Name: an orthotopic resectional mouse model of pancreatic cancer
Uploaded: 2020-09-24T14:45:00.000+00:00
Duration: 7 min 17 s
Description: Watch this Scientific Journal Video about An Orthotopic Resectional Mouse Model of Pancreatic Cancer at JoVE.com

作者得到了阿夫纳胰腺癌基金会的支持。

所有程序均由新南威尔士大学动物护理和伦理委员会（17/109A）批准。雌性水性巴尔布/c裸鼠，年龄8-10周，体重16-19克，用于此协议。老鼠被安置在微型隔离笼中，并喂养市售的辣椒食品和水。
1. 矫形胰腺癌植入
<ol><li>为植入细胞做好准备。首先，计算手术所需的细胞数量（每只动物需要1 x10 6 个路西法酶标记的AsPC-1细胞和1 x10 6 个与癌症相关的人类胰腺板状细胞[CAHPSC]）。<ol>
<li>将这些细胞保存在潮湿的温度控制 CO2 孵化器中，并执行常规支原体测试。用于 ASPC-1 和 CAhPSC 的文化介质为 RPMI 1640（含 300 毫克/升谷氨酰胺， 20% v/v 胎儿牛血清，1% v/v 青霉素/链霉素）和 IMDM（含 4 mM L-谷氨酰胺，10% v/v 胎儿牛血清，1% v/v 青霉素/链霉素）。</li>
		<li>使用标准的细胞培养技术尝试将细胞合成细胞悬架。使用各自的完整培养基中和三普辛，其体积是所用试穿素溶液的两倍。</li>
		<li>用磷酸盐缓冲盐水 （PBS） 清洗这些细胞两次，并重新注入含有 1 x 106 AsPC-1 细胞和 1 x 106 CAhPSC 的混合物中，在 50 μL 细胞悬架中。</li>
		<li>将此悬浮在冰上，直到使用。</li>
	</ol></li>
	<li>为该程序准备 II 类生物安全柜。使用由无菌塑料窗帘覆盖的加热垫。对于手术过程中的放大，使用一对2.5倍至3.5倍的放大手术窗格。</li>
	<li>将直径为 1 厘米的孔切成纱布拭子，准备钱包串拭子。用钱包串缝合固定这个孔。任何精细的编织缝合可用于此（例如，5/0 多糖酸缝合）。建议使用编织的缝合材料，因为它允许松结在拧紧后保持原位。图1a说明了这 一点。</li>
	<li>通过腹膜注射，用80毫克/千克氯胺酮和10毫克/千克的西拉津麻醉小鼠。</li>
	<li>以5毫克/千克的抗炎素抗生素预防，2.5毫克/千克氟辛镇痛和1毫升0.9%盐水皮下。</li>
	<li>麻醉后，将鼠标放在无菌田中，置于超标位置，然后施用波维酮碘，然后使用70%的乙醇进行皮肤准备。</li>
	<li>在腹部左颅象限的皮肤上做纵向切口，然后通过在钳子之间收缩肌肉层进入腹部。</li>
	<li>加载一个29 G胰岛素注射器与50μL的细胞悬浮-这相当于1 x 106 CAhPSC和1 x 106 路西法酶标记AsPC-1细胞每个注射。将其安装在注射装置上。该注射装置的设计和功能在 图1b 及其传说中作了详细解释。</li>
	<li>将钱包串拭子放在腹腔切除术切口上，然后通过打开这个拭子将脾脏和胰腺尾巴外化。拧紧钱包，轻轻地包围胰腺的身体，露出胰腺尾巴注射。重要的是要足够紧，纱布接触胰腺周长，同时不收缩它。</li>
	<li>使用一对钳子，抓住胰腺的尾巴，轻轻地将横向张力放在胰腺上。用针头在浅角刺穿腹膜表面，然后用注射装置缓慢而可控地将细胞悬浮液注射到胰腺中（超过10−15秒）。</li>
	<li>在注射过程中，仔细观察渗漏情况-注射部位（从回流）和胰腺球的另一侧（在透射和穿透的情况下）。如果发生可见泄漏，请停止注射，并通过检查注射器中剩余注射量来记录泄漏量。如果泄漏体积较小（<10 μL），然后用纱布吸收任何泄漏，并将针头重新定位到不同的胰腺球中以完成注射。</li>
	<li>更换脾脏和胰腺，并连续用 5/0 多糖酸缝合关闭腹壁。用夹子关闭皮肤。</li>
	<li>在加热的笼子里监视鼠标，直到从麻醉中恢复过来。一旦清醒和警觉，移动鼠标回到它的笼子。</li>
</ol>2. 癌症切除手术:切除胰腺切除术和切除术
<ol><li>与植入有关的切除时间可能因实验协议而异。一般来说，允许肿瘤在切除前至少生长3周，但从经验上优化此特定植入的癌细胞系。</li>
	<li>在切除手术前一天，对动物进行生物发光成像，以确认局部原发性肿瘤的存在。请注意，此成像研究仅用于将患有明显胰外疾病的小鼠排除在切除之外。尺寸和辐射通量都不应用作确定切除资格的阈值。<ol>
<li>称量小鼠，并在腹中注射D-露西芬（150毫克/千克）。</li>
		<li>通过路西法林动能曲线的性能确定每个实验与红素注射相关的成像步骤的时间。辐射通量超过其最大值 90% 的时间段代表生物发光成像的最佳时间（在此实验中，注射后 18 至 26 分钟）</li>
		<li>诱导麻醉和保持使用异氟素（分别为4%和3%的氧气），并使用生物发光成像设备（如IVIS Lumina II）进行成像。使用自动曝光和绑定设置（但是，这可以针对预期的辐射通量进行优化）。</li>
	</ol>
</li>
	<li>准备II类生物安全柜进行程序。使用由无菌塑料窗帘覆盖的加热垫。对于解剖过程中的放大倍数，使用一对 2.5 倍至 3.5 倍的放大手术窗格。</li>
	<li>通过腹膜注射，用80毫克/千克氯胺酮和10毫克/千克的西拉津麻醉小鼠。</li>
	<li>以5毫克/千克的抗炎素抗生素预防，2.5毫克/千克氟辛镇痛和1毫升0.9%盐水皮下。</li>
	<li>将鼠标放在无菌田地中，然后施用波维酮碘，然后使用70%的乙醇进行皮肤准备。</li>
	<li>在腹部左颅象限的皮肤上做纵向切口，最好通过以前的切口部位。</li>
	<li>模糊地解剖底层肌肉腹部壁上的皮肤，然后放置一个Alm自我保留的缩回器，以保持皮肤伤口打开。</li>
	<li>将钳子之间的肌肉层切入上次操作的缝合线的一侧，然后展开切口以切除整个以前的缝合线。</li>
	<li>将脾脏和胰腺外化，并疯狂地缩回。在胰腺的方面，结肠可以通过胶片粘附物找到。如果发现此情况，则直言不讳地解剖结肠。</li>
	<li>小心地将一对钳子后部传递到胰腺和脾脏血管的身上，并打开这个空间。这释放了一段胰腺，用于随后的连体。</li>
	<li>用钛结扣将胰腺靠近肿瘤的身体盖上，然后用烧灼将胰腺解剖到此。控制胰腺树桩的另一种方法是在切除前用5/0多糖酸缝合物连续地盖住胰腺树桩。</li>
	<li>以轻率的方式收回胰腺，使脾脏和胃的颅杆之间的胃质血管烧灼。</li>
	<li>取出标本并确认血吸虫病。</li>
	<li>连续用 5/0 聚糖酸缝合关闭腹壁。用夹子关闭皮肤。</li>
</ol>3. 术后管理
<ol><li>在麻醉后立即（上述两个程序），在加热的笼子里监测小鼠，直到从麻醉中恢复过来。一旦清醒和警觉，移动鼠标回到它的笼子。在术后期间，监测动物的疼痛和痛苦迹象。通过皮下注射管理0.05毫克/千克丁丙诺啡，并密切观察动物12小时。</li>
	<li>随后，每天监测小鼠的体重、食物摄入量和活动情况。检查切口部位并触觉肿瘤大小。在第七个术后日取出皮肤夹。</li>
	<li>如果达到人道的终点，则对鼠标实施安乐死。这些人道的终点包括:体重减轻>20%，无法治疗的窘迫（包括驼背姿势，缺乏运动或梳妆）和肿瘤大小大于1厘米3， 据外部心绞痛估计。</li>
</ol>

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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>

作者对这个项目没有透露任何信息。

胰腺癌的切除矫形小鼠模型很重要，因为它允许检测辅助剂和新辅助治疗。这在胰腺癌中尤为重要，因为手术仍然是最有效的治疗方法，但与复发的高风险有关。本文描述了一种可以可靠地产生胰腺癌的方法，这种方法通过切除可以治愈，复制了需要新辅助/辅助疗法的临床场景。
对现有方法的意义 尽管辅助疗法和新辅助疗法在胰腺癌中的重要性，但文献中很?...

胰腺管腺癌（胰腺癌[PC]）与不良预后1有关。手术切除术仍然是PC唯一潜在的治疗方法，对于患有早期疾病的患者应予以考虑。不幸的是，即使R0切除（即切除边缘没有肿瘤），复发率（局部或从未被发现的转移性疾病）是高2，3。因此，全身辅助疗法在几乎所有接受切除4的患者中都显示。此外，虽然新辅助疗法现在只推荐给边缘可切除的癌症，其适应症正在扩大，使其常规使用是许多临床研究的重点5，6，7，8。为了开发涉及切除的 PC 的新疗法，这些方法需要首先在临床前模型中进行评估，以准确回顾临床设置。
矫型小鼠型号的PC已经经常用于测试药物治疗9，10。其中许多是由单独注射癌细胞到小鼠胰腺产生的，导致肿瘤缺乏PC特有的突出的频闪。最近，联合注射正交模型，如我们最初通过注射人类PC和人类胰腺恒星细胞的混合物（PSC，PC中胶原体频闪的主要生产者）而开发的模型，已经进入常规使用11，12。这种癌症和频闪细胞的联合注射产生的肿瘤既表现出癌症元素和PC的特有的频闪（去塑性）成分，（二）增强癌细胞增殖和转移11。因此，这个模型非常类似于人类PC。虽然一些矫形PC的切除模型被描述为13，14，15，16，但没有一个反映临床现实的胰腺切除在人类的准确如这个模型，因此一直不尽如人意的测试辅助剂或新辅助治疗。
演示的小鼠模型的目的是演示如何:（i） 成功植入正交胰腺癌，同时最大限度地减少无意的腹内传播和 （ii） 随后完全切除癌症。本文强调了该技术的技巧和潜在陷阱。

<table><tbody><tr><td>&lt;strong&gt;Animals, Materials and Equipment for Implantation Procedure&lt;/strong&gt;</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&#039;s modified Dulbecco&#039;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 &amp;mu;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) &amp;times; 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>&lt;strong&gt;Equipment for Resection Procedure&lt;/strong&gt;</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 &amp;ldquo;Weck style&amp;rdquo; 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>&lt;strong&gt;Drugs for Procedures&lt;/strong&gt;</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 &amp;mu;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

在考虑接受胰腺癌手术（PC）的患者中，缺乏令人满意的动物模型来研究辅助疗法和/或新辅助疗法。为了解决这一缺陷，我们描述了一个涉及 PC 正交植入的鼠标模型，然后是切除胰腺切除术和切除术。该模型已被证明是安全的和适当的灵活，用于研究各种治疗方法的辅助和新辅助设置。
在这个模型中，胰腺肿瘤首先通过将人类胰腺癌细胞（路西法酶标记的AsPC-1）和人类癌症相关胰腺板状细胞的混合物植入Balb/c水生裸鼠的离散胰腺而生成。三周后，癌症通过切除术、切除胰腺切除术和切除术进行切除。在这个模型中，生物发光成像可用于跟踪癌症发展的进展和切除/治疗的影响。切除后，可给予辅助治疗。或者，在切除前可以进行新辅助治疗。
介绍了来自45只小鼠的代表数据。所有小鼠都成功地进行了胰腺切除术/切除术，没有血吸虫病问题。在 43 （96%） 中实现了大于 5 mm 的宏观近胰腺边缘小 鼠。胰腺切除技术成功率为100%，早期死亡率和发病率为0%。在切除后的一周内，没有一只动物死亡。
总之，我们描述了一个强大和可重复的技术，用于模拟临床场景的小鼠胰腺癌手术切除模型。该模型可能有助于测试辅助剂和新辅助治疗。

连续有59只老鼠接受了植入手术。总泄漏发生在八 （14%）小 鼠。注射时的泄漏程度估计如下协议部分所述。三周后，为了让这些植入的肿瘤生长，在切除前进行了预切除生物发光成像，排除了患有严重转移性疾病的小鼠。45 （76%）老鼠接受了手术切除手术。
所有 45 （100%）小鼠成功地进行了切除胰腺切除术/切除术，没有出血问题。在 43 （96%） 中实现了大于 5 mm 的宏观近胰?...

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

在临床背景下，局部胰腺癌患者将接受胰腺切除术，然后进行辅助治疗。这里报道的这个协议旨在建立一个安全和有效的方法，通过正交植入胰腺癌，然后进行分立胰腺切除术和切除术，在裸体小鼠中模拟这一临床场景。

胰腺癌矫形小鼠模型

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

10.2K 次观看.  University of New South Wales. 在临床背景下，局部胰腺癌患者将接受胰腺切除术，然后进行辅助治疗。这里报道的这个协议旨在建立一个安全和有效的方法，通过正交植入胰腺癌，然后进行分立胰腺切除术和切除术，在裸体小鼠中模拟这一临床场景。

视频: 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.

胰腺癌的荧光原位小鼠模型

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 ...

科研

癌症研究

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.

自体胰腺癌小鼠模型研究不可逆电穿孔的影响

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

Orthotopic Mouse Model of Colorectal Cancer

The traditional subcutaneous tumor model is less than ideal for studying colorectal cancer. Orthotopic mouse models of colorectal cancer, which feature cancer cells growing in their natural location, replicate human disease with high fidelity. Two techniques can be used to establish this model. Both techniques are similar and require mouse anesthesia and laparotomy for exposure of the cecum. One technique involves injection of a colorectal cancer cell suspension into the cecal wall. Cancer cells are first grown in culture, harvested when subconfluent and prepared as a single cell suspension. A small volume of cells is injected slowly to avoid leakage. The other technique involves transplantation of a piece of subcutaneous tumor onto the cecum. A mouse with a previously established subcutaneous colorectal tumor is euthanized and the tumor is removed using sterile technique. The tumor piece is divided into small pieces for transplantation to another mouse. Prior to transplantation, the cecal wall is lightly damaged to facilitate tumor cell infiltration. The time to developing primary tumors and liver metastases will vary depending on the technique, cell line, and mouse species used. This orthotopic mouse model is useful for studying the natural progression of colorectal cancer and testing new therapeutic agents against colorectal cancer.

Two techniques can be used to establish this model: injection of a cancer cell suspension into the cecal wall or transplantation of a piece of subcutaneous tumor onto the cecum. This model is useful for studying the natural progression of colorectal cancer and testing new therapeutic agents against colorectal cancer.

结直肠癌原位小鼠模型

The traditional subcutaneous tumor model is less than ideal for studying colorectal cancer. Orthotopic mouse models of colorectal cancer, which feature ...

生物学

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.

毛尿胰腺腺腺腺癌超声引导正射植入

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.

正交胰腺肿瘤的产生与肿瘤渗透T细胞细胞毒性的外体特征

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

Bioluminescent Orthotopic Model of Pancreatic Cancer Progression

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

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

生物荧光原位模型胰腺癌进展

Pancreatic cancer has an extremely poor five-year survival rate of 4-6%.  New therapeutic options are critically needed and depend on improved ...

医学

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.

通过超声引导注射产生胰腺癌细胞的原位异种移植物

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

Syngeneic Mouse Orthotopic Allografts to Model Pancreatic Cancer

Pancreatic ductal adenocarcinoma (PDAC) is a very complex disease characterized by a heterogeneous tumor microenvironment made up of a diverse stroma, immune cells, vessels, nerves, and extracellular matrix components. Over the years, different mouse models of PDAC have been developed to address the challenges posed by its progression, metastatic potential, and phenotypic heterogeneity. Immunocompetent mouse orthotopic allografts of PDAC have shown good promise owing to their fast and reproducible tumor progression in comparison to genetically engineered mouse models. Moreover, combined with their ability to mimic the biological features observed in autochthonous PDAC, cell line-based orthotopic allograft mouse models enable large-scale in vivo experiments. Thus, these models are widely used in preclinical studies for rapid genotype-phenotype and drug-response analyses. The aim of this protocol is to provide a reproducible and robust approach to successfully inject primary mouse PDAC cell cultures into the pancreas of syngeneic recipient mice. In addition to the technical details, important information is given that must be considered before performing these experiments.

Syngeneic mouse orthotopic allografts of pancreatic ductal adenocarcinoma (PDAC) recapitulate the biology, phenotypes, and therapeutic responses of disease subtypes. Owing to their fast, reproducible tumor progression, they are widely used in preclinical studies. Here, we show common practices to generate these models, injecting syngeneic murine PDAC cultures into the pancreas.

同系小鼠原位同种异体移植物模拟胰腺癌

Pancreatic ductal adenocarcinoma (PDAC) is a very complex disease characterized by a heterogeneous tumor microenvironment made up of a diverse stroma, ...

Dynamic Contrast Enhanced Magnetic Resonance Imaging of an Orthotopic Pancreatic Cancer Mouse Model

Dynamic contrast enhanced magnetic resonance imaging (DCE-MRI) has been limitedly used for orthotopic pancreatic tumor xenografts due to severe respiratory motion artifact in the abdominal area. Orthotopic tumor models offer advantages over subcutaneous ones, because those can reflect the primary tumor microenvironment affecting blood supply, neovascularization, and tumor cell invasion. We have recently established a protocol of DCE-MRI of orthotopic pancreatic tumor xenografts in mouse models by securing tumors with an orthogonally bent plastic board to prevent motion transfer from the chest region during imaging. The pressure by this board was localized on the abdominal area, and has not resulted in respiratory difficulty of the animals. This article demonstrates the detailed procedure of orthotopic pancreatic tumor modeling using small animals and DCE-MRI of the tumor xenografts. Quantification method of pharmacokinetic parameters in DCE-MRI is also introduced. The procedure described in this article will assist investigators to apply DCE-MRI for orthotopic gastrointestinal cancer mouse models.

The goal of this protocol is to apply dynamic contrast enhanced magnetic resonance imaging (DCE-MRI) for orthotopic pancreatic tumor xenografts in mice. DCE-MRI is a non-invasive method to analyze microvasculature in a target tissue, and useful to assess vascular response in a tumor following a novel therapy.

原位胰腺癌小鼠模型的动态对比增强磁共振成像

Dynamic contrast enhanced magnetic resonance imaging (DCE-MRI) has been limitedly used for orthotopic pancreatic tumor xenografts due to severe ...

Bioluminescent Orthotopic Pancreatic Cancer Mouse Model: A Non-invasive Technique to Monitor Cancer Progression in Mice

Source: Chai, M. G., et al. <a href="https://www.jove.com/v/50395/" target="_blank">Bioluminescent Orthotopic Model of Pancreatic Cancer Progression</a>.&#160;J. Vis. Exp.&#160;(2013).
This video demonstrates an orthotopic model of pancreatic cancer that utilizes Matrigel for localized cell delivery and&#160;in vivo&#160;bioluminescence imaging for non-invasive monitoring of tumor progression. The orthotopic model is suited to both syngeneic and xenograft models and could be used in pre-clinical trials to investigate the impact of novel anti-cancer therapeutics on the growth of the primary pancreatic tumor and metastasis.

生物发光原位胰腺癌小鼠模型:一种监测小鼠癌症进展的非侵入性技术

Source: Chai, M. G., et al. Bioluminescent Orthotopic Model of Pancreatic Cancer Progression.&#160;J. Vis. Exp.&#160;(2013).
This video demonstrates an ...