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 border="0" cellpadding="0" cellspacing="0" width="1195">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:339px;">50 ml conical tubes</td>
			<td style="width:159px;">Thermo Scientific (Nunc)</td>
			<td style="width:223px;">339652</td>
			<td style="width:475px;">Keep sterile</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:339px;">1X Phosphate-buffered saline</td>
			<td style="width:159px;">ThermoFisher Scientific</td>
			<td style="width:223px;">10010-023</td>
			<td>Keep sterile</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">Nude mice</td>
			<td style="width:159px;">Harlan Laboratories</td>
			<td style="width:223px;">Hsd:Athymic Nude-Foxn1nu</td>
			<td>Use at 6-12 weeks of age</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:43px;width:339px;">Polystyrene foam pad</td>
			<td style="width:159px;">N/A</td>
			<td style="width:223px;">N/A</td>
			<td style="width:475px;">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 height="23">
			<td height="23" style="height:23px;width:339px;">Forceps</td>
			<td style="width:159px;">Fine Science Tools</td>
			<td style="width:223px;">11050-10</td>
			<td style="width:475px;">Keep sterile</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:339px;">Scissors</td>
			<td style="width:159px;">Fine Science Tools</td>
			<td style="width:223px;">14058-11</td>
			<td style="width:475px;">Keep sterile</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:339px;">Gauze pads</td>
			<td style="width:159px;">Fisher Scientific</td>
			<td style="width:223px;">22-246069</td>
			<td style="width:475px;">Keep sterile</td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;width:339px;">60 mm2 glass petri dishes</td>
			<td style="width:159px;">Sigma-Aldrich</td>
			<td style="width:223px;">CLS7016560</td>
			<td style="width:475px;">Keep sterile</td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;width:339px;">Scalpel blades</td>
			<td style="width:159px;">Fisher Scientific</td>
			<td style="width:223px;">S95937A</td>
			<td style="width:475px;">Keep sterile</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">DMEM:F12</td>
			<td style="width:159px;">Life Technologies</td>
			<td style="width:223px;">21331-020</td>
			<td>Warm in 37 &#176;C water bath before use, keep sterile&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:339px;">1 x Mito+ Serum Extender</td>
			<td style="width:159px;">BD Biosciences</td>
			<td style="width:223px;">355006</td>
			<td style="width:475px;">Referred to as &#34;concentrated mitogen supplement&#34; in the manuscript. Keep sterile</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">Penicillin-Streptomycin (10,000 U/mL)</td>
			<td style="width:159px;">Life Technologies</td>
			<td style="width:223px;">15140-122</td>
			<td>Keep sterile</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:339px;">Rosewell Park Memorial Institute 1640 (RPMI 1640)</td>
			<td style="width:159px;">Life Technologies</td>
			<td style="width:223px;">11875-093</td>
			<td>Warm in 37 &#176;C water bath before use, keep sterile&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">Fetal Bovine Serum</td>
			<td style="width:159px;">Sigma-Aldrich</td>
			<td style="width:223px;">F1051-500ML</td>
			<td>Keep sterile</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:339px;">Trypsin/EDTA solution</td>
			<td style="width:159px;">ThermoFisher Scientific</td>
			<td style="width:223px;">R-001-100</td>
			<td>Warm in 37 &#176;C water bath before use, keep sterile&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">6-well tissue culture plates</td>
			<td style="width:159px;">Thermo Scientific (Nunc)</td>
			<td>140675</td>
			<td>Keep sterile</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">0.22 &#956;m syringe filters</td>
			<td>Sigma-Aldrich</td>
			<td>Z359904</td>
			<td>Keep sterile</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:339px;">T75 tissue culture flasks</td>
			<td style="width:159px;">Thermo Scientific (Nunc)</td>
			<td style="width:223px;">178905</td>
			<td>Keep sterile</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">Transwells</td>
			<td style="width:159px;">Sigma-Aldrich</td>
			<td style="width:223px;">CLS3464</td>
			<td>Keep sterile, use for migration assays</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:339px;">Anti-mouse Sca-1</td>
			<td>R&#38;D Systems</td>
			<td>FAB1226P</td>
			<td>use at 10 &#181;l/106 cells</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:339px;">Anti-mouse CD105</td>
			<td>R&#38;D Systems</td>
			<td style="width:223px;">FAB1320P</td>
			<td>use at 10 &#181;l/106 cells</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:339px;">Anti-mouse CD29</td>
			<td>R&#38;D Systems</td>
			<td style="width:223px;">FAB2405P-025</td>
			<td>use at 10 &#181;l/106 cells</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:339px;">Anti-mouse CD73</td>
			<td>R&#38;D Systems</td>
			<td style="width:223px;">FAB4488P</td>
			<td>use at 10 &#181;l/106 cells</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:339px;">Anti-mouse CD44</td>
			<td>R&#38;D Systems</td>
			<td style="width:223px;">MAB6127-SP</td>
			<td>use at 0.25 &#181;g/106 cells</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:339px;">Anti-mouse CD45</td>
			<td>eBioscience</td>
			<td style="width:223px;">11-0451-81</td>
			<td>use at 5 &#181;l/106 cells</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:339px;">Anti-mouse gp38</td>
			<td>eBioscience</td>
			<td style="width:223px;">12-5381-80</td>
			<td>use at 10 &#181;l/106 cells</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">&#946;-mercaptoethanol&#160;</td>
			<td style="width:159px;">Sigma-Aldrich</td>
			<td style="width:223px;">M6250&#160;</td>
			<td>Keep sterile</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">Protein arrays</td>
			<td style="width:159px;">RayBiotech Inc.</td>
			<td style="width:223px;">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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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

Nanyang Technological University

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

An In Vitro System to Study Tumor Dormancy and the Switch to Metastatic Growth

Recurrence of breast cancer often follows a long latent period in which there are no signs of cancer, and metastases may not become clinically apparent until many years after removal of the primary tumor and adjuvant therapy. A likely explanation of this phenomenon is that tumor cells have seeded metastatic sites, are resistant to conventional therapies, and remain dormant for long periods of time 1-4.
The existence of dormant cancer cells at secondary sites has been described previously as quiescent solitary cells that neither proliferate nor undergo apoptosis 5-7. Moreover, these solitary cells has been shown to disseminate from the primary tumor at an early stage of disease progression 8-10 and reside growth-arrested in the patients' bone marrow, blood and lymph nodes 1,4,11. Therefore, understanding mechanisms that regulate dormancy or the switch to a proliferative state is critical for discovering novel targets and interventions to prevent disease recurrence. However, unraveling the mechanisms regulating the switch from tumor dormancy to metastatic growth has been hampered by the lack of available model systems.
in vivo and ex vivo model systems to study metastatic progression of tumor cells have been described previously 1,12-14. However these model systems have not provided in real time and in a high throughput manner mechanistic insights into what triggers the emergence of solitary dormant tumor cells to proliferate as metastatic disease. We have recently developed a 3D in vitro system to model the in vivo growth characteristics of cells that exhibit either dormant (D2.OR, MCF7, K7M2-AS.46) or proliferative (D2A1, MDA-MB-231, K7M2) metastatic behavior in vivo . We demonstrated that tumor cells that exhibit dormancy in vivo at a metastatic site remain quiescent when cultured in a 3-dimension (3D) basement membrane extract (BME), whereas cells highly metastatic in vivo readily proliferate in 3D culture after variable, but relatively short periods of quiescence. Importantly by utilizing the 3D in vitro model system we demonstrated for the first time that the ECM composition plays an important role in regulating whether dormant tumor cells will switch to a proliferative state and have confirmed this in in vivo studies15-17. Hence, the model system described in this report provides an in vitro method to model tumor dormancy and study the transition to proliferative growth induced by the microenvironment.

An In Vitro System to Study Tumor Dormancy and the Switch to Metastatic Growth

A modified 3-D in vitro system is presented in which growth characteristics of several tumor cell lines in reconstituted basement membrane correlate with the dormant or proliferative behavior of the tumor cells at a metastatic secondary site in vivo.

一个<em在体外系统来研究肿瘤休眠和转移性增长

Recurrence of breast cancer often follows a long latent period in which there are no signs of cancer, and metastases may not become clinically apparent ...

科研

医学

Generation of Alginate Microspheres for Biomedical Applications

Alginate-based materials have received considerable attention for biomedical applications because of their hydrophilic nature, biocompatibility, and physical architecture. Applications include cell encapsulation, drug delivery, stem cell culture, and tissue engineering scaffolds. In fact, clinical trials are currently being performed in which islets are encapsulated in PLO coated alginate microbeads as a treatment of type I diabetes. However, large numbers of islets are required for efficacy due to poor survival following transplantation. The ability to locally stimulate microvascular network formation around the encapsulated cells may increase their viability through improved transport of oxygen, glucose and other vital nutrients. Fibroblast growth factor-1 (FGF-1) is a naturally occurring growth factor that is able to stimulate blood vessel formation and improve oxygen levels in ischemic tissues. The efficacy of FGF-1 is enhanced when it is delivered in a sustained fashion rather than a single large-bolus administration. The local long-term release of growth factors from islet encapsulation systems could stimulate the growth of blood vessels directly towards the transplanted cells, potentially improving functional graft outcomes. In this article, we outline procedures for the preparation of alginate microspheres for use in biomedical applications. In addition, we describe a method we developed for generating multilayered alginate microbeads. Cells can be encapsulated in the inner alginate core, and angiogenic proteins in the outer alginate layer. The release of proteins from this outer layer would stimulate the formation of local microvascular networks directly towards the transplanted islets.

In the following sections, we outline procedures for the preparation of alginate microspheres for use in biomedical applications. We specifically illustrate a technique for creating multilayered alginate microspheres for the dual purpose of cell and protein encapsulation as a potential treatment for type 1 diabetes.

生物医学应用海藻酸钠微球的一代

Alginate-based materials have received considerable attention for biomedical applications because of their hydrophilic nature, biocompatibility, and ...

Initiation of Metastatic Breast Carcinoma by Targeting of the Ductal Epithelium with Adenovirus-Cre: A Novel Transgenic Mouse Model of Breast Cancer

Breast cancer is a heterogeneous disease involving complex cellular interactions between the developing tumor and immune system, eventually resulting in exponential tumor growth and metastasis to distal tissues and the collapse of anti-tumor immunity. Many useful animal models exist to study breast cancer, but none completely recapitulate the disease progression that occurs in humans. In order to gain a better understanding of the cellular interactions that result in the formation of latent metastasis and decreased survival, we have generated an inducible transgenic mouse model of YFP-expressing ductal carcinoma that develops after sexual maturity in immune-competent mice and is driven by consistent, endocrine-independent oncogene expression. Activation of YFP, ablation of p53, and expression of an oncogenic form of K-ras was achieved by the delivery of an adenovirus expressing Cre-recombinase into the mammary duct of sexually mature, virgin female mice. Tumors begin to appear 6 weeks after the initiation of oncogenic events. After tumors become apparent, they progress slowly for approximately two weeks before they begin to grow exponentially. After 7-8 weeks post-adenovirus injection, vasculature is observed connecting the tumor mass to distal lymph nodes, with eventual lymphovascular invasion of YFP+ tumor cells to the distal axillary lymph nodes. Infiltrating leukocyte populations are similar to those found in human breast carcinomas, including the presence of &#945;&#946; and &#947;&#948; T cells, macrophages and MDSCs. This unique model will facilitate the study of cellular and immunological mechanisms involved in latent metastasis and dormancy in addition to being useful for designing novel immunotherapeutic interventions to treat invasive breast cancer.

Activation of latent mutations with adenovirus-Cre into the mammary ductal system results in a clinically relevant metastatic breast cancer. Incorporation of a YFP promoter allows tracking of distal metastatic tumor cells. This model is useful to study latent metastasis, anti-tumor immunity, and for designing novel immunotherapies to treat breast cancer.

乳腺癌的一种新的转基因小鼠模型:转移性乳腺癌通过瞄准导管上皮与腺病毒Cre重组酶的启动

Breast cancer is a heterogeneous disease involving complex cellular interactions between the developing tumor and immune system, eventually resulting in ...

Profiling of Estrogen-regulated MicroRNAs in Breast Cancer Cells

Estrogen plays vital roles in mammary gland development and breast cancer progression. It mediates its function by binding to and activating the estrogen receptors (ERs), ER&#945;, and ER&#946;. ER&#945; is frequently upregulated in breast cancer and drives the proliferation of breast cancer cells. The ERs function as transcription factors and regulate gene expression. Whereas ER&#945;&#39;s regulation of protein-coding genes is well established, its regulation of noncoding microRNA (miRNA) is less explored. miRNAs play a major role in the post-transcriptional regulation of genes, inhibiting their translation or degrading their mRNA. miRNAs can function as oncogenes or tumor suppressors&#160;and are also promising biomarkers. Among the miRNA assays available, microarray and quantitative real-time polymerase chain reaction (qPCR) have been extensively used to detect and quantify miRNA levels. To identify miRNAs regulated by estrogen signaling in breast cancer, their expression in ER&#945;-positive breast cancer cell lines were compared before and after estrogen-activation using both the &#181;Paraflo-microfluidic microarrays and Dual Labeled Probes-low density arrays. Results were validated using specific qPCR assays, applying both Cyanine dye-based and Dual Labeled Probes-based chemistry. Furthermore, a time-point assay was used to identify regulations over time. Advantages of the miRNA assay approach used in this study is that it enables a fast screening of mature miRNA regulations in numerous samples, even with limited sample amounts. The layout, including the specific conditions for cell culture and estrogen treatment, biological and technical replicates, and large-scale screening followed by in-depth confirmations using separate techniques, ensures a robust detection of miRNA regulations,&#160;and eliminates false positives and other artifacts. However, mutated or unknown miRNAs, or regulations at the primary and precursor transcript level, will not be detected. The method presented here represents a thorough investigation of estrogen-mediated miRNA regulation.

Molecular signaling through both estrogen and microRNAs are critical in breast cancer development and growth. Estrogen activates the estrogen receptors, which are transcription factors. Many transcription factors can regulate the expression of microRNAs, and estrogen-regulated microRNAs can be profiled using different large-scale techniques.

在乳腺癌细胞雌激素调节小分子RNA的剖析

Estrogen plays vital roles in mammary gland development and breast cancer progression. It mediates its function by binding to and activating the estrogen ...

Quantification of Breast Cancer Cell Invasiveness Using a Three-dimensional (3D) Model

It is now well known that the cellular and tissue microenvironment are critical regulators influencing tumor initiation and progression. Moreover, the extracellular matrix (ECM) has been demonstrated to be a critical regulator of cell behavior in culture and homeostasis in vivo. The current approach of culturing cells on two-dimensional (2D), plastic surfaces results in the disturbance and loss of complex interactions between cells and their microenvironment. Through the use of three-dimensional (3D) culture assays, the conditions for cell-microenvironment interaction are established resembling the in vivo microenvironment. This article provides a detailed methodology to grow breast cancer cells in a 3D basement membrane protein matrix, exemplifying the potential of 3D culture in the assessment of cell invasion into the surrounding environment. In addition, we discuss how these 3D assays have the potential to examine the loss of signaling molecules that regulate epithelial morphology by immunostaining procedures. These studies aid to identify important mechanistic details into the processes regulating invasion, required for the spread of breast cancer.

Quantification of Breast Cancer Cell Invasiveness Using a Three-dimensional 3D Model

This article provides detailed methodologies for the use of three-dimensional (3D) assays to quantify breast cancer cell invasion. Specifically, we discuss the procedures required to set up such assays, quantification, and data analysis, as well as methods to examine the loss of membrane integrity that occurs when cells invade.

乳腺癌细胞侵袭定量使用三维（3D）模型

It is now well known that the cellular and tissue microenvironment are critical regulators influencing tumor initiation and progression. Moreover, the ...

Voluntary Breath-hold Technique for Reducing Heart Dose in Left Breast Radiotherapy

Breath-holding techniques reduce the amount of radiation received by cardiac structures during tangential-field left breast radiotherapy. With these techniques, patients hold their breath while radiotherapy is delivered, pushing the heart down and away from the radiotherapy field. Despite clear dosimetric benefits, these techniques are not yet in widespread use. One reason for this is that commercially available solutions require specialist equipment, necessitating not only significant capital investment, but often also incurring ongoing costs such as a need for daily disposable mouthpieces. The voluntary breath-hold technique described here does not require any additional specialist equipment. All breath-holding techniques require a surrogate to monitor breath-hold consistency and whether breath-hold is maintained. Voluntary breath-hold uses the distance moved by the anterior and lateral reference marks (tattoos) away from the treatment room lasers in breath-hold to monitor consistency at CT-planning and treatment setup. Light fields are then used to monitor breath-hold consistency prior to and during radiotherapy delivery.

The current priority in breast cancer radiotherapy is to reduce cardiac doses without compromising target tissue coverage. The voluntary breath-hold technique described here is a simple, inexpensive solution to this problem and capable of being instituted widely without the need for specialized equipment.

自愿屏气技术减少心脏剂量的左乳房放射治疗

Breath-holding techniques reduce the amount of radiation received by cardiac structures during tangential-field left breast radiotherapy. With these ...

Tissue-simulating Phantoms for Assessing Potential Near-infrared Fluorescence Imaging Applications in Breast Cancer Surgery

Inaccuracies in intraoperative tumor localization and evaluation of surgical margin status result in suboptimal outcome of breast-conserving surgery (BCS). Optical imaging, in particular near-infrared fluorescence (NIRF) imaging, might reduce the frequency of positive surgical margins following BCS by providing the surgeon with a tool for pre- and intraoperative tumor localization in real-time. In the current study, the potential of NIRF-guided BCS is evaluated using tissue-simulating breast phantoms for reasons of standardization and training purposes.
Breast phantoms with optical characteristics comparable to those of normal breast tissue were used to simulate breast conserving surgery. Tumor-simulating inclusions containing the fluorescent dye indocyanine green (ICG) were incorporated in the phantoms at predefined locations and imaged for pre- and intraoperative tumor localization, real-time NIRF-guided tumor resection, NIRF-guided evaluation on the extent of surgery, and postoperative assessment of surgical margins. A customized NIRF camera was used as a clinical prototype for imaging purposes.
Breast phantoms containing tumor-simulating inclusions offer a simple, inexpensive, and versatile tool to simulate and evaluate intraoperative tumor imaging. The gelatinous phantoms have elastic properties similar to human tissue and can be cut using conventional surgical instruments. Moreover, the phantoms contain hemoglobin and intralipid for mimicking absorption and scattering of photons, respectively, creating uniform optical properties similar to human breast tissue. The main drawback of NIRF imaging is the limited penetration depth of photons when propagating through tissue, which hinders (noninvasive) imaging of deep-seated tumors with epi-illumination strategies.

Near-infrared fluorescence (NIRF) imaging may improve therapeutic outcome of breast cancer surgery by enabling intraoperative tumor localization and evaluation of surgical margin status. Using tissue-simulating breast phantoms containing fluorescent tumor-simulating inclusions, potential clinical applications of NIRF imaging in breast cancer patients can be assessed for standardization and training purposes.

组织鬼怪模拟，以评估乳癌手术潜在的近红外荧光成像中的应用

Inaccuracies in intraoperative tumor localization and evaluation of surgical margin status result in suboptimal outcome of breast-conserving surgery ...

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 ...

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 ...

An In Vitro Dormancy Model of Estrogen-sensitive Breast Cancer in the Bone Marrow: A Tool for Molecular Mechanism Studies and Hypothesis Generation

The study of breast cancer dormancy in the bone marrow is an exceptionally difficult undertaking due to the complexity of the interactions of dormant cells with their microenvironment, their rarity and the overwhelming excess of hematopoietic cells. Towards this end, we developed an in vitro 2D clonogenic model of dormancy of estrogen-sensitive breast cancer cells in the bone marrow. The model consists of a few key elements necessary for dormancy. These include 1) the use of estrogen sensitive breast cancer cells, which are the type likely to remain dormant for extended periods, 2) incubation of cells at clonogenic density, where the structural interaction of each cell is primarily with the substratum, 3) fibronectin, a key structural element of the marrow and 4) FGF-2, a growth factor abundantly synthesized by bone marrow stromal cells and heavily deposited in the extracellular matrix. Cells incubated with FGF-2 form dormant clones after 6 days, which consist of 12 or less cells that have a distinct flat appearance, are significantly larger and more spread out than growing cells and have large cytoplasm to nucleus ratios. In contrast, cells incubated without FGF-2 form primarily growing colonies consisting of >30 relatively small cells. Perturbations of the system with antibodies, inhibitors, peptides or nucleic acids on day 3 after incubation can significantly affect various phenotypic and molecular aspects of the dormant cells at 6 days and can be used to assess the roles of membrane-localized or intracellular molecules, factors or signaling pathways on the dormant state or survival of dormant cells. While recognizing the in vitro nature of the assay, it can function as a highly useful tool to glean significant information about the molecular mechanisms necessary for establishment and survival of dormant cells. This data can be used to generate hypotheses to be tested in vivo models.

An In Vitro Dormancy Model of Estrogen-sensitive Breast Cancer in the Bone Marrow: A Tool for Molecular Mechanism Studies and Hypothesis Generation

We developed an in vitro model of dormancy in the bone marrow for estrogen-sensitive breast cancer cells. The goal of this protocol is to demonstrate use of the model for the study of the molecular and cellular biology of dormancy and for generation of hypotheses for subsequent testing in vivo.

一个<em&gt;在体外</em&gt;对雌激素敏感的乳腺癌休眠模式在骨髓中:分子机理研究与假设生成的一个工具

The study of breast cancer dormancy in the bone marrow is an exceptionally difficult undertaking due to the complexity of the interactions of dormant ...