这项研究是由美国国立卫生研究院的资助校内程序支持。

一，电池的制备<ol><li>含10％胎牛血清的DMEM汇合处约80％汇合225厘米3瓶每5被植入小鼠的细胞生长。 </li><li>吸细胞的媒体;无钙或镁与10毫升的PBS洗。吸出PBS关闭细胞。 </li><li> Trypsinize细胞与胰蛋白酶的3毫升，等待10-15分钟，并消除胰蛋白酶，与15毫升的媒体。 </li><li>吸取到50毫升锥形细胞和媒体。两个大瓶放入一个50毫升锥形。 </li><li>在7分钟1600转的自旋细胞在4°C完全吸上清。在10毫升冷PBS悬浮细胞，并轻轻吹打上下冲洗细胞。确保细胞沉淀用PBS混合均匀。悬挂应该是阴天。 </li><li>在这个时候进行细胞计数，用库尔特计数器。在7分钟1600转离心细胞再次在4°C </li><li> 第二细胞离心，后海盗所有PBS和1毫升的PBS重悬。 </li><li>细胞纺在3000转每分钟为10分钟，在4°C的PBS1毫升PBS是吸气，PBS是加入终浓度为每5μLU87细胞调整到1x10 6，其他细胞株可能需要不同的最佳肿瘤生长的细胞数量和浓度。 </li><li>最终体积和浓度后得到的细胞保持在一个Eppendorf管中的冰。 </li></ol>二。异种移植<ol class="ualpha"><li>准备鼠标<ol><li>小鼠麻醉注射氯胺酮/甲苯​​噻嗪/生理盐水混合使用。混合物为120毫克/公斤氯胺酮，16毫克/公斤的Rompun和盐碱地。小鼠体重和0.1毫升每10克，根据自己的体重。麻醉用1毫升注射器IP连接到26G针。这将需要约15分钟小鼠达到无疼痛反应的睡眠阶段。你可以检查的疼痛反应小鼠捏他们的脚垫。 </li><li>鼠标是完全麻醉后，眼膏管理自己的眼睛，以防止干燥。 </li><li>鼠标被放置在缺口前上齿口栏上的立体床。在床上，然后放置在立体。 </li><li>鼻子警卫被放置在老鼠的鼻子，以防止头部运动，耳棒轻轻放置在老鼠的耳道。要小心，不要做任鼻子酒吧或耳酒吧太紧的位置，可能为颅骨骨折和/或防止呼吸鼠标。 </li><li>确保头鼠标是在这个过程中是有帮助的水平上立体刻度。 </li><li>要准备的鼠标，酒精和providine碘慷慨地应用于鼠标共3应用的碘的酒精和3应用交替的头部。其中有70％的酒精垫的最终应用适用于头。 </li></ol></li><li>手术<ol><li>通过高压灭菌或浸泡前开始的过程中70％的乙醇文书过程中的所有工具应消毒。 </li><li>头骨顶部背后右眼斜回左后颅骨切口。应该是约10毫米至15毫米长的切口。 </li><li>使用棉条，慢慢分开切口暴露的头骨和头骨和皮肤之间的膜擦去。使用一个Q尖端涂抹任何血和失血过多观看。 </li><li>暴露颅骨顶部的可视化前囟门。使用Hamilton注射器点，定位前囟门。在颅骨钻孔的正确位置是2毫米的权利和1毫米前囟门（图2）。一名外科医生的毡尖标记，购买无菌用来标记钻孔的位置。请注意，该洞是靠近了ANterior缝合线。 </li><li>接下来，使用在22G 18G针一针花瓶或通过美联储针点在显着位置，在头骨钻的小孔。 </li><li> 5μL细胞装入10μL玻璃汉密尔顿注射器。汉密尔顿注射器是10μl钝30G 33G固定针连接到它的。务必混合后装入他们的细胞，他们吸取1ml注射器或使用了25G附针1。 Hamilton注射器装入后，放置在手臂后面排队，它在洞的头骨。 </li><li>降低短打结束，一旦头骨表面的针针是与颅骨水平，降低针，直到它在3毫米以下的头骨表面的深度。 </li><li>降低针后，应保持2分钟，以避免任何重大创伤，影响大脑的针。植入的细胞是6-8分钟的速度在每分钟1μL，小心任何backflo的W。经过最后的细胞被植入，留针2分钟的地方，让所有的细胞，安顿下来。 </li><li>从大脑中取出针，清理具有Q-尖的孔。丙酮50％，100％乙醇和PBS，在每个解决方案和3倍的顺序进行清洁针头和注射器。 </li><li>皮肤上的头骨是准备在这一点上缝合。使用5-0 PBS关闭切口约3针。 </li><li>允许鼠标唤醒热垫，如果需要重新申请眼膏。鼠标可以返回到笼子里后，它显示了自身的运动（即抬起头或移动的肩膀）。 </li></ol></li></ol>第三。辐射和药物治疗的IC植入<ol class="ualpha"><li>放射治疗<ol><li>根据治疗的老鼠的细胞株，可以采取5至7天植入后。我们的部门使用放射治疗放射治疗单位orthovoltageMENT。 </li><li>如劳保局（发光成像），MRI或CT成像可用于可视化的肿瘤之前随机（图3A）。 </li><li>小鼠麻醉前氯胺酮/二甲苯胺噻嗪/盐水鸡尾酒，和眼膏适用于他们的眼睛。 </li><li>小鼠被放置在一个自定义的跳汰机的配置就像是一个馅饼轮，在小鼠的头朝向中心点辐射。小鼠体内铅辐射屏蔽。 </li><li>可给予药物治疗之前或辐射，通过不同的传递路线，即倚，IP和第四。 </li></ol></li></ol>四。结果鼠标唤醒的过程中，麻醉注射后45-60分钟内。如果鼠标已难起床后的程序和时间已超过1个半小时程序后，鼠标可能无法恢复和安乐死可能是一种有效的替代。如果日ERE是出血过多，或在任何时候，破获在手术过程中安乐死的头骨变得建议。如果一切顺利，取决于细胞系，肿瘤开始形成2-4个星期内，在手术后有发生。当使用颅内异种移植模型，疗效通常是衡量生存植入后5。然而，由于这是一个IC模型的症状和体征会发生之前死亡。鼠标可能会出现的体征有些驼背的姿势，体重减轻，和闲置。一些神经系统体征，可能是癫痫发作，头部倾斜，失去平衡。图3B显示他的肿瘤染色的死亡率。一个采用Kaplan-Meier曲线用于评估数据（图4）。存活天数的50％计算，比对照。如果联合组的生活比个人辐射和毒品军火此外较长的组合是成功的。 <img alT ="图1"的src ="/ files/ftp_upload/3397/3397fig1.jpg"/&gt; 图1。Stoelting立体定向设备。 <img alt="图2" src="/files/ftp_upload/3397/3397fig2.jpg" /> 图2:一个鼠标头骨钻洞的位置图。 <img alt="图3A" src="/files/ftp_upload/3397/3397fig3A.jpg" /> 图3A。劳保局成像植入后大紫荆勋章。 <img alt="图3B" src="/files/ftp_upload/3397/3397fig3B.jpg" /> 图3B。H和E在GBM的肿瘤死亡率。 <img alt="图4" src="/files/ftp_upload/3397/3397fig4.jpg" /> 图4。U87的IC与PARP抑制剂手术后7天内注射植入物的结果。

<ol>
	<li>Bleau, A. M., Holland, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trapping+the+mouse+genome+to+hunt+human+alternations.">Trapping the mouse genome to hunt human alternations.</a> Proc. Nat. Acad. Sci. U.S.A. 104, 7737-7738 (2007).</li><li>McGrady, B. J., McCormick, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+murine+model+of+intracranial+invasion:+morphological+observations+on+central+nervous+system+invasion+by+melanoma+cells.">A murine model of intracranial invasion: morphological observations on central nervous system invasion by melanoma cells.</a> Clin. Exp. Metastasis. 10, 387-393 (1992).</li><li>Fei, X., Zhang, Q., Dong, J., Diao, y., Wang, Z., Li, R., Wu, Z., Wang, A., Lan, Q., Zhang, S., Huang, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+clinically+relevant+orthotopic+xenograft+mouse+model+of+metastatic+lung+cancer+and+glioblastoma+through+surgical+tumor+tissues+injection+with+trocar.">Development of clinically relevant orthotopic xenograft mouse model of metastatic lung cancer and glioblastoma through surgical tumor tissues injection with trocar.</a> Journal of Experimental &#38; Clinical Cancer Research. 29, 84-84 (2010).</li><li>Fujita, M., Zhu, X., Sasaki, K., Ueda, R., Low, K., Pollack, I., Okada, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+STAT3+Promotes+the+Efficacy+of+Adoptive+Transfer+Therapy+Using+Type-1+CTLs+by+Modulation+of+the+Immunological+Microenvironment+in+a+Murine+Intracranial+Glioma.">Inhibition of STAT3 Promotes the Efficacy of Adoptive Transfer Therapy Using Type-1 CTLs by Modulation of the Immunological Microenvironment in a Murine Intracranial Glioma.</a> J. Immunol. 180, 2089-2098 (2008).</li><li>Candolfi, M., Curtin, J. F., Nichols, W. S., Muhammad, A. K. M. .. . G., King, G., Pluhar, G. D., McNiel, G. E., Ohlfest, E. A., Freese, J. R., Moore, P. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracranial+glioblastoma+models+in+preclinical+neuro-oncology:+neuropathlogical+characterization+and+tumor+progression.">Intracranial glioblastoma models in preclinical neuro-oncology: neuropathlogical characterization and tumor progression.</a> J. Neurooncol. 85, 133-148 (2007).</li><li>Kaye, A., Morstyn, G., Gardner, I., Pyke, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+xenograft+glioma+model+in+mouse+brain.">Development of a xenograft glioma model in mouse brain.</a> Cancer Research. 46, 1367-1373 (1986).</li><li>Zhao, Y., Xiao, A., diPierro, C., Carpenter, J., Abdel-Fattah, R., Redpath, G., Lopez, M., Hussaini, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+Extensive+Invasive+Intracranial+Human+Glioblastoma+Xenograft+Model.">An Extensive Invasive Intracranial Human Glioblastoma Xenograft Model.</a> American Journal of Pathology. 176, 3032-3049 (2010).</li><li>Learn, C., Grossi, P., Schmittling, R., Xie, W., Mitchell, D., Karikari, I., Wei, Z., Dressman, H., Sampson, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+Analysis+of+Intracranial+Tumors+in+a+Murine+Model+of+Glioma+Demonstrate+a+Shift+in+Gene+Expression+in+Response+to+Host+Immunity.">Genetic Analysis of Intracranial Tumors in a Murine Model of Glioma Demonstrate a Shift in Gene Expression in Response to Host Immunity.</a> J. Neuroimmunol. 182, 63-72 (2007).</li><li>Martens, T., Laabs, Y., Günther, H., Kemming, D., Zhu, Z., Witte, L., Hagel, C., Westphal, M., Lamszus, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+Glioblastoma+Growth+in+a+Highly+Invasive+Nude+Mouse+Model+Can+Be+Achieved+by+Targeting+Epidermal+Growth+Factor+Receptor+but+not+Vascular+Endothelial+Growth+Factor+Receptor-2.">Inhibition of Glioblastoma Growth in a Highly Invasive Nude Mouse Model Can Be Achieved by Targeting Epidermal Growth Factor Receptor but not Vascular Endothelial Growth Factor Receptor-2.</a> Clin. Cancer Res. 14, 5447-5458 (2008).</li><li>Purow, B., Schiff, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+the+genetics+of+glioblastoma:+are+we+reaching+critical+mass.">Advances in the genetics of glioblastoma: are we reaching critical mass.</a> Nat. Rev. Neurol. 5, 419-426 (2009).</li></ol>

我们什么都没有透露。

胶质母细胞瘤，恶性胶质瘤，如代表最常见的原发性脑肿瘤和预后极差4。大紫荆勋章的影响患者的生存期已大致维持不变（即诊断后9-12个月）在过去十年中，尽管在手术，放疗和化疗5垫款。应包括适当的治疗脑胶质瘤的研究模型的特点重现肿瘤的位置和生长的细胞作为一个相对独立的肿瘤细胞应类似于尽可能脑胶质瘤的组织学特征密切合作，应该有一个可预见的增长率，肿瘤的...

<table border="1"><tbody><tr><td>实验室标准立体定向Instrutment </td><td> Stoelting小鼠和大鼠适配器</td></tr><tr><td>优碘</td><td> 100％的乙醇</td></tr><tr><td> 70％乙醇</td><td> 50％的丙酮</td></tr><tr><td> 6的"Q-秘诀</td><td>酒精棉签</td></tr><tr><td>汉密尔顿注射器80008 </td><td>爱惜康5-0的PD </td></tr><tr><td> 31 1701的SN-33G / 1"/ PT3针</td><td> 1毫升注射器</td></tr><tr><td> 25G针头</td><td>加热垫</td></tr><tr><td>软膏人工泪液</td><td>尖很细的永久性标记</td></tr></tbody></table>

combination radiotherapy in an orthotopic mouse brain tumor model

胶质母细胞瘤（GBM），是最常见的和积极的成人原发性脑肿瘤1。近年来已取得实质性进展，在了解肿瘤浸润力学，直接脑内接种肿瘤提供了机会，在生理上适当的环境观测入侵过程。至于人类脑肿瘤有关，目前可用的原位模型建立立体定向注射细胞悬液或植入一个坚实的一块肿瘤，通过一个复杂的开颅手术过程3。在我们的技术，我们收获的细胞组织培养，创建一个用于直接植入到大脑细胞悬液。手术时间约30分钟，鼠标需要在一个恒定的手术平面，注射麻醉剂使用。鼠标被放置在了一个立体夹具Stoetling（图1）。前前后后ER被清除，并准备手术区，切口和前囟门位于确定开颅的位置。开颅手术的位置是2毫米的权利和延髓前囟门1毫米。深度为3毫米，从头骨的表面和细胞注入2μL，每2分钟的速度。皮肤缝合用5-0的PDS，允许唤醒了一个加热垫和鼠标。从我们的经验，根据细胞株，治疗可以采取手术后7-10天的地方。给药是依赖于药物组成。对于放射治疗的老鼠麻醉，并投入到一个做夹具定制。铅覆盖老鼠的身体和公开只老鼠的大脑。研究肿瘤发生和大紫荆勋章的新疗法的评价需要准确，重现性脑肿瘤动物模型。因此，我们使用这种原位脑模型研究的大脑和肿瘤微环境的相互作用，测试effectivenESS无辐射与不同的治疗药物。

Watch this Scientific Journal Video about Combination Radiotherapy in an Orthotopic Mouse Brain Tumor Model at JoVE.com

Combination Radiotherapy in an Orthotopic Mouse Brain Tumor Model

这篇文章的目的是描述使用原位神经母细胞瘤化疗研究模型。虽然本文将去细胞处理，植入，鼠标使用颅内模型放疗。

在原位小鼠脑肿瘤模型结合放疗

Glioblastoma multiforme (GBM) are the most common and aggressive adult primary brain tumors1. In recent years there has been substantial progress in the ...

combination-radiotherapy-in-an-orthotopic-mouse-brain-tumor-model

Research

JoVE Journal

Medicine

16.3K 次观看.  National Cancer Institute. 这篇文章的目的是描述使用原位神经母细胞瘤化疗研究模型。虽然本文将去细胞处理，植入，鼠标使用颅内模型放疗。

视频: Combination Radiotherapy in an Orthotopic Mouse Brain Tumor Model

An Orthotopic Model of Murine Bladder Cancer

In this straightforward procedure, bladder tumors are established in female C57 mice through the use of catheterization, local cauterization, and subsequent cell adhesion. After their bladders are transurethrally catheterized and drained, animals are again catheterized to permit insertion of a platinum wire into bladders without damaging the urethra or bladder. The catheters are made of Teflon to serve as an insulator for the wire, which will conduct electrical current into the bladder to create a burn injury. An electrocautery unit is used to deliver 2.5W to the exposed end of the wire, burning away extracellular layers and providing attachment sites for carcinoma cells that are delivered in suspension to the bladder through a subsequent catheterization. Cells remain in the bladder for 90 minutes, after which the catheters are removed and the bladders allowed to drain naturally. The development of tumor is monitored via ultrasound. Specific attention is paid to the catheterization technique in the accompanying video.

Bladder tumors are established in female mice in a minimally invasive fashion through catheterization, local cauterization, and subsequent adhesion of carcinoma cells to the burn sites.

一个小鼠膀胱癌原位模型

In this straightforward procedure, bladder tumors are established in female C57 mice through the use of catheterization, local cauterization, and ...

科研

医学

Multi-photon Imaging of Tumor Cell Invasion in an Orthotopic Mouse Model of Oral Squamous Cell Carcinoma

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient management strategies. Orthotopic mouse models of oral cancer have been developed to facilitate the study of factors that impact invasion and serve as model system for evaluating anti-tumor therapeutics. In these systems, visualization of disseminated tumor cells within oral cavity tissues has typically been conducted by either conventional histology or with in vivo bioluminescent methods. A primary drawback of these techniques is the inherent inability to accurately visualize and quantify early tumor cell invasion arising from the primary site in three dimensions. Here we describe a protocol that combines an established model for squamous cell carcinoma of the tongue (SCOT) with two-photon imaging to allow multi-vectorial visualization of lingual tumor spread. The OSC-19 head and neck tumor cell line was stably engineered to express the F-actin binding peptide LifeAct fused to the mCherry fluorescent protein (LifeAct-mCherry). Fox1nu/nu mice injected with these cells reliably form tumors that allow the tongue to be visualized by ex-vivo application of two-photon microscopy. This technique allows for the orthotopic visualization of the tumor mass and locally invading cells in excised tongues without disruption of the regional tumor microenvironment. In addition, this system allows for the quantification of tumor cell invasion by calculating distances that invaded cells move from the primary tumor site. Overall this procedure provides an enhanced model system for analyzing factors that contribute to SCOT invasion and therapeutic treatments tailored to prevent local invasion and distant metastatic spread. This method also has the potential to be ultimately combined with other imaging modalities in an in vivo setting.

A comprehensive overview of the techniques involved in generating a mouse model of oral cancer and quantitative monitoring of tumor invasion within the tongue through multi-photon microscopy of labeled cells is presented. This system can serve as a useful platform for the molecular assessment and drug efficacy of anti-invasive compounds.

多光子成像在口腔鳞状细胞癌原位小鼠模型的肿瘤细胞的侵袭

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient ...

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 ...

An Orthotopic Bladder Tumor Model and the Evaluation of Intravesical saRNA Treatment

We present a novel method for treating bladder cancer with intravesically delivered small activating RNA (saRNA) in an orthotopic xenograft mouse bladder tumor model. The mouse model is established by urethral catheterization under inhaled general anesthetic. Chemical burn is then introduced to the bladder mucosa using intravesical silver nitrate solution to disrupt the bladder glycosaminoglycan layer and allows cells to attach. Following several washes with sterile water, human bladder cancer KU-7-luc2-GFP cells are instilled through the catheter into the bladder to dwell for 2 hours. Subsequent growth of bladder tumors is confirmed and monitored by in vivo bladder ultrasound and bioluminescent imaging. The tumors are then treated intravesically with saRNA formulated in lipid nanoparticles (LNPs). Tumor growth is monitored with ultrasound and bioluminescence. All steps of this procedure are demonstrated in the accompanying video.

Establishing an orthotopic bladder tumor model to evaluate antitumor effects of intravesically delivered saRNA and monitoring tumor growth by ultrasound and bioluminescent imaging.

原位膀胱肿瘤模型和评价治疗膀胱萨纳

We present a novel method for treating bladder cancer with intravesically delivered small activating RNA (saRNA) in an orthotopic xenograft mouse bladder ...

An Orthotopic Mouse Model of Anaplastic Thyroid Carcinoma

Several types of animal models of human thyroid carcinomas have been established, including subcutaneous xenograft and orthotopic implantation of cancer cells into immunodeficient mice. Subcutaneous xenograft models have been valuable for preclinical screening and evaluation of new therapeutic treatments. There are a number of advantages to using a subcutaneous model; 1) rapid, 2) reproducible, and 3) tumor establishment, growth, and response to therapeutic agents may be monitored by visual inspection. However, substantial evidence has shed light on the short-comings of subcutaneous xenograft models1-3. For instance, medicinal treatments demonstrating curative properties in subcutaneous xenograft models often have no notable impact on the human disease. The microenvironment of the site of xenographic transplantation or injection lies at the heart of this dissimilarity.
Orthotopic tumor xenograft models provide a more biologically relevant context in which to study the disease. The advantages of implanting diseased cells or tissue into their anatomical origin equivalent within a host animal includes a suitable site for tumor-host interactions, development of disease-related metastases and the ability to examine site-specific influence on investigational therapeutic remedies. Therefore, orthotopic xenograft models harbor far more clinical value because they closely reproduce human disease. For these reasons, a number of groups have taken advantage of an orthotopic thyroid cancer model as a research tool4-7.
Here, we describe an approach that establishes an orthotopic model for the study of anaplastic thyroid carcinoma (ATC), which is highly invasive, resists treatment, and is virtually fatal in all diagnosed patients. Cultured ATC cells are prepared as a dissociated cellular suspension in a solution containing a basement membrane matrix. A small volume is slowly injected into the right thyroid gland. Overall appearance and health of the mice are monitored to ensure minimal post-operative complications and to gauge pathological penetrance of the cancer. Mice are sacrificed at 4 weeks, and tissue is collected for histological analysis. Animals may be taken at later time-points to examine more advance progression of the disease. Production of this orthotopic mouse model establishes a platform that accomplishes two objectives: 1) further our understanding of ATC pathology, and 2) screen current and future therapeutic agents for efficacy in combating ATC.

Generation of an orthotopic mouse model of anaplastic thyroid carcinoma is described here. This technique employs surgical placement of human anaplastic thyroid cancer cells into the thyroid of immunodeficient mice, thus creating a more clinically relevant setting to study disease progression as well as screen innovative therapeutic interventions.

甲状腺未分化癌的原位小鼠模型

Several types of animal models of human thyroid carcinomas have been established, including subcutaneous xenograft and orthotopic implantation of cancer ...

An Orthotopic Bladder Cancer Model for Gene Delivery Studies

Bladder cancer is the second most common cancer of the urogenital tract and novel therapeutic approaches that can reduce recurrence and progression are needed. The tumor microenvironment can significantly influence tumor development and therapy response. It is therefore often desirable to grow tumor cells in the organ from which they originated. This protocol describes an orthotopic model of bladder cancer, in which MB49 murine bladder carcinoma cells are instilled into the bladder via catheterization. Successful tumor cell implantation in this model requires disruption of the protective glycosaminoglycan layer, which can be accomplished by physical or chemical means. In our protocol the bladder is treated with trypsin prior to cell instillation. Catheterization of the bladder can also be used to deliver therapeutics once the tumors are established. This protocol describes the delivery of an adenoviral construct that expresses a luciferase reporter gene. While our protocol has been optimized for short-term studies and focuses on gene delivery, the methodology of mouse bladder catheterization has broad applications.

Implantation of cancer cells into the organ of origin can serve as a useful preclinical model to evaluate novel therapies. MB49 bladder carcinoma cells can be grown within the bladder following intravesical instillation. This protocol demonstrates catheterization of the mouse bladder for the purpose of tumor implantation and adenoviral delivery.

原位膀胱癌模型的基因传递研究

Bladder cancer is the second most common cancer of the urogenital tract and novel therapeutic approaches that can reduce recurrence and progression are ...

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

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

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

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

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

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

An Orthotopic Murine Model of Human Prostate Cancer Metastasis

Our laboratory has developed a novel orthotopic implantation model of human prostate cancer (PCa). As PCa death is not due to the primary tumor, but rather the formation of distinct metastasis, the ability to effectively model this progression pre-clinically is of high value. In this model, cells are directly implanted into the ventral lobe of the prostate in Balb/c athymic mice, and allowed to progress for 4-6 weeks. At experiment termination, several distinct endpoints can be measured, such as size and molecular characterization of the primary tumor, the presence and quantification of circulating tumor cells in the blood and bone marrow, and formation of metastasis to the lung. In addition to a variety of endpoints, this model provides a picture of a cells ability to invade and escape the primary organ, enter and survive in the circulatory system, and implant and grow in a secondary site. This model has been used effectively to measure metastatic response to both changes in protein expression as well as to response to small molecule therapeutics, in a short turnaround time.

This orthotopic model of human prostate cancer allows for quantification of tumor size, circulating tumor cells, and formation of distinct metastasis to the lung. As cells must escape the primary organ, enter the blood stream, and implant into a secondary site, this model effectively recapitulates the scenario in humans.

人类前列腺癌转移的原位小鼠模型

Our laboratory has developed a novel orthotopic implantation model of  human prostate cancer (PCa). As PCa death is not due to the primary tumor, but  ...

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 ...

Tumor Treating Field Therapy in Combination with Bevacizumab for the Treatment of Recurrent Glioblastoma

A novel device that employs TTF therapy has recently been developed and is currently in use for the treatment of recurrent glioblastoma (rGBM). It was FDA approved in April 2011 for the treatment of patients 22 years or older with rGBM. The device delivers alternating electric fields and is programmed to ensure maximal tumor cell kill1.
Glioblastoma is the most common type of glioma and has an estimated incidence of approximately 10,000 new cases per year in the United States alone2. This tumor is particularly resistant to treatment and is uniformly fatal especially in the recurrent setting3-5. Prior to the approval of the TTF System, the only FDA approved treatment for rGBM was bevacizumab6. Bevacizumab is a humanized monoclonal antibody targeted against the vascular endothelial growth factor (VEGF) protein that drives tumor angiogenesis7. By blocking the VEGF pathway, bevacizumab can result in a significant radiographic response (pseudoresponse), improve progression free survival and reduce corticosteroid requirements in rGBM patients8,9. Bevacizumab however failed to prolong overall survival in a recent phase III trial26. A pivotal phase III trial (EF-11) demonstrated comparable overall survival between physicians&#8217; choice chemotherapy and TTF Therapy but better quality of life were observed in the TTF arm10.
There is currently an unmet need to develop novel approaches designed to prolong overall survival and/or improve quality of life in this unfortunate patient population. One appealing approach would be to combine the two currently approved treatment modalities namely bevacizumab and TTF Therapy. These two treatments are currently approved as monotherapy11,12, but their combination has never been evaluated in a clinical trial. We have developed an approach for combining those two treatment modalities and treated 2 rGBM patients. Here we describe a detailed methodology outlining this novel treatment protocol and present representative data from one of the treated patients.

A novel methodology that is employed for the treatment of recurrent glioblastomas is described. This treatment approach employs the application of alternating electric tumor treating fields (TTFields), known as TTF Therapy in combination with bevacizumab, a targeted agent that is currently FDA approved as monotherapy.

肿瘤治疗领域的治疗与联合贝伐单抗治疗复发性胶质母细胞瘤的治疗

A novel device that employs TTF therapy has recently been developed and is currently in use for the treatment of recurrent glioblastoma (rGBM). It was FDA ...