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><tbody><tr><td>Xylene</td><td>Sigma-Aldrich</td><td>214736</td><td></td></tr><tr><td>Permount</td><td>Thermo Fisher Scientific</td><td>SP15-100</td><td>mounting media for coverslipping whole mounts and H&amp;amp;E sections</td></tr><tr><td>Leica automated processor</td><td>Leica Biosystems</td><td>Model # ST5020</td><td></td></tr><tr><td>HM 355S Automatic Microtome</td><td>Thermo Fisher Scientific&amp;nbsp;</td><td>905200</td><td></td></tr><tr><td>Paraffin</td><td>Leica Biosystems</td><td>EM-400</td><td></td></tr><tr><td>Superfrost plus microscope slides</td><td>Thermo Fisher Scientific</td><td>4951PLUS-001</td><td>electrostatically charged</td></tr><tr><td>Fisher Finest 24x60x1</td><td>Thermo Fisher Scientific</td><td>12-548-5P</td><td>coverslips</td></tr><tr><td>Varistain Gemini ES Automatic slide stainer</td><td>Thermo Fisher Scientific</td><td>A78000014</td><td></td></tr><tr><td>Sta-On</td><td>Leica Biosystems</td><td>3803107</td><td>liquid adhesive; used in water bath at sectioning</td></tr><tr><td>Modified Harris Hematoxylin 72711</td><td>Richard-Allan Scientific (Part of Thermo Scientific)</td><td>72711</td><td></td></tr><tr><td>Eosin</td><td>Leica Biosystems</td><td>3801600</td><td></td></tr><tr><td>Lithium Carbonate</td><td>SkyTech Laboratories</td><td>LCQ500</td><td></td></tr><tr><td>Glacial Acetic Acid</td><td>Thermo Fisher Scientific</td><td>A38-500</td><td>needed in Carnoy&#039;s fixative</td></tr><tr><td>Chloroform</td><td>Macron Fine Chemicals</td><td>4440-04</td><td>needed in Carnoy&#039;s fixative</td></tr><tr><td>100% Ethanol</td><td>The Warner Graham Company</td><td>6505001050000</td><td>needed in Carnoy&#039;s fixative and washing slides</td></tr><tr><td>95% Ethanol</td><td>The Warner Graham Company</td><td>6505011137320190</td><td></td></tr><tr><td>Carmine Alum</td><td>Sigma-Aldrich</td><td>C1022-25G</td><td></td></tr><tr><td>Aluminum potassium sulfate dodecahydrate, Sigma Ultra</td><td>Sigma-Aldrich</td><td>A7210-500G</td><td></td></tr><tr><td>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

Transfer of Mammary Gland-forming Ability Between Mammary Basal Epithelial Cells and Mammary Luminal Cells via Extracellular Vesicles/Exosomes

Cells can communicate via exosomes, ~100-nm extracellular vesicles (EVs) that contain proteins, lipids, and nucleic acids. Non-adherent/mesenchymal mammary epithelial cell (NAMEC)-derived extracellular vesicles can be isolated from NAMEC medium via differential ultracentrifugation. Based on their density, EVs can be purified via ultracentrifugation at 110,000 x g. The EV preparation from ultracentrifugation can be further separated using a continuous density gradient to prevent contamination with soluble proteins. The purified EVs can then be further evaluated using nanoparticle-tracking analysis, which measures the size and number of vesicles in the preparation. The extracellular vesicles with a size ranging from 50 to 150 nm are exosomes. The NAMEC-derived EVs/exosomes can be ingested by mammary epithelial cells, which can be measured by flow cytometry and confocal microscopy. Some mammary stem cell properties (e.g., mammary gland-forming ability) can be transferred from the stem-like NAMECs to mammary epithelial cells via the NAMEC-derived EVs/exosomes. Isolated primary EpCAMhi/CD49flo luminal mammary epithelial cells cannot form mammary glands after being transplanted into mouse fat pads, while EpCAMlo/CD49fhi basal mammary epithelial cells form mammary glands after transplantation. Uptake of NAMEC-derived EVs/exosomes by EpCAMhi/CD49flo luminal mammary epithelial cells allows them to generate mammary glands after being transplanted into fat pads. The EVs/exosomes derived from stem-like mammary epithelial cells transfer mammary gland-forming ability to EpCAMhi/CD49flo luminal mammary epithelial cells.

This protocol describes methods for purifying, quantitating, and characterizing extracellular vesicles (EVs)/exosomes from non-adherent/mesenchymal mammary epithelial cells and for using them to transfer mammary gland-forming ability to luminal mammary epithelial cells. EVs/exosomes derived from stem-like mammary epithelial cells can transfer this cell property to cells that ingest the EVs/exosomes.

通过细胞外囊泡/外来体转移乳腺基质上皮细胞和乳腺腔细胞之间的乳腺形成能力

Cells can communicate via exosomes, ~100-nm extracellular vesicles (EVs) that contain proteins, lipids, and nucleic acids. Non-adherent/mesenchymal ...

发育生物学

Quantifying Branching Density in Rat Mammary Gland Whole-mounts Using the Sholl Analysis Method

An increasing number of studies are utilizing the rodent mammary gland as an endpoint for assessing the developmental toxicity of a chemical exposure. The effects these exposures have on mammary gland development are typically evaluated using either basic dimensional measurements or by scoring morphological characteristics. However, the broad range of methods for interpreting developmental changes could lead to inconsistent translations across laboratories. A common method of assessment is needed so that proper interpretations can be formed from data being compared across studies. The present study describes the application of the Sholl analysis method to quantify mammary gland branching characteristics. The Sholl method was originally developed for use in quantifying neuronal dendritic patterns. By using ImageJ, an open-source image analysis software package, and a plugin developed for this analysis, the mammary gland branching density and the complexity of a mammary gland from a peripubertal female rat were determined. The methods described here will enable the use of the Sholl analysis as an effective tool for quantifying an important characteristic of mammary gland development.

Mammary gland development in the rodent has typically been evaluated using descriptive assessments or by measuring basic physical attributes. Branching density is an indicator of mammary development that is difficult to quantify objectively. This protocol describes a reliable method for the quantitative assessment of mammary gland branching characteristics.

使用Sholl分析方法量化大鼠乳腺组织中的分支密度

An increasing number of studies are utilizing the rodent mammary gland as an endpoint for assessing the developmental toxicity of a chemical exposure. The ...

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 ...

医学

A Label-Free Segmentation Approach for Intravital Imaging of Mammary Tumor Microenvironment

The ability to visualize complex and dynamic physiological interactions between numerous cell types and the extracellular matrix (ECM) within a live tumor microenvironment is an important step toward understanding mechanisms that regulate tumor progression. While this can be accomplished through current intravital imaging techniques, it remains challenging due to the heterogeneous nature of tissues and the need for spatial context within the experimental observation. To this end, we have developed an intravital imaging workflow that pairs collagen second harmonic generation imaging, endogenous fluorescence from the metabolic co-factor NAD(P)H, and fluorescence lifetime imaging microscopy (FLIM) as a means to non-invasively compartmentalize the tumor microenvironment into basic domains of the tumor nest, the surrounding stroma or ECM, and the vasculature. This non-invasive protocol details the step-by-step process ranging from the acquisition of time-lapse images of mammary tumor models to post-processing analysis and image segmentation. The primary advantage of this workflow is that it exploits metabolic signatures to contextualize the dynamically changing live tumor microenvironment without the use of exogenous fluorescent labels, making it advantageous for human patient-derived xenograft (PDX) models and future clinical use where extrinsic fluorophores are not readily applicable.

The intravital imaging method described here utilizes collagen second harmonic generation and endogenous fluorescence from the metabolic co-factor NAD(P)H to non-invasively segment an unlabeled tumor microenvironment into tumor, stromal, and vascular compartments for in-depth analysis of 4D intravital images.

一种用于乳腺肿瘤微环境活体内成像的无标记分割方法

The ability to visualize complex and dynamic physiological interactions between numerous cell types and the extracellular matrix (ECM) within a live tumor ...

Mammary Epithelial Transplant Procedure

This article describes and compares the fat pad clearance procedure developed by DeOme KB et al.1 and the sparing procedure developed by Brill B et al.2, followed by the mammary epithelial transplant procedure. The mammary transplant procedure is widely used by mammary biologists because it takes advantage of the fact that significant development of the mammary epithelium doesn't occur until after puberty. At 3 weeks of age, growth of the mammary epithelial tree is confined to the vicinity of the nipple and the fat pad is largely devoid of mammary epithelium, but by 7 weeks of age the epithelial ductal tree extends throughout the entire fat pad. Therefore, if this small portion of the fat pad containing epithelium, the region between the nipple and the lymph node, is removed at 3 weeks of age, the endogenous epithelium will never populate the mammary fat pad and the fat pad is described as "cleared". At this time, mammary epithelium from another source can be transplanted in the cleared fat pad where it has the potential to extend mammary ductal trees through out the fat pad. This procedure has been utilized in many experimental models including the examination of tumor phenotype in transgenic mammary epithelial tissue without the confounding effects of genotype on the entire animal3, in the identification of mammary stem cells by transplanting cells in limited dilution4,5, determining if hyperplastic nodules proceed to mammary tumors6, and to assess the effect of prior hormone exposure on the behavior of the mammary epithelium7,8.
Three week old host mice are anesthetized, cleaned and restrained on a surgical stage. A mid-sagittal incision is made through the skin, but not the peritoneum, extending from the pubis to the sternum. Oblique cuts are made through the skin from the mid-sagittal incision across the pelvis toward each leg. The skin is pulled away from the peritoneum to expose the 4th inguinal mammary gland. The fat pad is cleared by removing the fat pad tissue anterior to the lymph node. Epithelium fragments or epithelial cells are transplanted into the remaining cleared fat pad and the mouse is closed.

This article demonstrates the procedure developed by DeOme KB et al. (1959) and the sparing procedure developed by Brill B et al. (2008) for clearing the 4th inguinal mammary fat pad of a pubescent mouse in preparation for transplantation of mammary fragments, mammary epithelial cells, or mammary tumor cells.

乳腺上皮移植手术

This article describes and compares the fat pad clearance procedure developed by DeOme KB et al.1 and the sparing procedure developed by Brill B et al.2, ...

生物学

Changes in Mammary Gland Morphology and Breast Cancer Risk in Rats

Studies in rodent models of breast cancer show that exposures to dietary/hormonal factors during the in utero and pubertal periods, when the mammary gland undergoes extensive modeling and re-modeling, alter susceptibility to carcinogen-induced mammary tumors. Similar findings have been described in humans: for example, high birthweight increases later risk of developing breast cancer, and dietary intake of soy during childhood decreases breast cancer risk. It is thought that these prenatal and postnatal dietary modifications induce persistent morphological changes in the mammary gland that in turn modify breast cancer risk later in life. These morphological changes likely reflect epigenetic modifications, such as changes in DNA methylation, histones and miRNA expression that then affect gene transcription . In this article we describe how changes in mammary gland morphology can predict mammary cancer risk in rats. Our protocol specifically describes how to dissect and remove the rat abdominal mammary gland and how to prepare mammary gland whole mounts. It also describes how to analyze mammary gland morphology according to three end-points (number of terminal end buds, epithelial elongation and differentiation) and to use the data to predict risk of developing mammary cancer.

Our protocol describes how to dissect the rat abdominal mammary gland and how to prepare mammary gland whole mounts. It also describes how to analyze mammary gland morphology using three end-points (number of terminal end buds, epithelial elongation and differentiation) and to use these results to predict mammary cancer risk in rats which were exposed to dietary modifications in utero or during prepuberty.

变化在大鼠乳腺形态和患乳腺癌的风险

Studies in rodent models of breast cancer show that exposures to dietary/hormonal factors during the in utero and pubertal periods, when the mammary gland ...

Evaluation of Mammary Gland Development and Function in Mouse Models

The human mammary gland is composed of 15-20 lobes that secrete milk into a branching duct system opening at the nipple. Those lobes are themselves composed of a number of terminal duct lobular units made of secretory alveoli and converging ducts1. In mice, a similar architecture is observed at pregnancy in which ducts and alveoli are interspersed within the connective tissue stroma. The mouse mammary gland epithelium is a tree like system of ducts composed of two layers of cells, an inner layer of luminal cells surrounded by an outer layer of myoepithelial cells denoted by the confines of a basement membrane2. At birth, only a rudimental ductal tree is present, composed of a primary duct and 15-20 branches. Branch elongation and amplification start at the beginning of puberty, around 4 weeks old, under the influence of hormones3,4,5. At 10 weeks, most of the stroma is invaded by a complex system of ducts that will undergo cycles of branching and regression in each estrous cycle until pregnancy2. At the onset of pregnancy, a second phase of development begins, with the proliferation and differentiation of the epithelium to form grape-shaped milk secretory structures called alveoli6,7. Following parturition and throughout lactation, milk is produced by luminal secretory cells and stored within the lumen of alveoli. Oxytocin release, stimulated by a neural reflex induced by suckling of pups, induces synchronized contractions of the myoepithelial cells around the alveoli and along the ducts, allowing milk to be transported through the ducts to the nipple where it becomes available to the pups 8. Mammary gland development, differentiation and function are tightly orchestrated and require, not only interactions between the stroma and the epithelium, but also between myoepithelial and luminal cells within the epithelium9,10,11. Thereby, mutations in many genes implicated in these interactions may impair either ductal elongation during puberty or alveoli formation during early pregnancy, differentiation during late pregnancy and secretory activation leading to lactation12,13. In this article, we describe how to dissect mouse mammary glands and assess their development using whole mounts. We also demonstrate how to evaluate myoepithelial contractions and milk ejection using an ex-vivo oxytocin-based functional assay. The effect of a gene mutation on mammary gland development and function can thus be determined in situ by performing these two techniques in mutant and wild-type control mice.

This method describes how to dissect and assess mammary gland development and function from mice. Excised mammary glands are assessed for the degree of development using whole mount while milk ejection is evaluated using an oxytocin-based myoepithelial cell contraction assay.

乳腺的发育和功能在小鼠模型的评价

The human mammary gland is composed of 15-20 lobes that secrete milk into a branching duct system opening at the nipple. Those lobes are themselves ...

Reconstruction of 3-Dimensional Histology Volume and its Application to Study Mouse Mammary Glands

Histology volume reconstruction facilitates the study of 3D shape and volume change of an organ at the level of macrostructures made up of cells. It can also be used to investigate and validate novel techniques and algorithms in volumetric medical imaging and therapies. Creating 3D high-resolution atlases of different organs1,2,3 is another application of histology volume reconstruction. This provides a resource for investigating tissue structures and the spatial relationship between various cellular features. We present an image registration approach for histology volume reconstruction, which uses a set of optical blockface images. The reconstructed histology volume represents a reliable shape of the processed specimen with no propagated post-processing registration error. The Hematoxylin and Eosin (H&#38;E) stained sections of two mouse mammary glands were registered to their corresponding blockface images using boundary points extracted from the edges of the specimen in histology and blockface images. The accuracy of the registration was visually evaluated. The alignment of the macrostructures of the mammary glands was also visually assessed at high resolution.
This study delineates the different steps of this image registration pipeline, ranging from excision of the mammary gland through to 3D histology volume reconstruction. While 2D histology images reveal the structural differences between pairs of sections, 3D histology volume provides the ability to visualize the differences in shape and volume of the mammary glands.

We present an image registration approach for 3-dimensional (3D) histology volume reconstruction, which facilitates the study of the changes of an organ at the level of macrostructures made up of cells . Using this approach, we studied the 3D changes between wild-type and Igfbp7-null mammary glands.

3维组织学音量和其研究小鼠乳腺腺体的应用改造

Histology volume reconstruction facilitates the study of 3D shape and volume change of an organ at the level of macrostructures made up of cells. It can ...

Isolation of Intact, Whole Mouse Mammary Glands for Analysis of Extracellular Matrix Expression and Gland Morphology

The goal of this procedure was to harvest the #4 abdominal mammary glands from female nulliparous mice in order to assess ECM expression and ductal architecture. Here, a small pocket below the skin was created using Mayo scissors, allowing separation of the glands within the subcutaneous tissue from the underlying peritoneum. Visualization of the glands was aided by the use of 3.5x-R surgical micro loupes. The pelt was inverted and pinned back allowing identification of the intact mammary fat pads. Each of the #4 abdominal glands was bluntly dissected by sliding the scalpel blade laterally between the subcutaneous layer and the glands. Immediately post-harvest, glands were placed in 10% neutral buffered formalin for subsequent tissue processing. Excision of the entire gland is advantageous because it primarily eliminates the risk of excluding important tissue-wide interactions between ductal epithelial cells and other microenvironmental cellular populations that could be missed in a partial biopsy. One drawback of the methodology is the use of serial sections from fixed tissues which limits analyses of ductal morphogenesis and protein expression to discrete locations within the gland. As such, changes in ductal architecture and protein expression in 3 dimensions (3D) is not readily obtainable. Overall, the technique is applicable to studies requiring whole intact murine mammary glands for downstream investigations such as developmental ductal morphogenesis or breast cancer.

Here, we present a protocol for the isolation of whole, intact mouse mammary glands to investigate extracellular matrix (ECM) expression and ductal morphology. Mouse #4 abdominal glands were extracted from 8-10 week old female nulliparous mice, fixed in neutral buffered formalin, sectioned and stained using immunohistochemistry for ECM proteins.

完整、全小鼠乳腺的分离及细胞外基质表达及腺体形态学分析

The goal of this procedure was to harvest the #4 abdominal mammary glands from female nulliparous mice in order to assess ECM expression and ductal ...