我々は、この手順の開発で技術的なヘルプと専門知識のためのロバートS.カーベル（トロント大学、サニーブルック研究所、トロント、カナダ）の研究室に感謝しています。また、博士はサンドラ·セクストンや実験動物資源のロズウェルパークがん研究所部門に感謝したいと思います。この作品は、（JMLEへ）ロズウェルパークアライアンス財団から賞によってサポートされていました。

1。同所腎臓腫瘍移植<ol><li>細胞培養<ol><li>同所移植の前に、75％コンフルエントに単層としてマウスRENCA LUC細胞を成長させる。 </li><li> 5％FBS含有培地1,000 rpmで遠心分離細胞、5分間、4℃、PBS中で洗浄することを3回繰り返すことで、トリプシン処理および再懸濁以下。その後、5×10 4個のRENCA LUC / 5μlの無血清培地の濃度に無血清培地で細胞を懸濁します。 注：使用される変異体または細胞株に依存して、細胞が移植後に非常に攻撃的なマウス細胞増殖及び播種を遅らせる1:1マトリゲルとの比を無血清培地に再懸濁することができる。 </li></ol></li><li>手術と細胞移植 注：すべての動物実験は、施設内動物管理使用委員会（IACUC）Pに従って行った、実験的なエンドポイントの維持決定を含む、そこに記載されているロズウェルパークがん研究所によって承認rotocol。 <ol><li>イソフルランを用いたもののBalb / Cマウス（2-3％）で麻酔。足をつまんで、十分な麻酔を確保するために、反射的に確認。乾燥を防ぐために、目に獣医軟膏を適用します。右横臥位にマウスを置き、前後と後肢の間で左側を剃る。無菌的に切開するための領域を準備するために背側腰部領域に沿ってアルコールとヨウ素を適用します。 </li><li>最後の肋骨と股関節の間の縦方向に1cmの皮膚切開を開始するために、外科はさみを使用してください。鈍的切開のハサミを用いて皮膚の下に結合組織を緩めます。腹壁内の長手方向に0.5センチの切開を作るために、外科はさみを使用してください。そっと開いた傷口の周りにプッシュダウンする湾曲したピンセットを使用する - これは、腎臓を体外にし、注射前に臓器の穏やかな固定化を可能にする。 </li><li>調製した細胞混合物μlの5でハミルトンシリンジをロードします。サブ莢膜implan用tationsは、2つの埋め込み方法を用いることができる。 <ol><li>浅：縦方向に向い腎臓に平行して走り、、斜めの縁を上にして腎臓​​被膜下（しかし、実質上の）針を挿入します。針が配置されると、小さな白い泡が形成されるまで細胞の全てを注入する。ゆっくりとカプセルから針を外し、すぐに漏れを吸収し、細胞播種を防止するために、滅菌綿棒で注射にわたってブロット。 </li><li>傾斜端を上にして、腎臓の反対側から、最終的な移植部位に始まり、針は（パンクチャリングではなく）表示されるまで、腎臓の内部を通って針を挿入し、サブ莢スペース：内部。任意の漏れを防ぐために白い気泡フォームおよびスワブ注射部位まで、細胞の全てを注入する。 </li></ol></li><li>優しく体腔への腎臓を戻す。吸収性5-0ビクリル縫合糸を使用して腹部の体壁を閉じます。完了したら、一緒にスキン層を引き、wは2-3を配置クリップをound。クリップがしっかりと均等に全く傷の露出を確保しないように間隔をあけていることを確認してください。リカバリ前の0.9％NaCl500μlの皮下25 Gの針を用いてブプレノルフィン（0.01 mg / ml）を100μlの管理。日常のクリップの配置と安定性を監視し、10日後に削除します。 </li></ol></li></ol> 2。腎摘出/原発腫瘍の除去<ol><li>のセクション1.2.1と1.2.2で説明したように（上）マウス麻酔、固定化、切開し、腎臓の露出のための同一のプロトコールに従ってください。 </li><li>優しく尾方端から接続して脂肪組織、腎臓の頭側端から副腎を除去することによって、腎臓を分離するために鉗子の第二の対を使用してください。優しく腎臓を把握しながら、尿管、腎動脈、静脈を二重結び目を作るために、吸収性5-0ビクリル縫合糸を使用しています。ゆっくり確保してから腎臓を取り除くために、結び目の上にカット。すぐに血液の損失の兆候あれば、あるいは不十分なホールドの危険性がある尿管、動脈、静脈に縫合糸は、結び目の上にカットする代わりに、はさみを焼灼器を使用しています。 </li><li>腎臓が除去されると、慎重に縛ら動脈から出血を確認し、必要に応じて焼灼器を使用しています。 5-0ビクリルを使用して腹部の体壁を閉じます。一緒にスキン層を引き、1.2.4で説明したのと同様の注意事項を次の2〜3創傷クリップを配置します。密接に出血苦痛の徴候、あるいは限られた移動性のための手術後、毎日動物を監視します。安全に動物のための施設のガイドラインに従ってください。 </li></ol>エンドポイントで3。のモニタリング転移進行とローカリゼーション<ol><li>転移性疾患の定量化：生物発光モニタリング<ol><li>方法論は、以前にルシフェラーゼをトランスフェクトした細胞を用いて、転移をモニタリングするためJOVEに記載のゼノジェンIVISイメージングシステム11,12を用いて定量見る。 </li></ol></li><li>転移性分布の比較評価のための視覚的剖検ガイドのTiON <ol><li>犠牲の動物の苦痛の兆候、呼吸困難、体重減少などを含めたエンドポイントを使用して施設のガイドラインに従っ 注：自発的な転移性の進行が非常に変化し、急速なように、動物のクローズ監視は重要です。 </li><li>下顎骨に胸腔を続けて尿道上記切開を開始するために、外科はさみを使用してください。腹壁の筋肉から皮膚を分離するために、平滑末端はさみを使用してください。 </li><li>腕と脚に沿って切開を開始します。皮の皮離れて。密接に結節のための皮膚筋膜と腹壁を調べ、その後、表在リンパ節：鼠径、上腕、腋窩および表面的な子宮頸。 </li><li>腹壁の切開を行い、横隔膜までの左右の側面に沿って切断。 1水平カットと内臓を露出するために腹壁の前部を削除します。 </li><li>内臓fの邪魔されずに外観を調べまたは著しく顕著な腫瘍が。また、胃や腸の内容に注意してください。彼らが空の場合、マウスは食べることができなかった可能性があります。コンテンツが暗い場合、これは、上部/下部胃腸出血の証拠を示すことができる。 </li><li>視覚的に各マウス群の合計スコアを割り当てること、腫瘍の有無を各臓器のスコア（各群4匹の動物が図1D-iの中で例として使用される）。 注：1）リンパ節の拡大が必ずしもそれが腫瘍の成長であることを意味するものではありません。それは、大幅にサイズの非常にしっかりと、白っぽい色であれば、それは可能性が高い転移性結節である。 2）腹部の過剰な乳白色や曇り液体が腹水を示します。腹水は、特に肝臓と胃のローブ間腹部臓器に固体の蓄積を含むことができる。 3）肺などの器官のために、多数の小結節（例えば13を参照）に存在し、個々の動物間の比較にカウントすることができる。 </li><li>慎重に取り外し、噴出ボトルから、PBSで洗浄することにより、肝臓、脾臓、腎臓、膵臓：個々の腹部臓器の肉眼的腫瘍や異常を特定します。 </li><li>腹部臓器を除去した後、腰椎、仙骨、腎臓、および腸間膜リンパ節を含む腹部のリンパ節を検査する。 </li><li>転移拡散の証拠ダイアフラムを評価します。 注：ダイヤフラムに関する広範な普及はこじつけや呼吸の喪失につながる、それはその収縮性を失う可能性があります。 </li><li>ダイアフラムを外し、胸腔内に2の横切開を行い、肺と心臓を露出させ、正面部分を除去する。 </li><li>そっとつかんで持ち上げ、食道を、心臓の上に気管を切断することにより、肺と心臓を取り出します。結節のための組織を評価する。心を絞る。弾力の一貫性の欠如は、腫瘍の存在を示すことがあります。 </li><li>頭蓋骨を通して直線的にカットする外科はさみを使用してください。優しくトンまで頭蓋の小片を取り除く彼は、全脳を露出させる。目に見える結節や異常変色のために脳全体に見て。 </li></ol></li></ol>

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著者らは、開示することは何もありません。

このプロトコルの目的は、注入/切除技法を説明するために同系腫瘍マウスモデルを用いて、臨床的に関連する自発的な転移性疾患を評価することである。現在、新たな実験的な治療法を評価する前臨床試験の大半は、転移性疾患の研究が含まれていないと、わずか数は、原発腫瘍の増殖、外科的切除、および最終的な自発的な転移拡散の段階を再現。現在までに、遺伝子操作したマウスモデ...

腎細胞癌（RCC）を有する患者における死亡の主な原因は、典型的には、腎臓中で増殖する原発腫瘍の外科的切除後に生じる全身性の転移性疾患である。しかし、マウスでは実験的な治療法を評価する非常に少数の前臨床腫瘍モデルは、転移性疾患が含まれており、少数の、まだ忠実にローカライズされた成長、手術、および自発的なマイクロ転移開始および進行1-3の臨床病期を再現。テスト中のこのギャップは時々 、動物モデルで見られる顕著な抗腫瘍効果は、常に患者4の同様に治療の成功に翻訳されていない新しい治療法の評価においてますます重要になってきた。結果におけるこのような差異は、ローカライズされた異所性または同所原発腫瘍モデルと後期転移性疾患5-7間の差動薬効に由来する。 RCCの場合には、少数の研究のみが確立採用している一般的に、腫瘍を有する腎臓を受けた患者は全体的または部分的に2,3を削除模倣自発的再発が含まニマルプロトコル。マウスモデルのテストで、この不足の理由は様々である。まず、高コスト動物および腫瘍細胞の選択および転移能の固有の変動性が存在する。 （例えば、ヒト細胞株導出8-10の説明を参照）、例えば、ヒト腎細胞株はめったに転移していない傾向があり、一貫し普及および遠隔病変を形成する変異体を導出するために同所一次注入及び転移複数回の選択にわたって選択されなければならない。逆に、免疫応答性モデルでマウスの細胞では積極的に行動する傾向があり、低細胞数はすぐに全身スプレッド3を減らすためにマトリゲルを注入しなければならない。第二に、技術的な適切な注入、外科的切除（摘出術）を実施し、追跡の困難（かつ定量）自発metastatiCの成長は挑戦することができますし、（詳細については説明を参照）、この手法を採用する場合、いくつかの重要な変数は、検討が必要です。このプロトコルの目的は、同所移植、切除（腎摘出）、および自発的な転移性腎細胞癌疾患のモニタリングの重要なステップ（および潜在的な落とし穴）を記述するために、標準化され（そしてより広範な）のための指針を提供するために科学的な研究室の中で使用しているそれは、実験的治療法の有効性を評価する。

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			<td height="21" style="height:21px;width:397px;">DMEM-high glucose with Pyr. And L-Glutamine</td>
			<td style="width:147px;">Corning</td>
			<td style="width:119px;">10-013-CV</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:397px;">FBS</td>
			<td style="width:147px;">Invitrogen</td>
			<td style="width:119px;">10437-028</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:397px;">0.25% Trypsin EDTA</td>
			<td style="width:147px;">Corning</td>
			<td style="width:119px;">25-053-CL</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:397px;">1x DPBS without Calcium &#38; Magnesium</td>
			<td style="width:147px;">Corning</td>
			<td style="width:119px;">21-031-CV</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:397px;">Matrigel</td>
			<td style="width:147px;">BD Biosciences</td>
			<td style="width:119px;">354234</td>
			<td>must be kept on ice</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:397px;">Artifical tears-lubricant opthalmic ointment</td>
			<td style="width:147px;">Akorn Animal Health</td>
			<td style="width:119px;">17478-162-35</td>
			<td></td>
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			<td height="21" style="height:21px;width:397px;">Pocket pro pet trimmer</td>
			<td style="width:147px;">Braintree scientific</td>
			<td style="width:119px;">CLP9931B</td>
			<td></td>
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			<td height="24" style="height:24px;width:397px;">Alcohol swab</td>
			<td style="width:147px;">VWR</td>
			<td style="width:119px;">326895</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:397px;">Betadine solution swab</td>
			<td style="width:147px;">VWR</td>
			<td style="width:119px;">67618-152-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MICRO DISSECTING sissors straight,blunt - 25mm blades - 4.5&#34;&#160;</td>
			<td style="width:147px;">Southpointe surgical</td>
			<td style="width:119px;">RS-5982</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:397px;">Iris Forceps, serrated, curved, 10cm long</td>
			<td style="width:147px;">Kent scientific</td>
			<td style="width:119px;">INS15915</td>
			<td>need two of these</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:397px;">10&#181;l Hamilton syringe</td>
			<td style="width:147px;">Hamilton</td>
			<td style="width:119px;">7635-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:397px;">30G, 45 degree, RN needle</td>
			<td style="width:147px;">Hamilton</td>
			<td style="width:119px;">7803-07</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:397px;">Sterile cotton tipped appicator</td>
			<td style="width:147px;">VWR</td>
			<td style="width:119px;">10805-144</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:397px;">High temperature cautery kit</td>
			<td style="width:147px;">Kent scientific</td>
			<td style="width:119px;">INS500392</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:397px;">5-0 coated Vicryl, conventional cutting needle&#160;</td>
			<td style="width:147px;">Ethicon</td>
			<td style="width:119px;">J834</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:397px;">Reflex clip applier for 7mm clips</td>
			<td style="width:147px;">Kent scientific</td>
			<td style="width:119px;">INS500343</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:397px;">Reflex clips, 7mm, non-sterile</td>
			<td style="width:147px;">Kent scientific</td>
			<td style="width:119px;">INS500344</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:397px;">Removing forceps, 12cm lone</td>
			<td style="width:147px;">Kent scientific</td>
			<td style="width:119px;">INS500347</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:397px;">0.9% Sodium Chloride</td>
			<td style="width:147px;">Baxter Healthcare</td>
			<td style="width:119px;">2B1322</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:397px;">Buprenorphine 0.01mg/mL</td>
			<td style="width:147px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:397px;">25G 5/8&#34; needle</td>
			<td style="width:147px;">VWR</td>
			<td style="width:119px;">BD305122</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:397px;">1mL syringe w/out needle</td>
			<td style="width:147px;">VWR</td>
			<td style="width:119px;">BD309659</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:397px;">D-Luciferin</td>
			<td style="width:147px;">Gold Bio technology</td>
			<td style="width:119px;">LUCK-1G</td>
			<td></td>
		</tr>
	</tbody>
</table>

modeling spontaneous metastatic renal cell carcinoma (mrcc) in mice following nephrectomy

腎細胞癌（RCC）の新実験治療の改善されたテストの重要な課題の一つは、忠実に早期及び後期転移性疾患の進行を再現モデルの開発である。典型的な腫瘍移植モデルは、異所性または同所原発腫瘍移植を利用しますが、いくつかは、臨床設定を模倣全身自発転移性疾患が含まれる。このプロトコルは、患者さんに似腎細胞癌疾患の進行段階を開発するための重要な手順について説明します。まず、同所性腫瘍細胞移植を示すために同系モデルにおいて、高転移性マウス腫瘍細胞株を使用する。方法は、漏れや、早期の広がりを防ぐために、マトリゲルと組み合わせた細胞のサブ莢膜空間に表面的で、内部の移植が含まれています。その横には、重要な前と手術後のマウスの注意を払って、腫瘍を有する腎臓（腎摘出）の切除のための手順について説明します。最後に、監視し、評価するために必要な手順の概要を説明生物発光イメージングなどのミクロおよびマクロ転移性疾患の進行は、同様に全身性疾患の分布を獲得するための詳細な視覚的剖検ガイドを提供します。このプロトコル記述の目標は、将来の治療試験の予測値を改善するために臨床的に関連する転移性RCCモデルの普及を促進することである。

図1Aは、このプロトコルの概要で説明する手順を概説する回路図を示します。いくつかの重要な要因は、各ステップのために考慮されなければならない。たとえば、ステップ1で、それは、腎臓へのサブ莢腫瘍細胞移植のための二つの方法を示しています。腫瘍細胞は、針の慎重な除去と拭き取り過剰エスケープ流体（ 図1B-I）により防止漏れ細胞の局所的な配置を確...

Watch this Scientific Journal Video about Modeling Spontaneous Metastatic Renal Cell Carcinoma mRCC in Mice Following Nephrectomy at JoVE.com

Modeling Spontaneous Metastatic Renal Cell Carcinoma mRCC in Mice Following Nephrectomy

自発的な転移性腎細胞癌（RCC）、疾患進行のモデルは、臨床的に関連する設定で治療を評価するために使用することができる。このプロトコルは、同所腎臓腫瘍細胞移植、適切な腎摘出のためのさまざまな手順を示していて、最終的には転移性の負担とローカリゼーションの視覚的および生物発光採点のための剖検ガイドの概要を説明します。

腎摘出後のマウスにおける自発転移性腎細胞癌（MRCC）をモデル化する

One of the key challenges to improved testing of new experimental therapeutics in renal cell carcinoma (RCC) is the development of models that faithfully ...

modeling-spontaneous-metastatic-renal-cell-carcinoma-mrcc-mice

Research

JoVE Journal

Medicine

Modeling Spontaneous Metastatic Renal Cell Carcinoma (mRCC) in Mice Following Nephrectomy

16.4K ビュー.  Roswell Park Cancer Institute. 自発的な転移性腎細胞癌（RCC）、疾患進行のモデルは、臨床的に関連する設定で治療を評価するために使用することができる。このプロトコルは、同所腎臓腫瘍細胞移植、適切な腎摘出のためのさまざまな手順を示していて、最終的には転移性の負担とローカリゼーションの視覚的および生物発光採点のための剖検ガイドの概要を説明します。 

ビデオ: Modeling Spontaneous Metastatic Renal Cell Carcinoma mRCC in Mice Following Nephrectomy

Single Port Donor Nephrectomy

In 2007, Rane presented the first single port nephrectomy for a small non-functioning kidney at the World Congress of Endourology. Since that time, the use of single port surgery for nephrectomy has expanded to include donor nephrectomy. Over the next two years the technique was adopted for many others types of nephrectomies to include donor nephrectomy. We present our technique for single port donor nephrectomy using the Gelpoint device. We have successfully performed this surgery in over 100 patients and add this experience to our experience of over 1000 laparoscopic nephrectomies. With the proper equipment and technique, single port donor nephrectomy can be performed safely and effectively in the majority of live donors. We have found that our operative times and most importantly our transplant outcomes have not changed significantly with the adoption of the single port donor nephrectomy. We believe that single port donor nephrectomy represents a step forward in the care of living donors.

Single port laparoscopic surgery is changing the standard of care in surgical care like nothing since the laparoscopic technique was introduced 20 years ago. We present out technique of single port donor nephrectomy using the Gelpoint device. We have successfully performed this surgery in 100 patients.

シングルポートのドナー腎摘出

In 2007, Rane presented the first single port nephrectomy for a small non-functioning kidney at the World Congress of Endourology.  Since that time, the ...

医学

Modeling Stroke in Mice - Middle Cerebral Artery Occlusion with the Filament Model

Stroke is among the most frequent causes of death and adult disability, especially in highly developed countries. However, treatment options to date are very limited. To meet the need for novel therapeutic approaches, experimental stroke research frequently employs rodent models of focal cerebral ischaemia. Most researchers use permanent or transient occlusion of the middle cerebral artery (MCA) in mice or rats.
Proximal occlusion of the middle cerebral artery (MCA) via the intraluminal suture technique (so called filament or suture model) is probably the most frequently used model in experimental stroke research. The intraluminal MCAO model offers the advantage of inducing reproducible transient or permanent ischaemia of the MCA territory in a relatively non-invasive manner. Intraluminal approaches interrupt the blood flow of the entire territory of this artery. Filament occlusion thus arrests flow proximal to the lenticulo-striate arteries, which supply the basal ganglia. Filament occlusion of the MCA results in reproducible lesions in the cortex and striatum and can be either permanent or transient. In contrast, models inducing distal (to the branching of the lenticulo-striate arteries) MCA occlusion typically spare the striatum and primarily involve the neocortex. In addition these models do require craniectomy. In the model demonstrated in this article, a silicon coated filament is introduced into the common carotid artery and advanced along the internal carotid artery into the Circle of Willis, where it blocks the origin of the middle cerebral artery. In patients, occlusions of the middle cerebral artery are among the most common causes of ischaemic stroke. Since varying ischemic intervals can be chosen freely in this model depending on the time point of reperfusion, ischaemic lesions with varying degrees of severity can be produced. Reperfusion by removal of the occluding filament at least partially models the restoration of blood flow after spontaneous or therapeutic (tPA) lysis of a thromboembolic clot in humans.
In this video we will present the basic technique as well as the major pitfalls and confounders which may limit the predictive value of this model.

Filamentous occlusion of the Middle cerebral artery is a common model for studying ischemic stroke in mice.

マウスにおけるモデリングのストローク - フィラメントモデルと中大脳動脈閉塞

Stroke is among the most frequent causes of death and adult disability, especially in highly developed countries. However, treatment options to date are ...

Mammary Transplantation of Stromal Cells and Carcinoma Cells in C57BL/6J Mice

The influence of stromal cells, including fibroblasts on mammary tumor progression has been well documented through the use of mouse models, in particular through transplantation of stromal cells and epithelial cells in the mammary gland of mice. Current transplantation models often involve the use of immunocompromised mice due to the different genetic backgrounds of stromal cells and epithelial cells. Extracellular matrices are often used to embed the two different cell types for consistent cell-cell interactions, but involve the use of Matrigel or rat tail collagen, which are immunogenic substrates. The lack of functional T cells from immunocompromised mice prevents accurate assessment of stromal cells on mammary tumor progression in vivo, with important implications on drug development and efficacy. Moreover, immunocompromised mice are costly, hard to breed and require special care conditions. To overcome these obstacles, we have developed an approach to orthotopically transplant stromal cell and epithelial cells into mice from the same genetic background to induce consistent tumor formation. This system involves harvesting normal, carcinoma associated fibroblasts, PyVmT mammary carcinoma cells and collagen from donor C57BL/6J mice. The cells are then embedded in collagen and transplanted in the inguinal mammary glands of female C57BL/6J mice. Transplantation of PyVmT cells alone form palpable tumors 30-40 days post transplantation. Endpoint analysis at 60 days indicates that co-transplantation with fibroblasts enhances mammary tumor growth compared to PyVmT cells transplanted alone. While cells and matrix from C57BL/6J mice were used in these studies, the isolation of cells and matrix and transplantation approach may be applied towards mice from different genetic backgrounds demonstrating versatility. In summary, this system may be used to investigate molecular interactions between stromal cells and epithelial cells, and overcomes critical limitations in immunocompromised mouse models.

In this report, we demonstrate a system to isolate and culture donor cells from the mouse mammary gland, and orthotopically transplant these cells in recipient mice to analyze stromal: epithelial interactions during mammary tumor development.

C57BL/6Jマウスにおける間質細胞と癌細胞の乳腺移植

The influence of stromal cells, including fibroblasts on mammary tumor progression has been well documented through the use of mouse models, in particular ...

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 ...

Modeling Intracerebral Hemorrhage in Mice: Injection of Autologous Blood or Bacterial Collagenase

Spontaneous intracerebral hemorrhage (ICH) defines a potentially life-threatening neurological malady that accounts for 10-15% of all stroke-related hospitalizations and for which no effective treatments are available to date1,2. Because of the heterogeneity of ICH in humans, various preclinical models are needed to thoroughly explore prospective therapeutic strategies3. Experimental ICH is commonly induced in rodents by intraparenchymal injection of either autologous blood or bacterial collagenase4. The appropriate model is selected based on the pathophysiology of hemorrhage induction and injury progression. The blood injection model mimics a rapidly progressing hemorrhage. Alternatively, bacterial collagenase enzymatically disrupts the basal lamina of brain capillaries, causing an active bleed that generally evolves over several hours5. Resultant perihematomal edema and neurofunctional deficits can be quantified from both models. In this study, we described and evaluated a modified double injection model of autologous whole blood6 as well as an ICH injection model of bacterial collagenase7, both of which target the basal ganglia (corpus striatum) of male CD-1 mice. We assessed neurofunctional deficits and brain edema at 24 and 72 hr after ICH induction. Intrastriatal injection of autologous blood (30 &mu;l) or bacterial collagenase (0.075U) caused reproducible neurofunctional deficits in mice and significantly increased brain edema at 24 and 72 hr after surgery (p&lt;0.05). In conclusion, both models yield consistent hemorrhagic infarcts and represent basic methods for preclinical ICH research.

Clinically relevant animal models of intracerebral hemorrhage (ICH) are needed to extend our knowledge of hemorrhagic stroke and to examine novel therapeutic strategies. In this study, we describe and evaluate two ICH models that implement unilateral injections of either autologous whole blood or bacterial collagenase into the basal ganglia (corpus striatum) of mice.

マウスにおけるモデリング脳出血：自己血または細菌性コラゲナーゼの注入

Spontaneous intracerebral hemorrhage (ICH) defines a potentially life-threatening neurological malady that accounts for 10-15% of all stroke-related ...

Generation of Subcutaneous and Intrahepatic Human Hepatocellular Carcinoma Xenografts in Immunodeficient Mice

In vivo experimental models of hepatocellular carcinoma (HCC) that recapitulate the human disease provide a valuable platform for research into disease pathophysiology and for the preclinical evaluation of novel therapies. We present a variety of methods to generate subcutaneous or orthotopic human HCC xenografts in immunodeficient mice that could be utilized in a variety of research applications. With a focus on the use of primary tumor tissue from patients undergoing surgical resection as a starting point, we describe the preparation of cell suspensions or tumor fragments for xenografting. We describe specific techniques to xenograft these tissues i) subcutaneously; or ii) intrahepatically, either by direct implantation of tumor cells or fragments into the liver, or indirectly by injection of cells into the mouse spleen. We also describe the use of partial resection of the native mouse liver at the time of xenografting as a strategy to induce a state of active liver regeneration in the recipient mouse that may facilitate the intrahepatic engraftment of primary human tumor cells. The expected results of these techniques are illustrated. The protocols described have been validated using primary human HCC samples and xenografts, which typically perform less robustly than the well-established human HCC cell lines that are widely used and frequently cited in the literature. In comparison with cell lines, we discuss factors which may contribute to the relatively low chance of primary HCC engraftment in xenotransplantation models and comment on technical issues that may influence the kinetics of xenograft growth. We also suggest methods that should be applied to ensure that xenografts obtained accurately resemble parent HCC tissues.

Human tumor xenografts in immunodeficient mice are valuable tools to study cancer biology. Specific protocols to generate subcutaneous and intrahepatic xenografts from human hepatocellular carcinoma cells or tumor fragments are described. Liver regeneration induced by partial hepatectomy in recipient mice is presented as a strategy to facilitate intrahepatic engraftment.

免疫不全マウスの皮下および肝内ヒト肝細胞癌異種移植片の生成

In  vivo experimental models of hepatocellular carcinoma (HCC) that recapitulate  the human disease provide a valuable platform for research into disease  ...

Induction of Invasive Transitional Cell Bladder Carcinoma in Immune Intact Human MUC1 Transgenic Mice:  A Model for Immunotherapy Development

A preclinical model of invasive bladder cancer was developed in human mucin 1 (MUC1) transgenic (MUC1.Tg) mice for the purpose of evaluating immunotherapy and/or cytotoxic chemotherapy. To induce bladder cancer, C57BL/6 mice (MUC1.Tg and wild type) were treated orally with the carcinogen N-butyl-N-(4-hydroxybutyl)nitrosamine (OH-BBN) at 3.0 mg/day, 5 days/week for 12 weeks. To assess the effects of OH-BBN on serum cytokine profile during tumor development, whole blood was collected via submandibular bleeds prior to treatment and every four weeks. In addition, a MUC1-targeted peptide vaccine and placebo were administered to groups of mice weekly for eight weeks. Multiplex fluorometric microbead immunoanalyses of serum cytokines during tumor development and following vaccination were performed. At termination, interferon gamma (IFN-&#947;)/interleukin-4 (IL-4) ELISpot analysis for MUC1 specific T-cell immune response and histopathological evaluations of tumor type and grade were performed. The results showed that: (1) the incidence of bladder cancer in both MUC1.Tg and wild type mice was 67%; (2) transitional cell carcinomas (TCC) developed at a 2:1 ratio compared to squamous cell carcinomas (SCC); (3) inflammatory cytokines increased with time during tumor development; and (4) administration of the peptide vaccine induces a Th1-polarized serum cytokine profile and a MUC1 specific T-cell response. All tumors in MUC1.Tg mice were positive for MUC1 expression, and half of all tumors in MUC1.Tg and wild type mice were invasive. In conclusion, using a team approach through the coordination of the efforts of pharmacologists, immunologists, pathologists and molecular biologists, we have developed an immune intact transgenic mouse model of bladder cancer that expresses hMUC1.

An N-butyl-N-(4-hydroxybutyl)nitrosamine-induced bladder cancer model was developed in human mucin 1 (MUC1) transgenic mice for the purpose of testing MUC1-directed immunotherapy. After administering a MUC1-targeted peptide vaccine, a cytotoxic T lymphocyte response to MUC1 was confirmed by measuring serum cytokine levels and T-cell specific activity.

免疫無傷のヒトMUC1トランスジェニックマウスにおける侵襲移行細胞膀胱癌の誘導：免疫療法の開発のためのモデル

A preclinical model of invasive bladder cancer was developed in human mucin 1 (MUC1) transgenic (MUC1.Tg) mice for the purpose of evaluating immunotherapy ...

Initiation of Metastatic Breast Carcinoma by Targeting of the Ductal Epithelium with Adenovirus-Cre: A Novel Transgenic Mouse Model of Breast Cancer

Breast cancer is a heterogeneous disease involving complex cellular interactions between the developing tumor and immune system, eventually resulting in exponential tumor growth and metastasis to distal tissues and the collapse of anti-tumor immunity. Many useful animal models exist to study breast cancer, but none completely recapitulate the disease progression that occurs in humans. In order to gain a better understanding of the cellular interactions that result in the formation of latent metastasis and decreased survival, we have generated an inducible transgenic mouse model of YFP-expressing ductal carcinoma that develops after sexual maturity in immune-competent mice and is driven by consistent, endocrine-independent oncogene expression. Activation of YFP, ablation of p53, and expression of an oncogenic form of K-ras was achieved by the delivery of an adenovirus expressing Cre-recombinase into the mammary duct of sexually mature, virgin female mice. Tumors begin to appear 6 weeks after the initiation of oncogenic events. After tumors become apparent, they progress slowly for approximately two weeks before they begin to grow exponentially. After 7-8 weeks post-adenovirus injection, vasculature is observed connecting the tumor mass to distal lymph nodes, with eventual lymphovascular invasion of YFP+ tumor cells to the distal axillary lymph nodes. Infiltrating leukocyte populations are similar to those found in human breast carcinomas, including the presence of &#945;&#946; and &#947;&#948; T cells, macrophages and MDSCs. This unique model will facilitate the study of cellular and immunological mechanisms involved in latent metastasis and dormancy in addition to being useful for designing novel immunotherapeutic interventions to treat invasive breast cancer.

Activation of latent mutations with adenovirus-Cre into the mammary ductal system results in a clinically relevant metastatic breast cancer. Incorporation of a YFP promoter allows tracking of distal metastatic tumor cells. This model is useful to study latent metastasis, anti-tumor immunity, and for designing novel immunotherapies to treat breast cancer.

乳癌の新規トランスジェニックマウスモデル：アデノ-Creを持つ乳管上皮の標的とすることにより、転移性乳癌の開始

Breast cancer is a heterogeneous disease involving complex cellular interactions between the developing tumor and immune system, eventually resulting in ...

Modeling Stroke in Mice: Permanent Coagulation of the Distal Middle Cerebral Artery

Stroke is the third most common cause of death and a main cause of acquired adult disability in developed countries. Only very limited therapeutical options are available for a small proportion of stroke patients in the acute phase. Current research is intensively searching for novel therapeutic strategies and is increasingly focusing on the sub-acute and chronic phase after stroke because more patients might be eligible for therapeutic interventions in a prolonged time window. These delayed mechanisms include important pathophysiological pathways such as post-stroke inflammation, angiogenesis, neuronal plasticity and regeneration. In order to analyze these mechanisms and to subsequently evaluate novel drug targets, experimental stroke models with clinical relevance, low mortality and high reproducibility are sought after. Moreover, mice are the smallest mammals in which a focal stroke lesion can be induced and for which a broad spectrum of transgenic models are available. Therefore, we describe here the mouse model of transcranial, permanent coagulation of the middle cerebral artery via electrocoagulation distal of the lenticulostriatal arteries, the so-called &#8220;coagulation model&#8221;. The resulting infarct in this model is located mainly in the cortex; the relative infarct volume in relation to brain size corresponds to the majority of human strokes. Moreover, the model fulfills the above-mentioned criteria of reproducibility and low mortality. In this video we demonstrate the surgical methods of stroke induction in the &#8220;coagulation model&#8221; and report histological and functional analysis tools.

Various murine models of middle cerebral artery occlusion (MCAO) are widely used in experimental brain research. Here, we demonstrate the model of transcranial permanent distal MCAO which produces consistent cortical infarction of a size corresponding to damage imposed by the majority of human ischemic strokes.

マウスにおけるモデリングのストローク：遠位中大脳動脈の永久的な凝固

Stroke is the third most common cause of death and a main cause of acquired adult disability in developed countries. Only very limited therapeutical ...

Modeling Chemotherapy Resistant Leukemia In Vitro

It is well established that the bone marrow microenvironment provides a unique site of sanctuary for hematopoietic diseases that both initiate and progress in this site. The model presented in the current report utilizes human primary bone marrow stromal cells and osteoblasts as two representative cell types from the marrow niche that influence tumor cell phenotype. The in vitro co-culture conditions described for human leukemic cells with these primary niche components support the generation of a chemoresistant subpopulation of tumor cells that can be efficiently recovered from culture for analysis by diverse techniques. A strict feeding schedule to prevent nutrient fluxes followed by gel type 10 cross-linked dextran (G10) particles recovery of the population of tumor cells that have migrated beneath the adherent bone marrow stromal cells (BMSC) or osteoblasts (OB) generating a "phase dim" (PD) population of tumor cells, provides a consistent source of purified therapy resistant leukemic cells. This clinically relevant population of tumor cells can be evaluated by standard methods to investigate apoptotic, metabolic, and cell cycle regulatory pathways as well as providing a more rigorous target in which to test novel therapeutic strategies prior to pre-clinical investigations targeted at minimal residual disease.

Modeling Chemotherapy Resistant Leukemia In Vitro

The current report summarizes a protocol that can be utilized to model the influence of the bone marrow microenvironment niche on leukemic cells with emphasis placed on enrichment of the most chemoresistant subpopulation.