This work was supported by grants from the Canadian Breast Cancer Foundation-Ontario Region, the Canada Foundation for Innovation (No. 13199), and donor support from John and Donna Bristol through the London Health Sciences Foundation (to A.L.A.). Studentship and fellowship support were provided by the Ontario Graduate Scholarship program (Province of Ontario, to G.M.P. and J.E.C.), the Canada Graduate Scholarship-Master's program (to M.M.P), the Canadian Institutes of Health Research (CIHR)-Strategic Training Program (to M.M.P., G.M.P and J.E.C.) and the Pamela Greenaway-Kohlmeier Translational Breast Cancer Research Unit at the London Regional Cancer Program (to M.M.P., G.M.P., J.E.C. and Y.X.). A.L.A. is supported by a CIHR New Investigator Award and an Early Researcher Award from the Ontario Ministry of Research and Innovation.

所有动物研究均按照加拿大议会关于动物保护的建议进行的，根据受西方大学动物使用小组委员会批准的方案。 1.器官隔离（肺，脑，骨，淋巴结） <ol><li>制备四种无菌50ml锥形管中（每个器官中分离）含有大约30毫升无菌磷酸盐缓冲盐水（PBS）的。使用电子天平预称PBS中每管。 </li><li>安乐死6-12周龄小鼠用CO 2吸入。小鼠应中的CO 2室中放置约1 - 2分钟，或直至鼠标停止移动和呼吸。成功安乐死可以用手指手动检查时，可以进一步通过缺乏心跳的确认。避免颈椎脱臼因为这种方法可能会破裂颈部，​​导致难以去除腋下淋巴结的血管。 注:以前的工作专门用于健康的雌性裸鼠，HSD:无胸腺Nude- Foxn1 NU 13。 </li><li>在无菌组织培养罩，将鼠标放在其上的聚苯乙烯泡沫垫回，传播四肢和用针，以保持他们在的地方。 </li><li>使用无菌镊子和剪刀，剪开腹部皮肤在在生殖器中线向上朝切口。轻轻地拉了回来，从腹部肌肉腹部皮肤并在聚苯乙烯泡沫垫脚的地方。 </li><li>找到腋窝，上臂和腹股沟淋巴结肿大。 注意:淋巴结通常是由脂肪组织包围。腹股沟淋巴结是最容易定位，因为它们是两个血管上拉回腹部皮肤的交界表面找到。腋窝及肱淋巴结组织内位于更深并要求组织温柔操纵。 <ol><li>你已经找到淋巴结后，用剪刀轻轻地，小心地切淋巴结无德从皮肤，脂肪和血管，距鼠标删除它们。要确认正确的解剖，滚动镊子在去除的组织。如果滚过组织镊子时存在硬块，然后淋巴结可能已经被成功删除。 </li><li>放置在冰冷的PBS除去淋巴结。 </li></ol></li><li>使用镊子和剪刀，通过暴露的腹壁在朝向胸部向上运动切割打开腹腔。通过胸骨小心切开，露出胸腔。 </li><li>找到肺部下方的隔膜，减少隔膜。它应该拉向因紧张的肋骨。 </li><li>解除从下方肺和切向气管下面的组织。这使得肺部自由地从胸腔除去。在冰冷的PBS取出心脏和肺整块和地点。该心脏可以从这里或者只是在步骤2中称重前肺部被删除。 </li><li>去除销，保持小鼠就位在聚苯乙烯泡沫垫。打开鼠标和削减跨越从侧面侧翼后腰一路皮肤。 </li><li>使用无菌纱布按住鼠标的躯干，剥离鼠标在鼠标的腿和脚的背部皮肤。 </li><li>使用同一块无菌纱布，保持小腿到位，小心打破鼠标脚的踝关节和近侧剥离在关节皮肤朝着膝关节。 </li><li>用剪刀，除去从冰冷的PBS膝关节和地点免费胫骨。 </li><li>与对侧肢体重复步骤1.11）至1.12）。 </li><li>使用镊子，保持股骨的地方，切掉用剪刀肌肉组织周围并取出股骨，将其放置在冰冷的PBS。 </li><li>重复步骤1.14）与另一侧肢体。 </li><li>使用新的一块消毒纱布中，按住鼠标的头部到位。使用镊子和剪刀，轻轻去除皮肤暴露在S库尔。使用剪刀，小心地切枕骨从顶部中心在一条直线和向下线以暴露后大脑。 </li><li>使用镊子，舀朝前大脑的下面，并删除整个大脑。放置在冰冷的PBS大脑。 </li><li>重复1.1）至1.17）步骤至少四只小鼠。 </li></ol> 2.器官称重<ol><li>以下器官隔离，称量用电子天平含有肺和脑组织各个PBS管。 </li><li>通过减去隔离前体重（仅PBS）中从PBS管+器官（肺和脑）的重量计算的重量差。 </li><li>确定的，从所计算的重量差悬浮组织碎片所需要的介质的量。 注:肺和脑组织的重量重悬在4归:1介质:组织的比例（体积/重量）。 </li></ol> 3. Lung-和Brain-条件培养基的产生<ol><li>在无菌Ť问题培养罩，包含反转肺或脑三次从器官和吸PBS含血液清除残留血液PBS管。用新鲜的冷PBS重复，直到解决方案出现带有无血清晰。 </li><li>将肺和大脑在不同60毫米2的玻璃培养皿中。通过反复切片来回使用两个无菌手术刀片，肉肺或脑部，直至组织碎片约有约1 立方毫米大小。 </li><li>悬浮组织片段在适当体积的Dulbecco氏改良的Eagle氏培养基（DMEM）的（在步骤2.3预先确定的）:补充有1×F12培养基浓缩有丝分裂原的补充和青霉素（50微克/毫升）/链霉素（50微克/毫升）。 </li><li>添加再悬浮肺或脑组织碎片到6孔板的一个孔中。 </li><li>孵育组织片段中的媒体，在37℃下24小时和5％ 的 CO 2。孵育后，收集条件培养基对每个组织和further通过在50ml锥形管中加入新鲜的培养基三个等体积稀释。 </li><li>离心机在4℃下1000 XG在每个器官稀释条件培养基15分钟以除去大的组织碎片。收集介质上清并通过0.22μm注射过滤器进行过滤。 </li><li>池条件培养基从各器官（ 即 ，肺肺和大脑脑）从多个小鼠占鼠标对鼠标的可变性。分装和储存在-80°C，直到使用条件培养基。 </li></ol> 4.代骨髓条件培养基<ol><li>在无菌组织培养罩，从骨修剪多余的组织，并从骨头骨骺（端件）用剪刀。 </li><li>通过各骨的中心推1ml的PBS使用骨头27½ģ针，冲水髓腔。这将允许你收集骨髓基质细胞（BMSC）到含有PBS新管。 </li><li>离心1,000在4℃×g离心5分钟，并用PBS洗BMSC两次。在20ml骨基质细胞生长培养基的悬浮骨髓基质细胞（DMEM:补充有1×F12培养基浓缩有丝分裂原的补充，青霉素（50微克/毫升）/链霉素（50微克/毫升）和10％胎范博芬血清（FBS）的）。 </li><li>板10毫升悬浮骨髓基质细胞在一个T75烧瓶中。 注:在每个T75瓶中，每2只合并和板细胞;对四只小鼠，二T75烧瓶需要（约1×10 7个细胞/烧瓶）中。 </li><li>孵育在骨基质细胞生长培养基的骨髓基质细胞24小时，在37℃和5％的CO 2。孵育后，从两个T75瓶中取出媒体并投入新的T75瓶中，贴上标签这个烧瓶"浮瓶"。鲜骨基质细胞生长介质添加到以前的2瓶和培养所有3瓶在37℃和5％ 的 CO 2。 </li><li>后的细胞达到约70％汇合（约5 - 7天）传代细胞。要做到这一点，除去培养基并用PBS（3 ml的每种洗涤）洗涤细胞两次。移除PBS并加入3ml胰蛋白酶/ EDTA溶液，确保胰蛋白酶覆盖烧瓶的整个表面。细胞解除后离烧瓶（〜2 - 3分钟），通过加入3ml骨基质细胞生长培养基）停止胰蛋白酶反应。离心机900 xg离心在4℃下5分钟，弃去培养基，并悬浮细胞在10ml新鲜骨基质细胞生长培养基。 </li><li>从所有培养瓶中和通道1池细胞:5成三个新的T75烧瓶，并在37℃和5％的CO 2。经过细胞再次达到70％以上，重复步骤4.6和游泳池和通道都贴壁细胞第二次。重板的所有单元四个T75烧瓶孵育37℃和5％的CO 2。骨基质细胞生长培养基应当用于所有步骤。 </li><li>使细胞达到汇合，用PBS洗涤细胞三次，并添加骨基质细胞采集的媒体（DMEM:F12培养基+ 1X集中有丝分裂原苏pplement +青霉素（50微克/毫升）/链霉素（50微克/毫升）;以10ml / T75），确保该媒体是自由的FBS。采集骨髓条件培养基72小时后，在-80℃，直到使用0.22微米的过滤器，游泳池，分装和存储进行过滤。 </li><li>如果需要的话，确认使用针对小鼠的Sca-1，CD105，CD29，和CD73，CD44，抗体使用如先前13描述的标准流式细胞技术的分离的骨髓基质细胞（BMSC）的表型。 </li></ol> 5.代淋巴结空调传媒<ol><li>在无菌组织培养罩，包含反转淋巴结三次从淋巴结穿刺血腥PBS清除残留血液PBS管。用新鲜的冷PBS重复，直到解决方案出现带有无血清晰。 </li><li>放置60 平方毫米的玻璃培养皿中的淋巴结。使用两个无菌手术刀片，肉末淋巴结反复切片来回，直到Tissu酒店Ë片段约为1 立方毫米大小。 </li><li>在一个锥形管，在10ml罗斯韦尔园区纪念研究所1640（RPMI 1640），补充有青霉素（50微克/毫升）的介质/链霉素（50微克/毫升），5×10 -5 M的无菌β巯基乙醇的悬浮组织碎片（ 1.75微升/500毫升培养基）和10％FBS。 </li><li>离心机在4℃下900×g离心5分钟，重悬所有细胞在30毫升介质。添加5毫升介质/孔在6孔板中并孵育在37℃下7天，5％的CO 2。 </li><li>以下7天温育后，弃去培养基，洗涤贴壁细胞用5ml温PBS，加入5毫升的新鲜培养基到每个孔中。使细胞生长到汇合（约5 - 7天），trypsinize，池中的所有细胞，并通过如步骤4.6所述。重板的细胞到6孔板中。重复此步骤三次。 </li><li>经过三年的通道，让细胞生长至融合，用PBS洗孔三次，并加入2毫升/ DMEM的:F12 + 1X浓缩有丝分裂原的补充和青霉素（50微克/毫升）/链霉素（50微克/毫升），确保该媒体是免费的FBS。在72小时后，池，分装，并储存在-80°C，直到使用收集淋巴结条件培养基。 </li><li>如果需要的话，确认使用抗小鼠CD45和GP38，抗体使用如先前13描述的标准流式细胞技术的分离的淋巴结基质细胞（LNSC）的表型。 </li></ol> 6.使用器官的空调媒体对有关癌细胞的转移行为下游化验<ol><li>一旦器官条件培养基中的步骤1中描述已生成 - 5，用它来进行不同的下游细胞和分子生物学分析以确定可溶性器官衍生的因子对肿瘤细胞的转移行为的影响。标准测定法（在文献中其它地方描述的）的实例包括蛋白质阵列13，细胞生长测定&lt;SUP&gt; 13，细胞迁移测定法13，tumorsphere形成14，和RT-PCR 15。原始基础媒体用于生成器官条件培养基（ 即 ，无条件的DMEM:补充有1×F12培养基浓缩有丝分裂原的补充和青霉素（50微克/毫升）/链霉素（50微克/毫升）应该被用作阴性对照。 </li></ol>

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The authors declare that they have no competing financial interests.

转移是一个复杂的过程，其中一系列的细胞事件最终都是用于组织浸润和远处肿瘤形成4,30,31负责。这里提出的离体模型系统可用于转移进展的两个重要方面研究:癌细胞归巢或迁移到特定器官（"到达那里"）和生长在该机构（"生长在那里"）。先前许多研究都集中在识别与癌细胞本身有助于转移过程相关的关键分子的特性。例如，通过琼Massagué的组所做的工作已经确定肺特异性的，骨特异性的，和扩散10-12,32的脑特异性模式有关不同的基因签名。虽然这项工作提供了重要的见解与问候癌细胞对这一进程的贡献，更有人知道的器官特异性因素贡献的T中的作用他二次微环境，从而保持了相对具有挑战性的研究13。 这种体外模型系统先前已经通过展示器官-CM，乳腺癌细胞特异性线路迁移和增长验证作为辅助器官微环境的精确表示反映了转移扩散的格局体内 ，这些细胞系"。由楚和同事的研究使用了四种不同的人类乳腺癌细胞系在体内不同的转移潜力，包括MDA-MB-231和SUM-159（高转移性，转移至淋巴结，肺，肝，骨和脑的体内 ）和SUM-149和MDA-MB-468（适度转移，转移到在淋巴结和体内肺）。无论是MDA-MB-231和SUM-159的细胞系显示的增强的朝向骨髓，淋巴迁移并于节点肺-CM，而SUM-149和MDA-MB-468细胞系对优先迁移肺-CM 13。目前的研究表明，骨和肺寻求的MDA-MB-231变体都倾向于分别迁移到骨髓，肺-CM和暴露于器官CM能够促进骨和肺相关的转移基因的表达以前Massagué和他的同事11,12鉴定。两者合计，这些结果表明，器官-CM提供了一种离体代表的影响的表型和乳腺癌细胞的行为在体内发现的器官特异性可溶性因子的平台。 的特定器官中存在的可溶性因子和表达它们的相对水平鉴定提供了用于确定其对癌细胞行为的潜在影响的起点。此外，使用的技术，如免疫耗竭可以允许用户有效地消耗从器官-CM目的蛋白质，以确定其对体外细胞行为的影响。例如，特定器官微环境中存在许多可溶性因子作为趋化因子，具有诱导细胞迁移和增殖的潜力。使用肺-CM完成以前的工作已导致的存在于肺-CM许多可溶性因子可作为化学引诱物用于乳腺癌细胞，包括骨桥蛋白（OPN）和L-选择素（卖出）13作用的鉴定。通过从肺-CM immunodepleting这些因素，楚和他的同事能够证明减少细胞迁移和/或高转移性MDA-MB-231乳腺癌细胞13的生长。这些结果表明，条件培养基产生从整个器官提供了宝贵的资源用于研究对癌细胞行为的特定可溶性因素的影响，通过使用传统的体外技术。 尽管这是一个相当简单的模型系统中，有在实验方案的一些步骤是为事业有成临界升代器官条件培养基。首先，利用所定义的6-12周为器官收获年龄介于小鼠是为了控制为与年龄相关的影响重要，无论是涉及开发（6周前），或与在老年小鼠中老化。其次，不育是极为重要的整个协议，并且可以通过在无菌组织培养罩使用严格无菌技术和工作被最佳保证，使用在所有的培养基和补充剂灭菌组织培养耗材和试剂以及温和抗生素（ 即 ，青霉素/链霉素）。之一的使用在体内或离体模型系统的挑战之一是固有的动物对动物变性。如果此观察，故障排除步骤包括在下游实验使用之前，从小鼠的多批得到的条件培养基的池，以及确保lung-和脑空调媒体是准确的重量归一化在每个箱1媒体:在一个直径4集实验再悬浮组织的比例（体积/重量）。此外，主淋巴结和骨髓基质细胞不应该被允许在任何给定的通道，以超过70％汇合或待生长超过5总的通道，否则会最终分化。 修改这篇文章中提出的协议可以设想，允许应用程序不同的研究领域。虽然该模型迄今为止专门用于乳腺癌的研究，它可能会被用来研究在其他类型的癌症以及其它正常生理和疾病状态器官衍生的可溶性因子的作用。另外，尽管这种方法已经免疫缺陷裸鼠模型此处使用的，这种技术在理论上并不限于此物种小鼠甚至可能被与其它类型的动物模型的使用。 虽然这种模式提供性重要nt的见解，以特定的器官来源的可溶性因子对肿瘤细胞的行为的贡献，但也不是没有其局限性。首先这种模式仅仅着眼于器官微环境的可溶性成分，它忽略了细胞外基质（ECM）的重要性。对ECM是非蜂窝存在的所有组织类型和功能中的组件，以提供对细胞的物理支架，以及以诱导关键生化和生物力学线索关于各种细胞过程，包括形态，细胞分化和内环境稳定33所要求的能力-35。重要的是，细胞外基质的物理和生化组合物是广泛异构并且可以组织类型之间变化很大。因此，ECM的组织特异性组合物也可具有可与该模型忽略癌转移期间器官趋向性的影响。将ECM也可充当集中具体可溶性因子的方式，appropriatelŸ呈现他们某些细胞，没有ECM的这种微调，细胞信号的感应可能会减少或改变36。虽然在本文中提到的模型缺乏适当的ECM成分，许多研究可以通过使用同时利用与适当的ECM成分一起在这里提到的模型免费的方式得到加强。例如，合成的内源性ECM或重组ECM组分市售主要器官的成纤维细胞可在除了产生器官-CM被用于确定对细胞行为的整个器官微环境的全部范围。 这可能是由器官-CM生成的可溶性微环境可能无法恰当地模仿什么是转移的过程中，本生理学。癌细胞的生存，生长和发展高度依赖于癌细胞-宿主相互作用4,30,31。整个器官的CM代可能会导致在p的可溶性因子resence通常不由该转移进展过程中与肿瘤细胞相互作用的细胞类型中表达。此外，先前已经证明，原发肿瘤的存在可调节远处转移部位的微环境，从而为转移37"吸"它们。由于当前协议使用来自健康的，非肿瘤携带小鼠的器官，这将是有价值的，比较从器官从荷一个原发性乳腺肿瘤衍生的可溶性因子。 由于整个器官的各种器官CM的生成过程中机械分离，细胞内因素可能被释放到可能不正常地生理癌细胞遇到周围介质。此外，骨和淋巴结衍生的条件培养基需要来自这些器官的收集前分离基质细胞的培养。在体外文化前进存在于细胞类型环可能不准确地表示由癌细胞生理学遇到细胞类型的广度。而基质细胞主要分泌许多因素与各种器官微环境相关联，使用这些方法可能不占组织和器官38内存在的其它细胞类型的影响。最后，我们包括在用于产生器官条件培养基的基础介质有丝分裂原的补充，因为我们发现，这是需要的，以保持这两个培养器官（肺和脑）和分离的BMSC和LNSC的可行性。然而，我们认识到，需要提醒的是这种补充有丝分裂原的存在可能刺激器官人工产生可溶性因子，他们可能无法正常做， 总之，许多研究表明，特定器官微环境可以深刻影响肿瘤细胞生物学。尽管讨论局限， 体外模型系统这里介绍řepresents用于研究对细胞行为的许多实质器官的可溶性微环境的影响进行全面的技术。在作者的实验室，该模型系统已经和继续被用作研究许多与转移有关的细胞事件，包括侵入，迁移，增殖的手段，干样行为，以及基因表达的变化。从这些研究中获得的数据可以反过来通知，以确定其对表示潜在转移部位的各种微环境响应该模型系统，以从患者的肿瘤中分离原发性乳腺癌细胞的潜在的临床应用。两者合计，故希望转移性器官趋向性的过程中，微环境和肿瘤细胞之间的相互作用的进一步理解，将导致改善的未来的癌症的治疗。

乳腺癌是女性最常见的癌症和癌症相关死亡1的第二大原因。乳腺癌的死亡率高的主要原因是常规治疗，以减轻和消除转移性疾病的故障;癌症相关死亡的约90％是由于转移2。理解转移级联的分子机制是至关重要的，以有效地早期和晚期乳腺癌治疗剂的开发。 过去的研究已经帮助阐明乳腺癌转移的多步性质以及它是假设，既癌症进展和转移的结果在很大程度上取决于癌细胞和主机环境3之间的相互作用。临床观察表明，许多癌症显示器官趋向性， 即 ，该倾向优先转移到特定organs.In的CAS乳腺癌E，病人的疾病通常传播或转移至5个主要景点，包括骨，肺，淋巴结，肝，脑4-6。许多理论已​​经发展到解释这个过程，但只有少数经受住了时间的考验。尤文转移的理论，在20世纪20年代提出的，假设转移thatthe分布严格，由于机械因素;由此肿瘤细胞通过正常定义生理血流模式整个身体携带并在第一毛细管床简单地阻止他们遇到7。相比之下，斯蒂芬·佩吉特氏1889"种子和土壤"假说认为，额外的分子相互作用负责存活和转移生长，因此癌细胞（"种子"），只能建立自己和产生相应的分子因素proliferatein器官微环境（"土"）8。将近一个世纪之后，伦纳德在韦斯花了此前公布的尸检资料进行了荟萃分析，证实了尤文的预言，在尸检时发现许多转移性肿瘤的转移是否器官取向是由血流模式单独确定，将有望在预期的比例被发现了。然而，在manyinstances有在某些网站，然后会被尤文提出的力学因素9预期形成更少或更多的转移。这些帐户和理论认为特定器官的微环境起到传播模式和多种癌症，包括乳腺癌的随后的生长和存活的关键作用。 过去的研究工作主要集中于肿瘤细胞衍生因子及其在乳腺癌转移10-12观察到的器官向性的贡献，从器官微环境衍生但是很少有研究探讨因素，可能为建立提供了有利的利基乳腺癌转移。这主要归因于在体外研究器官微环境的组件的技术挑战。 当前文章描述了一种用于研究淋巴结，骨，肺和大脑对人乳腺癌细胞的转移行为的水溶性组分的影响的综合体外模型系统。通过证明不同乳腺癌细胞系显示响应于器官条件培养基器官特异性细胞系特异性和恶性行为对应于它们的体内转移潜能13以前的研究已经证实该模型的系统。该模型系统可用于识别和评估可在乳房和其他类型的癌细胞，包括生长，迁移影响的转移行为发挥作用器官特异性的可溶性因子，干样行为，以及基因表达，以及鉴定潜在的新的治疗靶点为癌症。这是可以用来详细研究器官特异性转移行为，并评价在驱动癌转移的方法的器官来源的可溶性因子的作用的第一个体外模型系统。

<table><tbody><tr><td>50 ml conical tubes</td><td>Thermo Scientific (Nunc)</td><td>339652</td><td>Keep sterile</td></tr><tr><td>1x Phosphate-buffered saline</td><td>ThermoFisher Scientific</td><td>10010-023</td><td>Keep sterile</td></tr><tr><td>Nude mice</td><td>Harlan Laboratories</td><td>Hsd:Athymic Nude-Foxn1nu</td><td>Use at 6 - 12 weeks of age</td></tr><tr><td>Polystyrene foam pad</td><td>N/A</td><td>N/A</td><td>The discarded lid (~ 4 x 8 inches or larger) of a polystyrene foam shipping container can be used for this purpose. Sterilize by wiping with ethanol.</td></tr><tr><td>Forceps</td><td>Fine Science Tools</td><td>11050-10</td><td>Keep sterile</td></tr><tr><td>Scissors</td><td>Fine Science Tools</td><td>14058-11</td><td>Keep sterile</td></tr><tr><td>Gauze pads</td><td>Fisher Scientific</td><td>22-246069</td><td>Keep sterile</td></tr><tr><td>60 mm&lt;sup&gt;2&lt;/sup&gt; glass petri dishes</td><td>Sigma-Aldrich</td><td>CLS7016560</td><td>Keep sterile</td></tr><tr><td>Scalpel blades</td><td>Fisher Scientific</td><td>S95937A</td><td>Keep sterile</td></tr><tr><td>DMEM:F12</td><td>Life Technologies</td><td>21331-020</td><td>Warm in 37 &amp;deg;C water bath before use, keep sterile&amp;nbsp;</td></tr><tr><td>1x Mito + Serum Extender</td><td>BD Biosciences</td><td>355006</td><td>Referred to as &quot;concentrated mitogen supplement&quot; in the manuscript. Keep sterile</td></tr><tr><td>Penicillin-Streptomycin (10,000 U/ml)</td><td>Life Technologies</td><td>15140-122</td><td>Keep sterile</td></tr><tr><td>Rosewell Park Memorial Institute 1640 (RPMI 1640)</td><td>Life Technologies</td><td>11875-093</td><td>Warm in 37 &amp;deg;C water bath before use, keep sterile&amp;nbsp;</td></tr><tr><td>Fetal Bovine Serum</td><td>Sigma-Aldrich</td><td>F1051-500ML</td><td>Keep sterile</td></tr><tr><td>Trypsin/EDTA solution</td><td>ThermoFisher Scientific</td><td>R-001-100</td><td>Warm in 37 &amp;deg;C water bath before use, keep sterile&amp;nbsp;</td></tr><tr><td>6-well tissue culture plates</td><td>Thermo Scientific (Nunc)</td><td>140675</td><td>Keep sterile</td></tr><tr><td>0.22 &amp;mu;m syringe filters</td><td>Sigma-Aldrich</td><td>Z359904</td><td>Keep sterile</td></tr><tr><td>T75 tissue culture flasks</td><td>Thermo Scientific (Nunc)</td><td>178905</td><td>Keep sterile</td></tr><tr><td>Transwells</td><td>Sigma-Aldrich</td><td>CLS3464</td><td>Keep sterile, use for migration assays</td></tr><tr><td>Anti-mouse Sca-1</td><td>R&amp;amp;D Systems</td><td>FAB1226P</td><td>use at 10 &amp;micro;l/10&lt;sup&gt;6&lt;/sup&gt; cells</td></tr><tr><td>Anti-mouse CD105</td><td>R&amp;amp;D Systems</td><td>FAB1320P</td><td>use at 10 &amp;micro;l/10&lt;sup&gt;6&lt;/sup&gt; cells</td></tr><tr><td>Anti-mouse CD29</td><td>R&amp;amp;D Systems</td><td>FAB2405P-025</td><td>use at 10 &amp;micro;l/10&lt;sup&gt;6&lt;/sup&gt; cells</td></tr><tr><td>Anti-mouse CD73</td><td>R&amp;amp;D Systems</td><td>FAB4488P</td><td>use at 10 &amp;micro;l/10&lt;sup&gt;6&lt;/sup&gt; cells</td></tr><tr><td>Anti-mouse CD44</td><td>R&amp;amp;D Systems</td><td>MAB6127-SP</td><td>use at 0.25 &amp;micro;g/10&lt;sup&gt;6&lt;/sup&gt; cells</td></tr><tr><td>Anti-mouse CD45</td><td>eBioscience</td><td>11-0451-81</td><td>use at 5 &amp;micro;l/10&lt;sup&gt;6&lt;/sup&gt; cells</td></tr><tr><td>Anti-mouse gp38</td><td>eBioscience</td><td>12-5381-80</td><td>use at 10 &amp;micro;l/10&lt;sup&gt;6&lt;/sup&gt; cells</td></tr><tr><td>&amp;beta;-mercaptoethanol&amp;nbsp;</td><td>Sigma-Aldrich</td><td>M6250&amp;nbsp;</td><td>Keep sterile</td></tr><tr><td>Protein arrays</td><td>RayBiotech Inc.</td><td>AAM-BLM-1-2</td><td>Use 1 array per media condition (including negative control), in triplicate</td></tr></tbody></table>

generation of organ-conditioned media and applications for studying organ-specific influences on breast cancer metastatic behavior

Breast cancer preferentially metastasizes to the lymph node, bone, lung, brain and liver in breast cancer patients. Previous research efforts have focused on identifying factors inherent to breast cancer cells that are responsible for this observed metastatic pattern (termed organ tropism), however much less is known about factors present within specific organs that contribute to this process. This is in part because of a lack of in vitro model systems that accurately recapitulate the organ microenvironment. To address this, an ex vivo model system has been established that allows for the study of soluble factors present within different organ microenvironments. This model consists of generating conditioned media from organs (lymph node, bone, lung, and brain) isolated from normal athymic nude mice. The model system has been validated by demonstrating that different breast cancer cell lines display cell-line specific and organ-specific malignant behavior in response to organ-conditioned media that corresponds to their in vivo metastatic potential. This model system can be used to identify and evaluate specific organ-derived soluble factors that may play a role in the metastatic behavior of breast and other types of cancer cells, including influences on growth, migration, stem-like behavior, and gene expression, as well as the identification of potential new therapeutic targets for cancer. This is the first ex vivo model system that can be used to study organ-specific metastatic behavior in detail and evaluate the role of specific organ-derived soluble factors in driving the process of cancer metastasis.

器官条件培养基的产生 器官分离和产生条件培养基的方法的概要图/示意图示于图1，与图2所示的过程的代表性照片图像。应该注意的是，当这个协议是第一下发展，肝被列入在我们的分析，因为它是乳腺癌转移的常见部位。然而，由于大量产生和由肝脏分泌的蛋白酶，它是非常困难的，以保持肝脏活足够长，以产生良好的质量的条件培养基。肝条件培养基也并不稳定，一旦分离，并且有趋向于大量的蛋白质沉淀物，可能出于同样的原因。因此肝不包括在任何进一步的分析。 图3A）和骨髓基质细胞展示较小的外观（ 图3C）。流式细胞分析证实，LNSCs基本上CD45 -和GP38 +，具有〜具有一个CD45细胞的60％ - GP38 +表示LNSCs 16（ 图3B）的表型。骨髓干细胞应表达CD44和CD29，弱阳性CD105和的Sca-1，和阴性CD73（ 图3D - 1H），指示性的BMSCs 17,18。 乳腺癌细胞RESPONSE器官相适应的移民模式与基因表达的变化使用这种体外模型系统，它先前已被证明是人类乳腺癌细胞具有不同遗传背景的展览微分迁移和生长模式向特定器官的条件。值得注意的是，这些模式反映体内 13这些细胞系的已知的转移性传播模式。在本研究中，骨（231-BOM） -和肺（231-LM2）-seeking的MDA-MB-231人乳腺癌细胞变异变种（从琼Massagué博士11,12一种礼物）也被在转孔迁移实验来确认器官相应的迁移模式。的结果表明，该骨寻求231-表变异优先迁移到超过brain-骨髓条件培养基和lung-条件培养基（ 图4A，左 ）。同样，肺寻求231-LM2变体优先迁移至过骨髓条件培养基和脑条件培养基肺条件培养基（ 图4A，右 ）。此外，RT-qPCR的分析表明，亲本的MDA-MB-231乳腺癌细胞暴露于骨或肺条件培养基导致与骨特异性转移相关基因的上调（CXCR4，IL-11，TGFß1）或lung-乳腺癌细胞在体内 11,12（ 图4B）的特定转移（VCAM1）。这些结果证明了体外系统的与问候体内乳腺癌细胞的预期器官趋向性的有效性，并表明在器官条件培养基本可溶性因子可以除了促进迁移影响转移性细胞表型。 器官空调介质包含转移行为的潜在可溶性介质 </stroNG&gt; 一种基于标签的生物素的小鼠抗体阵列被用来评估从不同器官的存在和常见可溶性因子身份在器官条件培养基。这个阵列包括针对最常见的可溶性小鼠proteins.This蛋白质阵列的308抗体先前已用于鉴定的转移性本肺和骨髓条件培养基内级联相关兴趣可溶性因子所示Chu 等人 （2014）并分别13,14皮奥等人 （2015）。在目前的研究中，蛋白质阵列结果示为相比淋巴于节点（ 图5B）和脑条件培养基（ 图5C）基础培养基（ 图5A）。每个阵列旁突出存在于媒体已被发现与在文献19-29中转移级联步骤相关联的可溶性因子。这些可溶的FAC器可能是值得研究，因为这些器官部位内的二次转移的潜在介质。 <img alt="图1" src="/files/ftp_upload/54037/54037fig1.jpg" /> 图1.条件培养基器官分离和生成过程的概述示意图。组织（肺，脑，股骨，胫骨和淋巴结）从鼠标的收获。肺，脑和淋巴结剁碎用手术刀。骨髓是从骨子里的髓腔通过与27½摹针腔推PBS收获。肺和脑组织片段孵育一段24小时的新鲜基础培养基三个等效体积稀释之前，收集使lung-和脑条件培养基。与基本培养基培养，使骨头和淋巴结空调在媒体面前的3倍 - 骨髓和淋巴结基质细胞传代2。 收获介质可以是pooled并储存在-80°C，直到准备在下游试验，例如蛋白质芯片，细胞生长实验，细胞迁移实验，tumorsphere形成和RT-PCR的使用。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54037/54037fig1large.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/54037/54037fig2.jpg" /> 图的程序条件培养基器官分离和代2.代表摄影作品。在第0天，肺，脑，股骨，胫骨和淋巴结是从小鼠取。肺，脑和淋巴结被放置在60毫米2的玻璃培养皿中，并剁碎两个手术刀刀片约1mm 3的尺寸。骨髓是从骨子里的髓腔通过用27½摹针推PBS通过腔到一个新的管收获PBS的。肺和脑组织碎片保温在37℃和5％CO 2的一个周期24小时用新鲜培养基三个等效体积稀释之前，收集使lung-和脑条件培养基后24小时。骨髓和淋巴结基质细胞在37℃下温育，5％ 的 CO 2和传代2 -用无血清基础培养基替换培养基，使骨和淋巴结条件培养基之前3次（〜3周内总计）。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54037/54037fig2large.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/54037/54037fig3.jpg" /> 图3.表征隔离淋巴结及骨髓基质干细胞。 （A）明亮的视野显微镜图像显示淋巴结基质细胞的形态（LNSCs）。（B）代表流流式细胞术用藻红蛋白（PE）缀合的GP38抗体和异硫氰酸荧光素（FITC）LNSCs的分析缀合CD45抗体（C）明场显微术图像，其显示骨髓基质细胞的形态（骨髓基质干细胞）。（DH）代表流流式细胞仪用PE共轭（黑色曲线）对抗体的骨髓基质干细胞分析（D）CD44，（E）CD106，（F）的Sca-1，（G）CD29，（H）CD73抗体相对于同型对照（白色曲线）。 10000可行的事件至少每个样品收集。这个数字已经从13修改。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54037/54037fig3large.jpg" target="_blank">请点击此处查看本图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/54037/54037fig4.jpg" /> 图4.或者甘相适应的移民模式与基因表达的变化响应器官条件培养基。 （A）骨寻求231-BOM变种（ 左，条纹条 ）或MDA-MB-231人乳腺癌细胞系肺寻求231-LM2变种（ 右，条纹条 ）以及父母MDA-MB- 231细胞（ 两个面板，实心柱 ）接种到明胶包被的插入物（8微米的孔）放置之前在基础培养基（DMEM:F12 +浓丝裂增刊）或器官-CM。平板在37℃保温和5％CO 2的18小时。（B）的所有人口父母的MDA-MB-231细胞暴露于任一骨-CM（ 左 ）或肺-CM（ 右 ）48小时。 RNA的分离并用RT-qPCR定量。响应于器官-CM（ 黑色条 ）的基因表达的变化进行标准化为GAPDH，并表示相对于在基础培养基（ 灰色柱 ）基线表达。数据表示为平均值±SEM（N = 3;折叠式的变化，从基底介质相应阴性对照）。 * =从基础培养基显著不同; δ=从用相同的培养基条件处理的亲代细胞系显著不同; P &lt;0.05，ANOVA。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54037/54037fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图5" src="/files/ftp_upload/54037/54037fig5.jpg" /> 图5.器官空调媒体包含转移行为的潜在可溶性介质，一个基于标签的生物素的小鼠抗体阵列被用来确定存在于淋巴结CM和脑-CM可溶性因子。 在 4 CO / N，（B）淋巴结-CM或（C）脑CM和使用chemilum可视化:膜用（A）基本培养基（F12 +浓丝裂原补充DMEM）的生物素标记的样品孵育inescence。蓝色椭圆表示内部阳性对照，内衬框表示内部阴性对照和固体框表示的那些可能参与乳腺癌细胞淋巴结或脑的转移行为兴趣可溶性因子， <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54037/54037fig5large.jpg" target="_blank">请点击这里查看这个大版本数字。</a>

Watch this Scientific Journal Video about Generation of Organ-conditioned Media and Applications for Studying Organ-specific Influences on Breast Cancer Metastatic Behavior at JoVE.com

Generation of Organ-conditioned Media and Applications for Studying Organ-specific Influences on Breast Cancer Metastatic Behavior

This manuscript describes an ex vivo model system comprised of organ-conditioned media derived from the lymph node, bone, lung, and brain of mice. This model system can be used to identify and study organ-derived soluble factors and their effects on the organ tropism and metastatic behavior of cancer cells.

器官空调，媒体和应用的产生对乳腺癌转移行为研究器官特异性的影响

Breast cancer preferentially metastasizes to the lymph node, bone, lung, brain and liver in breast cancer patients. Previous research efforts have focused ...

generation-organ-conditioned-media-applications-for-studying-organ

Universidad San Sebastian

Research

JoVE Journal

Medicine

8.4K 次观看.  London Health Sciences Centre. This manuscript describes an ex vivo model system comprised of organ-conditioned media derived from the lymph node, bone, lung, and brain of mice. This model system can be used to identify and study organ-derived soluble factors and their effects on the organ tropism and metastatic behavior of cancer cells. 

视频: Generation of Organ-conditioned Media and Applications for Studying Organ-specific Influences on Breast Cancer Metastatic Behavior

Generation of Tumor Organoids from Genetically Engineered Mouse Models of Prostate Cancer

Methods based on homologous recombination to modify genes have significantly furthered biological research. Genetically engineered mouse models (GEMMs) are a rigorous method for studying mammalian development and disease. Our laboratory has developed several GEMMs of prostate cancer (PCa) that lack expression of one or multiple tumor suppressor genes using the site-specific Cre-loxP recombinase system and a prostate-specific promoter. In this article, we describe our method for necropsy of these PCa GEMMs, primarily focusing on dissection of mouse prostate tumors. New methods developed over the last decade have facilitated the culture of epithelial-derived cells to model organ systems in vitro in three dimensions. We also detail a 3D cell culture method to generate tumor organoids from mouse PCa GEMMs. Pre-clinical cancer research has been dominated by 2D cell culture and cell line-derived or patient-derived xenograft models. These methods lack tumor microenvironment, a limitation of using these techniques in pre-clinical studies. GEMMs are more physiologically-relevant for understanding tumorigenesis and cancer progression. Tumor organoid culture is an in vitro model system that recapitulates tumor architecture and cell lineage characteristics. In addition, 3D cell culture methods allow for growth of normal cells for comparison to tumor cell cultures, rarely possible using 2D cell culture techniques. In combination, use of GEMMs and 3D cell culture in pre-clinical studies has the potential to improve our understanding of cancer biology.&#160;

We show a method for necropsy and dissection of mouse prostate cancer models, focusing on prostate tumor dissection. A step-by-step protocol for generation of mouse prostate tumor organoids is also presented.

从前列腺癌的遗传工程小鼠模型生成肿瘤有机体

Methods based on homologous recombination to modify genes have significantly furthered biological research. Genetically engineered mouse models (GEMMs) ...

科研

遗传学

In Vivo Imaging to Measure Spontaneous Lung Metastasis of Orthotopically-injected Breast Tumor Cells

Metastasis remains the primary cause of cancer-related death. The succession of events that characterize the metastatic cascade presents multiple opportunities for therapeutic intervention, and the ability to accurately model them in mice is critical to evaluate their effects. Here, a step-by-step protocol is presented for the establishment of orthotopic primary breast tumors and the subsequent monitoring of the establishment and growth of metastatic lesions in the lung using in vivo bioluminescence imaging. This methodology allows for the evaluation of treatment or its biological effects along the entire range of metastatic development, from primary tumor escape to outgrowth in the lungs. Breast orthotopic tumors are generated in mice via injection of a luciferase-labeled cell suspension in the 4th mammary gland. Tumors are allowed to grow and disseminate for a specific amount of time and are then surgically resected. Upon resection, spontaneous lung metastasis is detected, and the growth over time is monitored using in vivo bioluminescence imaging. At the desired experimental endpoint, lung tissue can be collected for downstream analysis. The treatment of established, clinically evident metastasis is critical to improve outcomes for stage IV cancer patients, and it can be evaluated through tail vein models of experimental lung metastasis. However, metastatic dissemination occurs early in breast cancer, and many patients have latent, subclinical disseminated disease after surgery. Utilization of spontaneous models such as this one provides the opportunity to study the whole spectrum of the disease, especially the systemic effects driven by treatment of the primary tumor such as pre-metastatic niche priming, and evaluate treatments on dormant and subclinical disease after surgery.

<meta charset="utf8"></head><body>体内成像测量原位注射乳腺肿瘤细胞的自发性肺转移

The manuscript describes a methodology for the establishment as well as longitudinal growth monitoring of spontaneous lung metastasis from orthotopically-injected breast tumors, amenable to intervention at all stages of the metastatic cascade.

体内研究 影像学检查测量原位注射乳腺肿瘤细胞的自发性肺转移

Metastasis remains the primary cause of cancer-related death. The succession of events that characterize the metastatic cascade presents multiple ...

癌症研究

Generating and Imaging Mouse and Human Epithelial Organoids from Normal and Tumor Mammary Tissue Without Passaging

Organoids are a reliable method for modeling organ tissue due to their self-organizing properties and retention of function and architecture after propagation from primary tissue or stem cells. This method of organoid generation forgoes single-cell differentiation through multiple passages and instead uses differential centrifugation to isolate mammary epithelial organoids from mechanically and enzymatically dissociated tissues. This protocol provides a streamlined technique for rapidly producing small and large epithelial organoids from both mouse and human mammary tissue in addition to techniques for organoid embedding in collagen and basement extracellular matrix. Furthermore, instructions for in-gel fixation and immunofluorescent staining are provided for the purpose of visualizing organoid morphology and density. These methodologies are suitable for myriad downstream analyses, such as co-culturing with immune cells and ex vivo metastasis modeling via collagen invasion assay. These analyses serve to better elucidate cell-cell behavior and create a more complete understanding of interactions within the tumor microenvironment.

This protocol discusses an approach for generating epithelial organoids from primary normal and tumor mammary tissue through differential centrifugation. Furthermore, instructions are included for three-dimensional culturing as well as immunofluorescent imaging of embedded organoids.

从正常和肿瘤乳腺组织中生成和成像小鼠和人上皮类器官，无需传代

Organoids are a reliable method for modeling organ tissue due to their self-organizing properties and retention of function and architecture after ...

Establishment and Culture of Patient-Derived Breast Organoids

Breast cancer is a complex disease that has been classified into several different histological and molecular subtypes. Patient-derived breast tumor organoids developed in our laboratory consist of a mix of multiple tumor-derived cell populations, and thus represent a better approximation of tumor cell diversity and milieu than the established 2D cancer cell lines. Organoids serve as an ideal in vitro model, allowing for cell-extracellular matrix interactions, known to play an important role in cell-cell interactions and cancer progression. Patient-derived organoids also have advantages over mouse models as they are of human origin. Furthermore, they have been shown to recapitulate the genomic, transcriptomic as well as metabolic heterogeneity of patient tumors; thus, they are capable of representing tumor complexity as well as patient diversity. As a result, they are poised to provide more accurate insights into target discovery and validation and drug sensitivity assays. In this protocol, we provide a detailed demonstration of how patient-derived breast organoids are established from resected breast tumors (cancer organoids) or reductive mammoplasty-derived breast tissue (normal organoids). This is followed by a comprehensive account of 3D organoid culture, expansion, passaging, freezing, as well as thawing of patient-derived breast organoid cultures.

A detailed protocol is provided here for establishing human breast organoids from patient-derived breast tumor resections or normal breast tissue. The protocol provides comprehensive step-by-step instructions for culturing, freezing, and thawing human patient-derived breast organoids.

患者来源乳腺类器官的建立和培养

Breast cancer is a complex disease that has been classified into several different histological and molecular subtypes. Patient-derived breast tumor ...

Generation of Multicellular Human Primary Endometrial Organoids

The human endometrium is one of the most hormonally responsive tissues in the body and is essential for the establishment of pregnancy. This tissue can also become diseased and cause morbidity and even death. Model systems to study human endometrial biology have been limited to in vitro culture systems of single cell types. In addition, the epithelial cells, one of the major cell types of the endometrium, do not propagate well or retain their physiological traits in culture, and thus our understanding of endometrial biology remains limited. We have generated, for the first time, endometrial organoids that consist of both epithelial and stromal cells of the human endometrium. These organoids do not require any exogenous scaffold materials and specifically organize so that epithelial cells encompass the spheroid-like structure and become polarized with stromal cells in the center that produce and secrete collagen. Estrogen, progesterone and androgen receptors are expressed in the epithelial and stromal cells and treatment with physiological levels of estrogen and testosterone promote the organization of the organoids. This new model system can be used to study normal endometrial biology and disease in ways that were not possible before.

A protocol to generate human primary endometrial organoids that consist of epithelial and stromal cells and retain characteristics of the native endometrial tissue is presented. This protocol describes methods from uterine tissue acquisition to the histologic processing of endometrial organoids.

多细胞人类原发性子宫内膜器官的生成

The human endometrium is one of the most hormonally responsive tissues in the body and is essential for the establishment of pregnancy. This tissue can ...

发育生物学

Generation of Mosaic Mammary Organoids by Differential Trypsinization

Organoids offer self-organizing, three-dimensional tissue structures that recapitulate physiological processes in the convenience of a dish. The murine mammary gland is composed of two distinct epithelial cell compartments, serving different functions: the outer, contractile myoepithelial compartment and the inner, secretory luminal compartment. Here, we describe a method by which the cells comprising these compartments are isolated and then combined to investigate their individual lineage contributions to mammary gland morphogenesis and differentiation. The method is simple and efficient and does not require sophisticated separation technologies such as fluorescence activated cell sorting. Instead, we harvest and enzymatically digest the tissue, seed the epithelium on adherent tissue culture dishes, and then use differential trypsinization to separate myoepithelial from luminal cells with ~90% purity. The cells are then plated in an extracellular matrix where they organize into bilayered, three-dimensional (3D) organoids that can be differentiated to produce milk after 10 days in culture. To test the effects of genetic mutations, cells can be harvested from wild type or genetically engineered mouse models, or they can be genetically manipulated prior to 3D culture. This technique can be used to generate mosaic organoids that allow investigation of gene function specifically in the luminal or myoepithelial compartment.

The mammary gland is a bilayered structure, comprising outer myoepithelial and inner luminal epithelial cells. Presented is a protocol to prepare organoids using differential trypsinization. This efficient method allows researchers to separately manipulate these two cell types to explore questions concerning their roles in mammary gland form and function.

通过微分试穿素生成马赛克乳腺有机体

Organoids offer self-organizing, three-dimensional tissue structures that recapitulate physiological processes in the convenience of a dish. The murine ...

Methods for Culturing Human Femur Tissue Explants to Study Breast Cancer Cell Colonization of the Metastatic Niche

Bone is the most common site of breast cancer metastasis. Although it is widely accepted that the microenvironment influences cancer cell behavior, little is known about breast cancer cell properties and behaviors within the native microenvironment of human bone tissue.We have developed approaches to track, quantify and modulate human breast cancer cells within the microenvironment of cultured human bone tissue fragments isolated from discarded femoral heads following total hip replacement surgeries. Using breast cancer cells engineered for luciferase and enhanced green fluorescent protein (EGFP) expression, we are able to reproducibly quantitate migration and proliferation patterns using bioluminescence imaging (BLI), track cell interactions within the bone fragments using fluorescence microscopy, and evaluate breast cells after colonization with flow cytometry. The key advantages of this model include: 1) a native, architecturally intact tissue microenvironment that includes relevant human cell types, and 2) direct access to the microenvironment, which facilitates rapid quantitative and qualitative monitoring and perturbation of breast and bone cell properties, behaviors and interactions. A primary limitation, at present, is the finite viability of the tissue fragments, which confines the window of study to short-term culture. Applications of the model system include studying the basic biology of&#160;breast cancer and other bone-seeking malignancies within the metastatic niche, and developing therapeutic strategies to effectively target breast cancer cells in bone tissues.

Protocols are described for studying breast cancer cell migration, proliferation and colonization in a human bone tissue explant model system.

方法培养人股骨组织外植体，究其利基转移性乳腺癌细胞定居

Bone is the most common site of breast cancer metastasis. Although it is widely accepted that the microenvironment influences cancer cell behavior, little ...

医学

Growth and Characterization of Irradiated Organoids from Mammary Glands

Organoids derived from the digested tissue are multicellular three-dimensional (3D) constructs that better recapitulate in vivo conditions than cell monolayers. Although they cannot completely model in vivo complexity, they retain some functionality of the original organ. In cancer models, organoids are commonly used to study tumor cell invasion. This protocol aims to develop and characterize organoids from the normal and irradiated mouse mammary gland tissue to evaluate the radiation response in normal tissues. These organoids can be applied to future in vitro cancer studies to evaluate tumor cell interactions with irradiated organoids. Mammary glands were resected, irradiated to 20 Gy and digested in a collagenase VIII solution. Epithelial organoids were separated via centrifugal differentiation, and 3D organoids were developed in 96-well low-adhesion microplates. Organoids expressed the characteristic epithelial marker cytokeratin 14. Macrophage interaction with the organoids was observed in co-culture experiments. This model may be useful for studying tumor-stromal interactions, infiltration of immune cells, and macrophage polarization within an irradiated microenvironment.

Organoids developed from mouse mammary glands were irradiated and characterized to assess epithelial traits and interactions with immune cells. Irradiated organoids can be used to better evaluate cell-cell interactions that may lead to tumor cell recruitment in irradiated normal tissue.

乳腺辐照有机体的生长与表征

Organoids derived from the digested tissue are multicellular three-dimensional (3D) constructs that better recapitulate in vivo conditions than cell ...

生物工程

Generating a Murine Orthotopic Metastatic Breast Cancer Model and Performing Murine Radical Mastectomy

In vivo mouse models to assess breast cancer progression are essential for cancer research, including preclinical drug developments. However, the majority of the practical and technical details are commonly omitted in published manuscripts which, therefore, makes it challenging to reproduce the models, particularly when it involves surgical techniques. Bioluminescence technology allows for the evaluation of small amounts of cancer cells even when a tumor is not palpable. Utilizing luciferase-expressing cancer cells, we establish a breast cancer orthotopic inoculation technique with a high tumorigenesis rate. Lung metastasis is assessed utilizing an ex vivo technique. We, then, establish a mastectomy model with a low local recurrence rate to assess the metastatic tumor burden. Herein, we describe, in detail, the surgical techniques of orthotopic implantation and mastectomy for breast cancer with a high tumorigenesis rate and low local recurrence rates, respectively, to improve breast cancer model efficiency.

We introduce a murine orthotopic breast cancer model and radical mastectomy model with bioluminescence technology to quantify the tumor burden to mimic human breast cancer progression.

小鼠原位转移性乳腺癌模型的建立及小鼠根治性乳房切除术

In vivo mouse models to assess breast cancer progression are essential for cancer research, including preclinical drug developments. However, the majority ...

器官空调，媒体和应用的产生对乳腺癌转移行为研究器官特异性的影响

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此视频中的章节

从前列腺癌的遗传工程小鼠模型生成肿瘤有机体

体内研究影像学检查测量原位注射乳腺肿瘤细胞的自发性肺转移

体内研究影像学检查测量原位注射乳腺肿瘤细胞的自发性肺转移

从正常和肿瘤乳腺组织中生成和成像小鼠和人上皮类器官，无需传代

患者来源乳腺类器官的建立和培养

多细胞人类原发性子宫内膜器官的生成

通过微分试穿素生成马赛克乳腺有机体

方法培养人股骨组织外植体，究其利基转移性乳腺癌细胞定居

乳腺辐照有机体的生长与表征

小鼠原位转移性乳腺癌模型的建立及小鼠根治性乳房切除术

器官空调，媒体和应用的产生对乳腺癌转移行为研究器官特异性的影响

摘要

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体内研究 影像学检查测量原位注射乳腺肿瘤细胞的自发性肺转移

从正常和肿瘤乳腺组织中生成和成像小鼠和人上皮类器官，无需传代

患者来源乳腺类器官的建立和培养

多细胞人类原发性子宫内膜器官的生成

通过微分试穿素生成马赛克乳腺有机体

方法培养人股骨组织外植体，究其利基转移性乳腺癌细胞定居

乳腺辐照有机体的生长与表征

小鼠原位转移性乳腺癌模型的建立及小鼠根治性乳房切除术

体内研究影像学检查测量原位注射乳腺肿瘤细胞的自发性肺转移

体内研究影像学检查测量原位注射乳腺肿瘤细胞的自发性肺转移