著者は、アヴナー膵臓癌財団からの支援を受けています。

すべての手続きは、ニューサウスウェールズ大学動物管理倫理委員会(17/109A)によって承認されました。雌の高胸腺Balb/cヌードマウスは、体重16〜19gの8〜10週齢、このプロトコルに使用された。マウスは、マイクロアイソレーターケージに収容され、市販のペレット化食品および水 のアドリビタムを供給した。
1. 正腸膵臓癌移植
<ol><li>細胞を移植のために準備します。まず、処置に必要な細胞数を計算します(1 x 106 ルシファーサタグ付きAsPC-1細胞と1 x 106 がん関連のヒト膵星細胞[CAhPSC]は各動物に必要です)。<ol>
<li>加湿温度制御CO2 インキュベーターでこれらの細胞を維持し、定期的なマイコプラズマ試験を行います。AsPC-1およびCAhPSCに使用される培地は、RPMI 1640(300mg/L-L-グルタミン、20%v/v胎児ウシ血清、1%v/vペニシリン/ストレプトマイシン)およびIMDM(4 mM L-グルタミン、10%対/胎児ウシ血清、1%v/胎児ウシ血清、1%v/胎児胎児スイシン/ストリン/ストリン/スクレビン/ストリン)です。</li>
		<li>標準的な細胞培養技術を用い、細胞を細胞懸濁液にトリプシン化する。使用するトリプシン溶液の2倍の体積でそれぞれの完全な培養培地を使用してトリプシンを中和する。</li>
		<li>リン酸緩衝生理食塩水(PBS)でこれらの細胞を2回洗浄し、50 μL細胞懸濁液中の1 x 106 AsPC-1細胞と1 x 106 CAhPSCを含む混合物に再懸濁します。</li>
		<li>使用するまで氷の上にこの懸濁液を保管してください。</li>
	</ol></li>
	<li>手順のためのクラスIIバイオセーフティキャビネットを準備します。無菌プラスチックドレープで重ねた加熱マットを使用してください。処置の間の拡大のために、2.5xから3.5倍の拡大の外科ルーペの対を使用する。</li>
	<li>直径1cmの穴をガーゼスワブに切り取って、財布ひも綿棒を準備します。この穴を財布の糸縫い糸で固定します。これのために任意の細かい編み縫合糸(例えば、5/0ポリグリコール酸縫合糸)を使用することができます。編組縫合材料は、締め付けた後に緩い結び目を所定の位置に保ち続けることを可能にするので推奨される。この例を図 1aに示します。</li>
	<li>腹腔内注射により80mg/kgのケタミンと10mg/kgのキシラジンでマウスを麻酔します。</li>
	<li>5 mg/kg エンフロフロキサシン抗生物質予防、2.5 mg/kg フルニシン鎮痛、皮下0.9%の生理食前の1 mLを投与する。</li>
	<li>麻酔をしたら、ステ無菌場にマウスを置き、ポビドンヨウ素を塗布し、続いて70%エタノールを皮膚調製に適用します。</li>
	<li>腹部の左頭蓋象限の皮膚に縦切開を行い、鉗子の間に筋肉層を切開して腹部に入る。</li>
	<li>29 G インシュリンシリンジを 50 μLの細胞懸濁液でロードし、これは1 x 106 の CAhPSC と 1 x 106 ルシメラーゼタグ付き AsPC-1 細胞を注射あたりに相当します。射出装置に取り付けます。この注入装置の設計および機能は 図1b およびその伝説で詳しく説明される。</li>
	<li>財布ひも綿棒を開口切開部の上に置き、この綿棒の開口部を通して脾臓と膵臓の尾を外装します。財布の紐を締めて膵臓の体を優しく囲み、膵臓の尾を露出して注射します。ガーゼが膵臓を周回に接触すると同時に収縮しないように十分にタイトであることが重要です。</li>
	<li>鉗子を使って、膵臓の尾を握り、横張り張りをそっと置きます。腹側腹膜表面を浅い角度で針で穿刺し、注射装置でゆっくりと制御された方法(10-15秒以上)で膵臓に細胞懸濁液を注入する。</li>
	<li>射出プロセス中は、注入部位の周り(逆流から)と膵臓の反対側(貫通貫した浸透の場合)の両方で、漏れについて注意深く観察します。目に見える漏れが発生した場合は、注射を停止し、シリンジ内の残りの注射量を確認して漏出量を確認してください。漏れが少量(<10 μL)の場合は、ガーゼで漏れを吸収し、注射を完了するために針を別の膵臓の小葉に再配置します。</li>
	<li>脾臓と膵臓を交換し、連続的な方法で5/0ポリグリコール酸縫合糸で腹壁を閉じます。クリップでスキンを閉じます。</li>
	<li>麻酔薬から回復するまで、温めたケージの中でマウスを監視します。目を覚まして警告したら、マウスをケージに戻します。</li>
</ol>2. がん切除手術:遠位膵切除術と脾臓摘出術
<ol><li>移植に関する切除のタイミングは、実験プロトコルによって異なる場合があります。一般に、腫瘍は切除前に少なくとも3週間は増殖するが、特定の移植癌細胞株に対して経験的に最適化する。</li>
	<li>切除手術の前日に、動物に生物発光イメージングを行い、局在した原発性腫瘍の存在を確認する。このイメージング研究は、明らかな膵外疾患を有するマウスを切除から除外するために使用されることに注意してください。サイズも放射フラックスも、切除の適格性を決定するための閾値として使用されるべきではありません。<ol>
<li>マウスの重量を量り、腹腔内にD-ルシフェリンを注射する(150mg/kg)。</li>
		<li>ルシフェリン運動曲線の性能により、各実験のルシフェリン注入に関するイメージングステップのタイミングを決定する。放射束が最大の90%を超える期間は、生物発光イメージングに最適な時間を表します(この実験では、18〜26分の注射後)</li>
		<li>麻酔を誘導し、イオブルラン(それぞれ酸素で4%と3%)を使用して維持し、生物発光イメージング装置(例えば、IVIS Lumina II)を用いてイメージングを行う。自動露出とビン分割の設定を使用します(ただし、これは予想される放射束に最適化することができます)。</li>
	</ol>
</li>
	<li>手順のためにクラスIIバイオセーフティキャビネットを準備します。無菌プラスチックドレープで重ねた加熱マットを使用してください。解剖中の倍率については、2.5x〜3.5倍の拡大手術ルーペのペアを使用してください。</li>
	<li>腹腔内注射により80mg/kgのケタミンと10mg/kgのキシラジンでマウスを麻酔します。</li>
	<li>5 mg/kg エンフロフロキサシン抗生物質予防、2.5 mg/kg フルニシン鎮痛、皮下0.9%の生理食前の1 mLを投与する。</li>
	<li>ステ無菌場にマウスを置き、ポビドンヨウ素を塗布し、続いて70%エタノールを皮膚調製に適用します。</li>
	<li>腹部の左頭蓋象限の皮膚に縦切開を行い、好ましくは前の切開部位を通して行う。</li>
	<li>下の筋肉の腹壁から皮膚を鈍く解剖し、皮膚の傷を開いたままにするためにAlm自己保持レトラクターを置く。</li>
	<li>前の操作の縫合線の片側に鉗子間の筋肉層を切開し、切開を延長して前の縫合線全体を切除する。</li>
	<li>脾臓と遠位膵臓を外装し、目ざまにそれを引き込む。膵臓の尾大の側面では、結腸は、フィルム接着によって付着して見つかり得る。これが見つかった場合は、コロンをぶっきらぼうに解剖します。</li>
	<li>慎重に膵臓と脾臓の容器の体に足を引く鉗子のペアを渡し、このスペースを開きます。これは、後の結紮のために膵臓のセグメントを解放します。</li>
	<li>膵臓の体をチタン結紮クリップで腫瘍に近似させ、これに近い膵臓を焼灼で伝達する。膵切り株を制御する別の方法は、横断術前に5/0ポリグリコール酸縫合糸で連続性にそれを液化することです。</li>
	<li>膵臓を引き込み、脾臓の頭蓋極と胃の間の胃血管を焼灼する。</li>
	<li>標本を取り除き、血体止を確認します。</li>
	<li>5/0ポリグリコール酸縫合糸で腹壁を閉じます。クリップでスキンを閉じます。</li>
</ol>3. 術後管理
<ol><li>即時麻酔後期間(上記の両方の手順)で、麻酔薬から回復するまで温めたケージでマウスを監視します。目を覚まして警告したら、マウスをケージに戻します。術後の期間に、動物の痛みや苦痛の兆候を監視します。0.05 mg/kg ブプレノルフィンを皮下注射で投与し、動物を12時間注意深く観察する。</li>
	<li>その後、体重、食物摂取および活性について毎日マウスを監視する。腫瘍の大きさについて切開部位と触診部位を調べる。術後7日目にスキンクリップを取り外します。</li>
	<li>人道的エンドポイントに到達した場合は、マウスを安楽死させる。これらの人道的エンドポイントには、体重>20%の喪失、治療不可能な苦痛の特徴(ハンチ姿勢、動きまたはグルーミングの欠如を含む)および外部触診によって推定される1cm3 を超える腫瘍サイズが含まれる。</li>
</ol>

<ol>
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P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hepatocyte+growth+factor+inhibition:+a+novel+therapeutic+approach+in+pancreatic+cancer.">Hepatocyte growth factor inhibition: a novel therapeutic approach in pancreatic cancer.</a> British Journal of Cancer. 114 (3), 269-280 (2016).</li><li>Giri, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+Immunocompetent+Model+of+Pancreatic+Cancer+Resection+and+Recurrence.">An Immunocompetent Model of Pancreatic Cancer Resection and Recurrence.</a> Journal of Gastrointestinal Surgery. , (2020).</li><li>Allen, V. B., Gurusamy, K. S., Takwoingi, Y., Kalia, A., Davidson, B. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diagnostic+accuracy+of+laparoscopy+following+computed+tomography+(CT)+scanning+for+assessing+the+resectability+with+curative+intent+in+pancreatic+and+periampullary+cancer.">Diagnostic accuracy of laparoscopy following computed tomography (CT) scanning for assessing the resectability with curative intent in pancreatic and periampullary cancer.</a> Cochrane Database of Systematic Reviews. (11), 009323 (2013).</li><li>Vaillant, F., Lindeman, G. J., Visvader, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Jekyll+or+Hyde:+does+Matrigel+provide+a+more+or+less+physiological+environment+in+mammary+repopulating+assays.">Jekyll or Hyde: does Matrigel provide a more or less physiological environment in mammary repopulating assays.</a> Breast Cancer Research. 13 (3), 108 (2011).</li><li>Jesnowski, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immortalization+of+pancreatic+stellate+cells+as+an+in+vitro+model+of+pancreatic+fibrosis:+deactivation+is+induced+by+matrigel+and+N-acetylcysteine.">Immortalization of pancreatic stellate cells as an in vitro model of pancreatic fibrosis: deactivation is induced by matrigel and N-acetylcysteine.</a> Laboratory Investigation. 85 (10), 1276-1291 (2005).</li><li>Phillips, P. 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=Cell+migration:+a+novel+aspect+of+pancreatic+stellate+cell+biology.">Cell migration: a novel aspect of pancreatic stellate cell biology.</a> Gut. 52 (5), 677-682 (2003).</li><li>Boj, S. F., 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=Organoid+models+of+human+and+mouse+ductal+pancreatic+cancer.">Organoid models of human and mouse ductal pancreatic cancer.</a> Cell. 160 (1-2), 324-338 (2015).</li><li>Egberts, J. 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=Superiority+of+extended+neoadjuvant+chemotherapy+with+gemcitabine+in+pancreatic+cancer:+a+comparative+analysis+in+a+clinically+adapted+orthotopic+xenotransplantation+model+in+SCID+beige+mice.">Superiority of extended neoadjuvant chemotherapy with gemcitabine in pancreatic cancer: a comparative analysis in a clinically adapted orthotopic xenotransplantation model in SCID beige mice.</a> Cancer Biology &amp; Therapy. 6 (8), 1227-1232 (2007).</li></ol>

著者らは、このプロジェクトに関して開示するものは何もありません。

膵臓癌の切除整形マウスモデルは、アジュバントおよびネオアジュバント治療の検査を可能にするため重要である。これは、手術が最も効果的な治療であり続けるが、再発のリスクが高いと関連している膵臓癌において特に重要である。本論文では、切除術で治癒可能な膵臓癌を確実に産生する方法について述べ、ネオアジュバント/アジュバント療法が必要な臨床シナリオを複製する。
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膵管腺癌(膵癌[PC])は予後不良1と関連している。外科的切除は、PCの唯一の潜在的治癒的治療であり、早期疾患を呈する患者に対して考慮されるべきである。残念なことに、R0切除術(すなわち、腫瘍のない切除マージン)を有しても、再発率(局所または未検出の転移性疾患から)は2、3である。したがって、全身アジュバント療法は、切除4を受けるほぼすべての患者に示される。さらに、ネオアジュバント療法は現在、境界分化可能な癌に対してのみ推奨されているが、その徴候は、その日常的な使用が多くの臨床研究5、6、7、8の焦点となるほど拡大している。切除を伴うPCの新しい治療アプローチを開発するためには、これらのアプローチは、臨床現場を正確に再現する前臨床モデルで最初に評価される必要があります。
PCの異所性マウスモデルは、薬物治療9、10をテストするために過去に頻繁に使用されてきた。これらの多くは、マウス膵臓に癌細胞を単独で注入することによって産生され、PCの特徴である顕著な間質を欠いた腫瘍をもたらした。さらに最近では、ヒトPCとヒト膵星細胞(PSC、PC中のコラーゲン質素の主要な生産者)の混合物を注入して最初に開発したもののような共噴射装具モデルが、11,12で定期的に使用されるようになってきた。癌と間質細胞の併用による腫瘍は、(i)PCの癌元素と特徴的な間質(デスモプラスチック)成分の両方を示し、かつ(ii)癌細胞増殖および転移を増強した。したがって、このモデルは人間のPCによく似ています。多くの整形体PCの切除モデルは13、14、15、16と記載されているが、このモデルほど正確なヒトにおける膵切除の臨床的実態を反映するものは一切ないので、アジュバントまたはネオアジュバント治療の検査に最適ではない。
提示されたマウスモデルの目的は、(i)不注意な腹膜播種を最小限に抑えながら、正腸膵臓癌を移植することに成功し、(ii)その後癌を完全に切断する方法を実証することであった。この論文は、この技術のヒントと潜在的な落とし穴を強調しています。

<table>
	<tbody>
		<tr>
			<td>Animals, Materials and Equipment for Implantation Procedure</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>AsPC-1 human pancreatic cancer cell line, luciferase tagged (luc+ gene from Promega PGL3 Basic plasmid)</td>
			<td>American Type Culture Collection, Manassas, VA, USA</td>
			<td></td>
			<td>supplied by Professor Takashi Murakami, Saitama Medical University, Saitama, Japan</td>
		</tr>
		<tr>
			<td>Autoclip wound clips, 9 mm</td>
			<td>Becton Dickson Pty Ltd, North Ryde, NSW, Australia</td>
			<td>500346</td>
			<td></td>
		</tr>
		<tr>
			<td>Basic Dressing Pack</td>
			<td>Multigate Medical Products Pty Ltd, Villawood, NSW, Australia</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cancer associated human pancreatic stellate cells</td>
			<td>Pancreatic Research Group cell bank</td>
			<td></td>
			<td>In house cell bank</td>
		</tr>
		<tr>
			<td>Cryogenic tubes, 1.0 mL</td>
			<td>Thermo Fisher Scientific Australia Pty Ltd, Scoresby, VIC, Australia</td>
			<td>366656</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable stainless-steel scalpel blade with handle, size 15</td>
			<td>Livingstone International, Mascot, NSW,</td>
			<td>SCP15</td>
			<td></td>
		</tr>
		<tr>
			<td>Foetal bovine serum (FBS)</td>
			<td>Life Technologies Corporation, Tullamarine, VIC, Australia</td>
			<td>16000044</td>
			<td></td>
		</tr>
		<tr>
			<td>Gilles fine tooth forceps 12 cm</td>
			<td></td>
			<td></td>
			<td>Generic stainless steel microsurgical instrument set</td>
		</tr>
		<tr>
			<td>Heated mats to maintain body temperature during surgery and postoperative recovery</td>
			<td>Generic</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Homozygous athymic nude mice: Strain BALB/c-Fox1nu/Ausb, female</td>
			<td>Australian Bioresources, Moss Vale, NSW, Australia</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Iscove&#39;s modified Dulbecco&#39;s medium (IMDM) with 4mM L-glutamine and no phenol red</td>
			<td>Life Technologies Corporation, Tullamarine, VIC, Australia</td>
			<td>21056023</td>
			<td></td>
		</tr>
		<tr>
			<td>Jewellers forceps 11.5 cm</td>
			<td></td>
			<td></td>
			<td>Generic stainless steel microsurgical instrument set</td>
		</tr>
		<tr>
			<td>Micro needle holder (round handle) 15 cm straight</td>
			<td></td>
			<td></td>
			<td>Generic stainless steel microsurgical instrument set</td>
		</tr>
		<tr>
			<td>Micro scissors (round handle) 15 cm straight</td>
			<td></td>
			<td></td>
			<td>Generic stainless steel microsurgical instrument set</td>
		</tr>
		<tr>
			<td>Penicillin 10,000 U/mL, streptomycin 10,000 &#956;g/mL</td>
			<td>Life Technologies Corporation, Tullamarine, VIC, Australia</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyglycolic acid suture, size USP 5/0 on 13mm half-circle round-bodied needle</td>
			<td>Braun Australia Pty Ltd, Bella Vista, NSW, Australia</td>
			<td>C1049407</td>
			<td></td>
		</tr>
		<tr>
			<td>Portable weighing scale</td>
			<td>Precision balances, Bradford, MA, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Reflex clip applier and clip remover</td>
			<td>World Precision Instruments, Sarasota, FL, USA</td>
			<td>500345</td>
			<td></td>
		</tr>
		<tr>
			<td>Roswell Park Memorial Institute (RPMI) 1640 with phenol red and 300 mg/L Lglutamine</td>
			<td>Life Technologies Corporation, Tullamarine, VIC, Australia</td>
			<td>11875085</td>
			<td></td>
		</tr>
		<tr>
			<td>Round bodied vessel dilator 15 cm, 0.1 mm tip</td>
			<td></td>
			<td></td>
			<td>Generic stainless steel microsurgical instrument set</td>
		</tr>
		<tr>
			<td>Trypsin 0.05%, EDTA 0.02%</td>
			<td>Life Technologies Corporation, Tullamarine, VIC, Australia</td>
			<td>25300054</td>
			<td>For pancreatic stellate cells</td>
		</tr>
		<tr>
			<td>Trypsin 0.25%, EDTA 0.02%</td>
			<td>Life Technologies Corporation, Tullamarine, VIC, Australia</td>
			<td>25200056</td>
			<td>For ASPC-1 cells</td>
		</tr>
		<tr>
			<td>U-100 insulin syringes, 0.5 mL with 29 G (0.33 mm) &#215; 13 mm needle</td>
			<td>Terumo Medical Corporation, Elkton, MD, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Equipment for Resection Procedure</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Alm self-retaining retractor</td>
			<td></td>
			<td></td>
			<td>Generic stainless steel microsurgical instrument set</td>
		</tr>
		<tr>
			<td>Autoclip wound clips 9 mm</td>
			<td>Becton Dickson Pty Ltd, North Ryde, NSW</td>
			<td>500346</td>
			<td></td>
		</tr>
		<tr>
			<td>Basic Dressing Pack</td>
			<td>Multigate Medical Products Pty Ltd, Villawood, NSW, Australia</td>
			<td>08-559NP</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable stainless-steel scalpel blade with handle, size 15</td>
			<td>Livingstone International, Mascot, NSW,</td>
			<td>SCP15</td>
			<td></td>
		</tr>
		<tr>
			<td>Gilles fine tooth forceps 12 cm</td>
			<td></td>
			<td></td>
			<td>Generic stainless steel microsurgical instrument set</td>
		</tr>
		<tr>
			<td>Hand-held high temperature fine tip cautery</td>
			<td>Bovie Medical Corporation, Melville, NY, USA</td>
			<td>AA01</td>
			<td></td>
		</tr>
		<tr>
			<td>Heated mats to maintain body temperature during surgery and postoperative recovery</td>
			<td>Generic</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>IVIS Lumina II Bioluminescent Imaging Device</td>
			<td>Caliper Life Sciences, Hopkinton, MA, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Jewellers forceps 11.5 cm</td>
			<td></td>
			<td></td>
			<td>Generic stainless steel microsurgical instrument set</td>
		</tr>
		<tr>
			<td>Micro needle holder (round handle) 15 cm straight</td>
			<td></td>
			<td></td>
			<td>Generic stainless steel microsurgical instrument set</td>
		</tr>
		<tr>
			<td>Micro scissors (round handle) 15 cm straight</td>
			<td></td>
			<td></td>
			<td>Generic stainless steel microsurgical instrument set</td>
		</tr>
		<tr>
			<td>Polyglycolic acid suture, size USP 5/0 on 13mm half-circle round-bodied needle</td>
			<td>Braun Australia Pty Ltd, Bella Vista, NSW, Australia</td>
			<td>C1049407</td>
			<td></td>
		</tr>
		<tr>
			<td>Portable weighing scale</td>
			<td>Precision balances, Bradford, MA, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Reflex wound clip applier and clip remover</td>
			<td>World Precision Instruments, Sarasota, FL, USA</td>
			<td>500345</td>
			<td></td>
		</tr>
		<tr>
			<td>Round bodied vessel dilator 15 cm, 0.1 mm tip</td>
			<td></td>
			<td></td>
			<td>Generic stainless steel microsurgical instrument set</td>
		</tr>
		<tr>
			<td>Titanium &#8220;Weck style&#8221; Ligaclip, small</td>
			<td>HZMIM, Hangzhou, China</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Titanium Ligaclip applier for open surgery, small</td>
			<td>HZMIM, Hangzhou, China</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Volatile anaesthetic machine, including vapouriser and induction chamber</td>
			<td>Generic</td>
			<td></td>
			<td>Generic vapouriser and induction chamber</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Drugs for Procedures</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>70% w/w ethanol solution</td>
			<td>Sigma-Aldrich Pty Ltd, Castle Hill, NSW, Australia</td>
			<td></td>
			<td>Applied topically as surgical skin preparation</td>
		</tr>
		<tr>
			<td>Buprenorphine 0.3 mg/mL</td>
			<td>Troy Laboratories Pty Ltd, Glendenning, NSW, Australia</td>
			<td></td>
			<td>Dose: 0.05 mg/kg s.c.</td>
		</tr>
		<tr>
			<td>D-Luciferin (1 U/g)</td>
			<td>PerkinElmer, Inc., Waltham, MA, USA</td>
			<td>122799</td>
			<td>diluted in PBS to 15 mg/mL. Dose: 150 mg/kg i.p</td>
		</tr>
		<tr>
			<td>Enrofloxacin 50 mg/mL</td>
			<td>Troy Laboratories Pty Ltd, Glendenning, NSW, Australia</td>
			<td></td>
			<td>Dose: 5 mg/kg s.c.</td>
		</tr>
		<tr>
			<td>Flunixin 50 mg/mL</td>
			<td>Norbrook Laboratories Australia, Tullamarine, VIC, Australia</td>
			<td></td>
			<td>Dose: 2.5 mg/kg s.c.</td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Zoetis Australia Pty Ltd., Rhodes, NSW, Australia</td>
			<td></td>
			<td>Dose (vapourised with oxygen): 4% induction, 3% maintenance</td>
		</tr>
		<tr>
			<td>Ketamine 100 mg/mL</td>
			<td>Maylab, Slacks Creek, QLD, Australia</td>
			<td></td>
			<td>Dose: 80 mg/kg i.p.</td>
		</tr>
		<tr>
			<td>Povidone-Iodine 10% w/v solution</td>
			<td>Perrigo Australia, Balcatta, WA, Australia</td>
			<td>RIO00802F</td>
			<td>Applied topically to the anterior abdomen as surgical skin preparation</td>
		</tr>
		<tr>
			<td>Refresh eye ointment (liquid paraffin 42.5% w/w, soft white paraffin 57.3% w/w)</td>
			<td>Allergan Australia Pty Ltd, Gordon, NSW, Australia</td>
			<td></td>
			<td>Applied to both eyes</td>
		</tr>
		<tr>
			<td>Sodium chloride 0.9% w/v</td>
			<td>Braun Australia Pty Ltd, Bella Vista, NSW, Australia</td>
			<td>9481P</td>
			<td>Dose: 900 &#956;L s.c.</td>
		</tr>
		<tr>
			<td>Water for injections BP</td>
			<td>Pfizer Australia, Sydney, NSW, Australia</td>
			<td></td>
			<td>For dilution of drugs</td>
		</tr>
		<tr>
			<td>Xylazine 20 mg/mL</td>
			<td>Troy Laboratories Pty Ltd, Glendenning, NSW, Australia</td>
			<td></td>
			<td>Dose: 10 mg/kg i.p.</td>
		</tr>
	</tbody>
</table>

an orthotopic resectional mouse model of pancreatic cancer

膵臓癌(PC)の手術を検討されている患者のアジュバントおよび/またはネオアジュバント療法を研究するための満足のいく動物モデルの欠如がある。この不全に対処するために、PCの異形移植を伴うマウスモデルを記述し、続いて遠位汎化切欠切および脾臓摘出術を行う。このモデルは、アジュバントおよびネオアジュバントの設定における様々な治療アプローチの研究に対して、安全かつ適切に柔軟であることが実証されています。
このモデルでは、膵臓腫瘍は、まず、ヒト膵臓癌細胞(ルシファーサタグ付きAsPC-1)とヒト癌関連膵星形細胞をBalb/cアチミックヌードマウスの遠位膵臓に移植することによって生成される。3週間後、癌は再ラパロトミー、遠位汎腎切除術および脾臓摘出術によって切除される。このモデルでは、生物発光イメージングを使用して、がんの発症の進行と切除/治療の効果に従うことができます。切除後、アジュバント療法を行うことができる。あるいは、切除前にネオアジュバント治療を施すことができる。
45匹のマウスからの代表的なデータが提示される。すべてのマウスは、止まり止めの問題のない遠位汎化切欠切術/脾臓摘出術に成功した。5mmを超える巨視近位膵臓マージンは43(96%)で達成されたマウス。膵切除の技術的成功率は100%で、早期死亡率と罹患率は0%であった。切除後の週に動物は誰も死ななかった。
要約すると、臨床シナリオを模倣したマウスにおける膵臓癌の外科的切除モデルに対する堅牢で再現可能な技術について述べた。このモデルは、アジュバントおよびネオアジュバントの両方の治療のテストに有用である可能性があります。

59匹の連続したマウスが移植手術を受けた。8件で発生した漏れ総件数(14%)マウス。注入時の漏出の程度は、プロトコルセクションで上述したように推定される。これらの移植された腫瘍が成長することを可能にする3週間後、切除前に大転移性疾患を有するマウスを除外するために前切除生物発光イメージングを行った。45人(76%)マウスは外科的切除を受けた。
全45(100%)マ?...

Watch this Scientific Journal Video about An Orthotopic Resectional Mouse Model of Pancreatic Cancer at JoVE.com

An Orthotopic Resectional Mouse Model of Pancreatic Cancer

臨床文脈では、局所性膵臓癌の患者は汎膵切除術を受け、続いてアジュバント治療を受ける。ここで報告されるこのプロトコルは、膵臓癌の正体性移植を経て、遠位汎化切欠切術および脾臓摘出術を通じて、ヌードマウスでこの臨床シナリオをモデル化する安全で効果的な方法を確立することを目的としている。

膵臓癌の正交性切除マウスモデル

There is a lack of satisfactory animal models to study adjuvant and/or neoadjuvant therapy in patients being considered for surgery of pancreatic cancer ...

an-orthotopic-resectional-mouse-model-of-pancreatic-cancer

Research

JoVE Journal

Cancer Research

10.0K ビュー.  University of New South Wales. 臨床文脈では、局所性膵臓癌の患者は汎膵切除術を受け、続いてアジュバント治療を受ける。ここで報告されるこのプロトコルは、膵臓癌の正体性移植を経て、遠位汎化切欠切術および脾臓摘出術を通じて、ヌードマウスでこの臨床シナリオをモデル化する安全で効果的な方法を確立することを目的としている。

ビデオ: An Orthotopic Resectional Mouse Model of Pancreatic Cancer

Fluorescent Orthotopic Mouse Model of Pancreatic Cancer

Pancreatic cancer remains one of the cancers for which survival has not improved substantially in the last few decades. Only 7% of diagnosed patients will survive longer than five years. In order to understand and mimic the microenvironment of pancreatic tumors, we utilized a murine orthotopic model of pancreatic cancer that allows non-invasive imaging of tumor progression in real time. Pancreatic cancer cells expressing green fluorescent protein (PANC-1 GFP) were suspended in basement membrane matrix, high concentration, (e.g., Matrigel HC) with serum-free media and then injected into the tail of the pancreas via laparotomy. The cell suspension in the high concentration basement membrane matrix becomes a gel-like substance once it reaches room temperature; therefore, it gels when it comes in contact with the pancreas, creating a seal at the injection site and preventing any cell leakage. Tumor growth and metastasis to other organs are monitored in live animals by using fluorescence. It is critical to use the appropriate filters for excitation and emission of GFP. The steps for the orthotopic implantation are detailed in this article so researchers can easily replicate the procedure in nude mice. The main steps of this protocol are preparation of the cell suspension, surgical implantation, and whole body fluorescent in vivo imaging. This orthotopic model is designed to investigate the efficacy of novel therapeutics on primary and metastatic tumors.

A procedure to implant green fluorescent protein-expressing pancreatic cancer cells (PANC-1 GFP) orthotopically into the pancreas of Balb-c Ola Hsd-Fox1nu mice to assess tumor progression and metastasis is presented here.

膵臓がんの蛍光同所性マウスモデル

Pancreatic cancer remains one of the cancers for which survival has not improved substantially in the last few decades. Only 7% of diagnosed patients will ...

がん研究

Isolation of Circulating Tumor Cells in an Orthotopic Mouse Model of Colorectal Cancer

Despite the advantages of easy applicability and cost-effectiveness, subcutaneous mouse models have severe limitations and do not accurately simulate tumor biology and tumor cell dissemination. Orthotopic mouse models have been introduced to overcome these limitations; however, such models are technically demanding, especially in hollow organs such as the large bowel. In order to produce uniform tumors which reliably grow and metastasize, standardized techniques of tumor cell preparation and injection are critical. 
We have developed an orthotopic mouse model of colorectal cancer (CRC) which develops highly uniform tumors and can be used for tumor biology studies as well as therapeutic trials. Tumor cells from either primary tumors, 2-dimensional (2D) cell lines or 3-dimensional (3D) organoids are injected into the cecum and, depending on the metastatic potential of the injected tumor cells, form highly metastatic tumors. In addition, CTCs can be found regularly. We here describe the technique of tumor cell preparation from both 2D cell lines and 3D organoids as well as primary tumor tissue, the surgical and injection techniques as well as the isolation of CTCs from the tumor-bearing mice, and present tips for troubleshooting.

We describe the establishment of orthotopic colorectal tumors via injection of tumor cells or organoids into the cecum of mice and the subsequent isolation of circulating tumor cells (CTCs) from this model.

結腸直腸癌モデルマウスにおける循環腫瘍細胞の単離

Despite the advantages of easy applicability and cost-effectiveness, subcutaneous mouse models have severe limitations and do not accurately simulate ...

A Syngeneic Pancreatic Cancer Mouse Model to Study the Effects of Irreversible Electroporation

Pancreatic cancer (PC), a disease which kills approximately 40,000 patients each year in the US, has successfully evaded several therapeutic approaches including the promising immunotherapeutic strategies. Irreversible electroporation (IRE) is a non-thermal ablation technique that induces tumor cell death without destruction of adjacent collagenous structures, thus enabling the procedure to be performed in tumors very close to blood vessels. Unlike thermal ablation techniques, IRE results in gradual apoptotic cell death, along with immediate ablation induced necrosis, and is currently in clinical use for selected patients with locally advanced PC. An ablative, non-target specific procedure like IRE can induce a myriad of responses in the tumor microenvironment. A few studies have addressed the effects of IRE on tumor growth in other tumor types, but none have focused on PC. We have developed a syngeneic mouse model of PC in which subcutaneous (SQ) and orthotopic tumors can be successfully treated with IRE in a highly controlled setting, facilitating various longitudinal studies post procedure. This animal model serves as a robust system to study the effects of IRE and ways to improve the clinical efficacy of IRE.

Irreversible electroporation (IRE) is a non-thermal ablation technique used for the treatment of locally advanced pancreatic cancer. Being a relatively new technique, the effects of IRE on the tumor growth are poorly understood. We have developed a syngeneic mouse model that facilitates studying the effects of IRE on pancreatic cancer.

不可逆エレクトロポレーションの効果を研究する同系膵癌マウス モデル

Pancreatic cancer (PC), a disease which kills approximately 40,000 patients each year in the US, has successfully evaded several therapeutic approaches ...

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 ...

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 ...

An Oncogenic Hepatocyte-Induced Orthotopic Mouse Model of Hepatocellular Cancer Arising in the Setting of Hepatic Inflammation and Fibrosis

The absence of a clinically relevant animal model addressing the typical immune characteristics of hepatocellular cancer (HCC) has significantly impeded elucidation of the underlying mechanisms and development of innovative immunotherapeutic strategies. To develop an ideal animal model recapitulating human HCC, immunocompetent male C57BL/6J mice first receive a carbon tetrachloride (CCl4) injection to induce liver fibrosis, then receive histologically-normal oncogenic hepatocytes from young male SV40 T antigen (TAg)-transgenic mice (MTD2) by intra-splenic (ISPL) inoculation. Androgen generated in recipient male mice at puberty initiates TAg expression under control of a liver-specific promoter. As a result, the transferred hepatocytes become cancer cells and form tumor masses in the setting of liver fibrosis/cirrhosis. This novel model mimics human HCC initiation and progression in the context of liver fibrosis/cirrhosis and reflects the most typical features of human HCC including immune dysfunction.

Here, we describe the development of a clinically relevant murine model of liver cancer recapitulating the typical immune features of hepatocellular cancer (HCC).

肝炎症と線維症の設定に起因する肝細胞癌の腫瘍性肝細胞誘発性オルコトピックマウスモデル

The absence of a clinically relevant animal model addressing the typical immune characteristics of hepatocellular cancer (HCC) has significantly impeded ...

Studying Triple Negative Breast Cancer Using Orthotopic Breast Cancer Model

Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype with limited therapeutic options. When compared to patients with less aggressive breast tumors, the 5-year survival rate of TNBC patients is 77% due to their characteristic drug-resistant phenotype and metastatic burden. Toward this end, murine models have been established aimed at identifying novel therapeutic strategies limiting TNBC tumor growth and metastatic spread. This work describes a practical guide for the TNBC orthotopic model where MDA-MB-231 breast cancer cells suspended in a basement membrane matrix are implanted in the fourth mammary fat pad, which closely mimics the cancer cell behavior in humans. Measurement of tumors by caliper, lung metastasis assessment via in vivo and ex vivo imaging, and molecular detection are discussed. This model provides an excellent platform to study therapeutic efficacy and is especially suitable for the study of the interaction between the primary tumor and distal metastatic sites.

This work presets an advanced protocol to accurately assess tumor loading by detection of green fluorescent protein and bioluminescence signals as well as the integration of quantitative molecular detection technique.

交感性乳癌モデルを用いたトリプルネガティブ乳癌の研究

Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype with limited therapeutic options. When compared to patients with less ...

Ultrasound-Guided Orthotopic Implantation of Murine Pancreatic Ductal Adenocarcinoma

The recent success of immune checkpoint blockade in melanoma and lung adenocarcinoma has galvanized the field of immuno-oncology as well as revealed the limitations of current treatments, as the majority of patients do not respond to immunotherapy. Development of accurate preclinical models to quickly identify novel and effective therapeutic combinations are critical to address this unmet clinical need. Pancreatic ductal adenocarcinoma (PDA) is a canonical example of an immune checkpoint blockade resistant tumor with only 2% of patients responding to immunotherapy. The genetically engineered KrasG12D+/-;Trp53R172H+/-;Pdx-1 Cre (KPC) mouse model of PDA recapitulates human disease and is a valuable tool for assessing therapies for immunotherapy resistant in the preclinical setting, but time to tumor onset is highly variable. Surgical orthotopic tumor implantation models of PDA maintain the immunobiological hallmarks of the KPC tissue-specific tumor microenvironment (TME) but require a time-intensive procedure and introduce aberrant inflammation. Here, we use an ultrasound-guided orthotopic tumor implantation model (UG-OTIM) to non-invasively inject KPC-derived PDA cell lines directly into the mouse pancreas. UG-OTIM tumors grow in the endogenous tissue site, faithfully recapitulate histological features of the PDA TME, and reach enrollment-sized tumors for preclinical studies by four weeks after injection with minimal seeding on the peritoneal wall. The UG-OTIM system described here is a rapid and reproducible tumor model that may allow for high throughput analysis of novel therapeutic combinations in the murine PDA TME.

We describe a protocol for the ultrasound guided implantation of murine-derived pancreatic ductal adenocarcinoma cell lines directly into the native tumor site. This approach resulted in pancreatic tumors detectable by ultrasound scanning within 2-4 weeks of injection, and significantly reduced the proportion tumor cell seeding on the peritoneal wall as compared to surgical orthotopic implantation.

マウス膵管腺癌の超音波誘導矯正着移植

The recent success of immune checkpoint blockade in melanoma and lung adenocarcinoma has galvanized the field of immuno-oncology as well as revealed the ...

Generation of Orthotopic Pancreatic Tumors and Ex vivo Characterization of Tumor-Infiltrating T Cell Cytotoxicity

In vivo models of pancreatic cancer provide invaluable tools for studying disease dynamics, immune infiltration and new therapeutic strategies. The orthotopic murine model can be performed on large cohorts of immunocompetent mice simultaneously, is relatively inexpensive and preserves the cognate tissue microenvironment. The quantification of T cell infiltration and cytotoxic activity within orthotopic tumors provides a useful indicator of an antitumoral response.
This protocol describes the methodology for surgical generation of orthotopic pancreatic tumors by injection of a low number of syngeneic tumor cells resuspended in 5 &#181;L basement membrane directly into the pancreas. Mice bearing orthotopic tumors take approximately 30 days to reach endpoint, at which point tumors can be harvested and processed for characterization of tumor-infiltrating T cell activity. Rapid enzymatic digestion using collagenase and DNase allows a single-cell suspension to be extracted from tumors. The viability and cell surface markers of immune cells extracted from the tumor are preserved; therefore, it is appropriate for multiple downstream applications, including flow-assisted cell sorting of immune cells for culture or RNA extraction, flow cytometry analysis of immune cell populations. Here, we describe the ex vivo stimulation of T cell populations for intracellular cytokine quantification (IFN&#947; and TNF&#945;) and degranulation activity (CD107a) as a measure of overall cytotoxicity. Whole-tumor digests were stimulated with phorbol myristate acetate and ionomycin for 5 h, in the presence of anti-CD107a antibody in order to upregulate cytokine production and degranulation. The addition of brefeldin A and monensin for the final 4 h was performed to block extracellular transport and maximize cytokine detection. Extra- and intra-cellular staining of cells was then performed for flow cytometry analysis, where the proportion of IFN&#947;+, TNF&#945;+ and CD107a+ CD4+ and CD8+ T cells was quantified.
This method provides a starting base to perform comprehensive analysis of the tumor microenvironment.

Generation of Orthotopic Pancreatic Tumors and Ex vivo Characterization of Tumor-Infiltrating T Cell Cytotoxicity

This protocol describes the surgical generation of orthotopic pancreatic tumors and the rapid digestion of freshly isolated murine pancreatic tumors. Following digestion, viable immune cell populations can be used for further downstream analysis, including ex vivo stimulation of T cells for intracellular cytokine detection by flow cytometry.

直交トピック膵腫瘍の生成と腫瘍浸潤T細胞細胞傷害のEx生体内特性評価

In vivo models of pancreatic cancer provide invaluable tools for studying disease dynamics, immune infiltration and new therapeutic strategies. The ...

Generation of an Orthotopic Xenograft of Pancreatic Cancer Cells by Ultrasound-Guided Injection

Pancreatic cancer (PCa) represents one of the deadliest cancer types worldwide. The reasons for PCa malignancy mainly rely on its intrinsic malignant behavior and high resistance to therapeutic treatments. Indeed, despite many efforts, both standard chemotherapy and innovative target therapies have substantially failed when moved from preclinical evaluation to the clinical setting. In this scenario, the development of preclinical mouse models better mimicking in vivo characteristics of PCa is urgently needed to test newly developed drugs. The present protocol describes a method to generate a mouse model of PCa, represented by an orthotopic xenograft obtained by ultrasound-guided injection of human pancreatic tumor cells. Using such a reliable and minimally invasive protocol, we also provide evidence of in vivo engraftment and development of tumor masses, which can be monitored by ultrasound (US) imaging. A noteworthy aspect of the PCa model described here is the slow development of the tumor masses over time, which allows precise identification of the starting point for pharmacological treatments and better monitoring of the effects of therapeutic interventions. Moreover, the technique described here is an example of implementation of the 3Rs principles since it minimizes pain and suffering and directly improves the welfare of animals in research.

We present a protocol to generate a minimally invasive orthotopic pancreatic cancer model by ultrasound-guided injection of human pancreatic cancer cells and the subsequent monitoring of tumor growth in vivo by ultrasound imaging.

超音波ガイド下注射による膵臓癌細胞の同所性異種移植片の作製

Pancreatic cancer (PCa) represents one of the deadliest cancer types worldwide. The reasons for PCa malignancy mainly rely on its intrinsic malignant ...