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...

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>1.5ml Eppendorf tubes</td>
			<td>Phenix</td>
			<td>MAX-815</td>
			<td></td>
		</tr>
		<tr>
			<td>50ml 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&#956;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?...

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

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

Generation of Comprehensive Thoracic Oncology Database - Tool for Translational Research

The Thoracic Oncology Program Database Project was created to serve as a comprehensive, verified, and accessible repository for well-annotated cancer specimens and clinical data to be available to researchers within the Thoracic Oncology Research Program. This database also captures a large volume of genomic and proteomic data obtained from various tumor tissue studies. A team of clinical and basic science researchers, a biostatistician, and a bioinformatics expert was convened to design the database. Variables of interest were clearly defined and their descriptions were written within a standard operating manual to ensure consistency of data annotation. Using a protocol for prospective tissue banking and another protocol for retrospective banking, tumor and normal tissue samples from patients consented to these protocols were collected. Clinical information such as demographics, cancer characterization, and treatment plans for these patients were abstracted and entered into an Access database. Proteomic and genomic data have been included in the database and have been linked to clinical information for patients described within the database. The data from each table were linked using the relationships function in Microsoft Access to allow the database manager to connect clinical and laboratory information during a query. The queried data can then be exported for statistical analysis and hypothesis generation.

A thoracic oncology database was developed to serve as a comprehensive repository for clinical and laboratory data for the purposes of translational research. The database will serve translational cancer researchers within the Thoracic Oncology Research Program. This database is adaptable to other cancer models, as well as other human diseases.

综合胸部肿瘤生成数据库 - 翻译研究的工具

The Thoracic Oncology Program Database Project was created to serve as a comprehensive, verified, and accessible repository for well-annotated cancer ...

科研

医学

Establishment and Propagation of Human Retinoblastoma Tumors in Immune Deficient Mice

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

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

在免疫缺陷小鼠的建立和传播人类视网膜母细胞瘤肿瘤

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

Ex Vivo Infection of Live Tissue with Oncolytic Viruses

Oncolytic Viruses (OVs) are novel therapeutics that selectively replicate in and kill tumor cells1. Several clinical trials evaluating the effectiveness of a variety of oncolytic platforms including HSV, Reovirus, and Vaccinia OVs as treatment for cancer are currently underway2-5. One key characteristic of oncolytic viruses is that they can be genetically modified to express reporter transgenes which makes it possible to visualize the infection of tissues by microscopy or bio-luminescence imaging6,7. This offers a unique advantage since it is possible to infect tissues from patients ex vivo prior to therapy in order to ascertain the likelihood of successful oncolytic virotherapy8. To this end, it is critical to appropriately sample tissue to compensate for tissue heterogeneity and assess tissue viability, particularly prior to infection9. It is also important to follow viral replication using reporter transgenes if expressed by the oncolytic platform as well as by direct titration of tissues following homogenization in order to discriminate between abortive and productive infection. The object of this protocol is to address these issues and herein describes 1. The sampling and preparation of tumor tissue for cell culture 2. The assessment of tissue viability using the metabolic dye alamar blue 3. Ex vivo infection of cultured tissues with vaccinia virus expressing either GFP or firefly luciferase 4. Detection of transgene expression by fluorescence microscopy or using an In Vivo Imaging System (IVIS) 5. Quantification of virus by plaque assay. This comprehensive method presents several advantages including ease of tissue processing, compensation for tissue heterogeneity, control of tissue viability, and discrimination between abortive infection and bone fide viral replication.

Ex Vivo Infection of Live Tissue with Oncolytic Viruses

Oncolytic viruses are promising for cancer therapeutics. The ability to ascertain the infectability of live tissue specimens obtained from patients prior to treatment is a unique advantage of this therapeutic approach. This protocol describes how to process tissues for ex vivo infection with oncolytic virus and subsequent viral quantification.

前溶瘤病毒的活组织的体内感染

Oncolytic Viruses (OVs) are novel therapeutics that selectively replicate in and kill tumor cells1. Several clinical trials evaluating the effectiveness ...

Experimental Generation of Carcinoma-Associated Fibroblasts (CAFs) from Human Mammary Fibroblasts

Carcinomas are complex tissues comprised of neoplastic cells and a non-cancerous compartment referred to as the 'stroma'. The stroma consists of extracellular matrix (ECM) and a variety of mesenchymal cells, including fibroblasts, myofibroblasts, endothelial cells, pericytes and leukocytes 1-3.
The tumour-associated stroma is responsive to substantial paracrine signals released by neighbouring carcinoma cells. During the disease process, the stroma often becomes populated by carcinoma-associated fibroblasts (CAFs) including large numbers of myofibroblasts. These cells have previously been extracted from many different types of human carcinomas for their in vitro culture. A subpopulation of CAFs is distinguishable through their up-regulation of &alpha;-smooth muscle actin (&alpha;-SMA) expression4,5. These cells are a hallmark of 'activated fibroblasts' that share similar properties with myofibroblasts commonly observed in injured and fibrotic tissues 6. The presence of this myofibroblastic CAF subset is highly related to high-grade malignancies and associated with poor prognoses in patients.
Many laboratories, including our own, have shown that CAFs, when injected with carcinoma cells into immunodeficient mice, are capable of substantially promoting tumourigenesis 7-10. CAFs prepared from carcinoma patients, however, frequently undergo senescence during propagation in culture limiting the extensiveness of their use throughout ongoing experimentation. To overcome this difficulty, we developed a novel technique to experimentally generate immortalised human mammary CAF cell lines (exp-CAFs) from human mammary fibroblasts, using a coimplantation breast tumour xenograft model.
In order to generate exp-CAFs, parental human mammary fibroblasts, obtained from the reduction mammoplasty tissue, were first immortalised with hTERT, the catalytic subunit of the telomerase holoenzyme, and engineered to express GFP and a puromycin resistance gene. These cells were coimplanted with MCF-7 human breast carcinoma cells expressing an activated ras oncogene (MCF-7-ras cells) into a mouse xenograft. After a period of incubation in vivo, the initially injected human mammary fibroblasts were extracted from the tumour xenografts on the basis of their puromycin resistance 11.
We observed that the resident human mammary fibroblasts have differentiated, adopting a myofibroblastic phenotype and acquired tumour-promoting properties during the course of tumour progression. Importantly, these cells, defined as exp-CAFs, closely mimic the tumour-promoting myofibroblastic phenotype of CAFs isolated from breast carcinomas dissected from patients. Our tumour xenograft-derived exp-CAFs therefore provide an effective model to study the biology of CAFs in human breast carcinomas. The described protocol may also be extended for generating and characterising various CAF populations derived from other types of human carcinomas.

Experimental Generation of Carcinoma-Associated Fibroblasts CAFs from Human Mammary Fibroblasts

Carcinoma-associated fibroblasts (CAFs) rich in myofibroblasts present within the tumour stroma, play a major role in driving tumour progression. We developed a coimplantation tumour xengraft model for experimentally generating CAFs from human mammary fibroblasts. The protocol describes how to establish CAF myofibroblasts that acquire an ability to promote tumourigenesis.

从人类乳腺成纤维细胞癌相关的成纤维细胞的实验发电（CAFS）

Carcinomas are complex tissues comprised of neoplastic cells and a non-cancerous compartment referred to as the 'stroma'. The stroma consists of ...

Processing of Human Reduction Mammoplasty and Mastectomy Tissues for Cell Culture

Experimental examination of normal human mammary epithelial cell (HMEC) behavior, and how normal cells acquire abnormal properties, can be facilitated by in vitro culture systems that more accurately model in vivo biology. The use of human derived material for studying cellular differentiation, aging, senescence, and immortalization is particularly advantageous given the many significant molecular differences in these properties between human and commonly utilized rodent cells1-2. Mammary cells present a convenient model system because large quantities of normal and abnormal tissues are available due to the frequency of reduction mammoplasty and mastectomy surgeries.
The mammary gland consists of a complex admixture of many distinct cell types, e.g., epithelial, adipose, mesenchymal, endothelial. The epithelial cells are responsible for the differentiated mammary function of lactation, and are also the origin of the vast majority of human breast cancers. We have developed methods to process mammary gland surgical discard tissues into pure epithelial components as well as mesenchymal cells3. The processed material can be stored frozen indefinitely, or initiated into primary culture. Surgical discard material is transported to the laboratory and manually dissected to enrich for epithelial containing tissue. Subsequent digestion of the dissected tissue using collagenase and hyaluronidase strips stromal material from the epithelia at the basement membrane. The resulting small pieces of the epithelial tree (organoids) can be separated from the digested stroma by sequential filtration on membranes of fixed pore size. Depending upon pore size, fractions can be obtained consisting of larger ductal/alveolar pieces, smaller alveolar clusters, or stromal cells. We have observed superior growth when cultures are initiated as organoids rather than as dissociated single cells. Placement of organoids in culture using low-stress inducing media supports long-term growth of normal HMEC with markers of multiple lineage types (myoepithelial, luminal, progenitor)4-5. Sufficient numbers of cells can be obtained from one individual's tissue to allow extensive experimental examination using standardized cell batches, as well as interrogation using high throughput modalities.
Cultured HMEC have been employed in a wide variety of studies examining the normal processes governing growth, differentiation, aging, and senescence, and how these normal processes are altered during immortal and malignant transformation4-15,16. The effects of growth in the presence of extracellular matrix material, other cell types, and/or 3D culture can be compared with growth on plastic5,15. Cultured HMEC, starting with normal cells, provide an experimentally tractable system to examine factors that may propel or prevent human aging and carcinogenesis.

A method to process human mammary surgical discard material is described. Processed tissue, in the form of organoids, can be stored frozen indefinitely or placed in culture for long-term growth. This method enables experimental examination of normal human epithelial cell biology, and the effects of exogenous perturbations.

处理人减少乳房整形和乳房切除术的组织细胞培养

Experimental  examination of normal human mammary epithelial cell (HMEC) behavior, and how  normal cells acquire abnormal properties, can be facilitated ...

A Modified Heterotopic Swine Hind Limb Transplant Model for Translational Vascularized Composite Allotransplantation (VCA) Research

Vascularized Composite Allotransplantation (VCA) such as hand and face transplants represent a viable treatment option for complex musculoskeletal trauma and devastating tissue loss. Despite favorable and highly encouraging early and intermediate functional outcomes, rejection of the highly immunogenic skin component of a VCA and potential adverse effects of chronic multi-drug immunosuppression continue to hamper widespread clinical application of VCA. Therefore, research in this novel field needs to focus on translational studies related to unique immunologic features of VCA and to develop novel immunomodulatory strategies for immunomodulation and tolerance induction following VCA without the need for long term immunosuppression.
This article describes a reliable and reproducible translational large animal model of VCA that is comprised of an osteomyocutaneous flap in a MHC-defined swine heterotopic hind limb allotransplantation. Briefly, a well-vascularized skin paddle is identified in the anteromedial thigh region using near infrared laser angiography. The underlying muscles, knee joint, distal femur, and proximal tibia are harvested on a femoral vascular pedicle. This allograft can be considered both a VCA and a vascularized bone marrow transplant with its unique immune privileged features. The graft is transplanted to a subcutaneous abdominal pocket in the recipient animal with a skin component exteriorized to the dorsolateral region for immune monitoring.
Three surgical teams work simultaneously in a well-coordinated manner to reduce anesthesia and ischemia times, thereby improving efficiency of this model and reducing potential confounders in experimental protocols. This model serves as the groundwork for future therapeutic strategies aimed at reducing and potentially eliminating the need for chronic multi-drug immunosuppression in VCA.

A Modified Heterotopic Swine Hind Limb Transplant Model for Translational Vascularized Composite Allotransplantation VCA Research

Vascularized Composite Allotransplantations (VCA) have become a clinical reality. However, broad clinical application of VCA is limited by chronic multi-drug immunosuppression. The authors present a reliable and reproducible large animal model to translate novel immunomodulatory strategies that can minimize or potentially eliminate the need of immunosuppression in VCA.

平移血管复合同种异体移植（VCA）研究一种改进的异位猪后肢移植模型

Vascularized Composite Allotransplantation (VCA) such as hand and face transplants represent a viable treatment option for complex musculoskeletal trauma ...

MR Molecular Imaging of Prostate Cancer with a Small Molecular CLT1 Peptide Targeted Contrast Agent

Tumor extracellular matrix has abundance of cancer related proteins that can be used as biomarkers for cancer molecular imaging. In this work, we demonstrated effective MR cancer molecular imaging with a small molecular peptide targeted Gd-DOTA monoamide complex as a targeted MRI contrast agent specific to clotted plasma proteins in tumor stroma. We performed the experiment of evaluating the effectiveness of the agent for non-invasive detection of prostate tumor with MRI in a mouse orthotopic PC-3 prostate cancer model. The targeted contrast agent was effective to produce significant tumor contrast enhancement at a low dose of 0.03 mmol Gd/kg. The peptide targeted MRI contrast agent is promising for MR molecular imaging of prostate tumor.

To demonstrate MR cancer molecular imaging with a small peptide targeted MRI contrast agent specific to clotted plasma proteins in tumor stroma in a mouse prostate cancer model.

前列腺癌与小分子肽CLT1靶向造影剂的磁共振分子成像

Tumor extracellular  matrix has abundance of cancer related proteins that can be used as biomarkers  for cancer molecular imaging. In this work,  we ...

Cell Death Associated with Abnormal Mitosis Observed by Confocal Imaging in Live Cancer Cells

Phenanthrene derivatives acting as potent PARP1 inhibitors prevented the bi-focal clustering of supernumerary centrosomes in multi-centrosomal human cancer cells in mitosis. The phenanthridine PJ-34 was the most potent molecule. Declustering of extra-centrosomes causes mitotic failure and cell death in multi-centrosomal cells. Most solid human cancers have high occurrence of extra-centrosomes. The activity of PJ-34 was documented in real-time by confocal imaging of live human breast cancer MDA-MB-231 cells transfected with vectors encoding for fluorescent &gamma;-tubulin, which is highly abundant in the centrosomes and for fluorescent histone H2b present in the chromosomes. Aberrant chromosomes arrangements and de-clustered &gamma;-tubulin foci representing declustered centrosomes were detected in the transfected MDA-MB-231 cells after treatment with PJ-34. Un-clustered extra-centrosomes in the two spindle poles preceded their cell death. These results linked for the first time the recently detected exclusive cytotoxic activity of PJ-34 in human cancer cells with extra-centrosomes de-clustering in mitosis, and mitotic failure leading to cell death. According to previous findings observed by confocal imaging of fixed cells, PJ-34 exclusively eradicated cancer cells with multi-centrosomes without impairing normal cells undergoing mitosis with two centrosomes and bi-focal spindles. This cytotoxic activity of PJ-34 was not shared by other potent PARP1 inhibitors, and was observed in PARP1 deficient MEF harboring extracentrosomes, suggesting its independency of PARP1 inhibition. Live confocal imaging offered a useful tool for identifying new molecules eradicating cells during mitosis.

The cytotoxic activity of the phenanthridine PJ-34 in cancer cells undergoing mitosis was documented in real time by live confocal imaging. PJ-34 eradicated human breast cancer MDA-MB-231 cells harboring extra-centrosomes in mitosis. Unlike normal bi-focal mitosis, the extra-centrosomes were not clustered in the two spindle poles in the presence of PJ-34.

共聚焦成像在肝癌细胞中观察到的异常有丝分裂细胞死亡相关

Phenanthrene derivatives acting as potent PARP1 inhibitors prevented the bi-focal clustering of supernumerary centrosomes in multi-centrosomal human ...

An Ex vivo Model to Study Hormone Action in the Human Breast

The study of hormone action in the human breast has been hampered by lack of adequate model systems. Upon in vitro culture, primary mammary epithelial cells tend to lose hormone receptor expression. Widely used hormone receptor positive breast cancer cell lines are of limited relevance to the in vivo situation. Here, we describe an ex vivo model to study hormone action in the human breast. Fresh human breast tissue specimens from surgical discard material such as reduction mammoplasties or mammectomies are mechanically and enzymatically digested to obtain tissue fragments containing ducts and lobules and multiple stromal cell types. These tissue microstructures kept in basal medium without growth factors preserve their intercellular contacts, the tissue architecture, and remain hormone responsive for several days. They are readily processed for RNA and protein extraction, histological analysis or stored in freezing medium. Fluorescence activated cell sorting (FACS) can be used to enrich for specific cell populations. This protocol provides a straightforward, standard approach for translational studies with highly complex, varied human specimens.

An Ex vivo Model to Study Hormone Action in the Human Breast

We have developed a novel ex vivo model to study hormone action in the human breast. It is based on tissue microstructures isolated from surgical breast tissue specimens which preserve tissue architecture, intercellular interactions, and paracrine signaling.

一个<em&gt;体外</em&gt;模型在乳腺癌研究激素的作用

The study of hormone action in the human breast has been hampered by lack of adequate model systems. Upon in vitro culture, primary mammary epithelial ...

Mammosphere Formation Assay from Human Breast Cancer Tissues and Cell Lines

Similar to healthy tissues, many blood and solid malignancies are now thought to be organised hierarchically, with a subset of stem-like cancer cells that self-renew while giving rise to more differentiated progeny. Understanding and targeting these cancer stem cells in breast cancer, which may possess enhanced chemo- and radio-resistance compared to the non-stem tumor bulk, has become an important research area. Markers including CD44, CD24, and ALDH activity can be assessed using fluorescence activated cell sorting (FACS) to prospectively isolate cells that display enhanced tumorigenicity when implanted into immunocompromised mice: the mammosphere assay has also become widely used for its ability to retrospectively identify sphere-forming cells that develop from single stem cell-like clones. Here we outline approaches for the appropriate culturing of mammospheres from cell lines or primary patient samples, their passaging, and calculations to estimate sphere forming efficiency (SFE). First we discuss key considerations and pitfalls in the appropriate planning and interpretation of mammosphere experiments.

Floating mammosphere assays can investigate the subset of stem-like breast cancer cells that survive in suspension conditions and show enhanced tumorigenesis when implanted into mice. This protocol provides a convenient in vitro measure of sphere-forming ability, a proxy for in vivo tumorigenesis, while facilitating analysis of the stem-associated transcriptional landscape.

从人乳腺癌组织及细胞系微球体形成分析

Similar to healthy tissues, many blood and solid malignancies are now thought to be organised hierarchically, with a subset of stem-like cancer cells that ...