这项研究是由亨利·谢泼德的AACR膀胱癌的研究资助（09-60-30-李）。

已批准制度的动物护理和使用委员会（IACUC），旧金山加州大学动物学科，涉及的程序。 1。细胞的制备<ol><li>是生长在人类膀胱癌细胞株KU-7-luc2-GFP（卡尺生命科学，霍普金顿，马）的RPMI-1640含10％胎牛血清和50微克/毫升的庆大霉素补充媒体。 </li><li>文化是保持在37℃，5％，与媒体的CO 2的变化，每两至三天。 </li><li>胰蛋白酶消化收获细胞，并计算使用血球。一个单细胞悬液2×10 6细胞在50μL准备在磷酸盐缓冲液放在冰上。 </li></ol> 2。原位膀胱肿瘤模型<ol><li>女性裸体（女/女）小鼠8-10周龄（西蒙森实验室，Gilory，CA）的使用。 </li><li>加热垫置于一个无菌的悬垂性服务SA手术平台。无菌润滑与管芯针，硝酸银溶液，无菌水果冻，24有瓜葛导管准备。 </li><li>全身麻醉诱导小鼠吸入异氟醚诱导（3％，为维护1-2％），并通过鼻锥保持。无菌眼药膏适用于动物的眼睛，热垫使用，以保持身体的热量。 </li><li>将鼠标在仰卧位。 </li><li>轻轻与稳定钳抓住动物的外阴。 </li><li>使用24有瓜葛静脉导管取出用针管芯，鼠标尿道插管。应使用足够的润滑。用鼠标仰卧，尿道口位于后外阴皱褶和阴道前。最初是一个45度的角度采取以cannulate尿道和耻骨下方通过。一个更浅的角度，然后通过尿道传入膀胱。温柔的骨盆压力确保整顿尿道往往可以帮助。必须小心，以避免推太硬反对阻力，因为这可能会导致尿道或膀胱穿孔。看到尿导管内确认其在膀胱腔的位置。 </li><li>从导管和膀胱流明使用管芯针吸尿。管芯用于所有后续的愿望和注射针，离开导管地方。这使得消除死空间内的导管注射用准确的卷，它使重复导管不必要的。 </li><li>膀胱尿路上皮损害聚糖层，注入10μL0.5 M的硝酸银和允许停留10秒。这可能会形成白色沉淀物与剩余尿。 </li><li>吸并放弃使用管芯针的沉淀和膀胱内容。 </li><li>注入100μL无菌水冲洗膀胱。吸出并丢弃的水。 REPE用无菌水冲洗3次，共4清洗。 </li><li>尿道口周围放置一个4-0真丝领带，准备拔除尿管尿道闭塞。 </li><li>注入事先准备好的2×10 6 KU-7-luc2-GFP的细胞，在50μL，使用管芯针。 </li><li>同时取出导管和管芯针，离开尿道闭塞的真丝领带。 </li><li>鼠标将保持2小时之前，真丝领带和动物被唤醒，在全身麻醉下。 </li></ol> 3。超声<ol><li>全身麻醉诱导与异氟醚蒸发和保持通过鼻锥。 </li><li> VEVO一个VisualSonics 体内770高分辨率显微成像系统（VisualSonics公司，多伦多，安大略省，加拿大）RMV-704 40 MHz探头使用。 </li><li>鼠标放置在超声平台上的仰卧姿势。 </li><li>确保动物的后腿用胶带，以避免在操纵的超声探头多余的动作。 </li><li>应用超声凝胶鼠标骨盆。 </li><li>鼠标骨盆降低到1 RMV-704（40 MHz）的超声探头获得横向或纵向的图像。 </li><li>如果发现肿瘤，肿瘤尺寸测量图像可以保存为。 </li></ol> 4。生物发光成像<ol><li>进行腹腔注射200μL（150毫克/公斤）荧光素底物。 </li><li>汽化异氟醚全身麻醉诱导和维持通过鼻锥。 </li><li>在仰卧位放置在发光成像的小鼠。 </li><li>五分钟后，荧光素注射，测量光子每秒动物发光。 </li></ol> 5。萨纳的制备<ol><li>合成的RNA寡核苷酸的规模在50-100μmol。 </li><li>退火互补的单链寡核苷酸我NTO双工saRNAs。 </li><li>脂质纳米粒（LNP）萨纳准备电离脂质1,2-dilinoleyl-4-（2 - 二甲氨基） - [1,3]二氧（DLinKC2，DMA），disteroylphosphatidyl胆碱，胆固醇和PEG-德马吉使用自发囊泡的形成制定过程如前所述。 </li></ol> 6。的膀胱政府的配方萨纳<ol><li>原位膀胱肿瘤模型的建立后，小鼠的治疗膀胱小活化脂质纳米粒（岭澳核电站）（ 见表1）RNA（萨纳）制定。 </li><li>全身麻醉，建立和保持与异氟醚蒸发，应用无菌眼科润滑剂。 </li><li>将动物仰卧位。 </li><li>执行导尿如前所述。 </li><li>尿道周围和导管放置一个4-0真丝领带。 </li><li>从导管吸出所有的尿液和膀胱使用一个空的注射器和管芯针。 </li><li>注入膀胱内的LNP-萨纳总剂量为50μL（3毫克/公斤）。同时取出导管和管芯针，离开尿道闭塞的真丝领带。 </li><li>鼠标将保持2小时闭塞尿道麻醉。 </li><li>治疗膀胱的LNP-萨纳开始肿瘤植入后第4天。小鼠连续治疗，每3天，共有14个治疗。 </li></ol> 7。代表结果可以监测膀胱肿瘤KU-7-luc2-GFP细胞产生荧光素酶生物发光成像（ 图1A和C）和膀胱超声（ 图1B和D）。肿瘤往往在3-5天内检测细胞接种后。我们发现，在超过90％的小鼠膀胱肿瘤成功植入。由于治疗膀胱双链RNA随之而来，肿瘤进展可以监视无线网络TH这些方式。动物牺牲后，肿瘤的生长，也可以得到证实使用GFP荧光。在一般情况下，平均发光与膀胱肿瘤的平均超声尺寸，直观地看到在图1C和1D。然而，后处死动物，证实GFP的肿瘤，我们发现存在可行的肿瘤比超声测量，生物发光与更加紧密。这是因为治疗小鼠残留瘢痕组织往往是通过超声波可视化，但不能从现场的肿瘤分化明显，尤其是在萨纳（相对于对照组）治疗小鼠。 <img src="/files/ftp_upload/4207/4207fig1.jpg" alt="图1" /> 图1。膀胱肿瘤的肿瘤生长动力学。 （一）在膀胱肿瘤生物发光成像测量荧光素酶活性，以确认他们的位置和生长。 （二） 在体内膀胱ultrasounð使用一个40MHz的探头产生肿瘤的图像，可以准确测量其尺寸。 （三）图说明肿瘤的发光和超声尺寸逐步增加。值代表的意思（+ / -标准差）6动物的测量。 <a href="http://www.jove.com/files/ftp_upload/4207/4207fig1large.jpg" target="_blank">点击这里查看大图</a> 。

<ol>
	<li>Semple, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rational+design+of+cationic+lipids+for+siRNA+delivery.">Rational design of cationic lipids for siRNA delivery.</a> Nature. 28, 172-176 (2010).</li><li>Li, L. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small+dsRNAs+induce+transcriptional+activation+in+human+cells.">Small dsRNAs induce transcriptional activation in human cells.</a> Proceedings of the National Academy of Sciences of the United States of America. 103, 17337-17342 (2006).</li><li>Chen, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antitumor+effect+of+dsRNA-induced+p21(WAF1/CIP1)+gene+activation+in+human+bladder+cancer+cells.">Antitumor effect of dsRNA-induced p21(WAF1/CIP1) gene activation in human bladder cancer cells.</a> Molecular cancer therapeutics. 7, 698-703 (2008).</li><li>Chancellor, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preliminary+results+of+myoblast+injection+into+the+urethra+and+bladder+wall:+a+possible+method+for+the+treatment+of+stress+urinary+incontinence+and+impaired+detrusor+contractility.">Preliminary results of myoblast injection into the urethra and bladder wall: a possible method for the treatment of stress urinary incontinence and impaired detrusor contractility.</a> Neurourology and urodynamics. 19, 279-287 (2000).</li><li>Hadaschik, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+validated+mouse+model+for+orthotopic+bladder+cancer+using+transurethral+tumour+inoculation+and+bioluminescence+imaging.">A validated mouse model for orthotopic bladder cancer using transurethral tumour inoculation and bioluminescence imaging.</a> BJU international. 100, 1377-1384 (2007).</li><li>Fu, C., Apelo, C. A., Torres, B., Thai, K. H., Hsieh, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+Bladder+Wall+Injection.">Mouse Bladder Wall Injection.</a> J. Vis. Exp. (53), e2523 (2011).</li><li>Dobek, G. L., Godbey, W. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+Orthotopic+Model+of+Murine+Bladder.">An Orthotopic Model of Murine Bladder.</a> Cancer. J. Vis. Exp. (48), 2535 (2011).</li><li>Dinney, C. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+characterization+of+metastatic+variants+from+human+transitional+cell+carcinoma+passaged+by+orthotopic+implantation+in+athymic+nude+mice.">Isolation and characterization of metastatic variants from human transitional cell carcinoma passaged by orthotopic implantation in athymic nude mice.</a> The Journal of urology. 154, 1532-1538 (1995).</li></ol>

拼箱上等候萨尔纳相关专利申请已提交由加州大学旧金山分校，并授权给Alnylam制药是名为发明者。

随后制定的LNP萨纳转录激活目标为P21 CIP1/WAF1（P21）启动肿瘤灌注治疗鼠标原位移植膀胱肿瘤模型的建立，我们提出了一个协议。2，由此产生的肿瘤可以确认和监测生物发光成像和膀胱超声。原位模型可能优于通过腹腔，皮下或静脉注射模型提供了一个环境，更接近内源性膀胱癌的尿液和尿路上皮。各种原位小鼠膀胱肿瘤已建立了直接的膀胱壁注射以及灌注4-8肿瘤灌注，?...

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论（可选） </td></tr><tr><td> KU-7-LUC-GFP细胞</td><td>卡尺</td><td> 128091 </td><td></td></tr><tr><td>胸腺裸鼠（女/女） </td><td>西蒙森实验室</td><td> 6-7周</td><td>辛:（NCR）NU / NU fisol的</td></tr><tr><td>超声波装置</td><td> visualsonics </td><td> VEVO 770 </td><td> RMV-704探针</td></tr><tr><td>发光单元</td><td>卡尺</td><td>的IVIS频谱</td><td></td></tr><tr><td> Exel的safelet导管</td><td>费舍尔</td><td> 14-841-21 </td><td> 24G X¾" </td></tr><tr><td>硝酸银</td><td>西格玛</td><td> S8157 </td><td></td></tr><tr><td>汉密尔顿注射器</td><td>汉密尔顿合作mpany </td><td> 80301 </td><td></td></tr><tr><td>携号转网的萨纳</td><td> Alnylam制药</td><td></td><td></td></tr></tbody></table> 表1。特定的试剂和设备。

an orthotopic bladder tumor model and the evaluation of intravesical sarna treatment

我们提出了一个新颖的方法治疗膀胱癌与膀胱内交付的小活化RNA（萨尔纳）的原位移植瘤小鼠膀胱肿瘤模型。小鼠模型的建立吸入全身麻醉下导尿。化学烧伤，然后介绍了使用膀胱内的硝酸银溶液膀胱粘膜破坏膀胱聚糖层，使细胞附着。用无菌水，人膀胱癌KU-7-luc2-GFP细胞经过几个清洗灌输通过导管进入膀胱停留2个小时。被确认和监测膀胱肿瘤的后续增长在体内的膀胱超声和生物发光成像。萨纳脂质纳米粒（岭澳核电站）制定的肿瘤治疗膀胱内。肿瘤的生长监测与超声和发光。此过程的所有步骤都体现在影随行。

Watch this Scientific Journal Video about An Orthotopic Bladder Tumor Model and the Evaluation of Intravesical saRNA Treatment at JoVE.com

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

建立原位膀胱肿瘤模型来评估膀胱内交付萨纳和超声监测肿瘤的生长和发光成像的抗肿瘤作用。

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-bladder-tumor-model-evaluation-intravesical-sarna

Research

JoVE Journal

Medicine

14.7K 次观看.  University of California, San Francisco. 建立原位膀胱肿瘤模型来评估膀胱内交付萨纳和超声监测肿瘤的生长和发光成像的抗肿瘤作用。

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

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 ...

Combination Radiotherapy in an Orthotopic Mouse Brain Tumor Model

Glioblastoma multiforme (GBM) are the most common and aggressive adult primary brain tumors1. In recent years there has been substantial progress in the understanding of the mechanics of tumor invasion, and direct intracerebral inoculation of tumor provides the opportunity of observing the invasive process in a physiologically appropriate environment2. As far as human brain tumors are concerned, the orthotopic models currently available are established either by stereotaxic injection of cell suspensions or implantation of a solid piece of tumor through a complicated craniotomy procedure3. In our technique we harvest cells from tissue culture to create a cell suspension used to implant directly into the brain. The duration of the surgery is approximately 30 minutes, and as the mouse needs to be in a constant surgical plane, an injectable anesthetic is used. The mouse is placed in a stereotaxic jig made by Stoetling (figure 1). After the surgical area is cleaned and prepared, an incision is made; and the bregma is located to determine the location of the craniotomy. The location of the craniotomy is 2 mm to the right and 1 mm rostral to the bregma. The depth is 3 mm from the surface of the skull, and cells are injected at a rate of 2 &mu;l every 2 minutes. The skin is sutured with 5-0 PDS, and the mouse is allowed to wake up on a heating pad. From our experience, depending on the cell line, treatment can take place from 7-10 days after surgery. Drug delivery is dependent on the drug composition. For radiation treatment the mice are anesthetized, and put into a custom made jig. Lead covers the mouse's body and exposes only the brain of the mouse. The study of tumorigenesis and the evaluation of new therapies for GBM require accurate and reproducible brain tumor animal models. Thus we use this orthotopic brain model to study the interaction of the microenvironment of the brain and the tumor, to test the effectiveness of different therapeutic agents with and without radiation.

The purpose of this article is to describe the use of an orthotopic glioblastoma model for chemoradiation studies. This article will go though cell processing, implanting, and radiotherapy of the mouse using an intracranial model.

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 ...

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 ...

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  ...

Minimally Invasive Establishment of Murine Orthotopic Bladder Xenografts

Orthotopic bladder cancer xenografts are the gold standard to study molecular cellular manipulations and new therapeutic agents&#160;in vivo. Suitable cell lines are inoculated either by intravesical instillation (model of nonmuscle invasive growth) or intramural injection into the bladder wall (model of invasive growth). Both procedures are complex and highly time-consuming. Additionally, the superficial model has its shortcomings due to the lack of cell lines that are tumorigenic following instillation. Intramural injection, on the other hand, is marred by the invasiveness of the procedure and the associated morbidity for the host mouse.
With these shortcomings in mind, we modified previous methods to develop a minimally invasive approach for creating orthotopic bladder cancer xenografts. Using ultrasound guidance we have successfully performed percutaneous inoculation of the bladder cancer cell lines UM-UC1, UM-UC3 and UM-UC13 into 50 athymic nude. We have been able to demonstrate that this approach is time efficient, precise and safe. With this technique, initially a space is created under the bladder mucosa with PBS, and tumor cells are then injected into this space in a second step. Tumor growth is monitored at regular intervals with bioluminescence imaging and ultrasound. The average tumor volumes increased steadily in in all but one of our 50 mice over the study period.
In our institution, this novel approach, which allows bladder cancer xenograft inoculation in a minimally-invasive, rapid and highly precise way, has replaced the traditional model.

The established technique to inoculate primary invasive orthotopic bladder cancer xenografts requires laparotomy and mobilization of the bladder. This procedure inflicts significant morbidity on the mice, is technically challenging and time-consuming. We therefore developed a high-precision, percutaneous approach utilizing ultrasound guidance.

微创建立小鼠原位膀胱移植瘤的

Orthotopic bladder cancer xenografts are the gold standard to study molecular cellular manipulations and new therapeutic agents&#160;in vivo. Suitable ...

Evaluation of Tumor-infiltrating Leukocyte Subsets in a Subcutaneous Tumor Model

Specialized immune cells that infiltrate the tumor microenvironment regulate the growth and survival of neoplasia.&#160; Malignant cells must elude or subvert anti-tumor immune responses in order to survive and flourish. Tumors take advantage of a number of different mechanisms of immune &#8220;escape,&#8221; including the recruitment of tolerogenic DC, immunosuppressive regulatory T cells (Tregs), and myeloid-derived suppressor cells (MDSC) that inhibit cytotoxic anti-tumor responses. Conversely, anti-tumor effector immune cells can slow the growth and expansion of malignancies: immunostimulatory dendritic cells, natural killer cells which harbor innate anti-tumor immunity, and cytotoxic T cells all can participate in tumor suppression. The balance between pro- and anti-tumor leukocytes ultimately determines the behavior and fate of transformed cells; a multitude of human clinical studies have borne this out. Thus, detailed analysis of leukocyte subsets within the tumor microenvironment has become increasingly important. Here, we describe a method for analyzing infiltrating leukocyte subsets present in the tumor microenvironment in a mouse tumor model. Mouse B16 melanoma tumor cells were inoculated subcutaneously in C57BL/6 mice. At a specified time, tumors and surrounding skin were resected en bloc and processed into single cell suspensions, which were then stained for multi-color flow cytometry. Using a variety of leukocyte subset markers, we were able to compare the relative percentages of infiltrating leukocyte subsets between control and chemerin-expressing tumors. Investigators may use such a tool to study the immune presence in the tumor microenvironment and when combined with traditional caliper size measurements of tumor growth, will potentially allow them to elucidate the impact of changes in immune composition on tumor growth. Such a technique can be applied to any tumor model in which the tumor and its microenvironment can be resected and processed.

This protocol describes a method for the detailed evaluation of leukocyte subsets within the tumor microenvironment in a mouse tumor model. Chemerin-expressing B16 melanoma cells were implanted subcutaneously into syngeneic mice. Cells from the tumor microenvironment were then stained and analyzed by flow cytometry, allowing for detailed leukocyte subset analyses.

在皮下肿瘤模型的评估肿瘤浸润白细胞亚群

Specialized immune cells that infiltrate the tumor microenvironment regulate the growth and survival of neoplasia.&#160; Malignant cells must elude or ...

Evaluation of the Efficacy of the H. pylori Protein HP-NAP as a Therapeutic Tool for Treatment of Bladder Cancer in an Orthotopic Murine Model

Bladder cancer is one of the most common malignancies of the urogenital tract. Intravesical injection of Bacillus Calmette-Gu&#233;rin (BCG) is the gold standard treatment for the high-grade non-muscle invasive bladder cancer (NMIBC). However, since the treatment-related side effects are relevant, newer biological response modifiers with a better benefit/side effects ratio are needed.
The tumour microenvironment can influence both tumour development and therapy efficacy. In order to obtain a good model, it is desirable to implant tumour cells in the organ from which the cancer originates.
In this protocol, we describe a method for establishing a tumour in the bladder cavity of female mice and subsequent delivery of therapeutic agents; the latter are exemplified by our use of Helicobacter pylori neutrophil activating protein (HP-NAP). A preliminary chemical burn of the mucosa, followed by the injection of mouse urothelial carcinoma cell line MB49 via urethral catheterization, enables the cells to attach to the bladder mucosa. After a period, required to allow an initial proliferation of the cells, mice are treated with HP-NAP, administrated again via catheterization. The anti-tumour activity of HP-NAP is evaluated comparing the tumour volume, the extent of necrosis and the degree of vascularization between vehicle- and HP-NAP-treated animals.

Evaluation of the Efficacy of the H. pylori Protein HP-NAP as a Therapeutic Tool for Treatment of Bladder Cancer in an Orthotopic Murine Model

Here the method to establish a syngeneic mouse model of orthotopic bladder tumour to evaluate the anti-tumour efficacy of the bacterial protein HP-NAP is described.

评价疗效<em&gt;小时。幽门螺杆菌</em&gt;蛋白HP-NAP为膀胱癌的治疗在原位小鼠模型的治疗工具

Bladder cancer is one of the most common malignancies of the urogenital tract. Intravesical injection of Bacillus Calmette-Gu&#233;rin (BCG) is the gold ...

A "Patient-Like" Orthotopic Syngeneic Mouse Model of Hepatocellular Carcinoma Metastasis

The majority of cancer-related deaths are caused by the metastasis of the cancer rather than the primary tumor itself. Yet, the underlying mechanisms of cancer metastasis are still unclear. Animal models are essential for elucidating the mechanisms and for evaluating novel strategies for the treatment of metastatic cancers. Here, an in-depth description of a &#8220;patient-like&#8221; orthotopic syngeneic mouse model for exploring the mechanisms of metastasis of solid organ tumors is provided. The survival surgical implantation of BNL 1ME A.7R.1 mouse hepatocellular carcinoma cells directly into the liver (the organ of origin) of the inbred wild-type immune competent laboratory mouse strain, BALB/c is described. The success and reproducibility of this methodology recommends it for widespread use in elucidating the biological mechanisms of solid organ cancer metastasis.

The metastatic spread of cancer is the major cause of cancer-related deaths. We provide an in-depth description of our survival surgery methodology for establishing a &#8220;patient-like&#8221; orthotopic syngeneic mouse model system for studying the mechanisms of metastasis in solid organ tumors.

"以病人为像"肝癌原位同源小鼠模型的癌转移

The majority of cancer-related deaths are caused by the metastasis of the cancer rather than the primary tumor itself. Yet, the underlying mechanisms of ...