著者は、博士に感謝したいと思います。手術の訓練での支援のためのWenjunのLiとダニエル独楽（セントルイスの医学のワシントン大学）。 RYLは健康グラントR01 DK068057とセントルイス大学の学長の研究基金の国立研究所によってサポートされています。

1。細胞調製 注 2）場所マトリゲル1）完全RPMI 1640ベースの文化·メディア·レシピ（500ミリリットル）435ミリリットルRPMI 1640、50ミリリットル熱不活性化FBS、5ミリリットル非必須アミノ酸、5ミリリットルのピルビン酸ナトリウム、5ミリリットル抗生物質抗真菌が含まれています4℃で一日のセルの収穫前に。 <ol><li>人間ATC細胞ラインTHJは-11T 8は 6ウェル培養プレートで増殖させた80〜90％コンフルエントに一度収穫される。 </li><li>室温で4分間200×gで遠心分離によって細胞をペレット化する。 </li><li> RPMI 1640培地[2ミリリットル/ 6ウェルプレート]でメディアと再懸濁した細胞を吸引。 </li><li> 0.4％トリパンブルー色素の一つのボリュームに追加し、TC10自動セルカウンターを用いて細胞密度を決定し、細胞懸濁液の小体積を取る。 </li><li>注入5×10 5細胞/マウス倍の数のマウスを得るために必要な細胞懸濁液の体積を計算する。 </li></ol> 注：広告を確保するため<html>あなたは、マウスの二重数を（ 例えば 5匹のマウスを注入する場合は、10匹のマウスを注入したかのように、その後、細胞懸濁液を準備）を注入されているかのように、注射用の細胞懸濁液を同一視する、細胞を調製。 <ol start="6"><li> 15ミリリットルコニカルチューブに細胞懸濁液の必要な量を転送します。 </li><li>室温で4分間200×gで遠心分離によって細胞をペレット化する。 </li><li>完全RPMI 1640培地でのメディア再懸濁した細胞を吸引。 </li><li> 1.6 mlチューブに細胞を移しマトリゲルの1ボリュームを追加して、徐々に内と外ペッティングにより穏やかに混合。 </li></ol> 注：各マウスは細胞懸濁液/マトリゲルカクテル10μlのを受け取ります。 10μlの注射量に懸濁し、5×10 5個の細胞が正常に以前のATC同所の研究9-10で使用されてきたので、本報告書でこれらのパラメータを使用していました。 <ol start="10"><li>使用する準備まで氷上で細胞懸濁液を置きます。 </li></ol>_title &quot;&gt; 2。マウスの準備<ol><li> 70％エタノールですべての表面を拭いて、解剖範囲と周辺地域を含む外科的なゾーンを、清掃してください。 </li><li>動物は手術から回復される加熱ランプ/パッドをオンにします。 </li><li> 1ミリリットル、27G½ &quot;ツベルクリン注射器を用いて10グラム体重腹腔あたり0.1ミリリットルケタミン/キシラジン（ケタミン9 mg / mlのとキシラジン1mg/mlのから成る薬物カクテル）を管理。 </li><li>手術のための準備マウス：1）マウス（あごのラインから胸骨と外のトップに腕に）、2の頸部を剃る）手術領域をスクラブ三回chorhexadine浸したガーゼの正方形と、3）ベタジン浸したガーゼで行う最後のスクラブ、4 ）優しく乾燥から目を防ぐために、眼軟膏を適用します。 </li><li>マウスが十分に鎮静されていることを確認するために反射ペダルチェック。 </li><li>解剖スコープの下に使い捨て無菌フィールドバリアに背側横臥に動物を置き、布テープで所定の位置に動物を固定します。 </li><li>スクラブ手とfingernailsその後無菌手術用手袋を置く。 </li><li>滅菌方法では、開腹手術はパックや楽器を手配。 </li><li>暴露だけ手術部位を残して、動物を介して無菌ドレープを置きます。 </li></ol> 3。甲状腺および細胞注入に外科用アクセス<ol><li>滅菌、使い捨てメスを使用して、喉の正中線に沿って1対1.5センチメートル縦切開を行います。 </li></ol> 注：正中線からの偏差は、気管にアクセスすることがより深いカッティングが複雑になります。かなり正確な正中切開は、あなたが一緒に左右の唾液腺を保持膜組織を介していじめるとカットすることができ、大規模な動脈ニッキングまたは切断の可能性を低減します。 <ol start="2"><li>気管周囲のストラップの筋肉に第二切開を行い、次に右甲状腺を公開する側に切開筋肉の右側を引っ張る。 </li><li>止血は、筋層を保持するために、この時点で使用することができる甲状腺（ 図1）に簡単にアクセスするためのパーツ。 </li></ol> 注：手順は、2つの手術スタッフによって実行された場合準備注射シリンジは支援スタッフが外科医に渡されている間あるいは、筋層が単に鉗子で戻って開催することができる。 <ol start="4"><li>ゆっくりゆっくり針を除去31G 5/16 &quot;インスリン注射器を使用して右の甲状腺に細胞懸濁液10μlを注入。 </li></ol> 4。手術部位とマウスの回復の閉鎖<ol><li>どちら連続または断続縫合スタイルを使用して6.0縫合材料と縫合筋層。 </li><li>同じ方法でステッチが皮膚。 </li><li>直接切開部位上トリプル抗生物質軟膏の層を適用します。 </li><li>動物は暖かい加熱パッドで背側横臥位で回復することができます。動物が支援なし胸骨横臥位を実現することができたら、それを他のマウスに戻って配置することができる。 </li></ol> &lt;Pクラス= &quot;jove_title&quot;&gt; 5。術後モニタリングとティッシュコレクション<ol><li>手術後一週間、毎日動物の切開部位と全体的な健康状態を確認し、週に一度確認してください。縫合糸は、14日後に無傷のままである場合、それらは除去される。 </li></ol> 注：健康の衰え（ 例えばかなりの減量、だらしない髪は、呼吸をこじつけ）の兆候を示す動物を安楽死させ、組織が ​​収集されるべきである。 <ol start="2"><li>一度マウスはキシラジン/ケタミン落ち着いた、上記説明するように管理し、所定の手術後の時点に達しています。 </li><li>心臓内に動物を灌流し、収穫甲状腺/気管や関心のある他の組織（ここでは詳細に説明しない）。 </li></ol>

<ol>
	<li>Bibby, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Orthotopic+models+of+cancer+for+preclinical+drug+evaluation:+advantages+and+disadvantages.">Orthotopic models of cancer for preclinical drug evaluation: advantages and disadvantages.</a> Eur. J. Cancer. 40, 852-857 (2004).</li><li>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=Orthotopic+metastatic+mouse+models+for+anticancer+drug+discovery+and+evaluation:+a+bridge+to+the+clinic.">Orthotopic metastatic mouse models for anticancer drug discovery and evaluation: a bridge to the clinic.</a> Invest. New Drugs. 17, 343-359 (1999).</li><li>Killion, J. J., Radinsky, R., Fidler, I. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Orthotopic+models+are+necessary+to+predict+therapy+of+transplantable+tumors+in+mice.">Orthotopic models are necessary to predict therapy of transplantable tumors in mice.</a> Cancer Metastasis Rev. 17, 279-284 (1998).</li><li>Kim, S., 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+model+of+anaplastic+thyroid+carcinoma+in+athymic+nude+mice.">An orthotopic model of anaplastic thyroid carcinoma in athymic nude mice.</a> Clin. Cancer Res. 11, 1713-1721 (2005).</li><li>Nucera, C., 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+novel+orthotopic+mouse+model+of+human+anaplastic+thyroid+carcinoma.">A novel orthotopic mouse model of human anaplastic thyroid carcinoma.</a> Thyroid. 19, 1077-1084 (2009).</li><li>Todaro, 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=Tumorigenic+and+metastatic+activity+of+human+thyroid+cancer+stem+cells.">Tumorigenic and metastatic activity of human thyroid cancer stem cells.</a> Cancer Res. 70, 8874-8885 (2010).</li><li>Tran Cao, H. S., 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=Real-time+imaging+of+tumor+progression+in+a+fluorescent+orthotopic+mouse+model+of+thyroid+cancer.">Real-time imaging of tumor progression in a fluorescent orthotopic mouse model of thyroid cancer.</a> Anticancer Res. 30, 4415-4422 (2010).</li><li>Marlow, L. A., 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=Detailed+molecular+fingerprinting+of+four+new+anaplastic+thyroid+carcinoma+cell+lines+and+their+use+for+verification+of+RhoB+as+a+molecular+therapeutic+target.">Detailed molecular fingerprinting of four new anaplastic thyroid carcinoma cell lines and their use for verification of RhoB as a molecular therapeutic target.</a> J. Clin. Endocrinol. Metab. 95, 5338-5347 (2010).</li><li>Nehs, M. A., 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=Late+Intervention+with+anti-BRAF+(V600E)+therapy+induces+tumor+regression+in+an+orthotopic+mouse+model+of+human+anaplastic+thyroid+cancer.">Late Intervention with anti-BRAF (V600E) therapy induces tumor regression in an orthotopic mouse model of human anaplastic thyroid cancer.</a> Endocrinology. 153, 985-994 (2012).</li><li>Nucera, C., 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=Targeting+BRAFV600E+with+PLX4720+displays+potent+antimigratory+and+anti-invasive+activity+in+preclinical+models+of+human+thyroid+cancer.">Targeting BRAFV600E with PLX4720 displays potent antimigratory and anti-invasive activity in preclinical models of human thyroid cancer.</a> Oncologist. 16, 296-309 (2011).</li></ol>

著者らは、開示することは何もありません。

我々のモデルでは、動物は、4週間後噴射によって悪液質および呼吸窮迫に関連甲状腺腫瘍転移および疾患を示した。細胞懸濁液の注射可能な送達を利用して他のほとんどの同所性モデルと同様に、周囲の組織への注射及び目標を超えて広がるポスト噴射の漏れが可能である。それが言われていると、この手順で生成1認識潜在不明、非疾病関連の転移や合併症へのオフターゲット暴露の効果である。これは関係なく、調査されて、病気の、ほとんどの異種移植移植の一般的な関心事である。それにもかかわらず、転移の意図しない結果が有意にぴったりと甲状腺を覆う膜を貫通した針先端の全体のベベルを確保し、徐々に細胞懸濁液を排出することによって最小化することができる。漏れが注入中または後に観察された場合、それらの動物を研究から省略してもよい。 <p c少女= &quot;jove_content&quot;&gt;それは動物が甲状腺/気管や他の組織を採取する前に灌流されることが不可欠である。利点は、組織形態と整合性が保持されます）赤血球がパージされ、組織標本と2を乱雑にしません）、1の二倍である。さらに、どちらかの腫瘍塊が添付されているか、その中に埋葬されて切除甲状腺/気管、解剖学的ランドマークは、組織学的準備のために埋め込まれる前に識別することができるような方法でカットする必要があります。これは劇的に標本作製時間を短縮できます。 本稿では、ATC細胞の同所注射後高度甲状腺腫瘍の局所成長および浸潤を記述します。 ATC転移は、ヘマトキシリン​​及びエオシンなどの標準的な組織学的汚れを使用して、他の組織の組織学的調製物を分析または免疫組織化学によって細胞または病理学関連マーカーを利用することによって調べることができる。このシステムはさらに、報告して開発することができるリアルタイム画像は、転移の治療処置の有効性をモニターするために使用することができるようにenetically蛍光記者と甲状腺癌細胞を修正する。

<table border="1">
 <tbody>
 <tr>
 <td>Name of Reagent/Material</td>
 <td>Company</td>
 <td>Catalogue Number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>RPMI 1640 cell culture media</td>
 <td>Mediatech</td>
 <td>10-041-CV</td>
 <td>Media and additives used depend on cell line</td>
 </tr>
 <tr>
 <td>TC10 Automated Cell Counter</td>
 <td>Bio-Rad</td>
 <td>145-0001</td>
 <td></td>
 </tr>
 <tr>
 <td>Matrigel</td>
 <td>Becton, Dickinson and Company</td>
 <td>354234</td>
 <td></td>
 </tr>
 <tr>
 <td>Graefe Forceps, Serrated; Slight Curve, 4"</td>
 <td>Roboz</td>
 <td>RS-5135</td>
 <td></td>
 </tr>
 <tr>
 <td>Disposable scalpel, No. 15</td>
 <td>Feather</td>
 <td>2975#15</td>
 <td></td>
 </tr>
 <tr>
 <td>Olsen Hegar Needle Holder - 4 &frac12;" delicate serrated</td>
 <td>gSource</td>
 <td>gS 21.5400</td>
 <td></td>
 </tr>
 <tr>
 <td>6-0 nylon black monofilament suture</td>
 <td>Surgical Specialties Corporation</td>
 <td>1279B</td>
 <td></td>
 </tr>
 <tr>
 <td>Heating Pad, reusable, 8" x 12"</td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Cloth tape, 1" X 10 yds</td>
 <td>Medline</td>
 <td>MIINON260101</td>
 <td>For restraining anesthetized mouse</td>
 </tr>
 <tr>
 <td>Peanut Clipper/Trimmer</td>
 <td>Wahl</td>
 <td>8655</td>
 <td>For removing hair from surgical site</td>
 </tr>
 <tr>
 <td>ChlorHex-Q SCRUB 2%</td>
 <td>Penn Veterinary Supply</td>
 <td>VED1222</td>
 <td></td>
 </tr>
 <tr>
 <td>Betadine</td>
 <td>Henry Schein Company</td>
 <td>6906727</td>
 <td></td>
 </tr>
 <tr>
 <td>Petrolatum ophthalmic ointment</td>
 <td>Dechra Veterinary Products</td>
 <td>17033-211-38</td>
 <td></td>
 </tr>
 <tr>
 <td>2 x 2 gauze, 12-ply</td>
 <td>Butler Animal Health Supply</td>
 <td>6936</td>
 <td></td>
 </tr>
 <tr>
 <td>Sterile Field, Barrier, 18" x 26"</td>
 <td>Busse</td>
 <td>696</td>
 <td></td>
 </tr>
 <tr>
 <td>Drapes (CSR Wrap)</td>
 <td>Cardinal Health</td>
 <td>AT 21 412</td>
 <td></td>
 </tr>
 <tr>
 <td>27G &frac12;" needle</td>
 <td>Becton, Dickinson and Company</td>
 <td>309623</td>
 <td></td>
 </tr>
 <tr>
 <td>31G 5/16" needle</td>
 <td>Becton, Dickinson and Company</td>
 <td>328438</td>
 <td></td>
 </tr>
 <tr>
 <td>Ketamine (9 mg/ml) / xylazine (1 mg/ml) solution</td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Triple antibiotic ointment</td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 </tbody>
</table>

an orthotopic mouse model of anaplastic thyroid carcinoma

ヒト甲状腺癌の動物モデルのいくつかのタイプは、免疫不全マウスへの癌細胞の皮下異種移植片と同所移植を含む、確立されている。皮下異種移植モデルは、前臨床スクリーニングおよび新規治療処置を評価するために貴重であった。皮下モデルを使用する多くの利点があり、1）急速、2）再現性、及び3）腫瘍の確立、成長、および治療剤に対する応答は、目視検査によって監視することができる。しかし、実質的な証拠は1-3皮下異種移植モデルの短期出入りに光を当てました。例えば、皮下異種移植モデルで治療特性を示す薬用治療は、多くの場合、人間の病気には顕著な影響を及ぼさない。異種移植移植または注射部位の微小環境は、この相違の中心に位置しています。 同所性腫瘍の異種移植モデルは、よりbioloを提供病気を研究する中でgically関連コンテキスト。宿主動物内での解剖学的起源と同等に病気の細胞や組織を移植することの利点は、適当な腫瘍 - 宿主相互作用のためのサイト、疾患関連転移の開発と治験治療の救済にサイト固有の影響を調べるための機能が含まれています。彼らは密接に人間の病気を再現するので、したがって、同所移植モデルは、はるかに多くの臨床的価値を抱く。これらの理由から、グループの数は、研究ツールとして4-7同所性甲状腺癌モデルを利用している。 ここでは、高度に侵襲的で未分化甲状腺癌（ATC）の研究のため同所モデルを確立し、治療に抵抗し、すべての診断された患者に事実上致命的であるアプローチを記述します。培養された細胞は、ATC基底膜マトリックスを含む溶液中で解離し、細胞懸濁液として調製される。小音量がゆっくりINJです右甲状腺にected。マウスの全体的な外観と健康最小限の術後合併症を確保し、癌の病理学的浸透度を測定するために監視される。マウスは、4週間後に屠殺し、そして組織学的分析のために回収される。動物は、疾患のより高度な進行を調べるために後での時点で採取してもよい。当社ATC病理の理解、および有効性のための2）の画面の現在および将来の治療薬ATCとの闘いでさらに1）：この同所マウスモデルの生産は、二つの目的を達成するプラットフォームを確立します。

私たちは、4週間後にATC細胞を注射したマウス20の合計から19浸潤性甲状腺腫瘍を検出しました。 図2Aに示す例では、添付された条件下で増殖5X10 5 ATC細胞の同所注射を受けたマウスは、4週間後噴射による甲状腺に癌細胞の有意な浸潤を示す。好酸性細胞質と大きな核を有する特性spindled細胞と中〜巨大サイズの細胞と侵入ATC細胞の性質は、ヘマトキシリン​​およびエオシン（H＆E）染色により確認した。比較のため、非注入された対照動物から甲状腺及び気管は、 図2Bに示されている。非注入動物の卵胞が近く、まだ緩い、関連付けを表示し、基本的に丸い形態を有する。 注射可能な送達の甲状腺および使用のサイズが小さいため、意図しない結果が生じる可能性がある。 図3Aは、例示する最も一般的に観察された、それは外の腫瘤の開発ですが、甲状腺を包含しない。細胞が排出されるとき、これは甲状腺を通じて以降針浸透に起因する可能性が高い以上のものです。 図3Bに示すように、時折、無腫瘍の成長、両方の肉眼および組織学的に、と提示したマウスが検出されています。検出可能な腫瘍発生の有無は、隣接組織およびキャビティへの注入部位と普及からの細胞懸濁液の放出によって引き起こされることができる。 <img src="/files/ftp_upload/50097/50097fig1.jpg" alt="図1"/> 図1。甲状腺を公開する。ラベルは、気管に隣接する左右の甲状腺を示す。 <img src="/files/ftp_upload/50097/50097fig2.jpg" alt="図2"/> 図2。腫瘍増殖のとで組織学的検査成功した甲状腺注射後vasion。組織標本は、前頭面に沿って切片。好酸性細胞質とヘマトキシリンおよびエオシンで大きな核（と特性spindled細胞と中〜巨大サイズのセルと実質的に腫瘍の成長を示すマウスの200Xで撮影）画像H＆E）は染色火は、腫瘍細胞が甲状腺に侵入し、そこから原発巣を示すB）気管および対照動物から甲状腺（100Xで撮影した画像） 火、腫瘍;。。。S、平滑筋、Trは 、気管、 汝 、甲状腺。 <img src="/files/ftp_upload/50097/50097fig3.jpg" alt="図3"/> 図3。同所注射の意図しない結果。組織標本は、前頭面に沿って区分された。）周辺腫瘍増殖thyroiで明らかにしない成長D（黒い矢印、100Xで撮影された画像）。B）総時腫瘍検出（図示せず）又は組織学的検査の欠如（50倍 ​​で撮影した画像）。 汝 、甲状腺。

Watch this Scientific Journal Video about An Orthotopic Mouse Model of Anaplastic Thyroid Carcinoma at JoVE.com

An Orthotopic Mouse Model of Anaplastic Thyroid Carcinoma

甲状腺未分化癌の同所性マウスモデルの生成は、ここで記載されている。この技術は、このように、疾患の進行、並びにスクリーン革新的な治療的介入を研究するための、より臨床的に関連する設定を作成し、免疫不全マウスの甲状腺にヒト甲状腺未分化癌細胞の外科的な配置を採用している。

甲状腺未分化癌の同所性マウスモデル

Several types of animal models of human thyroid carcinomas have been established, including subcutaneous xenograft and orthotopic implantation of cancer ...

an-orthotopic-mouse-model-of-anaplastic-thyroid-carcinoma

Research

JoVE Journal

Medicine

21.0K ビュー.  Saint Louis University School of Medicine. 甲状腺未分化癌の同所性マウスモデルの生成は、ここで記載されている。この技術は、このように、疾患の進行、並びにスクリーン革新的な治療的介入を研究するための、より臨床的に関連する設定を作成し、免疫不全マウスの甲状腺にヒト甲状腺未分化癌細胞の外科的な配置を採用している。

ビデオ: An Orthotopic Mouse Model of Anaplastic Thyroid Carcinoma

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 ...

医学

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 ...

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 ...

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 ...

Bioluminescent Orthotopic Model of Pancreatic Cancer Progression

Pancreatic cancer has an extremely poor five-year survival rate of 4-6%. New therapeutic options are critically needed and depend on improved understanding of pancreatic cancer biology. To better understand the interaction of cancer cells with the pancreatic microenvironment, we demonstrate an orthotopic model of pancreatic cancer that permits non-invasive monitoring of cancer progression. Luciferase-tagged pancreatic cancer cells are resuspended in Matrigel and delivered into the pancreatic tail during laparotomy. Matrigel solidifies at body temperature to prevent leakage of cancer cells during injection. Primary tumor growth and metastasis to distant organs are monitored following injection of the luciferase substrate luciferin, using in vivo imaging of bioluminescence emission from the cancer cells. In vivo imaging also may be used to track primary tumor recurrence after resection. This orthotopic model is suited to both syngeneic and xenograft models and may be used in pre-clinical trials to investigate the impact of novel anti-cancer therapeutics on the growth of the primary pancreatic tumor and metastasis.

Improved understanding of pancreatic cancer biology is critically needed to enable the development of better therapeutic options to treat pancreatic cancer. To address this need, we demonstrate an orthotopic model of pancreatic cancer that permits non-invasive monitoring of cancer progression using in vivo bioluminescence imaging.

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 ...

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  ...

An Orthotopic Glioblastoma Mouse Model Maintaining Brain Parenchymal Physical Constraints and Suitable for Intravital Two-photon Microscopy

Glioblastoma multiforme (GBM) is the most aggressive form of brain tumors with no curative treatments available to date.
Murine models of this pathology rely on the injection of a suspension of glioma cells into the brain parenchyma following incision of the dura-mater. Whereas the cells have to be injected superficially to be accessible to intravital two-photon microscopy, superficial injections fail to recapitulate the physiopathological conditions. Indeed, escaping through the injection tract most tumor cells reach the extra-dural space where they expand abnormally fast in absence of mechanical constraints from the parenchyma.
Our improvements consist not only in focally implanting a glioma spheroid rather than injecting a suspension of glioma cells in the superficial layers of the cerebral cortex but also in clogging the injection site by a cross-linked dextran gel hemi-bead that is glued to the surrounding parenchyma and sealed to dura-mater with cyanoacrylate. Altogether these measures enforce the physiological expansion and infiltration of the tumor cells inside the brain parenchyma. Craniotomy was finally closed with a glass window cemented to the skull to allow chronic imaging over weeks in absence of scar tissue development.
Taking advantage of fluorescent transgenic animals grafted with fluorescent tumor cells we have shown that the dynamics of interactions occurring between glioma cells, neurons (e.g. Thy1-CFP mice) and vasculature (highlighted by an intravenous injection of a fluorescent dye) can be visualized by intravital two-photon microscopy during the progression of the disease.
The possibility to image a tumor at microscopic resolution in a minimally compromised cerebral environment represents an improvement of current GBM animal models which should benefit the field of neuro-oncology and drug testing.

We have established a cortical orthotopic glioblastoma model in mice for intravital two-photon microscopy that recapitulates the biophysical constraints normally at play during the growth of the tumor. A chronic glass window replacing the skull above the tumor enables the follow-up of the tumor progression over time by two-photon microscopy.

同所神経膠芽腫モデルマウスは、脳実質細胞の物理的制約を維持し、生体内二光子顕微鏡に適し

Glioblastoma multiforme (GBM) is the most aggressive form of brain tumors with no curative treatments available to date.
Murine models of this pathology ...

The In Ovo Chick Chorioallantoic Membrane (CAM) Assay as an Efficient Xenograft Model of Hepatocellular Carcinoma

The chick chorioallantoic membrane (CAM) begins to develop by day 7 after fertilization and matures by day 12. The CAM is naturally immunodeficient and highly vascularized, making it an ideal system for tumor implantation. Furthermore, the CAM contains extracellular matrix proteins such as fibronectin, laminin, collagen, integrin alpha(v)beta3, and MMP-2, making it an attractive model to study tumor invasion and metastasis. Scientists have long taken advantage of the physiology of the CAM by using it as a model of angiogenesis. More recently, the CAM assay has been modified to work as an in vivo xenograft model system for various cancers that bridges the gap between basic in vitro work and more complex animal cancer models. The CAM assay allows for the study of tumor growth, anti-tumor therapies, and pro-tumor molecular pathways in a biologically relevant system that is both cost- and time-effective. Here, we describe the development of CAM xenograft model of hepatocellular carcinoma (HCC) with embryonic survival rates of up to 93% and reliable tumor take leading to growth of three-dimensional, vascularized tumors.

The In Ovo Chick Chorioallantoic Membrane CAM Assay as an Efficient Xenograft Model of Hepatocellular Carcinoma

The chick chorioallantoic membrane (CAM) is immunodeficient and highly vascularized, making it a natural in vivo model of tumor growth and angiogenesis. In this protocol, we describe a reliable method of growing three-dimensional, vascularized hepatocellular carcinoma (HCC) tumors using the CAM assay.

ザ・<em&gt;卵内</em肝細胞癌の効率的な異種移植モデルとして&gt;ニワトリ絨毛尿膜（CAM）アッセイ

The chick chorioallantoic membrane (CAM) begins to develop by day 7 after fertilization and matures by day 12. The CAM is naturally immunodeficient and ...

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.