作者要感谢Pam Ovwigho，Tenette Jones和NIEHS的Natasha Clayton在这个项目中的技术专长和支持。

本研究的所有动物使用和手术均由NIEHS实验动物批准护理和使用委员会，并在一个评估和认证协会进行实验动物护理认证设施。 1.乳腺全安装制备例2，3，6，7 <ol><li>如参考文献3所述，从非怀孕的CD-1雌性小鼠的一侧取下腹股沟乳腺，并将它们放在带静电的载玻片上。 </li><li>用不粘纸或胶片覆盖腺体，并在上面放置另一张幻灯片。向载玻片增加压力（ 即水重量）以将腺体平放，使腺体粘附到载玻片上进行固定。 </li><li>将载玻片浸入固定剂（ 例如， 100％乙醇，氯仿和6:3:1比例的冰醋酸）室温过夜。 </li><li>从固定剂中取出腺体，并在乙醇中洗涤15-30分钟。 30分钟洗涤后，倒出1/3的乙醇并加水，逐渐变成水。让洗澡每次静坐约5分钟，重复加水3次。 </li><li>染色（ 如胭脂红明矾溶液）12-24小时。 注意:较厚的组织需要较长的染色时间。染色细节参见参考3 。 </li><li>在分配时间后倒出污渍，并在水中冲洗30秒。通过在70％乙醇中洗涤载玻片15分钟使组织脱水，然后在95％乙醇中洗涤15分钟，最后在100％乙醇中洗涤20分钟。 </li><li>通过将组织中的脂肪置于二甲苯中至少24小时或更长时间，如果组织较厚，则可清除组织中的脂肪，以便去除任何不透明或白色区域。 </li><li>从二甲苯中取出组织，快速加入将unting培养液置于载玻片上避免组织脱水;在上面放置盖玻片。让载玻片干燥至少48小时。 </li><li>评估整体组织的异常情况，如2所述 。 注意:当整体检测到病变时，切片需要确定其身份，应遵循以下步骤。 </li></ol> 2.从玻璃片中去除乳房整体<ol><li>将乳化整体载玻片浸入装满二甲苯的玻璃染色瓶中，将盖玻片和固定介质置于一个晚上。 注意:使用过度安装溶液的较厚组织和载玻片可能需要额外的时间才能清除盖玻片。 </li><li>在初始过夜浸泡后，将载玻片置于新鲜的二甲苯中并浸泡6小时。在新鲜的二甲苯中一次最后浸泡过夜。 注意:最后一次浸泡后，盖玻片将很容易脱落。 </li><li>用ag亲手握住幻灯片，并用一对镊子小心地取出盖玻片，以确保其一体移除。 注意:如果盖玻片仍然连接到玻璃滑块上，请继续浸泡，直至能够轻松移除。 </li><li>一旦盖玻片被取出，将载玻片垂直地夹在含有二甲苯的玻璃培养皿中，小心地取出锋利的一次性剃刀刀片，并沿着幻灯片一次滑动刀片。 </li><li>一旦组织被移除，迅速将乳房浸入二甲苯填充的玻璃培养皿中，以确保组织不会空气干燥。 注意:注意这一步。整个安装件薄（≤1mm），二甲苯加工易碎。任何印象或眼泪可能会改变组织的形态，并使后续评估复杂化。 </li></ol> 3.组织加工<ol><li>一旦组织在培养皿中，仔细转移到标记的组织学盒。用钝头，锯齿状的镊子抓住脂肪垫的边缘，以尽量减少组织损伤。 <ol><li>使用剃须刀将较大的组织（特别是大鼠）切成两半，并将它们放在两个分开的标签盒中，以适应组织尺寸。将组织右侧放置（使用淋巴结作为参考）。确保没有淋巴结的组织在转移到盒中时保持在相同的直立位置。 </li></ol></li><li>将纸盒放入二甲苯容器中长达2小时，然后将其放入组织处理器中。 注意:建议在样品放入盒子的同一天处理样品。在二甲苯中的延长浸泡未经测试。 </li><li>用手将最大数量的纸盒装入纸巾处理器。 </li><li>在开始之前，将将用于此过程的所有试剂编入处理器的试剂列表。自动运动从一个站点到另一个站点，使用菜单选项并创建一个新的程序。所有步骤完成后，选择"确定"继续。 注意:处理器将允许在启动之前检查所有工作站的详细信息。 <ol><li>使处理器自动化，使其在二甲苯中在37℃下浸泡30分钟，然后在相同条件下在新鲜二甲苯中第二次浸泡。使处理器自动化以从二甲苯溶液中取出盒，并将组织转移到设置在60℃下的含有1:1二甲苯:熔融石蜡混合物的下一个工作站30分钟。 注意:处理器然后将盒子转移到含有熔融石蜡的新室中1小时。 1小时后，样品将自动转移到新的熔融石蜡室，并再加工2小时。两个步骤在60℃下进行。 </li><li>然后处理器将盒子转移到含有熔融对位的工位ffin 1小时。然后，将样品自动转移到新鲜熔融石蜡的最终工位并再加工2小时。两个步骤在60℃下进行。 </li></ol></li></ol> 4.包埋加工的乳腺组织<ol><li>将盒子放入嵌入站的58°C石蜡容器中，直到准备将组织嵌入模具中。 </li><li>取下盒盖确定组织的最佳模具尺寸。确保模具足够大以容纳组织，并留下足够的空间以在周边具有石蜡的无组织边界。 </li><li>向模具中加入3-4毫米的熔融石蜡，并将乳化的整体安装表面定向到与玻璃底部和药盒底部相对的模具中。 </li><li>将模具转移到冷却板上，并根据需要快速调整组织，使其平行于模具bottom。 注意:一旦石蜡硬化，组织将被置于适当位置。 </li><li>使用暖镊子，将带标签的下半部分放在模具顶部;用镊子牢牢地按住。 </li><li>将额外的熔融石蜡以连续的运动方式添加到模具中以覆盖整个盒。将模具从暖板移动到冷板，以完成块体的硬化。 </li><li>当石蜡完全固化以避免裂纹或气泡时，从模具中取出块体。 注意:将石蜡包埋的组织在室温下储存，直到准备好。 </li></ol> 5.在切片机上切片石蜡包埋的乳腺组织<ol><li>在切片前，将石蜡块在-20℃孵育1小时。 注意:从原来盖玻片的全贴纸组件中的块中将有残留的固定介质。冷却块改进块截面和色带。 </li><li>准备麦克风通过打开水浴，包含新鲜的蒸馏水，并将温度调节至42-45℃。将新鲜的，低调的刀片放置在切片机上，并将其设置为4μm。 注意:由于在组织中存在残留的固定介质，部分质量会减少，厚度大于4μm。 </li><li>将块插入切片机，蜡面朝向刀片并与垂直平面对齐。然后，在冷水中润湿一段纱布垫，并将其放在块上几分钟。 </li><li>通过与粗轮廓轮组合，以顺时针方向转动大轮，直到获得全面或代表性块的块来切块。 注意:由于在整个安装过程中二甲苯清除，组织很薄。将刀片正确对准刀片，以最大限度地减少获得代表部件的切割次数。 </li><li>挑丝带小心地将色带漂浮在温暖（45°C）的水浴（预先准备）中，使组织皱纹消散，并将部分小心地漂浮在干净的玻璃片上。 </li><li>将幻灯片放在干燥架上，以清除多余的水分。在37℃下将载玻片在稍微打开的载玻片盒中孵育过夜。 </li></ol> 6.分切乳房组织的自动和手动H＆E染色<ol><li>在染色之前，将载玻片在室温下储存。 </li><li>对于自动染色，请从主菜单上的染色选项中选择H＆E。脱蜡，染色和脱水都在自动染色机上完成。使用安装介质和盖玻片安装这些部分。 </li><li>如果手工染色载玻片，则将载玻片浸入二甲苯中5分钟，使切片脱蜡。转移到新鲜的二甲苯并重复5分钟。 </li><li>通过浸没来补充幻灯片在100％乙醇中分两次，每次3分钟，然后在新鲜的95％乙醇中重复两次，并在1X洗涤缓冲液中分别洗涤两次，每次5分钟。 </li><li>用蒸馏水冲洗载玻片。 </li><li>将载玻片加入苏木精1-2分钟。取出载玻片并用自来水冲洗5分钟。 注意:在每次使用前过滤苏木精。 </li><li>将载玻片置于1％冰醋酸中30秒以促进颜色分化，然后在自来水中冲洗1分钟。 </li><li>将幻灯片添加到1X PBS 30秒至1分钟以蓝色组织切片，然后在自来水中冲洗5分钟，然后在95％酒精中冲洗15-20秒。 </li><li>用曙红溶液复染30秒至1分钟。然后，将其在新鲜的95％乙醇中脱水两次，然后在100％乙醇中进行两次重复。进行每次洗涤5分钟。 </li><li>在二甲苯中清除载玻片，每次二甲苯变化5分钟。 </li><li>盖玻片组织部分与安装介质在室温干燥过夜。 </li></ol>

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作者与本文的研究，作者和/或出版相关的利益没有任何冲突。

乳腺整体是一个强大的工具，可用于说明暴露于化学物质（包括内分泌干扰物）后可能出现和持续的正常乳腺发育和形态学改变。当同一个动物的整个座位和H＆E部分被一起评估时，它们可以提供早期改变的准确视觉快照，这可以进入更多的疾病状态。 获得这种有用信息的能力在于确保在去除组织时要注意保持并不损害腺体形态。虽然啮齿类具有位于两侧沿背壁几个乳腺部位...

乳腺整体是一种有用和便宜的方法，在许多老鼠和小鼠研究中实现，以了解正常和化学诱导的异常发育。通常，在啮齿动物发育（ 即青春期，青春期，中期至晚期妊娠和退行期）各个阶段收集的乳腺将显示受旁分泌，内分泌和自分泌因素影响的组织和细胞结构的形态学变化1 。在衰老大鼠中，腺体的上皮和间质部分变得越来越致密，这使得测量形态参数在整体上难以测量。因此，通常的做法是收集用于组织学评估的腺体以鉴别微观水平上的变化。这两种方法在涉及化学暴露（ 即环境或药物）的研究中尤其有用，或者检查伴随遗传改变的形态学变化ations。 使用乳腺进行风险评估的技术和能力不断发展。虽然整个贴装制备是常规的和标准的，修改切片继续2,3。来自不同背景（ 即学术界，政府和行业）的许多研究小组已经将乳腺组织的冠状/纵向切片作为首选方法，而一些实验室仍然使用通过皮肤的横切部分4 。后一种方法导致表皮（皮肤）的过分表示，而不是感兴趣的组织:乳腺上皮2 。冠状或纵向切片对于表征正常组织5更有用，并且由于增加的表面积和存在的结构数量而大大改善了异常和损伤的检测。总的来说，这使整个人对于乳腺1，2的比较组织病理学评估UNT的合适方法。强制推荐使用小鼠或大鼠，检索和保存第4和第 5 个腹股沟腺体，以便将整个安全架与对侧H＆E部分进行比较。 当组合使用时，H＆E部分和整个安装座提供了由环境暴露引起的细胞和形态变化的准确描述。在特定的啮齿动物菌株中尤其如此，其中模型对肿瘤形成的敏感性低或肿瘤不是很明显。在某些情况下，使用两种技术（ 即，组织数量不足，资源不足或意外的实验结果）获得所需的组织可能并不总是可能的。在我们的情况下，在乳房整体上观察到异常，而组织病理学对侧H＆E腺的合理发现主要是正常的。对侧腺体之间的压倒性差异导致我们开发了一种经济有效的方法来识别整体的异常。使用改进的加工和嵌入程序，从石蜡包埋的整体安装中制备高质量的H＆E染色切片。我们设想这个协议成为化学暴露效应的强大的检测工具，增强了实验室间的比较。

<table border="0" cellpadding="0" cellspacing="0" width="1013">
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		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:385px;">Xylene</td>
			<td style="width:309px;">Sigma-Aldrich</td>
			<td style="width:152px;">214736</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:385px;">Permount</td>
			<td>Thermo Fisher Scientific</td>
			<td>SP15-100</td>
			<td>mounting media for coverslipping whole mounts and H&#38;E sections</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:385px;">Leica automated processor</td>
			<td>Leica Biosystems</td>
			<td>Model # ST5020</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:385px;">HM 355S Automatic Microtome</td>
			<td>Thermo Fisher Scientific&#160;</td>
			<td>905200</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:385px;">Paraffin</td>
			<td>Leica Biosystems</td>
			<td style="width:152px;">EM-400</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:385px;">Superfrost plus microscope slides</td>
			<td>Thermo Fisher Scientific</td>
			<td style="width:152px;">4951PLUS-001</td>
			<td>electrostatically charged</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:385px;">Fisher Finest 24x60x1</td>
			<td>Thermo Fisher Scientific</td>
			<td style="width:152px;">12-548-5P</td>
			<td>coverslips</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Varistain Gemini ES Automatic slide stainer</td>
			<td>Thermo Fisher Scientific</td>
			<td>A78000014</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:385px;">Sta-On</td>
			<td>Leica Biosystems</td>
			<td>3803107</td>
			<td>liquid adhesive; used in water bath at sectioning</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Modified Harris Hematoxylin 72711</td>
			<td>Richard-Allan Scientific (Part of Thermo Scientific)</td>
			<td>72711</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:385px;">Eosin</td>
			<td>Leica Biosystems</td>
			<td>3801600</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:385px;">Lithium Carbonate</td>
			<td>SkyTech Laboratories</td>
			<td style="width:152px;">LCQ500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:385px;">Glacial Acetic Acid</td>
			<td>Thermo Fisher Scientific</td>
			<td style="width:152px;">A38-500</td>
			<td>needed in Carnoy&#39;s fixative</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:385px;">Chloroform</td>
			<td>Macron Fine Chemicals</td>
			<td style="width:152px;">4440-04</td>
			<td>needed in Carnoy&#39;s fixative</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:385px;">100% Ethanol</td>
			<td>The Warner Graham Company</td>
			<td style="width:152px;">6505001050000</td>
			<td>needed in Carnoy&#39;s fixative and washing slides</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:385px;">95% Ethanol</td>
			<td>The Warner Graham Company</td>
			<td style="width:152px;">6505011137320190</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:385px;">Carmine Alum</td>
			<td>Sigma-Aldrich</td>
			<td style="width:152px;">C1022-25G</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:385px;">Aluminum potassium sulfate dodecahydrate, Sigma Ultra</td>
			<td>Sigma-Aldrich</td>
			<td style="width:152px;">A7210-500G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:385px;">Automation wash buffer, 20X</td>
			<td>Biocare Medical</td>
			<td>TWB945</td>
			<td></td>
		</tr>
	</tbody>
</table>

sectioning mammary gland whole mounts for lesion identification

正常的乳腺发育可能因暴露于环境有毒物质和药物产品，过度暴露于激素和遗传改变而改变。乳腺全身是一种廉价的方法来捕获暴露后可能出现的形态变化的进展。然而，在后来的生活中，当异常更容易发展时，仅依靠这一种方法可能不总是提供足够的信息来正确诊断异常。历史上，在化学测试指南研究中，在尸检时除去单个乳腺，并制备为苏木精和伊红（H＆E）染色部分。对侧乳腺整体收集和分析的并入减少了假阴性评估的可能性。整个安装座的评估受到幻灯片上一个或两个整个乳腺的存在的限制，在某些情况下，在整个安装座上观察到的异常不是在H＆E部分统一表示。这项研究的目的是制定一个方案，将覆盖乳房的整体安装转换为H＆E染色部分，以便可以识别否则将被遗漏或难以诊断的病变。在这里，我们详细介绍了一种从最初准备为整体的乳腺产生高品质，石蜡包埋的H＆E切片的方法。与有意为H＆E切片制备的组织相比，整个安装件需要额外的组织去除和加工准备。然而，这种方法被认为是便宜的，因为它需要常见的实验室试剂和少量额外的时间。因此，这种方法可以提供关于化学和环境暴露如何改变正常乳腺发育以及显示由于遗传修饰而发生的变化的宝贵信息。

如果乳房的原始对侧H＆E部分没有显示任何组织学变化，则该方法有助于协助可能会错过的诊断。然而，如果在初始整体准备过程中以及在组织学评估的组织准备过程中要注意，结果将是有用的。石蜡包埋将提供保护，并有助于保留组织以供将来切片。 为了确定乳房组织的理想厚度，制备了4μm和6μm的切片。我们测试了在切片?...

Watch this Scientific Journal Video about Sectioning Mammary Gland Whole Mounts for Lesion Identification at JoVE.com

Sectioning Mammary Gland Whole Mounts for Lesion Identification

我们开发了一种成功去除，处理，切片和染色的方法，用于组织病理学评估，原始固定在载玻片上的乳腺组织作为整体。这种方法可以促进生殖发育试验指南研究中乳腺整体的收集和评估。

切片乳腺全身为病变鉴别

Normal mammary gland development may be altered by exposure to environmental toxicants and pharmaceutical products, excessive exposure to hormones, and ...

sectioning-mammary-gland-whole-mounts-for-lesion-identification

Research

JoVE Journal

Cancer Research

10.3K 次观看.  University of North Carolina at Chapel Hill. 我们开发了一种成功去除，处理，切片和染色的方法，用于组织病理学评估，原始固定在载玻片上的乳腺组织作为整体。这种方法可以促进生殖发育试验指南研究中乳腺整体的收集和评估。 

视频: Sectioning Mammary Gland Whole Mounts for Lesion Identification

Surgical Procedures and Methodology for a Preclinical Murine Model of De Novo Mammary Cancer Metastasis

A rate-limiting aspect of transgenic mouse models of mammary adenocarcinoma is that primary tumor burden in mammary tissue typically defines study end-points. Thus, studies focused on elucidating mechanisms of late-stage de novo metastasis are compromised, as are studies examining efficacy of anti-cancer therapies targeting mediators of metastasis in the adjuvant setting. Numerous murine mammary cancer models have been developed via targeted expression of dominant oncoproteins to mammary epithelial cells yielding models variably mimicking histopathologic and transcriptome-defined breast cancer subtypes common in women1. While much has been learned regarding the biology of mammary carcinogenesis with these models, their utility in identifying molecules regulating growth of late-stage metastasis are compromised as mice are typically euthanized at earlier time points due to significant primary tumor burden. Moreover, since a significant percentage of women diagnosed with breast cancer receive adjuvant therapy after surgical resection of primary tumors and prior to presence of detectable metastatic disease, preclinical models of de novo metastasis are urgently needed as platforms to evaluate new therapies aimed at targeting metastatic foci. To address these deficiencies, we developed a murine model of de novo mammary cancer metastasis, wherein primary mammary tumors are surgically resected, and metastatic foci subsequently develop over a 115 day post-surgical period. This long latency provides a tractable model to identify functionally significant regulators of metastatic progression in mice lacking primary tumor, as well as a model to evaluate preclinical therapeutic efficacy of agents aimed at blocking functionally significant molecules aiding metastatic tumor survival and growth.

Surgical Procedures and Methodology for a Preclinical Murine Model of De Novo Mammary Cancer Metastasis

Pre-clinical models evaluating adjuvant therapy targeting breast cancer metastasis are lacking. To address this, we developed a murine model of de novo pulmonary mammary adenocarcinoma metastasis, wherein therapies administered in the adjuvant setting (post surgical resection of primary tumors) can be evaluated for efficacy in impacting previously seeded pulmonary metastases.

临床前小鼠模型的外科手术和方法<em&gt; De Novo</em&gt;乳腺癌转移

A rate-limiting aspect of transgenic mouse models of mammary adenocarcinoma is that primary tumor burden in mammary tissue typically defines study ...

科研

癌症研究

Target Cell Pre-enrichment and Whole Genome Amplification for Single Cell Downstream Characterization

Rare target cells can be isolated from a high background of non-target cells using antibodies specific for surface proteins of target cells. A recently developed method uses a medical wire functionalized with anti-epithelial cell adhesion molecule (EpCAM) antibodies for in vivo isolation of circulating tumor cells (CTCs)1. A patient-matched cohort in non-metastatic prostate cancer showed that the in vivo isolation technique resulted in a higher percentage of patients positive for CTCs as well as higher CTC counts as compared to the current gold standard in CTC enumeration. As cells cannot be recovered from current medical devices, a new functionalized wire (referred to as Device) was manufactured allowing capture and subsequent detachment of cells by enzymatic treatment. Cells are allowed to attach to the Device, visualized on a microscope and detached using enzymatic treatment. Recovered cells are cytocentrifuged onto membrane-coated slides and harvested individually by means of laser microdissection or micromanipulation. Single-cell samples are then subjected to single-cell whole genome amplification allowing multiple downstream analysis including screening and target-specific approaches. The procedure of isolation and recovery yields high quality DNA from single cells and does not impair subsequent whole genome amplification (WGA). A single cell&#39;s amplified DNA can be forwarded to screening and/or targeted analysis such as array comparative genome hybridization (array-CGH) or sequencing. The device allows ex vivo isolation from artificial rare cell samples (i.e. 500 target cells spiked into 5 mL of peripheral blood). Whereas detachment rates of cells are acceptable (50 - 90%), the recovery rate of detached cells onto slides spans a wide range dependent on the cell line used (&#60;10 - &#62;50%) and needs some further attention. This device is not cleared for the use in patients.

This protocol is to recover and prepare rare target cells from a mixture with non-target background cells for molecular genetic characterization at the single-cell level. DNA quality is equal to non-treated single cells and allows for single-cell application (both screening based and targeted analysis).

单细胞下游特征的靶细胞预富集与全基因组扩增

Rare target cells can be isolated from a high background of non-target cells using antibodies specific for surface proteins of target cells. A recently ...

Identification of OTX1 and OTX2 As Two Possible Molecular Markers for Sinonasal Carcinomas and Olfactory Neuroblastomas

OTX homeobox (HB) genes are expressed during embryonic morphogenesis and during the development of olfactory epithelium in adult organisms. Mutations occurring in these genes are often related to tumorigenesis in human. No data are available today regarding the possible correlation between OTX genes and tumors of the nasal cavity. The aim of this work is to understand if OTX1 and OTX2 can be considered as molecular markers in the development of nasal tumors. We selected nasal and sinonasal adenocarcinomas to investigate the expression of OTX1 and OTX2 genes through immunohistochemical and real-time PCR analyses.Both OTX1 and OTX2 were absent in all the samples of sinonasal Intestinal-Type Adenocarcinomas (ITACs). OTX1 mRNA was identified only in Non-Intestinal Type Adenocarcinomas (NITACs) while OTX2 mRNA was expressed only in Olfactory Neuroblastomas (ONs). We have demonstrated that the differential gene expression for both OTX1 and OTX2 genes might be a useful molecular marker to distinguish the different types of sinonasal tumors.

Homeobox genes are regulatory genes often associated with tumors in the adult organisms. We investigated their comparative expression by immunohistochemical and real-time PCR analysis, in normal and inflammatory nasal mucosae and in sinonasal neoplasms in order to use them as possible diagnostic and therapeutic targets.

otx1 和 otx2 作为鼻窦癌和嗅觉神经母细胞瘤的两种可能的分子标记物的鉴定

OTX homeobox (HB) genes are expressed during embryonic morphogenesis and during the development of olfactory epithelium in adult organisms. Mutations ...

Identification, Histological Characterization, and Dissection of Mouse Prostate Lobes for In Vitro 3D Spheroid Culture Models

Genetically engineered mouse models (GEMMs) serve as effective pre-clinical models for investigating most types of human cancers, including prostate cancer (PCa). Understanding the anatomy and histology of the mouse prostate is important for the efficient use and proper characterization of such animal models. The mouse prostate has four distinct pairs of lobes, each with their own characteristics. This article demonstrates the proper method of dissection and identification of mouse prostate lobes for disease analysis. Post-dissection, the prostate cells can be further cultured in vitro for mechanistic understanding. Since mouse prostate primary cells tend to lose their normal characteristics when cultured in vitro, we outline here a method for isolating the cells and growing them as 3D spheroid cultures, which is effective for preserving the physiological characteristics of the cells. These 3D cultures can be used for analyzing cell morphology and behavior in near-physiological conditions, investigating altered levels and localizations of key proteins and pathways involved in the development and progression of a disease, and looking at responses to drug treatments.

Genetically engineered mice are useful models for investigating prostate cancer mechanisms. Here we present a protocol to identify and dissect prostate lobes from a mouse urogenital system, differentiate them based on histology, and isolate and culture the primary prostate cells in vitro as spheroids for downstream analyses.

体外3D 球形培养模型小鼠前列腺叶的鉴定、组织学特征及解剖

Genetically engineered mouse models (GEMMs) serve as effective pre-clinical models for investigating most types of human cancers, including prostate ...

Orthotopic Injection of Breast Cancer Cells into the Mice Mammary Fat Pad

A proper animal model is crucial for a better understanding of diseases. Animal models established by different methods (subcutaneous injections, xenografts, genetic manipulation, chemical reagents induction, etc.) have various pathological characters and play important roles in investigating certain aspects of diseases. Although no single model can totally mimic the whole human disease progression, orthotopic organs disease models with a proper stromal environment play an irreplaceable role in understanding diseases and screening for potential drugs. In this article, we describe how to implant breast cancer cells into the mammary fat pad in a simple, less invasive, and easy-to-handle way, and follow the metastasis to distant organs. With the proper features of primary tumor growth, breast and nipple pathological changes, and a high occurrence of other organs&#39; metastasis, this model maximumly mimics human breast cancer progression. Primary tumor growth in situ, long-distant metastasis, and the tumor microenvironment of breast cancer can be investigated by using this model.

Here, we present a protocol to implant breast cancer cells into the mammary fat pad in a simple, less invasive, and easy-to-handle way, and this mouse orthotopic breast cancer model with a proper mammary fat pad environment can be used to investigate various aspects of cancer.

乳腺癌细胞原位注射到小鼠乳房脂肪板

A proper animal model is crucial for a better understanding of diseases. Animal models established by different methods (subcutaneous injections, ...

Modeling Breast Cancer via an Intraductal Injection of Cre-expressing Adenovirus into the Mouse Mammary Gland

Breast cancer is a heterogeneous disease, possibly due to complex interactions between different cells of origins and oncogenic events. Mouse models are instrumental in gaining insights into these complex processes. Although many mouse models have been developed to study contributions of various oncogenic events and cells of origin to breast tumorigenesis, these models are often not cell-type or organ specific or cannot induce the initiation of mammary tumorigenesis in a temporally controlled manner. Here we describe a protocol to generate a new type of breast cancer mouse models based on the intraductal injection of Cre-expressing adenovirus (Ad-Cre) into mouse mammary glands (MGs). Due to the direct injection of Ad-Cre into mammary ducts, this approach is MG specific, without any unwanted cancer induction in other organs. The intraductal injection procedure can be performed in mice at different stages of their MG development (thus, it permits temporal control of cancer induction, starting from ~3-4 weeks of age). The cell-type specificity can be achieved by using different cell-type-specific promoters to drive Cre expression in the adenoviral vector. We show that luminal and basal mammary epithelial cells (MECs) can be tightly targeted for Cre/loxP-based genetic manipulation via an intraductal injection of Ad-Cre under the control of the Keratin 8 or Keratin 5 promoter, respectively. By incorporating a conditional Cre reporter (e.g., Cre/loxP-inducible Rosa26-YFP reporter), we show that MECs targeted by Ad-Cre, and tumor cells derived from them, can be traced by following the reporter-positive cells after intraductal injection.

The goal of this protocol is to describe a new breast cancer modeling approach based on the intraductal injection of Cre-expressing adenovirus into mouse mammary glands. This approach allows both cell-type- and organ-specific manipulation of oncogenic events in a temporally controlled manner.

通过将表达的腺病毒注射到小鼠乳腺腺中,模拟乳腺癌

Breast cancer is a heterogeneous disease, possibly due to complex interactions between different cells of origins and oncogenic events. Mouse models are ...

Rat Mammary Epithelial Cell Transplantation into the Interscapular White Fat Pad

As early as the 1970s, researchers have successfully transplanted mammary epithelial cells into the interscapular white fat pad of rats. Grafting mammary epithelium using transplantation techniques takes advantage of the hormonal environment provided by the adolescent rodent host. These studies are ideally suited to explore the impact of various biological manipulations on mammary gland development and dissect many aspects of mammary gland biology. A common, but limiting, feature is that transplanted epithelial cells are strongly influenced by the surrounding stroma and outcompeted by endogenous epithelium; to utilize native mammary tissue, the abdominal-inguinal white fat pad must be cleared to remove host mammary epithelium prior to the transplantation. A major obstacle when using the rat model organism is that clearing the developing mammary tree in post-weaned rats is not efficient. When transplanted into gland-free fat pads, donor epithelial cells can repopulate the cleared host fat pad and form a functional mammary gland. The interscapular fat pad is an alternative location for these grafts. A major advantage is that it lacks ductal structures yet provides the normal stroma that is necessary to promote epithelial outgrowth and is easily accessible in the rat. Another major advantage of this technique is that it is minimally invasive, because it eliminates the need to cauterize and remove the growing endogenous mammary tree. Additionally, the interscapular fat pad contains a medial blood vessel that can be used to separate sites for grafting. Because the endogenous glands remain intact, this technique can also be used for studies comparing the endogenous mammary gland to the transplanted gland. This paper describes the method of mammary epithelial cell transplantation into the interscapular white fat pad of rats.

This article describes a transplantation method to graft donor rat mammary epithelial cells into the interscapular white fat pad of recipient animals. This method can be used to examine host and/or donor effects on mammary epithelium development and eliminates the need for pre-clearing, thereby extending the usefulness of this technique.

大鼠乳腺内皮细胞移植到间膜白脂肪垫

As early as the 1970s, researchers have successfully transplanted mammary epithelial cells into the interscapular white fat pad of rats. Grafting mammary ...

In Vitro Evaluation of Oncogenic Transformation in Human Mammary Epithelial Cells

Tumorigenesis is a multi-step process in which cells acquire capabilities&#160;that allow their growth, survival, and dissemination under hostile conditions. Different tests seek to identify and quantify these hallmarks of cancerous cells; however, they often focus on a single aspect of cellular transformation and, in fact, multiple tests are required for their proper characterization. The purpose of this work is to provide researchers with a set of tools to assess cellular transformation in vitro from a broad perspective, thereby making it possible to draw sound conclusions.
A sustained proliferative signaling activation is the major feature of tumoral tissues and can be easily monitored under in vitro conditions by calculating the number of population doublings achieved over time. Besides, the growth of cells in 3D cultures allows their interaction with surrounding cells, resembling what occurs in vivo. This enables the evaluation of cellular aggregation and, together with immunofluorescent labeling of distinctive cellular markers, to obtain information on another relevant feature of tumoral transformation: the loss of proper organization. Another remarkable characteristic of transformed cells is their capacity to grow without attachment to other cells and to the extracellular matrix, which can be evaluated with the anchorage assay.
Detailed experimental procedures to evaluate cell growth rate, to perform immunofluorescent labeling of cell lineage markers in 3D cultures, and to test anchorage-independent cell growth in soft agar are provided. These methodologies are optimized for Breast Primary Epithelial Cells (BPEC) due to its relevance in breast cancer; however, procedures can be applied to other cell types after some adjustments.

This protocol provides experimental in vitro tools to evaluate the transformation of human mammary cells. Detailed steps to follow-up cell proliferation rate, anchorage-independent growth capacity, and distribution of cell lineages in 3D cultures with basement membrane matrix are described.

人类乳腺上皮细胞致癌转化的体外评价

Tumorigenesis is a multi-step process in which cells acquire capabilities&#160;that allow their growth, survival, and dissemination under hostile ...

Identification of EGFR and RAS Inhibitors using Caenorhabditis elegans

The changes in the plasma membrane localization of the epidermal growth factor receptor (EGFR) and its downstream effector RAS have been implicated in several diseases including cancer. The free-living nematode C. elegans possesses an evolutionary and functionally conserved EGFR-RAS-ERK MAP signal cascade which is central for the development of the vulva. Gain of function mutations in RAS homolog LET-60 and EGFR homolog LET-23 induce the generation of visible nonfunctional ectopic pseudovulva along the ventral body wall of these worms. Previously, the multivulval (Muv) phenotype in these worms has been shown to be inhibited by small chemical molecules. Here we describe a protocol for using the worm in a liquid-based assay to identify inhibitors that abolish the activities of EGFR and RAS proteins. Using this assay, we show R-fendiline, an indirect inhibitor of K-RAS, suppresses the Muv phenotype expressed in the let-60(n1046) and let-23(sa62) mutant worms. The assay is simple, inexpensive, is not time consuming to setup, and can be used as an initial platform for the discovery of anticancer therapeutics.

Identification of EGFR and RAS Inhibitors using Caenorhabditis elegans

The genetically tractable nematode Caenorhabditis elegans can be used as a simple and inexpensive model for drug discovery. Described here is a protocol to identify anticancer therapeutics that inhibit the downstream signaling of RAS and EGFR proteins.

使用卡诺哈布迪炎埃莱甘斯识别 EGFR 和 RAS 抑制剂

The changes in the plasma membrane localization of the epidermal growth factor receptor (EGFR) and its downstream effector RAS have been implicated in ...

Mammary Epithelial and Endothelial Cell Spheroids as a Potential Functional In vitro Model for Breast Cancer Research

Breast cancer is the leading cause of mortality in women. The growth of breast cancer cells and their subsequent metastasis is a key factor for its progression. Although the mechanisms involved in promoting breast cancer growth have been intensively studied using monocultures of breast cancer cells such as MCF-7 cells, the contribution of other cell types, such as vascular and lymphatic endothelial cells that are intimately involved in tumor growth, has not been investigated in depth. Cell-cell interaction plays a key role in tumor growth and progression. Neoangiogenesis, or the development of vessels, is essential for tumor growth, whereas the lymphatic system serves as a portal for cancer cell migration and subsequent metastasis. Recent studies provide evidence that vascular and lymphatic endothelial cells can significantly influence cancer cell growth. These observations imply a need for developing in vitro models that would more realistically reflect breast cancer growth processes in vivo. Moreover, restrictions in animal research require the development of ex vivo models to elucidate better the mechanisms involved.
This article describes the development of breast cancer spheroids composed of both breast cancer cells (estrogen receptor-positive MCF-7 cells) and vascular and/or lymphatic endothelial cells. The protocol describes a detailed step-by-step approach in creating dual-cell spheroids using two different approaches, hanging drop (gold standard and cheap) and 96-well U-bottom plates (expensive). In-depth instructions are provided for how to delicately pick up the formed spheroids to monitor growth by microscopic sizing and assessing viability using dead and live cell staining. Moreover, procedures to fix the spheroids for sectioning and staining with growth-specific antibodies to differentiate growth patterns in spheroids are delineated. Additionally, details for preparing spheroids with transfected cells and methods to extract RNA for molecular analysis are provided. In conclusion, this article provides in-depth instructions for preparing multi-cell spheroids for breast cancer research.

Mammary Epithelial and Endothelial Cell Spheroids as a Potential Functional In vitro Model for Breast Cancer Research

Crosstalk between mammary epithelial cells and endothelial cells importantly contributes to breast cancer progression, tumor growth, and metastasis. In this study, spheroids have been made from breast cancer cells together with vascular and/or lymphatic endothelial cells and demonstrate their applicability as an in vitro system for breast cancer research.

乳腺上皮和内皮细胞球状体作为乳腺癌研究的潜在功能 体外 模型

Breast cancer is the leading cause of mortality in women. The growth of breast cancer cells and their subsequent metastasis is a key factor for its ...