become an urgent unmet medical need as its incidence has increased while therapeutic options have remained palliative. Creating experimental animal models of brain metastasis via intracarotid arterial injection of cancer cells facilitates mechanistic studies of the disease biology and evaluation of novel intervention regimens.

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. ...

Measuring Bone Remodeling and Recreating the Tumor-Bone Microenvironment Using Calvaria Co-culture and Histomorphometry

Bone is a connective tissue constituted of osteoblasts, osteocytes, and osteoclasts and a mineralized extracellular matrix, which gives it its strength and flexibility and allows it to fulfill its functions. Bone is continuously exposed to a variety of stimuli, which in pathological conditions can deregulate bone remodeling. To study bone biology and diseases and evaluate potential therapeutic agents, it has been necessary to develop in vitro and in vivo models.
This manuscript describes the dissection process and culture conditions of calvarias isolated from neonatal mice to study bone formation and the bone tumor microenvironment. In contrast to in vitro and in vivo models, this ex vivo model allows preservation of the three-dimensional environment of the tissue as well as the cellular diversity of the bone while culturing under defined conditions to simulate the desired microenvironment. Therefore, it is possible to investigate bone remodeling and its mechanisms, as well as the interactions with other cell types, such as the interactions between cancer cells and bone.
The assays reported here use calvarias from 5-7 day old BALB/C mice. The hemi-calvarias obtained are cultured in the presence of insulin, breast cancer cells (MDA-MB-231), or conditioned medium from breast cancer cell cultures. After analysis, it was established that insulin induced new bone formation, while cancer cells and their conditioned medium induced bone resorption. The calvarial model has been successfully used in basic and applied research to study bone development and cancer-induced bone diseases. Overall, it is an excellent option for an easy, informative, and low-cost assay.

The ex vivo culture of bone explants can be a valuable tool for the study of bone physiology and the potential evaluation of drugs in bone remodeling and bone diseases. The presented protocol describes the preparation and culture of calvarias isolated from newborn mice skulls, as well as its applications.

カルバリア共培養と組織病量を用いた骨リモデリングと腫瘍-骨微小環境の再現

Bone is a connective tissue constituted of osteoblasts, osteocytes, and osteoclasts and a mineralized extracellular matrix, which gives it its strength ...

Modeling Breast Cancer via an Intraductal Injection of Cre-expressing Adenovirus into the Mouse Mammary Gland

Breast cancer is a heterogeneous disease, possibly due to complex interactions between different cells of origins and oncogenic events. Mouse models are instrumental in gaining insights into these complex processes. Although many mouse models have been developed to study contributions of various oncogenic events and cells of origin to breast tumorigenesis, these models are often not cell-type or organ specific or cannot induce the initiation of mammary tumorigenesis in a temporally controlled manner. Here we describe a protocol to generate a new type of breast cancer mouse models based on the intraductal injection of Cre-expressing adenovirus (Ad-Cre) into mouse mammary glands (MGs). Due to the direct injection of Ad-Cre into mammary ducts, this approach is MG specific, without any unwanted cancer induction in other organs. The intraductal injection procedure can be performed in mice at different stages of their MG development (thus, it permits temporal control of cancer induction, starting from ~3-4 weeks of age). The cell-type specificity can be achieved by using different cell-type-specific promoters to drive Cre expression in the adenoviral vector. We show that luminal and basal mammary epithelial cells (MECs) can be tightly targeted for Cre/loxP-based genetic manipulation via an intraductal injection of Ad-Cre under the control of the Keratin 8 or Keratin 5 promoter, respectively. By incorporating a conditional Cre reporter (e.g., Cre/loxP-inducible Rosa26-YFP reporter), we show that MECs targeted by Ad-Cre, and tumor cells derived from them, can be traced by following the reporter-positive cells after intraductal injection.

The goal of this protocol is to describe a new breast cancer modeling approach based on the intraductal injection of Cre-expressing adenovirus into mouse mammary glands. This approach allows both cell-type- and organ-specific manipulation of oncogenic events in a temporally controlled manner.

マウス乳腺へのクレオンゲンデノウイルスの誘導注射による乳癌のモデル化

Breast cancer is a heterogeneous disease, possibly due to complex interactions between different cells of origins and oncogenic events. Mouse models are ...

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 ...

Establishment and Analysis of Three-Dimensional (3D) Organoids Derived from Patient Prostate Cancer Bone Metastasis Specimens and their Xenografts

Three-dimensional (3D) culture of organoids from tumor specimens of human patients and patient-derived xenograft (PDX) models of prostate cancer, referred to as patient-derived organoids (PDO), are an invaluable resource for studying the mechanism of tumorigenesis and metastasis of prostate cancer. Their main advantage is that they maintain the distinctive genomic and functional heterogeneity of the original tissue compared to conventional cell lines that do not. Furthermore, 3D cultures of PDO can be used to predict the effects of drug treatment on individual patients and are a step towards personalized medicine. Despite these advantages, few groups routinely use this method in part because of the extensive optimization of PDO culture conditions that may be required for different patient samples. We previously demonstrated that our prostate cancer bone metastasis PDX model, PCSD1, recapitulated the resistance of the donor patient&rsquo;s bone metastasis to anti-androgen therapy. We used PCSD1 3D organoids to characterize further the mechanisms of anti-androgen resistance. Following an overview of currently published studies of PDX and PDO models, we describe a step-by-step protocol for 3D culture of PDO using domed or floating basement membrane (e.g., Matrigel) spheres in optimized culture conditions. In vivo stitch imaging and cell processing for histology are also described. This protocol can be further optimized for other applications including western blot, co-culture, etc. and can be used to explore characteristics of 3D cultured PDO pertaining to drug resistance, tumorigenesis, metastasis and therapeutics.

Establishment and Analysis of Three-Dimensional 3D Organoids Derived from Patient Prostate Cancer Bone Metastasis Specimens and their Xenografts

Three-dimensional cultures of patient BMPC specimens and xenografts of bone metastatic prostate cancer maintain the functional heterogeneity of their original tumors resulting in cysts, spheroids and complex, tumor-like organoids. This manuscript provides an optimization strategy and protocol for 3D culture of heterogeneous patient derived samples and their analysis using IFC.

患者前立腺癌骨転移標本及びその異種移植片に由来する3次元(3D)オルガノイドの確立と解析

Three-dimensional (3D) culture of organoids from tumor specimens of human patients and patient-derived xenograft (PDX) models of prostate cancer, referred ...

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 Metastases Through Intracranial Injection and Magnetic Resonance Imaging

Metastatic spread of cancer is an unfortunate consequence of disease progression, aggressive cancer subtypes, and/or late diagnosis. Brain metastases are particularly devastating, difficult to treat, and confer a poor prognosis. While the precise incidence of brain metastases in the United States remains hard to estimate, it is likely to increase as extracranial therapies continue to become more efficacious in treating cancer. Thus, it is necessary to identify and develop novel therapeutic approaches to treat metastasis at this site. To this end, intracranial injection of cancer cells has become a well-established method in which to model brain metastasis. Previously, the inability to directly measure tumor growth has been a technical hindrance to this model; however, increasing availability and quality of small animal imaging modalities, such as magnetic resonance imaging (MRI), are vastly improving the ability to monitor tumor growth over time and infer changes within the brain during the experimental period. Herein, intracranial injection of murine mammary tumor cells into immunocompetent mice followed by MRI is demonstrated. The presented injection approach utilizes isoflurane anesthesia and a stereotactic setup with a digitally controlled, automated drill and needle injection to enhance precision, and reduce technical error. MRI is measured over time using a 9.4 Tesla instrument in The Ohio State University James Comprehensive Cancer Center Small Animal Imaging Shared Resource. Tumor volume measurements are demonstrated at each time point through use of ImageJ. Overall, this intracranial injection approach allows for precise injection, day-to-day monitoring, and accurate tumor volume measurements, which combined greatly enhance the utility of this model system to test novel hypotheses on the drivers of brain metastases.

Intracranial brain metastasis modeling is complicated by an inability to monitor tumor size and response to treatment with precise and timely methods. The presented methodology couples intracranial tumor injection with magnetic resonance imaging analysis, which when combined, cultivates precise and consistent injections, enhanced animal monitoring, and accurate tumor volume measurements.

頭蓋内注射と磁気共鳴画像による脳転移のモデル化

Metastatic spread of cancer is an unfortunate consequence of disease progression, aggressive cancer subtypes, and/or late diagnosis. Brain metastases are ...

Modeling Primary Bone Tumors and Bone Metastasis with Solid Tumor Graft Implantation into Bone

Primary bone tumors or bone metastasis from solid tumors result in painful osteolytic, osteoblastic, or mixed osteolytic/osteoblastic lesions. These lesions compromise bone structure, increase the risk of pathologic fracture, and leave patients with limited treatment options. Primary bone tumors metastasize to distant organs, with some types capable of spreading to other skeletal sites. However, recent evidence suggests that with many solid tumors, cancer cells that have spread to bone may be the primary source of cells that ultimately metastasize to other organ systems. Most syngeneic or xenograft mouse models of primary bone tumors involve intra-osseous (orthotopic) injection of tumor cell suspensions. Some animal models of skeletal metastasis from solid tumors also depend on direct bone injection, while others attempt to recapitulate additional steps of the bone metastatic cascade by injecting cells intravascularly or into the organ of the primary tumor. However, none of these models develop bone metastasis reliably or with an incidence of 100%. In addition, direct intra-osseous injection of tumor cells has been shown to be associated with potential tumor embolization of the lung. These embolic tumor cells engraft but do not recapitulate the metastatic cascade. We reported a mouse model of osteosarcoma in which fresh or cryopreserved tumor fragments (consisting of tumor cells plus stroma) are implanted directly into the proximal tibia using a minimally invasive surgical technique. These animals developed reproducible engraftment, growth, and, over time, osteolysis and lung metastasis. This technique has the versatility to be used to model solid tumor bone metastasis and can readily employ grafts consisting of one or multiple cell types, genetically-modified cells, patient-derived xenografts, and/or labeled cells that can be tracked by optical or advanced imaging. Here, we demonstrate this technique, modeling primary bone tumors and bone metastasis using solid tumor graft implantation into bone.

Bone metastasis models do not develop metastasis uniformly or with a 100% incidence. Direct intra-osseous tumor cell injection can result in embolization of the lung. We present our technique modeling primary bone tumors and bone metastasis using solid tumor graft implantation into bone, leading to reproducible engraftment and growth.

固形腫瘍移植による骨原発性骨腫瘍と骨転移のモデリング

Primary bone tumors or bone metastasis from solid tumors result in painful osteolytic, osteoblastic, or mixed osteolytic/osteoblastic lesions. These ...

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 ...