这项工作是由约翰·G·玛丽和肯尼迪斯特拉纪念基金（批准＃0727033）和黑色素瘤研究联盟的慈善捐款支助。作者要感谢得克萨斯大学MD安德森癌症中心的机构组织库（ITB）的大学协调组织分布和收集，促进转化研究，以及伊格纳西奥Wistuba博士维克多·普列托博士的MD安德森癌症中心部病理学和黑色素瘤登月计划。最后，作者希望承认受黑色素瘤的患者和家庭。

伦理声明:本协议经得克萨斯大学MD安德森癌症中心的大学。 1.样品采购<ol><li>集研究组织样本的剩余肿瘤及相关正常组织10-12。在湿冰储存标本，以避免自溶。 </li><li>发送所有的组织中，医疗器械和外科手术来病理学系为必要的检查过程中被除去的异物，以促进一个病理诊断。 </li><li>处理时要遵守适用的研究和组织银行的政策和程序分配的研究组织。 注:生物样本收集涉及病理学家的协调，病理学家"助理，histotechnologists和组织采购专家。 </li></ol> 2.病理质量保证（QA） <ol><li>完成并验证病历作为早期的质量保证措施，并咨询病理学家审查诊断PAtient记录（病理报告，组织切片等）和组织样品。判断组织样品上的感兴趣的形态特征，例如肿瘤的百分比，坏死，组织肿瘤界面等，这是按照biorepository最佳做法10-12 </li><li>在初步审查，进行biorepository数据和研究样本进行定期审查逗留生物样本的整个长度中。 </li><li>从组织病理学和其他调查的样本链接结果和说明，并通过一个专有biorepository软件系统提供该数据给研究者。 注:QA流程确保双方的临床和技术biorepository操作的各方面的完整性。 </li><li>执行的遗传和分子测试以验证和表征样品供临床使用。 </li><li>收集和分析的基础上反馈的研究人员一直在使用的样本进行实验室实验，谁的数据。跟踪采用条形码技术的样品。这种电子跟踪系统也主张在biorespository最佳实践10-12。 </li></ol> 3.标本/组织处理<ol><li>总是存储新鲜组织标本在湿冰上，并且过程尽可能快，以便限制致萃取自其天然环境中的组织样本的变化。流程正常肿瘤匹配和肿瘤组织标本分别以避免交叉污染。 注意:基于研究方案，生物试样的处理可以涉及收集试样中的一种或多种类型的媒体。 </li><li>在开始的过程中，制备无菌的陪替氏培养皿中，手术刀，以及针或镊子用于组织处理。确保管已经制备，标记，并以限制样本处理（图2）的时间敷设。 </li><li>所有相关的组织的信息，例如患者姓名，号码，治疗，日手术，因为可能适用于研究其他相关信息，应在机构组织的数据库容易接近。有限的患者信息应以书面或电子表格的形式实验室举行。 注意:只收集相关的研究即性质生物样本信息，基础生物医学研究，临床研究，转化研究等。对于疾病的具体信息，该biorepository人员应收集必要由当前文献资料和管理组织确保正确的诊断和有意义的研究。这包括但不限于肿瘤等级和癌症分段信息。 </li><li>打算所有期望的后续实验预先决定的需要为每个类型的分析，以及切除的位置的组织切片的尺寸。理想的情况下，虽然不可能的，所有的分析应代表整个组织切片以占组织heterogeneiTY和下游分析的极限偏差。铺陈所有必需项目提前，如图2中所描绘 。 </li><li>将组织标本在开放的Petri培养皿，平贴在表面上。 注:仅允许授权biorepository人员处理鉴定样本，并审查确定的健康数据。调查人员应该只访问所允许的签署知情同意书，并作为监管的IRB的患者标识。所有标本必须充分标记的，并应包括在最低限度，以独特的组织的识别号码和组织类型。不包括任何的PHI在标签中，按照HIPAA法规。 </li><li>从各个角度检查样本，以更好地了解它的结构和尺寸。引脚的样品与针盘的底部，并继续沿平分其最大和最佳代表轴的组织。 </li><li>切出一小块组织，并将其放置在无菌1.5毫升MICRocentrifuge管含有RNA稳定溶液1ml。该样品可以被放置在冰上的过程的其余部分，并储存在4℃下直到需要。 注意:当用RNA工作，手套应该总是被磨损，和无RNA酶的过滤嘴应当用于限制降解和RNA污染的机会。 </li><li>布置在陪替氏培养皿的盖子开放与预先标记的活检盒。切出组织的切片不大于3较厚，以5毫米，并将其转移到盒。放置在10％的盒中性缓冲的福尔马林（NBF）最少72小时的。石蜡包埋之前转移在70％乙醇盒用于长期贮存。 注意:标记盒的应用铅笔或与推荐标记作使用二甲苯的处理过程中，否则将擦除标签进行。 </li><li>切出一块组织的，并放置在组织模具。盖在最佳切割温度（OCT）化合物中的组织切片，并立即冻结其在-20℃下cryosectioning。本组织切片可被存储在-20℃，直到准备cryosectioning。 </li><li>切出的一个或多个组织片，并将它们转移到一个50ml管中含有的MACS缓冲液（1×磷酸盐缓冲盐水（PBS），0.5％胎牛血清（FBS），2mM的乙二胺四乙酸（EDTA）），用于后续的流式细胞分析。对于流式细胞术，较大的样品可优选作为细胞可以在肿瘤解离，细胞固定和透化需要时会丢失。一般地，此部分可采取在最后，一旦所有其他部分都已经被处理。剩的代表性肿瘤样品可以用于流式细胞仪。任选地，该管可以被存储在4℃的CO / N或用于流式细胞染色后立即消化成细胞悬浮液（见步骤4 - 流式细胞染色）。 </li><li>切割组织的剩余部分成片不大于0.5毫米×0.5毫米的最大尺寸，并将它们传送到低温小瓶在液氮立即冻结。这些快速冷冻样品可以用于未来的分析和合作目的。 注:应加倍小心用液氮工作时的直接接触可引起严重的伤害服用。眼睛应该被保护，安全眼镜带有侧面防护或防护面罩。低温手套也应佩戴液氮烧伤引起的飞溅和直接接触保护。手套应正确佩戴，但宽松，足以迅速取出应液氮溅入其中。 </li></ol> 4.流式细胞仪染色<ol><li> 组织均匀化 <ol><li>准备用DMEM（40毫升），DNA酶I（50U / ml）和胶原酶I（2毫克/毫升）消化培养基。将在MACS缓冲新鲜或O / N存储肿瘤在培养皿的盖子，并淹没消化中。 注:不同的消化方法都存在，各具优势（ 即细胞表面蛋白的前PRESSION，活力）。确保你有你想要的下游应用的正确方法。 </li><li>销肿瘤用针盘的底部，并从外部切割用手术刀居中，直到肿瘤具有糊状的质地。转移瘤组合，以50毫升管，密封并放在一个塑料袋，以防止泄漏。转移管至孵化器，并旋转以225RPM，37℃，1小时。 </li><li>通过一个70微米的细胞过滤器过滤肿瘤细胞悬浮液。离心细胞悬浮液在250×g离心，4℃，5分钟。悬浮细胞沉淀在1ml FACS缓冲液（500毫升PBS中，加入1ml的0.5M EDTA的库存，将10毫升的FCS或FBS）。 </li><li>使用血球计数细胞并重新悬浮于1×10 7个细胞/ ml的浓度。转移每孔100μl（10 6个细胞）在96孔圆底板进行染色。 </li></ol></li><li> 活力和抗体染色 <ol><li>加入200μl的生存能力染色液向每个孔并混合。 INCU软化30分钟，在4℃下，在黑暗中。降速细胞在250×g离心，4℃，5分钟，并吸出上清。 </li><li>在推荐/滴定浓度（根据制造商），用于在200μlFACS缓冲液表面抗原添加抗体混合物。孵育细胞，在4℃，30分钟，在黑暗中。降速细胞在250×g离心，4℃，5分钟，并吸出上清。 </li><li>用200微升FACS缓冲液洗，降速细胞在250 XG，4℃，5分钟，吸出上清液。悬浮细胞在200微升FACS缓冲液，并继续流式细胞仪进行采集和分析。 注:如果进行细胞内流式细胞染色，执行步骤4.2.2和4.2.3之间的固定和通透步骤和细胞内染色。如果执行因子染色，细胞应是预活化前染色的建议，并应活化过程中使用的细胞内蛋白质转运和分泌如莫能菌素的抑制剂确保保留的可溶性因子。固定的细胞可以被存储短期（一周），在4℃在黑暗中之前采集上的流式细胞仪。 </li></ol></li></ol>

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The authors have no conflicts of interest to disclose.

最终，处理的方法必须由所需的假说的支配和预期的技术测定，以进行（ 图1）。以下部分作为概述来描述可在这样的组织切片进行电位测定法。这决不是一个详尽的概述，但旨在帮助指导来样加工方法和选择下行测定所描述的技术，并列出他们的长处和短处。 流式细胞术染色可以执行上新鲜解剖组织切片，或片段存储的O / N的MACS缓冲液中于4℃。流式细胞仪便于高度特异性的表型分型和从组织样本中提取细胞群的分析，并允许高达17的细胞表面标志物和细胞内蛋白质，细胞因子和蛋白酶染色在单个细胞中。此外，某些流式细胞仪允许单细胞倍率，以便确定localizat细胞内的离子的蛋白质而不是仅阳性和信号强度。最后，质谱术允许超过35蛋白在同一样品中的量化，用下行在于细胞必须被裂解，因此，可能不进一步培养，排序，或分析的整体14。这种技术允许的许多标记和蛋白质分析以及详细的表征在单细胞水平。然而，流式细胞术需要组织均化和组织结构的损失，必须在新鲜组织上进行，以尽量减少改变细胞的表型。重要的是，延迟的存储组织用于流式细胞仪可能会影响细胞的含量，因为一些细胞亚群可能更容易缺血。磷酸化蛋白质也可以通过流中不良仪检测，如果处理动力学是次优15。 福尔马林固定和组织切片的石蜡包埋（FFPE）的可能的情况下的最佳选择关于未来的不确定性分析，因为它允许在其可进行的测定的类型的高度灵活性。传统上，FFPE部分被剖开上切片成5微米的部分为免疫组织化学（IHC）或免疫荧光（IF）染色。然而，FFPE组织被非常保守，并且因此可以用于多种目的。事实上，切割较厚部分从FFPE块允许DNA和RNA提取用市售的试剂盒，以及反相蛋白质阵列（RPPA）分析16-18。因此，处理的组织样本进入FFPE可以提供的优点是增加了灵活性，以及维持组织结构为免疫染色，因此提供了更精确的体内样组织结构和细胞群的描绘的优点。 FFPE允许是保守的长时间的组织切片。这种技术确实，但是，也有一些缺点。用于FFPE薄切片做没有考虑从顺序割伤和组织切片的组织的异质性开始发生氧化一旦暴露于周围空气中，从而损害RNA和DNA质量。最后，由于交联，共染色多种蛋白质的免疫组化对FFPE部分需要优化。 当通过IHC或IF显微镜是期望的，使用的OCT化合物是优选的。 OCT-存储部分必须用cryotome被放置在载玻片上，并用于IHC和IF之前被削减。十月保留抗原性，并使其成为显微镜的最佳技术降低背景。东部华侨城还将允许立即冻结及切割相比，FFPE这需要几天的NBF。尽管有这些好处，OCT是通常不太稳定，比FFPE因为只有IHC和IF可以由OCT-包埋组织切片进行不太灵活。 至于RNA分析，存储的样本在4℃下在硬化随时间的RNA稳定化的解决方案，从而允许更好的RNAëxtraction通过市售提取试剂盒和TRIzol试剂，等等。 RNA提取许可证的下游分析，如逆转录聚合酶链式反应（RT-PCR）来定量转录基因，RNA测序分析的表达，和调制的基因通过微阵列广谱分析，或nanoString分析。因此，从组织分离RNA可能非常高收益，通过提供对基因表达和影响治疗和疾病的途径大量信息。然而重要的是，RNA的表达可能会发生变化显着基于组织结构和采样大小，所以格外小心，应注意避免偏分析，由于取样的位置。此外，RNA的性质表明，在表达水平的动态变化，可能会发生，因此格外小心也应注意确保适当的动力学遵循研究基因和感兴趣19,20途径;处理应按照从RNA稳定小号提取被限制olution确保稳定。因此，存储互补的DNA的增加的稳定性的（cDNA的）是优选的RNA（如在与下游分析线）21。最后，因为RNA可以经常在下游分析扩增，污染细胞的分钟馏分可以导致大的偏差所获得的结果中。 DNA分析可以进行先前速冻组织样本，或在FFPE组织切片。这些方法使随后的整个外显子组或全基因组测序，其中两个已经表明，通过识别链接到多个病上下文病人的反应和预后突变和多态性的巨大承诺的技术。 DNA提取可以进一步允许鉴定的T细胞和B细胞受体（TCR和BCR）序列存在于样品中，为了取得关于抗原和在治疗和疾病20,21免疫应答知识。相比RNA，DNA是一旦提取高度稳定的，但是它考虑到转录和基因的翻译调控以及翻译后修饰和调节22。 蛋白可以直接从快速冷冻组织切片，或从FFPE样品中提取，以便执行蛋白质印迹分析，免疫共沉淀，以确定蛋白质相互作用和网络，以及RPPA，高产技术允许数百相对定量蛋白质在一个单一的样品23。然而，该技术允许鉴定已知的蛋白质仅通过特异性抗体，其要求。重要的是，进一步的翻译后修饰的分析，例如蛋白磷酸化，需要组织因不稳定15的快速处理。 快冷冻的样品很简单，只需有限的资源。它还允许长期贮存的样品时下游分析都尚未确定，除了在案例流式细胞仪必须新鲜样品进行。快速冷冻的样品也可以很容易地进行协作的目的共享。 最终，数据可从一个单一的一块组织中提取的量是无限的。没有通用的正确答案到检测应在任何给定的研究进行。因此，每个调查员必须确保他/她会考虑所有假设，所需的实验以及可利用的资源招募患者和处理组织之前，因为这可能会极大地影响了研究的设计，并获得其中的答案。

医学研究已经从两个人细胞系和动物模型的研究受益匪浅。细胞系允许在系统中更好控制元件，以及使用的细胞从适当的生物体。正与人体细胞，而更具有代表性，提出了这可能是负责健康和疾病机制大大简化快照，因为细胞系可能不概括其它细胞，基质组分，并且可能影响反应1器官的影响。另一方面，动物模型中，虽然不是完全准确的再现人类疾病和遗传异质性，提供通过允许整个动物系统在大多数型号2,3输入进一步洞察。 缺点不谈，这两种方法都有其优点和是互补的。然而，主要的目标仍然是研究人类细胞和器官，在一个完整的身体，多器官系统。因此，翻译研究具有德恩在促进人体器官和组织中提取患者的研究中，为了更好地把握机制潜在的疾病了长足的发展。转化型研究提供了研究的治疗和疾病导致的整个身体，在适当的细胞中，从而提供了两全其美的细胞和动物实验4之间的优势。 转化的研究也并非没有缺陷。从人类患者收获标本，在除去从该主机，立即经受缺血和从它们将被保护，否则其他外部因素。这可能导致应力诱导的分子和遗传变化，并导致偏压在随后的研究。因此，很多努力已投入通过严格的方法提取和处理组织，以最好的保护器官和细胞的完整性对下游的研究5。重要的是，未来的应用必须紧密选择方法□当考虑˚F处理人样品，如某些方法可能是有害的细胞成分和信号通路，同时保留其他。 对于涉及人体组织的任何研究，监管问题必须得到解决。之后塔斯基吉和贝尔蒙特报告，人们越来越接受了生物医学研究与固有的风险往往造成不可避免的伦理困境6有关的事实。需要国家标准被认可和联邦政府创建的机构审查委员会（IRB），以维护正当贝尔蒙特报告的原则（尊重个人，善行，正义）的手段。 任何机构的内部评级法是医生，研究人员和社区成员是谁负责保护研究参与者组成的委员会。它要求自愿同意从生物医学研究研究，所有参与者获得的，而这种同意被记录在FO知情同意书<html> RM。知情同意过程的目的是提供足够的信息，以一个参与者，从而使一个明智的决定可能会在是否要报名参加学习，还是要继续参加该项。在这方面，知情同意文件必须具有良好的可读性和应该写在，可以很容易理解（ 第 6至第 8次级读电平）由目标人群的语言。胁迫或不当影响的可能性必须最小化，并且对象必须给予充分的时间来考虑参与。参与者也必须允许行使其在任何阶段研究的过程中没有点球时撤回同意的权利。自愿知情同意是一个强制性的先决条件的受试者参与研究并且还具有法律效力7。 按照规定进行保护人类受试者21 CFR 50.25（/45cfr46.html&quot;&gt;http://www.hhs.gov/ohrp/humansubjects/guidance/45cfr46.html 8），研究参与者必须被告知的研究目的，参与这项研究的程序，替代品的参与，该研究所有可预见的风险和不适（不仅包括人身伤害，还可能出现的心理，社会或经济损害，不适或不便），福利社会，可能是为了个人，时间长度的主题，预计参加，联系人的问题的答案，或在研究相关的伤害或紧急情况下，声明，表示参与是自愿的，拒绝参加不会造成任何后果或利益损失，如在常规临床护理的妥协该参与者有权与其有关主体的权利，保密权和退出接收他/她的诊断结果，最后声明该研究在没有任何结果的任何时间。从与会者谁在研究的过程中撤回同意组织标本不应被存入银行，必须立即耗尽。的知情同意的一个或多个元件免除可从IRB获得一些可能实际上不可能没有改变完成到所需的元素或用于研究，其中所需的元素是不适用的研究项目。必须采取非常谨慎，以确保数据的保密性。健康保险流通与责任法案（HIPAA）是防止使用或披露"受保护的健康信息"（PHI）未经参与者书面同意的联邦法律。 PHI包括但不限于传输或维持在任何形式或媒介，并且包括可用于识别个人的9个字段的健康信息。 通过这项工作，我们的目标是勾勒必须Tissu酒店时，必须遵循协议ë处理 - 包括采购，并存储和强调以下这些严格的准则，以便尽量减少可能导致由于在提取的组织受到的应力的偏差的重要性。我们也将尽力提供下游分析或分析（ 图1）可以在这样的标本进行的简要介绍，使读者可以做出最适合他们需要什么样测定（S）合格的判断。

<table><tbody><tr><td>Needles</td><td>BD</td><td>305167</td><td></td></tr><tr><td>Stainless steel disposable scalpels</td><td>Miltex</td><td>4-410</td><td></td></tr><tr><td>Tissue culture dish</td><td>Corning</td><td>353003</td><td></td></tr><tr><td>Biopsy Cassettes</td><td>Thermo Fisher</td><td>58931</td><td></td></tr><tr><td>RNA-stabilizing solution</td><td>Ambion</td><td>AM7021</td><td></td></tr><tr><td>1.5 ml Eppendorf tubes</td><td>Phenix</td><td>MAX-815</td><td></td></tr><tr><td>50 ml tubes</td><td>Corning</td><td>430290</td><td></td></tr><tr><td>10% Neutral Buffered Formalin</td><td>StatLab Medical Products</td><td>28600-5</td><td></td></tr><tr><td>Formalin Containers</td><td>Fisher Scientific</td><td>23-032-059</td><td></td></tr><tr><td>Optimal cutting temperature solution (OCT)</td><td>Sakura Finetek</td><td>4583</td><td></td></tr><tr><td>Tissue-Tek Cryomold</td><td>Sakura Finetek</td><td>4557</td><td></td></tr><tr><td>Dnase I</td><td>Roche</td><td>10104159001</td><td></td></tr><tr><td>Collagenase I</td><td>Roche</td><td>11088793001</td><td></td></tr><tr><td>70&amp;mu;m cell strainers</td><td>BD Bioscience</td><td>352350</td><td></td></tr><tr><td>96 well round bottom plates</td><td>Corning</td><td>3799</td><td></td></tr><tr><td>Live/Dead stain solution</td><td>Life Technologies</td><td>L34957</td><td></td></tr></tbody></table>

working with human tissues for translational cancer research

Medical research for human benefit is greatly impeded by the necessity for human tissues and subjects. However, upon obtaining consent for human specimens, precious samples must be handled with the greatest care in order to ensure integrity of organs, tissues, and cells to the highest degree. Unfortunately, tissue processing by definition requires extraction of tissues from the host, a change which can cause great cellular stress and have major repercussions on subsequent analyses. These stresses could result in the specimen being no longer representative of the site from which it was retrieved. Therefore, a strict protocol must be adhered to while processing these specimens to ensure representativeness. The desired assay(s) must also be taken into consideration in order to ensure that an optimal technique is used for sample processing. Outlined here is a protocol for tissue retrieval, processing and various analyses which may be performed on processed tissue in order to maximize downstream production from limited tissue samples.

<html>下面显示的结果描绘流式细胞仪门控策略，在处理黑色素瘤广泛的免疫表型。染色的天，组织样品在225 RPM转动消化且用胶原酶I和DNA酶I温育60分钟，在37℃下。以下消化，细胞悬浮液通过70μm的细胞过滤滤波，以消除碎片和细胞用流式细胞仪抗体免疫细胞表型荧光标记流动。下面（图3）示出的是在MACS缓冲门控策略和染色对组织存储的O / N多个标记在4℃。如图所示，标本在4℃处理成所述和存储的O / N的MACS缓冲液显示极为有限的死亡率。此外，该图证明，如上所述的处理组织允许在黑素瘤肿瘤免疫细胞亚群的多色广泛表型。 S / ftp_upload / 53189 / 53189fig1.jpg"/&gt; 图1.分析可从人体血液和组织样本进行。这也可以进行以下处理实验的概要。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53189/53189fig1large.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/53189/53189fig2.jpg" /> 图2.项目需要进行适当的组织处理和存储。建立了组织处理的例子。组织可以被解剖的浅培养皿（A）与所述针和手术刀。代表性组织片应被转移到所述活检盒（B），那么它应该被转移至NBF容器（C）的三天固定在RT。另一块组织的应放置在一cryomold（D）和覆盖在OCT中溶液在b之前eing冷冻在-20℃。一大块组织的应转移到50ml管（E）流式细胞染色和可被储存在4℃下在MACS缓冲O / N。甲小块组织应转移到1.5ml管与RNA的稳定溶液（F），并储存在4℃直至RNA提取。最后，剩余的组织应该切成块，并转移到冻存管（G）为单元冷冻在液氮中，并长期保存。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53189/53189fig2large.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/53189/53189fig3.jpg" /> 图3.流式细胞仪门控策略进行广泛的免疫表型。广泛的免疫表型门控战略用流式细胞仪对黑色素瘤肿瘤存储的O / N在4°例; c。在MACS缓冲13 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53189/53189fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Working with Human Tissues for Translational Cancer Research at JoVE.com

Working with Human Tissues for Translational Cancer Research

Translational cancer research is dependent on extraction of human tissues. Much work has gone into optimizing extraction methods for ex vivo analysis. Here, we describe tissue processing methods allowing for maximal data output from limited samples.

与人体组织转化为癌症研究工作

Medical research for human benefit is greatly impeded by the necessity for human tissues and subjects. However, upon obtaining consent for human ...

working-with-human-tissues-for-translational-cancer-research

Universidad San Sebastian

Research

JoVE Journal

Medicine

9.8K 次观看.  University of Texas MD Anderson Cancer Center. Translational cancer research is dependent on extraction of human tissues. Much work has gone into optimizing extraction methods for ex vivo analysis. Here, we describe tissue processing methods allowing for maximal data output from limited samples.

视频: Working with Human Tissues for Translational Cancer Research

Utilization of Ultrasound Guided Tissue-directed Cellular Implantation for the Establishment of Biologically Relevant Metastatic Tumor Xenografts

Preclinical testing of anticancer therapies relies on relevant xenograft models that mimic the innate tendencies of cancer. Advantages of standard subcutaneous flank models include procedural ease and the ability to monitor tumor progression and response without invasive imaging. Such models are often inconsistent in translational clinical trials and have limited biologically relevant characteristics with low proclivity to produce metastasis, as there is a lack of a native microenvironment. In comparison, orthotopic xenograft models at native tumor sites have been shown to mimic the tumor microenvironment and replicate important disease characteristics such as distant metastatic spread. These models often require tedious surgical procedures with prolonged anesthetic time and recovery periods. To address this, cancer researchers have recently utilized ultrasound-guided injection techniques to establish cancer xenograft models for preclinical experiments, which allows for rapid and reliable establishment of tissue-directed murine models. Ultrasound visualization also provides a noninvasive method for longitudinal assessment of tumor engraftment and growth. Here, we describe the method for ultrasound-guided injection of cancer cells, utilizing the adrenal gland for NB and renal sub capsule for ES. This minimally invasive approach overcomes tedious open surgery implantation of cancer cells in tissue-specific locations for growth and metastasis, and abates morbid recovery periods. We describe the utilization of both established cell lines and patient derived cell lines for orthotopic injection. Pre-made commercial kits are available for tumor dissociation and luciferase tagging of cells. Injection of cell suspension using image-guidance provides a minimally invasive and reproducible platform for the creation of preclinical models. This method is utilized to create reliable preclinical models for other cancers such as bladder, liver and pancreas exemplifying its untapped potential for numerous cancer models.

Here, we present a protocol to utilize ultrasound-guided injection of neuroblastoma (NB) and Ewing's sarcoma (ES) cells (established cell lines and patient-derived tumor cells) at biologically relevant sites to create reliable preclinical models for cancer research.

超声引导组织定向细胞植入术在生物学相关转移性肿瘤移植中的应用

Preclinical testing of anticancer therapies relies on relevant xenograft models that mimic the innate tendencies of cancer. Advantages of standard ...

科研

癌症研究

Tissue Engineering of a Human 3D in vitro Tumor Test System

Cancer is one of the leading causes of death worldwide. Current therapeutic strategies are predominantly developed in 2D culture systems, which inadequately reflect physiological conditions in vivo. Biological 3D matrices provide cells an environment in which cells can self-organize, allowing the study of tissue organization and cell differentiation. Such scaffolds can be seeded with a mixture of different cell types to study direct 3D cell-cell-interactions. To mimic the 3D complexity of cancer tumors, our group has developed a 3D in vitro tumor test system.	
	Our 3D tissue test system models the in vivo situation of malignant peripheral nerve sheath tumors (MPNSTs), which we established with our decellularized porcine jejunal segment derived biological vascularized scaffold (BioVaSc). In our model, we reseeded a modified BioVaSc matrix with primary fibroblasts, microvascular endothelial cells (mvECs) and the S462 tumor cell line. For static culture, the vascular structure of the BioVaSc is removed and the remaining scaffold is cut open on one side (Small Intestinal Submucosa SIS-Muc). The resulting matrix is then fixed between two metal rings (cell crowns).	
	Another option is to culture the cell-seeded SIS-Muc in a flow bioreactor system that exposes the cells to shear stress. Here, the bioreactor is connected to a peristaltic pump in a self-constructed incubator. A computer regulates the arterial oxygen and nutrient supply via parameters such as blood pressure, temperature, and flow rate. This setup allows for a dynamic culture with either pressure-regulated pulsatile or constant flow.	
	In this study, we could successfully establish both a static and dynamic 3D culture system for MPNSTs. The ability to model cancer tumors in a more natural 3D environment will enable the discovery, testing, and validation of future pharmaceuticals in a human-like model.

Tissue Engineering of a Human 3D in vitro Tumor Test System

Methods to create human 3D tumor tissues as test systems are described. These technologies are based on a decellularized Biological Vascularized Scaffold (BioVaSc), primary human cells and a tumor cell line, which can be cultured under static as well as under dynamic conditions in a flow bioreactor.

一个人的3D组织工程<em&gt;在体外</em&gt;肿瘤测试系统

Cancer is one of the leading causes of death worldwide.  Current therapeutic strategies are predominantly developed in 2D culture  systems, which ...

生物工程

Preparation of Human Tissues Embedded in Optimal Cutting Temperature Compound for Mass Spectrometry Analysis

Sphingolipids are cellular components that have well-established roles in human metabolism and disease. Mass spectrometry can be used to determine whether sphingolipids are altered in a disease and investigate whether sphingolipids can be targeted clinically. However, properly powered prospective studies that acquire tissues directly from the surgical suite can be time consuming, and technically, logistically, and administratively challenging. In contrast, retrospective studies can take advantage of cryopreserved human specimens already available, usually in large numbers, at tissue biorepositories. Other advantages of procuring tissues from biorepositories include access to information associated with the tissue specimens including histology, pathology, and in some instances clinicopathological variables, all of which can be used to examine correlations with lipidomics data. However, technical limitations related to the incompatibility of optimal cutting temperature compound (OCT) used in the cryopreservation and mass spectrometry is a technical barrier for the analysis of lipids. However, we have previously shown that OCT can be easily removed from human biorepository specimens through cycles of washes and centrifugation without altering their sphingolipid content. We have also previously established that sphingolipids in human tissues cryopreserved in OCT are stable for up to 16 years. In this report, we outline the steps and workflow to analyze sphingolipids in human tissue specimens that are embedded in OCT, including washing tissues, weighing tissues for data normalization, the extraction of lipids, preparation of samples for analysis by liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS), mass spectrometry data integration, data normalization, and data analysis.

Sphingolipids are bioactive metabolites with well-established roles in human disease. Characterizing alterations in tissues with mass-spectrometry can reveal roles in disease etiology or identify therapeutic targets. However, the OCT-compound used for cryopreservation in biorepositories interferes with mass-spectrometry. We outline methods to analyze sphingolipids in human tissues embedded in OCT with LC-ESI-MS/MS.

用于质谱分析的最佳切割温度化合物中嵌入的人组织制备

Sphingolipids are cellular components that have well-established roles in human metabolism and disease. Mass spectrometry can be used to determine whether ...

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 ...

来自新鲜组织的胰腺癌来源的肿瘤类器官和成纤维细胞

Establishment of Pancreatic Cancer-Derived Tumor Organoids and Fibroblasts From Fresh Tissue

Tumor organoids are three-dimensional (3D) ex vivo tumor models that recapitulate the biological key features of the original primary tumor tissues. Patient-derived tumor organoids have been used in translational cancer research and can be applied to assess treatment sensitivity and resistance, cell-cell interactions, and tumor cell interactions with the tumor microenvironment. Tumor organoids are complex culture systems that require advanced cell culture techniques and culture media with specific growth factor cocktails and a biological basement membrane that mimics the extracellular environment. The ability to establish primary tumor cultures highly depends on the tissue of origin, the cellularity, and the clinical features of the tumor, such as the tumor grade. Furthermore, tissue sample collection, material quality and quantity, as well as correct biobanking and storage are crucial elements of this procedure. The technical capabilities of the laboratory are also crucial factors to consider. Here, we report a validated SOP/protocol that is technically and economically feasible for the culture of ex vivo tumor organoids from fresh tissue samples of pancreatic adenocarcinoma origin, either from fresh primary resected patient donor tissue or patient-derived xenografts (PDX). The technique described herein can be performed in laboratories with basic tissue culture and mouse facilities and is tailored for wide application in the translational oncology field.

作者聚焦:从新鲜组织中建立胰腺癌来源的肿瘤类器官和成纤维细胞  

Tumor organoids have revolutionized cancer research and the approach to personalized medicine. They represent a clinically relevant tumor model that allows researchers to stay one step ahead of the tumor in the clinic. This protocol establishes tumor organoids from fresh pancreatic tumor tissue samples and patient-derived xenografts of pancreatic adenocarcinoma origin.

从新鲜组织中建立胰腺癌来源的肿瘤类器官和成纤维细胞

Tumor organoids are three-dimensional (3D) ex vivo tumor models that recapitulate the biological key features of the original primary tumor tissues. ...

制备基于微流体的血管化人类微肿瘤模型

Establishing a Physiologic Human Vascularized Micro-Tumor Model for Cancer Research

A lack of validated cancer models that recapitulate the tumor microenvironment of solid cancers in vitro remains a significant bottleneck for preclinical cancer research and therapeutic development.&#160;To overcome this problem, we have developed the vascularized microtumor (VMT), or tumor chip, a microphysiological system that realistically models the complex human tumor microenvironment. The VMT forms de novo within a microfluidic platform by co-culture of multiple human cell types under dynamic, physiological flow conditions. This tissue-engineered micro-tumor construct incorporates a living perfused vascular network that supports the growing tumor mass just as newly formed vessels do in vivo. Importantly, drugs and immune cells must cross the endothelial layer to reach the tumor, modeling in vivo physiological barriers to therapeutic delivery and efficacy. Since the VMT platform is optically transparent, high-resolution imaging of dynamic processes such as immune cell extravasation and metastasis can be achieved with direct visualization of fluorescently labeled cells within the tissue. Further, the VMT retains in vivo tumor heterogeneity, gene expression signatures, and drug responses. Virtually any tumor type can be adapted to the platform, and primary cells from fresh surgical tissues grow and respond to drug treatment in the VMT, paving the way toward truly personalized medicine. Here, the methods for establishing the VMT and utilizing it for oncology research are outlined. This innovative approach opens new possibilities for studying tumors and drug responses, providing researchers with a powerful tool to advance cancer research.

作者聚焦：创建人类血管化微肿瘤作为转化癌症研究的模型 

This protocol presents a physiologically relevant tumor-on-a-chip model to perform high-throughput basic and translational human cancer research, advancing drug screening, disease modeling, and personalized medicine approaches with a description of loading, maintenance, and evaluation procedures.

建立用于癌症研究的生理性人血管化微肿瘤模型

A lack of validated cancer models that recapitulate the tumor microenvironment of solid cancers in vitro remains a significant bottleneck for preclinical ...

与人体组织转化为癌症研究工作

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一个人的3D组织工程

用于质谱分析的最佳切割温度化合物中嵌入的人组织制备

在携带人类肿瘤的人源化小鼠模型中测试癌症免疫疗法

从新鲜组织中建立胰腺癌来源的肿瘤类器官和成纤维细胞

从新鲜组织中建立胰腺癌来源的肿瘤类器官和成纤维细胞

从新鲜组织中建立胰腺癌来源的肿瘤类器官和成纤维细胞

建立用于癌症研究的生理性人血管化微肿瘤模型

建立用于癌症研究的生理性人血管化微肿瘤模型

建立用于癌症研究的生理性人血管化微肿瘤模型