Name: fluorescent orthotopic mouse model of pancreatic cancer
Uploaded: 2016-09-20T08:00:00.000+00:00
Duration: 6 min 48 s
Description: Watch this Scientific Journal Video about Fluorescent Orthotopic Mouse Model of Pancreatic Cancer at JoVE.com

We thank the Western University of Health Sciences for the Intramural Grant.

下面描述的协议的指导和西大学的动物护理和使用委员会的批准下进行。所有的实验都符合所有相关的准则，法规和监管机构执行。 1.细胞培养<ol><li> 完全培养基制备 <ol><li>使用II类生物安全柜，由无菌加入胎牛血清（FBS）和青霉素链霉素（P / S），以500毫升瓶的RPMI培养基的制备完全培养基。纷飞轻轻混匀。每个补充的终浓度为10％FBS和1％P / S（V / V）。例如，补充500毫升介质56毫升FBS和6ml P / S。 </li><li>放置完全培养基的水浴中在37℃。 </li></ol></li><li> 解冻和细胞传播 <ol><li>检索来自液氮PANC-1 GFP细胞。解冻通过温和搅拌小瓶在水浴中在37℃。 注意:解冻应迅速（约2 - 3分钟）。 </li><li>标签T-75烧瓶与（一个）细胞系的名称，（B）通道数，（C）的日期，和（d）研究者的缩写被使用。 </li><li>擦拭用70％乙醇和转移小瓶小瓶到生物安全柜中。 </li><li>以传播的细胞，加9.0毫升完全培养基至15毫升锥形管中。用1.0毫升吸管，小心地转移细胞悬液，9毫升完全培养基挂起。 </li><li>离心在125 xg离心在RT 8分钟。转移锥形小瓶至生物安全柜和吸出上清液，而不会干扰沉淀。 </li><li>轻轻上下吹打悬浮暂停在10ml新鲜的完全培养基的沉淀。 </li><li>计数细胞用血细胞计数器，并在2.1×10 6细胞/烧瓶的密度板他们在T-75烧瓶中。放置烧瓶在培养箱中在37℃和5％的CO 2。 </li><li>用肉眼和显微镜下监视每天的细胞。如果流感标识是需要更新，在无菌条件下吸从烧瓶中的完全生长培养基并丢弃。加入新鲜完全培养基等体积。 </li></ol></li><li> 细胞传播 <ol><li>在无菌条件下从烧瓶中删除网上平台。添加5毫升Dulbecco氏磷酸盐缓冲盐水（DPBS）中冲洗细胞并丢弃。 </li><li>加入3ml的0.25％胰蛋白酶的分离从烧瓶中的细胞。孵育含有在37℃的胰蛋白酶和5％CO 2的5至10分钟的烧瓶中。 </li><li>观察细胞的显微镜（放大10倍）下，并确保他们是圆和脱落。 </li><li>通过加入完全培养基等体积的中和胰蛋白酶和由向下轻轻吹打起来分手团块。 </li><li>使用血球计数细胞，并传送适当的细胞悬浮液体积在1.2.7中提到的细胞密度新瓶中;添加足够的新鲜培养基使终体积为每烧瓶10毫升</li></ol></li><li> 细胞Suspens离子制备注射 注:由于基底膜基质，浓度高，凝固在室温下，将所有材料冰上。放置在冰箱的所有无菌的枪头和小瓶过夜，并保持在冰上就暂停工作时。 <ol><li>保持了一瓶RPMI媒体无冰任何添加剂。解冻基底膜基质，浓度高，通过以下制造商的说明。优选地，分装成小小瓶避免多次冻融循环10。 </li><li>从所有培养瓶中吸出介质，并用5毫升的DPBS冲洗。重复上一节中的所有步骤，直至步骤1.3.4。 </li><li>在RT传送内容到15毫升锥形管和离心机在125×g离心8分钟。转移锥形小瓶生物安全柜，并重新暂停用10ml DPBS的沉淀。 </li><li>轻轻吸管悬架上下分手沉淀。算使用血球细胞。 </li><li>离心机再次作为以s概述TEP 1.4.3。吸去上清液，并根据细胞的数量，添加适当的稀释剂，得到3×10 6细胞到50微升的体积。稀释剂将是无血清培养基和高浓度的地下室基质膜的一个1的混合物:1（V / V）的比率。 </li><li>然后振荡悬挂，并保持在冰上在任何时候。该悬浮液现在已准备好用于注射。 </li></ol></li><li> 手术植入 注意:确保所有的手术材料和仪器是无菌的。实践在任何时候无菌技术。 <ol><li>放置在桌子上的加热垫，并用无菌披盖覆盖。设置麻醉机，并确保所有电源都触手可及。 </li><li>放置在动物的鼻子到麻醉掩模和麻醉设置为1升/分钟的氧气和2.5％的异氟醚。 </li><li>轻轻擦洗用碘动物的侧翼，并用70％乙醇冲洗干净。重复三次。 </li><li>确认动物按捏后肢完全麻醉。如果发现没有反应的动物是完全麻醉，准备手术。然而，如果动物退缩，确保有在蒸发器充分麻醉，并允许动物更多的时间来麻醉下完全去。 </li><li>负载大约200微升细胞悬浮液与一种18G的针头的1.0毫升结核病注射器（预先冷却）。更换一个27克针针，并返回到冰中待用。 </li><li>找到脾脏一般地区（腹部左上腹），并使用镊子捏在该区域上方的皮肤。用手术剪使大约为1.0厘米的切口创建口袋。同样，捏在脾脏顶部的平滑肌和为了访问腹膜腔切穿。 </li><li>轻轻抓住脾的尾端，将其排除体外。胰腺将附在脾脏。传播利用湿式ST胰腺erile棉条并找到胰尾。 </li><li>交付50微升注入胰尾，留针内，持续10秒，慢慢地旋转针头从胰腺。一个成功的植入看起来像没有任何泄漏肤浅的泡沫。 </li><li>返回胰腺和脾脏到腹膜腔。首先附上肌肉，然后分别附上皮肤。使用6-0缝合或钉关闭切口。为了避免在动物疼痛，辖酮洛芬皮下（SC）（5毫克/千克）经24小时。另外，管理丁丙诺啡SC（0.05 - 0.1毫克/千克），超过36小时的时间内，每12小时。 </li><li>从麻醉中恢复的动物，并返回到笼子里。同时监测的痛苦。手术 - 动物必须在（先发制人甚至之前）的时间被提供疼痛缓解。额外的疼痛缓解，必须提供给根据IACCUC协议或由在规定的经历疼痛的动物抚育兽医或指定人员。 </li></ol></li><li> 体内成像 注意:使用装备有暗室和GFP成像适当滤光片的商业成像系统进行图像捕捉。 （: - ;:513 - 557 nm发射495纳米455激发）图像采集，使用CCD照相机和由可互换的激发/发射透镜的光学系统来实现的。亮场图像，而不以1×1像素合并为每个时间点的过滤器被捕获。 GFP的激发利用氙多光谱光源。动物用麻醉机连接到一个气体麻醉歧集成到成像系统的暗室麻醉下保持整个成像过程。 <ol><li>麻醉动物如1.5.2所述。麻醉掩模是在商业成像系统的暗室的内部，以便在成像过程中，以保持动物麻醉下处理。 </li><li>建立正确的激发和发射滤波器。 </li><li>只用白光获得初始图像开始。保持动物的相同位置在整个成像会议并切换到GFP的过滤器，以采取第二图像。 </li><li>为了达到最佳效果叠加两个图像。分析荧光区域和强度的图像。 </li></ol></li></ol>

<ol>
	<li>Smyth, E., Cunningham, D., Kasper, D., 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> Harrison's Principles of Internal Medicine. , (2015).</li><li>Mahipal, A., Frakes, J., Hoffe, S., Kim, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Management+of+borderline+resectable+pancreatic+cancer.">Management of borderline resectable pancreatic cancer.</a> World J Gastrointest Oncol. 7, 241-249 (2015).</li><li>De La Cruz, M. S., Young, A. P., Ruffin, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diagnosis+and+management+of+pancreatic+cancer.">Diagnosis and management of pancreatic cancer.</a> Am Fam Physician. 89, 626-632 (2014).</li><li>Frese, K. K., Tuveson, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maximizing+mouse+cancer+models.">Maximizing mouse cancer models.</a> Nat Rev Cancer. 7, 645-658 (2007).</li><li>Hoffman, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patient-derived+orthotopic+xenografts:+better+mimic+of+metastasis+than+subcutaneous+xenografts.">Patient-derived orthotopic xenografts: better mimic of metastasis than subcutaneous xenografts.</a> Nat Rev Cancer. 15, 451-452 (2015).</li><li>Hoffman, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+multiple+uses+of+fluorescent+proteins+to+visualize+cancer+in+vivo.">The multiple uses of fluorescent proteins to visualize cancer in vivo.</a> Nat Rev Cancer. 5, 796-806 (2005).</li><li>Jiang, Y. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+of+an+orthotopic+pancreatic+cancer+mouse+model:+cells+suspended+and+injected+in+Matrigel.">Establishment of an orthotopic pancreatic cancer mouse model: cells suspended and injected in Matrigel.</a> World J Gastroenterol. 20, 9476-9485 (2014).</li><li>Arranz, A., Ripoll, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+optical+imaging+for+pharmacological+studies.">Advances in optical imaging for pharmacological studies.</a> Front Pharmacol. 6, 189 (2015).</li><li>Metildi, C. A., Kaushal, S., Hoffman, R. M., Bouvet, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+serial+selection+of+human+pancreatic+cancer+cells+in+orthotopic+mouse+models+produces+high+metastatic+variants+irrespective+of+Kras+status.">In vivo serial selection of human pancreatic cancer cells in orthotopic mouse models produces high metastatic variants irrespective of Kras status.</a> J Surg Res. 184, 290-298 (2013).</li><li>Kim, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+orthotopic+and+heterotopic+human+pancreatic+cancer+xenografts+in+immunodeficient+mice.">Generation of orthotopic and heterotopic human pancreatic cancer xenografts in immunodeficient mice.</a> Nat Protoc. 4, 1670-1680 (2009).</li><li>Katz, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Survival+efficacy+of+adjuvant+cytosine-analogue+CS-682+in+a+fluorescent+orthotopic+model+of+human+pancreatic+cancer.">Survival efficacy of adjuvant cytosine-analogue CS-682 in a fluorescent orthotopic model of human pancreatic cancer.</a> Cancer Res. 64, 1828-1833 (2004).</li><li>Bouvet, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+optical+imaging+of+primary+tumor+growth+and+multiple+metastatic+events+in+a+pancreatic+cancer+orthotopic+model.">Real-time optical imaging of primary tumor growth and multiple metastatic events in a pancreatic cancer orthotopic model.</a> Cancer Res. 62, 1534-1540 (2002).</li></ol>

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我们描述了胰腺癌的原位小鼠模型它表达绿色荧光蛋白，从而允许肿瘤生长的非侵入性监控用整个身体的体内荧光成像（ 图1）。这种技术使我们能够实时监测肿瘤发展（ 图3）;它可以为研究人员研究针对胰腺癌的新试剂的治疗功效的重要工具。这种模式的另一个重要方面是，GFP荧光提供了一种视觉提示指示成功植入和胰腺癌的生长;否则将难以在活的动物来衡量。...

胰腺癌诊断与增加的频率相对于其它癌症和是在美国癌症相关死亡的第 4主要原因。从诊断时，患者的90％以上，五年内1,2死亡。目前，手术切除肿瘤是胰腺癌唯一的治疗，但患者不足20％有资格接受手术，主要是因为在诊断时疾病是一个先进的阶段，并已转移3,4。缺乏特异性症状，使胰腺癌的一种无声的疾病;一些症状包括腹痛，背痛，食欲不振，黄疸和恶心的损失;可以很容易地解释为常见的消化系统疾病4。由于这个原因，重要的是开发新的药理学工具在胰腺癌的诊断和治疗，以帮助是重要的。 利用动物模型可以让我们了解pancre生物学ATIC癌症并提供了一​​个深入了解将这一知识用于人类。胰腺癌的异种移植原位模型是现实的，因为肿瘤起源于5器官生长。在对比异位模型，其中细胞系或肿瘤片段皮下植入，原位建模允许肿瘤微环境和模拟物的肿瘤细胞的相互作用的与其周围6的娱乐。此处所描述的异种移植物模型由人胰腺癌细胞系PANC-1的GFP，这是基因工程化以表达绿色荧光蛋白（GFP）派生的肿瘤。 GFP检测允许用于非侵入性成像和肿瘤生长和转移7的监视。肿瘤发展迅速发生，自发地和非常类似于人类胰腺癌患者8的原 ​​发肿瘤。原位模型提供药物疗效的一个更准确的预测响应于治疗剂，而模拟肿瘤微环境。 如上所述，这种动物模型可让肿瘤生长和转移的实时荧光检测。荧光检测允许相比，发光更直接/实时成像。荧光的发射光是由一较短波长的另一个光的激发的结果;而在发光，该发出的光的化学反应的结果，并且可能不具有强发射9。此外， 在体内荧光成像全身是不损害动物，并允许研究人员来监测肿瘤的生长随时间响应于治疗性治疗。

<table><tbody><tr><td>RPMI media 1640</td><td>Caisson Labs</td><td>RPL03-500ML</td><td></td></tr><tr><td>Fetal Bovine Serum&amp;nbsp;</td><td>Gibco</td><td>10437-077</td><td></td></tr><tr><td>Penicillin Streptomycin&amp;nbsp;&amp;nbsp;</td><td>Thermo Ficher Sci</td><td>15140-122</td><td></td></tr><tr><td>Matrigel HC basement membrane, high concentration</td><td>Corning</td><td>354248</td><td></td></tr><tr><td>SutureVet PGA 6-0 PGA</td><td>Henry Schein</td><td>39010</td><td></td></tr><tr><td>Alcare or Foamed Antiseptic Handrub</td><td>Steris</td><td>639680</td><td></td></tr><tr><td>DPBS (Dubelcco&#039;s Phosphate-Buffered saline)&amp;nbsp;</td><td>Thermo Ficher Sci</td><td>21300025</td><td></td></tr><tr><td>TB Syringe 27 G 1/2</td><td>Becton Dickinson</td><td>305620</td><td></td></tr><tr><td>Isoflurane</td><td>Blutler Schein</td><td>50562</td><td></td></tr><tr><td>Ketoprofen</td><td>Fort Dodge Animal Health</td><td></td><td></td></tr><tr><td>Surgical Scissors, 5.5&quot; straight mayo&amp;nbsp;</td><td>Henry Schein</td><td>22-1600</td><td></td></tr><tr><td>PANC-1 GFP cell line&amp;nbsp;</td><td>Anticancer, Inc</td><td></td><td></td></tr><tr><td>Small Animal Imaging System: iBox Scientia</td><td>UVP, LLC. Upland, CA.</td><td></td><td>Small Animal Imaging System to observe the fluorescent tumor in live animals</td></tr></tbody></table>

fluorescent orthotopic mouse model of pancreatic cancer

胰腺癌仍然是其中存活尚未在过去的几十年大大提高了癌症之一。只有确诊患者的7％将生存超过五年。为了理解和模仿胰腺肿瘤的微环境，我们利用胰腺癌的小鼠原位模型，允许肿瘤进展的非侵入性成像的实时性。表达绿色荧光蛋白（PANC-1，GFP）胰腺癌细胞悬浮于基底膜基质，浓度高，（ 例如 ，基质胶HC）与无血清培养基，然后注入通过剖腹胰腺的尾巴。在高浓度基底膜基质中的细胞悬浮液变成凝胶状物质一旦达到室温;因此，当它与胰腺接触，形成在注射部位的密封，防止任何细胞泄漏它胶凝。肿瘤生长和转移到其他器官活监视动物用荧光。它是使用适当的过滤器的GFP的激发和发射的关键。对于原位移植的步骤，在这篇文章中有详细因此，研究人员可以很容易地复制在裸鼠体内的过程。这个协议的主要步骤是制备细胞悬浮液，手术植入，和全身荧光体内成像。此原位模型被设计为调查对原发性和转移性肿瘤新的治疗的功效。

这个方法描述了荧光人胰腺癌细胞的外科原位移植，着眼于制备细胞悬浮液注射，对啮齿类动物适当麻醉，通过剖腹分娩细胞悬浮液，以及使用荧光体内小动物成像的。检测2和3星期之间的绿色荧光信号（GFP信号）植入后的，为研究人员提供视觉提示以确认显影胰腺癌肿瘤（ 图1）的存在。 图1是由一个鼠的三个图像的同PANC-1的GFP荧光的肿瘤...

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

Fluorescent Orthotopic Mouse Model of Pancreatic Cancer

胰腺癌的荧光原位小鼠模型

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.

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 ...

fluorescent-orthotopic-mouse-model-of-pancreatic-cancer

Research

JoVE Journal

Cancer Research

16.6K 次观看.  Western University of Health Sciences. 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. 

视频: Fluorescent Orthotopic Mouse Model of Pancreatic Cancer

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.

研究使用正交乳腺癌模型的三阴性乳腺癌

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

科研

癌症研究

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 ...

An Orthotopic Resectional Mouse Model of Pancreatic Cancer

There is a lack of satisfactory animal models to study adjuvant and/or neoadjuvant therapy in patients being considered for surgery of pancreatic cancer (PC). To address this deficiency, we describe a mouse model involving orthotopic implantation of PC followed by distal pancreatectomy and splenectomy. The model has been demonstrated to be safe and suitably flexible for the study of various therapeutic approaches in adjuvant and neo adjuvant settings.
In this model, a pancreatic tumor is first generated by implanting a mixture of human pancreatic cancer cells (luciferase-tagged AsPC-1) and human cancer associated pancreatic stellate cells into the distal pancreas of Balb/c athymic nude mice. After three weeks, the cancer is resected by re-laparotomy, distal pancreatectomy and splenectomy. In this model, bioluminescence imaging can be used to follow the progress of cancer development and effects of resection/treatments. Following resection, adjuvant therapy can be given. Alternatively, neoadjuvant treatment can be given prior to resection.
Representative data from 45 mice are presented. All mice underwent successful distal pancreatectomy/splenectomy with no issues of hemostasis. A macroscopic proximal pancreatic margin greater than 5 mm was achieved in 43 (96%) mice. The technical success rate of pancreatic resection was 100%, with 0% early mortality and morbidity. None of the animals died during the week after resection.
In summary, we describe a robust and reproducible technique for a surgical resection model of pancreatic cancer in mice which mimics the clinical scenario. The model may be useful for the testing of both adjuvant and neoadjuvant treatments.

In the clinical context, patients with localized pancreatic cancer will undergo pancreatectomy followed by adjuvant treatment. This protocol reported here aims to establish a safe and effective method of modelling this clinical scenario in nude mice, through orthotopic implantation of pancreatic cancer followed by distal pancreatectomy and splenectomy.

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 ...

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

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 ...

<meta charset="utf8"></head><body>在胰腺癌模型中使用肿瘤浸润淋巴细胞的过继治疗

Flow Cytometry-Based Isolation and Therapeutic Evaluation of Tumor-Infiltrating Lymphocytes in a Mouse Model of Pancreatic Cancer

Pancreatic cancer is an aggressive malignancy with a dismal prognosis and limited therapeutic options. Adoptive cell therapy, which involves isolating and activating a patient&#39;s own immune cells, such as tumor-infiltrating lymphocytes (TILs), before re-infusing them, represents a promising experimental approach. However, techniques for adoptive cell transfer in preclinical pancreatic cancer models are not well established. Here, we describe a detailed protocol for adoptive cell therapy using TILs from a syngeneic pancreatic cancer mouse model. The procedure involves implanting live or irradiated mouse pancreatic cancer cells in fluorescence-labeled reporter mice to initiate immune cell influx, then isolating lymphocytes from primary tumors via flow cytometry sorting and/or activating and expanding tumor-reactive T cells ex vivo, and adoptively transferring these activated T cells intraperitoneally into tumor-bearing mice, followed by interleukin-2 administration.&#160;Bioluminescent tumor imaging allows for longitudinal monitoring of orthotopic tumor growth and response to therapy, especially evaluating the tumor-specific cytotoxic effects. This approach recapitulates the logistics involved in developing adoptive cell transfer therapies for pancreatic cancer patients. The results demonstrate enhanced antitumor efficacy of adoptively transferred tumor-reactive T cells compared to irrelevant lymphocyte controls. This versatile methodology enables the in vivo study of adoptive immunotherapy in pancreatic cancer as well as the optimization of cell processing parameters and combination treatment regimens.

<meta charset="utf8"></head><body>基于流式细胞术的胰腺癌小鼠模型中肿瘤浸润淋巴细胞的分离和治疗评估

We describe a method to utilize tumor-infiltrating lymphocytes (TILs) from mice through flow cytometry for adoptive cell transfer. This protocol aims to verify the specific cytotoxicity of TILs against tumors in a syngeneic pancreatic cancer mouse model, providing insights into the development of adoptive cell therapies for pancreatic cancer.

基于流式细胞术的胰腺癌小鼠模型中肿瘤浸润淋巴细胞的分离和治疗评估

Pancreatic cancer is an aggressive malignancy with a dismal prognosis and limited therapeutic options. Adoptive cell therapy, which involves isolating and ...

Stabilized Longitudinal In Vivo Cellular-Level Visualization of the Pancreas in a Murine Model with a Pancreatic Intravital Imaging Window

Direct in vivo cellular-resolution imaging of the pancreas in a live small animal model has been technically challenging. A recent intravital imaging study, with an abdominal imaging window, enabled visualization of the cellular dynamics in abdominal organs in vivo. However, due to the soft sheet-like architecture of the mouse pancreas that can be easily influenced by physiologic movement (e.g., peristalsis and respiration), it was difficult to perform stabilized longitudinal in vivo imaging over several weeks at the cellular level to identify, track, and quantify islets or cancer cells in the mouse pancreas. Herein, we describe a method for implanting a novel supporting base, an integrated pancreatic intravital imaging window, that can spatially separate the pancreas from the bowel for longitudinal time-lapse intravital imaging of the pancreas microstructure. Longitudinal in vivo imaging with the imaging window enables stable visualization, allowing for the tracking of islets over a period of 3 weeks and high-resolution three-dimensional imaging of the microstructure, as evidenced here in an orthotopic pancreatic cancer model. With our method, further intravital imaging studies can elucidate the pathophysiology of various diseases involving the pancreas at the cellular level.

Stabilized Longitudinal In Vivo Cellular-Level Visualization of the Pancreas in a Murine Model with a Pancreatic Intravital Imaging Window

In vivo high-resolution imaging of the pancreas was facilitated with the pancreatic intravital imaging window.

稳定纵向 在Vivo 细胞级可视化胰腺在穆林模型与胰腺内活性成像窗口

Direct in vivo cellular-resolution imaging of the pancreas in a live small animal model has been technically challenging. A recent intravital imaging ...

胰腺癌的荧光原位小鼠模型

摘要

探索更多视频

此视频中的章节

研究使用正交乳腺癌模型的三阴性乳腺癌

毛尿胰腺腺腺腺癌超声引导正射植入

胰腺癌矫形小鼠模型

生物荧光原位模型胰腺癌进展

同系小鼠原位同种异体移植物模拟胰腺癌

原位胰腺癌小鼠模型的动态对比增强磁共振成像

基于流式细胞术的胰腺癌小鼠模型中肿瘤浸润淋巴细胞的分离和治疗评估

基于流式细胞术的胰腺癌小鼠模型中肿瘤浸润淋巴细胞的分离和治疗评估

基于流式细胞术的胰腺癌小鼠模型中肿瘤浸润淋巴细胞的分离和治疗评估

稳定纵向在Vivo 细胞级可视化胰腺在穆林模型与胰腺内活性成像窗口

胰腺癌的荧光原位小鼠模型

摘要

探索更多视频

此视频中的章节

研究使用正交乳腺癌模型的三阴性乳腺癌

毛尿胰腺腺腺腺癌超声引导正射植入

胰腺癌矫形小鼠模型

生物荧光原位模型胰腺癌进展

同系小鼠原位同种异体移植物模拟胰腺癌

原位胰腺癌小鼠模型的动态对比增强磁共振成像

基于流式细胞术的胰腺癌小鼠模型中肿瘤浸润淋巴细胞的分离和治疗评估

基于流式细胞术的胰腺癌小鼠模型中肿瘤浸润淋巴细胞的分离和治疗评估

基于流式细胞术的胰腺癌小鼠模型中肿瘤浸润淋巴细胞的分离和治疗评估

稳定纵向 在Vivo 细胞级可视化胰腺在穆林模型与胰腺内活性成像窗口

稳定纵向在Vivo 细胞级可视化胰腺在穆林模型与胰腺内活性成像窗口