A.S. was partially supported by grants from the National Institutes of Health (NIH) National Institute of Dental and Craniofacial Research (5R03DE021741-02) and the National Cancer Institute (1K08CA154963-01A1).&#160;
The Howard Hughes Medical Institute provided funding for M.L. through the HHMI Medical Research Fellows Program.

1.卵のインキュベーション<ol><li> 8日齢の特定病原体フリーの孵化卵を入手します。 </li><li>卵トレイを回転させるに卵を置き、上向きに刻印が終了し、卵のインキュベーターの内側に回転トレイを置きます。 36°Cおよび50％の湿度で48時間の卵をインキュベートします。 </li></ol> 2. CAMを削除し、卵を開きます<ol><li>インキュベーターから卵を収集します。卵のラックに卵を置き、刻印側を上にし、層流フードにもたらします。 </li><li>部屋とフードライトをオフにします。 </li><li> CAMの血管系だけでなく、気嚢を露出するために卵キャンドラーに軽く卵の刻印の端を持ってください。 注：卵キャンドラーは、発生中の胚および関連気嚢と血管系を可視化するために使用される光源です。 </li><li>二つの主要な血管の間に鉛筆マークを配置します。 </li><li>ライトの電源をオンにし、PEで気嚢（刻印終了）上記の卵の先端と一つの穴に一つの穴を作るために、滅菌プッシュピンを使用ncilマーク。すべての方法を介してプッシュピンを押さないでください。深い3ミリメートルの周りの穴は、通常は十分であろう。 </li><li>ライトをオフにします。気嚢上の穴にしっかりと開放端を押して、安全電球を絞ります。 CAMは、鉛筆のマークでシェルから離れて落下させる、鉛筆のマークの穴に空気を引っ張るために吸引を適用します。 </li><li>気嚢がキャンドラーを使用して、鉛筆でマークされた穴に卵の刻印端から移動したことを確認してください。それはしていない場合、安全バルブを使用して再適用する吸引両方の穴が若干深くするために押しピンを使用して。 </li><li>ライトをオンにします。添付15/16インチカットオフホイールでDREMEL回転工具をオンにし、シェルを切断するだけの十分な深2の横方向のカットを行いますが十分な深さではないが押さまですべての方法をカットするために、片手で卵を開催CAM。離間長さ2センチ、1センチメートル周りのカットを行います。直径1cmシリコーンリングが通過できるように十分な幅のカットを作りますしかし、標準的なスコッチテープの幅よりも薄いです。 </li><li>次に、間に軽くシェルにカットオフホイールをタッチして、2つの横方向のカットの端部の1の長手方向のカットを行います。 </li><li>シェルにシェル片と並列の下で滅菌ピンセットをスライドさせます。ピンセットで殻の部分をつかみ、きれいに作品全体を削除します。 </li><li>除去を容易にするため自体に一端を超える倍にしてくださいすること、新しく開いたウィンドウの上にスコッチテープを配置します。 </li><li>接種時まで（NOT回転卵ラックで）卵トレイにインキュベーターに戻って卵を置きます。 </li></ol> 3.接種<ol><li> （重合を防止するため）とフードに置き、氷上で、そのようなマトリゲルとして、基底膜タンパク質の混合物を保管してください。 </li><li> 5分間組織培養インキュベーター中で - （T75フラスコあたり5ミリリットル4）と場所フラスコの細胞を分離するために2 mMのEDTAを使用してください。 </li><li>メディアで剥離した細胞を再懸濁し、血球計およびトリパンブルーを用いて数えます。 50万トンの間で使用します細胞株に応じて、CAM上に移植するための2,000,000細胞O。予備実験で適切な数を最適化します。 </li><li> 5分間500×gで適切な細胞数と遠心分離のために必要な測定量。 </li><li>上清を捨て、次いで、PBS ++の40μLと基底膜の混合物の20μLに細胞ペレットを再懸濁します。 注：細胞/基底膜の混合懸濁液に再懸濁することにより実験のために必要に応じて、この時点で、細胞は、薬物または他の試験薬剤で処理することができます。あるいは、薬剤は、移植後の細胞/基底膜の混合懸濁液にピペッティングすることができます。 </li><li>層流フード内でインキュベーターと場所から卵と卵のラックを取得します。 </li><li>ウィンドウのオフピールテープ、滅菌ピンセットを用いて低温バイアルのキャップから1ミリメートルのシリコンリングを取得し、窓からリングをドロップします。 </li><li>ピペットでPBSの60μlの細胞溶液++および基底膜mixtur電子直接、CAM上に載っているシリコーンリングの中心部へ。 </li><li>ウィンドウを再テープとバックインキュベーターに卵のラックを移動します。この時点で、卵を移動する際、リング、細胞懸濁液として十分注意してください、簡単に変位することができます。 </li><li>腫瘍は5〜7日間増殖できるようにします。 </li></ol> 4.収穫腫瘍<ol><li>フード内の下パッドに置きます。組織学のために必要に応じて、35×10 mmプレート及びパラホルムアルデヒドコンテナを準備します。各35×10ミリメートルプレートに1mlのPBS ++を入れてください。 </li><li>フード内でバイオハザードバッグを置きます。無菌解剖はさみを取り、刻印端付近の穴に卵の中に先端を挿入します。 </li><li>縦方向全体の卵の周りをカットします。 注：リング付きCAMおよび腫瘍は、ウィンドウを持つシェルの上半分に固執する必要があります。 </li><li>解剖ハサミとピンセットを使用して、離れたCAMから腫瘍を分析し、35×10ミリメートルプレートに腫瘍を配置します。測定し、腫瘍の重量を量ります。 paraformaldehyで修正された腫瘍その後、脱パラフィン - 埋め込み、それらを組織学および免疫組織化学の実験で使用するため。 </li></ol> 5.（オプション）：腫瘍細胞解離<ol><li> 35×10ミリメートルプレートに腫瘍をミンチする解剖ハサミを使用してください。 15mlの遠心チューブにみじん切り腫瘍を注ぎ、ピペッティングを介して可能な限り多くのPBS ++を削除します。 </li><li> （PBS中1mg / mlの++）コラゲナーゼの2ミリリットルを加え、数回にわたってチューブを回して、（数時間まで）、少なくとも37℃で30分間チューブをインキュベートします。 </li><li>各チューブに5ミリリットルのメディアを追加し、ダウン20ピペッティングにより完全に再懸濁 - 40倍。徹底したピペット操作は、完全な細胞解離のために重要です。 </li><li>沈殿物が沈降することを許可し、上清を新しいチューブに移します。 5分間500×gで遠心分離します。解離細胞ペレットを残し、上清を取り除きます。 注：この時点で、細胞ペレットは、種々の実験のために使用することができる（フローサイトメトリー、ウェスタンブロッティング、RNAの単離などで使用するために溶解します）。全腫瘍細胞数はまた、血球計およびトリパンブルー染色を用いて得ることができます。小さく、楕円形の細胞は、CAMからの鶏の細胞であり、腫瘍細胞としてカウントすべきではないことに注意してください。腫瘍は慎重に、CAMから離れて解剖されている場合は非腫瘍（CAM）細胞の混入の最小数があるはずです。 </li></ol>

<ol>
	<li>Sangiovanni, 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=Increased+survival+of+cirrhotic+patients+with+a+hepatocellular+carcinoma+detected+during+surveillance.">Increased survival of cirrhotic patients with a hepatocellular carcinoma detected during surveillance.</a> Gastroenterology. 126 (4), 1005-1014 (2004).</li><li>Lopez, P. M., Villanueva, A., Llovet, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systematic+review:+evidence-based+management+of+hepatocellular+carcinoma--an+updated+analysis+of+randomized+controlled+trials.">Systematic review: evidence-based management of hepatocellular carcinoma--an updated analysis of randomized controlled trials.</a> Aliment Pharmacol Ther. 23 (11), 1535-1547 (2006).</li><li>Llovet, J. M., Burroughs, A., Bruix, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hepatocellular+carcinoma.">Hepatocellular carcinoma.</a> Lancet. 362 (9399), 1907-1917 (2003).</li><li>Folkman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=What+is+the+evidence+that+tumors+are+angiogenesis+dependent.">What is the evidence that tumors are angiogenesis dependent.</a> J Natl Cancer Inst. 82, 4-6 (1990).</li><li>Hagedorn, 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=Accessing+key+steps+of+human+tumor+progression+in+vivo+by+using+an+avian+embryo+model.">Accessing key steps of human tumor progression in vivo by using an avian embryo model.</a> Proc Natl Acad Sci U S A. 102 (5), 1643-1648 (2005).</li><li>Dumartin, L., 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=Netrin-1+mediates+early+events+in+pancreatic+adenocarcinoma+progression,+acting+on+tumor+and+endothelial+cells.">Netrin-1 mediates early events in pancreatic adenocarcinoma progression, acting on tumor and endothelial cells.</a> Gastroenterology. 138 (4), 1595-1606 (2010).</li><li>Aguirre-Ghiso, J. A., Estrada, Y., Liu, D., Ossowski, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ERK(MAPK)+activity+as+a+determinant+of+tumor+growth+and+dormancy;+regulation+by+p38(SAPK).">ERK(MAPK) activity as a determinant of tumor growth and dormancy; regulation by p38(SAPK).</a> Cancer Res. 63 (7), 1684-1695 (2003).</li><li>Baroni, T. 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=Ribonomic+and+short+hairpin+RNA+gene+silencing+methods+to+explore+functional+gene+programs+associated+with+tumor+growth+arrest.">Ribonomic and short hairpin RNA gene silencing methods to explore functional gene programs associated with tumor growth arrest.</a> Methods Mol Biol. 383, 227-244 (2007).</li><li>Lopez-Rivera, 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=Inducible+nitric+oxide+synthase+drives+mTOR+pathway+activation+and+proliferation+of+human+melanoma+by+reversible+nitrosylation+of+TSC2.">Inducible nitric oxide synthase drives mTOR pathway activation and proliferation of human melanoma by reversible nitrosylation of TSC2.</a> Cancer Res. 74 (4), 1067-1078 (2014).</li><li>Balke, 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=A+short-term+in+vivo+model+for+giant+cell+tumor+of+bone.">A short-term in vivo model for giant cell tumor of bone.</a> BMC Cancer. 11, 241 (2011).</li><li>Balke, 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=Morphologic+characterization+of+osteosarcoma+growth+on+the+chick+chorioallantoic+membrane.">Morphologic characterization of osteosarcoma growth on the chick chorioallantoic membrane.</a> BMC Res Notes. 3, 58 (2010).</li><li>Sys, G. 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=The+in+ovo+CAM-assay+as+a+xenograft+model+for+sarcoma.">The in ovo CAM-assay as a xenograft model for sarcoma.</a> J Vis Exp. (77), e50522 (2013).</li><li>Dohle, D. 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=Chick+ex+ovo+culture+and+ex+ovo+CAM+assay:+how+it+really+works.">Chick ex ovo culture and ex ovo CAM assay: how it really works.</a> J Vis Exp. (33), (2009).</li><li>Ossowski, L., Reich, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+model+for+quantitative+study+of+metastasis.">Experimental model for quantitative study of metastasis.</a> Cancer Res. 40 (7), 2300-2309 (1980).</li><li>Ghanekar, A., Ahmed, S., Chen, K., Adeyi, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+cells+do+not+arise+from+tumor-initiating+cells+in+human+hepatocellular+carcinoma.">Endothelial cells do not arise from tumor-initiating cells in human hepatocellular carcinoma.</a> BMC Cancer. 13, 485 (2013).</li><li>Ho, J. W., 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=Effects+of+a+novel+immunomodulating+agent,+FTY720,+on+tumor+growth+and+angiogenesis+in+hepatocellular+carcinoma.">Effects of a novel immunomodulating agent, FTY720, on tumor growth and angiogenesis in hepatocellular carcinoma.</a> Mol Cancer Ther. 4 (9), 1430-1438 (2005).</li><li>Hagedorn, 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=VEGF+coordinates+interaction+of+pericytes+and+endothelial+cells+during+vasculogenesis+and+experimental+angiogenesis.">VEGF coordinates interaction of pericytes and endothelial cells during vasculogenesis and experimental angiogenesis.</a> Dev Dyn. 230 (1), 23-33 (2004).</li></ol>

A.S.&#160;receives&#160;funding from the Advaxis Pharmaceutical company to support an investigator-initiated clinical trial.&#160;

このプロトコルのいくつかの重要なステップは、ほとんど改善された胚の生存率だけでなく、腫瘍増殖の信頼性向上を占めています。気嚢に吸引を適用することにより、離れたシェルからCAMを削除すると（卵、鈍的切開などからアルブミンを除去するために、針を使用して）他の既存の方法よりも低侵襲性です。この方法のために必要な2つの小さな穴を作成するために、無菌のプッシュピン...

肝細胞癌（HCC）は、世界1におけるがん死亡率の3 番目の主要な原因です。現在、HCC患者のわずか30％が治癒の可能性の外科的治療2、および全身化学療法の対象となる3有効ではありません。そのため、新たな肝細胞癌治療のための押し満たされていない臨床的必要性、および新しい薬剤の効率的な試験に適したモデル系の開発があります。ニワトリ絨毛尿膜（CAM）アッセイは、再現可能な、費用対効果、及びインビボでの潜在的な抗腫瘍薬を試験する迅速ミディアムスループット方法を提供します。 CAMアッセイは、血管新生4を研究するために広く使用されています。また、正常神経膠芽腫5、6膵臓癌、黒色腫7-9、および骨肉腫10-11含む、癌の腫瘍異種移植片モデルに開発されました。どちらOVO 12とEX卵内13の技術は、プロトコルからのプロトコルに様々な詳細は、文献に利用されてきました。 50％11-14 - CAM異種移植モデルに対する一つの大きな課題は、25に至るまで公表されたニワトリ胚死亡率で、卵の操作後胚死亡の発生率が比較的高いです。 この記事では、我々は確実に組織学的に未分化HCCに似ている三次元の、血管新生化腫瘍の増殖を生産HCCの卵内異種移植モデルの開発について説明します。まずオソウスキら 14によって記載されたプロトコルを適応していると非常に高い腫瘍の生着に93％までのヒヨコ胚の生存率を達成しています。

<table border="0" cellpadding="0" cellspacing="0" width="1094">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:401px;">Name of Reagent/ Equipment</td>
			<td style="width:108px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:467px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:401px;">Premium incubated eggs</td>
			<td style="width:108px;">Charles River</td>
			<td style="width:119px;">N/A</td>
			<td>http://www.criver.com/files/pdfs/avian/av_c_spf_egg_price_list.aspx</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:401px;">Egg incubators</td>
			<td style="width:108px;">GQF</td>
			<td style="width:119px;">Hova-Bator 2362N</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:401px;">Rotating egg trays</td>
			<td style="width:108px;">GQF</td>
			<td style="width:119px;">1611 Automatic Egg Turner</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:401px;">Egg candler</td>
			<td style="width:108px;">Lyon</td>
			<td style="width:119px;">Hi-Power 950-070</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:401px;">Dremel 100 rotary tool with 15/16 cut-off wheel</td>
			<td style="width:108px;">Dremel</td>
			<td style="width:119px;">100-N/7</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:401px;">Sterile forceps, push pin, dissection scissors, Scotch tape</td>
			<td style="width:108px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:401px;">Matrigel</td>
			<td style="width:108px;">BD Biosciences</td>
			<td style="width:119px;">356234</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:401px;">Cryogenic vials, external thread with silicone washer</td>
			<td style="width:108px;">Corning</td>
			<td style="width:119px;">430659</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:401px;">Collagenase from Clostridium histolyticum</td>
			<td style="width:108px;">Sigma</td>
			<td style="width:119px;">C9891</td>
			<td></td>
		</tr>
	</tbody>
</table>

the in ovo chick chorioallantoic membrane (cam) assay as an efficient xenograft model of hepatocellular carcinoma

ヒヨコ漿尿膜（​​CAM）は、CAMはその腫瘍の移植のための理想的なシステム作り、自然免疫及び高度に血管新生である受精後7日目までに開発を開始し、12日目により成熟します。さらに、CAMはその腫瘍の浸潤や転移を研究するための魅力的なモデル作り、フィブロネクチン、ラミニン、コラーゲン、インテグリンα（v）のベータ3、およびMMP-2のような細胞外マトリックスタンパク質が含まれています。科学者は、長い血管形成のモデルとして使用することによってCAMの生理機能の利点をとっています。より最近では、CAMアッセイは、基本的なインビトロでの作業がより複雑な動物癌モデル間のギャップを埋めるために、種々の癌のインビボ異種移植モデルシステムとして動作するように変更されています。 CAMアッセイは、コストと時間が効果的である生物学的に関連するシステムにおける腫瘍増殖、抗腫瘍療法、およびプロ腫瘍分子経路の研究を可能にします。ここでは、CAMのXenの開発を説明最大93％の胚の生存率と肝細胞癌（HCC）のograftモデルと信頼性の腫瘍は、三次元の、血管新生化腫瘍の成長につながる取ります。

プロトコルにおける重要なステップの代表的な画像がここに表示されます。 図1（a）は、胚、気嚢、およびCAMの血管系を可視化するキャンドラーの使用方法を示します。 図1B-1Cは、することによって、CAMをドロップするプロセスを示します二つの穴と、その後の安全電球を使用して負圧を加えると、 図1Dが正常に鉛筆でマークされた穴の下に大きな気泡?...

Watch this Scientific Journal Video about The In Ovo Chick Chorioallantoic Membrane CAM Assay as an Efficient Xenograft Model of Hepatocellular Carcinoma at JoVE.com

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 ...

the-ovo-chick-chorioallantoic-membrane-cam-assay-as-an-efficient

Research

JoVE Journal

Medicine

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

34.9K ビュー.  Icahn School of Medicine at Mount Sinai. 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.

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

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 ...

医学

An Orthotopic Mouse Model of Anaplastic Thyroid Carcinoma

Several types of animal models of human thyroid carcinomas have been established, including subcutaneous xenograft and orthotopic implantation of cancer cells into immunodeficient mice. Subcutaneous xenograft models have been valuable for preclinical screening and evaluation of new therapeutic treatments. There are a number of advantages to using a subcutaneous model; 1) rapid, 2) reproducible, and 3) tumor establishment, growth, and response to therapeutic agents may be monitored by visual inspection. However, substantial evidence has shed light on the short-comings of subcutaneous xenograft models1-3. For instance, medicinal treatments demonstrating curative properties in subcutaneous xenograft models often have no notable impact on the human disease. The microenvironment of the site of xenographic transplantation or injection lies at the heart of this dissimilarity.
Orthotopic tumor xenograft models provide a more biologically relevant context in which to study the disease. The advantages of implanting diseased cells or tissue into their anatomical origin equivalent within a host animal includes a suitable site for tumor-host interactions, development of disease-related metastases and the ability to examine site-specific influence on investigational therapeutic remedies. Therefore, orthotopic xenograft models harbor far more clinical value because they closely reproduce human disease. For these reasons, a number of groups have taken advantage of an orthotopic thyroid cancer model as a research tool4-7.
Here, we describe an approach that establishes an orthotopic model for the study of anaplastic thyroid carcinoma (ATC), which is highly invasive, resists treatment, and is virtually fatal in all diagnosed patients. Cultured ATC cells are prepared as a dissociated cellular suspension in a solution containing a basement membrane matrix. A small volume is slowly injected into the right thyroid gland. Overall appearance and health of the mice are monitored to ensure minimal post-operative complications and to gauge pathological penetrance of the cancer. Mice are sacrificed at 4 weeks, and tissue is collected for histological analysis. Animals may be taken at later time-points to examine more advance progression of the disease. Production of this orthotopic mouse model establishes a platform that accomplishes two objectives: 1) further our understanding of ATC pathology, and 2) screen current and future therapeutic agents for efficacy in combating ATC.

Generation of an orthotopic mouse model of anaplastic thyroid carcinoma is described here. This technique employs surgical placement of human anaplastic thyroid cancer cells into the thyroid of immunodeficient mice, thus creating a more clinically relevant setting to study disease progression as well as screen innovative therapeutic interventions.

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 ...

The In ovo CAM-assay as a Xenograft Model for Sarcoma

Sarcoma is a very rare disease that is heterogeneous in nature, all hampering the development of new therapies. Sarcoma patients are ideal candidates for personalized medicine after stratification, explaining the current interest in developing a reproducible and low-cost xenotransplant model for this disease. The chick chorioallantoic membrane is a natural immunodeficient host capable of sustaining grafted tissues and cells without species-specific restrictions. In addition, it is easily accessed, manipulated and imaged using optical and fluorescence stereomicroscopy. Histology further allows detailed analysis of heterotypic cellular interactions.
This protocol describes in detail the in ovo grafting of the chorioallantoic membrane with fresh sarcoma-derived tumor tissues, their single cell suspensions, and permanent and transient fluorescently labeled established sarcoma cell lines (Saos-2 and SW1353). The chick survival rates are up to 75%. The model is used to study graft- (viability, Ki67 proliferation index, necrosis, infiltration) and host (fibroblast infiltration, vascular ingrowth) behavior. For localized grafting of single cell suspensions, ECM gel provides significant advantages over inert containment materials. The Ki67 proliferation index is related to the distance of the cells from the surface of the CAM and the duration of application on the CAM, the latter determining a time frame for the addition of therapeutic products.

The In ovo CAM-assay as a Xenograft Model for Sarcoma

The in ovo chorioallantoic membrane (CAM) is grafted with fresh sarcoma-derived tumor tissues, their single cell suspensions, and permanent and transient fluorescently labeled established sarcoma cell lines. The model is used to study graft- (viability, Ki67 proliferation index, necrosis, infiltration) and host (fibroblast infiltration, vascular ingrowth) behavior.

ザ<em&gt;卵で</em肉腫のためXenograftmodelとして&gt; CAMアッセイ

Sarcoma is a very rare disease that is heterogeneous in nature, all hampering the development of new therapies. Sarcoma patients are ideal candidates for ...

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  ...

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As such, primary or &#39;neuronal-like&#39; cell culture systems are commonly utilized. While&#160;these systems are relatively easy to work with, and are useful model systems in which various functional outcomes (such as cell death) can be readily quantified, the examined outcomes and pathways in cultured immature neurons (such as excitotoxicity-mediated cell death pathways) are not necessarily the same as those observed in mature brain, or in intact tissue. Therefore, there is the need to develop models in which cellular mechanisms in mature neural tissue can be examined. We have developed an in vitro technique that can be used to investigate a variety of molecular pathways in intact nervous tissue. The technique described herein utilizes rat cortical tissue, but this technique can be adapted to use tissue from a variety of species (such as mouse, rabbit, guinea pig, and chicken) or brain regions (for example, hippocampus, striatum, etc.). Additionally, a variety of stimulations/treatments can be used (for example, excitotoxic, administration of inhibitors, etc.). In conclusion, the brain slice model described herein can be used to examine a variety of molecular mechanisms involved in excitotoxicity-mediated brain injury.

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

We have developed a brain slice model which can be used to examine molecular mechanisms involved in excitotoxicity-mediated brain injury.&#160;This technique generates viable mature brain tissue and reduces animal numbers required for experimentation, whilst keeping the neuronal circuitry, cellular interactions, and postsynaptic compartments partly intact.

脳Microslicesの興奮毒性刺激<em&gt;インビトロ</em&gt;脳卒中のモデル

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As ...

Confocal Time Lapse Imaging as an Efficient Method for the Cytocompatibility Evaluation of Dental Composites

It is generally accepted that in vitro cell material interaction is a useful criterion in the evaluation of dental material biocompatibility. The objective of this study was to use 3D CLSM time lapse confocal imaging to assess the in vitro biocompatibility of dental composites. This method provides an accurate and sensitive indication of viable cell rate in contact with dental composite extracts. The ELS extra low shrinkage, a dental composite used for direct restoration, has been taken as example. In vitro assessment was performed on cultured primary human gingival fibroblast cells using Live/Dead staining. Images were obtained with the FV10i confocal biological inverted system and analyzed with the FV10-ASW 3.1 Software. Image analysis showed a very slight cytotoxicity in the presence of the tested composite after 5 hours of time lapse. A slight decrease of cell viability was shown in contact with the tested composite extracts compared to control cells. The findings highlighted the use of 3D CLSM time lapse imaging as a sensitive method to qualitatively and quantitatively evaluate the biocompatibility behavior of dental composites.

The most studied aspect of the biocompatibility of dental composites is their cytotoxicity 3D CLSM time lapse imaging combined with fluorescent signal quantification allows sensitive evaluation of the cell fate over time. Utilizing this method could be an efficient tool to assess the cytocompatibility of dental composites.

歯科用複合材料の細胞適合性評価のための効率的な方法として、共焦点タイムラプスイメージング

It is generally accepted that in vitro cell material interaction is a useful criterion in the evaluation of dental material biocompatibility. The ...

A "Patient-Like" Orthotopic Syngeneic Mouse Model of Hepatocellular Carcinoma Metastasis

The majority of cancer-related deaths are caused by the metastasis of the cancer rather than the primary tumor itself. Yet, the underlying mechanisms of cancer metastasis are still unclear. Animal models are essential for elucidating the mechanisms and for evaluating novel strategies for the treatment of metastatic cancers. Here, an in-depth description of a &#8220;patient-like&#8221; orthotopic syngeneic mouse model for exploring the mechanisms of metastasis of solid organ tumors is provided. The survival surgical implantation of BNL 1ME A.7R.1 mouse hepatocellular carcinoma cells directly into the liver (the organ of origin) of the inbred wild-type immune competent laboratory mouse strain, BALB/c is described. The success and reproducibility of this methodology recommends it for widespread use in elucidating the biological mechanisms of solid organ cancer metastasis.

A &quot;Patient-Like&quot; Orthotopic Syngeneic Mouse Model of Hepatocellular Carcinoma Metastasis

The metastatic spread of cancer is the major cause of cancer-related deaths. We provide an in-depth description of our survival surgery methodology for establishing a &#8220;patient-like&#8221; orthotopic syngeneic mouse model system for studying the mechanisms of metastasis in solid organ tumors.

肝細胞癌転移の「患者様」同所同系マウスモデル

The majority of cancer-related deaths are caused by the metastasis of the cancer rather than the primary tumor itself. Yet, the underlying mechanisms of ...

Designing CAD/CAM Surgical Guides for Maxillary Reconstruction Using an In-house Approach

Computer-aided design/computer-assisted manufacturing (CAD/CAM) is now being evaluated as a preparative technique for maxillofacial surgery. Because this technique is expensive and available in only limited areas of the world, we developed a novel CAD/CAM surgical guide using an in-house approach. By using the CAD software, the maxillary resection area and cutting planes and the fibular cutting planes and angles are determined. Once the resection area is decided, the necessary faces are extracted using a Boolean modifier. These superficial faces are united to fit the surface of the bones and thickened to stabilize the solids. Not only the cutting guides for the fibula and maxilla but also the location arrangement of the transferred bone segments is defined by thickening the superficial faces. The CAD design is recorded as .stl files and three-dimensionally (3-D) printed as actual surgical guides. To check the accuracy of the guides, model surgery using 3-D-printed facial and fibular models is performed. These methods may be used to assist surgeons where commercial guides are not available.

Methods for designing a computer-aided design/computer-aided manufacturing (CAD/CAM) surgical guide are shown. Cutting planes are separated, united, and thickened to easily visualize the necessary bone transfer. These designs can be three-dimensional printed and checked for accuracy.

社内の取り組みを用いた上顎再建の CAD/CAM サージカル ガイドの設計

Computer-aided design/computer-assisted manufacturing (CAD/CAM) is now being evaluated as a preparative technique for maxillofacial surgery. Because this ...

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and drugs. It is widely used in many studies on the physiological functions of vascular smooth muscle, the pathogenesis of vascular diseases such as hypertension, and the development of smooth muscle relaxant drugs. The mouse is a widely used model animal with a large pool of disease models and genetically modified strains. We introduced this method to measure the isometric contraction of mouse mesenteric artery in detail. A 1.4-mm segment of mouse mesenteric resistance artery was isolated and mounted on a myograph chamber by passing two steel wires through its lumen. After equilibration and normalization steps, the vessel segment was potentiated by a high-K+ solution twice prior to the contraction assay. As an example of the application of this method in drug development, we measured the relaxant effect of a novel natural substance, neoliensinine, isolated from a Chinese herb, embryos of the lotus seed (Nelumbo nucifera Gaertn.) on mouse mesenteric arteries. The vessel segments mounted on the myograph chamber were stimulated with a high-K+ solution. When the force tension reached a stable sustained phase, cumulative doses of neoliensinine were added to the chamber. We found that neoliensinine had a dose-dependent relaxant effect on smooth muscle contraction, thus suggesting that it bears potential activity against hypertension. In addition, as the vessel segment can survive at least 4 hours after mounting and maintain contractility induced by the high-K+ solution for many times, we suggest that the wire myograph system may be used for the time-consuming process of drug screening.

The wire myograph technique is used to investigate vascular smooth muscle functions and screen new drugs. We report a detailed protocol for measuring the isometric contractility of the mouse mesenteric artery and for screening new relaxants of vascular smooth muscle.

ワイヤー図法を使用してマウスの腸間膜動脈の等尺性収縮力測定

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and ...

Predicting Treatment Response to Image-Guided Therapies Using Machine Learning: An Example for Trans-Arterial Treatment of Hepatocellular Carcinoma

Intra-arterial therapies are the standard of care for patients with hepatocellular carcinoma who cannot undergo surgical resection. The objective of this study was to develop a method to predict response to intra-arterial treatment prior to intervention.
The method provides a general framework for predicting outcomes prior to intra-arterial therapy. It involves pooling clinical, demographic and imaging data across a cohort of patients and using these data to train a machine learning model. The trained model is applied to new patients in order to predict their likelihood of response to intra-arterial therapy.
The method entails the acquisition and parsing of clinical, demographic and imaging data from N patients who have already undergone trans-arterial therapies. These data are parsed into discrete features (age, sex, cirrhosis, degree of tumor enhancement, etc.) and binarized into true/false values (e.g., age over 60, male gender, tumor enhancement beyond a set threshold, etc.). Low-variance features and features with low univariate associations with the outcome are removed. Each treated patient is labeled according to whether they responded or did not respond to treatment. Each training patient is thus represented by a set of binary features and an outcome label. Machine learning models are trained using N - 1 patients with testing on the left-out patient. This process is repeated for each of the N patients. The N models are averaged to arrive at a final model.
The technique is extensible and enables inclusion of additional features in the future. It is also a generalizable process that may be applied to clinical research questions outside of interventional radiology. The main limitation is the need to derive features manually from each patient. A popular modern form of machine learning called deep learning does not suffer from this limitation, but requires larger datasets.

Intra-arterial therapies are the standard of care for patients with hepatocellular carcinoma who cannot undergo surgical resection. A method for predicting response to these therapies is proposed. The technique uses pre-procedural clinical, demographic, and imaging information to train machine learning models capable of predicting response prior to treatment.

機械学習を用いた画像誘導治療治療に対する反応を予測: 肝細胞癌における経動脈的治療の例

Intra-arterial therapies are the standard of care for patients with hepatocellular carcinoma who cannot undergo surgical resection. The objective of this ...