这项工作是具有特别提到希拉里工艺和道格拉斯·迈尔斯爵士支持帝国BRC，国家卫生研究院的研究，并采取行动战胜癌症。

程序如下一直道德经伦敦帝国学院。 1.原发性代微球体从人乳腺癌细胞系注:执行下无菌培养罩以下步骤。 <ol><li>制备微球体媒体​​含有DMEM / F12补充了2mM L-谷氨酰胺，100U / ml青霉素，100U / ml链霉素。 ，10纳克/ ml重组人碱性成纤维细胞生长因子（bFGF; R＆D系统）和1x B27添加剂;通过加入20纳克/ ml重组人表皮生长因子（Sigma公司，EGF）在使用前立即制备完整的媒体。 </li><li> （或您选择的乳腺癌细胞系; 70​​-80％汇合）从瓶中含有粘附MCF-7或MDA-MB-231细胞吸媒体报道，在1X PBS洗两次，trypsinize细胞。 </li><li>离心细胞，在200×g离心，在室温下放置5分钟。倒出上清和悬浮细胞在1-5毫升的微球体介质。移液器上下，如有必要，使用一个40微米的细胞滤网过滤帽，以获得单细胞悬浮液。使用血球计数器，以确保单细胞悬浮液已形成（如果不是，则使用将25g针注射器，所述细胞悬液最多3次）。 </li><li>如果通过FACS必要分离CD44 + CD24-细胞的子集使用抗CD24藻红蛋白（PE）和抗CD44异硫氰酸荧光素（FITC）的单克隆抗体9。可替代地，使用磁激活细胞分选（MACS）系统用抗CD44和抗CD24的生物素合成微珠按制造商的说明分离这样的人口。用LD柱进行正选择使用LS列，并阴性选择和流确认所有分离细胞的表型流式细胞仪17。 </li><li>计算使用台盼蓝每毫升的活细胞的数目。 注:细胞活力被计算为活细胞通过细胞的总数的网格内的hemocy划分的数目垢仪。这样会占用台盼蓝细胞被认为是非可行的。下面的过程是用来精确地确定活细胞的比例。 <ol><li>制备在PBS中的台盼蓝的0.4％的溶液。加入0.1毫升锥虫蓝原液到1ml细胞。加载血球和显微镜低倍率下的立即检查。计数总细胞数和蓝染色的细胞的数目。 活菌= [1.00 - （蓝色细胞数÷总细胞数）]×100。 </li><li>计算每毫升培养物的活细胞的数量，使用下面的公式。记以校正稀释因子。 活细胞数×10 4×1.1 =细胞/ ml培养</li></ol></li><li>悬浮预先确定的细胞量在2毫升完全微球体媒体​​在6孔超低附着板的各孔中。接种密度通常为500-4,000个细胞/厘米每孔2细胞。可选择使用LO瓦特附件24孔板而在0.5ml的培养基接种的细胞在相同的密度。 注:我们推荐接种密度和文化的时间优化每个感兴趣的细胞系。 </li><li>而不干扰所述板孵育超低附件6孔板，在37℃和5％的CO 2为5-10天（取决于细胞系和乳腺球的大小），特别是在第5天的生长期。 </li></ol> 2.原代微球体从人乳腺癌的临床样本注意:人乳腺癌组织中可从接受手术切除乳腺肿瘤的患者中获得。在冰上商店组织长达24小时的50毫升无菌试管中的培养基含有100U / ml青霉素和100U / ml链霉素。执行下一个无菌培养罩以下步骤。 <ol><li>将样品转移到一个100mm组织培养皿用小体积的介质。祛瘀e。使用无菌剪刀，scapel和镊子的脂肪组织。 </li><li>添加2-3毫升的DMEM / F12和剁碎的样品成1mm 3枚无菌scapel或刀片，直到没有大块依然存在。 </li><li>重悬的组织片在预热10毫升的DMEM含有蛋白水解酶（3000单位/毫升胶原酶和1000 U / ml的透明质酸酶），并在旋转振荡器孵育在37℃，直到所有的组织碎片被消化。通常完全消化需要1-3小时。根据肿瘤的病理特征调整消化时间。 （例如乳腺癌通常是难以消化相比，粘液癌是比较脆弱的，需要更短的消化时间）。通过观察下血球20微升悬架评估每一个消化半小时的程度。 </li><li>允许片段沉降5分钟，然后在200×g离心，在室温统转移上清液在一个15毫升的锥形聚丙烯管中并离心10分钟perature。小心倒出上清，悬浮细胞在1 - 5毫升微球体介质。 </li><li>按照上面描述的用于细胞系，以获得和板单细胞悬浮液中的步骤1.1-1.4。穿过25 G细胞如果需要的最大的2倍注射器限制破坏细胞。 </li></ol> 3.微球体形成率（％）计算<ol><li>培养期结束后，算上微球体（大于40微米）的直径在显微镜下放大40倍。数字图像5无规字段使用数码相机上的光显微镜并使用采集软件确定的微球体的大小。使用任何图像分析软件。 </li><li>使用以下等式计算乳腺球形成率（MFE％）: MFE（％）=（＃每孔微球体）/（＃细胞每孔接种）×100 </li></ol>微球体4.自我更新的评估连续传代<ol><li&gt;移液器含有来自每个井的微球体入15ml管中的介质。用PBS洗涤各孔，并加入到收集介质。离心在115×g离心，在室温下10分钟。 </li><li>弃上清，悬浮颗粒在500微升预热的0.5％胰蛋白酶/ 0.2％EDTA的。孵育2-3分钟。 </li><li>加入500微升的FCS以中和胰蛋白酶。离心，在500×g离心5分钟。弃上清，重新悬浮颗粒在100微升微球体介质，移液，上下分列球。 </li><li>计数细胞用血细胞计数器，并确定是否细胞是在单细胞悬浮液。如果不通过将25g注射器最多3次，以获得单细胞。种子的细胞进入一个新的超低附件6孔板在在初级代中使用的相同的密度。 </li><li>经过5-10天，算球&gt; 40微米，使用前报道的公式计算球体形成效率。 </li></ol>

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The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of this article.

中小学微球体的成功评估依赖于细胞被镀在足够低的密度，从单一的克隆微球体的形式，以最小的领域聚集。然而，在密度过低，过少的微球体可能形成区分疗效的统计。播种密度应为使用，因为他们可以有很大的不同在他们的领域形成效率每个细胞系进行优化（那些表达低E-cadherin的可能形成不太稳定，短期微球体培养18）。这种优化阶段应确保从单细胞的克隆生长最初级和次级球形成的结果。电镀每孔一个细胞是完全确保克隆形成微球体的唯一方式，并且可以被推荐用于定量新鲜来源的样品13的自我更新/分化率。然而，这种方法还患有复杂的因素:一个微球体形成的1％的效率将产生每96孔板仅单个微球体，并且效率可与形成在没有旁分泌生长因子悬浮的细胞之间的信号更少微球体被低估。最后，辅助球传代后的频率可以估算自我更新的水平，并应当预期的次级比主球形成更高的效率，如果真实地选择用于细胞与癌干细胞的特性。再凝集是一个特别的问题导出次级球体和细胞类型合适极低的细胞密度应朝向进行优化时。 采取微球体的时间将增长至&gt; 40微米也将变化，并从临床样本微球体可能需要5-12天，取决于球体生长速度之间进行计数。聚集和融合领域中不可避免地发生是由于周围的介质细胞固有的运动，但过程似乎大幅提高前perimenters在生长阶段19移动的板，可避免的东西，因此。 而大球是经常认为干细胞都具有较高的长期复制能力这个假设应该谨慎对待。较大的球体可能反映祖细胞的增殖能力，响应于生长因子，或者是聚集或融合的产物。两者的干细胞和它们的过境放大的子细胞都能够引起微球体20，尽管这两种细胞类型的组合频率可能仍然反映源组织的致瘤性。 该微球体测定由未捕获的癌症干细胞的形成和行为的复杂性在它们的体内利基限制。为了真正评估长期在体外和体内自我更新和前瞻性分离乳腺癌干细胞的分化，MAMMO球试验，因此应与血统跟踪和异种器官移植实验相结合的理想进行。而谱系追踪依赖于标记物准确地反映了肿瘤干细胞亚群，它们大大受益从保持有助于动态形成"骨善意"癌症干细胞在体内的条件。评估免疫功能低下小鼠肿瘤形成的异种移植实验中也将提供许多利基成分的微球体检测缺失，同时还缺乏人体免疫和间质成分21。虽然更多的技术要求很高，这些技术应该尝试对乳腺癌干细胞的特性和致瘤性更完整和翔实的看法。 该微球体法确实提供了一个内容丰富和方便的工具，只要它进行了仔细的上述限制认为:确如我们体内康迪知识经历了癌症干细胞的发展系统蒸发散，检测微球体可以把分子和环境因素的范围越来越广。使用以上概述的方法中，球形形成率相当于肿瘤起始率在异种移植2,15,16，和自我更新球确实增加更具侵略性乳腺癌22和以下的干细胞增加的干预，如化疗23。同时，球本身能提供与研究表达的差异，并参与了干细胞的基因表达体系结构新颖的因素的宝贵资源。

通过干细胞样肿瘤干细胞为首的肿瘤细胞系的存在，极大地增加我们的肿瘤异质性的理解。而在一些肿瘤的遗传多样性并从基因不同克隆的克隆产物出现，大幅组件出现从后生差异导致:癌细胞可以通过激活特定基因或抑制干，祖和分化状态之间转换（有时可逆）表达程序1 - 3。这可能反映了细胞的内在或外在的因素，这反映当前正从邻近癌，间质或免疫细胞递送调节因子表达与在与旁分泌信号结合其所得自分泌信号传导的细胞中的基因表达程序，microenviromental条件如度缺氧2,4,5。 虽然创新的谱系追踪的方法在前进我们研究推定的癌干细胞在它们的体内利基6能力-如图8所示 ，球形成测定法仍估计乳腺癌细胞的潜力，以行为像干细胞，至少在所用的测定条件一种流行和方便的方法。它经常被用来一起回顾性方法用于净化癌症干细胞，通过对已提出的膜标记物CD44和CD24 9，和酶乙醛脱氢酶（醛脱氢酶）的活性水平10，标志物的表达，以对应于更mesenchymal-和上皮分别为11样癌干细胞。球体的形成方法首先被开发作为神经球测定法，使得能够从单个克隆在非粘附，无血清条件，增加了上皮生长因子（EGF）12推定的干细胞的生长，后来被有效地应用于正常和癌乳腺组织。 骨真正的干细胞，认为休息G0期，将不会遇到的因素将有利于激活体内的精确组合。所述微球体测定代替使细胞要么蓄势有丝分裂或已经除以13的生长。这些祖细胞，虽然不是一个真正的静态细胞，可能是细胞阶段，与在测定中使用的表皮生长因子和碱性成纤维细胞生长因子（bFGF）促分裂原增殖。然而，它们包含了一系列激活干细胞相关的信令的通路14。另外，它们的形成的速率涉及当通过在有限稀释测定法效力在小鼠异种移植2,15,16测定它们取自组织的致瘤&lt;/ SUP&gt;。 在这里，我们提供详细的协议，以分离单个细胞，并产生来自人乳腺癌细胞系和乳腺肿瘤的临床样品初级乳腺球。我们还描述了如何执行主微球体的连续传代，以评估自我更新，以及如何计算球体形成效率，允许在不同的接种密度比较（见方案在图1中）。

<table border="0" cellpadding="0" cellspacing="0" width="1049">
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			<td height="21" style="height:21px;width:625px;">Name of Material/ Equipment</td>
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			<td height="21" style="height:21px;">DMEM/F12</td>
			<td style="width:139px;">Lonza</td>
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			<td height="25" style="height:25px;width:625px;">2mM L-Glutamine</td>
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			<td>G8540</td>
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			<td height="22" style="height:22px;width:625px;">20ng/ml recombinant human basic fibroblast growth factor (bFGF)</td>
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			<td>233-FB-025</td>
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			<td height="22" style="height:22px;width:625px;">1x B27 supplement&#160;</td>
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			<td height="27" style="height:27px;">Phosphate buffered saline (PBS);</td>
			<td style="width:139px;">Thermo Scientific</td>
			<td>12399902</td>
			<td></td>
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			<td height="21" style="height:21px;">&#160;0.5% trypsin-0.2%EDTA;</td>
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			<td>59418C</td>
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			<td height="21" style="height:21px;">Fetal Calf Serum</td>
			<td style="width:139px;">First Link UK</td>
			<td>02-00-850</td>
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			<td>93595</td>
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			<td height="21" style="height:21px;">&#160;Low attachment 6 well plates</td>
			<td style="width:139px;">Corning</td>
			<td>CLS3814</td>
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			<td height="21" style="height:21px;">Collagenase type 1A</td>
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			<td>C9891</td>
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			<td height="21" style="height:21px;">Hyaluronidase</td>
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			<td>H3506</td>
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			<td style="width:139px;">Fisher Scientific</td>
			<td>12443170</td>
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			<td height="21" style="height:21px;width:625px;">Sterile scalpel</td>
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			<td style="width:119px;">11758353</td>
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			<td height="21" style="height:21px;width:625px;">Sterile micro-dissecting scissors</td>
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			<td>S3146</td>
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</table>

mammosphere formation assay from human breast cancer tissues and cell lines

类似的健康组织，许多血液和实体恶性肿瘤现在认为是分层组织的，与干细胞样癌细胞自我更新而引起更加分化的后代的一个子集。理解并针对这些癌干细胞在乳腺癌，其可以具有增强化疗和放射抗性相比非干肿瘤散装，已成为一个重要的研究领域。该微球体检测也成为广泛使用的，因为它能够识别追溯sphere-:标志物CD44包括，CD24和乙醛脱氢酶的活性可以用荧光活化细胞分选（FACS）来隔离前瞻性当细胞植入免疫缺陷小鼠显示增强的致瘤性进行评估形成细胞，从单一的干细胞样克隆的发展。在这里，我们列出的微球体从细胞系或小学患者样本，他们的传代，并计算估计范围f显示相应的培养方法orming效率（SFE）。首先我们讨论关键的考虑和陷阱在适当的规划和微球体的实验解释。

不同的样品或那些经受不同处理可以在微球体&gt;40μm的用于接种的初始细胞正火后形成的数目而变化。计算乳腺球形成率（MFE），用于在一式三份培养各处理。这使得能够用不同的接种密度实验以进行比较。数据最好显示在一条形图，理想地与阳性和阴性对照，并显示在整个一式三份孔的标准偏差。始终在60-80％汇合之间粘附细胞转移到非粘附板。小心细胞计数是必不可少的准确定量的治疗效果。估计变异，这关键的一步是由相同的待遇以及分别准备多个悬架促进结果的程度。如果测得的细胞浓度从同一井显著不同，考虑优化数量的方法（ 图2）。 <p类="jove_content"FO:保持together.within页="总是"&gt; <img alt="图1" src="/files/ftp_upload/52671/52671fig1.jpg" /> 图1:处理原发性乳腺癌和细胞系样品，以获得微球体培养物样品酶促消化，确认为单细胞，并在用生长因子的无血清培养基中微球体铺板于非粘附板。板不应在生长阶段处理，避免球体融合。右边图为代表MCF-7球5天之后拍下。 <a href="https://www.jove.com/files/ftp_upload/52671/52671fig1highres.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/52671/52671fig2.jpg" /> 图2:从上皮雌激素阳性形成微球体的代表性结果（MCF-7），和间充质三重negatiVE（MDA-MB-231）细胞系。最小细胞融合/聚集达到低密度镀，这里，在500个细胞/ cm 2的MCF-7，和1000个细胞/ cm 2的MDA-MB-231。

Watch this Scientific Journal Video about Mammosphere Formation Assay from Human Breast Cancer Tissues and Cell Lines at JoVE.com

Mammosphere Formation Assay from Human Breast Cancer Tissues and Cell Lines

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 ...

mammosphere-formation-assay-from-human-breast-cancer-tissues-cell

Nanyang Technological University

Research

JoVE Journal

Medicine

33.1K 次观看.  Imperial College London. 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.

视频: Mammosphere Formation Assay from Human Breast Cancer Tissues and Cell Lines

Human Neuroendocrine Tumor Cell Lines as a Three-Dimensional Model for the Study of Human Neuroendocrine Tumor Therapy

Neuroendocrine tumors (NETs) are rare tumors, with an incidence of two per 100,&#x200A;000 individuals per year, and they account for 0.5% of all human malignancies.1 Other than surgery for the minority of patients who present with localized disease, there is little or no survival benefit of systemic therapy. Therefore, there is a great need to better understand the biology of NETs, and in particular define new therapeutic targets for patients with nonresectable or metastatic neuroendocrine tumors. 3D cell culture is becoming a popular method for drug screening due to its relevance in modeling the in vivo tumor tissue organization and microenvironment.2,3 The 3D multicellular spheroids could provide valuable information in a more timely and less expensive manner than directly proceeding from 2D cell culture experiments to animal (murine) models.
To facilitate the discovery of new therapeutics for NET patients, we have developed an in vitro 3D multicellular spheroids model using the human NET cell lines. The NET cells are plated in a non-adhesive agarose-coated 24-well plate and incubated under physiological conditions (5% CO2, 37 &deg;C) with a very slow agitation for 16-24 hr after plating. The cells form multicellular spheroids starting on the 3rd or 4th day. The spheroids become more spherical by the 6th day, at which point the drug treatments are initiated. The efficacy of the drug treatments on the NET spheroids is monitored based on the morphology, shape and size of the spheroids with a phase-contrast light microscope. The size of the spheroids is estimated automatically using a custom-developed MATLAB program based on an active contour algorithm. Further, we demonstrate a simple method to process the HistoGel embedding on these 3D spheroids, allowing the use of standard histological and immunohistochemical techniques. 
This is the first report on generating 3D spheroids using NET cell lines to examine the effect of therapeutic drugs. We have also performed histology on these 3D spheroids, and displayed an example of a single drug's effect on growth and proliferation of the NET spheroids. Our results support that the NET spheroids are valuable for further studies of NET biology and drug development.

We present a simple agarose overlay platform to grow 3D multicellular spheroids using neuroendocrine cancer cell lines. This method provides a very convenient way to examine the effect of therapeutic drugs on the neuroendocrine tumor cells. It could also help us establish human neuroendocrine tumor spheroids for cancer therapy.

人类神经内分泌肿瘤细胞株为研究人类神经内分泌肿瘤治疗的三维模型

Neuroendocrine tumors (NETs) are rare tumors, with an incidence of two per 100,&#x200A;000 individuals per year, and they account for 0.5% of all human ...

科研

医学

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 ...

Time-lapse Imaging of Primary Preneoplastic Mammary Epithelial Cells Derived from Genetically Engineered Mouse Models of Breast Cancer

Time-lapse imaging can be used to compare behavior of cultured primary preneoplastic mammary epithelial cells derived from different genetically engineered mouse models of breast cancer. For example, time between cell divisions (cell lifetimes), apoptotic cell numbers, evolution of morphological changes, and mechanism of colony formation can be quantified and compared in cells carrying specific genetic lesions. Primary mammary epithelial cell cultures are generated from mammary glands without palpable tumor. Glands are carefully resected with clear separation from adjacent muscle, lymph nodes are removed, and single-cell suspensions of enriched mammary epithelial cells are generated by mincing mammary tissue followed by enzymatic dissociation and filtration. Single-cell suspensions are plated and placed directly under a microscope within an incubator chamber for live-cell imaging. Sixteen 650 &mu;m x 700 &mu;m fields in a 4x4 configuration from each well of a 6-well plate are imaged every 15 min for 5 days. Time-lapse images are examined directly to measure cellular behaviors that can include mechanism and frequency of cell colony formation within the first 24 hr of plating the cells (aggregation versus cell proliferation), incidence of apoptosis, and phasing of morphological changes. Single-cell tracking is used to generate cell fate maps for measurement of individual cell lifetimes and investigation of cell division patterns. Quantitative data are statistically analyzed to assess for significant differences in behavior correlated with specific genetic lesions.

Time-lapse imaging is used to assess behavior of primary preneoplastic mammary epithelial cells derived from genetically engineered mouse models of breast cancer risk to determine if there are correlations between specific behavioral parameters and distinct genetic lesions.

延时成像的主要癌前乳腺上皮细胞来源于遗传工程小鼠乳腺癌模型

Time-lapse  imaging can be used to compare behavior of cultured primary preneoplastic  mammary epithelial cells derived from different genetically ...

The Use of Reverse Phase Protein Arrays (RPPA) to Explore Protein Expression Variation within Individual Renal Cell Cancers

Currently there is no curative treatment for metastatic clear cell renal cell cancer, the commonest variant of the disease. A key factor in this treatment resistance is thought to be the molecular complexity of the disease 1. Targeted therapy such as the tyrosine kinase inhibitor (TKI)-sunitinib have been utilized, but only 40% of patients will respond, with the overwhelming majority of these patients relapsing within 1 year 2. As such the question of intrinsic and acquired resistance in renal cell cancer patients is highly relevant 3.
In order to study resistance to TKIs, with the ultimate goal of developing effective, personalized treatments, sequential tissue after a specific period of targeted therapy is required, an approach which had proved successful in chronic myeloid leukaemia 4. However the application of such a strategy in renal cell carcinoma is complicated by the high level of both inter- and intratumoral heterogeneity, which is a feature of renal cell carcinoma5,6 as well as other solid tumors 7. Intertumoral heterogeneity due to transcriptomic and genetic differences is well established even in patients with similar presentation, stage and grade of tumor. In addition it is clear that there is great morphological (intratumoral) heterogeneity in RCC, which is likely to represent even greater molecular heterogeneity. Detailed mapping and categorization of RCC tumors by combined morphological analysis and Fuhrman grading allows the selection of representative areas for proteomic analysis.
Protein based analysis of RCC8 is attractive due to its widespread availability in pathology laboratories; however, its application can be problematic due to the limited availability of specific antibodies 9. Due to the dot blot nature of the Reverse Phase Protein Arrays (RPPA), antibody specificity must be pre-validated; as such strict quality control of antibodies used is of paramount importance. Despite this limitation the dot blot format does allow assay miniaturization, allowing for the printing of hundreds of samples onto a single nitrocellulose slide. Printed slides can then be analyzed in a similar fashion to Western analysis with the use of target specific primary antibodies and fluorescently labelled secondary antibodies, allowing for multiplexing. Differential protein expression across all the samples on a slide can then be analyzed simultaneously by comparing the relative level of fluorescence in a more cost-effective and high-throughput manner.

The Use of Reverse Phase Protein Arrays RPPA to Explore Protein Expression Variation within Individual Renal Cell Cancers

RPPA enables the protein expression of hundreds of samples, printed on nitrocellulose slides to be interrogated simultaneously, using fluorescently labelled antibodies. This technique has been applied to study the effect of drug treatment heterogeneity within clear cell renal carcinoma.

使用反时相蛋白的阵列（RPPA）探索在个人的肾细胞癌蛋白表达的变化

Currently there is no curative treatment for metastatic clear cell renal cell cancer, the commonest variant of the disease. A key factor in this treatment ...

Formation of Human Prostate Epithelium Using Tissue Recombination of Rodent Urogenital Sinus Mesenchyme and Human Stem Cells

Progress in prostate cancer research is severely limited by the availability of human-derived and hormone-na&iuml;ve model systems, which limit our ability to understand genetic and molecular events underlying prostate disease initiation. Toward developing better model systems for studying human prostate carcinogenesis, we and others have taken advantage of the unique pro-prostatic inductive potential of embryonic rodent prostate stroma, termed urogenital sinus mesenchyme (UGSM). When recombined with certain pluripotent cell populations such as embryonic stem cells, UGSM induces the formation of normal human prostate epithelia in a testosterone-dependent manner. Such a human model system can be used to investigate and experimentally test the ability of candidate prostate cancer susceptibility genes at an accelerated pace compared to typical rodent transgenic studies. Since Human embryonic stem cells (hESCs) can be genetically modified in culture using inducible gene expression or siRNA knock-down vectors prior to tissue recombination, such a model facilitates testing the functional consequences of genes, or combinations of genes, which are thought to promote or prevent carcinogenesis.
The technique of isolating pure populations of UGSM cells, however, is challenging and learning often requires someone with previous expertise to personally teach. Moreover, inoculation of cell mixtures under the renal capsule of an immunocompromised host can be technically challenging. Here we outline and illustrate proper isolation of UGSM from rodent embryos and renal capsule implantation of tissue mixtures to form human prostate epithelium. Such an approach, at its current stage, requires in vivo xenografting of embryonic stem cells; future applications could potentially include in vitro gland formation or the use of induced pluripotent stem cell populations (iPSCs).

To unravel the earliest molecular mechanisms underlying prostate cancer initiation, novel and innovative human model systems and approaches are desperately needed. The potential of pre-prostatic urogenital sinus mesenchyme (UGSM) to induce pluripotent stem cell populations to form human prostate epithelium is a powerful experimental tool in prostate research.

人类前列腺上皮细胞形成的使用鼠类泌尿生殖窦间质细胞和人类干细胞的组织重组

Progress in prostate cancer research is severely  limited by the availability of human-derived and hormone-na&iuml;ve model systems,  which limit our ...

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 ...

Enrichment for Chemoresistant Ovarian Cancer Stem Cells from Human Cell Lines

Cancer stem cells (CSCs) are defined as a subset of slow cycling and undifferentiated cells that divide asymmetrically to generate highly proliferative, invasive, and chemoresistant tumor cells. Therefore, CSCs are an attractive population of cells to target therapeutically. CSCs are predicted to contribute to a number of types of malignancies including those in the blood, brain, lung, gastrointestinal tract, prostate, and ovary. Isolating and enriching a tumor cell population for CSCs will enable researchers to study the properties, genetics, and therapeutic response of CSCs. We generated a protocol that reproducibly enriches for ovarian cancer CSCs from ovarian cancer cell lines (SKOV3 and OVCA429). Cell lines are treated with 20 &#181;M cisplatin for 3 days. Surviving cells are isolated and cultured in a serum-free stem cell media containing cytokines and growth factors. We demonstrate an enrichment of these purified CSCs by analyzing the isolated cells for known stem cell markers Oct4, Nanog, and Prom1 (CD133) and cell surface expression of CD177 and CD133. The CSCs exhibit increased chemoresistance. This method for isolation of CSCs is a useful tool for studying the role of CSCs in chemoresistance and tumor relapse.

Cancer stem cells (CSCs) are&#160;implicated in tumor relapse due to chemoresistance. We have optimized a protocol for selection and expansion of CSCs from ovarian cancer cell lines. By treating cells with the chemotherapeutic cisplatin and culturing&#160;in a stem cell promoting media we enrich for non-adherent CSC cultures.

丰富的人力细胞系化疗耐药性卵巢癌干细胞

Cancer stem cells (CSCs) are defined as a subset of slow cycling and undifferentiated cells that divide asymmetrically to generate highly proliferative, ...

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 ...

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 ...