这些研究提供资金，部分通过从替代研究与发展基金会（107588），美国国立卫生研究院（1U54CA136465-04S1），和加州乳腺癌研究发展计划（201B-0141）资助。我们感谢博士。安德鲁·威尔伯和R.斯科特·麦基弗的慷慨捐赠自己<pkt2 lubig="">转座子和PK / hUbiC-SB11转座质粒。我们非常感谢约翰Tamaresis博士对统计方法的建议，蒂莫西·布朗的表演术流，乔其纱亨里奇上迁移试验的意见，和南希Bellagamba为促进THR标本采集。 </pkt2>

股骨头从择期THR手术矫形外科系在斯坦福大学医学院的患者收集。所有的组织收集的去鉴定标本按照斯坦福大学的研究合规办公室法规。 1。选择乳腺癌细胞系（S） <ol><li>获得或转染所需的乳腺癌细胞系（S）与萤光素酶-GFP报道构建。下面实验使用的MDA-MB-231和MCF-7乳腺癌细胞系工程改造为萤火虫荧光素酶的稳定表达（fLuc）和增强型绿色荧光蛋白（EGFP）的睡美人转座子的质粒PKT2 / LuBiG进行转染和转座质粒PK / hUbiC-SB11 45,46。 注：将转染的细胞系被称为MDA-MB-231-fLuc-EGFP和MCF-7-fLuc-EGFP。 </li></ol> 2.隔离组织˚F从股骨头ragments <ol><li>以下THR手术，收集废弃股骨头在无菌容器中填充有生理盐水。样品转移到实验室，并在1-3小时的手术处理。注意：如果在处理的延迟，将样品在4℃下。 </li><li>无菌手套，手术咬骨钳，镊子，烧杯，用70％异丙醇冲洗仪器，以及组织培养容器的规定如下为每个类型的实验：通过将下列项目上的吸收垫在层流组织培养罩准备的工作区域。 </li><li>穿着无菌手术手套以保持圆形股骨头在一方面，使用咬骨钳在另一方面，提取从股骨的暴露上部轴所需的尺寸（〜2-5毫米）的小梁骨碎片。转移碎骨入培养容器（ 例如，6-，12-，或24孔组织培养板或迁移室），这取决于扩散的类型，山口onization或迁移实验中，如在步骤3，4或5所述。 注：在完成对每个具体的增殖，定植或迁移实验的最初步骤后上述骨组织碎片应该有所收获。碎片将尺寸范围， 如图2。片段可以之前或实验为标准化的目的之后被测量。 </li></ol> 3.联合培养乳腺癌细胞毗邻碎骨来衡量乳房细胞增殖使用BLI <ol><li>在实验前，制备含2×10 5个乳腺癌细胞/ 50微升的悬浮液，并准备骨蜡的插头用于固定骨头碎片中培养。切P200枪头到3件，并使用最大的一块的窄端的“俗套”的方式来切小（〜35毫克）的骨片蜡。使用最小片的宽端以弹出骨蜡塞到培养皿进行存储。 </li><li>将一块骨蜡在每口井的12点钟位置，轻轻按下，用无菌手套的食指。 </li><li>吸移管将50μl含1×10 5个乳腺癌细胞在DMEM-10％FBS +青霉素-链霉素的6孔板的每个孔的中心的细胞悬浮液。 （可替换地，种子细胞，如果需要的分散图案。）将板在37℃组织培养孵化器45分钟，以促进细胞附着。 </li><li>放置1骨块到1片骨蜡的每个实验井并应用温和的压力与咬骨钳将其固定。一式三份，从一个给定的THR检体，使得3碎骨放入三甲孔进行实验，而在底部的三个孔中含有骨蜡仅作为对照。 </li><li>使用5毫升吸管，轻缓地交付5毫升的DMEM-10％FBS的每个的侧面，以避免撞出乳房细胞或骨碎片。将板在3776;Ç组织培养孵化20-24小时。 </li><li>执行生物发光成像（BLI）从培养箱取出板并添加荧光素底物（以达到300微克/ ml的浓度）至各孔中。图像使用下列参数立即将板的IVIS成像系统平台上：光圈1，小分级，1秒的曝光时间，水平D.测量每个的信号强度以及平均辐射率（光子/秒/平方厘米/球面度）。 </li><li>使用图像处理软件来定义感​​兴趣区域（ROI）的地区，以定量的平均四射的所有实验和对照孔。出口平均辐射值到Excel表进行统计并生成图表。 </li></ol>直接种植于碎骨4.联合培养乳腺癌细胞研究殖民化和细胞数量<ol><li>使用镊子直接将骨碎片在24孔组织培养板的空孔中（1片段/孔）。 </li><li>果仁ETTE的DMEM-10％FBS的含有直接在各个骨片段的顶部1×10月3日至6日的乳 ​​腺癌细胞的50微升细胞悬浮液液滴。将板在37℃组织培养孵化器45分钟，以促进细胞附着。轻轻加1-2毫升的DMEM-10％FBS的成的各孔，例如在一侧的片段是完全浸没在介质。孵育所述板在37℃组织培养箱为20-24小时。 </li><li>温育后，用钳子向每个骨组织片段转移到新鲜的24孔板中含有1ml的DMEM-10％FBS的每个孔中。要执行BLI，按照上面的步骤3.6-3.7。此外，片段可使用荧光显微镜进行观察，或冲刷如在步骤6中所述，得到的骨髓人口为乳腺癌细胞数量和性质进行分析。 </li><li>通过荧光显微镜下BLI，吸管5微升荧光标记的双膦酸盐溶液（预做准备完整的片段进行定性评估根据制造商的说明缩减）到每个孔来标记碎骨的矿化骨针。孵育板在组织培养培养箱中另外24小时。 </li><li>以下培养中的荧光标记的双膦酸盐溶液中的24小时后，用钳子向片段转移到含有酚红的培养基，并查看使用配置以图像的GFP（485纳米）和二膦酸标签（680 nm）的荧光显微镜组织培养板中。 </li></ol> 5.测量乳腺癌细胞移植到骨组织碎片和养殖骨头碎片上清<ol><li>迁移朝向骨组织培养上清液。 注：以一式三份进行实验，从一个给定的样品THR使得3个片段被用来产生3上清。对于每个试验，迁移横跨含有骨上清液与仅含对照培养基三口井三口井相比。 <ol><li>生成骨炎通过将骨碎片成12孔板每孔含有2.2毫升的DMEM-10％FBS中的孔中的上清液告。文化在37℃组织培养孵化24小时。退出介质在24小时，更换2.2毫升新鲜的DMEM-10％FBS。治疗对照孔含有DMEM-10％FBS的方式相同。 注：该介质是在24小时变更为除去分泌响应于组织创伤手术产生的因素。因为在生成骨组织上清FBS含含含FBS中只包括以控制FBS的乳腺癌细胞迁移的贡献中，水井。 </li><li>在48小时后，收集0.9毫升每种上清液/控制介质和吸管的一部接收机板的孔中。刀片将在后面被放入这些孔中。孵育接收板在组织培养孵化器同时播种的刀片。注意：剩余的上清液（〜蒸发后的1毫升）中，可收集的分泌组实际分析RS如果需要44。 </li><li>种子的Transwell小室，将它们放入一个标准的，空的24孔培养板。吸移管350微升细胞悬浮液含1×10 5个乳腺癌细胞在DMEM-10％FBS的到各刀片的上部，内膜表面， 如图1A所示 。放置含种子插入到37℃组织培养箱中在24孔板45分钟，以促进附着。 </li><li>一旦细胞已附连到插入件中，使用镊子根据制造商的说明将插入传送到接收器板。角度放置到接收器以及防止插入膜和接收机以及上清液之间形成气泡时每次插入。孵育接收机板与刀片20小时在37℃组织培养孵化器。 注意：检查接收板的底侧为插入膜和接收机以及supernatan之间的气泡吨。如果气泡是可见的，删除特定的插件，并再次将它们插入接收机很好，直到无气泡存在。 </li><li>经过20小时的使用镊子从接收机板取出刀片。在步骤使用程序3.6-3.7执行BLI图像已经迁移入并连接到接收器板孔底部的细胞。 </li></ol></li><li>移民对骨组织碎片。 注：以一式三份进行实验，从一个给定的样品THR使得3个片段被放入3个独立的刀片，和图3的玻璃珠放入3个独立的刀片作为对照。 <ol><li>吹打0.9毫升的DMEM-10％FBS的入每个孔制备接收机板。将其放入组织培养的孵化器而准备的刀片。 </li><li>种子的插入，颠倒它们的塑料离心管中，有一个盖的表面上。吸取的DMEM-10％FBS的含1×10 5个乳腺癌细胞D的50微升细胞悬浮液的液滴irectly到每个插入膜外，底表面（其朝上的反相插入）。覆盖离心管箱与盖裂了开，并将其传输到37℃组织培养箱中45分钟，以促进细胞附着。 </li><li>转移离心管方框，组织培养罩，并使用镊子打开种子插入右侧向上，并将它们传送到接收器板的孔中。吸取0.450毫升DMEM-10％FBS为每个插入杯中。使用镊子，传输一个骨组织片段（〜3毫米），为每个准备插入杯中。或者，用玻璃珠作为阴性对照额外的井。将板在组织培养培养箱中20小时。 </li><li>为了测量迁移，制备含有每孔1ml的DMEM-10％FBS的标准的24孔组织培养板。从孵化器中取出接收器板，然后使用镊子，从每个插入转移骨头碎片和珠子到标准板的不同孔。添加荧光素衬底（达到300微克/ ml的终浓度）至每个孔，并利用在步骤3.6-3.7中的过程执行BLI。 </li></ol></li></ol> 6.附加前和后的实验分析<ol><li>分析冲洗骨髓。 <ol><li>传送一个骨片段插入一个70微米的细胞滤网放置在50ml锥形管的顶部。使用10毫升注射器与地下25针大力冲洗片段用10ml的PBS中，以获得在锥形管骨髓细胞的悬浮液。离心细胞悬浮液3分钟，在300×g离心并21℃下，吸出上清，重悬沉淀的细胞在PBS或流式细胞其它所需的解决方案。 注：再悬浮量将取决于细胞沉淀后离心分离，和应用的大小而变化。 </li><li>对于存活测定的PBS〜75-300微升体积是合适的，并且这些混悬剂可以分析使用市售fluorescenc根据制造商的说明ë基于活力测定法，吸移到常规血球，并且使用倒置荧光显微镜计数。 </li><li>对于流式细胞仪分析，或基于检测GFP阳性乳腺癌细胞的分选，重悬含有2％FBS，2mM的EDTA的细胞沉淀在1ml PBS中，和10mM的HEPES，使得浓度为1×10 6个细胞/ ml。 </li></ol></li><li> H＆E染色及免疫组化。 <ol><li>处理用于苏木精 - 伊红（H＆E）染色和/或免疫组织化学的片段，放置在塑料盒标有一个号码2铅笔的片段，并淹没在容器中填充有10％的福尔马林缓冲液24小时。 </li><li>过程中组织进行石蜡包埋，切片和使用，其中包括一个脱钙一步占每个片段中的骨针矿化例行协议染色。 </li></ol></li></ol>

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梅拉等人以前描述了死后收集和骨标本分析转移性前列腺癌患者收获在尸检时，露出高质量组织和验证的使用人骨样品来研究转移过程47。在这里，我们已经描述了协议，用于收集，处理和使用从下面的THR手术在短期共培养系统来研究乳腺癌细胞的迁移，定居和增殖丢弃股骨头获得的人骨组织碎片。大约300,000髋关节置换手术是在美国（矫形外科医生的美国学院; Orthoinfo.aaos.org）每年...

肿瘤微环境是目前公认的癌细胞行为乳腺癌进展和转移过程中的关键决定因素传播1-3。这里提出的方法的目标是促进人体骨组织，乳腺癌转移4-6的最频繁的部位的微环境中的乳腺癌细胞的研究。骨是由矿化和骨髓室7-9，这两个怀有细胞和分泌的因子牵连转移进展10,11。尽管广泛使用在体内小鼠模型促进转移过程12-15系统性研究，骨架内细胞的相互作用是不进行观察和直接扰动容易获得。模型系统研究和培养小鼠骨髓组织和小鼠骨髓细胞提供更好的访问，并已经取得了对串扰许多见解和机制基本乳腺癌细胞米etastasis小鼠骨骼16-22。然而，研究表明，有可能是osteotropism的物种特异性的模式为骨组织23,24。这些模式可以反映固有物种特异性在骨组织性质的差异，从改变的骨髓群体中的免疫缺陷小鼠11产生的差异，和/或小鼠的相对年轻的年龄在实验中使用的，所有这些都在骨的可能的决定因素微环境。 体外方法用于人骨共培养研究乳腺癌细胞已经典型地集中于特定骨的细胞类型，如骨髓来源的成骨细胞或基质细胞作为单层培养的或工程化的三维模型系统25-35内。虽然2维细胞培养模型已形成的在体外的支柱接近为癌症研究，人们早就认识到，细胞的行为是从根本上改变在米onolayer培养系统18,36,37。这导致了设计的微环境，模仿的3-维活组织的复杂性，包括矩阵可与天然材料，如胶原蛋白构成的支架的基于模型的开发，或合成的聚合物接种有特定的细胞类型，以创建组织样的微环境36-41。设计方案还包括使用生物反应器平台的控制和研究微18,19,41-43体内的荷尔蒙环境。虽然仿生模型和生物反应器提供了一个复杂的组织微环境中的受控环境的许多元素，并且已成功地应用到推进乳腺癌转移到骨18,19,35,42,43的研究中，工程化的模型系统一般不掺入天然骨环境中存在的细胞类型和细胞外基质组分的全谱。 直到最近，乳腺癌细胞尚未研究天然的，完整的，三维人体骨组织的微环境中。我们最近报道使用人体骨组织碎片从全髋关节置换分离（THR）的手术标本44的共培养模型的发展。这些标本既怀有必要研究潜在的微转移在短期内培养机制的矿化和骨髓车厢。在以前的工作中，我们建立了验证性原则，使用生物发光成像（BLI）监测荧光素酶表达乳腺癌细胞（MDA-MB-231-fLuc）共培养骨碎片44的扩散。在这里，我们提出详细的实验方案为研究使用人骨组织的外植体的天然微环境的上下文中乳腺细胞癌的增殖，定植，和迁移。 首先，我们提出了一个协议相邻骨骼碎片来衡量乳房共培养乳腺癌细胞使用BLI细胞增殖。在这一节中，乳腺癌细胞悬液接种作为细胞的斑点在邻近骨头碎片，这是由骨蜡件固定6孔板中。对照孔含有骨蜡，但是没有骨片段。一旦细胞斑附着，培养基，并将该板是24小时，在这之后的生物发光信号的强度（与细胞数目相关联）的测量是用BLI培养。在接下来的步骤中，我们提出直接接种到骨碎片研究定居和增殖共培养的乳腺癌细胞的方法。这里的乳房细胞悬浮液被直接吸移到所述骨组织碎片，其通过BLI和荧光显微镜在培养监测随着时间的推移追踪定植和细胞数。在该方法中，骨髓隔室可以从骨碎片被刷新为在定植乳腺癌细胞通过流式细胞分析的任何时间点术，或骨髓存活测定。 此外，我们取消划线两种不同的方法，用于测量乳腺癌细胞迁移。在第一种方法的步骤进行了概述，用于测量朝向骨组织培养上清液迁移。在这个协议中的乳腺癌细胞接种到Transwell小室的内，上表面插入膜具有8微米的孔径（ 如图1A）。插入件，然后放入一个接收机板用含有骨组织上清液或对照培养基的孔中。超过20小时的温育期的过程中，少量的乳腺癌细胞通过插入膜迁移入下面的组织培养孔中，其中它们附着于检测通过BLI。在第二迁移方法的乳腺癌细胞接种到Transwell小室的插入件的膜的下部，外表面。骨组织碎片放入插入杯子和乳腺癌细胞向上迁移通过膜拓殖骨碎片（ 见图1B），其然后用成像BLI。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>DMEM (1X) + GlutaMAX-l</td>
			<td>Life Technologies</td>
			<td>10569-010</td>
			<td>Supplement all DMEM with 10% fetal bovine serum and 1% Pen/Strep</td>
		</tr>
		<tr>
			<td>McCoy&#39;s 5A Medium (1X)</td>
			<td>Life Technologies</td>
			<td>16600-082</td>
			<td>Supplement all McCoy&#39;s&#160; with 10% fetal bovine serum and 1% Pen/Strep</td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Life Technologies</td>
			<td>16000-044</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillen Streptomycin</td>
			<td>Life Technologies</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>Blastocidin (10 mgl/ml)</td>
			<td>InvivoGen&#160;</td>
			<td>ant-bl</td>
			<td>Supplement media used for transfected cell lines.</td>
		</tr>
		<tr>
			<td>Falcon&#174; Cell Culture Inserts for 24 Well Plate with Transparent PET Membrane (8.0 &#956;m pore size)</td>
			<td>Corning Incorporated</td>
			<td>353097</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon&#174; 24 Well TC-Treated Polystyrene Cell Culture Insert Companion Plate</td>
			<td>Corning Incorporated</td>
			<td>353504</td>
			<td></td>
		</tr>
		<tr>
			<td>LIVE/DEAD Viability/Cytotoxicity Kit *for mammalian cells*</td>
			<td>Invitrogen Detection Technologies</td>
			<td>L-3224</td>
			<td></td>
		</tr>
		<tr>
			<td>FluoroBrite DMEM</td>
			<td>Life Technologies</td>
			<td>A18967-01</td>
			<td></td>
		</tr>
		<tr>
			<td>D-luciferin firefly, potassium salt 5 grams (30 mg/mL)</td>
			<td>Life Technologies</td>
			<td>L-8220</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile Cell Strainer 70 &#956;m</td>
			<td>Fisher Scientific</td>
			<td>22363548</td>
			<td></td>
		</tr>
		<tr>
			<td>6 Well TC-Treated Cell Culture Cluster</td>
			<td>Corning Incorporated</td>
			<td>3516</td>
			<td></td>
		</tr>
		<tr>
			<td>12 Well TC-Treated Cell Culture Cluster</td>
			<td>Corning Incorporated</td>
			<td>3513</td>
			<td></td>
		</tr>
		<tr>
			<td>24 Well TC-Treated Cell Culture Cluster</td>
			<td>Corning Incorporated</td>
			<td>3526</td>
			<td></td>
		</tr>
		<tr>
			<td>Friedman-Pearson Rongeur</td>
			<td>Fine Science Tools</td>
			<td>16021-14</td>
			<td></td>
		</tr>
		<tr>
			<td>Bone wax</td>
			<td>Surgical Specialties Corporation</td>
			<td>903</td>
			<td></td>
		</tr>
		<tr>
			<td>IVIS 50 Imaging Platform</td>
			<td>Caliper Life Sciences/PerkinElmer</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Living Image Software Program&#160;</td>
			<td>Caliper Life Sciences/PerkinElmer</td>
			<td>133026</td>
			<td></td>
		</tr>
		<tr>
			<td>EVOS FL Imaging System</td>
			<td>Life Technologies</td>
			<td>Version 4.2</td>
			<td></td>
		</tr>
		<tr>
			<td>OsteoSense 680 EX</td>
			<td>PerkinElmer</td>
			<td>NEV10020EX</td>
			<td></td>
		</tr>
		<tr>
			<td>MCF-7 breast cancer cells</td>
			<td>ATCC</td>
			<td>HTB-22</td>
			<td></td>
		</tr>
		<tr>
			<td>MDA-MB-231 breast cancer cells</td>
			<td>ATCC</td>
			<td>HTB-26</td>
			<td></td>
		</tr>
		<tr>
			<td>Monoclonal mouse anti-human cytokeratin antibody</td>
			<td>DAKO Cytomation</td>
			<td>Clone (AE1/AE3)</td>
			<td></td>
		</tr>
	</tbody>
</table>

methods for culturing human femur tissue explants to study breast cancer cell colonization of the metastatic niche

骨是乳腺癌转移的最常见的部位。虽然它已被广泛接受的微环境影响癌细胞行为，知之甚少人体骨组织的天然微环境中的乳腺癌细胞的特性和行为。我们已开发的方法来跟踪，量化和的微环境中调节人乳腺癌细胞培养的人骨组织碎片从以下全髋关节置换手术丢弃股骨头隔离。使用设计用于萤光素酶和增强型绿色荧光蛋白（EGFP）表达的乳腺癌细胞中，我们能够后可重复地定量迁移和增殖型态使用生物发光成像（BLI），轨道细胞相互作用使用荧光显微镜的骨碎片内，并评估乳腺细胞定植流式细胞仪。这种模式的主要优势包括：1）一个人，建筑完好无损组织microenvironment包括有关人体的细胞类型，以及2）直接进入微环境，这有利于快速定量和定性监测和乳腺癌和骨细胞的特性，行为和交互作用扰动。一个主要的限制，目前，是组织片段，该组合限制住研究短期培养的窗口的有限的可行性。该模型系统的用途包括研究乳腺癌和其他骨寻求恶性肿瘤的基本生物学的转移性利基内，并且开发治疗策略，以有效地针对乳腺癌细胞在骨组织中。

从股骨头分离组织碎片股骨头从男性和女性患者（44-90岁）在接受矫形外科系选修全髋关节置换手术在斯坦福大学医学院的数量相等收集。每个丢弃股骨头含有股骨上端，其中窝藏小梁骨组织的矿化骨针和骨髓组成的一小部分。此组织是可透过轴（图2A）的使用外科咬骨钳提取小片段的外科手术暴露的横截面。所提取的片的组织学示于图2B中 ，揭示了?...

Watch this Scientific Journal Video about Methods for Culturing Human Femur Tissue Explants to Study Breast Cancer Cell Colonization of the Metastatic Niche at JoVE.com

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

协议描述为研究乳腺癌细胞的迁移，增殖和定植于人体骨组织外植体模型系统。

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 ...

methods-for-culturing-human-femur-tissue-explants-to-study-breast

Research

JoVE Journal

Medicine

12.4K 次观看.  Stanford University School of Medicine. 协议描述为研究乳腺癌细胞的迁移，增殖和定植于人体骨组织外植体模型系统。 

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

Method for Novel Anti-Cancer Drug Development using Tumor Explants of Surgical Specimens

The current therapies for malignant glioma have only palliative effect. For therapeutic development, one hurdle is the discrepancy of efficacy determined by current drug efficacy tests and the efficacy on patients. Thus, novel and reliable methods for evaluating drug efficacy are warranted in pre-clinical phase. In vitro culture of tumor tissues, including cell lines, has substantial phenotypic, genetic, and epigenetic alterations of cancer cells caused by artificial environment of cell culture, which may not reflect the biology of original tumors in situ. Xenograft models with the immunodeficient mice also have limitations, i.e., the lack of immune system and interspecies genetic and epigenetic discrepancies in microenvironment. Here, we demonstrate a novel method using the surgical specimens of malignant glioma as undissociated tumor blocks to evaluate treatment effects. To validate this method, data with the current first-line chemotherapeutic agent, temozolomide (TMZ), are described.


We used the freshly-removed surgical specimen of malignant glioma for our experiments. We performed intratumoral injection of TMZ or other drug candidates, followed by incubation and analysis on surgical specimens. Here, we sought to establish a tumor tissue explant method as a platform to determine the efficacy of novel anti-cancer therapies so that we may be able to overcome, at least, some of the current limitations and fill the existing gap between the current experimental data and the efficacy on an actual patient's tumor. This method may have the potential to accelerate identifying novel chemotherapeutic agents for solid cancer treatment.

Here, we established a method for drug efficacy testing with surgical specimens of brain tumors, termed &#x201C;tumor explant method&#x201D;. With this method, we can evaluate drug efficacy without breaking the microenvironment of solid tumors. To validate reliability of this method, we describe representative data with our glioma specimen treated with the current first-line chemotherapeutic agent, temozolomide.

新型抗癌药物使用手术标本肿瘤植的发展方法


The current therapies for malignant glioma have only palliative effect. For therapeutic development, one hurdle is the discrepancy of efficacy determined ...

科研

医学

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 ...

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 ...

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 ...

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 ...

Three Dimensional Cultures: A Tool To Study Normal Acinar Architecture vs. Malignant Transformation Of Breast Cells

Invasive breast carcinomas are a group of malignant epithelial tumors characterized by the invasion of adjacent tissues and propensity to metastasize. The interplay of signals between cancer cells and their microenvironment exerts a powerful influence on breast cancer growth and biological behavior1. However, most of these signals from the extracellular matrix are lost or their relevance is understudied when cells are grown in two dimensional culture (2D) as a monolayer. In recent years, three dimensional (3D) culture on a reconstituted basement membrane has emerged as a method of choice to recapitulate the tissue architecture of benign and malignant breast cells. Cells grown in 3D retain the important cues from the extracellular matrix and provide a physiologically relevant ex&#160;vivo system2,3. Of note, there is growing evidence suggesting that cells behave differently when grown in 3D as compared to 2D4. 3D culture can be effectively used as a means to differentiate the malignant phenotype from the benign breast phenotype and for underpinning the cellular and molecular signaling involved3. One of the distinguishing characteristics of benign epithelial cells is that they are polarized so that the apical cytoplasm is towards the lumen and the basal cytoplasm rests on the basement membrane. This apico-basal polarity is lost in invasive breast carcinomas, which are characterized by cellular disorganization and formation of anastomosing and branching tubules that haphazardly infiltrates the surrounding stroma. These histopathological differences between benign gland and invasive carcinoma can be reproduced in 3D6,7. Using the appropriate read-outs like the quantitation of single round acinar structures, or differential expression of validated molecular markers for cell proliferation, polarity and apoptosis in combination with other molecular and cell biology techniques, 3D culture can provide an important tool to better understand the cellular changes during malignant transformation and for delineating the responsible signaling.

Three dimensional culture of mammary epithelial cells on a reconstituted basement membrane is a useful method to recapitulate the in vivo architecture of the benign breast, and to differentiate the malignant phenotype from the benign breast phenotype. Importantly, this system can be applied to study invasive carcinomas in other tissues.

三维文化：一种工具来研究正常腺泡结构与恶性转化的乳腺细胞

Invasive breast carcinomas are a group of malignant epithelial tumors characterized by the invasion of adjacent tissues and propensity to metastasize. The ...

Orthotopic Injection of Breast Cancer Cells into the Mammary Fat Pad of Mice to Study Tumor Growth.

Breast cancer growth can be studied in mice using a plethora of models. Genetic manipulation of breast cancer cells may provide insights into the functions of proteins involved in oncogenic progression or help to discover new tumor suppressors. In addition, injecting cancer cells into mice with different genotypes might provide a better understanding of the importance of the stromal compartment. Many models may be useful to investigate certain aspects of disease progression but do not recapitulate the entire cancerous process. In contrast, breast cancer cells engraftment to the mammary fat pad of mice better recapitulates the location of the disease and presence of the proper stromal compartment and therefore better mimics human cancerous disease. In this article, we describe how to implant breast cancer cells into mice orthotopically and explain how to collect tissues to analyse the tumor milieu and metastasis to distant organs. Using this model, many aspects (growth, angiogenesis, and metastasis) of cancer can be investigated simply by providing a proper environment for tumor cells to grow.

Cancer is a complex disease that is influenced by the tissue surrounding the tumor as well as local pro- and anti-inflammatory mediators. Therefore, orthotropic injection models, rather than subcutaneous models may be useful to study cancer progression in a manner that better mimics human pathology.

注射原位乳腺癌细胞到小鼠乳腺脂肪垫来研究肿瘤的生长。

Breast cancer growth can be studied in mice using a plethora of models. Genetic manipulation of breast cancer cells may provide insights into the ...

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

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

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

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

一个<em&gt;体外</em&gt;模型在乳腺癌研究激素的作用

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

Mammosphere Formation Assay from Human Breast Cancer Tissues and Cell Lines

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

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

从人乳腺癌组织及细胞系微球体形成分析

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