The authors would like to acknowledge the Netherlands Organization for Scientific Research (NWO, grant 17.106.329)

动物实验批准了荷兰莱顿大学医学中心（LUMC）的动物福利委员会。 1.准备的细胞，仪器和小鼠<ol><li>手术前一天，从第四乳头中线刮胡子的NOD / SCID伽玛（NSG）小鼠，权衡小鼠验证所有小鼠有大致相当的权重。 </li><li>高压灭菌剪刀，镊子2和两个直的蚊钳（ 图1A）。 </li><li>对手术当天，洗人乳腺癌（MDA-MB-231）细胞中，将一次用磷酸缓冲盐水（PBS）被注入，pH为7.4和trypsinize细胞。通过在细胞的顶端加入10ml含血清DMEM培养基猝灭胰蛋白酶。离心将细胞在1,200rpm下在RT 7分钟通过重悬细胞无论在PBS或在没有血清的培养基以除去血清。再次离心他们彻底清除血的痕迹。将细胞重悬于PBS或媒体。 </li><li>计数细胞用血细胞计数器，并计算细胞的量。使用50万细胞/鼠标在不超过150微升。择，以PBS或媒体，重新悬浮在基质胶中的细胞。 注:确定取决于所使用的细胞类型的细胞的量。 </li><li>将细胞在无菌离心管，并让他们在冰。 </li><li>填充1 50ml锥形离心管中以35毫升的PBS pH 7.4的，另一个50ml的锥形离心管中，用70％的乙醇。浸泡棉签到每个试管中。 </li></ol> 2.注射原位<ol><li>在无菌罩下进行手术，以保持无菌气氛。通过皮下注射赛拉嗪/氯胺酮混合物在10毫克/千克，100毫克/公斤体重的剂量麻醉的小鼠。修复鼠标放在一个加热垫。确认麻醉由缺乏反应脚趾捏的成功。 注:如果一个人是不是很熟悉的外科手术，手术可能需要更长的时间。安排麻醉剂量相应。 </li><li>适用于小鼠的眼中的眼科软膏，以防止眼睛干燥。 </li><li>用棉签蘸取步骤1.6编写乙醇清洁剃光区域。交替使用优碘和酒精三次以循环的方式从计划切口部位旋转远离磨砂应该执行。另外，使用离子载体作为消毒剂。 </li><li>使第四奶嘴和中线之间的小切口用剪刀和通过将棉签浸湿，用PBS pH 7.4的做出一个口袋。 </li><li>使用镊子，露出乳房脂肪垫。观察脂肪垫它的白色。 </li><li>挤压脂肪垫从其基地等镊子;通过这样做，完全暴露的脂肪垫（ 图1B），以容易地进行注射。 </li><li>通过上下吹打均质的细胞混合物。轻轻吸50微升细胞悬浮液成胰岛素注射器和注入乳房脂肪垫由持针水平。通过检查脂肪垫肿胀确认成功注入。 </li><li>轻轻松开脂肪垫。 </li><li>通过使用针驱动缝合切口。使三个单结转动缝合周围的针驱动在顺时针，逆时针和顺时针方向转动。每缝合两个结应该被使用。 注:确保结紧张，否则小鼠可以打开缝合。 </li><li>手术后，在0.05-0.1毫克/公斤体重，皮下注射，以缓解疼痛注射止痛药，如temgesic。 </li><li>不要让动物无人看管，直到他们获得的意识，保持胸骨recumbence。将所有的动物分成不同的笼子里，直到他们完全康复。 </li><li>保持无菌，使用蒸压笼和水。改变笼，每三天要保持大气的清洁。 </li></ol> 3.摘器官的分析<ol><li>在收获的当天，细胞的植入8周后，填的15毫升锥形离心管中的每个小鼠3毫升Bouin氏溶液。此外，使用两个15ml试管充满每只小鼠5毫升福尔马林溶液。 </li><li>通过注射赛拉嗪/氯胺酮，以10mg的剂量麻醉动物/千克100毫克/公斤体重皮下​​注射。等到动物失去脚趾捏撤回反射。 </li><li>固定在基本的针的动物。做一个长期的，垂直的正中切口，用剪刀。使下方的前腿和后腿上面两横切口。由钉扎皮肤基底暴露肿瘤。测量肿瘤体积通过使用卡尺。 </li><li>分离使用剪刀皮肤肿瘤。卡扣冻结肿瘤在液氮中用于RNA分离的一部分。放置另一部分，进入锥形离心管中填充有福尔马林以下石蜡包埋，以进行免疫组化分析。 </li><li>轻轻取出肺。将左肺到布安的解决方案。保持在溶液中的肺3天。清楚地观察浅表转移灶，以肉眼。或者，将右肺到福尔马林使用苏木精和伊红（H＆E）染色，以验证微。 注:虽然，肺转移乳腺癌经常出现，人们可能还需要收集骨，肝，脑，脾，分析转移。将细胞在转移区是密度和形态不同，因此可以很容易地从肺组织区别开来。 </li><li>一天收获后，吸出从15ml试管的福尔马林溶液，并替换为70％的乙醇。嵌入在石蜡的组织和执行免疫组化研究。 </li><li>对于血液分析，开拓胸腔用剪刀。撤销450微升血液通过心脏穿刺。放置血液样本中含有50微升3.2％的柠檬酸钠微量离心管中。血液采集后，旋转它在2000 XG为10英里ñ。储存的血浆样品在-20℃直至进一步使用。 注意:有在实践20常用的几个其它血液收集技术，并且需要被使用的技术取决于实验设置和科学家的选择。如果不需要血液样品，根据动物的协议或按该机构的准则安乐死的动物。 </li></ol>

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The authors declare that they have no competing financial interests.

原位注射乳腺癌细胞是一个功能强大的模型来研究癌症生长的所有方面。应，以防止在变异的肿瘤生长进行仔细进行这些细胞在小鼠的乳房脂肪垫的注入。最重要的是，细胞注射相同量的给每只小鼠是至关重要的。要做到这一点，应该trypsinize细胞严格而不影响细胞的生存力。非存活细胞应在细胞计数和试剂（ 即台盼蓝），可以帮助人死亡，存活的细胞之间进行区分可能是用于确定活细胞数目有用期间被忽略。应通过上下吹打细胞避免细胞团块的形成作为形成细胞团块引起在细胞计数的问题，并导致的细胞数量计算错误。应当考虑的另一个重要方面是在细胞本身的体积。在协议部分，描述了细胞s的沉淀，以暂停它们在PBS或媒体。之前加入PBS或培养基，将细胞的体积可以通过比较与管填充有不同体积的介质中的沉淀来估计。随后，细胞体积可以从预期介质体积中减去，根据以下公式: 的介质体积需要被加入=计算体积 - 细胞体积试剂中使用的协议可能影响实验的结果;因此该协议应相应调整取决于研究的问题。举例来说，如果在实验的目的是阐明在肿瘤进展的膜蛋白的作用，胰蛋白酶可能导致膜蛋白的消化，并导致伪像9。如果这是一个问题，使用缓冲EDTA溶液或刮的细胞是一种替代分离细胞。如上所述，细胞可以在培养基，PBS或基质胶FO被再悬浮ř注入。其中，基质胶是一种方便的选择，因为基质胶聚合的脂肪垫，从而减少细胞漏出fatpad的。此外，在植入的基质胶的存在可提高肿瘤的生长和某些细胞系的转移潜能，如乳腺癌细胞系MDA-MB-435 2，人颌下癌A253，人表皮样癌KB和小鼠黑素瘤B16F10细胞7。请注意，一些基质胶制剂含有生长因子潜在地影响的实验结果。然而，生长因子耗尽基质胶可从不同的供应商。此外，基质胶作为细胞外基质（ECM），并激活整联子集。因此，如果所研究的问题涉及整合功能的肿瘤生长的作用，应该使用的介质或PBS作为对乳腺癌细胞的载体。 在我们的实验装置中，我们注射人乳腺癌细胞（MDA-MB-231）进NSG小鼠的乳腺脂肪垫。使用免疫缺陷小鼠明显使得它更难研究免疫调节基因在癌症发展的参与。然而，值得注意的是，这种技术也可在小鼠中具有完整的免疫系统所使用，如果需要被注入的细胞具有鼠源23是很重要的。此外，MDA-MB-231细胞系是雌激素受体阴性（ER-），因此，激素在乳腺癌发展的影响是丢失。尽管如此，我们以前的工作表明，这种技术也是ER +乳腺癌细胞，如MCF-7 13是可行的。利用这种方法，研究了常用的乳腺癌细胞系的肿瘤生长的论文如下: 过程本身包含一些关键步骤为好。与皮下注射，注射原位涉及对小鼠外科手术。因此，手术工具应清洗好，高压灭菌。注意，人体细胞在植入乳腺脂肪垫需要使用免疫缺陷小鼠（ 如 NOD / SCID）的。因此注射应优选地在生物安全柜，使用无菌仪器。此外，在注入过程中，针应保持在合适的角度与针开口朝上。保持在该位置，针减少泄漏并以这种方式成功地注射可以通过观察乳房脂肪垫的肿胀进行验证。另外，在进行手术时，至关重要的是，使一个切口一段距离的乳腺脂肪垫中，以避免伤口愈合过程中与肿瘤生长的干扰。然而，切口不应该从乳房脂肪垫太遥远要么作为露出乳房脂肪垫可能是困难的。 以下的方法，将动物应定期检查。由于手术和暴露到麻醉剂，老鼠可能会遇到大量的不适，甚至死。因此，在手术后的第一周是关键的和小鼠应仔细监测。取决于植入的细胞的生长速率，肿瘤体积，可以测量到，每周4次。注射的细胞也可标记有荧光蛋白或荧光素酶，从而使初级和转移性肿瘤细胞的跟踪，使用光敏感的照相机。肿瘤不应该被允许达到非常大的规模为溃疡可能发生的损害的肿瘤组织。这可能会妨碍肿瘤组织的适当的免疫组织化学分析。一旦肿瘤收获，人们可能会考虑切碎的肿瘤和培养该分散的肿瘤细胞，以建立一个新的乳腺癌细胞系，调查父母之间的差异和mammay脂肪垫传递（MFP）细胞系11。 总之，我们在本文中介绍的原位乳腺癌移植是为了研究癌症相关的进程非常有用的工具。一个CAn乘任上调操纵注射细胞的基因组或下调目的基因，并检查其对原发肿瘤生长，血管发生或转移的作用。这种方法是研究原癌基因，肿瘤抑制器或涉及EMT的基因有帮助的。此外，细胞系可以注入到转基因小鼠中，研究基质室的癌症进展的效果。我们强烈鼓励使用过上述乳腺癌模型原位注射技术由于其高的病理生理过程的概括。

癌症是一种非常复杂的疾病，一直受​​到研究了几个世纪。乳腺癌是最常见的癌症类型;它主要发生在女性，但可能偶尔也发生在男性27。该疾病主要是由控制机构的理事细胞分裂的损失，这又导致细胞在体内的无限增长。这些故障可引起几种机制:首先，健康的细胞需要为了增殖而癌细胞使自己的生长因子，并增加生长因子受体从而获得更高的增殖率1的表达从周围的细胞生长信号;第二，癌细胞不易受到抗增殖信号8;第三，为了平衡在体内的细胞死亡，还需要细胞数;然而，癌细胞程序性细胞死亡逃脱，被称为细胞凋亡14;第四，细胞粘附细胞外基质为了生存，但肿瘤细胞可以成长过程中没有依恋的需要，表现出抗失巢凋亡19;第五，激活端粒酶规避的端粒缩短，防止复制衰老21;最后但并非最不重要的，跳绳的DNA的质量控制下的有丝分裂的结果改变基因的内容15,16。为了确定在其中扮演放松管制的增殖作用，癌基因或抑癌基因，小鼠肿瘤生长的实验是至关重要的。 原发肿瘤的生长一般不会死亡的主要原因。癌细胞从原发部位，以辅助站点的迁移，被称为转移，是死亡的大多数癌症患者22主要原因。限嗣继承转移的肿瘤细胞浸润，血管内，通过循环行驶，避免免疫攻击，外渗和生长在辅助站点。上皮至间质转变（EMT）是一个关键工艺转移并涉及在基因表达谱的开关产生的细胞具有较高的运动和侵袭，这是先决条件为转移性细胞12。作为癌性过程的各种动作，包括癌症细胞，基质细胞和亲和抗炎细胞之间的相互交流的组合所得到的， 在体外的方法来癌症常常不提供完全的洞察癌变过程。同样地，抗癌治疗影响肿瘤脉管往往不能被研究在体外 ，从而利用在体内接近是不可避免的。 研究乳腺癌进展，不同的实验方法已经被开发出来。最广泛使用的模型是皮下注射乳腺癌细胞到小鼠中5。在本实验装置中，研究者可以引入一个广泛的改变，以选择在体外的细胞系（ 即上调，蛋白质的下调），并注入细胞在皮肤下。虽然这种方法非常简单，并在注射过程中，没有任何必要对小鼠进行手术简单，在该肿瘤被注入的部位并不代表本地乳腺肿瘤环境以及缺乏这种环境可能导致乳腺癌的发展是不同于在人类病理学观察。其次，基因工程小鼠经常被用作一个体内的工具来研究乳腺癌进展。在这个模型中，致癌基因（ 即 PyMT，NEU）的表达是由乳腺组织特异性启动子导致的自发乳腺肿瘤S中形成驱动。这个实验装置是有用的通过注射药物或抗体，而在时间3检查肿瘤的大小，以研究这种疾病的治疗方面。然而，繁殖这些小鼠与其他小​​鼠品系缺陷或突变的兴趣也可能会给插件的基因ights成不同的蛋白质在乳腺肿瘤生长24中的作用。这种模式的缺点是很容易产生变异肿瘤的大小和数量。此外，转基因表达的水平取决于整合位点在基因组中，并且可以从一个小鼠品系改变为另一个4。在这个模型中，转基因的表达可以通过所有细胞与上皮起源来实现，而在人类疾病，细胞仅一个亚群表达的原癌基因或下调的肿瘤抑制的水平26。研究转移，乳腺癌细胞也可以静脉注射（一种模式称为实验性转移）25。但是，这种方法只概括转移过程的部分;它避开对肿瘤细胞的侵入和intravasate的要求，并开始从该点处的肿瘤细胞很容易存在于循环中。 在我们的工作中，我们使用原位注入离子模型来研究中乳腺癌进展13感兴趣的基因的参与。我们过表达在人乳腺癌细胞中的蛋白质和它们注入NOD / SCID伽玛（NSG）小鼠的乳腺脂肪垫。这种方法在许多方面是有利的:它允许非常迅速和多样化的注射细胞系的基因变化，它覆盖了从原发肿瘤的生长，以转移乳腺癌进展的整个过程，在病理相关的网站，并且它也提供了一个很好的实验模型用于研究的治疗性治疗在疾病的早期或晚期阶段的影响。另外，使用这个模型1可以调查基质与疾病进展癌细胞来源的蛋白质通过使用遗传修饰的小鼠或细胞的作用。虽然皮下注射是更容易执行，原位模型产生一个更致瘤性和更转移性癌症细胞群。因此，通过皮下注射方式获得的结果S可能会发生是要么假阴性或假阳性6,17鼓励使用原位模型来研究肿瘤的生长。

<table border="0" cellpadding="0" cellspacing="0" width="1093">
	<tbody>
		<tr height="17">
			<td height="17" style="height:17px;width:185px;">Name of material/equipment</td>
			<td style="width:172px;">Company</td>
			<td style="width:95px;">Catalog number</td>
			<td style="width:641px;">Comments/Description</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Bouin&#39;s solution</td>
			<td>Sigma-Aldrich</td>
			<td>HT10132</td>
			<td>Used for investigating the metastasis on lungs</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Formalin solution</td>
			<td>Sigma-Aldrich</td>
			<td>HT501128</td>
			<td>Used to fix the tissues</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Matrigel, growth factor reduced</td>
			<td>Corning</td>
			<td>356230</td>
			<td>Cells can be resuspended in matrigel for injection</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Mosquito forceps</td>
			<td>Fine Science Tools</td>
			<td>13008-12</td>
			<td>Used for stiching</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Angled forceps</td>
			<td>Electron microscopy sciences</td>
			<td>72991-4c</td>
			<td>These make the exposure of mammary fat pad easier</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Scissors</td>
			<td>B Braun Medicals</td>
			<td>BC056R</td>
			<td>Used to cut open the mice</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Straight forceps</td>
			<td>B Braun Medicals</td>
			<td>BD025R</td>
			<td>This is used to open up the skin to expose mammary fat pad</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">NOD scid gamma mice</td>
			<td>Charles River</td>
			<td>005557</td>
			<td>Experimental animal used for experiment</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">MDA-MB-231</td>
			<td>Sigma-Aldrich</td>
			<td>92020424</td>
			<td>Experimental cells used for injections</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Oculentum simplex</td>
			<td>Teva Pharmachemie</td>
			<td></td>
			<td>Opthalmic ointment used to prevent drying out of eyes</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Betadine</td>
			<td>Fischer Scientific</td>
			<td>19-898-859</td>
			<td>Ionophore, used to disinfect the surgical area</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Xylazin/Ketamine</td>
			<td>Sigma-Aldrich</td>
			<td>X1251, K2753</td>
			<td>Use injected anesthesia as 10mg/kg and 100mg/kg body weight respectively</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Temgesic</td>
			<td>Schering-Plough</td>
			<td></td>
			<td>Use the painkiller as 0,05-0,1mg/kg body weight</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">DMEM</td>
			<td>Life sciences</td>
			<td>11995</td>
			<td>For trypsin neutralization,use media with serum(FBS:media 1:10 volume); for injection, use media with no serum</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Buffered sodium citrate</td>
			<td>Aniara</td>
			<td>A12-8480-10</td>
			<td>Use the volume ratio as citrate:blood; 1:9</td>
		</tr>
	</tbody>
</table>

orthotopic injection of breast cancer cells into the mammary fat pad of mice to study tumor growth.

<html>乳腺癌的生长可以通过使用过多的模型来研究小鼠。乳腺癌细胞的基因操纵可以洞察涉及致癌进展或有助于蛋白质的功能，发现新的抑癌基因。此外，注射癌细胞进入小鼠不同基因型可能提供更好的理解的基质隔室的重要性。许多模型可能是有用的，调查疾病进展的某些方面，但不重述整个癌变过程。与此相反，乳腺癌细胞移植到小鼠更好的乳房脂肪垫概括的疾病和适当的基质隔室的存在，因此，更好地模拟人类癌性疾病的位置。在这篇文章中，我们介绍了如何植入乳腺癌细胞注入小鼠原位，并解释如何收集组织分析:14px的;行高:28px;"&gt;肿瘤环境和转移到远处器官利用该模型，对癌症的许多方面（生长，血管生成和转移的）可以简单地通过提供用于肿瘤细胞生长的适当的环境进行调查。

成功应用了"原位乳腺癌模型"是基于适当的细胞注射到乳腺脂肪垫。实验误差如不精确接种细胞或泄漏的可能导致的变化的肿瘤大小或甚至不存在肿瘤这导致结构的形成寻找类似于一个乳房脂肪垫注射了对照缓冲液（ 图2A）的。肿瘤的生长速率依赖于所注入的细胞系的性质和在一般情况下，可以通过只小鼠（ 图2B）的皮肤中观察到。不像在体外实验中，对肿瘤细胞的生长速率可能不会在时间在体内 （ 图2C）常数。由于缺氧和营养素的缺乏，坏死区可形成和这些区域将影响肿瘤（ 图2D）的生长速率。此外，所关注的基因可能会影响一定相癌的进展的（早期或晚期），从而根据不同的实验装置，肿瘤生长可能会开始快但减慢在端，或反之亦然。 护理应肿瘤切除过程中服用;严格的处理可能影响肿瘤的结构，并导致在免疫组织化学分析的问题。肿瘤周围的区域可能会显示源于血管重构的大型船舶。这些血管中除去废物起着至关重要的作用，并提供在肿瘤组织与营养和氧气，从而促进肿瘤生长（ 图2B）。新生血管（血管）的形成可以通过染色肿瘤的新血管标志物进行调查（ 即 CD31，CD34）。 收集在Bouin氏溶液的肺部有助于可视化对肺浅表转移灶（ 图3A）。癌灶可从肺组织由苍白色的方式来区分d为容易检测18。转移可以被表示为形成在肺表面灶的数量。然而，形成灶是细胞系依赖性;非侵略性细胞产生仅微或无转移的。微转移可以容易地识别与在石蜡包埋的肺组织（ 图3C）一个苏木精/伊红染色。 <img alt="图1" src="/files/ftp_upload/51967/51967fig1highres.jpg" /> 图1:材料和方法（A）的手术所需的注射乳腺癌细胞到乳腺脂肪垫设备（B）的代表性的图像示出乳房脂肪垫的曝光。 <img alt="图2" src="/files/ftp_upload/51967/51967fig2highres.jpg" /> 图2:原位乳腺肿瘤生长的小鼠概述（一）媒体控制或<str翁&gt;（B）人类乳腺癌细胞（MDA-MB-231）注入雌性NSG小鼠的乳腺脂肪垫。小鼠安乐死和照片拍摄。肿瘤是由虚线圆表示。箭头指示的乳腺脂肪垫（C）的肿瘤体积的测定使用卡尺通过使用下面的公式:肿瘤体积= 0.5×长×宽×宽（D）的肿瘤的总体形态与H＆E染色进行分析。 I代表浸润细胞，T表示肿瘤细胞和N代表坏死区。 （比例尺= 200微米） <img alt="图3" src="/files/ftp_upload/51967/51967fig3highres.jpg" /> 图3:肿瘤转移至肺中（A）的小鼠的肺即分别原位注射肿瘤细胞（MDA-MB-231）。肺的布安的解决方案孵化公开转移灶的肺。（B）从对照组小鼠肺该行原位喷射控制媒体。正如可以清楚地看到，这些控制肺部不显示宏观转移。（℃），H＆E染色显示了肺组织中的转移区域。插入表示含有转移灶的肺组织的低倍率的概述。在将黑色虚线框表示，在大板。肿瘤细胞是由白虚线表示;注意做大密集的细胞核。 （黑色比例尺= 100微米，白色比例尺= 20微米）

Watch this Scientific Journal Video about Orthotopic Injection of Breast Cancer Cells into the Mammary Fat Pad of Mice to Study Tumor Growth. at JoVE.com

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

癌症是受肿瘤周围的组织，以及本地亲和抗炎介质影响的复杂疾病。因此，正交注入模式的，而不是皮下模型可能是有用的，研究癌症进展的方式，更好地模拟人类的病理。

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 ...

orthotopic-injection-breast-cancer-cells-into-mammary-fat-pad-mice-to

Nanyang Technological University

Research

JoVE Journal

Medicine

78.7K 次观看.  Leiden University Medical Center. 癌症是受肿瘤周围的组织，以及本地亲和抗炎介质影响的复杂疾病。因此，正交注入模式的，而不是皮下模型可能是有用的，研究癌症进展的方式，更好地模拟人类的病理。 

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

Performing and Processing FNA of Anterior Fat Pad for Amyloid

Historically, heart, liver, and kidney biopsies were performed to demonstrate amyloid deposits in amyloidosis. Since the clinical presentation of this disease is so variable and non-specific, the associated risks of these biopsies are too great for the diagnostic yield. Other sites that have a lower biopsy risk, such as skin or gingival, are also relatively invasive and expensive. In addition, these biopsies may not always have sufficient amyloid deposits to establish a diagnosis. Fat pad aspiration has demonstrated good clinical correlation with low cost and minimal morbidity. However, there are no standardized protocols for performing this procedure or processing the aspirated specimen, which leads to variable and nonreproducible results. The most frequently utilized modality for detecting amyloid in tissue is an apple-green birefringence on Congo red stained sections using a polarizing microscope. This technique requires cell block preparation of aspirated material. Unfortunately, patients presenting in early stage of amyloidosis have minimal amounts of amyloid which greatly reduces the sensitivity of Congo red stained cell block sections of fat pad aspirates. Therefore, ultrastructural evaluation of fat pad aspirates by electron microscopy should be utilized, given its increased sensitivity for amyloid detection. This article demonstrates a simple and reproducible procedure for performing anterior fat pad aspiration for the detection of amyloid utilizing both Congo red staining of cell block sections and electron microscopy for ultrastructural identification.

Fat pad aspiration is a preferred, minimally invasive, and low cost approach as compared to other methods to detect amyloid for diagnosis of systemic amyloidosis. This video article demonstrates a procedural outline for performing fat pad aspiration with appropriate processing of the specimen for the optimal diagnostic outcome.

表演和加工为淀粉样蛋白前脂肪垫活检

Historically, heart, liver, and kidney biopsies were performed to demonstrate amyloid deposits in amyloidosis.  Since the clinical presentation of this ...

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 ...

Orthotopic Aortic Transplantation in Mice for the Study of Vascular Disease

Vascular procedures involving anastomoses in the mouse are generally thought to be difficult and highly dependent on the skill of the individual surgeon. This is largely true, but there are a number of important principles that can reduce the difficulty of these procedures and enhance reproducibility. Orthotopic aortic transplantation is an excellent procedure in which to learn these principles because it involves only two end-to-end anastomoses, but requires good suturing technique and handling of the vessels for consistent success. This procedure begins with the procurement of a length of abdominal aorta from a donor animal, followed by division of the native aorta in the recipient. The procured aorta is then placed between the divided ends of the recipient aorta and sutured into place using end-to-end anastomoses. To accomplish this objective successfully requires a high degree of concentration, good tools, a steady hand, and an appreciation of how easily the vasculature of a mouse can be damaged, resulting in thrombosis. Learning these important principles is what occupies most of the beginner's time when learning microsurgery in small rodents. Throughout this protocol, we refer to these important points. This model can be used to study vascular disease in a variety of different experimental systems1-8. In the context shown here, it is most often used for the study of post-transplant vascular disease, a common long-term complication of solid organ transplantation in which intimal hyperplasia occurs within the allograft. The primary advantage of the model is that it facilitates quantitative morphometric analyses and the transplanted vessel lies contiguous to the endogenous vessel, which can serve as an additional control9. The technique shown here is most often used for mice weighing 18-25 grams. We have accumulated most of our experience using the C57BL/6J, BALB/cJ, and C3H/HeJ strains.

We describe a technique in which a section of the abdominal aorta from a mouse is transplanted orthotopically to just below the renal arteries in an allogeneic or syngeneic recipient. This technique can be useful in studies in which transplantation of large arteries of uniform size is deemed advantageous.

原位主动脉瓣移植在小鼠血管疾病的研究

Vascular procedures involving anastomoses in the mouse are generally thought to be difficult and highly dependent on the skill of the individual surgeon. ...

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 ...

A Novel High-resolution In vivo Imaging Technique to Study the Dynamic Response of Intracranial Structures to Tumor Growth and Therapeutics

We have successfully integrated previously established Intracranial window (ICW) technology 1-4 with intravital 2-photon confocal microscopy to develop a novel platform that allows for direct long-term visualization of tissue structure changes intracranially. Imaging at a single cell resolution in a real-time fashion provides supplementary dynamic information beyond that provided by standard end-point histological analysis, which looks solely at 'snap-shot' cross sections of tissue.
Establishing this intravital imaging technique in fluorescent chimeric mice, we are able to image four fluorescent channels simultaneously. By incorporating fluorescently labeled cells, such as GFP+ bone marrow, it is possible to track the fate of these cells studying their long-term migration, integration and differentiation within tissue. Further integration of a secondary reporter cell, such as an mCherry glioma tumor line, allows for characterization of cell:cell interactions. Structural changes in the tissue microenvironment can be highlighted through the addition of intra-vital dyes and antibodies, for example CD31 tagged antibodies and Dextran molecules.
Moreover, we describe the combination of our ICW imaging model with a small animal micro-irradiator that provides stereotactic irradiation, creating a platform through which the dynamic tissue changes that occur following the administration of ionizing irradiation can be assessed.
Current limitations of our model include penetrance of the microscope, which is limited to a depth of up to 900 &mu;m from the sub cortical surface, limiting imaging to the dorsal axis of the brain. The presence of the skull bone makes the ICW a more challenging technical procedure, compared to the more established and utilized chamber models currently used to study mammary tissue and fat pads 5-7. In addition, the ICW provides many challenges when optimizing the imaging.

A Novel High-resolution In vivo Imaging Technique to Study the Dynamic Response of Intracranial Structures to Tumor Growth and Therapeutics

We describe a novel in vivo imaging technique that couples fluorescent chimeric mice with intracranial windows and high-resolution 2-photon microscopy. This imaging platform aids studies of dynamic changes in brain tissue and microvasculature, at a single-cell level, following pathological insults and is adaptable to assess intracranial drug delivery and distribution.

一种新型的高分辨率<em&gt;在体内</em&gt;成像技术研究肿瘤生长及治疗颅内结构的动态响应

We have successfully integrated previously established Intracranial window (ICW) technology 1-4 with intravital 2-photon confocal microscopy to develop a ...

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 ...

Tracking the Mammary Architectural Features and Detecting Breast Cancer with Magnetic Resonance Diffusion Tensor Imaging

Breast cancer is the most common cause of cancer among women worldwide. Early detection of breast cancer has a critical role in improving the quality of life and survival of breast cancer patients. In this paper a new approach for the detection of breast cancer is described, based on tracking the mammary architectural elements using diffusion tensor imaging (DTI). 
The paper focuses on the scanning protocols and image processing algorithms and software that were designed to fit the diffusion properties of the mammary fibroglandular tissue and its changes during malignant transformation. The final output yields pixel by pixel vector maps that track the architecture of the entire mammary ductal glandular trees and parametric maps of the diffusion tensor coefficients and anisotropy indices. 
The efficiency of the method to detect breast cancer was tested by scanning women volunteers including 68 patients with breast cancer confirmed by histopathology findings. Regions with cancer cells exhibited a marked reduction in the diffusion coefficients and in the maximal anisotropy index as compared to the normal breast tissue, providing an intrinsic contrast for delineating the boundaries of malignant growth. Overall, the sensitivity of the DTI parameters to detect breast cancer was found to be high, particularly in dense breasts, and comparable to the current standard breast MRI method that requires injection of a contrast agent. Thus, this method offers a completely non-invasive, safe and sensitive tool for breast cancer detection.

We describe how to obtain parametric and vector maps of the diffusion tensor of the breast using magnetic resonance imaging. The protocol and final output following imaging processing are tailored for tracking breast architectural features and detecting breast malignancy.

跟踪乳腺建筑特色与检测乳腺癌的磁共振扩散张量成像

Breast cancer is the most common cause of cancer among women worldwide. Early detection of breast cancer has a critical role in improving the quality of ...

Assessment of Viability of Human Fat Injection into Nude Mice with Micro-Computed Tomography

Lipotransfer is a vital tool in the surgeon&#8217;s armamentarium for the treatment of soft tissue deficits of throughout the body. Fat is the ideal soft tissue filler as it is readily available, easily obtained, inexpensive, and inherently biocompatible.1 However, despite its burgeoning popularity, fat grafting is hampered by unpredictable results and variable graft survival, with published retention rates ranging anywhere from 10-80%. 1-3
To facilitate investigations on fat grafting, we have therefore developed an animal model that allows for real-time analysis of injected fat volume retention. Briefly, a small cut is made in the scalp of a CD-1 nude mouse and 200-400 &#181;l&#160;of processed lipoaspirate is placed over the skull. The scalp is chosen as the recipient site because of its absence of native subcutaneous fat, and because of the excellent background contrast provided by the calvarium, which aids in the analysis process. Micro-computed tomography (micro-CT) is used to scan the graft at baseline and every two weeks thereafter. The CT images are reconstructed, and an imaging software is used to quantify graft volumes.
Traditionally, techniques to assess fat graft volume have necessitated euthanizing the study animal to provide just a single assessment of graft weight and volume by physical measurement ex vivo. Biochemical and histological comparisons have likewise required the study animal to be euthanized. This described imaging technique offers the advantage of visualizing and objectively quantifying volume at multiple time points after initial grafting without having to sacrifice the study animal. The technique is limited by the size of the graft able to be injected as larger grafts risk skin and fat necrosis. This method has utility for all studies evaluating fat graft viability and volume retention. It is particularly well-suited to providing a visual representation of fat grafts and following changes in volume over time.

Fat grafting is an essential technique for reconstructing soft tissue deficits. However, it remains an unpredictable procedure characterized by variable graft survival. Our goal was to devise a mouse model that utilizes a novel imaging method to compare volume retention between differing techniques of fat graft preparation and delivery.

评估人体脂肪注射生存能力的裸鼠与微型计算机断层扫描

Lipotransfer is a vital tool in the surgeon&#8217;s armamentarium for the treatment of soft tissue deficits of throughout the body. Fat is the ideal soft ...

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 ...

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。

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