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>

<ol>
	<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 fo:keep-together.within-page="1">
	<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&#160;</td>
			<td>Bio-Rad</td>
			<td>172-5121&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>PrimePCR SYBR Green Assay: ERBB2, Human&#160;</td>
			<td>Bio-Rad</td>
			<td>100-25636&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>PrimePCR Template for SYBR Green Assay: ERBB2, Human&#160;</td>
			<td>Bio-Rad</td>
			<td>100-25716&#160;</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

Evaluation of Nanoparticle Uptake in Tumors in Real Time Using Intravital Imaging

Current technologies for tumor imaging, such as ultrasound, MRI, PET and CT, are unable to yield high-resolution images for the assessment of nanoparticle uptake in tumors at the microscopic level1,2,3, highlighting the utility of a suitable xenograft model in which to perform detailed uptake analyses. Here, we use high-resolution intravital imaging to evaluate nanoparticle uptake in human tumor xenografts in a modified, shell-less chicken embryo model. The chicken embryo model is particularly well-suited for these in vivo analyses because it supports the growth of human tumors, is relatively inexpensive and does not require anesthetization or surgery 4,5. Tumor cells form fully vascularized xenografts within 7 days when implanted into the chorioallantoic membrane (CAM) 6. The resulting tumors are visualized by non-invasive real-time, high-resolution imaging that can be maintained for up to 72 hours with little impact on either the host or tumor systems. Nanoparticles with a wide range of sizes and formulations administered distal to the tumor can be visualized and quantified as they flow through the bloodstream, extravasate from leaky tumor vasculature, and accumulate at the tumor site. We describe here the analysis of nanoparticles derived from Cowpea mosaic virus (CPMV) decorated with near-infrared fluorescent dyes and/or polyethylene glycol polymers (PEG) 7, 8, 9,10,11. Upon intravenous administration, these viral nanoparticles are rapidly internalized by endothelial cells, resulting in global labeling of the vasculature both outside and within the tumor7,12. PEGylation of the viral nanoparticles increases their plasma half-life, extends their time in the circulation, and ultimately enhances their accumulation in tumors via the enhanced permeability and retention (EPR) effect 7, 10,11. The rate and extent of accumulation of nanoparticles in a tumor is measured over time using image analysis software. This technique provides a method to both visualize and quantify nanoparticle dynamics in human tumors.

We present a novel approach to quantify nanoparticle localization in the vasculature of human xenografted tumors using dynamic, real-time intravital imaging in an avian embryo model.

使用实时活体成像在肿瘤纳米粒子吸收的评价

Current technologies for tumor imaging, such as ultrasound, MRI, PET and CT, are unable to yield high-resolution images for the assessment of nanoparticle ...

科研

医学

In vivo Bioluminescence Imaging of Tumor Hypoxia Dynamics of Breast Cancer Brain Metastasis in a Mouse Model

It is well recognized that tumor hypoxia plays an important role in promoting malignant progression and affecting therapeutic response negatively. There is little knowledge about in situ, in vivo, tumor hypoxia during intracranial development of malignant brain tumors because of lack of efficient means to monitor it in these deep-seated orthotopic tumors. Bioluminescence imaging (BLI), based on the detection of light emitted by living cells expressing a luciferase gene, has been rapidly adopted for cancer research, in particular, to evaluate tumor growth or tumor size changes in response to treatment in preclinical animal studies. Moreover, by expressing a reporter gene under the control of a promoter sequence, the specific gene expression can be monitored non-invasively by BLI. Under hypoxic stress, signaling responses are mediated mainly via the hypoxia inducible factor-1&alpha; (HIF-1&alpha;) to drive transcription of various genes. Therefore, we have used a HIF-1&alpha; reporter construct, 5HRE-ODD-luc, stably transfected into human breast cancer MDA-MB231 cells (MDA-MB231/5HRE-ODD-luc). In vitro HIF-1&alpha; bioluminescence assay is performed by incubating the transfected cells in a hypoxic chamber (0.1% O2) for 24 hr before BLI, while the cells in normoxia (21% O2) serve as a control. Significantly higher photon flux observed for the cells under hypoxia suggests an increased HIF-1&alpha; binding to its promoter (HRE elements), as compared to those in normoxia. Cells are injected directly into the mouse brain to establish a breast cancer brain metastasis model. In vivo bioluminescence imaging of tumor hypoxia dynamics is initiated 2 wks after implantation and repeated once a week. BLI reveals increasing light signals from the brain as the tumor progresses, indicating increased intracranial tumor hypoxia. Histological and immunohistochemical studies are used to confirm the in vivo imaging results. Here, we will introduce approaches of in vitro HIF-1&alpha; bioluminescence assay, surgical establishment of a breast cancer brain metastasis in a nude mouse and application of in vivo bioluminescence imaging to monitor intracranial tumor hypoxia.

In vivo Bioluminescence Imaging of Tumor Hypoxia Dynamics of Breast Cancer Brain Metastasis in a Mouse Model

Bioluminescence imaging of hypoxia inducible factor-1&alpha; activity is applied to monitor intracranial tumor hypoxia development in a breast cancer brain metastasis mouse model.

在小鼠模型体内生物发光成像乳腺癌脑转移的肿瘤乏氧动力学

It is well recognized that tumor hypoxia plays an important role in promoting malignant progression and affecting therapeutic response negatively. There ...

MicroRNA Detection in Prostate Tumors by Quantitative Real-time PCR (qPCR)

MicroRNAs (miRNAs) are single-stranded, 18&#x2013;24 nucleotide long, non-coding RNA molecules. They are involved in virtually every cellular process including development1, apoptosis2, and cell cycle regulation3. MiRNAs are estimated to regulate the expression of 30% to 90% of human genes4 by binding to their target messenger RNAs (mRNAs)5. Widespread dysregulation of miRNAs has been reported in various diseases and cancer subtypes6. Due to their prevalence and unique structure, these small molecules are likely to be the next generation of biomarkers, therapeutic agents and/or targets.
Methods used to investigate miRNA expression include SYBR green I dye- based as well as Taqman-probe based qPCR. If miRNAs are to be effectively used in the clinical setting, it is imperative that their detection in fresh and/or archived clinical samples be accurate, reproducible, and specific. qPCR has been widely used for validating expression of miRNAs in whole genome analyses such as microarray studies7. The samples used in this protocol were from patients who underwent radical prostatectomy for clinically localized prostate cancer; however other tissues and cell lines can be substituted in. Prostate specimens were snap-frozen in liquid nitrogen after resection. Clinical variables and follow-up information for each patient were collected for subsequent analysis8.
Quantification of miRNA levels in prostate tumor samples. The main steps in qPCR analysis of tumors are: Total RNA extraction, cDNA synthesis, and detection of qPCR products using miRNA-specific primers. Total RNA, which includes mRNA, miRNA, and other small RNAs were extracted from specimens using TRIzol reagent. Qiagen's miScript System was used to synthesize cDNA and perform qPCR (Figure 1). Endogenous miRNAs are not polyadenylated, therefore during the reverse transcription process, a poly(A) polymerase polyadenylates the miRNA. The miRNA is used as a template to synthesize cDNA using oligo-dT and Reverse Transcriptase. A universal tag sequence on the 5' end of oligo-dT primers facilitates the amplification of cDNA in the PCR step. PCR product amplification is detected by the level of fluorescence emitted by SYBR Green, a dye which intercalates into double stranded DNA. Specific miRNA primers, along with a Universal Primer that binds to the universal tag sequence will amplify specific miRNA sequences. 
The miScript Primer Assays are available for over a thousand human-specific miRNAs, and hundreds of murine-specific miRNAs. Relative quantification method was used here to quantify the expression of miRNAs. To correct for variability amongst different samples, expression levels of a target miRNA is normalized to the expression levels of a reference gene. The choice of a gene on which to normalize the expression of targets is critical in relative quantification method of analysis. Examples of reference genes typically used in this capacity are the small RNAs RNU6B, RNU44, and RNU48 as they are considered to be stably expressed across most samples. In this protocol, RNU6B is used as the reference gene.

MicroRNA Detection in Prostate Tumors by Quantitative Real-time PCR qPCR

Quantitative Real Time polymerase chain reaction (qPCR) is a rapid and sensitive method to investigate the expression levels of various microRNA (miRNA) molecules in tumor samples. Using this method expression of hundreds of different miRNA molecules can be amplified, quantified, and analyzed from the same cDNA template.

实时定量PCR（qPCR的microRNA检测在前列腺肿瘤）

MicroRNAs (miRNAs) are single-stranded, 18&#x2013;24 nucleotide long, non-coding RNA molecules. They are involved in virtually every cellular process ...

Models of Bone Metastasis

Bone metastases are a common occurrence in several malignancies, including breast, prostate, and lung. Once established in bone, tumors are responsible for significant morbidity and mortality1. Thus, there is a significant need to understand the molecular mechanisms controlling the establishment, growth and activity of tumors in bone. Several in vivo models have been established to study these events and each has specific benefits and limitations. The most commonly used model utilizes intracardiac inoculation of tumor cells directly into the arterial blood supply of athymic (nude) BalbC mice. This procedure can be applied to many different tumor types (including PC-3 prostate cancer, lung carcinoma, and mouse mammary fat pad tumors); however, in this manuscript we will focus on the breast cancer model, MDA-MB-231. In this model we utilize a highly bone-selective clone, originally derived in Dr. Mundy's group in San Antonio2, that has since been transfected for GFP expression and re-cloned by our group3. This clone is a bone metastatic variant with a high rate of osteotropism and very little metastasis to lung, liver, or adrenal glands. While intracardiac injections are most commonly used for studies of bone metastasis2, in certain instances intratibial4 or mammary fat pad injections are more appropriate. Intracardiac injections are typically performed when using human tumor cells with the goal of monitoring later stages of metastasis, specifically the ability of cancer cells to arrest in bone, survive, proliferate, and establish tumors that develop into cancer-induced bone disease. Intratibial injections are performed if focusing on the relationship of cancer cells and bone after a tumor has metastasized to bone, which correlates roughly to established metastatic bone disease. Neither of these models recapitulates early steps in the metastatic process prior to embolism and entry of tumor cells into the circulation. If monitoring primary tumor growth or metastasis from the primary site to bone, then mammary fat pad inoculations are usually preferred; however, very few tumor cell lines will consistently metastasize to bone from the primary site, with 4T1 bone-preferential clones, a mouse mammary carcinoma, being the exception 5,6.
This manuscript details inoculation procedures and highlights key steps in post inoculation analyses. Specifically, it includes cell culture, tumor cell inoculation procedures for intracardiac and intratibial inoculations, as well as brief information regarding weekly monitoring by x-ray, fluorescence and histomorphometric analyses.

Animal models are frequently utilized to study cancer metastasis to bone. In this protocol we will describe two common methods of tumor inoculation for bone metastasis studies and briefly describe some of the analyses utilized to monitor and quantify these models.

骨转移模型

Bone metastases are a common occurrence in several malignancies, including breast, prostate, and lung. Once established in bone, tumors are responsible ...

Live Imaging of Drug Responses in the Tumor Microenvironment in Mouse Models of Breast Cancer

The tumor microenvironment plays a pivotal role in tumor initiation, progression, metastasis, and the response to anti-cancer therapies. Three-dimensional co-culture systems are frequently used to explicate tumor-stroma interactions, including their role in drug responses. However, many of the interactions that occur in vivo in the intact microenvironment cannot be completely replicated in these in vitro settings. Thus, direct visualization of these processes in real-time has become an important tool in understanding tumor responses to therapies and identifying the interactions between cancer cells and the stroma that can influence these responses. Here we provide a method for using spinning disk confocal microscopy of live, anesthetized mice to directly observe drug distribution, cancer cell responses and changes in tumor-stroma interactions following administration of systemic therapy in breast cancer models. We describe procedures for labeling different tumor components, treatment of animals for observing therapeutic responses, and the surgical procedure for exposing tumor tissues for imaging up to 40 hours. The results obtained from this protocol are time-lapse movies, in which such processes as drug infiltration, cancer cell death and stromal cell migration can be evaluated using image analysis software.

We describe a method for imaging response to anti-cancer treatment in vivo and at single cell resolution.

实时成像在乳腺癌小鼠模型中的肿瘤微环境对药物的反应

The tumor microenvironment plays a pivotal role in tumor initiation, progression, metastasis, and the response to anti-cancer therapies. Three-dimensional ...

Using Quantitative Real-time PCR to Determine Donor Cell Engraftment in a Competitive Murine Bone Marrow Transplantation Model

Murine bone marrow transplantation models provide an important tool in measuring hematopoietic stem cell (HSC) functions and determining genes/molecules that regulate HSCs. In these transplant model systems, the function of HSCs is determined by the ability of these cells to engraft and reconstitute lethally irradiated recipient mice. Commonly, the donor cell contribution/engraftment is measured by antibodies to donor- specific cell surface proteins using flow cytometry. However, this method heavily depends on the specificity and the ability of the cell surface marker to differentiate donor-derived cells from recipient-originated cells, which may not be available for all mouse strains. Considering the various backgrounds of genetically modified mouse strains in the market, this cell surface/ flow cytometry-based method has significant limitations especially in mouse strains that lack well-defined surface markers to separate donor cells from congenic recipient cells. Here, we reported a PCR-based technique to determine donor cell engraftment/contribution in transplant recipient mice. We transplanted male donor bone marrow HSCs to lethally irradiated congenic female mice. Peripheral blood samples were collected at different time points post transplantation. Bone marrow samples were obtained at the end of the experiments. Genomic DNA was isolated and the Y chromosome specific gene, Zfy1, was amplified using quantitative Real time PCR. The engraftment of male donor-derived cells in the female recipient mice was calculated against standard curve with known percentage of male vs. female DNAs. Bcl2 was used as a reference gene to normalize the total DNA amount. Our data suggested that this approach reliably determines donor cell engraftment and provides a useful, yet simple method in measuring hematopoietic cell reconstitution in murine bone marrow transplantation models. Our method can be routinely performed in most laboratories because no costly equipment such as flow cytometry is required.

Determining donor cell engraftment presents a challenge in mouse bone marrow transplant models that lack well-defined phenotypical markers. We described a methodology to quantify male donor cell engraftment in female transplant recipient mice. This method can be used in all mouse strains for the study of HSC functions.

采用实时定量PCR检测供体细胞植入一个具有竞争力的小鼠骨髓移植模型

Murine bone marrow transplantation models provide an important tool in measuring hematopoietic stem cell (HSC) functions and determining genes/molecules ...

A Simple Method of Mouse Lung Intubation

A simple procedure to intubate mice for pulmonary function measurements would have several advantages in longitudinal studies with limited numbers or expensive animal. One of the reasons that this is not done more routinely is that it is relatively difficult, despite there being several published studies that describe ways to achieve it. In this paper we demonstrate a procedure that eliminates one of the major hurdles associated with this intubation, that of visualizing the trachea during the entire time of intubation. The approach uses a 0.5 mm fiberoptic light source that serves as an introducer to direct the intubation cannula into the mouse trachea. We show that it is possible to use this procedure to measure lung mechanics in individual mice over a time course of at least several weeks. The technique can be set up with relatively little expense and expertise, and it can be routinely accomplished with relatively little training. This should make it possible for any laboratory to routinely carry out this intubation, thereby allowing longitudinal studies in individual mice, thereby minimizing the number of mice needed and increasing the statistical power by using each mouse as its own control.

This paper describes a striaghforward and efficient method of intubating mice for pulmonary function measurements or pulmonary instillation, that allows the mice to recover and be studied at later times. The procedure involves an inexpensive fiberoptic light source that directly illuminates the trachea.

一个简单的方法小鼠肺插管

A  simple procedure to intubate mice for pulmonary function measurements would  have several advantages in longitudinal studies with limited numbers or  ...

Initiation of Metastatic Breast Carcinoma by Targeting of the Ductal Epithelium with Adenovirus-Cre: A Novel Transgenic Mouse Model of Breast Cancer

Breast cancer is a heterogeneous disease involving complex cellular interactions between the developing tumor and immune system, eventually resulting in exponential tumor growth and metastasis to distal tissues and the collapse of anti-tumor immunity. Many useful animal models exist to study breast cancer, but none completely recapitulate the disease progression that occurs in humans. In order to gain a better understanding of the cellular interactions that result in the formation of latent metastasis and decreased survival, we have generated an inducible transgenic mouse model of YFP-expressing ductal carcinoma that develops after sexual maturity in immune-competent mice and is driven by consistent, endocrine-independent oncogene expression. Activation of YFP, ablation of p53, and expression of an oncogenic form of K-ras was achieved by the delivery of an adenovirus expressing Cre-recombinase into the mammary duct of sexually mature, virgin female mice. Tumors begin to appear 6 weeks after the initiation of oncogenic events. After tumors become apparent, they progress slowly for approximately two weeks before they begin to grow exponentially. After 7-8 weeks post-adenovirus injection, vasculature is observed connecting the tumor mass to distal lymph nodes, with eventual lymphovascular invasion of YFP+ tumor cells to the distal axillary lymph nodes. Infiltrating leukocyte populations are similar to those found in human breast carcinomas, including the presence of &#945;&#946; and &#947;&#948; T cells, macrophages and MDSCs. This unique model will facilitate the study of cellular and immunological mechanisms involved in latent metastasis and dormancy in addition to being useful for designing novel immunotherapeutic interventions to treat invasive breast cancer.

Activation of latent mutations with adenovirus-Cre into the mammary ductal system results in a clinically relevant metastatic breast cancer. Incorporation of a YFP promoter allows tracking of distal metastatic tumor cells. This model is useful to study latent metastasis, anti-tumor immunity, and for designing novel immunotherapies to treat breast cancer.

乳腺癌的一种新的转基因小鼠模型:转移性乳腺癌通过瞄准导管上皮与腺病毒Cre重组酶的启动

Breast cancer is a heterogeneous disease involving complex cellular interactions between the developing tumor and immune system, eventually resulting in ...

Cerebrospinal Fluid MicroRNA Profiling Using Quantitative Real Time PCR

MicroRNAs (miRNAs) constitute a potent layer of gene regulation by guiding RISC to target sites located on mRNAs and, consequently, by modulating their translational repression. Changes in miRNA expression have been shown to be involved in the development of all major complex diseases. Furthermore, recent findings showed that miRNAs can be secreted to the extracellular environment and enter the bloodstream and other body fluids where they can circulate with high stability. The function of such circulating miRNAs remains largely elusive, but systematic high throughput approaches, such as miRNA profiling arrays, have lead to the identification of miRNA signatures in several pathological conditions, including neurodegenerative disorders and several types of cancers. In this context, the identification of miRNA expression profile in the cerebrospinal fluid, as reported in our recent study, makes miRNAs attractive candidates for biomarker analysis.
There are several tools available for profiling microRNAs, such as microarrays, quantitative real-time PCR (qPCR), and deep sequencing. Here, we describe a sensitive method to profile microRNAs in cerebrospinal fluids by quantitative real-time PCR. We used the Exiqon microRNA ready-to-use PCR human panels I and II V2.R, which allows detection of 742 unique human microRNAs. We performed the arrays in triplicate runs and we processed and analyzed data using the GenEx Professional 5 software.
Using this protocol, we have successfully profiled microRNAs in various types of cell lines and primary cells, CSF, plasma, and formalin-fixed paraffin-embedded tissues.

We describe a protocol of real time PCR to profile microRNAs in the cerebrospinal fluid (CSF). With the exception of RNA extraction protocols, the procedure can be extended to RNA extracted from other body fluids, cultured cells, or tissue specimens.

脑脊液的microRNA分析采用实时定量PCR技术

MicroRNAs (miRNAs) constitute a potent layer of gene regulation by guiding RISC to target sites located on mRNAs and, consequently, by modulating their ...

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 ...