この作品は、国立保健医療研究評議会、オーストラリア（1008865）、オーストラリアの研究評議会（LE110100125）、国立がん研究所（CA138687-01）によってサポートされていました、エリカ·スローンは、国立乳がんから早期キャリアフェローシップでサポートされています財団、オーストラリア。コリーナキム·フックスは、スイスのがんリーグと薬学部のモナッシュ研究所のHDRの奨学金からのフェローシップでサポートされています。エリアーヌ不安はベルンがんリーグからの助成金によってサポートされています。

実証されているプロトコルは、著者の機関の動物のケアと使用委員会の指導と承認の下で行われる。すべての実験は、関連するすべてのガイドライン、規制、規制機関に準拠して実行されます。 1。膵臓癌細胞株を形質導入<ol><li> 12,13 PANC-1。前述したように、ルシフェラーゼ発現させる膵臓癌細胞を形質導入し、ホタルルシフェラーゼを形質導入CAPAN-1膵臓癌細胞株は、ここで使用される。 </li></ol> 注：ウミシイタケルシフェラーゼ又は細菌ルシフェラーゼを用いてもよい。 2。膵臓癌細胞の調製<ol><li>培養は、70％コンフルエントになるまで膵臓癌細胞を形質。 </li><li>膵臓細胞を持ち上げて、生存能力が90％以上であることを確認してください。 </li><li>チルドマトリゲルの午前3時02混合物中の2×10 7細胞/ mlで再懸濁し：リンSALは、バッファINE（PBS）。 </li><li>事前の膵臓への注入に、氷上でマトリゲル細胞懸濁物を保管してください。 </li></ol> 注：マトリゲルの急冷凝固を確保するために、細胞ペレットの量を考慮して、PBS量を減らす。注入前に凝固を防ぐために、すべての回で氷冷楽器や注射器を使用したマトリゲルを扱う。提案された細胞数がガイドされ、各細胞株に対して経験的に決定されるべきである。 3。マウス準備<ol><li> 2〜3％イソフルラン吸入使用して、マウスを麻酔。穏やかなつま先のピンチにペダル反射の欠如によって麻酔の深さを決定します。 </li><li>乾燥を防ぐために、目には潤滑剤を適用します。 </li><li> 37°Cの加熱パッドでその背中の上にマウスを置き、そっと腹部の左側を上げるには、マウスの電源を入れます。 </li><li> 10％ポビドンヨード液で腹部を準備します。 </li></ol> 注：Injectabル麻酔は吸入麻酔の代わりに使用することができる。術前の絶食は必要ありません。 4。開腹<ol><li>無菌の手術器具を使用すると、約1cmが正中線からの横方向のままに皮膚は1.5センチ切開を行います。 </li><li>基礎腹筋1.5センチ切開を行います。 </li><li>ピンセットを用いて脾臓を見つけて、優しく腹腔から脾臓を除去します。基礎膵臓を公開するために滅菌綿棒に沿って脾臓を固定します。 </li><li>脾臓に隣接した膵尾部の位置を確認します。 </li><li> 29 G 0.3ミリリットルのインスリン注射器を用いて、膵臓にマトリゲル細胞懸濁液20μLを注入します。 </li><li>マトリゲルが固化するまで注入後、30〜60秒のために膵臓でシリンジを保持します。これは重要なステップは、セルの漏れを最小限に抑えます。 </li><li>漏れが発生していないことを確認するために注射部位を検査します。 </li><li>腹腔に脾臓と膵臓を返します。 </li></ol> 注意：薄いかもしれない膵臓の背側を穿刺しないように注意してください。 5。腹壁閉鎖<ol><li>連続的なステッチを使用してラウンド針で吸収性編組4-0縫合糸でマウスの腹部の筋肉を閉じます。 </li><li>連続的なステッチを使用してカッティング針で非吸収性モノフィラメント6-0縫合糸で皮膚外用を閉じます。 </li><li>吸入麻酔薬と皮下注射0.05 mg / kgでブプレノルフィンからマウスを外します。 </li><li>マウスは、食品や水への無料アクセスを37℃の加温パッド上に置かれ、そのケージに回復することができます。マウスは、このようなハンチングまたは身体障害などの疼痛の兆候を示す場合は、ブプレノルフィンを、36時間かけて12時間毎に与えられてもよい。 </li><li>創傷治癒（7-10日）の後、マウスを麻酔し、外部の縫合糸を除去する。 </li></ol> 6。膵臓CANCの生物発光トラッキングえー進行<ol><li>イソフルラン吸入使用して、マウスを麻酔。 </li><li>尾静脈を経由して150 mg / kgをD-ルシフェリンを注入。 </li><li>生物発光イメージングシステムでマウスを置き、前述のように白色光と生物発光の画像をキャプチャします。14,15は </li><li>吸入麻酔からマウスを削除し、それがそのホームケージで回復することができます。 </li></ol> 注：生物発光撮像では非侵襲的であり、腫瘍増殖動態を調査するために定期的に実施することができる。膵尾部の画像腫瘍に、その左側にマウスを置くことが重要である、カメラに向かって腫瘍ポイントそう。周波数が前FOV 12.5ビニング4とリビングイメージング4.3.1ソフトウェアを実行ルミナIIイメージングシステム（パーキンエルマー、以前はキャリパーライフサイエンス）を用いた実験のエンドポイントに週3回まで増加したと我々は、週一回撮像するF-ストップ1、露出1から60秒（最高EXPによって決定ピクセル飽和なしosure）。

<ol>
	<li>American Cancer Society. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Facts and Figures. , (2013).</li><li>Hariharan, D., Saied, A., Kocher, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+mortality+rates+for+pancreatic+cancer+across+the+world.">Analysis of mortality rates for pancreatic cancer across the world.</a> HPB (Oxford). 10, 58-62 (2008).</li><li>Li, D., Xie, K., Wolff, R., Abbruzzese, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pancreatic+cancer.">Pancreatic cancer.</a> Lancet. 363, 1049-1057 (2004).</li><li>Oettle, 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=Adjuvant+chemotherapy+with+gemcitabine+vs+observation+in+patients+undergoing+curative-intent+resection+of+pancreatic+cancer:+a+randomized+controlled+trial.">Adjuvant chemotherapy with gemcitabine vs observation in patients undergoing curative-intent resection of pancreatic cancer: a randomized controlled trial.</a> JAMA. 297, 267-277 (2007).</li><li>Andersson, R., 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=Gemcitabine+chemoresistance+in+pancreatic+cancer:+molecular+mechanisms+and+potential+solutions.">Gemcitabine chemoresistance in pancreatic cancer: molecular mechanisms and potential solutions.</a> Scand. J. Gastroenterol. 44, 782-786 (2009).</li><li>Angst, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioluminescence+imaging+of+angiogenesis+in+a+murine+orthotopic+pancreatic+cancer+model.">Bioluminescence imaging of angiogenesis in a murine orthotopic pancreatic cancer model.</a> Mol. Imaging Biol. 12, 570-575 (2010).</li><li>Angst, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=N-myc+downstream+regulated+gene-1+expression+correlates+with+reduced+pancreatic+cancer+growth+and+increased+apoptosis+in+vitro+and+in+vivo.">N-myc downstream regulated gene-1 expression correlates with reduced pancreatic cancer growth and increased apoptosis in vitro and in vivo.</a> Surgery. 149, 614-624 (2011).</li><li>Hotz, H. G., 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=An+orthotopic+nude+mouse+model+for+evaluating+pathophysiology+and+therapy+of+pancreatic+cancer.">An orthotopic nude mouse model for evaluating pathophysiology and therapy of pancreatic cancer.</a> Pancreas. 26, 89-98 (2003).</li><li>Partecke, L. I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+syngeneic+orthotopic+murine+model+of+pancreatic+adenocarcinoma+in+the+C57/BL6+mouse+using+the+Panc02+and+6606PDA+cell+lines.">A syngeneic orthotopic murine model of pancreatic adenocarcinoma in the C57/BL6 mouse using the Panc02 and 6606PDA cell lines.</a> Eur. Surg. Res. 47, 98-107 (2011).</li><li>Hingorani, S. R., 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=Preinvasive+and+invasive+ductal+pancreatic+cancer+and+its+early+detection+in+the+mouse.">Preinvasive and invasive ductal pancreatic cancer and its early detection in the mouse.</a> Cancer Cell. 4, 437-450 (2003).</li><li>Sloan, E. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+sympathetic+nervous+system+induces+a+metastatic+switch+in+primary+breast+cancer.">The sympathetic nervous system induces a metastatic switch in primary breast cancer.</a> Cancer Res. 70, 7042-7052 (2010).</li><li>Wang, X., McManus, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lentivirus+production.">Lentivirus production.</a> J. Vis. Exp. (32), e1499 (2009).</li><li>Morizono, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lentiviral+vector+retargeting+to+P-glycoprotein+on+metastatic+melanoma+through+intravenous+injection.">Lentiviral vector retargeting to P-glycoprotein on metastatic melanoma through intravenous injection.</a> Nat. Med. 11, 346-352 (2005).</li><li>Saha, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+bioluminescence+imaging+of+tumor+hypoxia+dynamics+of+breast+cancer+brain+metastasis+in+a+mouse+model.">In vivo bioluminescence imaging of tumor hypoxia dynamics of breast cancer brain metastasis in a mouse model.</a> J. Vis. Exp. (56), e3175 (2011).</li><li>Lim, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monitoring+tumor+metastases+and+osteolytic+lesions+with+bioluminescence+and+micro+CT+imaging.">Monitoring tumor metastases and osteolytic lesions with bioluminescence and micro CT imaging.</a> J. Vis. Exp. (52), e2775 (2011).</li><li>Burton, J. B., 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=Adenovirus-mediated+gene+expression+imaging+to+directly+detect+sentinel+lymph+node+metastasis+of+prostate+cancer.">Adenovirus-mediated gene expression imaging to directly detect sentinel lymph node metastasis of prostate cancer.</a> Nat Med. 14, 882-888 (2008).</li><li>Vezeridis, M. P., Doremus, C. M., Tibbetts, L. M., Tzanakakis, G., Jackson, B. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Invasion+and+metastasis+following+orthotopic+transplantation+of+human+pancreatic+cancer+in+the+nude+mouse.">Invasion and metastasis following orthotopic transplantation of human pancreatic cancer in the nude mouse.</a> J. Surg. Oncol. 40, 261-265 (1989).</li><li>Fu, X., Guadagni, F., Hoffman, R. 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+metastatic+nude-mouse+model+of+human+pancreatic+cancer+constructed+orthotopically+with+histologically+intact+patient+specimens.">A metastatic nude-mouse model of human pancreatic cancer constructed orthotopically with histologically intact patient specimens.</a> Proc. Natl. Acad. Sci. U.S.A. 89, 5645-5649 (1992).</li><li>Reyes, G., 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=Orthotopic+xenografts+of+human+pancreatic+carcinomas+acquire+genetic+aberrations+during+dissemination+in+nude+mice.">Orthotopic xenografts of human pancreatic carcinomas acquire genetic aberrations during dissemination in nude mice.</a> Cancer Res. 56, 5713-5719 (1996).</li><li>Kim, M. P., 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=Generation+of+orthotopic+and+heterotopic+human+pancreatic+cancer+xenografts+in+immunodeficient+mice.">Generation of orthotopic and heterotopic human pancreatic cancer xenografts in immunodeficient mice.</a> Nat Protoc. 4, 1670-1680 (2009).</li><li>Furukawa, T., Kubota, T., Watanabe, M., Kitajima, M., Hoffman, R. 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+novel+"patient-like"+treatment+model+of+human+pancreatic+cancer+constructed+using+orthotopic+transplantation+of+histologically+intact+human+tumor+tissue+in+nude+mice.">A novel "patient-like" treatment model of human pancreatic cancer constructed using orthotopic transplantation of histologically intact human tumor tissue in nude mice.</a> Cancer Res. 53, 3070-3072 (1993).</li><li>Lewis, C. E., Pollard, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+role+of+macrophages+in+different+tumor+microenvironments.">Distinct role of macrophages in different tumor microenvironments.</a> Cancer Res. 66, 605-612 (2006).</li><li>Brembeck, F. 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=The+mutant+K-ras+oncogene+causes+pancreatic+periductal+lymphocytic+infiltration+and+gastric+mucous+neck+cell+hyperplasia+in+transgenic+mice.">The mutant K-ras oncogene causes pancreatic periductal lymphocytic infiltration and gastric mucous neck cell hyperplasia in transgenic mice.</a> Cancer Res. 63, 2005-2009 (2003).</li></ol>

著者は、彼らが競合する経済的利益を持っていないことを宣言します。

ここでは、膵臓腫瘍発生および進行の長手方向の評価のための同所性モデルを記述。原発腫瘍増殖動態は、再現性がある（ 図3）と非侵襲的に新規抗膵臓癌治療に対する腫瘍の応答の分析のために、例えば 、ルシフェラーゼタグ付き細胞の生物発光撮像を用いてモニターすることができる。ヒトの疾患と一致して、モデルは、膵臓微小環境と腫瘍細胞との相互作用の調査?...

膵臓癌は4〜6％の5年生存率は、癌関連死因の第4位である。患者の1,2だけが15％、手術の対象になる病気の早い段階で十分に診断され、腫瘍が再発している&gt;でこれらの患者の80％は3,4ゲムシタビンは、膵臓腺癌の治療に使用されているが、化学療法耐性が一般的であり、多くの場合、薬物は、全体的な生存率にほとんど影響を与えない。膵臓癌を治療するために5新しい薬理学的戦略が決定的に必要とされる。その開発は、治療的介入に敏感かもしれない病気の進行の重要なステップの大幅に改善理解に依存します。 膵臓癌の同所性モデルは、膵臓癌の生物学を研究するための理想的なツールがそれらを作り、ヒトの疾患の重要な側面をエミュレートする。膵臓癌細胞の挙動のin vitroでの細胞ベースのアッセイとは対照的に6-9dは皮下膵臓癌のin vivoモデルにおいて 、同所性モデルは、膵臓腫瘍細胞微小環境との相互作用の調査を可能にする。病気の進行の速度論は同所性モデルで再現性の高いであり、それらはよく小説治療薬の前臨床試験に適しています短い時間枠（週）にわたって生じる。これにより、疾患発症が長く、より可変時間枠（ヶ月〜1年間）にわたって生じるトランスジェニックモデルとは対照的である。10は、より積極的な細胞株と一緒に使用すると、膵臓癌の同所性モデルは、に見られるものと同様の自然転移のパターンを有する患者はこのようなホタルルシフェラーゼが治療に転移性播種、再発および応答、腫瘍増殖の縦監視を容易にします。6,11のように生物発光レポーター遺伝子の発現は8 ここでは、MATRを利用膵臓癌の同所性モデルを記述腫瘍進行の非侵襲的なモニタリングのためのローカライズされた細胞送達およびin vivoの生物発光イメージングのためのIGEL。膵臓癌の同所このモデルは、疾患の進行および同系移植片または異種移植片モデルにおける治療的介入に対する応答の非侵襲的な分析を可能にする。

<table border="1">
 <tbody>
 <tr>
 <td>Equipment</td>
 <td>Company</td>
 <td>Catalog Number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>Clean Bench coat</td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Heating pad</td>
 <td></td>
 <td></td>
 <td>Set to 37 &deg;C </td>
 </tr>
 <tr>
 <td>Ivis Lumina ll Bioluminescent imager</td>
 <td>Caliper </td>
 <td></td>
 <td>Alternative bioluminescent imaging systems include In vivo F PRO (Carestream) and Photon Imager (Biospace Lab)</td>
 </tr>
 <tr>
 <td>Dissecting scissors</td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Iris forceps (serrated)</td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Needle holder</td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>27 G 0.3 ml insulin syringe</td>
 <td>Terumo</td>
 <td>T35525M2913</td>
 <td></td>
 </tr>
 </tbody>
</table>

bioluminescent orthotopic model of pancreatic cancer progression

<p class="jove_content"&gt;膵臓癌は4〜6％の極めて悪い5年生存率を有している。新たな治療オプションが批判​​的に必要であり、膵臓癌生物学の理解の向上に依存している。良好な膵微小環境とがん細胞との相互作用を理解するために、我々は、癌の進行の非侵襲的なモニタリングを可能に膵臓癌の同所性モデルを示す。ルシフェラーゼタグ付き膵臓癌細胞をマトリゲル中に再懸濁し、開腹時に膵尾部に送達される。マトリゲルは、注入時のがん細胞の漏れを防止するために体温で固化する。遠隔臓器に原発腫瘍の増殖や転移を使用して、ルシフェラーゼ基質ルシフェリンの注射後に監視されている<em生体内で&gt;</em癌細胞の生物発光の&gt;撮像。<em生体内で&gt;</em&gt;イメージングは​​また、切除後の原発腫瘍の再発を追跡するために使用することができる。このモデルは、同所性異種移植モデルと同系の両方に適しており、一次膵臓腫瘍および転移の増殖に対する新規抗癌治療の影響を調査するための前臨床試験で使用されてもよい。</p&gt;

この方法は、麻酔導入、開腹、マトリゲル、腹部閉止（ 図1A）中の癌細胞の注射を含む外科的処置を用いて膵臓癌の同所性モデルについて説明する。注入された細胞は、膵臓の表面の気泡（ 図1B）を形成する。膵臓癌の進行を非侵襲的に癌細胞の増殖と普及し（ 図2）を追跡するためにインビボ生物発光撮像で使用して監視することができ?...

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Bioluminescent Orthotopic Model of Pancreatic Cancer Progression

膵臓癌生物学の理解の向上は非常に重要膵臓癌を治療するための良好な治療選択肢の開発を可能にするために必要とされる。このニーズに対応するために、我々は使用して癌の進行の非侵襲的なモニタリングを可能に膵臓癌の同所性モデルを示す<em生体内で&gt;</em&gt;生物発光イメージング。

膵臓癌の進行の生物発光同所性モデル

Pancreatic cancer has an extremely poor five-year survival rate of 4-6%.  New therapeutic options are critically needed and depend on improved ...

bioluminescent-orthotopic-model-of-pancreatic-cancer-progression

Research

JoVE Journal

Medicine

26.8K ビュー.  Monash University. 膵臓癌生物学の理解の向上は非常に重要膵臓癌を治療するための良好な治療選択肢の開発を可能にするために必要とされる。このニーズに対応するために、我々は使用して癌の進行の非侵襲的なモニタリングを可能に膵臓癌の同所性モデルを示す生物発光イメージング。

ビデオ: Bioluminescent Orthotopic Model of Pancreatic Cancer Progression

Murine Bioluminescent Hepatic Tumour Model

This video describes the establishment of liver metastases in a mouse model that can be subsequently analysed by bioluminescent imaging. Tumour cells are administered specifically to the liver to induce a localised liver tumour, via mobilisation of the spleen and splitting into two, leaving intact the vascular pedicle for each half of the spleen. Lewis lung carcinoma cells that constitutively express the firefly luciferase gene (luc1) are inoculated into one hemi-spleen which is then resected 10 minutes later. The other hemi-spleen is left intact and returned to the abdomen. Liver tumour growth can be monitored by bioluminescence imaging using the IVIS whole body imaging system. Quantitative imaging of tumour growth using IVIS provides precise quantitation of viable tumour cells. Tumour cell death and necrosis due to drug treatment is indicated early by a reduction in the bioluminescent signal. This mouse model allows for investigating the mechanisms underlying metastatic tumour-cell survival and growth and can be used for the evaluation of therapeutics of liver metastasis. 

This article describes a procedure for the induction of orthotopic bioluminescent liver tumours in mice, and subsequent analysis of tumour growth confined to the liver using live whole body luminescence imaging.

マウス生物発光肝腫瘤モデル

This video describes the establishment of liver metastases in a mouse model that can be subsequently analysed by bioluminescent imaging. Tumour cells are ...

医学

An Orthotopic Model of Murine Bladder Cancer

In this straightforward procedure, bladder tumors are established in female C57 mice through the use of catheterization, local cauterization, and subsequent cell adhesion. After their bladders are transurethrally catheterized and drained, animals are again catheterized to permit insertion of a platinum wire into bladders without damaging the urethra or bladder. The catheters are made of Teflon to serve as an insulator for the wire, which will conduct electrical current into the bladder to create a burn injury. An electrocautery unit is used to deliver 2.5W to the exposed end of the wire, burning away extracellular layers and providing attachment sites for carcinoma cells that are delivered in suspension to the bladder through a subsequent catheterization. Cells remain in the bladder for 90 minutes, after which the catheters are removed and the bladders allowed to drain naturally. The development of tumor is monitored via ultrasound. Specific attention is paid to the catheterization technique in the accompanying video.

Bladder tumors are established in female mice in a minimally invasive fashion through catheterization, local cauterization, and subsequent adhesion of carcinoma cells to the burn sites.

マウス膀胱癌の同所性モデル

In this straightforward procedure, bladder tumors are established in female C57 mice through the use of catheterization, local cauterization, and ...

Multi-photon Imaging of Tumor Cell Invasion in an Orthotopic Mouse Model of Oral Squamous Cell Carcinoma

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient management strategies. Orthotopic mouse models of oral cancer have been developed to facilitate the study of factors that impact invasion and serve as model system for evaluating anti-tumor therapeutics. In these systems, visualization of disseminated tumor cells within oral cavity tissues has typically been conducted by either conventional histology or with in vivo bioluminescent methods. A primary drawback of these techniques is the inherent inability to accurately visualize and quantify early tumor cell invasion arising from the primary site in three dimensions. Here we describe a protocol that combines an established model for squamous cell carcinoma of the tongue (SCOT) with two-photon imaging to allow multi-vectorial visualization of lingual tumor spread. The OSC-19 head and neck tumor cell line was stably engineered to express the F-actin binding peptide LifeAct fused to the mCherry fluorescent protein (LifeAct-mCherry). Fox1nu/nu mice injected with these cells reliably form tumors that allow the tongue to be visualized by ex-vivo application of two-photon microscopy. This technique allows for the orthotopic visualization of the tumor mass and locally invading cells in excised tongues without disruption of the regional tumor microenvironment. In addition, this system allows for the quantification of tumor cell invasion by calculating distances that invaded cells move from the primary tumor site. Overall this procedure provides an enhanced model system for analyzing factors that contribute to SCOT invasion and therapeutic treatments tailored to prevent local invasion and distant metastatic spread. This method also has the potential to be ultimately combined with other imaging modalities in an in vivo setting.

A comprehensive overview of the techniques involved in generating a mouse model of oral cancer and quantitative monitoring of tumor invasion within the tongue through multi-photon microscopy of labeled cells is presented. This system can serve as a useful platform for the molecular assessment and drug efficacy of anti-invasive compounds.

口腔扁平上皮癌の同所性マウスモデルにおける腫瘍細胞の浸潤の多光子イメージング

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient ...

In vivo Dual Substrate Bioluminescent Imaging

Our understanding of how and when breast cancer cells transit from established primary tumors to metastatic sites has increased at an exceptional rate since the advent of in vivo bioluminescent imaging technologies 1-3. Indeed, the ability to locate and quantify tumor growth longitudinally in a single cohort of animals to completion of the study as opposed to sacrificing individual groups of animals at specific assay times has revolutionized how researchers investigate breast cancer metastasis. Unfortunately, current methodologies preclude the real-time assessment of critical changes that transpire in cell signaling systems as breast cancer cells (i) evolve within primary tumors, (ii) disseminate throughout the body, and (iii) reinitiate proliferative programs at sites of a metastatic lesion. However, recent advancements in bioluminescent imaging now make it possible to simultaneously quantify specific spatiotemporal changes in gene expression as a function of tumor development and metastatic progression via the use of dual substrate luminescence reactions. To do so, researchers take advantage for two light-producing luciferase enzymes isolated from the firefly (Photinus pyralis) and sea pansy (Renilla reniformis), both of which react to mutually exclusive substrates that previously facilitated their wide-spread use in in vitro cell-based reporter gene assays 4. Here we demonstrate the in vivo utility of these two enzymes such that one luminescence reaction specifically marks the size and location of a developing tumor, while the second luminescent reaction serves as a means to visualize the activation status of specific signaling systems during distinct stages of tumor and metastasis development. Thus, the objectives of this study are two-fold. First, we will describe the steps necessary to construct dual bioluminescent reporter cell lines, as well as those needed to facilitate their use in visualizing the spatiotemporal regulation of gene expression during specific steps of the metastatic cascade. Using the 4T1 model of breast cancer metastasis, we show that the in vivo activity of a synthetic Smad Binding Element (SBE) promoter was decreased dramatically in pulmonary metastasis as compared to that measured in the primary tumor 4-6. Recently, breast cancer metastasis was shown to be regulated by changes within the primary tumor microenvironment and reactive stroma, including those occurring in fibroblasts and infiltrating immune cells 7-9. Thus, our second objective will be to demonstrate the utility of dual bioluminescent techniques in monitoring the growth and localization of two unique cell populations harbored within a single animal during breast cancer growth and metastasis.

In vivo Dual Substrate Bioluminescent Imaging

Herein we describe the methods to construct, visualize, and quantify the bioluminescent reactions of both firefly and renilla luciferase enzymes expressed in metastatic breast cancer cells during their growth and metastasis in vivo.

生体デュアル基質生物発光イメージングにおける

Our understanding of how and when breast cancer cells transit from established primary tumors to metastatic sites has increased at an exceptional rate ...

Combination Radiotherapy in an Orthotopic Mouse Brain Tumor Model

Glioblastoma multiforme (GBM) are the most common and aggressive adult primary brain tumors1. In recent years there has been substantial progress in the understanding of the mechanics of tumor invasion, and direct intracerebral inoculation of tumor provides the opportunity of observing the invasive process in a physiologically appropriate environment2. As far as human brain tumors are concerned, the orthotopic models currently available are established either by stereotaxic injection of cell suspensions or implantation of a solid piece of tumor through a complicated craniotomy procedure3. In our technique we harvest cells from tissue culture to create a cell suspension used to implant directly into the brain. The duration of the surgery is approximately 30 minutes, and as the mouse needs to be in a constant surgical plane, an injectable anesthetic is used. The mouse is placed in a stereotaxic jig made by Stoetling (figure 1). After the surgical area is cleaned and prepared, an incision is made; and the bregma is located to determine the location of the craniotomy. The location of the craniotomy is 2 mm to the right and 1 mm rostral to the bregma. The depth is 3 mm from the surface of the skull, and cells are injected at a rate of 2 &mu;l every 2 minutes. The skin is sutured with 5-0 PDS, and the mouse is allowed to wake up on a heating pad. From our experience, depending on the cell line, treatment can take place from 7-10 days after surgery. Drug delivery is dependent on the drug composition. For radiation treatment the mice are anesthetized, and put into a custom made jig. Lead covers the mouse's body and exposes only the brain of the mouse. The study of tumorigenesis and the evaluation of new therapies for GBM require accurate and reproducible brain tumor animal models. Thus we use this orthotopic brain model to study the interaction of the microenvironment of the brain and the tumor, to test the effectiveness of different therapeutic agents with and without radiation.

The purpose of this article is to describe the use of an orthotopic glioblastoma model for chemoradiation studies. This article will go though cell processing, implanting, and radiotherapy of the mouse using an intracranial model.

同所性マウス脳腫瘍モデルのコンビネーション放射線治療

Glioblastoma multiforme (GBM) are the most common and aggressive adult primary brain tumors1. In recent years there has been substantial progress in the ...

An Orthotopic Bladder Tumor Model and the Evaluation of Intravesical saRNA Treatment

We present a novel method for treating bladder cancer with intravesically delivered small activating RNA (saRNA) in an orthotopic xenograft mouse bladder tumor model. The mouse model is established by urethral catheterization under inhaled general anesthetic. Chemical burn is then introduced to the bladder mucosa using intravesical silver nitrate solution to disrupt the bladder glycosaminoglycan layer and allows cells to attach. Following several washes with sterile water, human bladder cancer KU-7-luc2-GFP cells are instilled through the catheter into the bladder to dwell for 2 hours. Subsequent growth of bladder tumors is confirmed and monitored by in vivo bladder ultrasound and bioluminescent imaging. The tumors are then treated intravesically with saRNA formulated in lipid nanoparticles (LNPs). Tumor growth is monitored with ultrasound and bioluminescence. All steps of this procedure are demonstrated in the accompanying video.

Establishing an orthotopic bladder tumor model to evaluate antitumor effects of intravesically delivered saRNA and monitoring tumor growth by ultrasound and bioluminescent imaging.

同所性膀胱腫瘍モデルと膀胱内Sarnaの治療の評価

We present a novel method for treating bladder cancer with intravesically delivered small activating RNA (saRNA) in an orthotopic xenograft mouse bladder ...

Surgical Procedures for a Rat Model of Partial Orthotopic Liver Transplantation with Hepatic Arterial Reconstruction

Orthotopic liver transplantation (OLT) in rats using a whole or partial graft is an indispensable experimental model for transplantation research, such as studies on graft preservation and ischemia-reperfusion injury 1,2, immunological responses 3,4, hemodynamics 5,6, and small-for-size syndrome 7. The rat OLT is among the most difficult animal models in experimental surgery and demands advanced microsurgical skills that take a long time to learn. Consequently, the use of this model has been limited. Since the reliability and reproducibility of results are key components of the experiments in which such complex animal models are used, it is essential for surgeons who are involved in rat OLT to be trained in well-standardized and sophisticated procedures for this model.
While various techniques and modifications of OLT in rats have been reported 8 since the first model was described by Lee et al. 9 in 1973, the elimination of the hepatic arterial reconstruction 10 and the introduction of the cuff anastomosis technique by Kamada et al. 11 were a major advancement in this model, because they simplified the reconstruction procedures to a great degree. In the model by Kamada et al., the hepatic rearterialization was also eliminated. Since rats could survive without hepatic arterial flow after liver transplantation, there was considerable controversy over the value of hepatic arterialization. However, the physiological superiority of the arterialized model has been increasingly acknowledged, especially in terms of preserving the bile duct system 8,12 and the liver integrity 8,13,14.
In this article, we present detailed surgical procedures for a rat model of OLT with hepatic arterial reconstruction using a 50% partial graft after ex vivo liver resection. The reconstruction procedures for each vessel and the bile duct are performed by the following methods: a 7-0 polypropylene continuous suture for the supra- and infrahepatic vena cava; a cuff technique for the portal vein; and a stent technique for the hepatic artery and the bile duct.

Orthotopic liver transplantation in rats is an indispensable experimental model for biomedical research. Here we present our surgical procedures for orthotopic rat liver transplantation with hepatic arterial reconstruction using a 50% partial graft.

肝動脈再建を用いた部分同所性肝移植のラットモデルのための外科的処置

Orthotopic liver transplantation (OLT) in rats using a whole or partial graft is an indispensable experimental model for transplantation research, such as ...

An Orthotopic Bladder Cancer Model for Gene Delivery Studies

Bladder cancer is the second most common cancer of the urogenital tract and novel therapeutic approaches that can reduce recurrence and progression are needed. The tumor microenvironment can significantly influence tumor development and therapy response. It is therefore often desirable to grow tumor cells in the organ from which they originated. This protocol describes an orthotopic model of bladder cancer, in which MB49 murine bladder carcinoma cells are instilled into the bladder via catheterization. Successful tumor cell implantation in this model requires disruption of the protective glycosaminoglycan layer, which can be accomplished by physical or chemical means. In our protocol the bladder is treated with trypsin prior to cell instillation. Catheterization of the bladder can also be used to deliver therapeutics once the tumors are established. This protocol describes the delivery of an adenoviral construct that expresses a luciferase reporter gene. While our protocol has been optimized for short-term studies and focuses on gene delivery, the methodology of mouse bladder catheterization has broad applications.

Implantation of cancer cells into the organ of origin can serve as a useful preclinical model to evaluate novel therapies. MB49 bladder carcinoma cells can be grown within the bladder following intravesical instillation. This protocol demonstrates catheterization of the mouse bladder for the purpose of tumor implantation and adenoviral delivery.

遺伝子送達研究のための同所膀胱癌モデル

Bladder cancer is the second most common cancer of the urogenital tract and novel therapeutic approaches that can reduce recurrence and progression are ...

An Orthotopic Murine Model of Human Prostate Cancer Metastasis

Our laboratory has developed a novel orthotopic implantation model of human prostate cancer (PCa). As PCa death is not due to the primary tumor, but rather the formation of distinct metastasis, the ability to effectively model this progression pre-clinically is of high value. In this model, cells are directly implanted into the ventral lobe of the prostate in Balb/c athymic mice, and allowed to progress for 4-6 weeks. At experiment termination, several distinct endpoints can be measured, such as size and molecular characterization of the primary tumor, the presence and quantification of circulating tumor cells in the blood and bone marrow, and formation of metastasis to the lung. In addition to a variety of endpoints, this model provides a picture of a cells ability to invade and escape the primary organ, enter and survive in the circulatory system, and implant and grow in a secondary site. This model has been used effectively to measure metastatic response to both changes in protein expression as well as to response to small molecule therapeutics, in a short turnaround time.

This orthotopic model of human prostate cancer allows for quantification of tumor size, circulating tumor cells, and formation of distinct metastasis to the lung. As cells must escape the primary organ, enter the blood stream, and implant into a secondary site, this model effectively recapitulates the scenario in humans.

ヒト前立腺癌転移の同所マウスモデル

Our laboratory has developed a novel orthotopic implantation model of  human prostate cancer (PCa). As PCa death is not due to the primary tumor, but  ...

Dynamic Contrast Enhanced Magnetic Resonance Imaging of an Orthotopic Pancreatic Cancer Mouse Model

Dynamic contrast enhanced magnetic resonance imaging (DCE-MRI) has been limitedly used for orthotopic pancreatic tumor xenografts due to severe respiratory motion artifact in the abdominal area. Orthotopic tumor models offer advantages over subcutaneous ones, because those can reflect the primary tumor microenvironment affecting blood supply, neovascularization, and tumor cell invasion. We have recently established a protocol of DCE-MRI of orthotopic pancreatic tumor xenografts in mouse models by securing tumors with an orthogonally bent plastic board to prevent motion transfer from the chest region during imaging. The pressure by this board was localized on the abdominal area, and has not resulted in respiratory difficulty of the animals. This article demonstrates the detailed procedure of orthotopic pancreatic tumor modeling using small animals and DCE-MRI of the tumor xenografts. Quantification method of pharmacokinetic parameters in DCE-MRI is also introduced. The procedure described in this article will assist investigators to apply DCE-MRI for orthotopic gastrointestinal cancer mouse models.

The goal of this protocol is to apply dynamic contrast enhanced magnetic resonance imaging (DCE-MRI) for orthotopic pancreatic tumor xenografts in mice. DCE-MRI is a non-invasive method to analyze microvasculature in a target tissue, and useful to assess vascular response in a tumor following a novel therapy.