NINDS补助NS49720和NS65819和NCI授予CA97257

1。肿瘤细胞的制备。 源自人类大脑肿瘤异种移植的细胞可以从肿瘤在裸鼠皮下生长，或从细胞培养繁殖。在下面讨论这两种细胞来源的利用，随着细胞移植的方法示范。 为了准备皮下肿瘤细胞转移到颅内车厢，切除侧翼肿瘤置于培养皿中，组织最初是用手术刀，然后重复吹打创建一个细胞总悬浮 1机械打乱剁碎。细胞总悬浮液，然后通过一个70μm的尼龙网过滤，生产单细胞悬液，适合颅内注射。细胞悬液，离心1000转10分钟，在4 ° C，并resuspending适当体积的无血清培养基中的细胞沉淀，获得了最后的工作浓度（见下文）前吸气上清。对于准备建立的细胞系颅内植入，细胞的收获trypsinizing单层，或通过收集神经球悬浮培养，然后离心和resuspending 以上2所示的细胞。注射细胞的数量是注射神经解剖学位置上的变量的依赖。幕注射，我们经常注入3μL无血清培养基（DMEM）3-5 × 10 5细胞，而脑干注射 3，如5 × 10 4细胞数在0.5μL注入。注射比建议大量可导致肿瘤细胞通过针道返流，由此exophytic（图1），而不是颅内肿瘤的生长。撤销颅内注射样品后，剩余的细胞悬液，应放在冰，混合，经常保持适当的浓度，同时完成注塑系列成员之间的颅内肿瘤建立的内容。 2。肿瘤细胞移植请注意，下面描述的所有程序已经审查和批准的机构使用动物，并在加州大学旧金山分校护理委员会。 <ol><li>手术区应做好所有的表面，如2％洗必泰溶液，用消毒剂喷洒。使用消毒剂后，应放在以下耗材在手术区： <ul><li>加热垫，以保持鼠标体温</li><li>两个小培养皿;一个含有3％的双氧水，和一个含有2％洗必泰</li><li>无菌纱布和棉签</li><li>无菌一次性手术刀</li><li>蒸压手术吻合器</li></ul></li><li>麻醉应采用注射麻醉剂，通常是氯胺酮，甲苯噻嗪的混合物。 </li><li>一旦鼠标被麻醉，头皮是准备用一块无菌纱布蘸洗必泰溶液擦拭几次。眼药膏应在手术过程中保持充足的水分。使用无菌手术刀，完成了parieto枕骨一个矢状切口，约1厘米长。暴露颅骨表面，然后用棉签在3％的双氧水溶液浸泡清洗。前囟门应在这一点上明显（见视频）。 </li><li>肿瘤细胞注射的坐标会有所不同，根据肿瘤的建立所需的站点。下面我们使用脑肿瘤建立在神经解剖学位置，在许多脑肿瘤患者的经验肿瘤的发生发展的过程。在脑肿瘤研究感兴趣的其他地点包括脑桥，脑干肿瘤 3解剖模型，并硬膜下注射建模脑膜肿瘤的位置。肿瘤细胞注射前，用无菌的25计利针穿刺的头骨在2 mm至前囟门，冠状缝前1毫米的权利，从而创造一个注射肿瘤细胞（见视频）开放。此过程可以很好地用于小鼠和大鼠的老鼠（22号针头）。 </li><li>进入注射器吸取细胞之前，用手指轻敲混合细胞悬液的内容。与所需数量的细胞悬液装入注射器，小心，以避免产生气泡。注射器外，然后用酒精棉球清洗擦拭外观无任何贴壁细胞，这将有助于防止颅外肿瘤的建立和成长（图1A）。为了确保适当的注射深度取得，使用手术刀切割3mm的关闭了P20的pipetteman提示指出。本节针尖可以装在注射器，将采取措施限制注射深度，此外，并会确保该TI注射器针头P是从3mm的头骨底部。注射器垂直放置的头骨，并在以前创建的孔，慢慢注入细胞悬液（超过1分钟的时间内注入一个3μL应暂停）。注射器插入一个不恰当的角度，可能会导致在完成注射后的细胞和随后脊髓传播（图1B：右）脑室注射，针留在一分钟的地方，然后慢慢退出（见视频）：这些步骤将有助于减少肿瘤细胞的回流。 </li><li>作为替代求告无门或手绘的方法颅内肿瘤细胞植入 5，可以使用小动物脑立体定向仪（面板的示意图F）的，一般促进更一致的注射位置，但在大量的费用程序的时间。在我们的经验，两个手术人员可以注入多达60只小鼠/小时，在使用自由的做法，而用小动物脑立体定向仪的最大注射率约为15只/小时。程序权宜之计，是一个重要的考虑因素时，小鼠注射大系列，并有助于减少肿瘤细胞，被注入，在冰上剩余的时间。 </li><li> 3％双氧水和使用无菌干棉签干燥清洁的头骨。应用无菌骨蜡孔。使用镊子，共同绘制头皮以上的头骨和主食关闭。为了达到最佳的愈合，头皮应装订，每一方对对方的头皮的真皮层（对底面底面）。装订头皮应清理干净，用洗必泰溶液，然后通过手术后的疼痛缓解皮下注射管理和丁丙诺啡。 </li><li>手术后，直到他们成为园内，并保留正常活动监视所有小鼠。通常情况下，恢复时间约为30分钟。 </li></ol> 3。生物发光监测肿瘤生长<ol><li>背景。生物发光成像（劳保局）是基于荧光素的氧化[D -（ - ）-2 - （60 - 羟基- 20 -苯并噻唑） - 噻酮-4 - 羧酸的氧气和三磷酸腺苷（ATP）的存在。这是催化反应的酶的荧光素酶，转换成化学能，由此产生的排放光的光子。人体细胞表达荧光素酶与荧光素酶表达的细胞能够发光的荧光素底物的存在（见下文），可以修改。是从动物的最小的背景发光的主机荧光素治疗，例如，有一个非常有利的信号噪声比检测荧光素酶修饰的肿瘤细胞发光的排放量，使高度敏感的肿瘤的生长和体内治疗监测的。此外，荧光素酶及其底物，荧光素，已被证明非哺乳动物细胞的毒性，和我们所观察到相应的未修改的细胞表达荧光素酶相对的细胞之间功能上没有区别。荧光素很容易穿过细胞膜和后腹腔内的血 - 脑屏障（IP）或静脉（IV）注射液在小鼠体内，从而使每一个车厢，其中包含荧光素酶基因细胞成像。光子发射和荧光素酶基因细胞所发出的光频谱的水平是足够的穿透组织的研究小动物，如小鼠和大鼠，，并与低光成像摄像机可以检测外部。生物发光成像的非侵入性的性质，允许反复监测个别受试动物治疗肿瘤的生长和响应。 </li><li>萤火虫荧光素酶的表达，植入的细胞来源应该是稳定的修改。可以购买这种细胞来源，或个人利用已构建荧光素酶的组成型表达的慢病毒实验室。我们强烈建议使用的荧光素酶编码的慢病毒，而不是质粒，修饰细胞，以确保稳定的荧光素酶的细胞在体内的表达，这是必要的定量发光成像在肿瘤负荷提供准确地反映动物个体的课题，反复的变化在实验过程成像。 </li><li>劳保局监测。我们建议进行定量生物发光成像（qBLI）1X - 2X每周，肿瘤细胞注射后的一个星期开始。我们qBLI进行使用IVIS Lumina的成像站（卡尺生命科学版），和我们的研究结果表明，类似的数据是在使用IVIS Lumina的，IVIS 150，或IVIS 200成像站。在准备用于成像，老鼠同时通过腹腔注射麻醉与氯胺酮/甲苯​​噻嗪和管理的荧光素（D -荧光素盐，钾150毫克/公斤，卡尺生命科学），成像在注射后12分钟的小鼠。在荧光素的药代动力学表明，adminis之间的时间细胞发光的荧光素和决心tration应在10-15分钟之间的荧光素注射后，以获得最大的发光排放和最大的检测灵敏度。 10-15分钟的时间间隔内选定的时间长度应保持常数正在成像动物之间。这是非常重要的是保持在注射荧光素和获得BLI读数之间的时间长度的一致性。包括颅内信号区域利益的定义区域使用的生活图像软件（图2），总photons/s/sr/cm2（每平方厘米每秒每球面度的光子）的记录（见视频）。 </li><li>数据分析。个别动物进行监测，而肿瘤的生长和治疗的反应，我们强烈建议增加肿瘤反应的统计结论的确定性至少有8个治疗组，或缺乏，治疗。方面介绍qBLI结果，发光读数转换为标准化值除以每个鼠标小号发光，期间获得的治疗方案完成后，其相应的最大预处理读6,7发光。生存分析，采用Kaplan - Meier估计8使用，并从中产生生存曲线，中位生存期值确定。使用log - rank检验比较生存曲线之间的差异。 </li><li>随后的分析处理和未经处理的肿瘤脑检索。动物安乐死后，应迅速切除鼠脑（见视频），并在福尔马林放在肿瘤的形态学和免疫组织化学的后续分析，或应安装在华侨城标本冻结。 </li></ol> 4。代表性的成果如图3a所示的例子中，接受颅内肿瘤细胞注射的小鼠进行了监测颅内发光，直到连续平均发光值表示进步的肿瘤生长，此时治疗开始（第34天开始的灰色箭头：厄洛替尼每日150毫克管理直到要求安乐死/公斤）。每个鼠标的发光值设置为1的归一化值，在开始治疗时，每个鼠标以后归其最终的预处理成像价值发光读数。作为一个例子，鼠标与治疗前的2.0 × 10 7光子/秒34天，其发光已上升至6.0最后的发光读数× 10 7光子/天38秒，每天38归发光值3.0。平均正常化生物发光和相应的控制和治疗组的标准误差为每个成像时间点绘制。在这个例子中，均归发光的显着性差异明显，在第一成像时间点后开始治疗（38天），平均组在随后的时间点进一步提高发光的区别。在大多数情况下，抗肿瘤治疗，qBLI表示，活动是伴随着相应的生存显着性差异（即P &lt;0.05），是这里的情况（图3B）。面板3C和3D显示相邻的苏木精和曙红和鼠脑的抗EGFR染色切片获得的“安乐死”时，福尔马林和随后的石蜡包埋后切片手术切除脑安置。 <img src="/files/ftp_upload/1986/1986fig1.jpg" alt="图1" /> 图1。颅内注射错误的迹象。一）Exophytic（颅外）肿瘤的生长（红色圆圈），可以造成太大的注射量，剩余的细胞悬液注射器，或从注射器注射肿瘤细胞后，撤回太快。二）注入脑室肿瘤细胞，可导致脊柱肿瘤的传播（鼠标右），相反，正确注射的肿瘤细胞，为保持局部注射部位（鼠标左）信号。 <img src="/files/ftp_upload/1986/1986fig2.jpg" alt="图2" /> 图2（右）代表从控制老鼠的生物发光强度交涉热图图像（左）和治疗组治疗的反应实验。生活图像软件可以设置定义感兴趣区域（红色圆圈），或仪器操作员可以手动定义感兴趣区域。对于使用如施工图这些图像，我们建议，仪器操作员显示热图的图像，使用相同的生物发光的热图范围（图上半部分），提供的可视化表示的生物发光受试动物之间的差异程度。 <img src="/files/ftp_upload/1986/1986fig3.jpg" alT =“图3”/&gt; 图3。生物发光，生存和肿瘤组织分析，从实验中，治疗反应是显而易见的。剧情的控制和治疗组小鼠的平均生物发光读数与标准错误，的），表示每个成像点。 B）相同的小鼠的生存情节; P -值确定通过使用log - rank 检验 7 。 C）H＆E染色部分的鼠脑肿瘤。四）EGFR染色部分。 E和F）表示地区倍率板彗星和D，分别与面板显示肿瘤抑制蛋白PTEN的负染发送。

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原位（颅内）脑肿瘤异种移植的建立提供了一个适当的微环境建模中枢神经系统癌症治疗反应测试 10 。这种类型的造型，另外还提供关于治疗获得脑和脑肿瘤，这是非常重要的，以确定是否应提前在患者的临床试验评价实验代理的信息。由于颅内移植瘤的金额不能直接测量，如卡尺，纵向监测的颅内肿瘤的生长和对治疗的反应要求与我们的经验表明生物发光成像的非侵入性的成像，最实用的方法进行实验，其主要目标是评估肿瘤对治疗的反应程度。当生物发光成像的结果与动物受生存分析相结合，这两个数据集提供了一个强大和可靠的实验疗效评估的方法。 最后，这是极为重要的颅内脑肿瘤异种移植，安乐死受试动物的收获，以评估治疗的形态学和分子的影响，为此，我们希望在实施安乐死时全脑切除术，切除大脑保存以供后续分析，。 鉴于前面的介绍已经取得了具体的脑瘤研究，概念肯定generalizeable适合在啮齿类动物中的原位模型​​的其他人类癌症。

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establishing intracranial brain tumor xenografts with subsequent analysis of tumor growth and response to therapy using bioluminescence imaging

使用人脑肿瘤细胞的移植模型，有担任多年在神经肿瘤学研究的重要功能。在过去，最常用的程序，包括为人类肿瘤异种移植建立的细胞培养瓶中收集，收集细胞免疫功能低下小鼠皮下注射。鉴于这种做法仍然认为在许多实验室的频繁使用，出现了一个强调在过去十年中的重大转变，对原位移植瘤的建立，其中，在脑肿瘤的实例，需要到相应的神经解剖结构的肿瘤细胞注射。由于颅内移植瘤的建立，消除了监控能力通过直接测量肿瘤的生长，如使用卡钳，原位脑肿瘤异种移植模型中的重点转向转变，有必要利用的非侵入性的成像的评估宿主动物的肿瘤负担。目前可用的成像方法，被普遍认为是生物发光监测提供了灵敏度，权宜之计，和成本的最佳组合。在这里，我们将展示原位脑肿瘤的建立，为监测肿瘤的生长和实验疗法对治疗的反应时测试的程序。

Watch this Scientific Journal Video about Establishing Intracranial Brain Tumor Xenografts With Subsequent Analysis of Tumor Growth and Response to Therapy using Bioluminescence Imaging at JoVE.com

Establishing Intracranial Brain Tumor Xenografts With Subsequent Analysis of Tumor Growth and Response to Therapy using Bioluminescence Imaging

可以建立荧光素酶修饰人脑肿瘤异种移植，颅内无胸腺小鼠，随后的监测肿瘤的生长和治疗用生物发光成像。在与生存分析相结合，生物发光监测是一个重要的研究工具疗法被认为是治疗脑肿瘤的临床前测试。

建立颅内脑肿瘤异种移植肿瘤的生长和对治疗的反应与后续利用生物发光成像分析

Transplantation models using human brain tumor cells have served an essential function in neuro-oncology research for many years. In the past, the most ...

establishing-intracranial-brain-tumor-xenografts-with-subsequent

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

39.2K 次观看.  University of California, San Francisco - UCSF. 可以建立荧光素酶修饰人脑肿瘤异种移植，颅内无胸腺小鼠，随后的监测肿瘤的生长和治疗用生物发光成像。在与生存分析相结合，生物发光监测是一个重要的研究工具疗法被认为是治疗脑肿瘤的临床前测试。

视频: Establishing Intracranial Brain Tumor Xenografts With Subsequent Analysis of Tumor Growth and Response to Therapy using Bioluminescence Imaging

Orthotopic Xenografting of Human Luciferase-Tagged Malignant Peripheral Nerve Sheath Tumor Cells for in vivo Testing of Candidate Therapeutic Agents

Although in vitro screens are essential for the initial identification of candidate therapeutic agents, a rigorous assessment of the drug's ability to inhibit tumor growth must be performed in a suitable animal model. The type of animal model that is best for this purpose is a topic of intense discussion. Some evidence indicates that preclinical trials examining drug effects on tumors arising in transgenic mice are more predictive of clinical outcome1and so candidate therapeutic agents are often tested in these models. Unfortunately, transgenic models are not available for many tumor types. Further, transgenic models often have other limitations such as concerns as to how well the mouse tumor models its human counterpart, incomplete penetrance of the tumor phenotype and an inability to predict when tumors will develop.
Consequently, many investigators use xenograft models (human tumor cells grafted into immunodeficient mice) for preclinical trials if appropriate transgenic tumor models are not available. Even if transgenic models are available, they are often partnered with xenograft models as the latter facilitate rapid determination of therapeutic ranges. Further, this partnership allows a comparison of the effectiveness of the agent in transgenic tumors and genuine human tumor cells. Historically, xenografting has often been performed by injecting tumor cells subcutaneously (ectopic xenografts). This technique is rapid and reproducible, relatively inexpensive and allows continuous quantitation of tumor growth during the therapeutic period2. However, the subcutaneous space is not the normal microenvironment for most neoplasms and so results obtained with ectopic xenografting can be misleading due to factors such as an absence of organ-specific expression of host tissue and tumor genes. It has thus been strongly recommended that ectopic grafting studies be replaced or complemented by studies in which human tumor cells are grafted into their tissue of origin (orthotopic xenografting)2. Unfortunately, implementation of this recommendation is often thwarted by the fact that orthotopic xenografting methodologies have not yet been developed for many tumor types.
Malignant peripheral nerve sheath tumors (MPNSTs) are highly aggressive sarcomas that occur sporadically or in association with neurofibromatosis type 13and most commonly arise in the sciatic nerve4. Here we describe a technically straightforward method in which firefly luciferase-tagged human MPNST cells are orthopically xenografted into the sciatic nerve of immunodeficient mice. Our approach to assessing the success of the grafting procedure in individual animals and subsequent non-biased randomization into study groups is also discussed.

Orthotopic Xenografting of Human Luciferase-Tagged Malignant Peripheral Nerve Sheath Tumor Cells for in vivo Testing of Candidate Therapeutic Agents

A method for reliably grafting luciferase-tagged human malignant peripheral nerve sheath tumor cells into the sciatic nerve of immunodeficient mice is described. The use of bioluminescence imaging to demonstrate proper establishment of tumor grafts and criteria for random segregation of animals into study groups are also discussed.

原位Xenografting人类荧光素酶标记的恶性外周神经鞘瘤细胞在体内测试候选治疗药物

Although in vitro screens are essential for the initial identification of candidate therapeutic agents, a rigorous assessment of the drug's ability to ...

科研

医学

In vivo Bioluminescence Imaging of Tumor Hypoxia Dynamics of Breast Cancer Brain Metastasis in a Mouse Model

It is well recognized that tumor hypoxia plays an important role in promoting malignant progression and affecting therapeutic response negatively. There is little knowledge about in situ, in vivo, tumor hypoxia during intracranial development of malignant brain tumors because of lack of efficient means to monitor it in these deep-seated orthotopic tumors. Bioluminescence imaging (BLI), based on the detection of light emitted by living cells expressing a luciferase gene, has been rapidly adopted for cancer research, in particular, to evaluate tumor growth or tumor size changes in response to treatment in preclinical animal studies. Moreover, by expressing a reporter gene under the control of a promoter sequence, the specific gene expression can be monitored non-invasively by BLI. Under hypoxic stress, signaling responses are mediated mainly via the hypoxia inducible factor-1&alpha; (HIF-1&alpha;) to drive transcription of various genes. Therefore, we have used a HIF-1&alpha; reporter construct, 5HRE-ODD-luc, stably transfected into human breast cancer MDA-MB231 cells (MDA-MB231/5HRE-ODD-luc). In vitro HIF-1&alpha; bioluminescence assay is performed by incubating the transfected cells in a hypoxic chamber (0.1% O2) for 24 hr before BLI, while the cells in normoxia (21% O2) serve as a control. Significantly higher photon flux observed for the cells under hypoxia suggests an increased HIF-1&alpha; binding to its promoter (HRE elements), as compared to those in normoxia. Cells are injected directly into the mouse brain to establish a breast cancer brain metastasis model. In vivo bioluminescence imaging of tumor hypoxia dynamics is initiated 2 wks after implantation and repeated once a week. BLI reveals increasing light signals from the brain as the tumor progresses, indicating increased intracranial tumor hypoxia. Histological and immunohistochemical studies are used to confirm the in vivo imaging results. Here, we will introduce approaches of in vitro HIF-1&alpha; bioluminescence assay, surgical establishment of a breast cancer brain metastasis in a nude mouse and application of in vivo bioluminescence imaging to monitor intracranial tumor hypoxia.

In vivo Bioluminescence Imaging of Tumor Hypoxia Dynamics of Breast Cancer Brain Metastasis in a Mouse Model

Bioluminescence imaging of hypoxia inducible factor-1&alpha; activity is applied to monitor intracranial tumor hypoxia development in a breast cancer brain metastasis mouse model.

在小鼠模型体内生物发光成像乳腺癌脑转移的肿瘤乏氧动力学

It is well recognized that tumor hypoxia plays an important role in promoting malignant progression and affecting therapeutic response negatively. There ...

Fluorescence Molecular Tomography for In Vivo Imaging of Glioblastoma Xenografts

Tumorigenicity is the capability of cancer cells to form a tumor mass. A widely used approach to determine if the cells are tumorigenic is by injecting immunodeficient mice subcutaneously with cancer cells and measuring the tumor mass after it becomes visible and palpable. Orthotopic injections of cancer cells aim to introduce the xenograft in the microenvironment that most closely resembles the tissue of origin of the tumor being studied. Brain cancer research requires intracranial injection of cancer cells to allow the tumor formation and analysis in the unique microenvironment of the brain. The in vivo imaging of intracranial xenografts monitors instantaneously the tumor mass of orthotopically engrafted mice. Here we report the use of fluorescence molecular tomography (FMT) of brain tumor xenografts. The cancer cells are first transduced with near infrared fluorescent proteins and then injected in the brain of immunocompromised mice. The animals are then scanned to obtain quantitative information about the tumor mass over an extended period of time. Cell pre-labeling allows for cost effective, reproducible, and reliable quantification of the tumor burden within each mouse. We eliminated the need for injecting imaging substrates, and thus reduced the stress on the animals. A limitation of this approach is represented by the inability to detect very small masses; however, it has better resolution for larger masses than other techniques. It can be applied to evaluate the efficacy of a drug treatment or genetic alterations of glioma cell lines and patient-derived samples.

Fluorescence Molecular Tomography for In Vivo Imaging of Glioblastoma Xenografts

Orthotopic intracranial injection of tumor cells has been used in cancer research to study brain tumor biology, progression, evolution, and therapeutic response. Here we present fluorescence molecular tomography of tumor xenografts, which provides real-time intravital imaging and quantification of a tumor mass in preclinical glioblastoma models.

胶质瘤移植中体内成像的荧光分子层析成像研究

Tumorigenicity is the capability of cancer cells to form a tumor mass. A widely used approach to determine if the cells are tumorigenic is by injecting ...

癌症研究

Intracranial Implantation with Subsequent 3D In Vivo Bioluminescent Imaging of Murine Gliomas

The mouse glioma 261 (GL261) is recognized as an in vivo model system that recapitulates many of the features of human glioblastoma multiforme (GBM). The cell line was originally induced by intracranial injection of 3-methyl-cholantrene into a C57BL/6 syngeneic mouse strain 1; therefore, immunologically competent C57BL/6 mice can be used. While we use GL261, the following protocol can be used for the implantation and monitoring of any intracranial mouse tumor model. GL261 cells were engineered to stably express firefly luciferase (GL261-luc). We also created the brighter GL261-luc2 cell line by stable transfection of the luc2 gene expressed from the CMV promoter. C57BL/6-cBrd/cBrd/Cr mice (albino variant of C57BL/6) from the National Cancer Institute, Frederick, MD were used to eliminate the light attenuation caused by black skin and fur. With the use of albino C57BL/6 mice; in vivo imaging using the IVIS Spectrum in vivo imaging system is possible from the day of implantation (Caliper Life Sciences, Hopkinton, MA). The GL261-luc and GL261-luc2 cell lines showed the same in vivo behavior as the parental GL261 cells. Some of the shared histological features present in human GBMs and this mouse model include: tumor necrosis, pseudopalisades, neovascularization, invasion, hypercellularity, and inflammation 1.
Prior to implantation animals were anesthetized by an intraperitoneal injection of ketamine (50 mg/kg), xylazine (5 mg/kg) and buprenorphine (0.05 mg/kg), placed in a stereotactic apparatus and an incision was made with a scalpel over the cranial midline. A burrhole was made 0.1mm posterior to the bregma and 2.3mm to the right of the midline. A needle was inserted to a depth of 3mm and withdrawn 0.4mm to a depth of 2.6mm. Two &mu;l of GL261-luc or GL261-luc2 cells (107 cells/ml) were infused over the course of 3 minutes. The burrhole was closed with bonewax and the incision was sutured.
Following stereotactic implantation the bioluminescent cells are detectable from the day of implantation and the tumor can be analyzed using the 3D image reconstruction feature of the IVIS Spectrum instrument. Animals receive a subcutaneous injection of 150&mu;g luciferin /kg body weight 20 min prior to imaging. Tumor burden is quantified using mean tumor bioluminescence over time. Tumor-bearing mice were observed daily to assess morbidity and were euthanized when one or more of the following symptoms are present: lethargy, failure to ambulate, hunched posture, failure to groom, anorexia resulting in &gt;10% loss of weight. Tumors were evident in all of the animals on necropsy.

Intracranial Implantation with Subsequent 3D In Vivo Bioluminescent Imaging of Murine Gliomas

Intracranial implantation of GL261 cells into C57BL/6 mice produces malignant gliomas that recapitulate many of the hallmarks of human glioblastoma multiforme. We used GL261 cells stably expressing luciferase to allow us to use in vivo imaging to follow tumor progression. The surgery and 3D in vivo imaging are demonstrated.

随后的三维颅内植入在体内</em生物发光成像鼠脑胶质瘤

The mouse glioma 261 (GL261) is recognized as an in vivo model system that recapitulates many of the features of human glioblastoma multiforme (GBM). The ...

Longitudinal Intravital Imaging of Brain Tumor Cell Behavior in Response to an Invasive Surgical Biopsy

Biopsies are standard of care for cancer treatment and are clinically beneficial as they allow solid tumor diagnosis, prognosis, and personalized treatment determination. However, perturbation of the tumor architecture by biopsy and other invasive procedures has been associated with undesired effects on tumor progression, which need to be studied in depth to further improve the clinical benefit of these procedures. Conventional static approaches, which only provide a snapshot of the tumor, are limited in their ability to reveal the impact of biopsy on tumor cell behavior such as migration, a process closely related to tumor malignancy. In particular, tumor cell migration is the key in highly aggressive brain tumors, where local tumor dissemination makes total tumor resection virtually impossible. The development of multiphoton imaging and chronic imaging windows allows scientists to study this dynamic process in living animals over time. Here, we describe a method for the high-resolution longitudinal imaging of brain tumor cells before and after a biopsy in the same living animal. This approach makes it possible to study the impact of this procedure on tumor cell behavior (migration, invasion, and proliferation). Furthermore, we discuss the advantages and limitations of this technique, as well as the ability of this methodology to study changes in the cancer cell behavior for other surgical interventions, including tumor resection or the implantation of chemotherapy wafers.

Here we describe a method for high-resolution time-lapse multiphoton imaging of brain tumor cells before and after invasive surgical intervention (e.g., biopsy) within the same living animal. This method allows studying the impact of these invasive surgical procedures on tumor cells&#39; migratory, invasive, and proliferative behavior at a single cell level.

脑肿瘤细胞行为的纵向生命成像,以响应侵入性手术活检

Biopsies are standard of care for cancer treatment and are clinically beneficial as they allow solid tumor diagnosis, prognosis, and personalized ...

Stereotactic Intracranial Implantation and In vivo Bioluminescent Imaging of Tumor Xenografts in a Mouse Model System of Glioblastoma Multiforme

Glioblastoma multiforme (GBM) is a high-grade primary brain cancer with a median survival of only 14.6 months in humans despite standard tri-modality treatment consisting of surgical resection, post-operative radiation therapy and temozolomide chemotherapy 1. New therapeutic approaches are clearly needed to improve patient survival and quality of life. The development of more effective treatment strategies would be aided by animal models of GBM that recapitulate human disease yet allow serial imaging to monitor tumor growth and treatment response. In this paper, we describe our technique for the precise stereotactic implantation of bio-imageable GBM cancer cells into the brains of nude mice resulting in tumor xenografts that recapitulate key clinical features of GBM 2. This method yields tumors that are reproducible and are located in precise anatomic locations while allowing in vivo bioluminescent imaging to serially monitor intracranial xenograft growth and response to treatments 3-5. This method is also well-tolerated by the animals with low perioperative morbidity and mortality.

Stereotactic Intracranial Implantation and In vivo Bioluminescent Imaging of Tumor Xenografts in a Mouse Model System of Glioblastoma Multiforme

We describe an integrated method for the precise, stereotactic implantation of human glioblastoma multiforme cells into the brains of nude mice and subsequent serial in vivo imaging to monitor growth and response to treatment of the resultant xenografts.

立体定向颅内植入和<em在体内</em生物发光成像的肿瘤异种移植的小鼠模型系统的多形性胶质母细胞瘤

Glioblastoma multiforme (GBM) is a high-grade primary brain cancer with a median survival of only 14.6 months in humans despite standard tri-modality ...

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 ...

An Orthotopic Glioblastoma Mouse Model Maintaining Brain Parenchymal Physical Constraints and Suitable for Intravital Two-photon Microscopy

Glioblastoma multiforme (GBM) is the most aggressive form of brain tumors with no curative treatments available to date.
Murine models of this pathology rely on the injection of a suspension of glioma cells into the brain parenchyma following incision of the dura-mater. Whereas the cells have to be injected superficially to be accessible to intravital two-photon microscopy, superficial injections fail to recapitulate the physiopathological conditions. Indeed, escaping through the injection tract most tumor cells reach the extra-dural space where they expand abnormally fast in absence of mechanical constraints from the parenchyma.
Our improvements consist not only in focally implanting a glioma spheroid rather than injecting a suspension of glioma cells in the superficial layers of the cerebral cortex but also in clogging the injection site by a cross-linked dextran gel hemi-bead that is glued to the surrounding parenchyma and sealed to dura-mater with cyanoacrylate. Altogether these measures enforce the physiological expansion and infiltration of the tumor cells inside the brain parenchyma. Craniotomy was finally closed with a glass window cemented to the skull to allow chronic imaging over weeks in absence of scar tissue development.
Taking advantage of fluorescent transgenic animals grafted with fluorescent tumor cells we have shown that the dynamics of interactions occurring between glioma cells, neurons (e.g. Thy1-CFP mice) and vasculature (highlighted by an intravenous injection of a fluorescent dye) can be visualized by intravital two-photon microscopy during the progression of the disease.
The possibility to image a tumor at microscopic resolution in a minimally compromised cerebral environment represents an improvement of current GBM animal models which should benefit the field of neuro-oncology and drug testing.

We have established a cortical orthotopic glioblastoma model in mice for intravital two-photon microscopy that recapitulates the biophysical constraints normally at play during the growth of the tumor. A chronic glass window replacing the skull above the tumor enables the follow-up of the tumor progression over time by two-photon microscopy.

原位胶质母细胞瘤小鼠模型的维护脑实质的物理约束，并适用于活体双光子显微镜

Glioblastoma multiforme (GBM) is the most aggressive form of brain tumors with no curative treatments available to date.
Murine models of this pathology ...

Creating Anatomically Accurate and Reproducible Intracranial Xenografts of Human Brain Tumors

Orthotopic tumor models are currently the best way to study the characteristics of a tumor type, with and without intervention, in the context of a live animal &#8211;&#160;particularly in sites with unique physiological and architectural qualities such as the brain. In vitro and ectopic models cannot account for features such as vasculature, blood brain barrier, metabolism, drug delivery and toxicity, and a host of other relevant factors. Orthotopic models have their limitations too, but with proper technique tumor cells of interest can be accurately engrafted into tissue that most closely mimics conditions in the human brain. By employing methods that deliver precisely measured volumes to accurately defined locations at a consistent rate and pressure, mouse models of human brain tumors with predictable growth rates can be reproducibly created and are suitable for reliable analysis of various interventions. The protocol described here focuses on the technical details of designing and preparing for an intracranial injection, performing the surgery, and ensuring successful and reproducible tumor growth and provides starting points for a variety of conditions that can be customized for a range of different brain tumor models.

The brain is a unique site with qualities that are not well represented by in vitro or ectopic analyses. Orthotopic mouse models with reproducible location and growth characteristics can be reliably created with intracranial injections using a stereotaxic fixation instrument and a low pressure syringe pump.

创建精确的解剖学和人脑肿瘤的重现性颅内移植瘤

Orthotopic tumor models are currently the best way to study the characteristics of a tumor type, with and without intervention, in the context of a live ...

Bioluminescence Imaging of an Immunocompetent Animal Model for Glioblastoma

In contrast to commonly reported human glioma xenograft animal models, GL261 murine glioma xenografts recapitulate nearly all relevant clinical and histopathologic features of the human disease. When GL261 cells are implanted intracranially in syngeneic C57BL/6 mice, the model has the added advantage of maintaining an intact immune microenvironment. Stable expression of luciferase in GL261 cells allows non-invasive cost effective bioluminescence monitoring of intracranial tumor growth. We have recently demonstrated that luciferase expression in GL261 cells does not affect the tumor growth properties, tumor cell immunomodulatory cytokine expression, infiltration of immune cells into the tumor, or overall survival of animals bearing the intracranial tumor. Therefore, it appears that the GL261 luciferase glioma model can be useful in the study of novel chemotherapeutic and immunotherapeutic modalities. Here we report the technique for generating stable luciferase expression in GL261 cells and how to study the in vitro and in vivo growth of the tumor cells by bioluminescence imaging.

GL261 glioma cells provide a useful immunocompetent animal model of glioblastoma. The goals of this protocol are to demonstrate proper techniques for monitoring intracranial tumor growth using in vivo bioluminescence imaging, and to verify the utility of luciferase-modified GL261 cells for studying tumor immunology and immunotherapeutic approaches for treating glioblastoma.

免疫活性动物模型胶质母细胞瘤的生物发光成像

In contrast to commonly reported human glioma xenograft animal models, GL261 murine glioma xenografts recapitulate nearly all relevant clinical and ...

建立颅内脑肿瘤异种移植肿瘤的生长和对治疗的反应与后续利用生物发光成像分析

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