著者たちは、組織病理学的支援のためのジョー・ヒル、手術技法指導のジョン・グリースティン博士、ビデオ撮影支援のためのテッサ・ダイエル、重要な洞察と議論のためのウォン・クーセンズ研究所の全メンバー、財政支援のためのOHSU騎士癌研究所に感謝する。著者らは、TEGGM071388-10およびT32CA106195-11からCEG、NCI / NIH、国防乳癌研究プログラム、スーザンGコメン財団、乳がん研究財団、およびスタンドアップトゥーガン - ラスガルテン財団への支援を認めている膵臓癌コンバージェンスドリームチーム翻訳研究グラント（SU2C-AACR-DT14-14）を、LWMC、女性ヘルス・サークル・オブ・ヘルス・ファンデーション・オブ・ヘルス・グラント、MHWおよびLMCに対するBrenden-Colson膵臓ヘルス・センターに寄贈しました。

以下のプロトコルで使用される動物は、動物福祉法規制および公衆衛生サービス（PHS）ポリシーに準拠するように設計された、オレゴン保健科学大学の施設内動物管理および使用委員会（IACUC）の対象となります。 滅菌条件の維持：滅菌済みの器具を使用し、マウス間で滅菌ガーゼで拭き取り、PBSですすぎ、次に消毒剤70％エタノールで少なくとも15分間滅菌する必要があります。生存手術のためには、外科用キャップ、フェイスマスク、ガウン、手袋を着用する必要があります。生存手術のための動物の手術前の準備は、以下のプロトコールに含まれる。試薬および機器のリストについては、表1を参照してください。 原発性乳腺腫瘍からの単細胞浮遊液の分離および調製<ol><li>ドナーの雌100日齢のトランスジェニックMMTV-PyMT（FVB / n）マウスに麻酔をかけ、c2％イソフルランを麻酔マスクを介して投与することにより、連続的な鎮静を行う。フットピンチ反射がない状態でマウスが正しく麻酔されていることを確認します。 </li><li>滅菌設定では、100日齢のトランスジェニック雌性MMTV-PyMT（FVB / n）マウス由来の原発性乳腺腫瘍を、無菌ハサミを用いて上皮および周囲の脂肪組織および/またはリンパ節から乳房腫瘍を分離することによって切除する。頸部脱臼により麻酔したマウスを安楽死させる。 </li><li> 滅菌はさみまたはメスを使用して、原発腫瘍を手動で小片（約1.0mm 3 ）に切ります。腫瘍片を3.0mg / mLのコラゲナーゼAおよび4.0U / mLのDNアーゼIをDMEMに溶解したものに入れる。直径1.0cmの腫瘍あたり約10mLの上記消化培地を使用する。 <ol><li> 〜125rpm、37℃で40分間無菌攪拌棒を用いて滅菌25mLボトルで消化を行います。 </li></ol></li><li>ウシ胎児血清（FBS）を最終希釈10％に加えて消化を停止し、混合物全体をwそれは維持されています。 </li><li>消化した腫瘍懸濁液を0.7μmのナイロンストレーナーで50mLコニカルチューブに濾過し、ストレーナーに残っている腫瘍を捨てる。 4℃で300 xgで上清を遠心分離する。 </li><li>ペレットを1.0cmの腫瘍あたり10mLのDMEMに再懸濁し、0.7μmのナイロンストレーナーで再濾過する。細胞濃度を数え、4℃で300 xgで遠心分離します。 </li><li>ペレットを2×10 7個/ mLの濃度で90％FBSを含む10％ジメチルスルホキシドに再懸濁する。 -80℃で全原発腫瘍の単細胞懸濁液を保存する。 </li></ol> 2.乳腺腫瘍の同所性注射<ol><li>凍結したペレットを極低温チューブから20mLのDMEMに放出し、細胞を計数するまで37℃で凍結した原発腫瘍懸濁液を部分的に解凍する。 4℃で300 xgで遠心分離し、1：1 DMEM：増殖因子で還元された基底膜調製物eで細胞を再懸濁する。Engelbreth-Holm-Swarm（EHS）マウス肉腫から1×10 7細胞/ mLの濃度で採取した（ 表の表を参照）。 </li><li>麻酔をかけたレシピエントの同系マウスの腹側を上に置き、麻酔マスクを介して2％イソフルランを投与することにより、連続鎮静下に維持する。 </li><li>エアロゾル化した70％エタノールを使用して右第4乳腺注射部位を滅菌し、ポリ（ビニルピロリドン） - ヨウ素を滅菌綿棒で塗布する。 </li><li> 29 G 0.3 mLインスリン注射器を使用して、6〜10週齢の女性のFVB / nマウスの右胸部第4乳腺に100μL（凍結原発腫瘍懸濁液から1×10 6個の生存細胞）を斜面側に注入する。 </li></ol> 3.同所性乳腺腫瘍の外科的切除<ol><li>腫瘍細胞注入の38〜60日後、172〜450mm 3の範囲の同所性腫瘍体積を示さないマウスを安楽死させる[長さx（幅2 </）/ 2]（注射したマウスの約75％）。 </li><li>麻酔マスクを介して2％イソフルランを投与することにより、連続鎮静下で適切な腫瘍サイズの腹側を上にして麻酔したマウスを配置し、マウスが不足足ピンチ反射で適切に麻酔されていることを確認する。鎮静下での乾燥を防ぐために、眼に獣医軟膏を塗布する。 </li><li>原発腫瘍を取り囲む手術領域を滅菌するために70％エタノールをスプレーし、ポリ（ビニルピロリドン） - ヨウ素を滅菌綿棒で塗布する。 注：外科部位での脱毛は〜5％のマウスの皮膚擦過傷および/または感染をもたらし、したがってこの工程から除外した（データは示していない）。 </li><li> 図1Cに示すように、腫瘍の内側尾の鈍いはさみを使用して、最初の皮膚切開を行う。 </li><li>次に、腫瘍の内側の皮膚（ 図1C ）の優れた切除を行う。任意の血管系を焼灼する必要性に注意を払う切開部を拡張する前に皮膚上に位置する腫瘍を切除する。 </li><li>横方向（腫瘍の後方）で皮膚切開を継続し、優れた皮膚切除（腫瘍に対して側方）および内側切除（腫瘍に対して優位）を行う（ 図1C ）。 </li><li>皮膚を腫瘍の周りに円周方向に切除した後（ 図1C ）、腫瘍に付着した皮膚を鉗子で持ち上げ、腹壁筋肉から腫瘍を摘出し、乳腺をそのままにする。 </li><li> 第 4および第 5乳腺を通っている大きな血管を（鈍的な切開によって）同定し、焼灼する。 </li><li>焼灼部位で4 番目と5 番目の乳腺のおよそ半分を切除して、腫瘍、上皮、および乳腺のセグメントを解放する（ 図1C ）。 </li><li>出血の場合は、積極的に出血を確認するすぐにそれらを焼灼する。 250μL以上の血液が失われた場合は、マウスを研究から除外し安楽死させる。 </li><li>創傷クリップアプライヤを用いて創傷クリップを用いて切除部位を閉じる（ 図1C ）。 注：創傷クリップ除去剤で外科手術の10日後に創傷クリップを除去する。 </li><li> 暖かい滅菌生理食塩水を皮下に投与し（動物の体重の4％）、加熱ランプで暖かい動物を保つ。麻酔からの回復の間、動物を5〜10分ごとに点検する。 注：ブピバカインまたはリドカインの投与は術後死亡率を増加させた（データは示さず）。手術後1時間で、痛みの徴候を示すマウス（鈍感、動かない、身づくろい）を安楽死させた。 <ol><li>マウスが完全に回復したら、それを他のマウスの会社に戻します。 </li></ol></li></ol>フローサイトメトリーおよび組織学のための血液および肺の単離および処理 &lt;ol&gt; <li>研究のエンドポイントでは、必要に応じて様々な組織病理学的評価のためにマウスを準備する。安楽死の90分前に、各マウスに1x PBS中6.25μg/μLの濃度のブロモデオキシウリジン（BrdU、50μg/ gマウス重量）の腹腔内注射を行う。 注：溶解したブロモデオキシウリジンの凍結ストックは、調製後1ヶ月以内に使用する。 </li><li>マウスを麻酔した後、安楽死の10分前に、ヘパリン処理された毛管を用いて眼窩後血液（&gt;500μL）を収集し、続いて氷上に保持されたEDTA二カリウムで被覆したチューブに移す。 </li><li>肺および残りの乳房組織を除去するために、胸郭および腹腔（ 図2A）を露出させ、また、乳房番目の残りの右4 番目と5を露出させるために横方向に皮膚を剥離するために口に下腹部からハサミで正中切開を行います腺。 </li><li>残りの乳腺組織を切除し、iを検査する。ヘマトキシリンおよびエオシン（H＆E）染色（ 補助的な 図1 ）による連続切片ホルマリン固定パラフィン包埋（FFPE）組織を評価することにより、原発腫瘍再増殖を除外する。 </li><li>肺を除去するには、はさみで隔壁に隔壁を開き、肺の灌流の前に血液を排出するために腹部大動脈を切断して動物を安楽死させる（ 図2B -2C ）。 </li><li>肺と心臓を避けるように腹部アプローチから胸郭に沿って横隔膜を切る。次に、胸郭の側面を切断して胸郭を露出させます （ 図2D ）。 </li><li> 20mLシリンジで23ゲージの針を使用し、肺が完全に白くなるまで心臓の右心室を通して約5​​.0mLのDPBS（約10mL /分）で肺を灌流する（ 図2E -2F ）。メインの船からすぐに心臓を切り離す血液は肺に再灌流しません。 </li><li>肺組織を固定し、組織病理学的評価のために処理されるために、23ゲージ針（ - 2H 図2G）を使用して肺に向かって露出した気管ベベル側に4℃〜1.0mLの10％ホルマリンを注入します。肺が完全に拡張され、固定剤で満たされると注射を止める（ 図2I ）。 </li><li>標準的な組織病理学的手順に従って、その後のパラフィン包埋またはOCT凍結培地のために、中空緩衝ホルマリン中の肺組織を気管から排出し、エマルジョンで固定する。 </li><li> FFPE肺組織の連続切片化により肺における転移性負荷を定量的に評価し、H＆E染色（ 図1B ）によってミクロトーム切片（100μm、13切片）を評価する。 </li></ol>

<ol>
	<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 Res. 8, 212 (2006).</li><li>Howlader, N. N. A., Krapcho, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> SEER Cancer Statistics Review 1975-2008. , (2011).</li><li>National Comprehensive Cancer Network. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Breast Cancer (Version 3.2014). , (2016).</li><li>Kataja, V., Castiglione, M., Group, E. G. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Locally+recurrent+or+metastatic+breast+cancer:+ESMO+clinical+recommendations+for+diagnosis,+treatment+and+follow-up.">Locally recurrent or metastatic breast cancer: ESMO clinical recommendations for diagnosis, treatment and follow-up.</a> Ann Oncol. 19 Suppl 2, 11-13 (2008).</li><li>Margolese, R. G., Hortobagyi, G. N., Buchholz, T. A., Kufe, D. W., Pollock, R. E., Weichselbaum, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Management of Metastatic Breast Cancer. Holland-Frei Cancer Medicine. , (2003).</li><li>Guy, C. T., Cardiff, R. D., Muller, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+mammary+tumors+by+expression+of+polyomavirus+middle+T+oncogene:+a+transgenic+mouse+model+for+metastatic+disease.">Induction of mammary tumors by expression of polyomavirus middle T oncogene: a transgenic mouse model for metastatic disease.</a> Mol Cell Biol. 12, 954-961 (1992).</li><li>Kouros-Mehr, 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=GATA-3+links+tumor+differentiation+and+dissemination+in+a+luminal+breast+cancer+model.">GATA-3 links tumor differentiation and dissemination in a luminal breast cancer model.</a> Cancer Cell. 13, 141-152 (2008).</li><li>Minn, A. J., 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=Genes+that+mediate+breast+cancer+metastasis+to+the+lung.">Genes that mediate breast cancer metastasis to the lung.</a> Nature. 436, 518-524 (2005).</li><li>Qian, B. 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=FLT1+signalling+in+metastasis-associated+macrophages+activates+an+inflammatory+signature+that+promotes+breast+cancer+metastasis.">FLT1 signalling in metastasis-associated macrophages activates an inflammatory signature that promotes breast cancer metastasis.</a> J Exp Med. 212 (9), 1433-1448 (2015).</li><li>Verkooijen, H. 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=Patients'+refusal+of+surgery+strongly+impairs+cancer+survival.">Patients' refusal of surgery strongly impairs cancer survival.</a> Ann Surg. 242 (2), 276-280 (2005).</li><li>Peinado, 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=Pre-metastatic+niches:+organ-specific+homes+for+metastasis.">Pre-metastatic niches: organ-specific homes for metastasis.</a> Nat Rev Cancer. 17 (5), 302-317 (2017).</li></ol>

著者らはここに提示されたデータとの矛盾については開示していない。

変更とトラブルシューティング： 鈍的な腫瘍を腹壁から摘出するとき、腫瘍は腹壁に付着したままである可​​能性がある。これは腫瘍を注射したマウスの5％未満で観察された（データは示さず）。腹壁に付着した腫瘍を有するマウスの場合、原発腫瘍の再増殖なしには切除が困難であるため、マウスを安楽死させるべきである。 <str...

北アメリカの女性は、乳がん2を発症する生涯リスクが〜12％です。これらの個体の大多数は、手術により摘出された原発腫瘍を有し、がんサブタイプに応じて、アジュバント設定3で標的、内分泌、化学療法および/または放射線療法を受ける。何の標的療法は、トリプルネガティブ腫瘍の3のためにまだ利用できないのに対し、例としては、放射線/化学療法とHER2標的治療を受けたHER2陽性腫瘍を抗エストロゲン療法と女性を受けるホルモン受容体陽性癌と診断された女性が含まれます。外科的切除を補完する放射線、化学療法、パーソナライズされたホルモン療法の進歩にもかかわらず、病気はステージIIまたはIIIの病気と診断された女性の30〜70％で再発する4 、遠隔臓器における転移性疾患の根絶には効果がほとんどない肺、骨、b雨および/または肝臓5 。これは、転移性疾患が原発腫瘍再増殖の不在下で起こる場合、これは、播種性悪性細胞が確定手術の時点で二次臓器にすでに存在している可能性があることを意味するので、特に重要である。したがって、転移性腫瘍の根絶または緩徐化が可能な治療法が急務である。 乳腺発がんの新規のマウスモデルは、腫瘍性の進行1を制御するメカニズムを明らかに著しく有益であったが、既存のモデルにもいくつかの制限があります。これらのうちの1つは、 新規のトランスジェニックモデルが典型的には、原発腫瘍負荷が研究の期間を制限する複数の乳腺において原発腫瘍を発症するという事実である。原発腫瘍細胞の逃避および転移播種は、これらのモデルにおける新生物の進行の早期に起こる可能性が高いが、転移性腫瘍の率直な発達は遅く起こり、マウスモデルと株のバックグラウンドは、しばしば部分的に浸透している1 。これは、二次器官における転移を制御する分子の発見のための新規モデルの有用性をさらに制限し、アジュバント設定における治療薬の臨床前の有効性を評価するためにさらに制限する。 これらの制限を回避するために、我々は、肺への乳癌の転移の新たな土着モデルを開発しました。後期デノボ乳腺腫瘍を担持（FVB / N本明細書に記載の研究のための株バックグラウンドで、すなわち 、MMTV-PyMT）親トランスジェニック雌は〜100日6歳であり、それらの原発腫瘍をその時点で外科的に切除し、酵素的に解離されています単一細胞懸濁液。単一の原発性乳腺腫瘍が38〜60日の期間に亘って発生する6〜7週齢のレシピエント同系雌性マウスに懸濁液（1×10 6細胞）を順化させる3 ）で、レシピエントマウスを麻酔し、原発腫瘍を外科的切除して、外科手術部位での腫瘍再増殖を最小限に抑え、女性の手術と一致させる（ 補足図1 ）。 FVB / n株のバックグラウンドでは、マウスは手術後約115日までに45％の浸透率を有する肺で組織学的に検出可能な転移病巣を発症する（ 図1B ）。転移腫瘍増殖の延長された潜伏期間により、このモデルは、補助的治療の送達、および原発腫瘍の外科的除去後の転移性進行に影響を及ぼす基礎となる生物学の解明および評価のために独特に位置付けられる。

<table border="0" cellpadding="0" cellspacing="0" width="718">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:292px;">Isofluorane</td>
			<td style="width:140px;">Piramal Healthcare</td>
			<td style="width:119px;">N/A</td>
			<td style="width:167px;">Prescription order</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Collagenase A</td>
			<td>Roche</td>
			<td>11088793001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DNase I</td>
			<td>Roche</td>
			<td>10104159001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMEM</td>
			<td>ThermoFisher</td>
			<td>12634010</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">25 mL Pyrex bottle</td>
			<td>Sigma-Aldrich</td>
			<td>CLS139525</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fetal Bovine Serum</td>
			<td>Atlanta Bio&#160;</td>
			<td>S11150</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">0.7 &#181;m nylon strainer&#160;</td>
			<td>Corning</td>
			<td>352350</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">50 mL conical tube&#160;</td>
			<td>VWR</td>
			<td>89039-658</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dimethyl sulfoxide</td>
			<td>Sigma-Aldrich</td>
			<td>D2650</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Growth factor-reduced Matrigel&#160;</td>
			<td>BD</td>
			<td>354230</td>
			<td>Growth factor-reduced solubilized basement membrane preparation extracted from the Engelbreth-Holm-Swarm (EHS) mouse sarcoma</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Poly(vinylpyrrolidone)&#8211;Iodine complex</td>
			<td>Sigma-Aldrich</td>
			<td>PVP1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">29 gauge 0.3 mL insulin syringe</td>
			<td>BD</td>
			<td>324702</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Small Vessel Cauterizer Kit</td>
			<td>FST</td>
			<td>18000-00</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Wound clips</td>
			<td>Texas Scientific</td>
			<td>205016</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">AutoClip wound clip applier&#160;</td>
			<td>BD</td>
			<td>427630</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">AutoClip wound clip remover&#160;</td>
			<td>BD</td>
			<td>427637</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bromodeoxyuridine</td>
			<td>Roche</td>
			<td>10280879</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Heparinized capillary tubes&#160;</td>
			<td>Fisher</td>
			<td>22362566</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microtainer tubes with dipotassium EDTA&#160;</td>
			<td>BD</td>
			<td>365974</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">20 mL syringe&#160;</td>
			<td>BD</td>
			<td>309661</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DPBS</td>
			<td>Thermo-Fisher</td>
			<td>14190-250</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">OCT-freezing medium&#160;</td>
			<td>VWR</td>
			<td>25608930</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Formalin, Buffered, 10% (Phosphate Buffer)</td>
			<td>Fisher</td>
			<td>SF100-4</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">23g needle</td>
			<td>Fisher</td>
			<td>14-826-6B</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FVB/n mouse</td>
			<td>Jackson Laboratories</td>
			<td>001800</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

MMTV-PyMTマウス由来の原発性乳腺腫瘍由来の1×10 6細胞を受けるレシピエントマウスの75％超が、38〜60 日以内にサイズが172〜450mm 3の単一の乳腺腺癌を発症する（データ示さず）。無作為化の可能性のあるマウスを、次に示されるように原発腫瘍の外科的切除後に研究グループに登録する（ 図1C ）。原発腫瘍の外科的切除?...

Watch this Scientific Journal Video about Surgical Procedures and Methodology for a Preclinical Murine Model of De Novo Mammary Cancer Metastasis at JoVE.com

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

乳癌の転移を標的としたアジュバント療法を評価する前臨床モデルは欠けている。これに対処するために、本発明者らは、アジュバント設定（原発腫瘍の外科的切除後）で投与された治療が、以前に播種された肺転移に影響を及ぼす有効性について評価され得る、 新規な肺乳腺腺癌転移のマウスモデルを開発した。

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 ...

surgical-procedures-methodology-for-preclinical-murine-model-de-novo

Research

JoVE Journal

Cancer Research

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

13.7K ビュー.  Oregon Health & Science University. 乳癌の転移を標的としたアジュバント療法を評価する前臨床モデルは欠けている。これに対処するために、本発明者らは、アジュバント設定（原発腫瘍の外科的切除後）で投与された治療が、以前に播種された肺転移に影響を及ぼす有効性について評価され得る、 新規な肺乳腺腺癌転移のマウスモデルを開発した。 

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

A Detailed Protocol for Characterizing the Murine C1498 Cell Line and its Associated Leukemia Mouse Model

The intravenous injection of C1498 cells into syngeneic or congenic mice has been performed since 1941. These injections result in the development of acute leukemia. However, the nature of this disease has not been well documented in the literature. Here, we provide a technical protocol for characterizing C1498 cells in vitro and for determining the nature of the induced leukemia in vivo. The first part of this procedure is focused on determining the hematopoietic lineage and the stage of differentiation of cultured C1498 cells. To achieve this, multi-parametric flow cytometric staining is used to detect hematopoietic cell markers. Immunofluorescence microscopy, cytochemistry and a May-Gr&#252;nwald Giemsa staining are then performed to assess the expression of myeloperoxidase, the activity of esterases and cellular morphology, respectively. The second part of this protocol is dedicated to describing the leukemia disease that is induced in vivo. The latter can be achieved by determining the frequencies of leukemic and inherent cells in the blood, hematopoietic organs (e.g., bone marrow and spleen) and non-lymphoid tissues (e.g., the liver and lungs) using specific staining and flow cytometry analyses. The nature of the leukemia is then confirmed using May-Gr&#252;nwald Giemsa staining and staining for specific esterases in the bone marrow. Here, we present the results that were obtained using this protocol in age-matched C1498- and PBS-injected mice.

This manuscript provides a technical procedure that can be used to characterize C1498 cell cultures in vitro and the acute leukemia induced in mice after their injection. Phenotypic and functional analyses are performed using flow cytometry, immunofluorescence microscopy, cytochemistry and May-Gr&#252;nwald Giemsa staining.

マウスC1498細胞株およびその関連白血病マウスモデルを特徴付けるための詳細なプロトコル

The intravenous injection of C1498 cells into syngeneic or congenic mice has been performed since 1941. These injections result in the development of ...

がん研究

Murine Experimental Model of Original Tumor Development and Peritoneal Metastasis via Orthotopic Inoculation with Ovarian Carcinoma Cells

Epithelial ovarian carcinoma (EOC) is associated with a poor prognosis because it shows peritoneal dissemination. To improve the prognosis, it is important to control peritoneal dissemination. However, it is still unclear how tumor cells detach from primary lesions and attach to the mesothelium. The establishment of an appropriate animal model is needed to gain an understanding of the mechanism of peritoneal dissemination in vivo. In the current study, we introduce the process from the local injection of EOC cells into the murine ovarian surface to the development of metastasis, including the peritoneum and distant organs. Female nude mice (BALB/c nu/nu) at 8 weeks of age were used. Under a microscopic field of view, EOC cells (1 x 105 cells/&#181;l of medium-extracellular matrix (ECM)-based hydrogel/unilateral ovary/mouse) were injected into murine ovaries through a retroperitoneal approach from the dorsal flank. This proposed method is a less invasive procedure for the mouse and minimizes damage to the ovary. Here, we describe the methodological steps in the development of the original and metastatic tumor formation of EOC.

Murine Experimental Model of Original Tumor Development and Peritoneal Metastasis via Orthotopic Inoculation with Ovarian Carcinoma Cells

To clarify the multistep process of peritoneal dissemination of ovarian carcinoma, the current study presents a murine experimental model of original tumor development and peritoneal metastasis via orthotopic inoculation with these tumor cells.

オリジナル腫瘍の開発のマウス実験モデルと腹膜転移<em&gt;経由で</em卵巣癌細胞で&gt;同所接種

Epithelial ovarian carcinoma (EOC) is associated with a poor prognosis because it shows peritoneal dissemination. To improve the prognosis, it is ...

Development and Maintenance of a Preclinical Patient Derived Tumor Xenograft Model for the Investigation of Novel Anti-Cancer Therapies

Patient derived tumor xenograft (PDTX) models provide a necessary platform in facilitating anti-cancer drug development prior to human trials. Human tumor pieces are injected subcutaneously into athymic nude mice (immunocompromised, T cell deficient) to create a bank of tumors and subsequently are passaged into different generations of mice in order to maintain these tumors from patients. Importantly, cellular heterogeneity of the original tumor is closely emulated in this model, which provides a more clinically relevant model for evaluation of drug efficacy studies (single agent and combination), biomarker analysis, resistant pathways and cancer stem cell biology. Some limitations of the PDTX model include the replacement of the human stroma with mouse stroma after the first generation in mice, inability to investigate treatment effects on metastasis due to the subcutaneous injections of the tumors, and the lack of evaluation of immunotherapies due to the use of immunocompromised mice. However, even with these limitations, the PDTX model provides a powerful preclinical platform in the drug discovery process.

Utilizing patient-derived tumors in a subcutaneous preclinical model is an excellent way to study the efficacy of novel therapies, predictive biomarker discovery, and drug resistant pathways. This model, in the drug development process, is essential in determining the fate of many novel anti-cancer therapies prior to clinical investigation.

前臨床患者由来の腫瘍新規抗がん療法の調査のための異種移植モデルの開発とメンテナンス

Patient derived tumor xenograft (PDTX) models provide a necessary platform in facilitating anti-cancer drug development prior to human trials. Human tumor ...

In Vivo Model for Testing Effect of Hypoxia on Tumor Metastasis

Hypoxia has been implicated in the metastasis of Ewing sarcoma (ES) by clinical observations and in vitro data, yet direct evidence for its pro-metastatic effect is lacking and the exact mechanisms of its action are unclear. Here, we report an animal model that allows for direct testing of the effects of tumor hypoxia on ES dissemination and investigation into the underlying pathways involved. This approach combines two well-established experimental strategies, orthotopic xenografting of ES cells and femoral artery ligation (FAL), which induces hindlimb ischemia. Human ES cells were injected into the gastrocnemius muscles of SCID/beige mice and the primary tumors were allowed to grow to a size of 250 mm3. At this stage either the tumors were excised (control group) or the animals were subjected to FAL to create tumor hypoxia, followed by tumor excision 3 days later. The efficiency of FAL was confirmed by a significant increase in binding of hypoxyprobe-1 in the tumor tissue, severe tumor necrosis and complete inhibition of primary tumor growth. Importantly, despite these direct effects of ischemia, an enhanced dissemination of tumor cells from the hypoxic tumors was observed. This experimental strategy enables comparative analysis of the metastatic properties of primary tumors of the same size, yet significantly different levels of hypoxia. It also provides a new platform to further assess the mechanistic basis for the hypoxia-induced alterations that occur during metastatic tumor progression in vivo. In addition, while this model was established using ES cells, we anticipate that this experimental strategy can be used to test the effect of hypoxia in other sarcomas, as well as tumors orthotopically implanted in sites with a well-defined blood supply route.

In Vivo Model for Testing Effect of Hypoxia on Tumor Metastasis

This manuscript describes the development of an animal model that allows for the direct testing of the effects of tumor hypoxia on metastasis and the deciphering the mechanisms of its action. Although the experiments described here focus on Ewing sarcoma, a similar approach can be applied to other tumor types.

腫瘍転移に対する低酸素のテストの効果のためのインビボモデル

Hypoxia has been implicated in the metastasis of Ewing sarcoma (ES) by clinical observations and in vitro data, yet direct evidence for its pro-metastatic ...

Operating Procedures of the Electrochemotherapy for Treatment of Tumor in Dogs and Cats

Electrochemotherapy (ECT) is a local approach which is used for treating solid tumors of different histologies. Its mechanism is based on cell membrane permeabilization by means of &#34;electroporation&#34;. To achieve the &#34;electroporation&#34; of the cells, electric pulses are generated by a generator and delivered to the target tissue by the use of electrodes. Electroporation is a physical method which is used to introduce molecules, like cytostatic drugs, into the cells that could not pass the cell membrane on their own. In electrochemotherapy, currently, cisplatin and bleomycin are clinically used. Electrochemotherapy antitumor effectiveness is high, for example up to 100% complete response of canine mast cell tumors smaller than 2 cm3 was achieved. Additionally, electrochemotherapy can be used for the treatment of inoperable tumors. One of the important characteristics of electrochemotherapy is that it can be effective as a one-time treatment only. However, in the case of failure or partial tumor response it can be repeated several times with equal or improved effectiveness. Electrochemotherapy is already a standard treatment for cutaneous and subcutaneous tumors of various histologies in human and veterinary oncology. Furthermore, several clinical studies exploiting electrochemotherapy for deep-seated tumors are on-going.

Electrochemotherapy is the local treatment of solid tumors. The main mechanism is electroporation-mediated permeabilization of the tumor cells' membrane which enables increased internalization of cytostatics like bleomycin and cisplatin. Thus, antitumor effectiveness is potentiated at the site of the application of electric pulses to the tumors.

犬と猫における腫瘍の治療のための電気化学の操作手順

Electrochemotherapy (ECT) is a local approach which is used for treating solid tumors of different histologies. Its mechanism is based on cell membrane ...

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 ...

A Syngeneic Mouse Model of Metastatic Renal Cell Carcinoma for Quantitative and Longitudinal Assessment of Preclinical Therapies

Renal cell carcinoma (RCC) affects &#62; 60,000 people in the United States annually, and ~ 30% of RCC patients have multiple metastases at the time of diagnosis. Metastatic RCC (mRCC) is incurable, with a median survival time of only 18 months. Immune-based interventions (e.g., interferon (IFN) and interleukin (IL)-2) induce durable responses in a fraction of mRCC patients, and multikinase inhibitors (e.g., sunitinib or sorafenib) or anti-VEGF receptor monoclonal antibodies (mAb) are largely palliative, as complete remissions are rare. Such shortcomings in current therapies for mRCC patients provide the rationale for the development of novel treatment protocols. A key component in the preclinical testing of new therapies for mRCC is a suitable animal model. Beneficial features that recapitulate the human condition include a primary renal tumor, renal tumor metastases, and an intact immune system to investigate any therapy-driven immune effector responses and the formation of tumor-induced immunosuppressive factors. This report describes an orthotopic mRCC mouse model that has all of these features. We describe an intrarenal implantation technique using the mouse renal adenocarcinoma cell line Renca, followed by the assessment of tumor growth in the kidney (primary site) and lungs (metastatic site).

Implementation of an orthotopic model of renal cell carcinoma in immunocompetent mice affords the investigator a clinically-relevant system defined by the presence of a primary renal tumor and lung metastases in the same animal. This system can be used to preclinically test a variety of treatments in vivo.

定量的および前臨床治療の縦評価のための転移性腎細胞癌の同系マウスモデル

Renal cell carcinoma (RCC) affects &#62; 60,000 people in the United States annually, and ~ 30% of RCC patients have multiple metastases at the time of ...

A Mouse Model of Fatigue Induced by Peripheral Irradiation

Cancer-related fatigue (CRF) is a distressing and costly condition that often affects patients receiving cancer treatments, including radiation therapy. Here we describe a method using targeted peripheral irradiation to induce fatigue-like behavior in mice. With appropriate shielding, the irradiation targets the lower abdominal/pelvic region of the mouse, sparing the brain, in an effort to model radiation treatment received by individuals with pelvic cancers. We deliver an irradiation dose that is sufficient to induce fatigue-like behavior in mice, measured by voluntary wheel-running activity (VWRA), without causing obvious morbidity. Since wheel running is a normal, voluntary behavior in mice, its use should have little confounding effect on other behavioral tests or biological measures. Hence, wheel running can be used as a feasible outcome measure in understanding the behavioral and biological correlates of fatigue. CRF is a complex condition with frequent comorbidities, and likely has causes related both to cancer and its various treatments. The methods described in this paper are useful for investigating radiation-induced changes that contribute to the development of CRF and, more generally, to explore the biological networks that can explain the development and persistence of a peripherally-triggered but centrally-driven behavior like fatigue.

We describe a method using targeted peripheral irradiation to induce fatigue-like behavior in mice. The selected non-lethal irradiation dose leads to a week-long reduction in voluntary wheel-running activity.

周辺照射による疲労のマウスモデル

Cancer-related fatigue (CRF) is a distressing and costly condition that often affects patients receiving cancer treatments, including radiation therapy. ...

Development of a Human Preclinical Model of Osteoclastogenesis from Peripheral Blood Monocytes Co-cultured with Breast Cancer Cell Lines

The crosstalk between tumor cells and bone cells in the bone microenvironment is crucial to understanding the mechanism of bone metastasis formation. We developed an in vitro fully human preclinical model of a co-culture of breast cancer cells and monocytes undergoing differentiation towards osteoclasts. We optimized a model of osteoclastogenesis starting from a sample of peripheral blood collected from healthy donors. Peripheral blood mononuclear cells (PBMCs) were first separated by density gradient centrifugation, seeded at a high density and induced to differentiate by adding two growth factors (GFs): receptor activator of nuclear factor-&#954;B ligand (RANKL) and macrophage colony-stimulating factor (MCSF). The cells were left in culture for 14 days and then fixed and analyzed by downstream analysis. In osteolytic bone metastases, one of the effects of cancer cell arrival in bone is the induction of osteoclastogenesis. We thus challenged our model with co-cultures of breast cancer cells to study the differentiation power of cancer cells with respect to GFs. A straightforward way of studying cancer cell-osteoclast interaction is to perform indirect co-cultures based on the use of conditioned medium collected from breast cancer cell cultures and mixed with fresh medium. This mixture is then used to induce osteoclast differentiation. We also optimized a method of direct co-culture in which cancer cells and monocytes undergoing differentiation share the medium and exchange secreted factors. This is a significant improvement over the original indirect co-culture method as researchers can observe the reciprocal interactions of the two cell types and perform downstream analyses for both cancer cells and osteoclasts. This method enables us to study the effect of drugs on the metastatic bone microenvironment and to seed cell lines other than those derived from breast cancer. The model can also be used to study other diseases such as osteoporosis or other bone conditions.

This protocol describes development of an in vitro human preclinical model of osteoclastogenesis from peripheral blood monocytes cultured with breast cancer cell lines to mimic the cancer cell-osteoclast interaction. The model could be used to further our understanding of bone metastasis formation and improve therapeutic options.

破骨細胞形成乳房癌細胞と共培養末梢血単球からの人間の前臨床モデルの開発

The crosstalk between tumor cells and bone cells in the bone microenvironment is crucial to understanding the mechanism of bone metastasis formation. We ...

Important Endpoints and Proliferative Markers to Assess Small Intestinal Injury and Adaptation using a Mouse Model of Chemotherapy-Induced Mucositis

Intestinal adaptation is the natural compensatory mechanism that occurs when the bowel is lost due to trauma. The adaptive responses, such as crypt cell proliferation and increased nutrient absorption, are critical in recovery, yet poorly understood. Understanding the molecular mechanism behind the adaptive responses is crucial to facilitate the identification of nutrients or drugs to enhance adaptation. Different approaches and models have been described throughout the literature, but a detailed descriptive way to essentially perform the procedures is needed to obtain reproducible data. Here, we describe a method to estimate important endpoints and proliferative markers of small intestinal injury and compensatory hyperproliferation using a model of chemotherapy-induced mucositis in mice. We demonstrate the detection of proliferating cells using a cell cycle specific marker, as well as using small intestinal weight, crypt depth, and villus height as endpoints. Some of the critical steps within the described method are the removal and weighing of the small intestine and the rather complex software system suggested for the measurement of this technique. These methods have the advantages that they are not time-consuming, and that they are cost-effective and easy to carry out and measure.

Here, we present a protocol to establish important endpoints and proliferative markers of small intestinal injury and compensatory hyperproliferation using a model of chemotherapy-induced mucositis. We demonstrate the detection of proliferating cells using a cell cycle specific marker and using small intestinal weight, crypt depth, and villus height as endpoints.

化学療法誘発性粘膜炎のマウスモデルを用いて小腸損傷と適応を評価するための重要なエンドポイントと増殖マーカー

Intestinal adaptation is the natural compensatory mechanism that occurs when the bowel is lost due to trauma. The adaptive responses, such as crypt cell ...