这项工作是支持的，一部分由苏 ​​格兰拨款委员会（项目编号HR07005; <a href="http://www.sfc.ac.uk/">http://www.sfc.ac.uk/</a> ），医学研究苏格兰（ <a href="http://www.medicalresearchscotland.org.uk/">http://www.medicalresearchscotland.org.uk/</a> ），癌症英国的实验研究癌症医学中心（ <a href="http://www.cancerresearchuk.org/">http://www.cancerresearchuk.org/</a> ），并突破乳腺癌（ <a href="http://breakthrough.org.uk/">http://breakthrough.org.uk/</a> ）。

1。组织编制<ol class="lalpha"><li>超薄切片<ol class="lroman"><li>第福尔马林固定石蜡包埋的肿瘤在4微米厚的块，用旋转切片。 </li><li>放置到带正电的微观幻灯片的部分。 </li><li>在37 ° C烘箱过夜孵育的幻灯片。 </li></ol></li><li>存储<ol><li>存储部分在-18 ° C至-25 ° C冰箱，以防止损失的抗原性。 </li></ol></li></ol> 2。免疫荧光组织切片<ol class="lalpha"><li>脱蜡和补液<ol class="lroman"><li>脱蜡二甲苯两次5分钟的幻灯片。 </li><li>补充水分在99％，99％，80％，50％的乙醇和运行每2分钟，以自来水的幻灯片。 </li></ol></li><li>抗原修复<ol class="lroman"><li>执行热诱导抗原检索，使用压力锅在MI 5分钟0.15 mM柠檬酸钠，pH 6.0的缓冲微波。 </li><li>降温20分钟的幻灯片。 </li><li>摇杆5分钟的0.05％PBST冲洗的幻灯片。 </li></ol></li><li>阻止程序<ol class="lroman"><li>在10分钟的3％的过氧化氢治疗部分。 </li><li>摇杆5分钟的0.05％PBST冲洗的幻灯片。 </li><li>新增部分Sequenza免疫染色架。 </li><li>在血清蛋白块10分钟的免费治疗部分。 </li></ol></li><li>初级抗体孵育<ol class="lroman"><li>孵育一抗稀释Dako公司抗体稀释液最佳稀释，在室温下1小时。 </li><li>在0.05％PBST冲洗3次，每次5分钟。 </li></ol></li><li>上皮面具（角蛋白）孵化<ol class="lroman"><li>孵育小鼠抗-泛角蛋白，Dako公司抗体稀释液1:50稀释，4℃过夜° C。 </li></ol></li><li>上皮面具可视化<ol class="lroman"><li>准备1:25稀释的羊抗鼠Alexa555小号在预稀释econdary抗体设想山羊兔HRP抗体溶液。在室温下为1.5小时孵育在黑暗中的幻灯片。 </li><li> 0.05％PBST冲洗3次各为5min。 </li></ol></li><li>目标可视化<ol class="lroman"><li>将幻灯片Sequenza湿度试验箱。 </li><li>结合目标信号放大的稀释剂（HistoRx浴缸五）在1:50的浓度和Cy5的酪胺（HistoRx管F）的。涡旋调匀。在室温下10分钟的黑暗中孵育的幻灯片。 </li><li>在0.05％PBST冲洗3次，每次5分钟。 </li><li>在1分80％乙醇脱水幻灯片。 </li><li>在黑暗中的空气干燥的幻灯片。 </li></ol></li><li> Counterstaining和coverslipping <ol class="lroman"><li>申请上盖玻片的DAPI安装介质和地方在组织切片中的盖玻片。 </li><li>在黑暗中通宵干幻灯片。 </li><li>幻灯片后晾干，用指甲油安全EN确保长期保存，并保持在4℃冰箱。 </li></ol></li></ol> 3。采用全断面扫描的图像捕捉<ol class="lalpha"><li>负载五个幻灯片在幻灯片卡带Aperio ScanScope FL幻灯片扫描仪。 </li><li>对于每一个幻灯片，选择图像在ScanScope控制台中使用的接口一般地区。选择该地区的所有组织，无论染色样品的图案或观察到的其他方面，包括在幻灯片上。 </li><li>使用ScanScope控制台，为每个通道（DAPI，Cy3和Cy5）确定最佳曝光如下： <ol class="lroman"><li>选择一个区域组织询问 - 理想情况下，这方面应该在肿瘤区域组织提供最佳的代表性。 </li><li>使用ScanScope控制台的自动曝光功能，评估每个通道的建议的曝光时间，随着图像的焦点。 </li><li>重复以上3-5每个样品的地区合作并将通过稳定的价值。 </li></ol></li><li>选择一个额外的地区（如由蓝色钻石ScanScope控制台形象所示），远离组织，但仍然在幻灯片coverslipped地区，定义每一个过滤器，平场校正的图像将被收购的背景区域。 </li><li>图像采集前审查这些图像。如果图像出现高背景或其他图像伪影，图像区域应移动和收购新的图像。 </li><li>收购的数字幻灯片图像自动上传到Aperio频谱数据库。 </li></ol> 4。 AQUA的自动图像分析<ol class="lalpha"><li>频谱数据库中的数字整个幻灯片图像和注释肿瘤领域的兴趣，在整个组织和染色模式（S）的范围内。 </li><li>选择的幻灯片，从频谱数据库中的数字幻灯片列表分析双击拇指AQUAnalysis钉形象（或者，选择图片旁边的复选框，然后选择“查看图片”，在列表的顶部菜单）。这会自动打开ImageScope软件和合并后的图像色彩的组织样本。 </li><li>用这个软件使用的工具提供导航图像。 </li><li>使用随附的H＆E（物理滑动或注明的形象），标注的荧光图像的利息区域。多个地区的利益，个别地区上空盘旋，可以选择单个样品。还有一个“负面”的工具，它是用来排除选定区域内的地区（即受损的组织，穷人组织学等领域，非肿瘤区，泥石流等）。 </li><li>保存在图像上标注层（S）。 </li><li>返回到主菜单频谱和选择的形象，只是注明旁边的复选框。选择“分析”从整个列表的顶部的菜单条。 </li><li>在Analysis窗口上来了，塞莱CT“HistoRx AQUA的分析”，从下拉菜单。 </li><li>如果有多个层次的注解，使用复选框以选择适当的层（S）进行分析。 </li><li>按下“分析”按钮，当准备开始。 </li><li>此时，一个最小化的控制台窗口将出现在Windows任务栏上。这将显示从频谱数据库的图像传输到本地PC（“拉”操作）的进展。 </li><li>一旦数据传输，将推出AQUAnalysis。然后，用户可以使用该软件的内部功能来查看，浏览和分析图像。 </li><li>注明地区的形象从波谱数据库被分解成512x512像素的图像砖 - 每个单独取得。 </li><li>在这项研究中，基于聚类的无监督的Aqua得分算法，用于8。在这个算法中，AQUA的分数产生如下： <ol class="lroman"><li>泛角蛋白形象SI上述背景及gnal是阈值的像素是用来定义一个“面具”，然后用于后续计算肿瘤。泛高表达细胞角蛋白像素定义的肿瘤细胞的细胞质/无核武器地区。 </li><li>在DAPI通道的高信号，肿瘤内面具的像素确定为核的性质。 </li><li>聚类算法还考察要么DAPI染色或角蛋白的表达，并尝试部分归咎于他们要么核或细胞质车厢，或背景进行低强度的像素。 </li><li>一旦所有像素被分配到肿瘤的车厢或背景，从靶蛋白（ER或HER2）的信号强度，然后计算每个车厢内车厢的大小和规范化，从而产生AQUA的分数。 </li><li>没有足够的肿瘤代表性的图片，没有进球（定义同乐SS代表肿瘤的像素超过5％）。这些地区也产生了“最终结果”的价值，所谓“不及格”，并因此可以在随后的统计分析中删除。 </li><li>此外，如果经审查，确定运营商领域，不适合进行打分，由于样品或成像构件（如折叠的组织，碎片），这些领域的节录得分，并制作了“最终结果”所谓“不及格”。 </li></ol></li><li> AQUA的评分结果，是与每个地区的利益在整个组织切片样本中的512x512像素的瓷砖的分数表。 。csv格式，这些文件，然后可以被用来为后续的统计分析。 </li></ol> 5。统计分析：所有在SPSS进行统计分析（IBM公司） <ol><li>在可能的情况下，大作业，生成SPSS语法代码文件（SPS）进行数据处理和分析步骤。例如语法的代码是提供补充文件。 </li><li&gt;读入SPSS数据文件。删除未使用的变量，在原始数据（如文书的具体参数，从非利用车厢AQUA的分数）。 </li><li>凡地区有一个“最终结果”，“不及格”（见上文），标记SYSMIS，从而消除他们从数值分析这些值。 </li><li>每到一个单一的主SPSS格式的数据集标记（ER或HER2）的所有幻灯片总的所有数据。使用这个主文件为所有随后的计算。 </li><li>使用SPSS语法文件（每个标记：ER或HER2（见补充语法文件）的数据进一步验证和辛普森多样性指数的计算方法如下： <ol class="lroman"><li>产生主文件的分析，以防止修改先前生成的数据集的主。 </li><li>验证对产生的原始数据和图像数据集的主。 </li><li>生产AQUA的分数，每个样品（计数字段，汇总统计数据的平均得分，平均得分，minimu米的得分，最高得分，和几十个样本的标准偏差）。 </li><li>随机抽样检查，对原始数据输出文件和结果，对原始图像数据得分。 </li></ol></li><li>要产生的热图图像（图2和图3所示）： <ol class="lroman"><li> “斌”的AQUA的得分数据（使用的“视觉分级”功能在SPSS）到8个人层面。对于急诊室，被用于核分数，为HER2基因，细胞质分数。 </li><li>推导每个斌代表可用的情况下从整个数据集等百分比，垃圾箱等。 </li><li>使用SPSS功能“分割文件”，使所有后续的输出将是一个“每张幻灯片/样本”的水平上。 </li><li>每个区域（​​对应到512 × 512瓦）画出其x，y坐标和对应的值分配它的垃圾桶的颜色（见图2和图3）。 </li></ol></li><li>要生成的异质性分数，写代码生成一个辛普森的使用AQUA的分数多样性指数。 <ol class="lroman"><li> Simpson指数的多样性，这里所用，被定义为： <img alt="公式1" src="/files/ftp_upload/3334/3334eq1.jpg" /> 其中N是用于一个给定的样本，n是分数/领域的标准化分数（如下所述产生）的分数/领域的总数。 </li></ol></li><li>使用上述分析的主文件中描述的数据源。在所示的例子中，所有的ER计算中使用的核AQUA的得分和HER2的计算使用细胞质的Aqua得分。执行下面的计算，每个样品/幻灯片。 </li><li>由于荧光数据方差不稳定，这样，误差的大小与强度传播，所有旱厕成绩申请数（基数为2）正常化。 </li><li>进一步转变归一化数据（日志转​​换）到Z（标准）分数。 <ol class="lroman"><li> Z - score模型转换计算的小号如下：Z =（AQUA的得分 - 所有样品平均分数）/分数为样本的标准偏差。这转换成分数的分布，围绕中央的零偏差值分布的偏差。 </li></ol></li><li>斌这些Z评分值分为六组（&lt;-2，-2 - -1，-1 - 0,0 - 1，1 - 2&gt; 2）。 </li><li>计算分配到每个容器和每个容器的总和值。使用此值，随着总数的领域，计算值用于索引的6箱。 </li><li>这些值相加，并减去从一个生产多样性指数。 </li><li>多样性指数的围绕一个特定的样本平均得分的传播提供了一个指标。因此，较高的指数显示的分数更广泛的传播，或表达的异质性增加。相反，较低的指数，表明表达测量和样品均质表达的变化。这说明在FigurES 2和3。 </li></ol> 6。代表性的成果： 有针对性的治疗与毒性相对较低的药物如他莫昔芬和曲妥珠单抗，是不是卵巢癌患者的护理的标准。有足够的证据，铂，紫杉类一线化疗优于其他化疗方案卵巢癌的9-13，而70-80％的患者初步回应，大部分会复发，死于他们的疾病。预测替代疗法的生物标志物的测量，这可能会选择为每一个病人的个体化治疗，因为在肿瘤化疗施加选择压力，幸存的肿瘤细胞可能具有不同的特点，代表了肿瘤治疗前的亚群;量化这变化可能是有用的。我们应用我们的异质性映射方法ER和HER2的表达在卵巢癌化疗前后标本，在奥德r来衡量中表达量的变化，并评估治疗前后之间的生物标志物的表达的关系。 ER和HER2表现出明显的异质性，在个别肿瘤和某些肿瘤，治疗后的Simpson指数从异质性比较高低异质性的变化，通过减少Simpson指数得分（参见图2和3）代表。这支持克隆选择发生在细胞毒性化疗的概念，更重要的是，这可能是利用对剩余人口有针对性的治疗，为了更好的个性化癌症患者的治疗。 <img alt="图1" src="/files/ftp_upload/3334/3334fig1.jpg" /> 图1。苏木精和曙红染色显微照片显示变量形态相同的卵巢癌的不同领域。 （一）X40显微照片显示了两种不同形态的肿瘤邻近地区。高功率的意见（X200）揭示细胞的细胞质结算和固体肿瘤的上部（二）增长格局，而下部（三）组织更均匀的嗜酸性胞浆和乳头状生长模式。然而，这种肿瘤，浆液性乳头状为进一步管理的目的组织学亚型进行分类。 <img alt="图2" src="/files/ftp_upload/3334/3334fig2.jpg" /> 图2角蛋白阳性的卵巢癌组织中ER蛋白表达的异质性heatmaps前（上图）（下半部分）治疗后。 <img alt="图3" src="/files/ftp_upload/3334/3334fig3.jpg" /> 图3角蛋白阳性的卵巢癌组织中的表达，HER2的蛋白质前（上图）后（下图）治疗的异质性heatmaps。

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T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stability+of+phosphoprotein+as+a+biological+marker+of+tumor+signaling.">Stability of phosphoprotein as a biological marker of tumor signaling.</a> Clin. Cancer. Res. 11, 4338-4340 (2005).</li><li>Espina, V., Edmiston, K. H., Heiby, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+portrait+of+tissue+phosphoprotein+stability+in+the+clinical+tissue+procurement+process.">A portrait of tissue phosphoprotein stability in the clinical tissue procurement process.</a> Mol. Cell. Proteomics. 7, 1998-2018 (2008).</li><li>Klymkowsky, M. W., Savagner, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epithelial-mesenchymal+transition:+a+cancer+researcher's+conceptual+friend+and+foe.">Epithelial-mesenchymal transition: a cancer researcher's conceptual friend and foe.</a> Am. J. Pathol. 174, 1588-1593 (2009).</li><li>Kurrey, N. K., Jalgaonkar, S. P., Joglekar, A. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Snail+and+slug+mediate+radioresistance+and+chemoresistance+by+antagonizing+p53-mediated+apoptosis+and+acquiring+a+stem-like+phenotype+in+ovarian+cancer+cells.">Snail and slug mediate radioresistance and chemoresistance by antagonizing p53-mediated apoptosis and acquiring a stem-like phenotype in ovarian cancer cells.</a> Stem. Cells. 27, 2059-2068 (2009).</li><li>Creighton, C. J., Li, X., Landis, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Residual+breast+cancers+after+conventional+therapy+display+mesenchymal+as+well+as+tumor-initiating+features.">Residual breast cancers after conventional therapy display mesenchymal as well as tumor-initiating features.</a> Proc. Natl. Acad. Sci. U. S. A. 106, 13820-13825 (2009).</li><li>Aitken, S. J., Thomas, J. S., Langdon, S. P., Harrison, D. J., Faratian, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+analysis+of+changes+in+ER,+PR+and+HER2+expression+in+primary+breast+cancer+and+paired+nodal+metastases.">Quantitative analysis of changes in ER, PR and HER2 expression in primary breast cancer and paired nodal metastases.</a> Ann. Oncol. 21, 1254-1261 (2010).</li><li>Sayagues, J. M., Abad, M. M., Melchor, H. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intratumoural+cytogenetic+heterogeneity+of+sporadic+colorectal+carcinomas+suggests+several+pathways+to+liver+metastasis.">Intratumoural cytogenetic heterogeneity of sporadic colorectal carcinomas suggests several pathways to liver metastasis.</a> J. Pathol. 221, 308-319 (2010).</li></ol>

JC，MG名，终审法院首席法官，和CS HistoRx，公司的员工。

本网站所描述的方法允许量化标准福尔马林固定，石蜡包埋肿瘤材料的组织切片的分子异质性。该方法允许一个被分配到的异质性程度在组织部分，所以这可能是考虑到在生物标志物的开发和分析的变量的值。虽然在一般情况下最好是组织的生物标志物来表达，都是同质化，使检测不容易受到抽样误差或偏见，在某些情况下，它可能是有用的量化参数的异质性。例如，在ER和HER2的例证表明，常见的生物标志物显示相当大的异质性表达，目前尚不清楚这是否代表一个独立的预后预测因子与临床结果或对治疗的反应，分别。同样，作为说明，治疗本身可能动态改变细胞组成的人群，因此，测量目标富集可能是有用的指导治疗决定。 然而，技术的限制传统的病理组织学或生物标志物（如免疫组化）分析同样的因素是有限的。结果是依赖于分析组织的类型，大小和质量，并因此，一个单一的组织部分，可能无法代表整个肿瘤。事实上，Simpson指数可能过分偏袒组织的大小（帧捕获和测量AQUA的分数）。被测量表位的免疫原性，可能会受到无法控制的分析前因素artifactually改变整个组织切片（如冷缺血时间，或固定的长度，和边缘神器）的表达。这是特别为磷酸化表位，这是出了名的不稳定14，15的问题。因此，该技术可能更适合切除标本，而不是小的活组织切片检查，以及对多个部分而不仅仅是一个进行。最终，三维成像技术，可以提供一个机会，以更好地量化这个参数。 所提交的技术也可能是由生物因素，如变性的角蛋白掩蔽抗原表位的表达，有限的。特别关注，这可能是这些癌症，包括卵巢癌和乳腺癌，接受上皮间质转化（EMT），或丰富的非上皮成分或干细胞16，最近被证明在发生化疗在体外培养 17和乳腺癌患者在治疗18的治疗卵巢癌。使用替代掩蔽抗体（或抗体鸡尾酒），如角蛋白加波形，这是在EMT 的 16上调，可以克服这个限制。此外，由于该组织的其他组件（麦克风roenvironment），如成纤维细胞或血管内皮细胞也越来越多地被生物疗法的目标，该技术可能也将扩大到得分这些组件，使用车厢的利益，如PECAM / CD31，血管内皮细胞染色具体口罩。 虽然已适应Simpson指数作为衡量异质性，在目前的协议由于其简单，可用于异质性的其他措施，如简单的测量集中趋势，（平均值，方差，中位数，等）。此外，Simpson指数计算可以修改，以更好地满足特定的测试人群。允许使用的Z - score模型转换的得分，以更适用多个标记，由于异质性是围绕一个数据集的平均值的偏差的基础上。然而，整体得分范围可以限制在这种类型的转换。有些设计可能更适合简单bin中的一个特定的标记集所有样品的实际AQUA的分数。箱和截止的选择也是一个范围值，这可能会导致的限制，可用于替代实验设计优化。 我们使用ER和HER2在目前的候选生物标志物研究，因为它们已经在临床上使用，帮助回答与靶向治疗的疗效有关的临床问题，并表现出相当大的异质性和变化，在小学和遥远疾病19。然而，异质性的最佳指标仍有待确定。其他的可能性包括克隆的细胞遗传学标记，如以前在间期FISH 研究 20 。这种方法需要在大，以及注明的临床同伙或临床试验的进一步验证，以进一步建立此参数在癌症生物学相关。

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td> ScanScope佛罗里达州</td><td> Aperio </td><td> ScanScope佛罗里达州</td><td>从供应商使用配备</td></tr><tr><td>频谱</td><td> Aperio </td><td>频谱</td><td>地方机构如何设置自己的数据库和IT结构配置和目录的数目会有所不同。 </td></tr><tr><td> AQUAnalysis，AQUAjustment结合</td><td> HistoRx </td><td> V2.3版</td><td> AQUAjustment包，允许AQUAnalysis使用存储在频谱中的图像。 </td></tr><tr><td> HER2基因</td><td> Dako公司</td><td> A0485 </td><td> 1 400稀释</td></tr><tr><td>鼠抗角蛋白泛</td><td> Dako公司</td><td> M3515 </td><td> 1 50的稀释</td></tr><tr><td>抗体稀释液</td><td> Dako公司</TD&gt; <td> S0809 </td><td></td></tr><tr><td>设想山羊兔HRP </td><td> Dako公司</td><td> K4003 </td><td></td></tr><tr><td>蛋白质块</td><td> Dako公司</td><td> X0909 </td><td></td></tr><tr><td>羊抗小鼠Alexa555 </td><td> Invitrogen公司</td><td> A21422 </td><td></td></tr><tr><td> DAPI安装介质</td><td> Invitrogen公司</td><td> P36931 </td><td></td></tr><tr><td> Cy5的酪胺</td><td> HistoRx </td><td> AQ - EMR1 - 00001 </td><td></td></tr><tr><td> Sequenza免疫染色中心</td><td>热科学Shandon </td><td> 73300001 </td><td>这里使用的台式免疫荧光染色</td></tr></tbody></table>

heterogeneity mapping of protein expression in tumors using quantitative immunofluorescence

在个别肿瘤的形态学异质性，在外科实习histopathologists公认。虽然这往​​往需要形成鲜明的差异化领域，为公认的组织学亚型或不同的病理分级，往往存在表型更细微的差别，无法准确的分类（图1）。最终，由于形态是由潜在的分子表型决定的，明显的差别的地区可能会在协调细胞的功能和行为的蛋白表达的差异，因此，外观陪同。有形及无形（分子）预后的异质性的意义是未知的，但最近的证据表明，至少在基因水平，异质性的存在在原发肿瘤1,2，和一些这些子克隆引起转移性（因此致命）的疾病。 此外，一些蛋白质作为生物标志物的测量，因为它们是治疗的目标（例如ER和HER2的他莫昔芬和曲妥珠单抗（赫赛汀），分别）。如果这些蛋白质显示变量的表达式，然后在肿瘤治疗的反应也可能是变量。广泛用于免疫组化病理组织学评分计划，要么忽略，或数字均质蛋白表达的定量。同样，在破坏性的技术，均质肿瘤样本（如基因表达分析），定量信息可以澄清，但空间信息丢失。胰腺癌的遗传异质性的映射方法都依赖一个单细胞悬液 3代，或macrodissection 4 。最近的一项研究量子点，以图在前列腺癌组织5中的形态学和分子异质性，提供证据原则，形态学和分子映射是可行的，但下降小号园艺量化的异质性。由于免疫组化，充其量只是半定量和内和观察者间的偏见，更敏感和定量方法是需要以准确的地图和量化组织在原地的异质性。 我们已经开发和应用实验和统计方法，以系统量化的基础上的自动定量分析系统（水族）6，在整个肿瘤组织切片中的蛋白表达的异质性。组织切片是针对角蛋白和利益目标的特异性抗体，再加荧光标记的二抗标记。使用整个幻灯片的荧光扫描仪成像幻灯片。图片分为几百到成千上万的瓷砖，每个tile，然后分配一个AQUA的得分，这是一种蛋白浓度的措施，组织内的上皮细胞（肿瘤）组件。 HEAtmaps产生代表组织表达的蛋白质和分配异质性得分，使用的统计异质性措施最初用于在生态学，基于辛普森多样性指数7。 到目前为止，一直没有尝试系统地图和量化这种变化与蛋白表达的同时，在组织学的准备。在这里，我们首先使用说明ER和HER2基因在卵巢癌的生物标志物表达方法。使用这种方法铺平了道路，分析异质性翻译研究的生物标志物表达的研究作为一个独立的变量的方式，以建立异质性的预后和对治疗的反应预测的意义。

Watch this Scientific Journal Video about Heterogeneity Mapping of Protein Expression in Tumors using Quantitative Immunofluorescence at JoVE.com

Heterogeneity Mapping of Protein Expression in Tumors using Quantitative Immunofluorescence

在这里，我们描述了一种方法来量化分子异质性肿瘤的材料组织部分采用定量免疫荧光图像分析，和一个异质性的统计措施。该方法用于临床生物标志物的开发和分析。

在使用定量免疫肿瘤的异质性蛋白表达的映射

Morphologic heterogeneity within an individual tumor is well-recognized by histopathologists in surgical practice. While this often takes the form of ...

heterogeneity-mapping-protein-expression-tumors-using-quantitative

Research

JoVE Journal

Medicine

18.5K 次观看.  University of Edinburgh. 在这里，我们描述了一种方法来量化分子异质性肿瘤的材料组织部分采用定量免疫荧光图像分析，和一个异质性的统计措施。该方法用于临床生物标志物的开发和分析。

视频: Heterogeneity Mapping of Protein Expression in Tumors using Quantitative Immunofluorescence

Establishment and Propagation of Human Retinoblastoma Tumors in Immune Deficient Mice

Culturing retinoblastoma tumor cells in defined stem cell media gives rise to primary tumorspheres that can be grown and maintained for only a limited time. These cultured tumorspheres may exhibit markedly different cellular phenotypes when compared to the original tumors. Demonstration that cultured cells have the capability of forming new tumors is important to ensure that cultured cells model the biology of the original tumor. 
Here we present a protocol for propagating human retinoblastoma tumors in vivo using Rag2-/- immune deficient mice. Cultured human retinoblastoma tumorspheres of low passage or cells obtained from freshly harvested human retinoblastoma tumors injected directly into the vitreous cavity of murine eyes form tumors within 2-4 weeks. These tumors can be harvested and either further passaged into murine eyes in vivo or grown as tumorspheres in vitro. Propagation has been successfully carried out for at least three passages thus establishing a continuing source of human retinoblastoma tissue for further experimentation. 
Wesley S. Bond and Lalita Wadhwa are co-first authors.

A method is described to propagate human retinoblastoma tumors in mice. Tumor cells are directly injected into the eyes of immune deficient mice. Secondary tumors have been successfully established using both cells directly harvested from human tumors and cultured tumorspheres.

在免疫缺陷小鼠的建立和传播人类视网膜母细胞瘤肿瘤

Culturing retinoblastoma tumor cells in defined stem cell media gives rise to primary tumorspheres that can be grown and maintained for only a limited ...

科研

医学

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 ...

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 ...

Identification of Sleeping Beauty Transposon Insertions in Solid Tumors using Linker-mediated PCR

Genomic, proteomic, transcriptomic, and epigenomic analyses of human tumors indicate that there are thousands of anomalies within each cancer genome compared to matched normal tissue. Based on these analyses it is evident that there are many undiscovered genetic drivers of cancer1. Unfortunately these drivers are hidden within a much larger number of passenger anomalies in the genome that do not directly contribute to tumor formation. Another aspect of the cancer genome is that there is considerable genetic heterogeneity within similar tumor types. Each tumor can harbor different mutations that provide a selective advantage for tumor formation2. Performing an unbiased forward genetic screen in mice provides the tools to generate tumors and analyze their genetic composition, while reducing the background of passenger mutations. The Sleeping Beauty (SB) transposon system is one such method3. The SB system utilizes mobile vectors (transposons) that can be inserted throughout the genome by the transposase enzyme. Mutations are limited to a specific cell type through the use of a conditional transposase allele that is activated by Cre Recombinase. Many mouse lines exist that express Cre Recombinase in specific tissues. By crossing one of these lines to the conditional transposase allele (e.g. Lox-stop-Lox-SB11), the SB system is activated only in cells that express Cre Recombinase. The Cre Recombinase will excise a stop cassette that blocks expression of the transposase allele, thereby activating transposon mutagenesis within the designated cell type. An SB screen is initiated by breeding three strains of transgenic mice so that the experimental mice carry a conditional transposase allele, a concatamer of transposons, and a tissue-specific Cre Recombinase allele. These mice are allowed to age until tumors form and they become moribund. The mice are then necropsied and genomic DNA is isolated from the tumors. Next, the genomic DNA is subjected to linker-mediated-PCR (LM-PCR) that results in amplification of genomic loci containing an SB transposon. LM-PCR performed on a single tumor will result in hundreds of distinct amplicons representing the hundreds of genomic loci containing transposon insertions in a single tumor4. The transposon insertions in all tumors are analyzed and common insertion sites (CISs) are identified using an appropriate statistical method5. Genes within the CIS are highly likely to be oncogenes or tumor suppressor genes, and are considered candidate cancer genes. The advantages of using the SB system to identify candidate cancer genes are: 1) the transposon can easily be located in the genome because its sequence is known, 2) transposition can be directed to almost any cell type and 3) the transposon is capable of introducing both gain- and loss-of-function mutations6. The following protocol describes how to devise and execute a forward genetic screen using the SB transposon system to identify candidate cancer genes (Figure 1).

Identification of Sleeping Beauty Transposon Insertions in Solid Tumors using Linker-mediated PCR

A method of identifying unknown drivers of carcinogenesis using an unbiased approach is described. The method uses the Sleeping Beauty transposon as a random mutagen directed to specific tissues. Genomic mapping of transposon insertions that drive tumor formation identifies novel oncogenes and tumor suppressor genes

鉴定<em&gt;睡美人</em&gt;转座子插入连接器介导的PCR实体瘤

Genomic, proteomic, transcriptomic, and epigenomic analyses of human tumors indicate that there are thousands of anomalies within each cancer genome ...

In vitro Organoid Culture of Primary Mouse Colon Tumors

Several human and murine colon cancer cell lines have been established, physiologic integrity of colon tumors such as multiple cell layers, basal-apical polarity, ability to differentiate, and anoikis are not maintained in colon cancer derived cell lines. The present study demonstrates a method for culturing primary mouse colon tumor organoids adapted from Sato T et al. 1, which retains important physiologic features of colon tumors. This method consists of mouse colon tumor tissue collection, adjacent normal colon epithelium dissociation, colon tumor cells digestion into single cells, embedding colon tumor cells into matrigel, and selective culture based on the principle that tumor cells maintain growth on limiting nutrient conditions compared to normal epithelial cells.
The primary tumor organoids if isolated from genetically modified mice provide a very useful system to assess tumor autonomous function of specific genes. Moreover, the tumor organoids are amenable to genetic manipulation by virus meditated gene delivery; therefore signaling pathways involved in the colon tumorigenesis could also be extensively investigated by overexpression or knockdown. Primary tumor organoids culture provides a physiologic relevant and feasible means to study the mechanisms and therapeutic modalities for colon tumorigenesis.

In vitro Organoid Culture of Primary Mouse Colon Tumors

A simple method to establish primary murine colon tumor organoid is described. This method utilizes the feature that colon tumor cells survive and grow into organoids in media containing limited growth factors, whereas normal colon epithelial do not.

在体外化培养基层小鼠结肠肿瘤

Several  human and murine colon cancer cell lines have been established, physiologic  integrity of colon tumors such as multiple cell layers, basal-apical ...

The Use of Reverse Phase Protein Arrays (RPPA) to Explore Protein Expression Variation within Individual Renal Cell Cancers

Currently there is no curative treatment for metastatic clear cell renal cell cancer, the commonest variant of the disease. A key factor in this treatment resistance is thought to be the molecular complexity of the disease 1. Targeted therapy such as the tyrosine kinase inhibitor (TKI)-sunitinib have been utilized, but only 40% of patients will respond, with the overwhelming majority of these patients relapsing within 1 year 2. As such the question of intrinsic and acquired resistance in renal cell cancer patients is highly relevant 3.
In order to study resistance to TKIs, with the ultimate goal of developing effective, personalized treatments, sequential tissue after a specific period of targeted therapy is required, an approach which had proved successful in chronic myeloid leukaemia 4. However the application of such a strategy in renal cell carcinoma is complicated by the high level of both inter- and intratumoral heterogeneity, which is a feature of renal cell carcinoma5,6 as well as other solid tumors 7. Intertumoral heterogeneity due to transcriptomic and genetic differences is well established even in patients with similar presentation, stage and grade of tumor. In addition it is clear that there is great morphological (intratumoral) heterogeneity in RCC, which is likely to represent even greater molecular heterogeneity. Detailed mapping and categorization of RCC tumors by combined morphological analysis and Fuhrman grading allows the selection of representative areas for proteomic analysis.
Protein based analysis of RCC8 is attractive due to its widespread availability in pathology laboratories; however, its application can be problematic due to the limited availability of specific antibodies 9. Due to the dot blot nature of the Reverse Phase Protein Arrays (RPPA), antibody specificity must be pre-validated; as such strict quality control of antibodies used is of paramount importance. Despite this limitation the dot blot format does allow assay miniaturization, allowing for the printing of hundreds of samples onto a single nitrocellulose slide. Printed slides can then be analyzed in a similar fashion to Western analysis with the use of target specific primary antibodies and fluorescently labelled secondary antibodies, allowing for multiplexing. Differential protein expression across all the samples on a slide can then be analyzed simultaneously by comparing the relative level of fluorescence in a more cost-effective and high-throughput manner.

The Use of Reverse Phase Protein Arrays RPPA to Explore Protein Expression Variation within Individual Renal Cell Cancers

RPPA enables the protein expression of hundreds of samples, printed on nitrocellulose slides to be interrogated simultaneously, using fluorescently labelled antibodies. This technique has been applied to study the effect of drug treatment heterogeneity within clear cell renal carcinoma.

使用反时相蛋白的阵列（RPPA）探索在个人的肾细胞癌蛋白表达的变化

Currently there is no curative treatment for metastatic clear cell renal cell cancer, the commonest variant of the disease. A key factor in this treatment ...

In Vivo Optical Imaging of Brain Tumors and Arthritis Using Fluorescent SapC-DOPS Nanovesicles

We describe a multi-angle rotational optical imaging (MAROI) system for in vivo monitoring of physiopathological processes labeled with a fluorescent marker. Mouse models (brain tumor and arthritis) were used to evaluate the usefulness of this method. Saposin C (SapC)-dioleoylphosphatidylserine (DOPS) nanovesicles tagged with CellVue Maroon (CVM) fluorophore were administered intravenously. Animals were then placed in the rotational holder (MARS) of the in vivo imaging system. Images were acquired in 10&#176; steps over 380&#176;. A rectangular&#160;region of interest (ROI) was placed across the full image width at the model disease site. Within the ROI, and for every image, mean fluorescence intensity was computed after background subtraction. In the mouse models studied, the labeled nanovesicles were taken up in both the orthotopic and transgenic brain tumors, and in the arthritic sites (toes and ankles). Curve analysis of the multi angle image ROIs determined the angle with the highest signal. Thus, the optimal angle for imaging each disease site was characterized. The MAROI method applied to imaging of fluorescent compounds is a noninvasive, economical, and precise tool for in vivo quantitative analysis of the disease states in the described mouse models.

In Vivo Optical Imaging of Brain Tumors and Arthritis Using Fluorescent SapC-DOPS Nanovesicles

We describe a multi-angle rotational optical imaging (MAROI) system for in vivo quantitation of a fluorescent marker delivered by saposin C (SapC)-dioleoylphosphatidylserine (DOPS) nanovesicles. Employing mouse models of cancer and arthritis, we demonstrate how the MAROI signal curve analysis can be used for the precise mapping and biological characterization of disease processes.

在脑肿瘤和关节炎的用荧光SAPC-DOPS纳米囊泡体光学成像

We describe a multi-angle rotational optical imaging (MAROI) system for in vivo monitoring of physiopathological processes labeled with a fluorescent ...

Coordinate Mapping of Hyolaryngeal Mechanics in Swallowing

Characterizing hyolaryngeal movement is important to dysphagia research. Prior methods require multiple measurements to obtain one kinematic measurement whereas coordinate mapping of hyolaryngeal mechanics using Modified Barium Swallow (MBS) uses one set of coordinates to calculate multiple variables of interest. For demonstration purposes, ten kinematic measurements were generated from one set of coordinates to determine differences in swallowing two different bolus types. Calculations of hyoid excursion against the vertebrae and mandible are correlated to determine the importance of axes of reference.
To demonstrate coordinate mapping methodology, 40 MBS studies were randomly selected from a dataset of healthy normal subjects with no known swallowing impairment. A 5 ml thin-liquid bolus and a 5 ml pudding swallows were measured from each subject. Nine coordinates, mapping the cranial base, mandible, vertebrae and elements of the hyolaryngeal complex, were recorded at the frames of minimum and maximum hyolaryngeal excursion. Coordinates were mathematically converted into ten variables of hyolaryngeal mechanics.
Inter-rater reliability was evaluated by Intraclass correlation coefficients (ICC). Two-tailed t-tests were used to evaluate differences in kinematics by bolus viscosity. Hyoid excursion measurements against different axes of reference were correlated. Inter-rater reliability among six raters for the 18 coordinates ranged from ICC = 0.90 - 0.97. A slate of ten kinematic measurements was compared by subject between the six raters. One outlier was rejected, and the mean of the remaining reliability scores was ICC = 0.91, 0.84 - 0.96, 95% CI. Two-tailed t-tests with Bonferroni corrections comparing ten kinematic variables (5 ml thin-liquid vs. 5 ml pudding swallows) showed statistically significant differences in hyoid excursion, superior laryngeal movement, and pharyngeal shortening (p &#60; 0.005). Pearson correlations of hyoid excursion measurements from two different axes of reference were: r = 0.62, r2 = 0.38, (thin-liquid); r = 0.52, r2 = 0.27, (pudding).
Obtaining landmark coordinates is a reliable method to generate multiple kinematic variables from video fluoroscopic images useful in dysphagia research.

Coordinate mapping is a method of documenting salient features of hyolaryngeal biomechanics in the pharyngeal phase of swallowing. This methodology uses image analysis software to record coordinates of anatomical landmarks. These coordinates are imported into an excel macro and translated into kinematic variables of interest useful in dysphagia research.

协调Hyolaryngeal力学映射在吞咽

Characterizing hyolaryngeal movement is important to dysphagia research. Prior methods require multiple measurements to obtain one kinematic measurement ...

Quantitative Mass Spectrometric Profiling of Cancer-cell Proteomes Derived From Liquid and Solid Tumors

In-depth analyses of cancer cell proteomes are needed to elucidate oncogenic pathomechanisms, as well as to identify potential drug targets and diagnostic biomarkers. However, methods for quantitative proteomic characterization of patient-derived tumors and in particular their cellular subpopulations are largely lacking. Here we describe an experimental set-up that allows quantitative analysis of proteomes of cancer cell subpopulations derived from either liquid or solid tumors. This is achieved by combining cellular enrichment strategies with quantitative Super-SILAC-based mass spectrometry followed by bioinformatic data analysis. To enrich specific cellular subsets, liquid tumors are first immunophenotyped by flow cytometry followed by FACS-sorting; for solid tumors, laser-capture microdissection is used to purify specific cellular subpopulations. In a second step, proteins are extracted from the purified cells and subsequently combined with a tumor-specific, SILAC-labeled spike-in standard that enables protein quantification. The resulting protein mixture is subjected to either gel electrophoresis or Filter Aided Sample Preparation (FASP) followed by tryptic digestion. Finally, tryptic peptides are analyzed using a hybrid quadrupole-orbitrap mass spectrometer, and the data obtained are processed with bioinformatic software suites including MaxQuant. By means of the workflow presented here, up to 8,000 proteins can be identified and quantified in patient-derived samples, and the resulting protein expression profiles can be compared among patients to identify diagnostic proteomic signatures or potential drug targets.

In-depth analyses of cancer cell proteomes facilitate identification of novel drug targets and diagnostic biomarkers. We describe an experimental workflow for quantitative analysis of (phospho-)proteomes in cancer cell subpopulations derived from liquid and solid tumors. This is achieved by combining cellular enrichment strategies with quantitative Super-SILAC-based mass spectrometry.

对源自液体和固体肿瘤癌症细胞定量蛋白质组质谱剖析

In-depth analyses of cancer cell proteomes are needed to elucidate oncogenic pathomechanisms, as well as to identify potential drug targets and diagnostic ...

Quantitative Mapping of Specific Ventilation in the Human Lung using Proton Magnetic Resonance Imaging and Oxygen as a Contrast Agent

Specific ventilation imaging (SVI) is a functional magnetic resonance imaging technique capable of quantifying specific ventilation &#8213; the ratio of the fresh gas entering a lung region divided by the region&#8217;s end-expiratory volume &#8213; in the human lung, using only inhaled oxygen as a contrast agent. Regional quantification of specific ventilation has the potential to help identify areas of pathologic lung function. Oxygen in solution in tissue shortens the tissue&#8217;s longitudinal relaxation time (T1), and thus a change in tissue oxygenation can be detected as a change in T1-weighted signal with an inversion recovery acquired image. Following an abrupt change between two concentrations of inspired oxygen, the rate at which lung tissue within a voxel equilibrates to a new steady-state reflects the rate at which resident gas is being replaced by inhaled gas. This rate is determined by specific ventilation. To elicit this sudden change in oxygenation, subjects alternately breathe 20-breath blocks of air (21% oxygen) and 100% oxygen while in the MRI scanner. A stepwise change in inspired oxygen fraction is achieved through use of a custom three-dimensional (3D)-printed flow bypass system with a manual switch during a short end-expiratory breath hold. To detect the corresponding change in T1, a global inversion pulse followed by a single shot fast spin echo sequence was used to acquire two-dimensional T1-weighted images in a 1.5&#160;T MRI scanner, using an eight-element torso coil. Both single slice and multi-slice imaging are possible, with slightly different imaging parameters. Quantification of specific ventilation is achieved by correlating the time-course of signal intensity for each lung voxel with a library of simulated responses to the air/oxygen stimulus. SVI estimations of specific ventilation heterogeneity have been validated against multiple breath washout and proved to accurately determine the heterogeneity of the specific ventilation distribution.

Specific ventilation imaging is a functional magnetic resonance imaging technique that allows for quantification of regional specific ventilation in the human lung, using inhaled oxygen as a contrast agent. Here, we present a protocol to collect and analyze specific ventilation imaging data.

以质子磁共振成像和氧为造影剂对人肺特异性通气进行定量定位

Specific ventilation imaging (SVI) is a functional magnetic resonance imaging technique capable of quantifying specific ventilation &#8213; the ratio of ...