Name: orthotopic injection of breast cancer cells into the mammary fat pad of mice to study tumor growth.
Uploaded: 2015-02-08T14:00:00.000+00:00
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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?...

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><tbody><tr><td>Bouin&#039;s solution</td><td>Sigma-Aldrich</td><td>HT10132</td><td>Used for investigating the metastasis on lungs</td></tr><tr><td>Formalin solution</td><td>Sigma-Aldrich</td><td>HT501128</td><td>Used to fix the tissues</td></tr><tr><td>Matrigel, growth factor reduced</td><td>Corning</td><td>356230</td><td>Cells can be resuspended in matrigel for injection</td></tr><tr><td>Mosquito forceps</td><td>Fine Science Tools</td><td>13008-12</td><td>Used for stiching</td></tr><tr><td>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><td>Scissors</td><td>B Braun Medicals</td><td>BC056R</td><td>Used to cut open the mice</td></tr><tr><td>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><td>NOD scid gamma mice</td><td>Charles River</td><td>005557</td><td>Experimental animal used for experiment</td></tr><tr><td>MDA-MB-231</td><td>Sigma-Aldrich</td><td>92020424</td><td>Experimental cells used for injections</td></tr><tr><td>Oculentum simplex</td><td>Teva Pharmachemie</td><td></td><td>Opthalmic ointment used to prevent drying out of eyes</td></tr><tr><td>Betadine</td><td>Fischer Scientific</td><td>19-898-859</td><td>Ionophore, used to disinfect the surgical area</td></tr><tr><td>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><td>Temgesic</td><td>Schering-Plough</td><td></td><td>Use the painkiller as 0.05-0.1 mg/kg body weight</td></tr><tr><td>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><td>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?...

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

Research

JoVE Journal

Medicine

78.8K 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.

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, ...

科研

癌症研究

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 ...

Studying Triple Negative Breast Cancer Using Orthotopic Breast Cancer Model

Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype with limited therapeutic options. When compared to patients with less aggressive breast tumors, the 5-year survival rate of TNBC patients is 77% due to their characteristic drug-resistant phenotype and metastatic burden. Toward this end, murine models have been established aimed at identifying novel therapeutic strategies limiting TNBC tumor growth and metastatic spread. This work describes a practical guide for the TNBC orthotopic model where MDA-MB-231 breast cancer cells suspended in a basement membrane matrix are implanted in the fourth mammary fat pad, which closely mimics the cancer cell behavior in humans. Measurement of tumors by caliper, lung metastasis assessment via in vivo and ex vivo imaging, and molecular detection are discussed. This model provides an excellent platform to study therapeutic efficacy and is especially suitable for the study of the interaction between the primary tumor and distal metastatic sites.

This work presets an advanced protocol to accurately assess tumor loading by detection of green fluorescent protein and bioluminescence signals as well as the integration of quantitative molecular detection technique.

研究使用正交乳腺癌模型的三阴性乳腺癌

Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype with limited therapeutic options. When compared to patients with less ...

Orthotopic Transplantation of Breast Tumors as Preclinical Models for Breast Cancer

Preclinical models that faithfully recapitulate tumor heterogeneity and therapeutic response are critical for translational breast cancer research. Immortalized cell lines are easy to grow and genetically modify to study molecular mechanisms, yet the selective pressure from cell culture often leads to genetic and epigenetic alterations over time. Patient-derived xenograft (PDX) models faithfully recapitulate the heterogeneity and drug response of human breast tumors. PDX models exhibit a relatively short latency after orthotopic transplantation that facilitates the investigation of breast tumor biology and drug response. The transplantable genetically engineered mouse models allow the study of breast tumor immunity. The current protocol describes the method to orthotopically transplant breast tumor fragments into the mammary fat pad followed by drug treatments. These preclinical models provide valuable approaches to investigate breast tumor biology, drug response, biomarker discovery and mechanisms of drug resistance.

Patient-derived xenograft (PDX) models and transplantable genetically engineered mouse models faithfully recapitulate human disease and are preferred models for basic and translational breast cancer research. Here, a method is described to orthotopically transplant breast tumor fragments into the mammary fat pad to study tumor biology and evaluate drug response.

乳腺癌临床前模型乳腺肿瘤的矫形移植

Preclinical models that faithfully recapitulate tumor heterogeneity and therapeutic response are critical for translational breast cancer research. ...

Ultrasound Imaging-guided Intracardiac Injection to Develop a Mouse Model of Breast Cancer Brain Metastases Followed by Longitudinal MRI

Breast cancer brain metastasis, occurring in 30% of breast cancer patients at stage IV, is associated with high mortality. The median survival is only 6 months. It is critical to have suitable animal models to mimic the hemodynamic spread of the metastatic cells in the clinical scenario. Here, we are introducing the use of small animal ultrasound imaging to guide an accurate injection of brain tropical breast cancer cells into the left ventricle of athymic nude mice. Longitudinal MRI is used to assessing intracranial initiation and growth of brain metastases. Ultrasound-guided intracardiac injection ensures not only an accurate injection and hereby a higher successful rate but also significantly decreased mortality rate, as compared to our previous manual procedure. In vivo high resolution MRI allows the visualization of hyperintense multifocal lesions, as small as 310 &#181;m in diameter on T2-weighted images at 3 weeks post injection. Follow-up MRI reveals intracranial tumor growth and increased number of metastases that distribute throughout the whole brain.

A breast cancer brain metastasis mouse model is established with ultrasound imaging-guided intracardiac injection of MDA-MB231/Br-GFP cells. Development of multifocal intracranial metastases has been monitored longitudinally using high-resolution 9.4 T MRI.

超声成像引导下腔内注射到患乳腺癌脑转移其次是纵向的MRI的小鼠模型

Breast cancer brain metastasis, occurring in 30% of breast cancer patients at stage IV, is associated with high mortality. The median survival is only 6 ...

医学

In Vivo Imaging to Measure Spontaneous Lung Metastasis of Orthotopically-injected Breast Tumor Cells

Metastasis remains the primary cause of cancer-related death. The succession of events that characterize the metastatic cascade presents multiple opportunities for therapeutic intervention, and the ability to accurately model them in mice is critical to evaluate their effects. Here, a step-by-step protocol is presented for the establishment of orthotopic primary breast tumors and the subsequent monitoring of the establishment and growth of metastatic lesions in the lung using in vivo bioluminescence imaging. This methodology allows for the evaluation of treatment or its biological effects along the entire range of metastatic development, from primary tumor escape to outgrowth in the lungs. Breast orthotopic tumors are generated in mice via injection of a luciferase-labeled cell suspension in the 4th mammary gland. Tumors are allowed to grow and disseminate for a specific amount of time and are then surgically resected. Upon resection, spontaneous lung metastasis is detected, and the growth over time is monitored using in vivo bioluminescence imaging. At the desired experimental endpoint, lung tissue can be collected for downstream analysis. The treatment of established, clinically evident metastasis is critical to improve outcomes for stage IV cancer patients, and it can be evaluated through tail vein models of experimental lung metastasis. However, metastatic dissemination occurs early in breast cancer, and many patients have latent, subclinical disseminated disease after surgery. Utilization of spontaneous models such as this one provides the opportunity to study the whole spectrum of the disease, especially the systemic effects driven by treatment of the primary tumor such as pre-metastatic niche priming, and evaluate treatments on dormant and subclinical disease after surgery.

<meta charset="utf8"></head><body>体内成像测量原位注射乳腺肿瘤细胞的自发性肺转移

The manuscript describes a methodology for the establishment as well as longitudinal growth monitoring of spontaneous lung metastasis from orthotopically-injected breast tumors, amenable to intervention at all stages of the metastatic cascade.

体内研究 影像学检查测量原位注射乳腺肿瘤细胞的自发性肺转移

Metastasis remains the primary cause of cancer-related death. The succession of events that characterize the metastatic cascade presents multiple ...

<meta charset="utf8"></head><body>乳腺癌患者来源的异种移植物的高通量解离和原位植入

High-Throughput Dissociation and Orthotopic Implantation of Breast Cancer Patient-Derived Xenografts

Patient-derived xenografts (PDXs) provide a clinically relevant method for recapitulating tumor-involved cell types and the tumor microenvironment, which is essential for advancing knowledge of breast cancer (BC). Additionally, PDX models enable the study of BC systemic effects, which is not possible using in vitro models. Traditional methods for implanting BC xenografts typically involve anesthesia and sterile surgical procedures, which are time-consuming, invasive, and limit the scalability of PDX models in BC research. This protocol describes a simple and scalable method for the orthotopic implantation of BC PDXs in mice. The immunodeficient mouse strain NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) was used for PDX engraftment. Human BC samples obtained from IRB-consented patients were mechanically and enzymatically dissociated, then resuspended in a solution of basement membrane extract (BME) and RPMI 1640. Animals were restrained by scruffing, and depilatory cream was applied to remove hair from the fat pads at the fourth inguinal nipple, followed by injection. Approximately 2 million cells in a 100 &#181;L suspension were bilaterally injected orthotopically into the mammary fat pads using a 26 G needle. Notably, no anesthetic was required, and the total procedure time was under 5 min, from cell preparation to injection. After a growth period of several months, tumors were excised and processed for authentication. Validation included receptor status assessment using immunohistochemistry with specific antibodies for traditional BC receptors (i.e., ER, PR, HER2). Tumor morphology was confirmed with hematoxylin and eosin (H&amp;E) staining, which was interpreted by a pathologist. Genetic similarity to the patient sample was verified through bulk RNA sequencing and short tandem repeat (STR) analysis. This approach to PDX engraftment and validation supports the rigorous development of models and high-throughput tumor implantation, enabling well-powered studies across various BC subtypes.

This protocol describes an efficient, non-surgical method for the orthotopic implantation of breast cancer patient-derived xenografts in mice. The technique involves enzymatic tumor dissociation followed by direct injection into the mammary fat pads, enabling high-throughput implantation. Comprehensive validation ensures model fidelity, facilitating rigorous studies across various breast cancer subtypes.

乳腺癌患者来源的异种移植物的高通量解离和原位植入

Patient-derived xenografts (PDXs) provide a clinically relevant method for recapitulating tumor-involved cell types and the tumor microenvironment, which ...

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。

摘要

探索更多视频

此视频中的章节

乳腺癌细胞原位注射到小鼠乳房脂肪板

通过将表达的腺病毒注射到小鼠乳腺腺中,模拟乳腺癌

研究使用正交乳腺癌模型的三阴性乳腺癌

乳腺癌临床前模型乳腺肿瘤的矫形移植

超声成像引导下腔内注射到患乳腺癌脑转移其次是纵向的MRI的小鼠模型

体内研究影像学检查测量原位注射乳腺肿瘤细胞的自发性肺转移

体内研究影像学检查测量原位注射乳腺肿瘤细胞的自发性肺转移

乳腺癌患者来源的异种移植物的高通量解离和原位植入

乳腺癌患者来源的异种移植物的高通量解离和原位植入

乳腺癌患者来源的异种移植物的高通量解离和原位植入

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。

摘要

探索更多视频

此视频中的章节

乳腺癌细胞原位注射到小鼠乳房脂肪板

通过将表达的腺病毒注射到小鼠乳腺腺中,模拟乳腺癌

研究使用正交乳腺癌模型的三阴性乳腺癌

乳腺癌临床前模型乳腺肿瘤的矫形移植

超声成像引导下腔内注射到患乳腺癌脑转移其次是纵向的MRI的小鼠模型

体内研究 影像学检查测量原位注射乳腺肿瘤细胞的自发性肺转移

体内研究 影像学检查测量原位注射乳腺肿瘤细胞的自发性肺转移

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