著者らは、分子細胞診施設、メモリアルスローンケタ リング癌センターの動物施設によって提供される技術サービスをご了承ください。この作品は、NIH 助成金 CA157686 (r. j. ウォン) と P30 CA008748 によって支持された (メモリアルスローンケタ リングがんセンター助成金)。

すべての動物の主題のプロシージャは機関動物ケア、メモリアルスローンケタ リング癌センターで利用委員会によって承認されました。
1. がん細胞の調製
<ol><li>37 ° C で 5 分間 0.25% トリプシン サブ合流する Panc02 H7 セルを収穫します。15 mL 遠心管の細胞を収集します。 注: セルは、80% の confluency と 4 mL のトリプシンでフラスコあたり約 12 × 106セル T-225 フラスコで育つ/フラスコを使用します。</li>
	<li>5 分洗って再細胞による細胞 (でピペッティングを上下 2 回)、1 mL の PBS でペレット、氷の懸濁液を 1.5 mL 遠心チューブに転送のための 4 ° C で 900 g x で細胞を遠心します。</li>
	<li>4 ° C、5 分で 900 g x で再び細胞を遠心し、ペレットを乱すことがなく上澄みを廃棄します。注射まで氷の上小球形にされた細胞を保ちます。</li>
</ol>2. マウスや手術の準備
メモ: この研究では 8 週齢、男性と女性の c57bl/6 j マウス使用します。手術条件当院の IACUC 規則に従います。器具を滅菌、手術の作業面の消毒、動物の消毒、外科医は、滅菌手袋を着用します。
<ol><li>手術前に日に 2% イソフルランを使用してマウスを麻酔、薄いかみそりまたは化学毛の除去剤で背側の大腿骨の長さに沿う毛皮を取り外します。</li>
	<li>手術の日、部屋に誘導 2 %isofluorane を用いたマウスを麻酔します。つま先ピンチ刺激と応答の欠如によって anesthetization を確認します。</li>
	<li>麻酔下での乾燥を防ぐため目に獣医軟膏を適用します。</li>
	<li>腹側に麻酔下のマウスを置き、ゆっくり注入するのには、手足に軽い張力を作成する低刺激性のテープで各のリムを保護します。麻酔はイソフルレン ノーズコーンと精度気化器を介して配信を使用して維持されます。</li>
	<li>その後、再び 70% アルコールで Betadine で注射部位をきれい。さらに 2 回繰り返します。手術野の緩い毛が残っていないことを確認します。</li>
	<li>小さなハサミと大腿骨平行に下約 2 mm で 1 cm の切開を確認します。下にある筋肉を公開するために横方向にピンセットで皮膚を撤回します。</li>
	<li>坐骨神経は、大殿筋と大腿二頭筋の筋への深い実行されます。小さなハサミで筋膜平面に沿ってこれらの 2 つの筋肉を分離し、下に坐骨神経を公開します。鈍的切離を使用して周囲の筋肉から神経を解放します。</li>
	<li>10 μ L のシリンジにペレットから癌細胞 (約 50,000 細胞) の 3 μ L を描画します。 注: 別の方法として、コントロールとして 3 μ L の PBS を描画します。</li>
	<li>サポートのために注射の時点で神経の下に小さな金属のヘラを配置します。解剖顕微鏡で観察、下で挿入時に可能な限り神経を並列として針を維持する、金属ベースに対して神経に針を挿入します。神経の背面を穿刺しないように注意します。このプロセス全体を通じて、できるだけ神経の処理を最小限に抑えます。</li>
	<li>神経 5 以上にゆっくりと注入 s。噴射領域での球根の形成は良い注入を示します。3 のための場所で針を残してそっと針を削除する前に s。3 のための場所で針を維持する s 神経から細胞の逆流を最小限に抑えます。 注: 末梢神経や脊髄に向かって注入が行えます。実験のセット内で一貫性を維持することが重要です。場合は、細胞は、外流出、実験における動物を含めない。経験と、これらのイベントは非常にまれです。</li>
	<li>神経を元の位置に戻ります。覆っている筋肉と神経をカバーします。適切な鎮痛を持つマウスを治療し、5-0 ナイロン縫合糸で皮膚を閉じます。</li>
	<li>それは完全に麻酔から覚醒するまで、復旧中に観察のためきれいなケージで一人でマウスを配置します。 注: それは完全な意識を取り戻すために動物のための 5-15 分かかります。それは胸骨の横臥を維持するために十分な意識を取り戻したまで動物は放置しません。動物は他の動物を完全に回復するまでの会社に返されません。その後、少なくとも 72 時間のすべての 24 h の回復を評価します。</li>
</ol>3. 坐骨神経抽出
<ol><li>Postop 日 #7、CO2を持つマウスを安楽死させます。腹側にマウスを置き、ピンを使用して遠位四肢を安定します。</li>
	<li>注入された手足と胴体の背側の皮膚を削除します。</li>
	<li>鈍的切離を使用、深い筋肉に坐骨神経を公開します。</li>
	<li>腸骨と仙骨の間の神経のコース。脊髄領域における神経へのアクセスを持っている、2 つの骨を区切ります。まず坐骨神経がある狭い領域で閉じたハサミを挿入し、マウスを押しながらはさみを開きます。繊細し、解剖中坐骨神経の整合性を維持します。</li>
	<li>慎重に、大腿骨骨端を遠位と近位脊髄に坐骨神経を解剖します。</li>
	<li>注: 侵略の神経は非常に壊れやすく、引張り破断や強制的な処理になりやすいです。</li>
	<li>最初の遠位端を切断することによって神経を収穫します。隣接組織から解放しながら神経を慎重に持ち上げます。近位端、脊柱から出口にできるだけ近くで神経を切る。</li>
	<li>記録し、ノギスを使用して侵入の総長さ。このマクロ的推定は示しています。</li>
</ol>4. 神経処理と定量
<ol><li>10 月化合物に切り裂かれた神経を埋め込みます。神経を縦に配置することを確認して、できるだけモールドの底部の平らを確認します。</li>
	<li>カセットの P と D の文字をマークすることによって神経の近位および遠位側を示す</li>
	<li>彼らはすぐに区分がない場合は、ドライの氷の上に埋め込まれた神経を配置します。サンプルは、数週間-80 ° C で保存可能性があります。</li>
	<li>クライオスタット ミクロトームを使用してスライド ガラスに 5 μ m の厚さと場所セクションでセクションのサンプル。可能であれば、合わせて 2 神経セクションごとにスライド。神経の近位側を示します。</li>
	<li>H & E 染色12を使用してスライドを染色します。</li>
	<li>デジタル高解像度デジタル データを提供するスライド スキャナーで汚された神経のセクションをスキャンします。</li>
	<li>測定距離ボタンをクリックして侵略の長さを定量化するイメージング ソフトウェアを使用して、侵入、または他の領域はパラメーターを希望します。侵略の長さの良い推定、同じ神経の複数のセクション (2、4) を使用します。</li>
</ol>

<ol>
	<li>Liebig, C., Ayala, G., Wilks, J. A., Berger, D. H., Albo, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Perineural+invasion+in+cancer.">Perineural invasion in cancer.</a> Cancer. 115 (15), 3379-3391 (2009).</li><li>Bapat, A. A., Hostetter, G., Von Hoff, D. D., Han, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Perineural+invasion+and+associated+pain+in+pancreatic+cancer.">Perineural invasion and associated pain in pancreatic cancer.</a> Nat Rev Cancer. 11 (10), 695-707 (2011).</li><li>Deborde, S., Wong, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+Schwann+cells+facilitate+cancer+progression+in+nerves.">How Schwann cells facilitate cancer progression in nerves.</a> Cell Mol Life Sci. 341 (177-186), 1236361-1236416 (2017).</li><li>Abiatari, 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=Consensus+transcriptome+signature+of+perineural+invasion+in+pancreatic+carcinoma.">Consensus transcriptome signature of perineural invasion in pancreatic carcinoma.</a> Mol Cancer Ther. 8 (6), 1494-1504 (2009).</li><li>Ayala, G. 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=In+vitro+dorsal+root+ganglia+and+human+prostate+cell+line+interaction:+redefining+perineural+invasion+in+prostate+cancer.">In vitro dorsal root ganglia and human prostate cell line interaction: redefining perineural invasion in prostate cancer.</a> Prostate. 49 (3), 213-223 (2001).</li><li>Gil, Z., Cavel, O., 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=Paracrine+regulation+of+pancreatic+cancer+cell+invasion+by+peripheral+nerves.">Paracrine regulation of pancreatic cancer cell invasion by peripheral nerves.</a> J Natl Cancer Inst. 102 (2), 107-118 (2010).</li><li>Deborde, S. T., 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=Schwann+cells+induce+cancer+cell+dispersion+and+invasion.">Schwann cells induce cancer cell dispersion and invasion.</a> J Clin Invest. 126 (4), 1538-1554 (2016).</li><li>Pour, P. M., Egami, H., Takiyama, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patterns+of+growth+and+metastases+of+induced+pancreatic+cancer+in+relation+to+the+prognosis+and+its+clinical+implications.">Patterns of growth and metastases of induced pancreatic cancer in relation to the prognosis and its clinical implications.</a> Gastroenterology. 100 (2), 529-536 (1991).</li><li>Eibl, G., Reber, H. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+xenograft+nude+mouse+model+for+perineural+invasion+and+recurrence+in+pancreatic+cancer.">A xenograft nude mouse model for perineural invasion and recurrence in pancreatic cancer.</a> Pancreas. 31 (3), 258-262 (2005).</li><li>Stopczynski, R. 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=Neuroplastic+changes+occur+early+in+the+development+of+pancreatic+ductal+adenocarcinoma.">Neuroplastic changes occur early in the development of pancreatic ductal adenocarcinoma.</a> Cancer Res. 74 (6), 1718-1727 (2014).</li><li>Gil, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nerve-sparing+therapy+with+oncolytic+herpes+virus+for+cancers+with+neural+invasion.">Nerve-sparing therapy with oncolytic herpes virus for cancers with neural invasion.</a> Clin Cancer Res. 13 (21), 6479-6485 (2007).</li><li>Cardiff, R. D., Miller, C. H., Munn, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Manual+hematoxylin+and+eosin+staining+of+mouse+tissue+sections.">Manual hematoxylin and eosin staining of mouse tissue sections.</a> Cold Spring Harb Protoc. 2014 (6), 655-658 (2014).</li><li>Cremer, 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=Inactivation+of+the+N-CAM+gene+in+mice+results+in+size+reduction+of+the+olfactory+bulb+and+deficits+in+spatial+learning.">Inactivation of the N-CAM gene in mice results in size reduction of the olfactory bulb and deficits in spatial learning.</a> Nature. 367 (6462), 455-459 (1994).</li><li>He, 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=The+chemokine+(CCL2-CCR2)+signaling+axis+mediates+perineural+invasion.">The chemokine (CCL2-CCR2) signaling axis mediates perineural invasion.</a> Mol Cancer Res. 13 (2), 380-390 (2015).</li><li>He, 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=GFR&#945;1+released+by+nerves+enhances+cancer+cell+perineural+invasion+through+GDNF-RET+signaling.">GFR&#945;1 released by nerves enhances cancer cell perineural invasion through GDNF-RET signaling.</a> P Natl Acad Sci USA. , 02944 (2014).</li></ol>

著者が明らかに何もありません。

このプロトコルでは膵臓癌細胞による坐骨神経浸潤の定量化は、浸潤の体内マウス モデルをについて説明します。このモデルにより、神経浸潤の分子メカニズムの研究です。この技術を用いた実験の成功プロセスで 3 つの重要な手順に慎重なアプローチが必要: 2) 侵略神経 (ステップ 3.4) の抽出と収穫された神経 (手順 4.1) の処理 3) 癌細胞 (手順 2.7、2.8) の 1) の注入。
<p class="jove_con...

神経は、がんの成長と移行1,2,3を刺激する特定の腫瘍微小環境を形成します。浸潤 (PNI) は、神経周辺を介して、がん細胞に侵入するプロセスです。それは、癌の浸潤が神経に沿って元のサイトから延びてので転移のユニークなルートとして考慮されるかもしれない。PNI は 22% から 1001,2に至るまで発生率と膵臓、前立腺、頭と首、唾液、子宮頚部、大腸癌を含むいくつかのがんの種類で発見されます。PNI 痛みに関連付けられ、予後不良と悪い生存率1,2と相関します。
神経周囲浸潤のモデルの開発は、このプロセスの細胞および分子メカニズムを明らかにし、梗数を減らす治療剤の候補物質をテストするのには不可欠です。癌と神経間相互作用を勉強の in vitro方法があります神経外植片4後根罹5,6、7、または特定のセル癌細胞の共培養神経からシュワンなど微小細胞7をです。生体内でのアプローチは、ただし、もっと生理学的に関連して、癌マウスモデルでがんが誘発または移植の使用を含む、会計全体の神経の微小環境の利点があります。同所性同種膵や前立腺癌、PNI のモデルはずっと報告された8,9,10 PNI の発生率が記録される可能性がありますがこれら器官の神経のサイズが小さいため参照してくださいすることは困難です。全体の神経とそのため梗数の程度を定量化します。ここで述べるモデルは単純な手術11を介してマウスの坐骨神経に注入される細胞の癌 PNI の体内モデルです。異所性移植は、脊髄へ神経内に侵入します。脊髄注射のサイトから神経の侵略の長さ測定される、神経中の癌のボリュームだけでなく。重要なは、侵略の神経は様々 なアッセイを顕微鏡、分子解析などのも収集できます。様々 な癌細胞をテストことができますと遺伝的に変更または特定化合物処理ホスト マウスを使用することもことがあります。この強力なアッセイとは、がん細胞とホスト微小環境 PNI のメカニズムを調査用に変更します。

<table border="0" cellpadding="0" cellspacing="0" style="width:973px;" width="973">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:340px;">Mouse</td>
			<td style="width:123px;"></td>
			<td style="width:344px;"></td>
			<td style="width:167px;">Number and age variable depending on experimental needs</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Cell culture media (PBS, Trypsin, and DMEM+10% FBS)</td>
			<td>Any</td>
			<td></td>
			<td>Steps 1.1, 1.2, 1.3.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Conical centrifuge tube, 50 mL</td>
			<td>Falcon</td>
			<td>352098</td>
			<td>Step 1.1</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Microcentrifuge tube 1.5 mL</td>
			<td>Axygen</td>
			<td>MCT-150-C-S</td>
			<td>Step 1.2</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Electric razor</td>
			<td>WAHL</td>
			<td>9962</td>
			<td>Step 2.1. Can be substituted with commercial hair removal agent</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Isoflurane, 250 mL</td>
			<td>Baxter</td>
			<td>1001936060</td>
			<td>Step 2.2</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Hypoallergenic surgical tape</td>
			<td>3M Blenderm</td>
			<td>70200419342</td>
			<td>Step 2.3</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Betadine Swapsticks</td>
			<td>PDI</td>
			<td>SKU 41350</td>
			<td>Step 2.4</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Webcol Alcohol Preps</td>
			<td>Covidien</td>
			<td>5110</td>
			<td>Step 2.4</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Sterile surgical tools (scissors and forceps)</td>
			<td></td>
			<td></td>
			<td>Steps 2.4, 2.5, 3.3, 3.4, 3.5</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">10 &#956;L Hamilton syringe</td>
			<td>Hamilton</td>
			<td>80308</td>
			<td>Steps 2.7, 2.8</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Steel Micro spatula</td>
			<td>Fisher Scientific</td>
			<td>S50823</td>
			<td>Step 2.7</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Dissecting microscope</td>
			<td></td>
			<td></td>
			<td>Step 2.7</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Bupivacine, 1 g</td>
			<td>Enzo Life Sciences</td>
			<td>BML-NA139-0001</td>
			<td>Step 2.9. Reconstitute to 0.5%</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">5-0 Nylon suture</td>
			<td>Ethicon</td>
			<td>698H</td>
			<td>Step 2.9</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Tissue-Tek O.C.T. Compound</td>
			<td>VWR</td>
			<td>25608-930</td>
			<td>Step 4.1</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Tissue-Tek Cryomold Molds</td>
			<td>VWR</td>
			<td>25608-916</td>
			<td>Step 4.1</td>
		</tr>
	</tbody>
</table>

an in vivo murine sciatic nerve model of perineural invasion

癌細胞浸潤 (PNI)、がん細胞が増殖し、神経の微小環境の移行と呼ばれるプロセスを介して神経に侵入します。このタイプの侵略は様々 な癌の種類は、展示し、膵臓癌の発見は非常に頻繁。マウスの膵臓の神経線維の微細なサイズは、直交異方性マウスモデルで難しい PNI の研究をレンダリングします。ここでは、マウスの坐骨神経に同系膵癌細胞株 Panc02 H7 を注入我々 PNI の生体内異所性モデルについて述べる.このモデルでは、麻酔下マウスの坐骨神経を公開され、がん細胞を注入します。挿入ポイントから脊髄に向かって近位の神経の癌細胞に侵入します。侵略の坐骨神経は抽出し、10 月冷凍区分のため処理します。H & E と以下の免疫蛍光染色は、蛋白質の表現の侵略の度に変化の定量化を許可します。このモデルは、その汎用性を与え PNI に関する研究各種に適用できます。PNI の細胞および分子メカニズムの調査のため、別のがんの種類によりさまざまな遺伝的変更および/または異なったタイプの癌細胞とマウスを使用します。さらに、これらのマウスに治療を適用することによって神経浸潤に対する治療薬の効果を学ぶことができます。

このメソッドは、定量化可能な神経浸潤の生体内でモデルを作成するマウスの坐骨神経に膵臓癌細胞の外科的移植について説明します。図 1は、坐骨神経の解剖学的位置と注射部位を示しています。図 2は、裸のマウスの 2 つの坐骨神経を示しています。(右) MiaPaCa 2 がん細胞を注入した神経に注入 PBS 神経 (左) が比?...

Watch this Scientific Journal Video about An In Vivo Murine Sciatic Nerve Model of Perineural Invasion at JoVE.com

An In Vivo Murine Sciatic Nerve Model of Perineural Invasion

神経周囲浸潤のIn Vivoマウスの坐骨神経モデル

坐骨神経に同系膵癌細胞を注入することによって浸潤の体内マウス モデルについて述べる。モデル神経浸潤の程度の定量化と浸潤細胞および分子メカニズムの調査をサポートしています。

Cancer cells invade nerves through a process termed perineural invasion (PNI), in which cancer cells proliferate and migrate in the nerve ...

an-in-vivo-murine-sciatic-nerve-model-of-perineural-invasion

Research

JoVE Journal

Cancer Research

An In Vivo Murine Sciatic Nerve Model of Perineural Invasion

11.9K ビュー.  Memorial Sloan Kettering Cancer Center. 坐骨神経に同系膵癌細胞を注入することによって浸潤の体内マウス モデルについて述べる。モデル神経浸潤の程度の定量化と浸潤細胞および分子メカニズムの調査をサポートしています。

ビデオ: An In Vivo Murine Sciatic Nerve Model of Perineural Invasion

Coculture Assays to Study Macrophage and Microglia Stimulation of Glioblastoma Invasion

Glioblastoma multiforme (grade IV glioma) is a very aggressive human cancer with a median survival of 1 year post diagnosis. Despite the increased understanding of the molecular events that give rise to glioblastomas, this cancer still remains highly refractory to conventional treatment. Surgical resection of high grade brain tumors is rarely complete due to the highly infiltrative nature of glioblastoma cells. Therapeutic approaches which attenuate glioblastoma cell invasion therefore is an attractive option. Our laboratory and others have shown that tumor associated macrophages and microglia (resident brain macrophages) strongly stimulate glioblastoma invasion. The protocol described in this paper is used to model glioblastoma-macrophage/microglia interaction using in vitro culture assays. This approach can greatly facilitate the development and/or discovery of drugs that disrupt the communication with the macrophages that enables this malignant behavior. We have established two robust coculture invasion assays where microglia/macrophages stimulate glioma cell invasion by 5 - 10 fold. Glioblastoma cells labelled with a fluorescent marker or constitutively expressing a fluorescent protein are plated without and with macrophages/microglia on matrix-coated polycarbonate chamber inserts or embedded in a three dimensional matrix. Cell invasion is assessed by using fluorescent microscopy to image and count only invasive cells on the underside of the filter. Using these assays, several pharmacological inhibitors (JNJ-28312141, PLX3397, Gefitinib, and Semapimod), have been identified which block macrophage/microglia stimulated glioblastoma invasion.

Understanding the malignant behavior of cancer requires creating accurate models of how tumor cells interact with components of the tumor microenvironment, such as macrophages. Here we describe two methods to study glioblastoma cell interaction with tumor associated macrophages and microglia where the effect on glioblastoma invasion is assessed.

共培養アッセイは、神経膠芽腫の浸潤のマクロファージとミクログリアの刺激を研究します

Glioblastoma multiforme (grade IV glioma) is a very aggressive human cancer with a median survival of 1 year post diagnosis. Despite the increased ...

がん研究

In Vivo Model for Testing Effect of Hypoxia on Tumor Metastasis

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

In Vivo Model for Testing Effect of Hypoxia on Tumor Metastasis

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

腫瘍転移に対する低酸素のテストの効果のためのインビボモデル

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

A Model for Perineural Invasion in Head and Neck Squamous Cell Carcinoma

Perineural invasion (PNI) is found in approximately 40% of head and neck squamous cell carcinomas (HNSCC). Despite multimodal treatment with surgery, radiation, and chemotherapy, locoregional recurrences and distant metastases occur at higher rates, and overall survival is decreased by 40% compared to HNSCC without PNI. In vitro studies of the pathways involved in HNSCC PNI have historically been challenging given the lack of a consistent, reproducible assay. Described here is the adaptation of the dorsal root ganglion (DRG) assay for the examination of PNI in HNSCC. In this model, DRG are harvested from the spinal column of a sacrificed nude mouse and placed within a semisolid matrix. Over the subsequent days, neurites are generated and grow in a radial pattern from the cell bodies of the DRG. HNSCC cell lines are then placed peripherally around the matrix and invade preferentially along the neurites toward the DRG. This method allows for rapid evaluation of multiple treatment conditions, with very high assay success rates and reproducibility.

Perineural invasion (PNI) is a common feature of head and neck squamous cell carcinoma (HNSCC), conferring lower survival rates. Its mechanisms are poorly understood. Utilizing neurites generated from murine dorsal root ganglia confined to a semisolid matrix, the pathways involved in the PNI of HNSCC cell lines can be investigated.

頭頸部扁平上皮癌における神経周囲の浸潤のためのモデル

Perineural invasion (PNI) is found in approximately 40% of head and neck squamous cell carcinomas (HNSCC). Despite multimodal treatment with surgery, ...

A Mouse Model of Fatigue Induced by Peripheral Irradiation

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

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

周辺照射による疲労のマウスモデル

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

Moving Upwards: A Simple and Flexible In Vitro Three-dimensional Invasion Assay Protocol

Although 3D invasion assays have been developed, the challenge remains to study cells without affecting the integrity of their microenvironment. Traditional 3D assays such as the Boyden Chamber require that cells are displaced from the original culture location and moved to a new environment. Not only does this disrupt the cellular processes that are intrinsic to the microenvironment, but it often results in a loss of cells. These problems are especially challenging when dealing with cells that are either rare, or extremely sensitive to their microenvironment. Here, we describe the development of a 3D invasion assay that avoids both concerns. In this assay, cells are plated within a small well and an ECM matrix containing a chemoattractant is laid atop the cells. This requires no cell displacement, and allows the cells to invade upwards into the matrix. In this assay, cell invasion as well as cell morphology can be assessed within the collagen gel. Using this assay, we characterize the invasive capacity of rare and sensitive cells; the hybrid cells resulting from fusion between breast cancer cells MCF7 and mesenchymal/multipotent stem/stroma cells (MSCs).

Moving Upwards: A Simple and Flexible In Vitro Three-dimensional Invasion Assay Protocol

This protocol describes an inverted vertical invasion assay that could be used to quantify the migration and invasion capabilities of cells in a three-dimensional setting while preserving the cell microenvironment. This assay is suitable for rare and/or sensitive cells.

上に移動: シンプルかつ柔軟なIn Vitro三次元侵略試金のプロトコル

Although 3D invasion assays have been developed, the challenge remains to study cells without affecting the integrity of their microenvironment. ...

In Vivo EPR Assessment of pH, pO2, Redox Status, and Concentrations of Phosphate and Glutathione in the Tumor Microenvironment

This protocol demonstrates the capability of low-field electron paramagnetic resonance (EPR)-based techniques in combination with functional paramagnetic probes to provide quantitative information on the chemical tumor microenvironment (TME), including pO2, pH, redox status, concentrations of interstitial inorganic phosphate (Pi), and intracellular glutathione (GSH). In particular, an application of a recently developed soluble multifunctional trityl probe provides unsurpassed opportunity for in vivo concurrent measurements of pH, pO2 and Pi in Extracellular space (HOPE probe). The measurements of three parameters using a single probe allow for their correlation analyses independent of probe distribution and time of the measurements.

In Vivo EPR Assessment of pH, pO2, Redox Status, and Concentrations of Phosphate and Glutathione in the Tumor Microenvironment

Low-field (L-band, 1.2 GHz) electron paramagnetic resonance using soluble nitroxyl and trityl probes is demonstrated for assessment of physiologically important parameters in the tumor microenvironment in mouse models of breast cancer.

生体内でPH、 pO2、酸化還元状態とリン酸濃度および腫瘍微小環境におけるグルタチオンの EPR 評価

This protocol demonstrates the capability of low-field electron paramagnetic resonance (EPR)-based techniques in combination with functional paramagnetic ...

Immunophenotyping of Orthotopic Homograft (Syngeneic) of Murine Primary KPC Pancreatic Ductal Adenocarcinoma by Flow Cytometry

Homograft (syngeneic) tumors are the workhorse of today&#39;s immuno-oncology (I/O) preclinical research. The tumor microenvironment (TME), particularly its immune-components, is vital to the prognosis and prediction of treatment outcomes, especially those of immunotherapy. TME immune-components are composed of different subsets of tumor-infiltrating immune cells assessable by multi-color FACS. Pancreatic ductal adenocarcinoma (PDAC) is among the deadliest malignances lacking good treatment options, thus an urgent and unmet medical need. One important reason for its non-responsiveness to various therapies (chemo-, targeted, I/O) has been its abundant TME, consisting of fibroblasts and leukocytes that protect tumor cells from these therapies. Orthotopically implanted PDAC is believed to more accurately recapture the TME of human pancreatic cancers than conventional subcutaneous (SC) models.
Homograft tumors (KPC) are transplants of mouse spontaneous PDAC originating from genetically engineered KPC-mice (KrasG12D/+/P53-/-/Pdx1-Cre) (KPC-GEMM). The primary tumor tissue is cut into small fragments (~2 mm3) and transplanted subcutaneously (SC) to the syngeneic recipients (C57BL/6, 7&#8211;9 weeks old). The homografts were then surgically orthotopically transplanted onto the pancreas of new C57BL/6 mice, along with SC-implantation, which reached tumor volumes of 300&#8211;1,000 mm3 by 17 days. Only tumors of 400&#8211;600 mm3 were harvested per approved autopsy procedure and cleaned to remove the adjacent non-tumor tissues. They were dissociated per protocol using a tissue dissociator into single-cell suspensions, followed by staining with designated panels of fluorescently-labeled antibodies for various markers of different immune cells (lymphoid, myeloid and NK, DCs). The stained samples were analyzed using multi-color FACS to determine numbers of immune cells of different lineages, as well as their relative percentage within tumors. The immune profiles of orthotopic tumors were then compared to those of SC tumors. The preliminary data demonstrated significantly elevated infiltrating TILs/TAMs in tumors over the pancreas, and higher B-cell infiltration into orthotopic rather than SC tumors.

Immunophenotyping of Orthotopic Homograft Syngeneic of Murine Primary KPC Pancreatic Ductal Adenocarcinoma by Flow Cytometry

The experimental procedure on the immunophenotyping of murine orthotopic PDAC homografts aims at profiling the tumor immuno-microenvironment. Tumors are orthotopically implanted via surgery. Tumors of 200&#8211;600 mm3 in size were harvested and dissociated to prepare single-cell suspensions, followed by multi-immune marker FACS analysis using different fluorescently-labeled antibodies.

同所性同種 (同系) マウスのプライマリ KPC 膵管腺癌のフローサイトメトリーによる診療

Homograft (syngeneic) tumors are the workhorse of today&#39;s immuno-oncology (I/O) preclinical research. The tumor microenvironment (TME), particularly ...

A Simple Migration/Invasion Workflow Using an Automated Live-cell Imager

Cancer cell mobility is crucial for the initiation of metastasis. Therefore, investigation of the cell movement and invasive capacity is of great significance. Migration assays provide basic insight of cell movement at a 2D level, whereas invasion assays are more physiologically relevant, mimicking in vivo cancer cell dislodgment from the original site and invading through the extracellular matrix. The current protocol provides a single workflow for migration and invasion assays. Together with the integrated automated microscopic camera for real-time HD images and built-in analysis module, it gives researchers a time-efficient, simple and reproducible experimental option. This protocol also includes substitutions for the consumables and alternative analysis methods for users to choose from.

The current protocol describes an integrated method investigating cancer cell migration and invasion on a single platform in real-time, providing an easily reproducible and time-efficient option to study cell mobility and morphology.

自動生細胞固体撮像素子を使用して単純な移行/侵略ワークフロー

Cancer cell mobility is crucial for the initiation of metastasis. Therefore, investigation of the cell movement and invasive capacity is of great ...

An Orthotopic Endometrial Cancer Model with Retroperitoneal Lymphadenopathy Made From In Vivo Propagated and Cultured VX2 Cells

Endometrial cancer is the most common gynecologic malignancy in North America and the incidence is rising worldwide. Treatment consists of surgery with or without adjuvant therapy depending on lymph node involvement as determined by lymphadenectomy. Lymphadenectomy is a morbid procedure, which has not been shown to have a therapeutic benefit in many patients, and thus a new method to diagnose lymph node metastases is required. To test novel imaging agents, a reliable model of endometrial cancer with retroperitoneal lymph node metastases is needed. The VX2 endometrial cancer model has been described frequently in the literature; however, significant variation exists with respect to the method of model establishment. Furthermore, no studies have reported on the use of cultured VX2 cells to create this model as only cells propagated in vivo have been previously used. Herein, we present a standardized surgical method and post-operative monitoring method for the establishment of the VX2 endometrial cancer model and report on the first use of cultured VX2 cells to create this model.

This protocol presents a standardized method to grow VX2 cells in culture and to create an orthotopic VX2 model of endometrial cancer with retroperitoneal lymph node metastases in rabbits. Orthotopic endometrial cancer models are important for the pre-clinical study of novel imaging modalities for the diagnosis of lymph node metastases.

生体内伝播および培養VX2細胞から作られた後腹部リンパ管リンパデノパシーを有するオルトトピック子宮内膜癌モデル

Endometrial cancer is the most common gynecologic malignancy in North America and the incidence is rising worldwide. Treatment consists of surgery with or ...

Spatial and Temporal Control of Murine Melanoma Initiation from Mutant Melanocyte Stem Cells

Cutaneous melanoma is well known as the most aggressive skin cancer. Although the risk factors and major genetic alterations continue to be documented with increasing depth, the incidence rate of cutaneous melanoma has shown a rapid and continuous increase during recent decades. In order to find effective preventative methods, it is important to understand the early steps of melanoma initiation in the skin. Previous data has demonstrated that follicular melanocyte stem cells (MCSCs) in the adult skin tissues can act as melanoma cells of origin when expressing oncogenic mutations and genetic alterations. Tumorigenesis arising from melanoma-prone MCSCs can be induced when MCSCs transition from a quiescent to active state. This transition in melanoma-prone MCSCs can be promoted by the modulation of either hair follicle stem cells&#39; activity state or through extrinsic environmental factors such as ultraviolet-B (UV-B). These factors can be artificially manipulated in the laboratory by chemical depilation, which causes transition of hair follicle stem cells and MCSCs from a quiescent to active state, and by UV-B exposure using a benchtop light. These methods provide successful spatial and temporal control of cutaneous melanoma initiation in the murine dorsal skin. Therefore, these in vivo model systems will be valuable to define the early steps of cutaneous melanoma initiation and could be used to test potential methods for tumor prevention.

The following procedure describes a method for spatial and temporal control of melanocytic tumor initiation in murine dorsal skin, using a genetically engineered mouse model. This protocol describes macroscopic as well as microscopic cutaneous melanoma initiation.

変異体メラノサイト幹細胞からのマウス黒色腫開始の空間的および時間的制御

Cutaneous melanoma is well known as the most aggressive skin cancer. Although the risk factors and major genetic alterations continue to be documented ...