Authors thank Jeffrey Sellers to assist orthotopic pancreatic cancer mouse modeling. This work was supported by Research Initiative Pilot Awards from the Department of Radiology at UAB and NIH grants 2P30CA013148 and P50CA101955.

所有的程序批准的机构动物护理和使用委员会在阿拉巴马大学伯明翰分校。 1.原位胰腺肿瘤的老鼠模型<ol><li>在Dulbecco改良的Eagle培养基（DMEM）中培养的标准人胰腺癌肿瘤细胞系在补充有10％胎牛血清。保持所有培养物在37℃下在湿润的气氛中，用5％的CO 2。 </li><li>使用8-10周龄雌性重症联合免疫缺陷小鼠。放置动物笼子在12小时光照和12小时黑暗周期在RT（21±2℃）和60％的湿度。 </li><li>麻醉用通风所有动物用异氟烷与氧气（2L /分钟）在整个手术混合的2％。确认麻醉通过脚趾捏反射性的深度。上的加热垫（37℃）处的动物，以保持体温。取适量涂抹于眼睛兽医眼药膏，以防止干燥，而在麻醉下。 ＆＃160; </li><li>除去毛在各小鼠的腹部的左上象限，并给予止痛药（carpofen，5毫克/公斤体重皮下​​）进入该区。应用优碘解决暴露的皮肤。制备蒸压手术器械。 </li><li>使1cm的切口，在皮肤和腹膜使用虹膜直剪刀。轻轻地取出使用手术镊子腹部的胰腺。 </li><li>插入0.5 ml胰岛素注射器28克针入胰尾，然后慢慢注入2500000人胰腺癌细胞的溶液在30微升DMEM中。确认的小疱在胰头通过溶液创建。 </li><li>轻轻将胰腺回用手术镊子腹部。关闭腹膜和皮肤中1层2间断5-0普理灵缝线，然后终止麻醉。不返回已经历手术给其他动物的公司，直到完全恢复的动物。60; 7〜10日手术后取出缝合。 </li><li>给予止痛药（carpofen，5毫克/公斤体重皮下​​）的另一剂量在手术后24小时。 </li><li>检查肿瘤大小通过触诊用两个手指手术区域。肿瘤通常感觉比周围组织器官密度和颠簸。通常需要1〜 - 2周开始感觉肿瘤。 </li><li>每天监控动物疾病的迹象。当动物出现不适（缺乏正常的疏导和回避行为），我们终止它们使用颈椎脱位，而在麻醉下。 </li></ol> 2.磁共振成像<ol><li>适用的MRI当肿瘤大小为约5 - 7毫米直径通常在2〜4周后，细胞植入。使用磁共振扫描仪专门用于小动物成像或临床磁共振扫描仪配备了专门的线圈小动物成像。 注意：我们使用了9.4T小动物磁共振扫描仪有1小时容量谐振器的组合/发射机和表面线圈接收器（30毫米直径）（布鲁克BIOSPIN股份有限公司，比尔里卡，MA）中。表面线圈提供更好的信噪比（SNR），14。 </li><li>制备钆基MRI对比剂注入〜0.1 - 0.2毫摩尔/千克，以每只动物在〜0.1 - 0.2毫升的PBS（磷酸盐缓冲盐水）。 注意：我们使用钆特醇，并在一段持续15秒（0.1毫升/秒）注入0.2毫摩尔/千克0.15毫升的PBS。 </li><li>制备微聚乙烯管（长度：7.62毫米，内径：0.45毫米，外径0.64毫米）。插入地下30针（12.7毫米长）插入管的一端，和一个地下30的钝头针头（9.5毫米长）到另一端。连接1ml注射器含有MR造影剂的钝头针头，并慢慢地推注射器填满整个管与MR造影剂。 </li><li>在整个制备和成像异氟烷与氧气（2L /分钟）混合的2％ - 麻醉用通气〜1动物。确认ANEST的深度hesia由脚趾捏反射。取适量涂抹于眼睛兽医眼药膏，以防止干燥，而在麻醉下。扩张尾静脉用烤灯针头插入前。抢用凯利镊子地下30针的中间，并且将其插入到尾静脉。胶带二者的尾管上的一块塑料或纸板纸（10毫米宽×100毫米长），以保持尾直。 </li><li>放置在仰卧位的动物在装有循环热水（或暖空气）成像期间调节体温的动物床。设置在床的温度至37℃。插入直肠温度探头监控成像期间的体温。 </li><li>应用的垂直弯曲的塑料板到腹部。确保肿瘤位于板的上端身后，然后拉下板略（〜2毫米），确保肿瘤被捉住由董事会。大盘董事会动物睡觉坚决。 </li><li>大盘呼吸垫转导R（SA仪器公司，石溪，NY）的胸部成像过程中监测动物的呼吸。放置在肿瘤区域的顶部表面线圈，并将其用胶带动物床牢牢。推动物床进入MR扫描仪放置肿瘤区域中心处的体积线圈（内径72毫米）。 </li><li>为接收器和发射器进行匹配和调整，其次是垫补。 </li><li>开头的解剖磁共振序列来定位肿瘤。使用T2加权（T2W）快速自旋回波序列，以获得轴向图像与下列采集参数。重复时间（TR）/回波时间（TE）= 3000/34毫秒，128×128矩阵，图30×30mm的场，平均值的数= 1，回波串长度= 4，和20个连续1mm厚的片在隔行模式，以覆盖整个肿瘤区域（总扫描时间：1.6分钟）。 注意：由于原位胰腺肿瘤更难位于比皮下的人，常规locali具有较低分辨率图像ZER未必有用。 </li><li>获得T1加权（T1W）与各种翻转角度的图像检索T1的地图。为了这个目的，可使用回波使用以下参数multiflip角的方法的梯度：重复时间（TR）/回波时间（TE）=三分之一百一十五毫秒，128×128矩阵，来看一个30×30mm的场，数平均值= 4，〜5 - 7个连续1mm厚的片在交错模式以覆盖肿瘤区域，和10个7翻转角，20，30，40，50，60，和每翻转角70（总扫描时间：1分钟）。 注意：不过，multiflip角度的方法是有效的，只有当B1场均匀性是很高的。如果没有，T1的地图可以与多个TR方法，而不是15获得。 </li><li>之前基于钆磁共振造影剂注射后，即可获得T1W图像。使用相同的采集参数和几何形状为T1的映射，但与30使用线性编码的固定的翻转角，以确保获得的k空间的中心时稳定状态下，尤ecially当短TR和低度翻转角被使用。收购注射造影剂前5的基准图像。那么收购后注射造影剂40张图片（共扫描时间：45分钟）。使用注射泵以恒定速率（0.01毫升/秒）注入造影剂。 </li><li>持续监测动物的呼吸，并调整异氟醚浓度，保持呼吸频率50 - 每分钟100次呼吸。监控整个成像动物体温。 </li><li>完成DCE-MRI之后，脱下针和其他探测器，并把动物层状用纸巾空笼子。轻轻按摩下腹部区域。笼应放置在热灯下一半，以允许动物进出，因为它恢复移动的热量梯度。不要让动物无人看管，直到它恢复足够的意识，保持胸骨斜卧。 </li></ol> 3.图像处理与分析<ol><li>段肿瘤REGI<html>在T2W图像。在T2W成像，在肿瘤区域中的信号强度比周围组织更亮，所以肿瘤边界可以手动圈定。 注：如全局阈值或活性轮廓半自动分割技术可以用来16,17，但不均匀背景强度应当纠正特别是当一个表面线圈被使用。 </li><li>创建T1和质子密度图。在用梯度回波序列获得T1W图像，假定回波时间（TE）比T2 *值，像素值由下式确定要少得多 <img alt="式（1）" fo:content-width="3in" src="/files/ftp_upload/52641/52641eq1.jpg" /> 其中S 0是质子密度，T 1为T 1弛豫时间常数，TR为重复时间，θ是一翻转角。公式（1）可重写为 641eq2.jpg“/&gt; 当S（θ）/SINθ替换为Y，和S（θ）/tanθ被替换的X.等式（2）是线性方程，其斜率和截距可用于检索的T1和S 0的值，分别为。 </li><li>计算DCE-MR图像的磁共振造影剂浓度。当基于钆MR对比剂注射，T 1弛豫时间常数随时间变化。所以，方程（1）可重写为 <img alt="公式3" fo:content-width="3in" src="/files/ftp_upload/52641/52641eq3.jpg" /> Ť1（t）的相关与MR对比浓度C（T），如下， <img alt="公式4" fo:content-width="3in" src="/files/ftp_upload/52641/52641eq4.jpg" /> 其中 ，r 1是磁共振造影剂的纵向弛豫。这样，通过结合方程式（3）和（4），MR造影浓度由下式确定 <img aLT =“式5”FO：内容宽度=“3英寸”SRC =“/文件/ ftp_upload / 52641 / 52641eq5.jpg”/&gt; </li><li>发起对比度注射后定量的MR造影剂。 中c p（t）的呈现在血浆MR造影浓度的药代动力学参数在时间 t。 中c p（t）被所谓的动脉输入功能（AIF）。如果AIF是可用的，MR造影剂的药代动力学参数可通过计算 <img alt="方程6" fo:content-width="4in" src="/files/ftp_upload/52641/52641eq6.jpg" /> 其中，C T（t）是MR造影剂浓度在靶组织，V p是小数血浆容量，V e是分数血管外细胞外液容量， 和 K 跨量是转移常数。通量速率常数，k EP，等于 k TRANs除以V 即如果AIF不可用，则参考区模型可以用来代替18,19。基准区域模型是基于所述流限制凯蒂模型20，并且使用对比度浓度在基准区域如下来除去的必要性AIF中， <img alt="公式7" fo:content-width="5in" src="/files/ftp_upload/52641/52641eq7.jpg" /> 其中，C T，投资回报率 （T），K 反，投资回报率 ，以及v E，ROI是造影剂浓度，体积转移常数和分数血管外，细胞外液容量，分别在感兴趣区域（ROI）的区域，而C T，RR （吨），K 反式，RR和 v 即，RR是那些在该基准区域。椎旁肌常被选择为基准区域， 和 v 即，RR小鼠巴ravertebral肌肉被假定为恒定在0.08 21。我们使用的参考区域模型。 </li></ol>

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J., 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=Experimentally-derived+functional+form+for+a+population-averaged+high-temporal-resolution+arterial+input+function+for+dynamic+contrast-enhanced+MRI.">Experimentally-derived functional form for a population-averaged high-temporal-resolution arterial input function for dynamic contrast-enhanced MRI.</a> Magn Reson Med. 56 (5), 993-1000 (2006).</li><li>Tseng, W., Leong, X., Engleman, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Orthotopic+mouse+model+of+colorectal+cancer.">Orthotopic mouse model of colorectal cancer.</a> J Vis Exp. (10), 484 (2007).</li><li>Bhullar, J. 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=A+true+orthotopic+gastric+cancer+murine+model+using+electrocoagulation.">A true orthotopic gastric cancer murine model using electrocoagulation.</a> J Am Coll Surg. 217 (1), 64-70 (2013).</li></ol>

Authors do not have any conflicts of interest to declare.

我们已经介绍了原位胰腺肿瘤模型使用免疫缺陷小鼠，小鼠的DCE-MRI腹部肿瘤，以及其动力学参数量化的具体方法。在原位胰腺肿瘤模型中，必须将针插入胰尾时服用。如果成功的话，将细胞将被转移到胰头创建一个小疱。当施加一个垂直弯曲的塑料板，关键是要确认肿瘤位于下方板的上端。由于胰腺肿瘤附近至该膜片，所述基板也可以是不能够牢固地保持它，特别是当肿瘤大小小于5毫米的直径?...

该方法的总的目标是要应用动态对比增强磁共振成像（DCE-MRI）对小鼠原位胰腺肿瘤异种移植物。 DCE-MRI是一种非侵入性的方法，通过监测MR对比的变化超过一定时间内注射后，以评估微脉管的目标组织。 DCE-MRI已经被用于诊断恶性肿瘤，并评估肿瘤对各种疗法1-4。定量DCE-MRI已经呈现高重复性5。定量的靶组织的磁共振造影剂的药代动力学参数，在注射造影剂前获得不同的时间点和T1地图获得的所有DCE-MR图像必须配准6。然而，由于在腹部呼吸和蠕动运动，定量的DCE-MRI已经具备了胃肠道肿瘤应用有限。 原位胰腺肿瘤模型已被用于评估胰腺肿瘤反应下的生物治疗和化疗7,8。原位肿瘤模型被认为是优于传统的皮下模型中，由于在原来的肿瘤部位的微环境被反射并由此人肿瘤对治疗的反应，可以更准确地预测。然而，小鼠胰腺位于腹部的左上象限，在小鼠中，以便定量的DCE-MRI的原位胰腺肿瘤异种移植物还未容易地实现。 我们已经建立了小鼠腹部肿瘤中的DCE-MRI的协议通过使用正交弯曲的塑料板，以防止从胸部区域9运动传递定影的肿瘤。通过这款主板所施加的压力是局部的腹部，并没有导致呼吸困难。一种自动化图像配准技术已经被验证为在自由呼吸模式腹部器官的DCE-MRI，但它执行effectivelY仅在目标地区缓慢移动，并定期10。动物的呼吸速率成像期间可变的，在腹部区域，以便物理约束将是必要的，以检索在原位胰腺肿瘤小鼠模型可靠的药物动力学参数。我们已经使用正交弯曲的塑料板DCE-MRI中11-13成功定量在原位胰腺肿瘤异种移植物的MR造影剂的药动学参数。在这里，我们提出了原位胰腺肿瘤模型的详细过程，对移植瘤小鼠和药代动力学参数量化的DCE-MRI。

<table border="0" cellpadding="0" cellspacing="0" width="1275">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Name of Material/ Equipment</td>
			<td style="width:199px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:712px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMEM</td>
			<td>Invitrogen</td>
			<td>11965-118</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fetal bovine serum</td>
			<td>Harlan Laboratories</td>
			<td>BT-9501</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Betadine</td>
			<td style="width:199px;">Purdue products</td>
			<td style="width:119px;">67618-153-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">5-0 Prolene sutures</td>
			<td style="width:199px;">Ethicon</td>
			<td style="width:119px;">8720H</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">9.4T MR scanner</td>
			<td>Bruker Biospin Corporation</td>
			<td style="width:119px;">BioSpec 94/20 USR</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Gadoteridol</td>
			<td>Bracco Diagnostics Inc</td>
			<td style="width:119px;">NDC 0270-1111-03</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Micro-polyethelene tube</td>
			<td style="width:199px;">Strategic Applications, Inc</td>
			<td style="width:119px;">#PE-10-25</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">30G blunt tip needle</td>
			<td style="width:199px;">Strategic Applications, Inc</td>
			<td style="width:119px;">89134-194</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Monitoring and gating system</td>
			<td style="width:199px;">SA instruments, Inc</td>
			<td style="width:119px;">Model 1030</td>
			<td>This is an MR compatiable system to measure resiratory rating and body temperature of small animals at the same time.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Syringe pump</td>
			<td>New Era Pump Systems, Inc.</td>
			<td>NE-1600</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

人类胰腺肿瘤细胞在小鼠胰腺创建实体瘤成功生长。 图1显示（A）的正常的胰腺，其中肿瘤细胞溶液被注入，和（B）的照片的代表性小鼠附有是胰腺肿瘤异种移植物原位（MIA PACA -2- ）。肿瘤位于腹部的左上腹，旁边的脾脏。它通常需要2 - 4周的肿瘤长到5 - 后细胞植入直径7毫米。 原位胰腺肿瘤异种移植的议案被大幅抑制，尽管运动伪影存在于MR图像有一定幅度?...

Watch this Scientific Journal Video about Dynamic Contrast Enhanced Magnetic Resonance Imaging of an Orthotopic Pancreatic Cancer Mouse Model at JoVE.com

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

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.

原位胰腺癌小鼠模型的动态对比增强磁共振成像

Dynamic contrast enhanced magnetic resonance imaging (DCE-MRI) has been limitedly used for orthotopic pancreatic tumor xenografts due to severe ...

dynamic-contrast-enhanced-magnetic-resonance-imaging-an-orthotopic

Research

JoVE Journal

Medicine

15.0K 次观看.  University of Alabama at Birmingham. 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. 

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

Contrast Enhanced Vessel Imaging using MicroCT

Microscopic computed tomography (microCT) offers high-resolution volumetric imaging of the anatomy of living small animals. However, the contrast between different soft tissues and body fluids is inherently poor in micro-CT images 1. Under these circumstances, visualization of blood vessels becomes a nearly impossible task. To overcome this and to improve the visualization of blood vessels exogenous contrast agents can be used. Herein, we present a methodology for visualizing the vascular network in a rodent model. By using a long-acting aqueous colloidal polydisperse iodinated blood-pool contrast agent, eXIA 160XL, we optimized image acquisition parameters and volume-rendering techniques for finding blood vessels in live animals. Our findings suggest that, to achieve a superior contrast between bone and soft tissue from vessel, multiple-frames (at least 5-8/ frames per view), and 360-720 views (for a full 360&deg; rotation) acquisitions were mandatory. We have also demonstrated the use of a two-dimensional transfer function (where voxel color and opacity was assigned in proportion to CT value and gradient magnitude), in visualizing the anatomy and highlighting the structure of interest, the blood vessel network. This promising work lays a foundation for the qualitative and quantitative assessment of anti-angiogenesis preclinical studies using transgenic or xenograft tumor-bearing mice.

Contrast enhanced small animal vessel imaging by microCT is a rapid, cost-effective and high-throughput technique for serial in situ examination for tumor development, for analyzing the network of blood vessels that nourish them, and for following the response of tumors to preclinical therapeutic intervention(s).

增强容器成像MicroCT

Microscopic computed tomography (microCT) offers high-resolution volumetric imaging of the anatomy of living small animals. However, the contrast between ...

科研

医学

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 ...

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 ...

Multi-modal Imaging of Angiogenesis in a Nude Rat Model of Breast Cancer Bone Metastasis Using Magnetic Resonance Imaging, Volumetric Computed Tomography and Ultrasound

Angiogenesis is an essential feature of cancer growth and metastasis formation. In bone metastasis, angiogenic factors are pivotal for tumor cell proliferation in the bone marrow cavity as well as for interaction of tumor and bone cells resulting in local bone destruction. Our aim was to develop a model of experimental bone metastasis that allows in vivo assessment of angiogenesis in skeletal lesions using non-invasive imaging techniques.
For this purpose, we injected 105 MDA-MB-231 human breast cancer cells into the superficial epigastric artery, which precludes the growth of metastases in body areas other than the respective hind leg1. Following 25-30 days after tumor cell inoculation, site-specific bone metastases develop, restricted to the distal femur, proximal tibia and proximal fibula1. Morphological and functional aspects of angiogenesis can be investigated longitudinally in bone metastases using magnetic resonance imaging (MRI), volumetric computed tomography (VCT) and ultrasound (US).
MRI displays morphologic information on the soft tissue part of bone metastases that is initially confined to the bone marrow cavity and subsequently exceeds cortical bone while progressing. Using dynamic contrast-enhanced MRI (DCE-MRI) functional data including regional blood volume, perfusion and vessel permeability can be obtained and quantified2-4. Bone destruction is captured in high resolution using morphological VCT imaging. Complementary to MRI findings, osteolytic lesions can be located adjacent to sites of intramedullary tumor growth. After contrast agent application, VCT angiography reveals the macrovessel architecture in bone metastases in high resolution, and DCE-VCT enables insight in the microcirculation of these lesions5,6. US is applicable to assess morphological and functional features from skeletal lesions due to local osteolysis of cortical bone. Using B-mode and Doppler techniques, structure and perfusion of the soft tissue metastases can be evaluated, respectively. DCE-US allows for real-time imaging of vascularization in bone metastases after injection of microbubbles7.
In conclusion, in a model of site-specific breast cancer bone metastases multi-modal imaging techniques including MRI, VCT and US offer complementary information on morphology and functional parameters of angiogenesis in these skeletal lesions.

In the pathogenesis of bone metastasis, angiogenesis is a crucial process and therefore represents a target for imaging and therapy. Here, we present a rat model of site-specific breast cancer bone metastasis and describe strategies to non-invasively image angiogenesis in vivo using magnetic resonance imaging, volumetric computed tomography and ultrasound.

利用磁共振成像，容积电脑断层扫描及超音波的裸鼠乳腺癌骨转移大鼠模型血管生成的多模态成像

Angiogenesis is an essential feature of cancer growth and metastasis formation. In bone metastasis, angiogenic factors are pivotal for tumor cell ...

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 ...

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  ...

Simultaneous PET/MRI Imaging During Mouse Cerebral Hypoxia-ischemia

Dynamic changes in tissue water diffusion and glucose metabolism occur during and after hypoxia in cerebral hypoxia-ischemia reflecting a bioenergetics disturbance in affected cells. Diffusion weighted magnetic resonance imaging (MRI) identifies regions that are damaged, potentially irreversibly, by hypoxia-ischemia. Alterations in glucose utilization in the affected tissue may be detectable by positron emission tomography (PET) imaging of 2-deoxy-2-(18F)fluoro-&#7429;-glucose ([18F]FDG) uptake. Due to the rapid and variable nature of injury in this animal model, acquisition of both modes of data must be performed simultaneously in order to meaningfully correlate PET and MRI data. In addition, inter-animal variability in the hypoxic-ischemic injury due to vascular differences limits the ability to analyze multi-modal data and observe changes to a group-wise approach if data is not acquired simultaneously in individual subjects. The method presented here allows one to acquire both diffusion-weighted MRI and [18F]FDG uptake data in the same animal before, during, and after the hypoxic challenge in order to interrogate immediate physiological changes.

The method presented here uses simultaneous positron emission tomography and magnetic resonance imaging. In the cerebral hypoxia-ischemia model, dynamic changes in diffusion and glucose metabolism occur during and after injury. The evolving and irreproducible damage in this model necessitates simultaneous acquisition if meaningful multi-modal imaging data are to be acquired.

同时PET / MRI成像在小鼠脑缺氧缺血

Dynamic changes in tissue water diffusion and glucose metabolism occur during and after hypoxia in cerebral hypoxia-ischemia reflecting a bioenergetics ...