在星爷的实验室是支持由美国癌症协会的机构研究基金（IRG-97-219-14）从美国国防部的资助奖励给霍林斯癌症中心MUSC，由科研经费（W81XWH-11-2-科研经费0229） 在MUSC，并通过从关怀基金会（到ESY）裁决。

该协议遵循的准则和南卡罗来纳州（MUSC）和它的机构动物护理和使用委员会的医科大学（IACUC）动物保健标准。 1.肺解剖<ol><li>乳腺肿瘤切除（可选取决于学习的目标和是否进行原发肿瘤的分析或尾静脉注射）。 <ol><li>使用根据IACUC和大学调控致死剂量异氟醚麻醉安乐死鼠标。确认安乐死手术脱位。 </li><li>在泡沫板，针与解剖针通过把鼠标在它的后面有四肢蔓延。 </li><li>喷小鼠用70％的乙醇。 </li><li>使用镊子，把握动物的下部在中心。 </li><li>使用剪刀，剪向上朝颈部穿过皮肤，小心不要刺破腹膜鼠标腔。 </li><li>减少皮肤四肢揭示肿瘤组织。 </li><li>解剖出肿瘤组织和公关通过优选的方法eserve，如冷冻或固定，以供进一步分析（未进一步在这个协议中所讨论的）。 </li></ol></li><li>肺组织清扫。 <ol><li>如果上述肿瘤组织收获，牵制任何多余的皮肤。 </li><li>使用镊子，把握腹膜在动物的下端，并开始朝向颈部的底部切削向上。 </li><li>沿着肋笼小心，以避免切断任何血管可能出血进入胸腔的左，右两侧小心切开。通过切割到左边，并通过肋骨的膜片和上部右去除肋骨。 </li><li>使用镊子抓住老鼠的气管，向前拉。 </li><li>断绝与解剖剪刀气管和鼠标删除肺部。 注意:心脏可能仍然附着到肺。如果发生这种情况，小心地从肺部解剖心脏远小心翼翼地收集所有五个叶片（ 见图 1 </stroNG&gt;）。 </li><li>用PBS轻轻冲洗，去除多余血液。 </li></ol></li><li>肺组织的总评价。 <ol><li>选项1: 在体内成像 。 <ol><li>萤光素成像，〜10分钟之前成像，给动物150毫克/公斤剂量的萤光素通过腹膜内（IP）注射。 在体内在麻醉动物或在除去后解剖（图2）5图像肺部。 </li></ol></li><li>选项​​2:总的评价。 <ol><li>检查体视显微镜下肺可视化的肿瘤结节。注意:如果细胞是绿色荧光蛋白标记的，可以使用含有（邻近五百十分之四百七十毫微米例如 ）一个光立方与适当的激发/发射最大值的荧光立体显微镜能够以检测GFP的检查肺立体对加工前的荧光。 </li></ol></li><li>如果肺要立即进行分析，进入"RNA提取"部分。否则，使用理屈扣冻结的组织的id氮气或干冰。储存在-80℃。 </li></ol></li></ol> 2. RNA分离注:对于代表分析，RNA是使用RNA提取试剂盒（见材料清单）隔离。而当前的分析使用一个特定制造商的产品，各种分离试剂盒的可以从多个信誉的供应商。此外，非试剂盒基于离心的方法也是一种选择。的cDNA也应该从一个阳性对照RNA样品制备，例如一个细胞系或质粒，对于标准曲线分析。另外，具体的引物探针组阳性对照可以购买（见材料清单）。 <ol><li>如果使用以前冰冻组织，收集干冰​​冷冻组织。 </li><li>均质化使用以下方法如之一组织:研钵和杵研磨冷冻的组织在液氮中后由手工进行过度干冰;组织匀浆器或机械分离装置按照制造商的建议;超声处理，或其他的优选的方法。 注:对于代表分析，同质化的首选方法是超声波。 <ol><li> 对于图3中所作的分析，制备裂解缓冲液（在RNA分离试剂盒中提供）和2-巯基乙醇的比率20微升2-巯基乙醇，每1毫升的裂解缓冲液。重悬在组织300微升裂解缓冲液补充有2-巯基乙醇，每30克组织和超声的10秒用超声波仪设定在30％的幅度。 </li><li>如果使用研钵和杵，重悬地面组织在300μl的裂解缓冲液研磨后。 </li></ol></li><li>加10微升蛋白酶K至590微升的TE缓冲液（10mM的Tris-HCl，pH值8.0; 1mM EDTA）中。加入600微升蛋白酶K的解决方案，以每300微升（ 即每30毫克）的组织。下室温温育10分钟。 注:消化时间可能有所不同。 </li><li>离心样品在≥13000克10分钟以除去碎片。 </li><li>上清转移到新管。 </li><li>添加450微升乙醇（96％-100％），每900微升上清液来沉淀RNA。 </li><li>传输700微升溶胞产物/乙醇混合到柱上。 </li><li>离心样品在≥13,000克1分钟，从而结合核糖核酸柱。 </li><li>弃废液，加入剩余的上清液列，并再次旋转。 </li><li>一旦所有的溶胞产物通过柱纺丝，添加350微升洗涤缓冲液1到列和离心机样品的上部在≥13,000克30秒。 </li><li>从塔倒掉液体，更换上部。 </li><li>通过混合5单位DNA酶I酶与5微升10×DNA酶和40μlDNA酶/ RNA酶的水每样品（50微升/样品总体积）制备DNA酶。 </li><li>加入50微升DNA酶的组合，以每一列并孵育15分钟，在室温。 </li><li>加入350微升洗涤液1列和离心机SAM普莱斯在≥13,000克30秒。 </li><li>从塔倒掉液体，更换上部。 </li><li>添加600μl的洗涤缓冲液2到列和自旋30秒≥13,000克。 </li><li>从塔倒掉液体，更换上部。 </li><li>加入250微升洗涤液2，离心2分钟≥13,000克</li><li>把列的一部分进入新的收集管并弃去老收集管。 </li><li>加入50-100微升RNA酶/ DNA酶的游离水柱并孵育1分钟。 </li><li>自旋为1分钟≥13,000克。 </li><li>重新运行，通过柱收集洗脱液，以增加RNA产量。 </li><li>为了立即使用，保持在冰上的样品，并继续定量。以备后用，扣冻结的干冰或液氮。直到需要储存在-80℃冰箱。 </li></ol> 3.第一链合成注意:对于代表分析，逆转录酶（RT）进行反应，一架FIRST链cDNA合成试剂盒专为荧光定量PCR（见材料清单）。 <ol><li>如果以前的样品收集，解冻冰上管。 </li><li>使用分光光度计测量RNA的量。 </li><li>使用0.5-2微克总RNA，使用逆转录酶，以产生cDNA的库存进行第一链合成。 <ol><li>在无菌的，无RNase /无DNase的PCR管/板，准备反应混合物（ 表1）。轻轻混匀离心。编程一个标准PCR机（见表2）。 </li></ol></li><li>稀释成品反应使用无菌无核酸酶水所需体积（通常50-100微升）。 </li></ol> 4.实时荧光定量PCR 注:对于代表分析，SYBR绿色使用。然而，任何优选的方法可以在用户的​​决定被取代。 <ol><li>使用阳性对照基因，建立了一个标准曲线各引物组。 <ol><li>为了显示我的分析Q图3，做标准曲线，使用厂家人 HER2推荐阳性对照基因（见材料清单）。对于小鼠GAPDH，使用阴性对照肺cDNA，以产生标准曲线。对于每个探针，串联稀释的cDNA 1:4至5生成的标准。 </li></ol></li><li>计算总样品的数量，其中应包括标准曲线和实验样品。 注意:这里的分析说明，每个样品2-3实验重复进行了分析。进行标准曲线分析，以产生可以应用到每个样品，计算最终归量确定HER2的相对量和 GAPDH一个简单的线性回归模型。这种分析也将确保引物效率是足够的分析在手。 </li><li>在无菌的，无RNase / DNase的自由管，制备主混合物（表3）。 <ol><li>计算主的Amoun组合牛逼每个样品。向上扩展超过一个样本。使用一个主混合物对于每个实验基因的兴趣，以及作为内部对照如GAPDH。使用的引物为GAPDH以200nM的最终浓度。 注意:尝试人和小鼠细胞来源区分时，内部控制是特别有用的。 注意:引物浓度对 HER2进行优化，由制造商确定。 </li></ol></li><li>制备试验板包含对应于每个标准或实验样品1微升的cDNA。至少分配1 cDNA样品/孔为每个引物探测器。添加19微升每孔主混合物的每种引物探针。 注意:对于图 3中的有代表性的数据，将cDNA样品一式两份每个引物探针进行了分析和作图作为两个样品的平均值。 </li><li>使用推荐的或以前测试PCR方法在荧光定量PCR仪运行的反应。 见表4 </s仲&gt;为用于在图 3和图 4中的表示数据的PCR方案。 </li></ol>

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	<li>Gupta, G. P., Massague, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+metastasis:+building+a+framework.">Cancer metastasis: building a framework.</a> Cell. 127 (4), 679-695 (2006).</li><li>Bos, P. D., Nguyen, D. X., Massague, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modeling+metastasis+in+the+mouse.">Modeling metastasis in the mouse.</a> Curr Opin Pharmacol. 10 (5), 571-577 (2010).</li><li>Kim, I. S., Baek, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+models+for+breast+cancer+metastasis.">Mouse models for breast cancer metastasis.</a> Biochem Biophys Res Commun. 394 (3), 443-447 (2010).</li><li>Saxena, M., Christofori, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rebuilding+cancer+metastasis+in+the+mouse.">Rebuilding cancer metastasis in the mouse.</a> Mol Oncol. 7 (2), 283-296 (2013).</li><li>Yang, S., Zhang, J. J., Huang, X. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+models+for+tumor+metastasis.">Mouse models for tumor metastasis.</a> Methods Mol Biol. 928, 221-228 (2012).</li><li>Rampetsreiter, P., Casanova, E., Eferl, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetically+modified+mouse+models+of+cancer+invasion+and+metastasis.">Genetically modified mouse models of cancer invasion and metastasis.</a> Drug Discov Today Dis Models. 8 (2-3), 67-74 (2011).</li><li>Condeelis, J., Segall, 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=Intravital+imaging+of+cell+movement+in+tumours.">Intravital imaging of cell movement in tumours.</a> Nat Rev Cancer. 3 (12), 921-930 (2003).</li><li>Massoud, T. F., Gambhir, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+imaging+in+living+subjects:+seeing+fundamental+biological+processes+in+a+new+light.">Molecular imaging in living subjects: seeing fundamental biological processes in a new light.</a> Genes Dev. 17 (5), 545-580 (2003).</li><li>Zinn, K. 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=Noninvasive+bioluminescence+imaging+in+small+animals.">Noninvasive bioluminescence imaging in small animals.</a> ILAR J. 49 (1), 103-115 (2008).</li><li>Tseng, J. C., Kung, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+bioluminescence+imaging+of+mouse+tumor+models.">Quantitative bioluminescence imaging of mouse tumor models.</a> Cold Spring Harb Protoc. 2015 (1), (2015).</li><li>Radonic, 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=Guideline+to+reference+gene+selection+for+quantitative+real-time+PCR.">Guideline to reference gene selection for quantitative real-time PCR.</a> Biochem Biophys Res Commun. 313 (4), 856-862 (2004).</li></ol>

ESY和MAA不受FirstString研究公司聘用，不抱任何经济利益的公司。 FirstString研究公司提供的是在这个手稿提出的荧光素酶成像数据。 GSG是FirstString Research的总裁兼首席执行官。 CLG是FirstString研究的雇员。 GSG和CLG必须通过FirstString研究发行股票期权。

这个协议描述使用QRT-PCR，以评估乳腺肿瘤细胞转移到使用异种移植物小鼠模型中肺。应当承认，其他技术，包括生物发光成像，都可以，也是有效的，尽管有自己的价值观和限制检测转移。提出的数据表明，QRT-PCR提供了检测肺组织内的肿瘤来源的mRNA的总转移的定量的有效措施。所以建议的QRT-PCR分析提供了，可以补充或代替这些成像技术来执行辅助方法。这种技术可能最适合用于研究有限的环境...

QRT-PCR分析，提出作为评估肿瘤转移的方法。这种方法被提出作为该有兴趣评估转移，但可能无法获得特定设备，例如在体内成像设备或荧光能立体镜用户的替代。的常用方法的讨论，提出后跟一个示范如何QRT-PCR分析可以使用，也可以作为一个单独的或作为伴侣方法来评价转移。此过程有可能提供的转移性负担进行定量分析。 总分析的标准方法，包括肺的立体显微镜下的可视化以及连续切片随后苏木精和曙红肺组织（H＆E）染色，可以量化但在很大程度上依赖于用户定义的参数，用于计数2-5。在评估整个肺使用立体显微镜，只有大面积转移是VISI竹叶提取和分析需要研究者具有肺解剖结构的合理的知识来确定什么构成转移性病灶。肿瘤细胞与一个标记例如GFP和使用包含光立方与适当的激发/发射最大值（如邻近五百一十分之四百七毫微米的GFP）在立体显微镜的荧光标记协助在此过程中，但只有表面肿瘤结节是可检测的。此外，从血液污染，这是根据相同的参数的GFP可见荧光，可能会导致可能的转移病灶误识别。 切片肺其次是H＆E染色可视化肺转移是评价微转移等微观过程，包括免疫细胞浸润的有效方法，但往往需要利用整个肺组织石蜡包埋，切片，和染色程序。因此，下游的过程是不理想的下面这个实现方法具ð。虽然量化，这个过程需要研究者评估了大量的每只动物染色肺切片，以确保分析占肺的整个3D结构。因此，这种类型的检查非常耗时，可能会导致计数误差，并分析在很大程度上依赖于研究者决定。 几个体内成像 技术 （例如MRI，PET，SPECT）目前用于执行或在实验啮齿动物模型中进行测试8的生物过程。 体内生物发光成像是用于获取转移9的总图的常用方法。这种技术通常被应用于评估荧光素酶报告活性的存在，由于肿瘤细胞，其工程化以包含一个萤光素酶应答元件，驻留在像后肿瘤细胞植入乳腺以及在自发转移肺特定器官的积累10。可视化的荧光素酶报告活性的由荧光素底物（D-萤光素）的存在下诱发。萤光素酶催化的D-荧光素的氧化脱羧以产生氧化萤光素生物发光。而信息量大，这种方法是由几个因素，包括基片的稳定性（ 即半衰期短），基板，取决于它是如何传递到实验动物的适当分配，并检测9的低灵敏度的限制。一个主要的优点在该技术是，它是非侵入性的，可以在活的动物进行的，并可能导致在多个器官肿瘤细胞转移的检测可能没有被正常收获解剖9,10。 体内成像技术之一积极方面是肺组织不受干扰允许像石蜡包埋次级步骤或如这里提出，QRT-PCR分析。但是，因为QRT-PCR是theoreticaLLY检测的更灵敏的测量，总评价可能不能揭示低号码存在于肺的肿瘤细胞。虽然有用，上述的成像技术可以被取代或补充有目前描述的QRT-PCR法。 QRT-PCR有潜力肺内提供的肿瘤来源的mRNA的一个敏感量度。

<table><tbody><tr><td>2-mercaptoethanol</td><td>Fisher</td><td>BP176-100</td><td></td></tr><tr><td>DNAse</td><td>Thermo Scientific</td><td>EN0521</td><td></td></tr><tr><td>GeneJet RNA Isolation Kit</td><td>Thermo Scientific</td><td>K0732</td><td></td></tr><tr><td>iScript Reverse Transcription Supermix for QRT-PCR</td><td>Bio-Rad</td><td>1708841</td><td></td></tr><tr><td>iTaq Universal SYBR Green Supermix&amp;nbsp;</td><td>Bio-Rad</td><td>172-5121&amp;nbsp;</td><td></td></tr><tr><td>PrimePCR SYBR Green Assay: ERBB2, Human&amp;nbsp;</td><td>Bio-Rad</td><td>100-25636&amp;nbsp;</td><td></td></tr><tr><td>PrimePCR Template for SYBR Green Assay: ERBB2, Human&amp;nbsp;</td><td>Bio-Rad</td><td>100-25716&amp;nbsp;</td><td></td></tr><tr><td>gapdh Primer Sequence</td><td></td><td></td><td>For-ATGGTGAAGGTCGGTGTGAACG Rev-CGCTCCTGGAAGATGGTGATGG</td></tr></tbody></table>

evaluation of lung metastasis in mouse mammary tumor models by quantitative real-time pcr

转移性疾病是从主癌症部位 ​​到远处器官恶性肿瘤细胞的扩散和是癌症相关死亡1的首要原因。转移扩散共同位点包括肺，淋巴结，脑，骨和2。机制，推动转移是癌症研究的激烈的领域。因此，有效的试验来测量转移性负担转移遥远的站点是有助于为癌症研究。一般是由以下解剖定性观察肺组织的总评价进行乳腺肿瘤模型肺转移。评估转移的定量方法目前仅限于体外 ，并在需要的用户定义的参数基于活体成像技术。许多这些技术是在整个生物体水平，而不是在细胞水平3-6。虽然利用多光子显微镜较新的成像方法能够评估METASTASIS在细胞水平7，这些高度优雅的程序更适合于传播评估机制，而不是转移负担的量化评估。这里，一个简单的体外方法定量评估转移呈现。利用定量实时PCR（QRT-PCR），肿瘤细胞特异性mRNA可以内的小鼠肺组织中检测到。

超出所花费的时间来执行初始接种肿瘤细胞向实验动物和如果执行，主体内肿瘤分析，组织收获，RNA分离，和QRT-PCR分析是一个1-2天的过程（图1） 。 总分析的一个实例是生物发光成像内的肺组织评估肿瘤细胞。这里，一个乳腺肿瘤实验以评估在研药物靶向间隙连接蛋白43，称为ACT1，是否会削弱4T1-的luc乳腺?...

Watch this Scientific Journal Video about Evaluation of Lung Metastasis in Mouse Mammary Tumor Models by Quantitative Real-time PCR at JoVE.com

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

这个协议描述了如何使用定量实时PCR（QRT-PCR），以检测表示内的小鼠肺组织转移的肿瘤细胞特异性mRNA。

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 ...

evaluation-lung-metastasis-mouse-mammary-tumor-models-quantitative

Universidad de Cartagena UDEC

Research

JoVE Journal

Medicine

9.8K 次观看.  Medical University of South Carolina. 这个协议描述了如何使用定量实时PCR（QRT-PCR），以检测表示内的小鼠肺组织转移的肿瘤细胞特异性mRNA。 

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

Surgical Procedures and Methodology for a Preclinical Murine Model of De Novo Mammary Cancer Metastasis

A rate-limiting aspect of transgenic mouse models of mammary adenocarcinoma is that primary tumor burden in mammary tissue typically defines study end-points. Thus, studies focused on elucidating mechanisms of late-stage de novo metastasis are compromised, as are studies examining efficacy of anti-cancer therapies targeting mediators of metastasis in the adjuvant setting. Numerous murine mammary cancer models have been developed via targeted expression of dominant oncoproteins to mammary epithelial cells yielding models variably mimicking histopathologic and transcriptome-defined breast cancer subtypes common in women1. While much has been learned regarding the biology of mammary carcinogenesis with these models, their utility in identifying molecules regulating growth of late-stage metastasis are compromised as mice are typically euthanized at earlier time points due to significant primary tumor burden. Moreover, since a significant percentage of women diagnosed with breast cancer receive adjuvant therapy after surgical resection of primary tumors and prior to presence of detectable metastatic disease, preclinical models of de novo metastasis are urgently needed as platforms to evaluate new therapies aimed at targeting metastatic foci. To address these deficiencies, we developed a murine model of de novo mammary cancer metastasis, wherein primary mammary tumors are surgically resected, and metastatic foci subsequently develop over a 115 day post-surgical period. This long latency provides a tractable model to identify functionally significant regulators of metastatic progression in mice lacking primary tumor, as well as a model to evaluate preclinical therapeutic efficacy of agents aimed at blocking functionally significant molecules aiding metastatic tumor survival and growth.

Surgical Procedures and Methodology for a Preclinical Murine Model of De Novo Mammary Cancer Metastasis

Pre-clinical models evaluating adjuvant therapy targeting breast cancer metastasis are lacking. To address this, we developed a murine model of de novo pulmonary mammary adenocarcinoma metastasis, wherein therapies administered in the adjuvant setting (post surgical resection of primary tumors) can be evaluated for efficacy in impacting previously seeded pulmonary metastases.

临床前小鼠模型的外科手术和方法<em&gt; De Novo</em&gt;乳腺癌转移

A rate-limiting aspect of transgenic mouse models of mammary adenocarcinoma is that primary tumor burden in mammary tissue typically defines study ...

科研

癌症研究

A Portal Vein Injection Model to Study Liver Metastasis of Breast Cancer

Breast cancer is the leading cause of cancer-related mortality in women worldwide. Liver metastasis is involved in upwards of 30% of cases with breast cancer metastasis, and results in poor outcomes with median survival rates of only 4.8 - 15 months. Current rodent models of breast cancer metastasis, including primary tumor cell xenograft and spontaneous tumor models, rarely metastasize to the liver. Intracardiac and intrasplenic injection models do result in liver metastases, however these models can be confounded by concomitant secondary-site metastasis, or by compromised immunity due to removal of the spleen to avoid tumor growth at the injection site. To address the need for improved liver metastasis models, a murine portal vein injection method that delivers tumor cells firstly and directly to the liver was developed. This model delivers tumor cells to the liver without complications of concurrent metastases in other organs or removal of the spleen. The optimized portal vein protocol employs small injection volumes of 5 - 10 &#956;l, &#8805; 32 gauge needles, and hemostatic gauze at the injection site to control for blood loss. The portal vein injection approach in Balb/c female mice using three syngeneic mammary tumor lines of varying metastatic potential was tested; high-metastatic 4T1 cells, moderate-metastatic D2A1 cells, and low-metastatic D2.OR cells. Concentrations of &#8804; 10,000 cells/injection results in a latency of ~ 20 - 40 days for development of liver metastases with the higher metastatic 4T1 and D2A1 lines, and &#62; 55 days for the less aggressive D2.OR line. This model represents an important tool to study breast cancer metastasis to the liver, and may be applicable to other cancers that frequently metastasize to the liver including colorectal and pancreatic adenocarcinomas.

A surgical procedure was developed to deliver mammary tumor cells to the murine liver via portal vein injection. This model permits investigation of late stages of liver metastasis in a fully immune competent host, including tumor cell extravasation, seeding, survival, and metastatic outgrowth in the liver.

门静脉注射模型来研究肝转移乳腺癌

Breast cancer is the leading cause of cancer-related mortality in women worldwide. Liver metastasis is involved in upwards of 30% of cases with breast ...

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 ...

Combined Use of Tail Vein Metastasis Assays and Real-Time In Vivo Imaging to Quantify Breast Cancer Metastatic Colonization and Burden in the Lungs

Metastasis is the main cause of cancer-related deaths and there are limited therapeutic options for patients with metastatic disease. The identification and testing of novel therapeutic targets that will facilitate the development of better treatments for metastatic disease requires preclinical in vivo models. Demonstrated here is a syngeneic mouse model for assaying breast cancer metastatic colonization and subsequent growth. Metastatic cancer cells are stably transduced with viral vectors encoding firefly luciferase and ZsGreen proteins. Candidate genes are then stably manipulated in luciferase/ZsGreen-expressing cancer cells and then the cells are injected into mice via the lateral tail vein to assay metastatic colonization and growth. An in vivo imaging device is then used to measure the bioluminescence or fluorescence of the tumor cells in the living animals to quantify changes in metastatic burden over time. The expression of the fluorescent protein allows the number and size of metastases in the lungs to be quantified at the end of the experiment without the need for sectioning or histological staining. This approach offers a relatively quick and easy way to test the role of candidate genes in metastatic colonization and growth, and provides a great deal more information than traditional tail vein metastasis assays. Using this approach, we show that simultaneous knockdown of Yes associated protein (YAP) and transcriptional co-activator with a PDZ-binding motif (TAZ) in breast cancer cells leads to reduced metastatic burden in the lungs and that this reduced burden is the result of significantly impaired metastatic colonization and reduced growth of metastases.

The described approach combines experimental tail vein metastasis assays with in vivo live animal imaging to allow real-time monitoring of breast cancer metastasis formation and growth in addition to the quantification of metastasis number and size in the lungs.

结合使用尾静脉转移分析与实时 Vivo 成像，以量化乳腺癌转移殖民化和肺负担

Metastasis is the main cause of cancer-related deaths and there are limited therapeutic options for patients with metastatic disease. The identification ...

Pathological Analysis of Lung Metastasis Following Lateral Tail-Vein Injection of Tumor Cells

Metastasis, the primary cause of morbidity and mortality for most cancer patients, can be challenging to model preclinically in mice. Few spontaneous metastasis models are available. Thus, the experimental metastasis model involving tail-vein injection of suitable cell lines is a mainstay of metastasis research. When cancer cells are injected into the lateral tail-vein, the lung is their preferred site of colonization. A potential limitation of this technique is the accurate quantification of the metastatic lung tumor burden. While some investigators count macrometastases of a pre-defined size and/or include micrometastases following sectioning of tissue, others determine the area of metastatic lesions relative to normal tissue area. Both of these quantification methods can be exceedingly difficult when the metastatic burden is high. Herein, we demonstrate an intravenous injection model of lung metastasis followed by an advanced method for quantifying metastatic tumor burden using image analysis software. This process allows for investigation of multiple end-point parameters, including average metastasis size, total number of metastases, and total metastasis area, to provide a comprehensive analysis. Furthermore, this method has been reviewed by a veterinary pathologist board-certified by the American College of Veterinary Pathologists (SEK) to ensure accuracy.

Intravenous injection of cancer cells is often used in metastasis research, but the metastatic tumor burden can be difficult to analyze. Herein, we demonstrate a tail-vein injection model of metastasis and include a novel approach to analyze the resulting metastatic lung tumor burden.

肿瘤细胞外侧尾静脉注射后肺转移的病理分析

Metastasis, the primary cause of morbidity and mortality for most cancer patients, can be challenging to model preclinically in mice. Few spontaneous ...

Using Computer-based Image Analysis to Improve Quantification of Lung Metastasis in the 4T1 Breast Cancer Model

Breast cancer is a devastating malignancy, accounting for 40,000 female deaths and 30% of new female cancer diagnoses in the United States in 2019 alone. The leading cause of breast cancer related deaths is the metastatic burden. Therefore, preclinical models for breast cancer need to analyze metastatic burden to be clinically relevant. The 4T1 breast cancer model provides a spontaneously-metastasizing, quantifiable mouse model for stage IV human breast cancer. However, most 4T1 protocols quantify the metastatic burden by manually counting stained colonies on tissue culture plates. While this is sufficient for tissues with lower metastatic burden, human error in manual counting causes inconsistent and variable results when plates are confluent and difficult to count. This method offers a computer-based solution to human counting error. Here, we evaluate the protocol using the lung, a highly metastatic tissue in the 4T1 model. Images of methylene blue-stained plates are acquired and uploaded for analysis in Fiji-ImageJ. Fiji-ImageJ then determines the percentage of the selected area of the image that is blue, representing the percentage of the plate with metastatic burden. This computer-based approach offers more consistent and expeditious results than manual counting or histopathological evaluation for highly metastatic tissues. The consistency of Fiji-ImageJ results depends on the quality of the image. Slight variations in results between images can occur, thus it is recommended that multiple images are taken and results averaged. Despite its minimal limitations, this method is an improvement to quantifying metastatic burden in the lung by offering consistent and rapid results.

We describe a more consistent and expeditious method to quantify lung metastasis in the 4T1 breast cancer model by using Fiji-ImageJ.

利用计算机图像分析提高4T1乳腺癌模型中肺转移的定量

Breast cancer is a devastating malignancy, accounting for 40,000 female deaths and 30% of new female cancer diagnoses in the United States in 2019 alone. ...

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

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 ...

Primary Tumor and MEF Cell Isolation to Study Lung Metastasis

In breast tumorigenesis, the metastatic stage of the disease poses the greatest threat to the affected individual. Normal breast cells with altered genotypes now possess the ability to invade and survive in other tissues. In this protocol, mouse mammary tumors are removed and primary cells are prepared from tumors. The cells isolated from this procedure are then available for gene profiling experiments. For successful metastasis, these cells must be able to intravasate, survive in circulation, extravasate to distant organs, and survive in that new organ system. The lungs are the typical target of breast cancer metastasis. A set of genes have been discovered that mediates the selectivity of metastasis to the lung. Here we describe a method of studying lung metastasis from a genetically engineered mouse model.. Furthermore, another protocol for analyzing mouse embryonic fibroblasts (MEFs) from the mouse embryo is included. MEF cells from the same animal type provide a clue of non-cancer cell gene expression. Together, these techniques are useful in studying mouse mammary tumorigenesis, its associated signaling mechanisms and pathways of the abnormalities in embryos.

The goal of this protocol is to study breast tumorigenesis. With this technique, mouse mammary tumors are removed and primary cells are prepared from tumors. A lung extraction protocol is included for studying lung metastasis. Furthermore, another protocol for analyzing mouse embryonic fibroblasts from the mouse embryo is included.

原发灶和MEF细胞分离，研究肺转移

In breast tumorigenesis, the metastatic stage of the disease poses the greatest threat to the affected individual. Normal breast cells with altered ...

评价肺转移的小鼠乳腺肿瘤模型定量实时荧光定量PCR

摘要

探索更多视频

此视频中的章节

临床前小鼠模型的外科手术和方法

门静脉注射模型来研究肝转移乳腺癌

研究使用正交乳腺癌模型的三阴性乳腺癌

结合使用尾静脉转移分析与实时 Vivo 成像，以量化乳腺癌转移殖民化和肺负担

肿瘤细胞外侧尾静脉注射后肺转移的病理分析

利用计算机图像分析提高4T1乳腺癌模型中肺转移的定量

人类前列腺癌转移的原位小鼠模型

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

原发灶和MEF细胞分离，研究肺转移

评价肺转移的小鼠乳腺肿瘤模型定量实时荧光定量PCR

摘要

探索更多视频

此视频中的章节

临床前小鼠模型的外科手术和方法

门静脉注射模型来研究肝转移乳腺癌

研究使用正交乳腺癌模型的三阴性乳腺癌

结合使用尾静脉转移分析与实时 Vivo 成像，以量化乳腺癌转移殖民化和肺负担

肿瘤细胞外侧尾静脉注射后肺转移的病理分析

利用计算机图像分析提高4T1乳腺癌模型中肺转移的定量

人类前列腺癌转移的原位小鼠模型

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

原发灶和MEF细胞分离，研究肺转移

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

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