We are grateful to Janina Kuhl, Nadine Chelius, Petra Kussmann, Lena Willnow and Dorothee Lenhard for excellent technical assistance.

所有动物实验均按照Regierungspräsidium弗赖堡Referat 35的当地伦理和法律准则进行。 1.试剂准备注:在开始试验之前，准备从离解包以下试剂: <ol><li>为了制备酶ħ溶解冻干粉末的每个小瓶在3毫升的RPMI 1640罗斯韦尔园区纪念研究所1640（RPMI 1640）或Dulbecco改进的Eagle培养基（DMEM）的。为了避免反复冻融制备合适等份（ 例如 ，200微升），并将它们存储在- 20℃下进行长达6个月。 </li><li>为了制备酶ř溶解冻干粉末的小瓶中2.7毫升RPMI或DMEM培养基。为了避免反复冻融制备合适等份（ 例如，100微升），并将它们存储在- 20℃下进行长达6个月。 </li><li>为了准备酶溶解在V在1ml缓冲液A的冻干粉末IAL供给与没有涡旋的试剂盒。为了避免反复冻融制备合适等份并在存储（ 例如 ，25微升。） - 20℃长达6个月。请务必要彻底退出所需要的反应体积在此之前暂停，立即混合。 </li><li>制备250毫升缓冲液（1×PBS中的0.5％BSA）中进行细胞分离过程和抗体染色。 注意:当除去小鼠细胞后培养细胞通过0.22微米的过滤器过滤所有缓冲液和酶溶液。 </li></ol> 2.肿瘤离解协议注意:在此研究中的非小细胞肺癌（NSCLC）的患者来源异种移植物，肾癌和膀胱中使用。如前所述13 6周龄雌性NMRI nu / nu小鼠-通过注入肾癌非小细胞肺癌的异种移植物（NSCLC）和膀胱中4分别产生的肿瘤</s了&gt;。 注意:作为该协议是完全独立于肿瘤类型的，它可用于任何类型的患者或细胞系衍生的肿瘤以及其他类型的人类异种移植组织的未经协议的修改。 <ol><li>制备5-毫升，每组织样品消化混合（高达1克）新鲜加入4.7毫升的RPMI 1640或DMEM，200微升酶H型，100微升酶的R，和25微升酶A的成蜂窝解离管（ C封装Tube）。确保收回所需要的卷之前调匀酶 - [R停牌。 </li><li>牺牲麻醉下颈椎脱臼的动物。通过用80％V / V乙醇少量喷雾消毒鼠标。 </li><li>嘉实皮下肿瘤使用镊子和剪刀在组织培养罩鼠标的侧翼。切开皮肤代替用剪刀肿瘤的开放，然后使用镊子和剪刀从邻近的健康组织的肿瘤中分离出来。 注:根据的目的该实验在无菌条件下执行此和以下步骤。在这项研究中的所有步骤都在顺序在无菌条件下的细胞培养罩进行，以能够培育细胞。 </li><li>通过去除脂肪（因为这会使示例float）和坏死区（因为这会导致活力下降），将其截走使用镊子和手术刀准备消化肿瘤样品。用手术刀和镊子4毫米 - 剁碎组织成2块。 </li><li>转移组织块放入含有消化混合的C管。 </li><li>关闭C封装Tube并确保它被紧紧关闭。附加它倒置到台式组织离解器的套筒，并确保该组织片位于转子/定子的面积。 </li><li>按"向上"或"向下"按钮，直到模式出现在显示屏上，然后按"开始"按钮，选择模式"h_tumor_01"。 <ol><li>如果你唱台式组织离解的加热功能，确保还附上加热器。然后通过点击触摸屏上的文件夹符号直到文件夹"美天旎"出现运行模式37C_h_TDK_1（软肿瘤），37C_h_TDK_2（用于中等肿瘤）或37C_h_TDK_3（硬肿瘤）。 </li><li>要选择模式，请单击向上或向下箭头，直到离解模式37C_h_TDK_2高亮显示。的台式组织离解的套筒被描述为在设备的显示器上的位置。选择样本通过单击它们在触摸屏上运行的位置。按下"开始"运行模式，并继续执行步骤2.16。 注:根据不同的肿瘤类型另一个分离程序可能是合适的（见步骤2.7.1）。 </li></ol></li><li>观察显示在屏幕上的"节目的终止"的窗口。节目结束后，分离从台式组织离解C封装Tube。 </li><li>孵育样品在37 30分钟°℃下在连续旋转， 例如 ，通过使用一管肩。 </li><li>孵化后附上C封装Tube倒挂到台式组织离解的袖子。再次确保样品材料位于转子/定子的面积。 </li><li>运行程序分解h_tumor_02（软，中肿瘤）或h_tumor_01（硬瘤），按步骤2.7.1所述。 </li><li>节目结束后，分离从台式组织离解C封装Tube。 </li><li>孵育样品下连续旋转，在37℃30分钟。 </li><li>附加C封装Tube倒挂到孵化后的台式组织离解的袖子。再次确保样品材料位于转子/定子的面积。 </li><li>运行的解离方案h_tumor_03（软肿瘤），h_tumor_02（用于中等肿瘤）或h_tumor_01（硬肿瘤）如在步骤2.7中描述。 </li><li>收集样品材料在试管底部进行抄室温离心步骤（在100×g离心10秒）。 </li><li>重悬细胞，并适用于细胞过滤，用70微米置于50ml管NESH大小。用20ml的RPMI 1640或DMEM中洗涤细胞过滤。 </li><li>在300×g离心7分钟离心细胞悬浮液。吸完全上清。 </li><li>悬浮细胞在5ml缓冲的计数。通过磁性细胞分离（第3节）小鼠细胞耗竭继续。 </li></ol> 3.磁性细胞分离技术小鼠细胞耗竭注意:下面给出的卷为磁标记是长达2×10 6个肿瘤细胞或10 7个总细胞包括红细胞。根据肿瘤大小更低或更高的细胞数，将获得。在较少的细胞的情况下，使用相同的卷所示。当使用更高的细胞数量，扩大规模的所有试剂卷和卷总额相应（ 例如，4×10 6肿瘤细胞或2×10 <suP&gt; 7个细胞，利用一切表明试剂体积和总体积）的两倍音量。 <ol><li>确定从细胞悬浮液，在步骤2.21使用用于计数显微镜和合适的腔室中的等分试样的细胞数。 注意:此时的细胞悬浮液由人肿瘤细胞的异质混合物的以及小鼠基质和血细胞，包括红细胞。细胞的组合物在很大程度上取决于肿瘤类型和区域用于移植。 </li><li>在进行流式细胞仪分析后去除小鼠细胞中撤出100微升等分适合用于管未染色和两个单色彩控制染色的流式细胞仪分析。 8°C，直到进一步的染色步骤（第4节） - 在2冰箱保存。 </li><li>在300×g离心10分钟，离心细胞悬浮液。弃去上清液。 </li><li>在80微升每2×10 6个肿瘤细胞或10 7缓冲液重悬细胞沉淀总细胞加入20μl磁性标记试剂的小鼠细胞。 </li><li>拌匀，孵育在冰箱15分钟（2 - 8°C）。根据制造商的方案中的磁性标记，制备用于磁分离大规模柱（LS柱），通过将其放置在一个合适的磁体的磁场（见材料和​​设备的数据表）。用3毫升缓冲液冲洗该柱。 </li><li>调节使用缓冲器的样品体积为500微升长达2×10 6个肿瘤细胞或高达10 7总细胞。 （最多1×10 7个肿瘤细胞或高达5×10 7个总细胞可以在一个LS柱进行处理。当与多个小区工作分裂样本到多个LS柱）。 <ol><li>在进行流式细胞仪分析，分子生物学分析或文化比较分数，取消50微升等分作为未分类的样本。 8°C，直到进一步的处理 - 在2冰箱储存。 </li></ol> &lt;/ LI&gt; <li>应用500微升的细胞悬液到预先制备的柱。收集流过含有未标记的靶细胞，分别代表富集的人肿瘤细胞。 注意:当具有更高的细胞数的工作至2.5 ml的细胞悬浮液被按照制备步骤3.5可应用于一列。 </li><li>用2×1毫升缓冲液洗柱。收集穿过所述细胞，并与流过来自步骤3.6结合。通过只要柱储层是空加入1毫升缓冲液的等分试样进行清洗步骤。 注意:通流包含纯化的人肿瘤细胞。 </li><li>为了还收集保留在柱子的小鼠细胞，从分离器中删除列，并将其放置在合适的管子。吸管3毫升缓冲液上柱，并立即用柱塞由力推入柱冲洗掉的小鼠细胞。 </li><li>降速的级分和对照样品在300×g离心10分钟。吸苏佩完全rnatant和在缓冲液，培养基悬浮细胞或在液氮中冷冻沉淀，这取决于所期望的下游应用。 </li></ol> 4.下游分析<ol><li> 染色流式细胞仪分析 注:在进行进一步下游分析之前，从小鼠细胞耗竭过程获得的细胞中的一部分可以被分析（ 例如 ，评估的纯度和产率），在流式细胞仪。 <ol><li>用于流式细胞分析，在300 XG自旋3.5从磁性细胞分离过程（部分3）中得到的阴性和阳性分数，以及来自步骤3.2的对照样品和至少10％的10分钟。 </li><li>弃去上清液。在90微升缓冲液中重悬染色的对照样品的细胞沉淀从步骤3.2。重悬在80μl的缓冲液中从磁性细胞分离过程中得到的级分。 </li><li>加10μl鼠标FcR阻断剂的所有样品。拌匀，孵育在2 5分钟 - 8℃的冰箱中。 </li><li>添加抗人EpCAM-PE的10微升（等于1:10抗体稀释）和25μl的抗小鼠-APC鸡尾酒（等于1抗体稀释度:4），以从部分3的样品中添加10微升抗 - 人EpCAM的聚乙烯对单一颜色对照样品（等于1:10抗体稀释）和10μl抗人EpCAM-APC到其他的（等于1:10抗体稀释）。混合所有样品，孵育在2 10分钟 - 8℃的冰箱中。 注:EpCAM的不适合作为任何肿瘤类型的肿瘤细胞的标志物。在这项研究的EpCAM表达式中使用的肿瘤是众所周知的。 </li><li>洗样品，在300 xg离心加各1ml缓冲液和自旋5分钟。 </li><li>弃上清，重悬细胞沉淀用于流式细胞仪分析的浓度。以排除死细胞添加碘化丙啶（1微克/毫升）。 </li><li>使用合适˚F进行流式细胞仪分析根据制造商的协议低血细胞计数。 </li></ol></li><li> 人类肿瘤细胞的培养 注意:除了流式细胞仪，从分离过程得到的细胞可以培养并用于体外进一步的测定。 <ol><li>重悬在培养基中部分3的排序馏分的5×10 5细胞/ ml的浓度。 注意:根据肿瘤类型涂层，适合接种密度和培养条件可有所不同。 </li><li>加入500微升细胞悬浮液到涂有0.1％明胶24孔板的孔中。 </li><li>孵育在细胞培养孵化器细胞在37℃和5％。 注:最佳培养条件取决于所用的肿瘤类型。因此，申请适合使用的肿瘤类型的文化条件。 </li></ol></li><li> 培养细胞的免疫荧光染色 <ol><li>制备通过添加10％FCS和0.1％封闭溶液的Triton X-100的1X PBS。 </li><li>丢弃培养基。加入500μl1X PBS，每孔洗细胞。孵育在室温（RT）下2分钟，然后完全丢弃1×PBS中。 </li><li>通过加入300微升4％PFA溶液固定细胞。孵育在室温下进行15分钟。 </li><li>丢弃PFA解决方案。洗固定的细胞3次，如在步骤4.3.2指示。 </li><li>加入300微升每孔堵塞的执行通透性和阻塞。孵育在室温下进行30分钟。 </li><li>丢弃封闭溶液。洗涤细胞如步骤4.3.2指示。 </li><li>添加第一抗体以300μl阻断溶液至细胞稀释。抗人EpCAM抗体稀释1:40，波形蛋白抗体稀释1:200。 8°C - 在2孵育过夜。 注意:使用的抗体不适合任何肿瘤类型如EpCAM的可能不会对肿瘤细胞和/或波形蛋白表达也由肿瘤细胞中表达。确保使用的抗体是适合于异种移植物莫德尔。 </li><li>丢弃第一抗体溶液。洗涤细胞3次，如在步骤4.3.2指示。 </li><li>添加二级抗体以300μl阻断溶液至细胞稀释。两种抗体，抗兔IgG-Alexe594和抗小鼠IgG-Alexa488稀释1:400。孵育在室温黑暗1小时。 </li><li>丢弃第二抗体溶液。洗涤细胞2次，如步骤4.3.2指示。 </li><li>加的DAPI溶液300微升（0.1微克/毫升）至各孔中。孵育在室温黑暗中10分钟。 </li><li>丢弃DAPI解决方案。洗涤细胞3次，如在步骤4.3.2指示。 </li><li>添加500μl的1×PBS中的每个孔中。使用合适的显微镜进行荧光显微镜。 </li></ol></li><li> 全基因组测序 <ol><li>对于从步骤3.9液氮全外显子测序（WES）冻结颗粒。存储和在-80℃收集样本。 </li><li>收集细胞后，执行外显子组捕获和整个外显子组以次根据对各试剂盒的制造商的说明uencing（见材料和​​设备的数据表）。 </li></ol></li></ol>

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M., Lohse, M., Usadel, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trimmomatic:+a+flexible+trimmer+for+Illumina+sequence+data.">Trimmomatic: a flexible trimmer for Illumina sequence data.</a> Bioinformatics. 30 (15), 2114-2120 (2014).</li><li>Li, H., Durbin, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fast+and+accurate+short+read+alignment+with+Burrows-Wheeler+transform.">Fast and accurate short read alignment with Burrows-Wheeler transform.</a> Bioinformatics. 25 (14), 1754-1760 (2009).</li><li>Koboldt, D. C., Larson, D. E., Wilson, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+VarScan+2+for+Germline+Variant+Calling+and+Somatic+Mutation+Detection.">Using VarScan 2 for Germline Variant Calling and Somatic Mutation Detection.</a> Curr Protoc Bioinformatics. 44, (2013).</li><li>Robinson, J. T., 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=Integrative+genomics+viewer.">Integrative genomics viewer.</a> Nat Biotechnol. 29 (1), 24-26 (2011).</li><li>Aloia, 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=The+sialyl-glycolipid+stage-specific+embryonic+antigen+4+marks+a+subpopulation+of+chemotherapy-resistant+breast+cancer+cells+with+mesenchymal+features.">The sialyl-glycolipid stage-specific embryonic antigen 4 marks a subpopulation of chemotherapy-resistant breast cancer cells with mesenchymal features.</a> Breast Cancer Res. 17 (1), 146 (2015).</li></ol>

David Agorku, Stefan Tomiuk, Stefan Wild, Silvia R&#252;berg, Lisa Zatrieb, Andreas Bosio and Olaf Hardt are or were employees of Miltenyi Biotec GmbH, Germany. Kerstin Klingner and Julia Sch&#252;ler are employees of Oncotest GmbH, Germany.

我们已经开发出一种快速和容易的方法来隔离异种移植肿瘤组织不变人肿瘤细胞。此过程是通过结合自动组织解离和磁性细胞分选基于小鼠来源的细胞的全面枯竭。除了所有小鼠的细胞也死细胞和碎片的量的耗尽靶细胞级分显著降低。总之，这种方法显著提高分子下游分析和人类肿瘤细胞的培养。 在对比的替代方法，也没有必要对肿瘤细胞特异性标记，调整到不同的小鼠细胞或建立的算法的组合物的知识。该方法是独立于小鼠品系和肿瘤类型。因此，通过单人的特定标志物的基础可在表达发生变化，如图对EpCAM的11对人肿瘤亚群的分离偏压，是避免编辑。此外，它并不限于肿瘤材料，但可用于任何类型的异种移植组织的小鼠细胞靶向代替特定人肿瘤亚群（未示出数据）。 全面去除小鼠细胞通过靶向小鼠来源的细胞只表达表面表位促进。除了如在本研究中提出的肿瘤分解行之有效的方法，有使用不同种类的酶的许多程序。细胞表面表位的保存为这种新方法，而且对人肿瘤细胞群的准确分析的先决条件。因此，至关重要的是，平缓的酶用于肿瘤组织的消化。因此，小鼠细胞耗竭只能在与酶促消化程序，显然不影响小鼠细胞耗竭过程期间目标表位组合使用。在有疑问的情况下，这只能通过各自的制造商进行验证。 <p类="jove_content"&gt;这个方法也适用于人类的循环肿瘤细胞（的CTC）。然而，作为靶细胞的频率通常&lt;0.1切除的小鼠细胞的％另外需要红细胞溶解和的50％以上的纯度不能期待。在这种情况下，小鼠细胞耗竭可以用作的CTC接着使用正标记富集的预富集（数据未显示）。 小鼠细胞的去除通过避免小鼠成纤维细胞的培养长满显著改善人类肿瘤细胞的培养。此外，通过新一代测序人肿瘤异种移植物的分析是显著改善和标准化。如观察到这种效果，尽管外显子组富集过程中靶向人类序列特异性选择已经进行，在整个基因组和全转录组测序的影响预计将更为突出。 此外，所提出的方法便于ACCURA人类异种移植肿瘤内的肿瘤细胞亚群的TE分子研究。从在第一步骤中的相应的样品的小鼠细胞的去除和随后在两个不同的亚群排序人肿瘤细胞使感兴趣的人的大块肿瘤细胞20的肿瘤亚群的直接分子的比较。肿瘤细胞亚型之间的差异基因表达，可以更可靠地评估，因为它不会受到源于小鼠的分子的交叉杂交。

固体人肿瘤包括多个生理以及肿瘤细胞类型。他们形成复杂的生物结构的异质性的组织。像肿瘤的形成，发展生物过程和对治疗的反应都还没有完全理解。 在体外细胞培养模型代表一个有用的工具来研究和理解肿瘤生物学。但是，它们仅能部分地反映在肿瘤组织中发现的结构和过程。可靠，稳定的临床前的人肿瘤模型是抗肿瘤的药物和疗法4,6的发展以及对理解肿瘤生物学和肿瘤细胞和他们的环境的相互作用的一个先决条件。 原发性肿瘤患者来源的人肿瘤异种移植模型显示出高的关系起源组织有关的组织学结构，相互作用与微环境搭建，转移潜能和应对药品7 </sup&gt;。即使当肿瘤异种移植物是从培养的细胞或细胞系，他们更接近地模拟患者肿瘤衍生的，因此示出了相比于在体外细胞培养模型中大多数测定更高有效性。这些特点使他们临床前模型4的黄金标准。除了 ​​其在癌症研究中的应用，人类细胞的异种移植到小鼠中也经常在干细胞研究用于确定一个目标人口8的干性和分化潜能。 已经表明，人类微血管和人免疫细胞在体内的人类肿瘤2,9-传播代替。如肿瘤亚型，生长速率，和移植的区域因素对浸润的全球主要的影响，以及在小鼠细胞类型中发现的组合物。然而，即使当这些因素保持不变，所述量和小鼠细胞的组合物是高度可变。 异种移植组织的下游分析通常由鼠起源的细胞的挑战。从移植人类肿瘤细胞原代培养经常由成纤维细胞杂草丛生。除了 ​​妨碍的体外肿瘤细胞系的产生，下游分析如药物的细胞毒性测试或药代动力学，在硅片校正自偏压用于从污染小鼠细胞是在大多数情况下是不可能始发的效果。除此之外，小鼠成纤维细胞和人肿瘤细胞之间的仅部分阐明串扰对研究10个实验的结果有直接的影响。此外，浸润小鼠细胞中最广泛不可预知的变化加剧准确分子下游分析。在NGS或蛋白质组分析，而不是测量的人肿瘤信号的每个小鼠衍生的信号直接降低测序的灵敏度。也是基于微阵列表达分析是由鼠类NUC挑战leic酸可能交叉杂交人类探测器。 为了规避在从异种移植模型中不同的方法，已经提出的人类肿瘤细胞的下​​游分析污染小鼠细胞的障碍。在许多研究中用于下游分析所需靶细胞通过利用对人类肿瘤细胞只表达标记或标志物的组合分离。然而，由于缺乏对人类肿瘤细胞阳性鉴定可靠指标常常代表一个很大的障碍。即使是广泛表达的标志物，如对人体癌EpCAM的，经常表现出肿瘤的内在表达差异11。这增加了低表达的亚群， 例如，肿瘤细胞经历上皮至间质转变，隔离过程中丢失的风险。此外，直接结合选择剂的靶细胞的可能影响随后的分析结果。通过耗竭小鼠细胞的尝试使用特异于鼠CD45和MHC I类表位的抗体的组合也已提出了12。然而，这种标记组合仅检测小鼠细胞的子集。另一种方法是用软件来增强分析流程。然而，所有这些方法要么是不适合于任何种类的实验装置或任何肿瘤实体和移植方法。 在这项研究中，我们提出了一种新的，快速的方法，为全面独立的小鼠品系和原产地组织异种移植的组织耗竭小鼠细胞。在关于异种移植物（包括皮肤，肺，脑，肾和骨骼肌）的移植多个目标器官和组织放映我们能够识别的抗体，允许全面检测小鼠细胞的组合。这些抗体偶联到超顺磁性粒子，滴定并用磁性细胞分选对消耗优化。由于只有特定的鼠标抗体用于人类靶细胞留"不变"，并且该方法不限定于异种移植肿瘤组织。我们表明，全面去除小鼠细胞异种移植从肿瘤样本标准化并促进人类肿瘤细胞的培养。此外，我们表明，人肿瘤异种移植物的由下一代测序分析是显著改善。如观察到这种效果，尽管外显子组富集期间人序列特定选择已经进行，在整个基因组和全转录组测序的影响预计将更为突出。两者合计，除去使用这种新方法的小鼠细胞的促进人类肿瘤细胞的培养和显著改善人类肿瘤异种移植物的下游分析。

<table border="0" cellpadding="0" cellspacing="0" width="1111">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Tumor Dissociatio Kit, human</td>
			<td style="width:185px;">Miltenyi Biotec</td>
			<td style="width:119px;">130-095-929</td>
			<td style="width:593px;">contains enzymes H, R and A; see data sheet for reconstitution instructions</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">RPMI 1640</td>
			<td style="width:185px;">Biowest</td>
			<td style="width:119px;">L0501-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">gentleMACS C-Tubes</td>
			<td style="width:185px;">Miltenyi Biotec</td>
			<td style="width:119px;">130-096-334</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">gentleMACS Octo Dissociator with Heaters</td>
			<td style="width:185px;">Miltenyi Biotec</td>
			<td style="width:119px;">130-096-427</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">MACS SmartStrainer (70 &#181;m)</td>
			<td style="width:185px;">Miltenyi Biotec</td>
			<td style="width:119px;">130-098-462</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Petri dish 100 mm</td>
			<td style="width:185px;">Various</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Scalpel</td>
			<td style="width:185px;">Various</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Mouse Cell Depletion Kit</td>
			<td style="width:185px;">Miltenyi Biotec</td>
			<td style="width:119px;">130-104-694</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">PBS</td>
			<td style="width:185px;">Various</td>
			<td style="width:119px;"></td>
			<td>use 1x PBS for PEB buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">EDTA</td>
			<td style="width:185px;">Sigma-Aldrich</td>
			<td style="width:119px;">3610</td>
			<td>use final concentration of 2 mM in PEB buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">BSA</td>
			<td style="width:185px;">Miltenyi Biotec</td>
			<td style="width:119px;">130-091-376</td>
			<td>or use 5 mg/L BSA in PEB buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">MACS LS columns</td>
			<td style="width:185px;">Miltenyi Biotec</td>
			<td style="width:119px;">130-042-401</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">QuadroMACS Separator</td>
			<td style="width:185px;">Miltenyi Biotec</td>
			<td style="width:119px;">130-090-976</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">MACS MultiStand</td>
			<td style="width:185px;">Miltenyi Biotec</td>
			<td style="width:119px;">130-042-303</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">PreSep filters (70 &#181;m)</td>
			<td style="width:185px;">Miltenyi Biotec</td>
			<td style="width:119px;">130-095-823</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">MACSmix Tube Rotator</td>
			<td style="width:185px;">Miltenyi Biotec</td>
			<td style="width:119px;">130-090-753</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">CD326-FITC, human</td>
			<td style="width:185px;">Miltenyi Biotec</td>
			<td style="width:119px;">130-080-301</td>
			<td>use 1:40 for immunofluorescence staining&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">anti-Vimentin antibody</td>
			<td style="width:185px;">abcam</td>
			<td style="width:119px;">ab92547</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">goat-anti-mouse IgG-Alexa488</td>
			<td style="width:185px;">Life Technologies</td>
			<td style="width:119px;">A11029</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">goat-anti-rabbit IgG-Alexa594</td>
			<td style="width:185px;">Life Technologies</td>
			<td style="width:119px;">A11012</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">DAPI</td>
			<td style="width:185px;">Sigma-Aldrich</td>
			<td style="width:119px;">D9542</td>
			<td>dilute to a final concentration of 0.1 &#181;g/ml in 0.1 M sodium bicarbonate&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Inside Stain Kit</td>
			<td style="width:185px;">Miltenyi Biotec</td>
			<td style="width:119px;">130-090-477</td>
			<td>InsideFix from this kit can be used for fixation step during immunofluorescence staining&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">FBS</td>
			<td style="width:185px;">Various</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Triton X-100&#160;</td>
			<td style="width:185px;">Sigma-Aldrich</td>
			<td style="width:119px;">93418</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">FcR Blocking Reagent, mouse</td>
			<td style="width:185px;">Miltenyi Biotec</td>
			<td style="width:119px;">130-092-575</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Gelatin</td>
			<td style="width:185px;">Sigma-Aldrich</td>
			<td style="width:119px;">G2500-100g</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">Nextera Rapid Capture Enrichment Kit</td>
			<td style="width:185px;">Illumina</td>
			<td style="width:119px;">FC-140-1000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">MiSeq Reagent Kit v3</td>
			<td style="width:185px;">Illumina</td>
			<td style="width:119px;">MS-102-3001</td>
			<td></td>
		</tr>
	</tbody>
</table>

depletion of mouse cells from human tumor xenografts significantly improves downstream analysis of target cells

癌症研究中使用的体外细胞系模型的一直是有用的工具。然而，已经表明，这些模型不能可靠地模仿患者的肿瘤中不同的测定1。人肿瘤异种移植物代表相对于肿瘤生物学，药物发现和转移研究2-4的黄金标准。肿瘤异种移植物可以从不同的类型，如肿瘤细胞系，从初级患者肿瘤4或连续移植肿瘤的肿瘤组织的材料中获得。 当体内传播，异种移植组织渗透和通过小鼠来源的细胞血管化。多种因素，例如肿瘤实体，异种移植材料，生长速度和区域移植的原点影响组合物和目前在肿瘤异种移植物的小鼠细胞的量。然而，即使当这些因素保持恒定，小鼠细胞污染的程度是高度可变的。 有限公司ntaminating小鼠细胞显著损害人体肿瘤异种移植的下游分析。由于小鼠成纤维细胞表现出高疗效的电镀和增殖率，他们往往过度生长的人类肿瘤细胞的培养，尤其是缓慢增殖亚群。小鼠细胞来源的DNA，mRNA表达，以及蛋白质成分可以偏压下游基因表达分析，新一代测序，以及蛋白质组分析5。为了克服这些限制，我们已开发了一种快速和容易的方法，以隔离从异种移植肿瘤组织不变人肿瘤细胞。此过程由自动组织离解与台式组织离解器和磁性细胞分选组合基于小鼠来源的细胞的全面枯竭。这里，我们表明，人靶细胞可以是能够与纯度高于96％，小于20分钟的独立的肿瘤类型的内获得。

不同的方法已被提出，以从异种移植的人肿瘤的鉴定或耗尽小鼠细胞，例如针对CD45和MHC I类然而，尽管所提出的组合检测到的CD45的使用这些组合仅检出小鼠的细胞亚群小鼠特异性抗体的组合和MHC I类表达的小鼠细胞，以及在靶组织中发现用于移植的小鼠细胞的其余部分（ 图1）。利用这种新颖的用于磁性细胞分选的抗体的组合，人类肿瘤细胞可以分离独立的肿瘤类型（ 图2）的。 从磁性细胞分离基于过程的小鼠细胞去除得到的细胞可以是培养的，导致人类肿瘤细胞（ 图4）的纯培养物。此外，可以得到和从培养存活的小鼠细胞同样的样品。除了 ​​去除小鼠的细胞，也碎片在小鼠细胞耗竭（MCD）（ 图3）中除去。 小鼠细胞的全面枯竭以及碎片清除导致改善分子下游分析。这是通过比较来自三个不同患者来源的异种移植肿瘤模型（ - 9图5）上大块肿瘤块和小鼠细胞耗尽样品上进行从全基因组测序（WES）得到的结果证实。我们的数据表明，在异种移植肿瘤样本执行WES之前除去小鼠细胞不仅显著增加读（图5）的合计量也大大降低衍生主机的读取数映射到人类参照基因组（图6B）。由于这些错误映射鼠标读经常与SNP通话干扰，我们观察到的错误预测强还原单核苷酸多态性MCD后（图7 - 8）。最后，我们发现，在硅片去除鼠源性通过生物信息学方法读取不能完全代替实验过程中，作为一个明确的序列为基础的分配来源的种类是不可能的所有读取。此外，在硅片过程不能正确的提高的质量和体外耗尽样品伴随更高的读取范围（图9）。 <img alt="图1" src="/files/ftp_upload/54259/54259fig1.jpg" /> 图1. 建立一个抗体混合物认识所有小鼠细胞跨越多器官14。掩护在多种器官，包括皮肤，肺，脑，肾和骨骼肌，已执行。这些器官代表异种器官移植的主要靶组织。组合，例如那些识别谋本身CD45和MHC I类抗原表位已经为了异种移植后耗尽小鼠细胞使用。然而，这些标志物的组合确认在所有分析的组织只小鼠细胞的子集。在前面的掩护5抗体的组合已被确定，允许综合检测从小鼠来源的所有细胞。该面板包括红血细胞，并独立起源组织的（A，B和数据未显示）。从14修改。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54259/54259fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/54259/54259fig2.jpg" /> 小鼠细胞图 2. 可靠和快速的消耗与超顺磁性的n个抗体结合的15。偶联物anoparticles被用来通过磁性细胞分选（磁性细胞分离）（A）中制定用于从人肿瘤异种移植物的小鼠细胞的耗竭的优化协议。这种新颖的协议允许消除在不到20分钟的污染的小鼠细胞的&gt; 99％，而与肿瘤类型。从磁性细胞分离过程得到的细胞级分，标有泛小鼠抗体鸡尾酒和抗CD326（EpCAM的）人特异性抗体（B）。来自15个修改。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54259/54259fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/54259/54259fig3.jpg" /> 图3. 人类成胶质细胞瘤细 ​​胞的分离。小鼠细胞耗竭试剂盒被用于人脑胶质瘤细胞从成年备忘录隔离Ë大脑。在神经组织​​中大量碎片的分解通常产生。 MCDK有效地消耗通过显示单元尺寸与电池粒度的情节所看到的死细胞和碎片。通过这种结合的鸡尾酒，有可能消除污染的小鼠细胞&gt; 99％，碎片&gt; 60％。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54259/54259fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/54259/54259fig4.jpg" /> 图4. 小鼠细胞的耗竭便于人肿瘤细胞14的下游培养 。小鼠成纤维细胞经常阻碍人肿瘤细胞的培养移植组织的离解后，或者导致异质培养物。成纤维细胞附着并展开更有效，从而长满目标C<html>厄尔。此扰乱体外细胞培养测定（ 例如，药物的细胞毒性测试），由于用于从污染小鼠细胞是在大多数情况下是不可能产生的效应的数学校正。小鼠细胞耗竭后的负极（B）和阳性的级分（C）的培养三天，固定和染色的小鼠成纤维细胞特异性标记物（波形蛋白）和特定的人类肿瘤标志物CD326（的EpCAM）。作为对照，含有未分离的细胞（A）的原始分数为培养和染色为好。甚至在培养三天，在负部分（B）中观察到人肿瘤细胞的几乎纯的人口，而无序组分（A）是由成纤维细胞几乎长满。靶细胞仅一小部分被输给正级分（C）。从14修改。ge.jpg"目标="_空白"&gt;点击此处查看该图的放大版本。 <img alt="图5" src="/files/ftp_upload/54259/54259fig5.jpg" /> 图5. 全外显子测序肿瘤样本（WES）之前和小鼠细胞耗竭（MCD）15日之后 ，我们对人的肾脏，肺和膀胱癌衍生三种不同的异种移植模型，以评估对MCD的影响进行WES下一代测序数据的质量。从大块肿瘤和来自小鼠细胞耗竭后，分离的肿瘤细胞的DNA用于产生施加外显子组捕获试剂盒外显子组捕获测序文库。有关台式仪器序器，桌面序试剂盒150次测序，被用来产生75个碱基配对末端读。在集群密度的增加显著（P &lt;0.05） （A），以及作为AVE观察到耗尽的小鼠细胞的样品中的33％的（B）中读出的计数愤怒增加，表明提高了样品的质量。相应地，碎片和死细胞时MCD强烈降低也可以通过流式细胞术分析表明（ 见图3）。来自15个修改。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54259/54259fig5large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图6" src="/files/ftp_upload/54259/54259fig6.jpg" /> 图6. 小鼠细胞耗竭（MCD）强烈减少错误地映射小鼠总数所有外显子测序（WES）15后读取 。作为用于蛋白质编码序列的靶向富集捕获寡核苷酸是基于人类基因组，初始预先设计人源fr的DNA片段的富集嗡预计小鼠和人类细胞的混合物。为了评估捕获寡核苷酸可能与小鼠基因组DNA交叉杂交的数量，我们进行了对小鼠基因组中的每个单独的快速捕捉外显子组探头的BLAST搜索。所得比对参数来测定可能交叉杂交。 （。对准长度，无错配的，没有间隙的）根据选择的阈值，我们预测的5交叉反应 - 与小鼠转录捕获探针的10％（未显示数据）。适配器削波（trimmomatic v0.32 16）后，我们映射针对人类和小鼠基因组中的所有样本（BWA v0.7.12 17）的读取和确定基于各对准参数的推定的原点（LINUX的外壳，命令行的Perl） （A）。的读取从大块肿瘤样品衍生的12％，平均是由于小鼠细胞。这一数额可以通过鼠标细胞前被消耗降低到0.3％（B </strong&gt;的）。作为上的平均15％的小鼠衍生的读取错误映射到人类基因组中，对应于总的1.9％读取大块肿瘤样品中，并在分离的肿瘤细胞，小鼠细胞耗竭的上下游分析强正影响0.04％可以预期的。 图6B例示了大块肿瘤和从膀胱癌异种移植物衍生的分离的人肿瘤细胞中的详细读取分配。来自15个修改。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54259/54259fig6large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图7" src="/files/ftp_upload/54259/54259fig7.jpg" /> 图7. MCD强降低错误地预测的SNP 15的数目 。为了确定小鼠的影响读取映射到人类基因组，我们确定predicte数之前和MCD d后的SNP的异种移植物样品。因为没有健康组织是可用于比较，SNP被定义为测序样品和参照基因组（hg19）之间的差。删除重复的读取后，SNP和INDEL通话进行了18只限于由外显子组捕获试剂盒指定的区域。所有SNP的63±10％的小鼠细胞耗竭后大块肿瘤样品不再检测到，18±1％的特异性分离的人肿瘤细胞（A）的预测。而前者主要引起误映射鼠标读取后者似乎由于分离的人类肿瘤细胞的样品中更高的读取计数，并相应地更高的覆盖被检测到。这种效应也对预测插入缺失（B）中可见。来自15个修改。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54259/54259fig7large.jpg" target="_blank">请点击此处查看这个大版本</a>数字。 <img alt="图8" src="/files/ftp_upload/54259/54259fig8.jpg" /> 图8. MCD提高影响较大的15个SNPs预测 。（A）MCD的POLA1基因19例示的蛋白编码外显子上的SNP预测的影响。尽管错误地映射到鼠标读取大部分肾癌异种移植引起了一些错误预测的SNP，这些SNP进行了MCD后失踪。此外，MCD还改进高影响力的SNP位点的预测。例如，鼠标读取映射到大块肿瘤样品中的人类基因组参考导致GRIA3基因（B）的起始密码子的错误预测的破坏。来自15个修改。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54259/54259fig8large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> S ="jove_content"FO: - together.within页保留="1"&gt; <img alt="图9" src="/files/ftp_upload/54259/54259fig9.jpg" /> 图9 中 的小鼠衍生的 硅铝 耗竭读取不能完全代替I N体外 MCD 15。该读取预测为小鼠来源的被计算从测序数据中除去，并重复SNP呼叫。通过这种方法，预测的SNPs的数目为大块肿瘤样品从63％的显着减少到SNP的总数的8.5％（与图8相比较）。然而，这在硅片的方法不能完全体外 MCD取代，尤其是如果改进的样品质量和在读计数随之增加和覆盖被考虑。来自15个修改。"_blank"&gt;请点击此处查看该图的放大版本。

Watch this Scientific Journal Video about Depletion of Mouse Cells from Human Tumor Xenografts Significantly Improves Downstream Analysis of Target Cells at JoVE.com

Depletion of Mouse Cells from Human Tumor Xenografts Significantly Improves Downstream Analysis of Target Cells

人类肿瘤异种移植是血管，并在体内生长阶段由鼠源细胞浸润。为了规避下游分析过程中造成这些污染的细胞偏差，我们开发了允许通过磁性细胞分选所有小鼠细胞的全面枯竭的方法。

从人类肿瘤异种移植的小鼠细胞的消耗显着改善靶细胞下游分析

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic ...

depletion-mouse-cells-from-human-tumor-xenografts-significantly

Nanyang Technological University

Research

JoVE Journal

Medicine

12.6K 次观看.  Miltenyi Biotec GmbH. 人类肿瘤异种移植是血管，并在体内生长阶段由鼠源细胞浸润。为了规避下游分析过程中造成这些污染的细胞偏差，我们开发了允许通过磁性细胞分选所有小鼠细胞的全面枯竭的方法。 

视频: Depletion of Mouse Cells from Human Tumor Xenografts Significantly Improves Downstream Analysis of Target Cells

Stereotactic Intracranial Implantation and In vivo Bioluminescent Imaging of Tumor Xenografts in a Mouse Model System of Glioblastoma Multiforme

Glioblastoma multiforme (GBM) is a high-grade primary brain cancer with a median survival of only 14.6 months in humans despite standard tri-modality treatment consisting of surgical resection, post-operative radiation therapy and temozolomide chemotherapy 1. New therapeutic approaches are clearly needed to improve patient survival and quality of life. The development of more effective treatment strategies would be aided by animal models of GBM that recapitulate human disease yet allow serial imaging to monitor tumor growth and treatment response. In this paper, we describe our technique for the precise stereotactic implantation of bio-imageable GBM cancer cells into the brains of nude mice resulting in tumor xenografts that recapitulate key clinical features of GBM 2. This method yields tumors that are reproducible and are located in precise anatomic locations while allowing in vivo bioluminescent imaging to serially monitor intracranial xenograft growth and response to treatments 3-5. This method is also well-tolerated by the animals with low perioperative morbidity and mortality.

Stereotactic Intracranial Implantation and In vivo Bioluminescent Imaging of Tumor Xenografts in a Mouse Model System of Glioblastoma Multiforme

We describe an integrated method for the precise, stereotactic implantation of human glioblastoma multiforme cells into the brains of nude mice and subsequent serial in vivo imaging to monitor growth and response to treatment of the resultant xenografts.

立体定向颅内植入和<em在体内</em生物发光成像的肿瘤异种移植的小鼠模型系统的多形性胶质母细胞瘤

Glioblastoma multiforme (GBM) is a high-grade primary brain cancer with a median survival of only 14.6 months in humans despite standard tri-modality ...

科研

医学

Isolation, Characterization and Comparative Differentiation of Human Dental Pulp Stem Cells Derived from Permanent Teeth by Using Two Different Methods

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human pulp-derived stem cells (hDPSCs) possess high proliferation potential with multi-lineage differentiation capacity compare to the ordinary source of adult stem cells1-8; therefore, hDPSCs could be the good candidates for autologous transplantation in tissue engineering and regenerative medicine. Along with these benefits, possessing the mesenchymal stem cells (MSC) features, such as immunolodulatory effect, make hDPSCs more valuable, even in the case of allograft transplantation6,9,10. Therefore, the primary step for using this source of stem cells is to select the best protocol for isolating hDPSCs from pulp tissue. In order to achieve this goal, it is crucial to investigate the effect of various isolation conditions on different cellular behaviors, such as their common surface markers &amp; also their differentiation capacity.
Thus, here we separate human pulp tissue from impacted third molar teeth, and then used both existing protocols based on literature, for isolating hDPSCs,11-13 i.e. enzymatic dissociation of pulp tissue (DPSC-ED) or outgrowth from tissue explants (DPSC-OG). In this regards, we tried to facilitate the isolation methods by using dental diamond disk. Then, these cells characterized in terms of stromal-associated Markers (CD73, CD90, CD105 &amp; CD44), hematopoietic/endothelial Markers (CD34, CD45 &amp; CD11b), perivascular marker, like CD146 and also STRO-1. Afterwards, these two protocols were compared based on the differentiation potency into odontoblasts by both quantitative polymerase chain reaction (QPCR) &amp; Alizarin Red Staining. QPCR were used for the assessment of the expression of the mineralization-related genes (alkaline phosphatase; ALP, matrix extracellular phosphoglycoprotein; MEPE &amp; dentin sialophosphoprotein; DSPP).14

The method described isolation and characterization of human Dental Pulp Stem Cells (hDPSCs) by using either enzymatic dissociation of pulp (DPSC-ED) or direct outgrowth of stem cells from pulp tissue explants (DPSC-OG). Then followed by in vitro comparative differentiation of both types of hDPSCs into odontoblasts.

人牙髓干细胞的分离，鉴定和比较性恒牙使用两种不同的方法，得出

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human ...

Molecular Imaging to Target Transplanted Muscle Progenitor Cells

Duchenne muscular dystrophy (DMD) is a severe genetic neuromuscular disorder that affects 1 in 3,500 boys, and is characterized by progressive muscle degeneration1, 2. In patients, the ability of resident muscle satellite cells (SCs) to regenerate damaged myofibers becomes increasingly inefficient4. Therefore, transplantation of muscle progenitor cells (MPCs)/myoblasts from healthy subjects is a promising therapeutic approach to DMD. A major limitation to the use of stem cell therapy, however, is a lack of reliable imaging technologies for long-term monitoring of implanted cells, and for evaluating its effectiveness. Here, we describe a non-invasive, real-time approach to evaluate the success of myoblast transplantation. This method takes advantage of a unified fusion reporter gene composed of genes (firefly luciferase [fluc], monomeric red fluorescent protein [mrfp] and sr39 thymidine kinase [sr39tk]) whose expression can be imaged with different imaging modalities9, 10. A variety of imaging modalities, including positron emission tomography (PET), single-photon emission computed tomography (SPECT), magnetic resonance imaging (MRI), optical imaging, and high frequency 3D-ultrasound are now available, each with unique advantages and limitations11. Bioluminescence imaging (BLI) studies, for example, have the advantage of being relatively low cost and high-throughput. It is for this reason that, in this study, we make use of the firefly luciferase (fluc) reporter gene sequence contained within the fusion gene and bioluminescence imaging (BLI) for the short-term localization of viable C2C12 myoblasts following implantation into a mouse model of DMD (muscular dystrophy on the X chromosome [mdx] mouse)12-14. Importantly, BLI provides us with a means to examine the kinetics of labeled MPCs post-implantation, and will be useful to track cells repeatedly over time and following migration. Our reporter gene approach further allows us to merge multiple imaging modalities in a single living subject; given the tomographic nature, fine spatial resolution and ability to scale up to larger animals and humans10,11, PET will form the basis of future work that we suggest may facilitate rapid translation of methods developed in cells to preclinical models and to clinical applications.

A non-invasive means to evaluate the success of myoblast transplantation is described. The method takes advantage of a unified fusion reporter gene composed of genes whose expression can be imaged with different imaging modalities. Here, we make use of a fluc reporter gene sequence to target cells via bioluminescence imaging.

目标移植的肌肉祖细胞的分子成像

Duchenne muscular dystrophy (DMD) is a severe genetic neuromuscular disorder that affects 1 in 3,500 boys, and is characterized by progressive muscle ...

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for progression-free and overall survival. Therefore, translational research needs to provide further molecular evidence to improve targeted therapies for malignant melanomas. In the past, oncogenic mechanisms related to melanoma were extensively studied in established cell lines. On the way to more personalized treatment regimens based on individual genetic profiles, we propose to use patient-derived cell lines instead of generic cell lines. Together with high quality clinical data, especially on patient follow-up, these cells will be instrumental to better understand the molecular mechanisms behind melanoma progression.
Here, we report the establishment of primary melanoma cultures from dissected fresh tumor tissue. This procedure includes mincing and dissociation of the tissue into single cells, removal of contaminations with erythrocytes and fibroblasts as well as primary culture and reliable verification of the cells' melanoma origin.
Recent reports revealed that melanomas, like the majority of tumors, harbor a small subpopulation of cancer stem cells (CSCs), which seem to exclusively fuel tumor initiation and progression towards the metastatic state. One of the key markers for CSC identification and isolation in melanoma is CD133. To isolate CD133+ CSCs from primary melanoma cultures, we have modified and optimized the Magnetic-Activated Cell Sorting (MACS) procedure from Miltenyi resulting in high sorting purity and viability of CD133+ CSCs and CD133- bulk, which can be cultivated and functionally analyzed thereafter.

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

This article describes the preparation of freshly obtained melanoma tissue into primary cell cultures, and how to remove contaminations of erythrocytes and fibroblasts from the tumor cells. Finally, we describe how CD133+ putative melanoma stem cells are sorted from the CD133- bulk using Magnetic Activated Cell Sorting (MACS).

来自患者的细胞培养和分离的CD133<sup&gt; +</sup&gt;假定的肿瘤干细胞的恶性黑色素瘤

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for ...

Isolation of Cancer Stem Cells From Human Prostate Cancer Samples

The cancer stem cell (CSC) model has been considerably revisited over the last two decades. During this time CSCs have been identified and directly isolated from human tissues and serially propagated in immunodeficient mice, typically through antibody labeling of subpopulations of cells and fractionation by flow cytometry. However, the unique clinical features of prostate cancer have considerably limited the study of prostate CSCs from fresh human tumor samples. We recently reported the isolation of prostate CSCs directly from human tissues by virtue of their HLA class I (HLAI)-negative phenotype. Prostate cancer cells are harvested from surgical specimens and mechanically dissociated. A cell suspension is generated and labeled with fluorescently conjugated HLAI and stromal antibodies. Subpopulations of HLAI-negative cells are finally isolated using a flow cytometer. The principal limitation of this protocol is the frequently microscopic and multifocal nature of primary cancer in prostatectomy specimens. Nonetheless, isolated live prostate CSCs are suitable for molecular characterization and functional validation by transplantation in immunodeficient mice.

The isolation of cancer stem cells (CSCs) directly from human tissues is requisite for their biological characterization. This manuscript describes a methodology for the isolation of prostate CSCs from human tissues, while also providing tips on troubleshooting difficult steps.

癌症干细胞从人类前列腺癌样本隔离

The cancer stem cell (CSC) model has been considerably revisited over the last two decades. During this time CSCs have been identified and directly ...

In vitro Method to Observe E-selectin-mediated Interactions Between Prostate Circulating Tumor Cells Derived From Patients and Human Endothelial Cells

Metastasis is a process in which tumor cells shed from the primary tumor intravasate blood vascular and lymphatic system, thereby, gaining access to extravasate and form a secondary niche.&#160;The extravasation of tumor cells from the blood vascular system can be studied using endothelial cells (ECs) and tumor cells obtained from different cell lines.&#160;Initial studies were conducted using static conditions but it has been well documented that ECs behave differently under physiological flow conditions.&#160;Therefore, different flow chamber assemblies are currently being used to studying cancer cell interactions with ECs.&#160;Current flow chamber assemblies offer reproducible results using either different cell lines or fluid at different shear stress conditions.&#160;However, to observe and study interactions with rare cells such as circulating tumor cells (CTCs), certain changes are required to be made to the conventional flow chamber assembly.&#160;CTCs are a rare cell population among millions of blood cells. Consequently, it is difficult to obtain a pure population of CTCs.&#160;Contamination of CTCs with different types of cells normally found in the circulation is inevitable using present enrichment or depletion techniques.&#160;In the present report, we describe a unique method to fluorescently label circulating prostate cancer cells and study their interactions with ECs in a self-assembled flow chamber system.&#160;This technique can be further applied to observe interactions between prostate CTCs and any protein of interest.

In vitro Method to Observe E-selectin-mediated Interactions Between Prostate Circulating Tumor Cells Derived From Patients and Human Endothelial Cells

Our report describes a unique method to visualize and analyze CTC/EC interactions in prostate cancer under physiological flow conditions.

在体外的方法来观察派生病人和人内皮细胞前列腺循环肿瘤细胞之间的E-选择素介导的相互作用

Metastasis is a process in which tumor cells shed from the primary tumor intravasate blood vascular and lymphatic system, thereby, gaining access to ...

Creating Anatomically Accurate and Reproducible Intracranial Xenografts of Human Brain Tumors

Orthotopic tumor models are currently the best way to study the characteristics of a tumor type, with and without intervention, in the context of a live animal &#8211;&#160;particularly in sites with unique physiological and architectural qualities such as the brain. In vitro and ectopic models cannot account for features such as vasculature, blood brain barrier, metabolism, drug delivery and toxicity, and a host of other relevant factors. Orthotopic models have their limitations too, but with proper technique tumor cells of interest can be accurately engrafted into tissue that most closely mimics conditions in the human brain. By employing methods that deliver precisely measured volumes to accurately defined locations at a consistent rate and pressure, mouse models of human brain tumors with predictable growth rates can be reproducibly created and are suitable for reliable analysis of various interventions. The protocol described here focuses on the technical details of designing and preparing for an intracranial injection, performing the surgery, and ensuring successful and reproducible tumor growth and provides starting points for a variety of conditions that can be customized for a range of different brain tumor models.

The brain is a unique site with qualities that are not well represented by in vitro or ectopic analyses. Orthotopic mouse models with reproducible location and growth characteristics can be reliably created with intracranial injections using a stereotaxic fixation instrument and a low pressure syringe pump.

创建精确的解剖学和人脑肿瘤的重现性颅内移植瘤

Orthotopic tumor models are currently the best way to study the characteristics of a tumor type, with and without intervention, in the context of a live ...

Studying Pancreatic Cancer Stem Cell Characteristics for Developing New Treatment Strategies

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor initiation, metastasis and resistance to radio- and chemotherapy. Here we describe a specific methodology for culturing primary human pancreatic CSCs as tumor spheres in anchorage-independent conditions. Cells are grown in serum-free, non-adherent conditions in order to enrich for CSCs while their more differentiated progenies do not survive and proliferate during the initial phase following seeding of single cells. This assay can be used to estimate the percentage of CSCs present in a population of tumor cells. Both size (which can range from 35 to 250 micrometers) and number of tumor spheres formed represents CSC activity harbored in either bulk populations of cultured cancer cells or freshly harvested and digested tumors 1,2. Using this assay, we recently found that metformin selectively ablates pancreatic CSCs; a finding that was subsequently further corroborated by demonstrating diminished expression of pluripotency-associated genes/surface markers and reduced in vivo tumorigenicity of metformin-treated cells. As the final step for preclinical development we treated mice bearing established tumors with metformin and found significantly prolonged survival. Clinical studies testing the use of metformin in patients with PDAC are currently underway (e.g., NCT01210911, NCT01167738, and NCT01488552). Mechanistically, we found that metformin induces a fatal energy crisis in CSCs by enhancing reactive oxygen species (ROS) production and reducing mitochondrial transmembrane potential. In contrast, non-CSCs were not eliminated by metformin treatment, but rather underwent reversible cell cycle arrest. Therefore, our study serves as a successful example for the potential of in vitro sphere formation as a screening tool to identify compounds that potentially target CSCs, but this technique will require further in vitro and in vivo validation to eliminate false discoveries.

Pancreatic cancer stem cells (CSCs) can be expanded in vitro using the anchorage-independent sphere culture technique, which represents a powerful tool to study CSC biology and can serve as the first step to develop novel CSC-targeting therapies. Here the methodology for expanding, analyzing and targeting of pancreatic CSCs is provided.

研究胰腺癌干细胞特性开发新的治疗策略

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor ...

Isolation and Propagation of Circulating Tumor Cells from a Mouse Cancer Model

Cancer metastasis is the foremost cause of cancer-associated deaths. Recent studies have shown that circulating tumor cells (CTCs) are important in cancer metastasis. Indeed, the number of CTCs correlates with tumor size. Here, a detailed description is provided of a methodology for isolation and propagation of CTCs from a syngeneic mouse model of hepatocellular carcinoma (HCC) which allows for downstream analysis of potentially important molecular mechanisms of solid organ tumor metastasis. This method is efficient and reproducible. It is a non-invasive technique and, therefore, has potential to replace the invasive biopsy of tissues from humans which may be associated with complications. Therefore, the method discussed here allows for the isolation and propagation of CTCs from whole blood samples such that they can be examined and characterized. This has potential for future adaptation for clinical applications such as diagnosis, and personalized targeted therapy.

Circulating tumor cells (CTCs) have been shown to play an important role in tumor metastasis. Here, a method for the isolation and propagation of CTCs from the whole blood of a syngeneic mouse tumor model of hepatocellular carcinoma (HCC) metastasis is described.

从小鼠癌症模型的循环肿瘤细胞的分离和繁殖

Cancer metastasis is the foremost cause of cancer-associated deaths. Recent studies have shown that circulating tumor cells (CTCs) are important in cancer ...

Generation of Prostate Cancer Patient Derived Xenograft Models from Circulating Tumor Cells

Patient derived xenograft (PDX) models are gaining popularity in cancer research and are used for preclinical drug evaluation, biomarker identification, biologic studies, and personalized medicine strategies. Circulating tumor cells (CTC) play a critical role in tumor metastasis and have been isolated from patients with several tumor types. Recently, CTCs have been used to generate PDX experimental models of breast and prostate cancer. This manuscript details the method for the generation of prostate cancer PDX models from CTCs developed by our group. Advantages of this method over conventional PDX models include independence from surgical sample collection and generating experimental models at various disease stages. Density gradient centrifugation followed by red blood cell lysis and flow cytometry depletion of CD45 positive mononuclear cells is used to enrich CTCs from peripheral blood samples collected from patients with metastatic disease. The CTCs are then injected into immunocompromised mice; subsequently generated xenografts can be used for functional studies or harvested for molecular characterization. The primary limitation of this method is the negative selection method used for CTC enrichment. Despite this limitation, the generation of PDX models from CTCs provides a novel experimental model to be applied to prostate cancer research.

This manuscript details a method used to generate prostate cancer patient derived xenografts (PDXs) from circulating tumor cells (CTCs). The generation of PDX models from CTCs provides an alternative experimental model to study prostate cancer; the most commonly diagnosed tumor and a frequent cause of death from cancer in men.

前列腺癌患者产生派生异种移植模型从循环肿瘤细胞

Patient derived xenograft (PDX) models are gaining popularity in cancer research and are used for preclinical drug evaluation, biomarker identification, ...