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>

作者什么都没有透露。

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>RPMI media 1640&#160;</td>
			<td>Caisson Labs&#160;</td>
			<td>RPL03-500ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum&#160;</td>
			<td>Gibco</td>
			<td>10437-077</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin Streptomycin&#160;&#160;</td>
			<td>Thermo Ficher Sci</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>Matrigel HC&#160;</td>
			<td>Corning&#160;</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&#39;s Phosphate-Buffered saline)&#160;</td>
			<td>Thermo Ficher Sci</td>
			<td>21300025</td>
			<td></td>
		</tr>
		<tr>
			<td>TB Syringe 27G1/2</td>
			<td>Becton Dickinson</td>
			<td>305620</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane&#160;</td>
			<td>Blutler Schein</td>
			<td>50562</td>
			<td></td>
		</tr>
		<tr>
			<td>Ketoprofen&#160;</td>
			<td>Fort Dodge Animal Health&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical Scissors, 5.5&#34;straight mayo&#160;</td>
			<td>Henry Schein</td>
			<td>22-1600</td>
			<td></td>
		</tr>
		<tr>
			<td>PANC-1 GFP cell line&#160;</td>
			<td>Anticancer, Inc</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Small Animal Imaging System:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>iBOx Scientia, UVP :</td>
			<td>UVP, LLC&#160; Upland, CA.&#160;</td>
			<td colspan="2">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 ...

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Cancer Research

16.5K 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

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.

在直肠癌原位小鼠模型中循环肿瘤细胞的分离

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

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 ...

A Bioluminescent and Fluorescent Orthotopic Syngeneic Murine Model of Androgen-dependent and Castration-resistant Prostate Cancer

Orthotopic tumor modeling is a valuable tool for pre-clinical prostate cancer research, as it has multiple advantages over both subcutaneous and transgenic genetically engineered mouse models. Unlike subcutaneous tumors, orthotopic tumors contain more clinically accurate vasculature, tumor microenvironment, and responses to multiple therapies. In contrast to genetically engineered mouse models, orthotopic models can be performed with lower cost and in less time, involve the use of highly complex and heterogeneous mouse or human cancer cell lines, rather that single genetic alterations, and these cell lines can be genetically modified, such as to express imaging agents. Here, we present a protocol to surgically injecting a luciferase- and mCherry-expressing murine prostate cancer cell line into the anterior prostate lobe of mice. These mice developed orthotopic tumors that were non-invasively monitored in vivo and further analyzed for tumor volume, weight, mouse survival, and immune infiltration. Further, orthotopic tumor-bearing mice were surgically castrated, leading to immediate tumor regression and subsequent recurrence, representing castration-resistant prostate cancer. Although technical skill is required to carry out this procedure, this syngeneic orthotopic model of both androgen-dependent and castration-resistant prostate cancer is of great use to all investigators in the field.

The goal of this protocol is to demonstrate the intra-prostatic injection of prostate cancer cells, with subsequent castration. Orthotopic pre-clinical models of androgen-dependent and castration-resistant prostate cancer are critical to study the disease in the context of a clinically relevant tumor microenvironment and an immunocompetent host.

雄激素依赖性和去势抗性前列腺癌的荧光自体小鼠模型

Orthotopic tumor modeling is a valuable tool for pre-clinical prostate cancer research, as it has multiple advantages over both subcutaneous and ...

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.

流式细胞术对小鼠原发性胰腺导管腺癌原位同种异体 (自体) 免疫分型的研究

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

Patient-derived Heterogeneous Xenograft Model of Pancreatic Cancer Using Zebrafish Larvae as Hosts for Comparative Drug Assessment

Patient-derived tumor xenograft (PDX) and cell-derived tumor xenograft (CDX) are important techniques for preclinical assessment, medication guidance and basic cancer researches. Generations of PDX models in traditional host mice are time-consuming and only working for a small proportion of samples. Recently, zebrafish PDX (zPDX) has emerged as a unique host system, with the characteristics of small-scale and high efficiency. Here, we describe an optimized methodology for generating a dual fluorescence-labeled tumor xenograft model for comparative chemotherapy assessment in zPDX models. Tumor cells and fibroblasts were enriched from freshly-harvested or frozen pancreatic cancer tissue at different culture conditions. Both cell groups were labeled by lentivirus expressing green or red fluorescent proteins, as well as an anti-apoptosis gene BCL2L1. The transfected cells were pre-mixed and co-injected into the 2 dpf larval zebrafish that were then bred in modified E3 medium at 32 &#176;C. The xenograft models were treated by chemotherapy drugs and/or BCL2L1 inhibitor, and the viabilities of both tumor cells and fibroblasts were investigated simultaneously. In summary, this protocol allows researchers to quickly generate a large amount of zPDX models with a heterogeneous tumor microenvironment and provides a longer observation window and a more precise quantitation in assessing the efficiency of drug candidates.

This protocol describes optimization procedures in a virus-based dual fluorescence-labeled tumor xenograft model using larval zebrafish as hosts. This heterogeneous xenograft model mimics the tissue composition of pancreatic cancer microenvironment in vivo and serves as a more precise tool for assessing drug responses in personalized zPDX (zebrafish patient-derived xenograft) models.

患者衍生的胰腺癌异质异种移植模型,使用斑马鱼幼虫作为比较药物评估的宿主

Patient-derived tumor xenograft (PDX) and cell-derived tumor xenograft (CDX) are important techniques for preclinical assessment, medication guidance and ...

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 ...

Culture, Manipulation, and Orthotopic Transplantation of Mouse Bladder Tumor Organoids

The development of advanced tumor models has long been encouraged because current cancer models have shown limitations such as lack of three-dimensional (3D) tumor architecture and low relevance to human cancer. Researchers have recently developed a 3D in vitro cancer model referred to as tumor organoids that can mimic the characteristics of a native tumor in a culture dish. Here, experimental procedures are described in detail for the establishment of bladder tumor organoids from a carcinogen-induced murine bladder tumor, including culture, passage, and maintenance of the resulting 3D tumor organoids in vitro. In addition, protocols to manipulate the established bladder tumor organoid lines for genetic engineering using lentivirus-mediated transduction are described, including optimized conditions for the efficient introduction of new genetic elements into tumor organoids. Finally, the procedure for orthotopic transplantation of bladder tumor organoids into the wall of the murine bladder for further analysis is laid out. The methods described in this article can facilitate the establishment of an in vitro model for bladder cancer for the development of better therapeutic options.

This protocol provides detailed experimental steps to establish a three-dimensional in vitro culture of bladder tumor organoids derived from carcinogen-induced murine bladder cancer. Culture methods including passaging, genetic engineering, and orthotopic transplantation of tumor organoids are described.

小鼠膀胱肿瘤器官的培养、操作和正交移植

The development of advanced tumor models has long been encouraged because current cancer models have shown limitations such as lack of three-dimensional ...

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 ...

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 ...