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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<li>Agorku, D., et al. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aAgorku%2C+D+%22Next-generation+sequencing+of+human+tumor+xenografts+is+significantly+improved+by+prior+depletion+of+mouse+cells.+Abstract+1455%22">Next-generation sequencing of human tumor xenografts is significantly improved by prior depletion of mouse cells. Abstract 1455</a>. , AACR. (2015).</li>
<li>Bolger, A. M., Lohse, M., Usadel, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Trimmomatic%3A+a+flexible+trimmer+for+Illumina+sequence+data%5BTitle%5D)+AND+%22Bioinformatics%22%5BJournal%5D)">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/pubmed?term=((Fast+and+accurate+short+read+alignment+with+Burrows-Wheeler+transform%5BTitle%5D)+AND+%22Bioinformatics%22%5BJournal%5D)">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/pubmed?term=((Using+VarScan+2+for+Germline+Variant+Calling+and+Somatic+Mutation+Detection%5BTitle%5D)+AND+%22Curr+Protoc+Bioinformatics%22%5BJournal%5D)">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/pubmed?term=((Integrative+genomics+viewer%5BTitle%5D)+AND+%22Nat+Biotechnol%22%5BJournal%5D)">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/pubmed?term=((The+sialyl-glycolipid+stage-specific+embryonic+antigen+4+marks+a+subpopulation+of+chemotherapy-resistant+breast+cancer+cells+with+mesenchymal+features%5BTitle%5D)+AND+%22Breast+Cancer+Res%22%5BJournal%5D)">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>
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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><tbody><tr><td>Tumor Dissociation Kit, human</td><td>Miltenyi Biotec</td><td>130-095-929</td><td>contains enzymes H, R and A; see data sheet for reconstitution instructions</td></tr><tr><td>RPMI 1640</td><td>Biowest</td><td>L0501-500</td><td></td></tr><tr><td>gentleMACS C-Tubes</td><td>Miltenyi Biotec</td><td>130-096-334</td><td></td></tr><tr><td>gentleMACS Octo Dissociator with Heaters</td><td>Miltenyi Biotec</td><td>130-096-427</td><td></td></tr><tr><td>MACS SmartStrainer (70 &amp;micro;m)</td><td>Miltenyi Biotec</td><td>130-098-462</td><td></td></tr><tr><td>Petri dish 100 mm</td><td>Various</td><td></td><td></td></tr><tr><td>Scalpel</td><td>Various</td><td></td><td></td></tr><tr><td>Mouse Cell Depletion Kit</td><td>Miltenyi Biotec</td><td>130-104-694</td><td></td></tr><tr><td>PBS</td><td>Various</td><td></td><td>use 1x PBS for PEB buffer</td></tr><tr><td>EDTA</td><td>Sigma-Aldrich</td><td>3610</td><td>use final concentration of 2 mM in PEB buffer</td></tr><tr><td>BSA</td><td>Miltenyi Biotec</td><td>130-091-376</td><td>or use 5 mg/L BSA in PEB buffer</td></tr><tr><td>MACS LS columns</td><td>Miltenyi Biotec</td><td>130-042-401</td><td></td></tr><tr><td>QuadroMACS Separator</td><td>Miltenyi Biotec</td><td>130-090-976</td><td></td></tr><tr><td>MACS MultiStand</td><td>Miltenyi Biotec</td><td>130-042-303</td><td></td></tr><tr><td>PreSep filters (70 &amp;micro;m)</td><td>Miltenyi Biotec</td><td>130-095-823</td><td></td></tr><tr><td>MACSmix Tube Rotator</td><td>Miltenyi Biotec</td><td>130-090-753</td><td></td></tr><tr><td>CD326-FITC, human</td><td>Miltenyi Biotec</td><td>130-080-301</td><td>use 1:40 for immunofluorescence staining&amp;nbsp;</td></tr><tr><td>anti-Vimentin antibody</td><td>abcam</td><td>ab92547</td><td></td></tr><tr><td>goat-anti-mouse IgG-Alexa488</td><td>Life Technologies</td><td>A11029</td><td></td></tr><tr><td>goat-anti-rabbit IgG-Alexa594</td><td>Life Technologies</td><td>A11012</td><td></td></tr><tr><td>DAPI</td><td>Sigma-Aldrich</td><td>D9542</td><td>dilute to a final concentration of 0.1 &amp;micro;g/ml in 0.1 M sodium bicarbonate&amp;nbsp;</td></tr><tr><td>Inside Stain Kit</td><td>Miltenyi Biotec</td><td>130-090-477</td><td>InsideFix from this kit can be used for fixation step during immunofluorescence staining&amp;nbsp;</td></tr><tr><td>FBS</td><td>Various</td><td></td><td></td></tr><tr><td>Triton X-100&amp;nbsp;</td><td>Sigma-Aldrich</td><td>93418</td><td></td></tr><tr><td>FcR Blocking Reagent, mouse</td><td>Miltenyi Biotec</td><td>130-092-575</td><td></td></tr><tr><td>Gelatin</td><td>Sigma-Aldrich</td><td>G2500-100g</td><td></td></tr><tr><td>Nextera Rapid Capture Enrichment Kit</td><td>Illumina</td><td>FC-140-1000</td><td></td></tr><tr><td>MiSeq Reagent Kit v3</td><td>Illumina</td><td>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/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/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/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/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/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/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/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

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

Development and Maintenance of a Preclinical Patient Derived Tumor Xenograft Model for the Investigation of Novel Anti-Cancer Therapies

Patient derived tumor xenograft (PDTX) models provide a necessary platform in facilitating anti-cancer drug development prior to human trials. Human tumor pieces are injected subcutaneously into athymic nude mice (immunocompromised, T cell deficient) to create a bank of tumors and subsequently are passaged into different generations of mice in order to maintain these tumors from patients. Importantly, cellular heterogeneity of the original tumor is closely emulated in this model, which provides a more clinically relevant model for evaluation of drug efficacy studies (single agent and combination), biomarker analysis, resistant pathways and cancer stem cell biology. Some limitations of the PDTX model include the replacement of the human stroma with mouse stroma after the first generation in mice, inability to investigate treatment effects on metastasis due to the subcutaneous injections of the tumors, and the lack of evaluation of immunotherapies due to the use of immunocompromised mice. However, even with these limitations, the PDTX model provides a powerful preclinical platform in the drug discovery process.

Utilizing patient-derived tumors in a subcutaneous preclinical model is an excellent way to study the efficacy of novel therapies, predictive biomarker discovery, and drug resistant pathways. This model, in the drug development process, is essential in determining the fate of many novel anti-cancer therapies prior to clinical investigation.

开发和维护的临床前患者来源的肿瘤异种移植模型为新的抗癌疗法的调查

Patient derived tumor xenograft (PDTX) models provide a necessary platform in facilitating anti-cancer drug development prior to human trials. Human tumor ...

科研

癌症研究

Tumor Engraftment in a Xenograft Mouse Model of Human Mantle Cell Lymphoma

B lymphocytes are key players in immune cell circulation and they mainly home to and reside in lymphoid organs. While normal B cells only proliferate when stimulated by T lymphocytes, oncogenic B cells survive and expand autonomously in undefined organ niches. Mantle cell lymphoma (MCL) is one such B cell disorder, where the median survival rate of patients is 4 - 5 years. This calls for the need of effective mechanisms by which the homing and engraftment of these cells are blocked in order to increase the survival and longevity of patients. Therefore, the effort to develop a xenograft mouse model to study the efficacy of MCL therapeutics by blocking the homing mechanism in vivo is of utmost importance. Development of animal recipients for human cell xenotransplantation to test early stage drugs have long been pursued, as relevant preclinical mouse models are crucial to screen new therapeutic agents. This animal model is developed to avoid human graft rejection and to establish a model for human diseases, and it may be an extremely useful tool to study disease progression of different lymphoma types and to perform preclinical testing of candidate drugs for hematologic malignancies, like MCL. We established a xenograft mouse model that will serve as an excellent resource to study and develop novel therapeutic approaches for MCL.

Mantle cell lymphoma (MCL) is a difficult to treat B cell disorder and it is equally difficult to establish a xenograft mouse model of primary MCL to study and develop therapeutics. Here, we describe the successful establishment of MCL xenografts in mice to help understand its underlying biology.

人地幔细胞淋巴瘤移植小鼠模型中肿瘤植入的研究

B lymphocytes are key players in immune cell circulation and they mainly home to and reside in lymphoid organs. While normal B cells only proliferate when ...

A Human Peripheral Blood Mononuclear Cell (PBMC) Engrafted Humanized Xenograft Model for Translational Immuno-oncology (I-O) Research

The discovery and development of immuno-oncology (I-O) therapy in recent years represents a milestone in the treatment of cancer. However, treatment challenges persist. Robust and disease-relevant animal models are vital resources for continued preclinical research and development in order to address a range of additional immune checkpoints. Here, we describe a human peripheral blood mononuclear cell (PBMC) &#8212; based humanized xenograft model. BGB-A317 (Tislelizumab), an investigational humanized anti-PD-1 antibody in late-stage clinical development, is used as an example to discuss platform set-up, model characterization and drug efficacy evaluations. These humanized mice support the growth of most human tumors tested, thus allowing the assessment of I-O therapies in the context of both human immunity and human cancers. Once established, our model is comparatively time- and cost-effective, and usually yield highly reproducible results. We suggest that the protocol outlined in this article could serve as a general guideline for establishing mouse models reconstituted with human PBMC and tumors for I-O research.

A Human Peripheral Blood Mononuclear Cell PBMC Engrafted Humanized Xenograft Model for Translational Immuno-oncology I-O Research

We describe a human peripheral blood mononuclear cell (PBMC) &#8212; based humanized xenograft mouse model for translational immuno-oncology research. This protocol could serve as a general guideline for establishing and characterizing similar models for I-O therapy assessment.

用于转化免疫肿瘤学(I-O)研究的人类外周血单核细胞(PBMC)的人工化异种移植模型

The discovery and development of immuno-oncology (I-O) therapy in recent years represents a milestone in the treatment of cancer. However, treatment ...

Generation of Subcutaneous and Intrahepatic Human Hepatocellular Carcinoma Xenografts in Immunodeficient Mice

In vivo experimental models of hepatocellular carcinoma (HCC) that recapitulate the human disease provide a valuable platform for research into disease pathophysiology and for the preclinical evaluation of novel therapies. We present a variety of methods to generate subcutaneous or orthotopic human HCC xenografts in immunodeficient mice that could be utilized in a variety of research applications. With a focus on the use of primary tumor tissue from patients undergoing surgical resection as a starting point, we describe the preparation of cell suspensions or tumor fragments for xenografting. We describe specific techniques to xenograft these tissues i) subcutaneously; or ii) intrahepatically, either by direct implantation of tumor cells or fragments into the liver, or indirectly by injection of cells into the mouse spleen. We also describe the use of partial resection of the native mouse liver at the time of xenografting as a strategy to induce a state of active liver regeneration in the recipient mouse that may facilitate the intrahepatic engraftment of primary human tumor cells. The expected results of these techniques are illustrated. The protocols described have been validated using primary human HCC samples and xenografts, which typically perform less robustly than the well-established human HCC cell lines that are widely used and frequently cited in the literature. In comparison with cell lines, we discuss factors which may contribute to the relatively low chance of primary HCC engraftment in xenotransplantation models and comment on technical issues that may influence the kinetics of xenograft growth. We also suggest methods that should be applied to ensure that xenografts obtained accurately resemble parent HCC tissues.

Human tumor xenografts in immunodeficient mice are valuable tools to study cancer biology. Specific protocols to generate subcutaneous and intrahepatic xenografts from human hepatocellular carcinoma cells or tumor fragments are described. Liver regeneration induced by partial hepatectomy in recipient mice is presented as a strategy to facilitate intrahepatic engraftment.

皮下及肝内人肝癌裸鼠移植瘤的免疫缺陷小鼠的产生

In  vivo experimental models of hepatocellular carcinoma (HCC) that recapitulate  the human disease provide a valuable platform for research into disease  ...

医学

An Orthotopic Murine Model of Human Prostate Cancer Metastasis

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

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

人类前列腺癌转移的原位小鼠模型

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

Testing Cancer Immunotherapeutics in a Humanized Mouse Model Bearing Human Tumors

Reversing the immunosuppressive nature of the tumor microenvironment is critical for the successful treatment of cancers with immunotherapy drugs. Murine cancer models are extremely limited in their diversity and suffer from poor translation to the clinic. To serve as a more physiological preclinical model for immunotherapy studies, this protocol has been developed to evaluate the treatment of human tumors in a mouse reconstituted with a human immune system. This unique protocol demonstrates the development of human immune system (HIS, &#34;humanized&#34;) mice, followed by implantation of a human tumor, either a cell-line derived xenograft (CDX) or a patient derived xenograft (PDX). HIS mice are generated by injecting CD34+ human hematopoietic stem cells isolated from umbilical cord blood into neonatal BRGS (BALB/c Rag2-/- IL2R&#947;C-/- NODSIRP&#945;) highly immunodeficient mice that are also capable of accepting a xenogeneic tumor. The importance of the kinetics and characteristics of the human immune system development and tumor implantation is emphasized. Finally, an in-depth evaluation of the tumor microenvironment using flow cytometry is described. In numerous studies using this protocol, it was found that the tumor microenvironment of individual tumors is recapitulated in HIS-PDX mice; &#34;hot&#34; tumors exhibit large immune infiltration while &#34;cold&#34; tumors do not. This model serves as a testing ground for combination immunotherapies for a wide range of human tumors and represents an important tool in the quest for personalized medicine.

This protocol outlines the generation of human immune system (HIS) mice for immuno-oncology studies. Instructions and considerations in the use of this model for testing human immunotherapeutics on human tumors implanted in this model are presented with an emphasis on characterizing the response of the human immune system to the tumor.

在携带人类肿瘤的人源化小鼠模型中测试癌症免疫疗法

Reversing the immunosuppressive nature of the tumor microenvironment is critical for the successful treatment of cancers with immunotherapy drugs. Murine ...

Isolation and Characterization of Neutrophils with Anti-Tumor Properties

Neutrophils, the most abundant of all white blood cells in the human circulation, play an important role in the host defense against invading microorganisms. In addition, neutrophils play a central role in the immune surveillance of tumor cells. They have the ability to recognize tumor cells and induce tumor cell death either through a cell contact-dependent mechanism involving hydrogen peroxide or through antibody-dependent cell-mediated cytotoxicity (ADCC). Neutrophils with anti-tumor activity can be isolated from peripheral blood of cancer patients and of tumor-bearing mice. These neutrophils are termed tumor-entrained neutrophils (TEN) to distinguish them from neutrophils of healthy subjects or na&#239;ve mice that show no significant tumor cytotoxic activity. Compared with other white blood cells, neutrophils show different buoyancy making it feasible to obtain a &#62; 98% pure neutrophil population when subjected to a density gradient. However, in addition to the normal high-density neutrophil population (HDN), in cancer patients, in tumor-bearing mice, as well as under chronic inflammatory conditions, distinct low-density neutrophil populations (LDN) appear in the circulation. LDN co-purify with the mononuclear fraction and can be separated from mononuclear cells using either positive or negative selection strategies. Once the purity of the isolated neutrophils is determined by flow cytometry, they can be used for in vitro and in vivo functional assays. We describe techniques for monitoring the anti-tumor activity of neutrophils, their ability to migrate and to produce reactive oxygen species, as well as monitoring their phagocytic capacity ex vivo. We further describe techniques to label the neutrophils for in vivo tracking, and to determine their anti-metastatic capacity in vivo. All these techniques are essential for understanding how to obtain and characterize neutrophils with anti-tumor function.

Neutrophils play an important role not only in host defense against invading microorganisms, but are also involved in the immune surveillance of tumor cells. Here, we describe techniques related to the isolation of neutrophils with anti-tumor properties and methods for monitoring anti-tumor neutrophil function in vitro and in vivo.

隔离和中性粒细胞的表征与抗肿瘤性质

Neutrophils, the most abundant of all white blood cells in the human circulation, play an important role in the host defense against invading ...

免疫与感染

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 ...

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, ...

Mouse Models of Cancer Study

Mice have long served as models for studying human biology and pathology because of their phylogenetic and physiological similarity with humans. They are also easy to maintain and breed in the laboratory, and hence, many inbred strains are now available for research. Studies on mice have contributed immeasurably to our understanding of cancer biology.
The development of transgenic, knockout, and knock-in mice has led to an exponential increase in their use as model organisms in research, including cancer research. A gene-of-interest can be added or deleted from a mouse&#8217;s genome using genetic engineering techniques, and if it causes tumor initiation in mice, it is highly likely to be a cancer-critical gene in humans. In addition, mouse models such as reporter mice can also be used to study the crucial stages of tumor initiation and their progression to aggressive cancers. In such mice, a reporter gene with luminescence or fluorescence tags is inserted into the cells under study to monitor their growth and proliferation.
Pre-clinical studies in mouse models serve as a perfect step between in vitro studies on cell cultures and clinical studies in humans. They can be used for investigating in vivo pharmacokinetics, toxicity, and anti-tumor efficacy of numerous chemotherapeutic agents before any of the drugs go to clinical trials. However, since mouse tumor cells or immune responses may not exactly represent the tumorigenic process in humans, mouse models have been developed that can closely mimic the tumor progression and immune responses in humans, for example, human tumor xenografts in immunocompromised mice or humanized mouse models.
In vivo testing in animals, including mice, can often raise ethical concerns. While it is true that experimentation in animals causes suffering, the benefits of animal experimentation in drug discovery are undeniable. Therefore, animal studies should be strictly avoided wherever alternative testing methods are available. In addition, experiments on animals should only be conducted after in vitro studies have been successfully performed on suitable cell lines.

Mouse Models of Cancer and Pre-clinical Testing of Cancer Drugs

癌症小鼠模型研究

Mice have long served as models for studying human biology and pathology because of their phylogenetic and physiological similarity with humans. They are ...