笔者想感谢博士。 ：李文军和丹尼尔KREISEL的（在圣路易斯华盛顿大学医学院）他们协助手术培训。 RYL由美国国立卫生格兰特R01 DK068057和总统的美国圣路易斯大学研究基金支持。

1。细胞的制备 注释：1）的完全RPMI 1640的培养基配方（500毫升）包括435毫升的RPMI 1640，加入50ml热灭活的胎牛血清，5毫升的非必需的氨基酸，5毫升丙酮酸钠，5毫升抗生素，抗真菌剂; 2）这里基底膜在4℃的前一天收获细胞。 <ol><li>人力ATC细胞株THJ-11T 8 6孔培养板，生长在80-90％汇合收获一次。 </li><li>离心沉淀细胞，在200×g离心4分钟，在室温下。 </li><li>吸媒体和悬浮细胞在RPMI 1640媒体[2毫升/ 6孔板]。 </li><li>举一个小体积的细胞悬液，加一体积的0.4％台盼蓝染色，并确定使用TC10自动细胞计数的细胞密度。 </li><li>需要得到5×10 5细胞/小鼠倍的数量注入小鼠的细胞悬浮液的体积计算。 </li></ol> 注：为了确保广告<html>等同于注射细胞悬液，准备细胞好像你是双注射的小鼠数量（ 例如，如果注入5只小鼠，然后，仿佛你准备细胞悬液注入10只老鼠）。 <ol start="6"><li>所需体积的细胞悬液转移到15毫升锥形管。 </li><li>离心沉淀细胞，在200×g离心4分钟，在室温下。 </li><li>吸媒体和悬浮细胞在完全RPMI 1640媒体。 </li><li>细胞转移到1.6毫升管，加入1倍体积的基底膜，轻轻混匀，慢慢吹打和。 </li></ol> 注：每个鼠标将收到10μl的细胞悬液/基底膜鸡尾酒的。由于5×10 5细胞悬浮在10微升注射量已成功地应用在先前的ATC原位研究9-10，我们使用本报告中的这些参数。 <ol start="10"><li>将细胞悬液在冰上，直到准备使用。 </li></ol>_title“&gt; 2。鼠标准备<ol><li>清洁手术的区域，其中包括解剖范围和周边地区的，用70％乙醇擦拭所有表面。 </li><li>打开加热灯/垫动物将被从手术中恢复。 </li><li>管理0.1毫升氯胺酮/甲苯​​噻嗪（包括氯胺酮9毫克/毫升和甲苯噻嗪1mg/ml的药物鸡尾酒），每10克体重腹腔注射1毫升，27G½“结核菌素注射器。 </li><li>准备手术鼠标：1）鼠标（从下颌线，上方的胸骨和出武器），2）颈部区域刮胡子，擦洗手术区三次chorhexadine浸湿纱布广场，3）最终完成擦洗用优碘浸湿纱布，4 ）轻轻涂抹眼膏，以防止眼睛干涩。 </li><li>检查踏板反射，以确保鼠标充分镇静。 </li><li>背斜卧在一次性无菌区障碍的解剖范围下将动物和保护动物的地方布胶带。 </li><li>磨砂手和fingernails然后把无菌手术手套。 </li><li>在一个无菌的方式，打开包装，并安排手术仪器。 </li><li>将无菌的悬垂性动物，只留下手术部位暴露。 </li></ol> 3。外科手术进入甲状腺细胞注射<ol><li>使用无菌的，一次性手术刀，做一个1到1.5厘米的纵向切口，沿中线的咽喉。 </li></ol> 注：从中线偏差复杂更深的切削访问气管。正中切口相当精确，让你逗左右唾液腺划破膜组织，减少划伤或切断大动脉的机会。 <ol start="2"><li>拨打第二个切口进气管周围的带状肌，然后右侧阴刻肌肉拉向一边，露出右甲状腺。 </li><li>持有的肌肉层，在这一点上，可以使用止血为方便甲状腺（ 图1）的一部分。 </li></ol> 注：另外，如果进行两个手术人员，肌肉层可能只是回用钳子而准备的注射针筒交给外科医生的协助人员。 <ol start="4"><li>缓缓注入10微升细胞悬液进入右甲状腺使用31G 5/16“然后轻轻取出针胰岛素注射器。 </li></ol> 4。手术部位和鼠标恢复闭幕<ol><li>缝合肌肉层6.0缝合材料，使用一个连续或间断缝合风格。 </li><li>以同样的方式缝合皮肤。 </li><li>三联抗生素软膏，直接在切口部位涂抹上一层。 </li><li>允许动物复原在背斜卧上暖加热垫。一旦动物可以实现胸骨斜卧没有援助，它可以与其他老鼠放回。 </li></ol> &lt;P类=“j​​ove_title”&gt; 5。手术后的监测和组织收集<ol><li>检查切口部位和整体健康的动物每天手术后一个星期，然后检查，每周一次。如果缝线14天之后仍然完好，它们将被删除。 </li></ol> 注：动物健康状况下降（ 如大幅减肥，邋遢的头发，呼吸困难）表现应被人道毁灭，收集和组织。 <ol start="2"><li>一旦老鼠已经达到了预定的手术后的时间点，管理氯胺酮/甲苯​​噻嗪上述稳重。 </li><li>心脏灌注动物和收获甲状腺/气管和其他组织（未在这里详细描述）。 </li></ol>

<ol>
	<li>Bibby, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Orthotopic+models+of+cancer+for+preclinical+drug+evaluation:+advantages+and+disadvantages.">Orthotopic models of cancer for preclinical drug evaluation: advantages and disadvantages.</a> Eur. J. Cancer. 40, 852-857 (2004).</li><li>Hoffman, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Orthotopic+metastatic+mouse+models+for+anticancer+drug+discovery+and+evaluation:+a+bridge+to+the+clinic.">Orthotopic metastatic mouse models for anticancer drug discovery and evaluation: a bridge to the clinic.</a> Invest. New Drugs. 17, 343-359 (1999).</li><li>Killion, J. J., Radinsky, R., Fidler, I. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Orthotopic+models+are+necessary+to+predict+therapy+of+transplantable+tumors+in+mice.">Orthotopic models are necessary to predict therapy of transplantable tumors in mice.</a> Cancer Metastasis Rev. 17, 279-284 (1998).</li><li>Kim, S., et al. <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+anaplastic+thyroid+carcinoma+in+athymic+nude+mice.">An orthotopic model of anaplastic thyroid carcinoma in athymic nude mice.</a> Clin. Cancer Res. 11, 1713-1721 (2005).</li><li>Nucera, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+orthotopic+mouse+model+of+human+anaplastic+thyroid+carcinoma.">A novel orthotopic mouse model of human anaplastic thyroid carcinoma.</a> Thyroid. 19, 1077-1084 (2009).</li><li>Todaro, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumorigenic+and+metastatic+activity+of+human+thyroid+cancer+stem+cells.">Tumorigenic and metastatic activity of human thyroid cancer stem cells.</a> Cancer Res. 70, 8874-8885 (2010).</li><li>Tran Cao, H. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+imaging+of+tumor+progression+in+a+fluorescent+orthotopic+mouse+model+of+thyroid+cancer.">Real-time imaging of tumor progression in a fluorescent orthotopic mouse model of thyroid cancer.</a> Anticancer Res. 30, 4415-4422 (2010).</li><li>Marlow, L. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detailed+molecular+fingerprinting+of+four+new+anaplastic+thyroid+carcinoma+cell+lines+and+their+use+for+verification+of+RhoB+as+a+molecular+therapeutic+target.">Detailed molecular fingerprinting of four new anaplastic thyroid carcinoma cell lines and their use for verification of RhoB as a molecular therapeutic target.</a> J. Clin. Endocrinol. Metab. 95, 5338-5347 (2010).</li><li>Nehs, M. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Late+Intervention+with+anti-BRAF+(V600E)+therapy+induces+tumor+regression+in+an+orthotopic+mouse+model+of+human+anaplastic+thyroid+cancer.">Late Intervention with anti-BRAF (V600E) therapy induces tumor regression in an orthotopic mouse model of human anaplastic thyroid cancer.</a> Endocrinology. 153, 985-994 (2012).</li><li>Nucera, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+BRAFV600E+with+PLX4720+displays+potent+antimigratory+and+anti-invasive+activity+in+preclinical+models+of+human+thyroid+cancer.">Targeting BRAFV600E with PLX4720 displays potent antimigratory and anti-invasive activity in preclinical models of human thyroid cancer.</a> Oncologist. 16, 296-309 (2011).</li></ol>

什么都没有透露。

在我们的模型中，动物表现出甲状腺肿瘤的转移和疾病相关的恶病质和呼吸窘迫，注射后4周。与大多数其他原位模型，利用注射交付的细胞悬浮液，注射到周围组织和后喷射漏超越的目标扩大是可能的。有了这样说，一个辨认潜在的未知此过程中产生的脱靶暴露于非疾病相关的转移或并发症的效果。这是一个普遍关注与大多数xenographic移植，而不管被调查的疾病。然而，无意转移的后果可能会显着地最小化，确保整个斜面针尖已经渗透膜紧贴地覆盖在甲状腺细胞悬浮液慢慢排出。如果观察到泄漏注射过程中或之后，也可以省略这些动物在研究之外。 在这份报告中，我们描述了当地的晚期甲状腺肿瘤的生长和侵袭后ATC细胞原位注射。 ATC转移可以调查分析其他组织使用标准的组织污渍，如苏木精伊红的组织准备工作，或通过免疫组化利用细胞或病理相关标记。该系统能够进一步发展，由g荧光记者甲状腺癌细胞enetically修改，以便实时成像可被用来监测转移和治疗性治疗的功效。

<table border="1">
 <tbody>
 <tr>
 <td>Name of Reagent/Material</td>
 <td>Company</td>
 <td>Catalogue Number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>RPMI 1640 cell culture media</td>
 <td>Mediatech</td>
 <td>10-041-CV</td>
 <td>Media and additives used depend on cell line</td>
 </tr>
 <tr>
 <td>TC10 Automated Cell Counter</td>
 <td>Bio-Rad</td>
 <td>145-0001</td>
 <td></td>
 </tr>
 <tr>
 <td>Matrigel</td>
 <td>Becton, Dickinson and Company</td>
 <td>354234</td>
 <td></td>
 </tr>
 <tr>
 <td>Graefe Forceps, Serrated; Slight Curve, 4"</td>
 <td>Roboz</td>
 <td>RS-5135</td>
 <td></td>
 </tr>
 <tr>
 <td>Disposable scalpel, No. 15</td>
 <td>Feather</td>
 <td>2975#15</td>
 <td></td>
 </tr>
 <tr>
 <td>Olsen Hegar Needle Holder - 4 &frac12;" delicate serrated</td>
 <td>gSource</td>
 <td>gS 21.5400</td>
 <td></td>
 </tr>
 <tr>
 <td>6-0 nylon black monofilament suture</td>
 <td>Surgical Specialties Corporation</td>
 <td>1279B</td>
 <td></td>
 </tr>
 <tr>
 <td>Heating Pad, reusable, 8" x 12"</td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Cloth tape, 1" X 10 yds</td>
 <td>Medline</td>
 <td>MIINON260101</td>
 <td>For restraining anesthetized mouse</td>
 </tr>
 <tr>
 <td>Peanut Clipper/Trimmer</td>
 <td>Wahl</td>
 <td>8655</td>
 <td>For removing hair from surgical site</td>
 </tr>
 <tr>
 <td>ChlorHex-Q SCRUB 2%</td>
 <td>Penn Veterinary Supply</td>
 <td>VED1222</td>
 <td></td>
 </tr>
 <tr>
 <td>Betadine</td>
 <td>Henry Schein Company</td>
 <td>6906727</td>
 <td></td>
 </tr>
 <tr>
 <td>Petrolatum ophthalmic ointment</td>
 <td>Dechra Veterinary Products</td>
 <td>17033-211-38</td>
 <td></td>
 </tr>
 <tr>
 <td>2 x 2 gauze, 12-ply</td>
 <td>Butler Animal Health Supply</td>
 <td>6936</td>
 <td></td>
 </tr>
 <tr>
 <td>Sterile Field, Barrier, 18" x 26"</td>
 <td>Busse</td>
 <td>696</td>
 <td></td>
 </tr>
 <tr>
 <td>Drapes (CSR Wrap)</td>
 <td>Cardinal Health</td>
 <td>AT 21 412</td>
 <td></td>
 </tr>
 <tr>
 <td>27G &frac12;" needle</td>
 <td>Becton, Dickinson and Company</td>
 <td>309623</td>
 <td></td>
 </tr>
 <tr>
 <td>31G 5/16" needle</td>
 <td>Becton, Dickinson and Company</td>
 <td>328438</td>
 <td></td>
 </tr>
 <tr>
 <td>Ketamine (9 mg/ml) / xylazine (1 mg/ml) solution</td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Triple antibiotic ointment</td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 </tbody>
</table>

an orthotopic mouse model of anaplastic thyroid carcinoma

已经建立了人甲状腺癌的动物模型中有几种类型的，包括皮下移植和原位移植到免疫缺陷小鼠的癌细胞。皮下移植瘤模型一直宝贵的临床前筛选和评估新的治疗方法。有很多的优点，使用皮下模型; 1）快速，2）重现性好，和3）肿瘤的建立，生长，治疗剂，可通过目测进行监测。然而，大量证据揭示缺憾皮下移植瘤模型1-3。例如，皮下移植瘤模型中证明疗效的药物治疗往往没有显着影响，对人类疾病。微移植或注射xenographic在于该网站在此相异的心脏。 原位移植瘤模型提供了更多的生物学家gically相关的上下文中来研究这种疾病。到他们的的解剖起源相当于内宿主动物植入病变的细胞或组织的优势，包括肿瘤 - 宿主相互作用的一个合适的地点，与疾病相关的转移的发展和能力的考查特定网站的影响力调查治疗的补救措施。因此，原位移植瘤模型怀有更多的临床价值，因为它们密切重现人类疾病。由于这些原因，一些团体已经采取原位甲状腺癌模型的优势，作为一种研究工具4-7。 在这里，我们描述了一种方法，，建立原位模型的研究甲状腺未分化癌（ATC），它具有很强的侵袭，抗拒治疗，几乎是致命的，在所有确诊患者。养空管细胞制备，作为含有基底膜基质的溶液中解离的细胞悬浮液。小体积慢慢注入连结到正确的甲状腺影响。整体外观和健康的小鼠进行监控，以确保最小的手术后并发症，以了解癌症的病理渗透率。在第4周，处死小鼠，收集和组织进行组织学分析。动物可能会在稍后的时间点，研究更先进的病情发展。生产这种原位小鼠模型建立了一个平台，实现两个目标：1）进一步了解的ATC病理，和2）屏幕当前和未来的治疗药物疗效ATC打击。

我们检测到19共20只小鼠注射4周后ATC细胞的侵入性甲状腺肿瘤。在图2A中所示的例子中，接收5X10 5 ATC所附的条件下生长的细胞原位注射的小鼠，表现出显着的癌细胞浸润进入甲状腺4周后喷射。经苏木精伊红（H＆E）染色性质的入侵ATC细胞特征的梭形细胞中巨型的细胞，嗜酸性粒细胞细胞质和细胞核大。为了进行比较，非注射的对照动物的甲状腺，气管从图2B中所示。卵泡未注射动物显示接近尾声，尚未松动，结社和本质上有一个圆形的形态。 无意的结果由于甲状腺和使用小尺寸的可注射的发送，可能出现问题。 图3A示出了最常观察到的，这是发展的肿瘤以外的质量，但不涵盖，甲状腺。这是可能多针穿透和超越甲状腺，由于当细胞被开除。有时候，小鼠表现为无肿瘤的生长，大体，组织学，检测到如图3B所示。没有检测肿瘤发展，可引起驱逐从注射部位和传播到邻近的组织和空腔的细胞悬液。 <img src="/files/ftp_upload/50097/50097fig1.jpg" alt="图1"/> 图1。暴露甲状腺。标签表示左，右甲状腺气管两侧。 <img src="/files/ftp_upload/50097/50097fig2.jpg" alt="图2"/> 图2。组织学检查肿瘤的生长和成功后甲状腺注射vasion。组织标本切片沿冠状面A）拍摄的图像在200X的小鼠表现出大量的肿瘤生长特性梭形细胞中巨型的细胞，嗜酸性粒细胞的细胞质和细胞核大，苏木精伊红（ ħ＆ē）染色。 涂表示从肿瘤细胞侵入甲状腺的初级病变。B）气管和甲状腺从控制动物（图像100X）。 涂，肿瘤;Ş，平滑肌; TR，气管; 你 ，甲状腺。 <img src="/files/ftp_upload/50097/50097fig3.jpg" alt="图3"/> 图3。意外的结果原位注射组织标本切片沿冠状面A）外设肿瘤的生长没有明显增长的thyroi。（黑色箭头，拍摄的图像在100X）B）缺乏肿瘤检测总额时（图中未示出）或组织学检查（放大50倍拍摄的图像）。 你的甲状腺。

Watch this Scientific Journal Video about An Orthotopic Mouse Model of Anaplastic Thyroid Carcinoma at JoVE.com

An Orthotopic Mouse Model of Anaplastic Thyroid Carcinoma

这里描述的甲状腺未分化癌的原位小鼠模型生成。这种技术采用的人甲状腺未分化癌的细胞免疫缺陷小鼠进入甲状腺手术放置，从而创造了更多的临床相关的设置，研究疾病的进展，以及屏幕创新的治疗干预。

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 ...

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20.9K 次观看.  Saint Louis University School of Medicine. 这里描述的甲状腺未分化癌的原位小鼠模型生成。这种技术采用的人甲状腺未分化癌的细胞免疫缺陷小鼠进入甲状腺手术放置，从而创造了更多的临床相关的设置，研究疾病的进展，以及屏幕创新的治疗干预。

视频: An Orthotopic Mouse Model of Anaplastic Thyroid Carcinoma

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 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 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 ...

Bioluminescent Orthotopic Model of Pancreatic Cancer Progression

Pancreatic cancer has an extremely poor five-year survival rate of 4-6%. New therapeutic options are critically needed and depend on improved understanding of pancreatic cancer biology. To better understand the interaction of cancer cells with the pancreatic microenvironment, we demonstrate an orthotopic model of pancreatic cancer that permits non-invasive monitoring of cancer progression. Luciferase-tagged pancreatic cancer cells are resuspended in Matrigel and delivered into the pancreatic tail during laparotomy. Matrigel solidifies at body temperature to prevent leakage of cancer cells during injection. Primary tumor growth and metastasis to distant organs are monitored following injection of the luciferase substrate luciferin, using in vivo imaging of bioluminescence emission from the cancer cells. In vivo imaging also may be used to track primary tumor recurrence after resection. This orthotopic model is suited to both syngeneic and xenograft models and may be used in pre-clinical trials to investigate the impact of novel anti-cancer therapeutics on the growth of the primary pancreatic tumor and metastasis.

Improved understanding of pancreatic cancer biology is critically needed to enable the development of better therapeutic options to treat pancreatic cancer. To address this need, we demonstrate an orthotopic model of pancreatic cancer that permits non-invasive monitoring of cancer progression using in vivo bioluminescence imaging.

生物荧光原位模型胰腺癌进展

Pancreatic cancer has an extremely poor five-year survival rate of 4-6%.  New therapeutic options are critically needed and depend on improved ...

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 ...

The In Ovo Chick Chorioallantoic Membrane (CAM) Assay as an Efficient Xenograft Model of Hepatocellular Carcinoma

The chick chorioallantoic membrane (CAM) begins to develop by day 7 after fertilization and matures by day 12. The CAM is naturally immunodeficient and highly vascularized, making it an ideal system for tumor implantation. Furthermore, the CAM contains extracellular matrix proteins such as fibronectin, laminin, collagen, integrin alpha(v)beta3, and MMP-2, making it an attractive model to study tumor invasion and metastasis. Scientists have long taken advantage of the physiology of the CAM by using it as a model of angiogenesis. More recently, the CAM assay has been modified to work as an in vivo xenograft model system for various cancers that bridges the gap between basic in vitro work and more complex animal cancer models. The CAM assay allows for the study of tumor growth, anti-tumor therapies, and pro-tumor molecular pathways in a biologically relevant system that is both cost- and time-effective. Here, we describe the development of CAM xenograft model of hepatocellular carcinoma (HCC) with embryonic survival rates of up to 93% and reliable tumor take leading to growth of three-dimensional, vascularized tumors.

The In Ovo Chick Chorioallantoic Membrane CAM Assay as an Efficient Xenograft Model of Hepatocellular Carcinoma

The chick chorioallantoic membrane (CAM) is immunodeficient and highly vascularized, making it a natural in vivo model of tumor growth and angiogenesis. In this protocol, we describe a reliable method of growing three-dimensional, vascularized hepatocellular carcinoma (HCC) tumors using the CAM assay.

该<em&gt;卵内</em&gt;鸡胚尿囊膜（CAM）含量为肝癌的有效异种移植模型

The chick chorioallantoic membrane (CAM) begins to develop by day 7 after fertilization and matures by day 12. The CAM is naturally immunodeficient and ...

Dynamic Contrast Enhanced Magnetic Resonance Imaging of an Orthotopic Pancreatic Cancer Mouse Model

Dynamic contrast enhanced magnetic resonance imaging (DCE-MRI) has been limitedly used for orthotopic pancreatic tumor xenografts due to severe respiratory motion artifact in the abdominal area. Orthotopic tumor models offer advantages over subcutaneous ones, because those can reflect the primary tumor microenvironment affecting blood supply, neovascularization, and tumor cell invasion. We have recently established a protocol of DCE-MRI of orthotopic pancreatic tumor xenografts in mouse models by securing tumors with an orthogonally bent plastic board to prevent motion transfer from the chest region during imaging. The pressure by this board was localized on the abdominal area, and has not resulted in respiratory difficulty of the animals. This article demonstrates the detailed procedure of orthotopic pancreatic tumor modeling using small animals and DCE-MRI of the tumor xenografts. Quantification method of pharmacokinetic parameters in DCE-MRI is also introduced. The procedure described in this article will assist investigators to apply DCE-MRI for orthotopic gastrointestinal cancer mouse models.

The goal of this protocol is to apply dynamic contrast enhanced magnetic resonance imaging (DCE-MRI) for orthotopic pancreatic tumor xenografts in mice. DCE-MRI is a non-invasive method to analyze microvasculature in a target tissue, and useful to assess vascular response in a tumor following a novel therapy.