Name: a syngeneic mouse model of metastatic renal cell carcinoma for quantitative and longitudinal assessment of preclinical therapies
Uploaded: 2017-04-12T14:00:00.000+00:00
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Description: Watch this Scientific Journal Video about A Syngeneic Mouse Model of Metastatic Renal Cell Carcinoma for Quantitative and Longitudinal Assessment of Preclinical Therapies at JoVE.com

这项工作是由美国国家癌症研究所（R15CA173657到AW和R01CA109446到TSG），西蒙斯癌症研究所（至AW），以及Minnestoa大学助学金爬上4肾脏癌症基金会（TSG到）的支持。我们感谢克丽斯廷·安德森博士与免疫援助。

以下方案描述用于诱导和监测实验原位肾肿瘤和任何潜在自发转移的生长的实验程序。下面的所有程序都是按照有关人性化的使用实验动物的体制政策和批准的程序进行。 1.细胞系的维护<ol><li>维持鼠肾腺癌细胞系的Renca，在的Roswell Park Memorial研究所（RPMI）1640培养基补充有10％胎牛血清（FCS），1％青霉素 - 链霉素，和各1mM的非必需氨基酸，L-谷氨酰胺的和丙酮酸钠（简称完全RPMI）。培养细胞在培养箱中在37℃和5％CO 2。 注意:与任何建立的细胞系，则建议使用低传代数RENCA细胞用于转染和随后的注入，以最小化长期崇拜后可能发生的任何表型或遗传变化URE。 <ol><li>使用转座子系统工程师RENCA细胞稳定表达绿色荧光蛋白（GFP）和萤火虫荧光素酶。 <ol><li>使用阳离子基于脂质体的转染试剂，共转染的Renca细胞与转座酶表达载体（PPGK-SB11）和萤火虫萤光素酶的表达的转座子载体编码，以及一个编码GFP融合基因和zeocin抗性基因，两者的双向启动子（PKT2 / LuBiG）19的转录控制下。 </li><li>两天后，板转染的细胞以有限稀释并允许细胞扩大的完全RPMI补充有博莱霉素（300微克/毫升）。确认在通过流式细胞术每个克隆GFP表达。这些细胞被称为的Renca-GL。 注:限制稀释克隆将允许从单个细胞衍生的所选单元格。 </li></ol></li></ol></li><li>植入RENCA细胞肾内，制备RENCA细胞我n要在Hank氏平衡盐溶液（HBSS）以2×10 6个细胞/ mL的单细胞悬浮液。 <ol><li>使用0.25％胰蛋白酶在HBSS中的组织培养瓶中除去粘附在表面的Renca细胞。约5分钟后，完全RPMI添加到该烧瓶中以中和胰蛋白酶，将细胞转移到一个离心管中进行洗涤。离心将细胞在300相对离心克力（RCF×g）离心5分钟，在25℃下。 </li><li>倒出上清液和重悬细胞，在HBSS中。计数使用血球的细胞，并用HBSS调节音量，得到2×10 6细胞/ mL。 </li><li>绘制细胞成1毫升注射器装配有18号针头，以防止对细胞的剪切应力。替换前的肿瘤细胞的注射一个28号针头，18号针头。 0.1毫升的注射递送2×10 5个细胞入肾。 </li></ol></li></ol> 2.手术区准备<ol><li>准备无菌手术区在生物安全柜通过将无菌手术单过一个加热垫。收集所有用品，包括镊子，解剖刀，剪刀，滴眼剂，5％聚维酮碘防腐剂，和无菌纱布。高压灭菌器使用前所有仪器。 </li><li>执行使用无菌尖端技术的手术。动物之间重新消毒文书，珠灭菌。戴上无菌手套，白大褂和口罩。 </li></ol> 3.动物的制备<ol><li>麻醉用根据机构指南氯胺酮/赛拉嗪（87.5 mg / kg和12.5毫克/千克，分别，腹膜内）的小鼠。通过执行脚趾捏确定麻醉深度。 </li><li>当动物被认为是无反应，由拔除（用手指）或剃须（使用裁切机）的皮毛上准备Balb / c小鼠注射部位（ 例如，左侧面）。 </li><li>从手术准备区移动小鼠（ 例如，房栊）到手术区和报考兽医药膏给眼睛，防止干燥。宽松拭制备的切口部位用5％聚维酮碘防腐剂。 </li><li>注入盐酸布比卡因（0.1毫升2.5毫克/毫升溶液，SC的）插入其中的切口将被制成为手术前的镇痛的区域。 </li><li>使单个切口（〜1.0 - 1.5厘米）对动物的左翼使用无菌解剖刀或剪刀，小心以免穿透腹膜。用无菌剪刀腹膜分离真皮。民建联切口部位用无菌纱布，以消除任何血液。 </li><li>按住鼠标，用双手，用左手持有的头部和右手支撑侧翼。 </li><li>识别脾通过腹膜和，用右手的一个手指，从下方触诊鼠标;肾脏现在应该通过腹膜可见。保持动物的坚定持有，以提供跨腹膜和肾脏紧张。 </li><li>有第二人注入肿瘤CELLS（1.2节中制备的。）通过完整腹膜成使用1毫升注射器装配有28号针头的肾脏的中心。慢慢地递送细胞的0.1毫升（2×10 5）。一旦细胞注射，持针到位5 - 10秒，以减少细胞的回流。注意在此步骤非常谨慎的需要，因为注射细胞的人就会把针头插入接近按住鼠标的人的手指是很重要的。 </li><li>卸下针和用氰基丙烯酸酯组织粘合剂的薄层关闭切口。 </li><li>将动物放回一个干净的笼子消毒纸巾。让动物在加热垫上恢复。不要离开无人看管的动物在恢复期。不要将动物返回到标准的住房条件，直到他们正直和胸骨。按照使用实验动物的所有程序制度的指导方针。 </li></ol> 4.生物发光成像<ol><li>监测由生物发光成像的肿瘤生长。 注意:用于执行生物发光成像程序的细节可以从Lim 等来获得。 20荧光素酶信号可以在肿瘤细胞植入后早在24小时进行检测。由于有在该模型中没有外部可见的肿瘤，它周期性地监视肿瘤的生长是必不可少的。根据不同的设计的实验，原位肿瘤生长可以进行不同的时间长度。使用荧光素酶表达的Renca细胞的一个好处是，肿瘤的生长可反复在相同的动物定期监测在实验的持续时间，和荷瘤和无肿瘤的器官的特定询问可以在完成定义端点。 <ol><li>注入荧光素（0.1毫升在无菌PBS中有15毫克/毫升溶液）ip和允许其将鼠标到生物发光成像系统之前循环10分钟。将莫<html>其背部朝上使用，在其一侧略微卷起，才能有朝上注入的肾。 </li><li>测量使用图像分析软件确定的关注区域（ROI）的区域内的每秒（光子通量）发出的光的光子的肿瘤负荷。 </li></ol></li><li>在个别器官的肿瘤负荷的定量。 注:定量可以在确定的时间点在个别器官从安乐死的小鼠其取出后进行。 <ol><li>注入小鼠荧光素，如步骤4.1。 </li><li> 10分钟后，安乐死使用经批准的过程的小鼠。 </li><li>解剖出感兴趣的器官（ 例如，肾和肺;见下文第5节）。转移组织以35×10毫米的培养皿并将其放置在生物发光成像系统。 </li><li>测量使用图像分析软件定义的ROI内每秒（光子通量）发出的光的光子的肿瘤负荷。 </li></ol></li></ol><html> e_title"&gt; 5。器官收获<ol><li>在用于各个实验所定义的终点，根据优选机构准则（ 例如，CO 2，颈椎脱臼，麻醉过量）安乐死荷瘤小鼠。然而，要避免颈椎脱位，如果肺是印度墨通货膨胀之后被收集。 </li><li>通过测量湿器官重量执行在所注入的肾原发性肾肿瘤生长的毛评估。 <ol><li>为了确定肿瘤块（以克计），隔离21和称重荷瘤肾脏和从同一小鼠，其中在肾重量差定义了肿瘤质量的对侧肾。测量它们的重量之前，请仔细剖析远离切除肾脏任何多余的结缔组织。 </li></ol></li><li>通过用墨汁之前切除充气肺并立即固定在的Fekete的溶液肺评估肿瘤转移至肺的程度小姑娘= "外部参照"&gt; 22。然后可以执行的肺表面上的转移病灶的人工计算。 <ol><li>请使用开始于-腹部中期剪刀正中切口，通过胸腔，并且向上通过颈部/唾液腺。小心地取出用剪刀和镊子胸腔，而不会损害肺组织。 </li><li>通过去除结缔组织暴露气管。 </li><li>制备3mL注射器填充有在HBSS 10％印度油墨溶液的18号针头。 </li><li>使用镊子，小心翼翼地拧气管下丝线。这将用于打结在充气后的气管。 </li><li>小心地将针头顺着气管，慢慢压低注射器膨胀与印度墨液肺部。大约1 - 1.5毫升的印度油墨溶液将完全膨胀肺部。 </li><li>捏通过把几节，与以下针缝合，以抑制墨逆流关闭气管。 </li><li>祛瘀E中的针，小心地剪去结缔组织去除膨胀肺部。 </li><li>在化学通风橱中，将肺成含有5的Fekete的溶液中的溶液15毫升锥形管中（35毫升95％乙醇，15毫升卫生署2 O，5毫升的甲醛，和2.5毫升冰酸性酸的中一个100毫升的玻璃瓶）23。 </li><li>允许肺修复/脱色24 - 在室温下48小时之前手动计数脱色肿瘤结节。 </li><li>脱色后取出完整肺和仔细叶在35×10毫米的培养皿解剖固定肺。算肿瘤结节（白色结节）上在解剖显微镜下每个叶的数目。 </li></ol></li></ol>

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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=Diet-induced+obesity+alters+dendritic+cell+function+in+the+presence+and+absence+of+tumor+growth.">Diet-induced obesity alters dendritic cell function in the presence and absence of tumor growth.</a> J. Immunol. 189, 1311-1321 (2012).</li><li>Brincks, E. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Triptolide+enhances+the+tumoricidal+activity+of+TRAIL+against+renal+cell+carcinoma.">Triptolide enhances the tumoricidal activity of TRAIL against renal cell carcinoma.</a> FEBS J. 282, 4747-4765 (2015).</li><li>Anderson, K. G., 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=Cutting+edge:+intravascular+staining+redefines+lung+CD8+T+cell+responses.">Cutting edge: intravascular staining redefines lung CD8 T cell responses.</a> J. Immunol. 189, 2702-2706 (2012).</li><li>Anderson, K. G., 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=Intravascular+staining+for+discrimination+of+vascular+and+tissue+leukocytes.">Intravascular staining for discrimination of vascular and tissue leukocytes.</a> Nat. Protocols. 9, 209-222 (2014).</li></ol>

The authors declare no conflicts of interest.

我们提出了一个原位RCC小鼠模型的协议。小鼠肾腺癌肿瘤细胞植入小鼠肾脏提供海上救援协调中心临床相关模型。该模型导致在肺中，这两者都是晚期RCC的标志的肾脏和远处转移中的原发肿瘤。本文中所呈现的描述是具体的在免疫活性Balb / c小鼠的的Renca细胞系。使用这种原位模型，我们已经表明的Renca肿瘤响应免疫，表明治疗性测试<sup class="xref...

肾细胞癌（RCC）占全世界大多数恶性肾肿瘤的和约3％所有成年恶性肿瘤的1，2。由于缺乏症状，误诊，和不足的筛选工具，用于RCC，患者几乎30％将呈现与转移性RCC（MRCC）在诊断时，与患者进展至转移期2 20-30％的额外。这些情况导致每年〜13500人死亡1，3。虽然早期检测和治疗原发性RCC的有所改善，RCC相关的死亡的速率持续增加，这表明转移性疾病是死亡3的主要原因。治疗发展RCC已经发展了与发现并实施有针对性的治疗和免疫治疗的最后十年。不幸的是，无进展surviv人与晚期病例总生存率仍远低于2年，大多数患者4，5，6。这些统计数据为保证进一步研究的识别和对RCC有效的治疗方法发展的需要。为了充分促进治疗性干预在一项针对mRCC，首先需要疾病的翻译相关的临床前模型。 相关的和一致的模型的发展概括晚期RCC人的条件应解决几个关键问题:1）是模型解剖相关; 2）是否肿瘤进展类似于人体病理学; 3）执行转移从原发肿瘤出现;及4）可原发性和转移性肿瘤进展随时间监测？根据治疗的类型被调查，小鼠RCC肿瘤细胞系，人肾细胞癌的肿瘤细胞系，和人RCC患者来源xenog筏可在免疫活性或免疫缺陷的小鼠中。具有完整和功能性免疫系统中的主机需要这些研究评估免疫疗法的一些方面中，需要使用来自Balb / c小鼠7的自发肾腺癌导出的充分描述的Renca细胞系。大多数研究注入RENCA细胞皮下（sc），其形成一个易于度量局部肿瘤，或静脉内（IV）到Balb / c小鼠，以产生实验性肺"转移" 8，9，10，11，12。的SC-植入的Renca肿瘤模型人RCC的使用具有许多限制，包括脉管系统13的不准确的神经支配，在微环境14，15，和缺乏器官/肿瘤细胞COMMUNIC差异通货膨胀13,16。此外，许多皮下肿瘤（特别的Renca）不会转移到远端器官，抑制这是一种常见的临床特征17事件的研究。为了研究mRCC的，RENCA细胞可以静脉注射以建立在RCC患者转移肺-主要位置肿瘤负荷。发起转移性肿瘤的静脉内注射的方法，然而，不允许的侦查如何，何时，或为什么细胞已经从初级器官（ 即，肾）到远端部位迁移。与原发性和转移性疾病明显在相同的动物模型是研究晚期疾病的进展和治疗是至关重要的，尤其是考虑到转移通常在这些患者中的死亡率的原因。 我们的实验室已开发海上救援协调中心的鼠模型，结合了所有上述功能。要建立公关imary，原位肿瘤，的Renca细胞直接植入到动物的透过半透明腹膜肾。在左侧面一个小切口允许脾（如地标）和左肾的可视化。使用小规格针，的Renca肿瘤细胞通过腹膜用于原位移植直接注射到肾脏。相比植入肾囊18的下方的Renca细胞的其它方法中，注入的这个方法允许更高的吞吐量，因为它是相当的非侵入性的，并不需要缝合，是一种低疼痛类的，而且是时间有效时实践。 这充分表征的模型的结果在注射肾再现的原发性肿瘤负担（〜99％的接受率），以及转移性肿瘤负荷于肺部。这种模式的优点显著包括它的同源性质，允许免疫治疗调查;其自发转移，以研究疾病的先进适用的;其原位移植，来模拟对疾病进展和治疗解剖的影响。旨在针对RCC的治疗会从这个模型的临床前开发过程中的利用率大大受益。

<table><tbody><tr><td>Artificial tears ophthalmic ointment</td><td>Akorn</td><td>NDC 17478-162-35</td><td></td></tr><tr><td>Vetbond Tissue Adhesive</td><td>3M</td><td>CBGBIW011019</td><td></td></tr><tr><td>Betadine Solution (Povidone-iodine, 5%)</td><td>Purdue Products, L.P.</td><td>NDC 67618-155-32</td><td></td></tr><tr><td>0.25% Marcaine (bupivavaine HCl injection)</td><td>Hospira</td><td>0409-1587-50&amp;nbsp;</td><td></td></tr><tr><td>Heating pad</td><td>Sunbeam</td><td></td><td></td></tr><tr><td>Syringes</td><td>BD</td><td>309659</td><td></td></tr><tr><td>Gauze</td><td>Venture</td><td>908291</td><td></td></tr><tr><td>Ketamine</td><td>Phoenix Pharmaceuticals</td><td>NDC 57319-609-02</td><td></td></tr><tr><td>Xylazine&amp;nbsp;</td><td>Akorn</td><td>NDC 59399-111</td><td></td></tr><tr><td>Hank&amp;rsquo;s Balanced Salts</td><td>Sigma-Aldrich</td><td>H2387</td><td></td></tr><tr><td>IVIS</td><td>Caliper</td><td></td><td></td></tr><tr><td>D-Luciferin, Potassium Salt</td><td>GoldBio</td><td>LUCK-1G</td><td></td></tr><tr><td>India Ink</td><td>Chartpak Inc</td><td>44201</td><td></td></tr><tr><td>Scissors</td><td></td><td></td><td></td></tr><tr><td>Forceps</td><td></td><td></td><td></td></tr><tr><td>Sleeping Beauty (SB) transposon system</td><td>Neuromics</td><td>SBT0200</td><td></td></tr><tr><td>Renca</td><td>ATCC</td><td>CRL-2947</td><td></td></tr></tbody></table>

a syngeneic mouse model of metastatic renal cell carcinoma for quantitative and longitudinal assessment of preclinical therapies

肾细胞癌（RCC）每年影响&gt; 60000人在美国，和RCC〜30％的患者在诊断时多发转移。转移性肾细胞癌（MRCC）是不治之症，与只有18个月的中位生存时间。基于免疫的干预（ 例如，干扰素（IFN）和白细胞介素（IL）-2）诱导的mRCC患者的一小部分持续反应，和多激酶抑制剂（ 例如，舒尼替尼或索拉非尼）或抗VEGF受体的单克隆抗体（单抗）是主要是治标不治本，因为完全缓解是罕见的。在目前的治疗对于患者mRCC的这些缺点提供的新颖的治疗方案的开发的理由。在新的疗法用于mRCC的临床前检测的关键部件是一个合适的动物模型。重演人的条件有益特征包括原发性肾肿瘤，肾肿瘤转移，和一个完整的免疫系统，以调查任何治疗驱动的免疫效:ř反应和肿瘤诱导的免疫抑制因子的形成。这份报告描述了具有所有这些特征的原位mRCC的小鼠模型。我们描述使用鼠标肾腺癌细胞系的Renca一个肾内注入技术，随后在肾（原发部位）肿瘤生长和肺（转移部位）的评估。

<html> RENCA细胞的成功植入会导致肿瘤的发展在肾脏和转移到小鼠肺。荷瘤和对侧肾脏切除后，湿组织重量（克）进行测定，以证实基于增加的重量（ 图1）的肿瘤负荷。 RENCA肿瘤可以通过免疫组化通过细胞角蛋白8和18染色（ 图2）来识别。对于纵向研究中，荧光素酶表达的Renca细胞系植入。使用体内成像系统（IVIS）来跟踪肿瘤负荷和对治疗?...

Watch this Scientific Journal Video about A Syngeneic Mouse Model of Metastatic Renal Cell Carcinoma for Quantitative and Longitudinal Assessment of Preclinical Therapies at JoVE.com

A Syngeneic Mouse Model of Metastatic Renal Cell Carcinoma for Quantitative and Longitudinal Assessment of Preclinical Therapies

在免疫活性小鼠肾细胞癌的原位模型的实现，得到研究者通过初级肾肿瘤和肺转移瘤的在相同的动物的存在所定义的临床相关系统。该系统可用于临床前测试各种体内治疗。

转移性的型号同源小鼠肾细胞癌的临床前疗法的定量和纵向评估

Renal cell carcinoma (RCC) affects &#62; 60,000 people in the United States annually, and ~ 30% of RCC patients have multiple metastases at the time of ...

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Cancer Research

13.5K 次观看.  University of Minnesota. 在免疫活性小鼠肾细胞癌的原位模型的实现，得到研究者通过初级肾肿瘤和肺转移瘤的在相同的动物的存在所定义的临床相关系统。该系统可用于临床前测试各种体内治疗。 

视频: A Syngeneic Mouse Model of Metastatic Renal Cell Carcinoma for Quantitative and Longitudinal Assessment of Preclinical Therapies

Comparing Metastatic Clear Cell Renal Cell Carcinoma Model Established in Mouse Kidney and on Chicken Chorioallantoic Membrane

Metastatic clear cell renal cell carcinoma (ccRCC) is the most common subtype of kidney cancer. Localized ccRCC has a favorable surgical outcome. However, one third of ccRCC patients will develop metastases to the lung, which is related to a very poor outcome for patients. Unfortunately, no therapy is available for this deadly stage, because the molecular mechanism of metastasis remains unknown. It has been known for 25 years that the loss of function of the von Hippel-Lindau (VHL) tumor suppressor gene is pathognomonic of ccRCC. However, no clinically relevant transgenic mouse model of ccRCC has been generated. The purpose of this protocol is to introduce and compare two newly established animal models for metastatic ccRCC. The first is renal implantation in the mouse model. In our laboratory, the CRISPR gene editing system was utilized to knock out the VHL gene in several RCC cell lines. Orthotopic implantation of heterogeneous ccRCC populations to the renal capsule created novel ccRCC models that develop robust lung metastases in immunocompetent mice. The second model is the chicken chorioallantoic membrane (CAM) system. In comparison to the mouse model, this model is more time, labor, and cost-efficient. This model also supported robust tumor formation and intravasation. Due to the short 10 day period of tumor growth in CAM, no overt metastasis was observed by immunohistochemistry (IHC) in the collected embryo tissues. However, when tumor growth was extended by two weeks in the hatched chicken, micrometastatic ccRCC lesions were observed by IHC in the lungs. These two novel preclinical models will be useful to further study the molecular mechanism behind metastasis, as well as to establish new, patient-derived xenografts (PDXs) toward the development of novel treatments for metastatic ccRCC.

Metastatic clear cell renal cell carcinoma is a disease without a comprehensive animal model for thorough preclinical investigation. This protocol illustrates two novel animal models for the disease: the orthotopically implanted mouse model and the chicken chorioallantoic membrane model, both of which demonstrate lung metastasis resembling clinical cases.

比较在小鼠肾脏和鸡胆膜上建立的转移性透明细胞肾细胞癌模型

Metastatic clear cell renal cell carcinoma (ccRCC) is the most common subtype of kidney cancer. Localized ccRCC has a favorable surgical outcome. However, ...

科研

癌症研究

Discovery of Metastatic Regulators using a Rapid and Quantitative Intravital Chick Chorioallantoic Membrane Model

Recent advances in cancer research has illustrated the highly complex nature of cancer metastasis. Multiple genes or genes networks have been found to be involved in differentially regulating cancer metastatic cascade genes and gene products dependent on the cancer type, tissue, and individual patient characteristics. These represent potentially important targets for genetic therapeutics and personalized medicine approaches. The development of rapid screening platforms is essential for the identification of these genetic targets.
The chick chorioallantoic membrane (CAM) is a highly vascularized, collagen rich membrane located under the eggshell that allows for gas exchange in the developing embryo. Due to the location and vascularization of the CAM, we developed it as an intravital human cancer metastasis model that allows for robust human cancer cell xenografting and real-time imaging of cancer cell interactions with the collagen rich matrix and vasculature. 
Using this model, a quantitative screening platform was designed for the identification of novel drivers or suppressors of cancer metastasis. We transduced a pool of head and neck HEp3 cancer cells with a complete human genome shRNA gene library, then injected the cells, at low density, into the CAM vasculature. The cells proliferated and formed single-tumor cell colonies. Individual colonies that were unable to invade into the CAM tissue were visible as a compact colony phenotype and excised for identification of the transduced shRNA present in the cells. Images of individual colonies were evaluated for their invasiveness. Multiple rounds of selections were performed to decreases the rate of false positives. Individual, isolated cancer cell clones or newly engineered clones that express genes of interest were subjected to primary tumor formation assay or cancer cell vasculature co-option analysis. In summary we present a rapid screening platform that allows for anti-metastatic target identification and intravital analysis of a dynamic and complex cascade of events.

This is an effective method to screen for suppressors or drivers of cancer metastasis. Cells, transduced with an expression library, are injected into the chicken chorioallantoic membrane vasculature to form metastatic colonies. Colonies having decreased or increased invasiveness are excised, expanded, reinjected to confirm their phenotype, and finally, analyzed using high throughput sequencing.

使用快速和定量的细胞内胆碱膜模型发现转移调节器

Recent advances in cancer research has illustrated the highly complex nature of cancer metastasis. Multiple genes or genes networks have been found to be ...

An Orthotopic Murine Model of Human Prostate Cancer Metastasis

Our laboratory has developed a novel orthotopic implantation model of human prostate cancer (PCa). As PCa death is not due to the primary tumor, but rather the formation of distinct metastasis, the ability to effectively model this progression pre-clinically is of high value. In this model, cells are directly implanted into the ventral lobe of the prostate in Balb/c athymic mice, and allowed to progress for 4-6 weeks. At experiment termination, several distinct endpoints can be measured, such as size and molecular characterization of the primary tumor, the presence and quantification of circulating tumor cells in the blood and bone marrow, and formation of metastasis to the lung. In addition to a variety of endpoints, this model provides a picture of a cells ability to invade and escape the primary organ, enter and survive in the circulatory system, and implant and grow in a secondary site. This model has been used effectively to measure metastatic response to both changes in protein expression as well as to response to small molecule therapeutics, in a short turnaround time.

This orthotopic model of human prostate cancer allows for quantification of tumor size, circulating tumor cells, and formation of distinct metastasis to the lung. As cells must escape the primary organ, enter the blood stream, and implant into a secondary site, this model effectively recapitulates the scenario in humans.

人类前列腺癌转移的原位小鼠模型

Our laboratory has developed a novel orthotopic implantation model of  human prostate cancer (PCa). As PCa death is not due to the primary tumor, but  ...

医学

A Robust Discovery Platform for the Identification of Novel Mediators of Melanoma Metastasis

Metastasis is a complex process, requiring cells to overcome barriers that are only incompletely modeled by in vitro assays. A systematic workflow was established using robust, reproducible in vivo models and standardized methods to identify novel players in melanoma metastasis. This approach allows for data inference at specific experimental stages to precisely characterize a gene's role in metastasis. Models are established by introducing genetically modified melanoma cells via intracardiac, intradermal, or subcutaneous injections into mice, followed by monitoring with serial in vivo imaging. Once preestablished endpoints are reached, primary tumors and/or metastases-bearing organs are harvested and processed for various analyses. Tumor cells can be sorted and subjected to any of several 'omics' platforms, including single-cell RNA sequencing. Organs undergo imaging and immunohistopathological analyses to quantify the overall burden of metastases and map their specific anatomic location. This optimized pipeline, including standardized protocols for engraftment, monitoring, tissue harvesting, processing, and analysis, can be adopted for patient-derived, short-term cultures and established human and murine cell lines of various solid cancer types.

This article describes a workflow of techniques employed for testing novel candidate mediators of melanoma metastasis and their mechanism(s) of action.

用于鉴定黑色素瘤转移新介质的强大发现平台

Metastasis is a complex process, requiring cells to overcome barriers that are only incompletely modeled by in vitro assays. A systematic workflow was ...

In Vivo Imaging to Measure Spontaneous Lung Metastasis of Orthotopically-injected Breast Tumor Cells

Metastasis remains the primary cause of cancer-related death. The succession of events that characterize the metastatic cascade presents multiple opportunities for therapeutic intervention, and the ability to accurately model them in mice is critical to evaluate their effects. Here, a step-by-step protocol is presented for the establishment of orthotopic primary breast tumors and the subsequent monitoring of the establishment and growth of metastatic lesions in the lung using in vivo bioluminescence imaging. This methodology allows for the evaluation of treatment or its biological effects along the entire range of metastatic development, from primary tumor escape to outgrowth in the lungs. Breast orthotopic tumors are generated in mice via injection of a luciferase-labeled cell suspension in the 4th mammary gland. Tumors are allowed to grow and disseminate for a specific amount of time and are then surgically resected. Upon resection, spontaneous lung metastasis is detected, and the growth over time is monitored using in vivo bioluminescence imaging. At the desired experimental endpoint, lung tissue can be collected for downstream analysis. The treatment of established, clinically evident metastasis is critical to improve outcomes for stage IV cancer patients, and it can be evaluated through tail vein models of experimental lung metastasis. However, metastatic dissemination occurs early in breast cancer, and many patients have latent, subclinical disseminated disease after surgery. Utilization of spontaneous models such as this one provides the opportunity to study the whole spectrum of the disease, especially the systemic effects driven by treatment of the primary tumor such as pre-metastatic niche priming, and evaluate treatments on dormant and subclinical disease after surgery.

<meta charset="utf8"></head><body>体内成像测量原位注射乳腺肿瘤细胞的自发性肺转移

The manuscript describes a methodology for the establishment as well as longitudinal growth monitoring of spontaneous lung metastasis from orthotopically-injected breast tumors, amenable to intervention at all stages of the metastatic cascade.

体内研究 影像学检查测量原位注射乳腺肿瘤细胞的自发性肺转移

Metastasis remains the primary cause of cancer-related death. The succession of events that characterize the metastatic cascade presents multiple ...

Modeling Spontaneous Metastatic Renal Cell Carcinoma (mRCC) in Mice Following Nephrectomy

One of the key challenges to improved testing of new experimental therapeutics in renal cell carcinoma (RCC) is the development of models that faithfully recapitulate early- and late-stage metastatic disease progression. Typical tumor implantation models utilize ectopic or orthotopic primary tumor implantation, but few include systemic spontaneous metastatic disease that mimics the clinical setting. This protocol describes the key steps to develop RCC disease progression stages similar to patients. First, it uses a highly metastatic mouse tumor cell line in a syngeneic model to show orthotopic tumor cell implantation. Methods include superficial and internal implantation into the sub-capsular space with cells combined with matrigel to prevent leakage and early spread. Next it describes the procedures for excision of tumor-bearing kidney (nephrectomy), with critical pre- and post- surgical mouse care. Finally, it outlines the steps necessary to monitor and assess micro-and macro-metastatic disease progression, including bioluminescent imaging as well provides a detailed visual necropsy guide to score systemic disease distribution. The goal of this protocol description is to facilitate the widespread use of clinically relevant metastatic RCC models to improve the predictive value of future therapeutic testing.&#160;

Modeling Spontaneous Metastatic Renal Cell Carcinoma mRCC in Mice Following Nephrectomy

Models of spontaneous metastatic renal cell carcinoma (RCC) disease progression can be used for evaluating treatments in a clinically relevant setting. This protocol demonstrates different procedures for orthotopic kidney tumor cell implantation, proper nephrectomy, and finally outlines a necropsy guide for visual and bioluminescent scoring of metastatic burden and localization.

在小鼠模型自发转移性肾细胞癌（MRCC）肾切除

One of the key challenges to improved testing of new experimental therapeutics in renal cell carcinoma (RCC) is the development of models that faithfully ...

A Preclinical Murine Model of Hepatic Metastases

Numerous murine models have been developed to study human cancers and advance the understanding of cancer treatment and development. Here, a preclinical, murine pancreatic tumor model of hepatic metastases via a hemispleen injection of syngeneic murine pancreatic tumor cells is described. This model mimics many of the clinical conditions in patients with metastatic disease to the liver. Mice consistently develop metastases in the liver allowing for investigation of the metastatic process, experimental therapy testing, and tumor immunology research.

A preclinical, murine model of hepatic metastases performed via a hemispleen injection technique.

肝转移的临床前小鼠模型

Numerous murine models have been developed to study human cancers and advance the understanding of cancer treatment and development. Here, a preclinical, ...

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

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

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

"以病人为像"肝癌原位同源小鼠模型的癌转移

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

Evaluation of Lung Metastasis in Mouse Mammary Tumor Models by Quantitative Real-time PCR

Metastatic disease is the spread of malignant tumor cells from the primary cancer site to a distant organ and is the primary cause of cancer associated death 1. Common sites of metastatic spread include lung, lymph node, brain, and bone 2. Mechanisms that drive metastasis are intense areas of cancer research. Consequently, effective assays to measure metastatic burden in distant sites of metastasis are instrumental for cancer research. Evaluation of lung metastases in mammary tumor models is generally performed by gross qualitative observation of lung tissue following dissection. Quantitative methods of evaluating metastasis are currently limited to ex vivo and in vivo imaging based techniques that require user defined parameters. Many of these techniques are at the whole organism level rather than the cellular level 3-6. Although newer imaging methods utilizing multi-photon microscopy are able to evaluate metastasis at the cellular level 7, these highly elegant procedures are more suited to evaluating mechanisms of dissemination rather than quantitative assessment of metastatic burden. Here, a simple in vitro method to quantitatively assess metastasis is presented. Using quantitative Real-time PCR (QRT-PCR), tumor cell specific mRNA can be detected within the mouse lung tissue.

This protocol describes how to use quantitative Real-time PCR (QRT-PCR) to detect tumor cell specific mRNA representing metastasis within the mouse lung tissue.

评价肺转移的小鼠乳腺肿瘤模型定量实时荧光定量PCR

Metastatic disease is the spread of malignant tumor cells from the primary cancer site to a distant organ and is the primary cause of cancer associated ...

转移性的型号同源小鼠肾细胞癌的临床前疗法的定量和纵向评估

摘要

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比较在小鼠肾脏和鸡胆膜上建立的转移性透明细胞肾细胞癌模型

使用快速和定量的细胞内胆碱膜模型发现转移调节器

人类前列腺癌转移的原位小鼠模型

用于鉴定黑色素瘤转移新介质的强大发现平台

体内研究影像学检查测量原位注射乳腺肿瘤细胞的自发性肺转移

体内研究影像学检查测量原位注射乳腺肿瘤细胞的自发性肺转移

在小鼠模型自发转移性肾细胞癌（MRCC）肾切除

肝转移的临床前小鼠模型

"以病人为像"肝癌原位同源小鼠模型的癌转移

评价肺转移的小鼠乳腺肿瘤模型定量实时荧光定量PCR

转移性的型号同源小鼠肾细胞癌的临床前疗法的定量和纵向评估

摘要

探索更多视频

此视频中的章节

比较在小鼠肾脏和鸡胆膜上建立的转移性透明细胞肾细胞癌模型

使用快速和定量的细胞内胆碱膜模型发现转移调节器

人类前列腺癌转移的原位小鼠模型

用于鉴定黑色素瘤转移新介质的强大发现平台

体内研究 影像学检查测量原位注射乳腺肿瘤细胞的自发性肺转移

体内研究 影像学检查测量原位注射乳腺肿瘤细胞的自发性肺转移

在小鼠模型自发转移性肾细胞癌（MRCC）肾切除

肝转移的临床前小鼠模型

"以病人为像"肝癌原位同源小鼠模型的癌转移

评价肺转移的小鼠乳腺肿瘤模型定量实时荧光定量PCR

体内研究影像学检查测量原位注射乳腺肿瘤细胞的自发性肺转移

体内研究影像学检查测量原位注射乳腺肿瘤细胞的自发性肺转移