作者承认以下资金来源:P01CA040035（FE / JAS）和VA职业发展奖（JAS）。

1。池的维护<ol><li>许多骨转移亚克隆MDA-MB-231的存在有变骨的定植和生长的倾向。对于我们的实验中，我们使用来自GFP标记的MDA-MB-231 UTHSCA 2开发克隆。这种特殊的子克隆在大约3周接种后，将形成明显的溶骨性病变，在第4周与牺牲。 </li><li>维持细胞在DMEM中含有10％FBS和0.1毫克/毫升青霉素-链霉素，在37℃下，在5-8％CO 2 7。 </li><li>传代细胞在80-90％汇合，并重新在1:10稀释板（这取决于细胞的类型和尺寸的印版。对于MDA-MB-231细胞中的T-75，这是约6×10 5），这是大约每72小时。 </li><li> MDA-MB-231细胞保持一般成纤维细胞的外观与众多伪。任何舍入指示过度拥挤或其它不希望的应力，并应避免，因为这可能影响体内转移潜力 。 </li><li>理想的情况下，电池应维持在文化，只是简单地在接种前（小于1周的实验，直到你有足够多的细胞），和一个新的小瓶应解冻，如果四舍五入或细胞的生长率的变化发生。 </li></ol> 2。细胞制备<ol><li> Trypsinize 80-90％汇合的细胞用0.15％胰蛋白酶/ EDTA（稀释0.5％Gibco公司，在PBS中的胰蛋白酶EDTA 1:2）。该浓度允许快速脱离（&lt;2分钟），并降低的细胞聚集。 </li><li>立即除去细胞从板与10毫升冰冷的DMEM含10％FBS，吸移管到15毫升锥形管，在200×g离心5分钟离心。 </li><li>重悬沉淀在50ml的锥形管中在25毫升冰冷的PBS（无钙和镁）和计数。 </li><li>离心重新沉淀，并在每毫升（腔内）的10 6个细胞，或在250000 cells/10微升（骨肿瘤引起的）在冰冷的PBS重悬。温度TURE PBS中重要的是在注射后的细胞和随后的栓塞，以防止结块。将细胞悬浮液应保持在冰上，同时执行注射液和将是可行的，并保持30分钟，未结块。虽然确切的数量会有所不同，从研究者到所需的板研究者，我们一般使用8-10小鼠的T-75。 </li></ol> 3。腔内接种<ol><li> 4-6周龄雌性（Foxn核/ - :哈伦）小鼠用于这些实验中，虽然其他的免疫妥协小鼠（RAG1 - / - ，RAG2 - / - ，SCID的XID，等）也可以是与人类肿瘤细胞的实验中使用。 </li><li>裸鼠，应喂给Teklad 2920x饮食5-10天注射前降低死亡率从注射过程中，减少背景荧光成像程序（自食苜蓿免费）。 </li><li>如果使用小鼠以外的其他女/女，消除头发剃或使用奈尔（根据我们的经验奈尔效果更好）上腹部AREA开始前的程序。 </li><li>麻醉小鼠用异氟醚蒸发器（2.5％异:2-3升/分钟O 2）或其它优选的麻醉（根据我们的经验，异氟醚是最好的用于心内接种的耐受性）的隔室。 </li><li>一个鼠标移动在一个不锈钢通风罩的麻醉机鼻锥的时间。 </li><li>每个鼠标放置在其背部，胸部朝上。 </li><li>洗胸部有10％的聚维酮/碘棉签/用70％的乙醇溶液中，然后，重复2次。 </li><li>马克鼠标的胸部注射液（用消毒过的标记或标记稍微向一侧，以保持注射部位的无菌）。我们使用标记位置的胸骨切迹和剑突过程顶部的中间，稍微左胸骨（解剖）。绘制一个小的气泡的空气进入注射器创建300微升28克½胰岛素注射器柱塞和弯液面之间的空间（重要的看到心脏的脉冲），和吸取100μl的细胞。 </li><li>保持直立的针。按住鼠标用另一只手拉紧皮肤，插入针。 </li><li>成功插入左心室，导致一个明显的亮红色的脉搏，血液的注射器。在这个深度，小心地压下活塞的注射器（100微升量），没有显着运动的针，以避免穿刺入胸腔内的心脏或溢出细胞。 </li><li>细胞被注射后，小幅拉涨柱塞创建少量的负压力，以减少入胸腔酣畅淋漓的细胞。 </li><li>直接拉针的胸部，而在拆卸过程中，它可以衬里的心撕裂，引起出血，一定要避免倾斜针。 </li><li>轻轻推压胸部注射部位，以减少出血。 </li><li>删除鼠标从鼻锥体，并继续施加压力约1分钟的光。 </li><li>移动鼠标到加热垫，直到完全CONscious。 </li><li>如果鼠标开始转动或抽动恢复后，可能的原因是栓塞。小鼠腹膜内注射80〜120毫克/公斤的氯胺酮，同时降低血管阻力，增加血流量和心输出量。这应该可以帮助的鼠标通过血块。发生此问题后，它是最好的改变的细胞制剂，因为它表明，细胞聚集。我们改变细胞制剂后约8只小鼠，或30分钟。 </li></ol> 4。骨肿瘤引起的<ol><li>注入鼠标Buprenex（0.1毫克/千克）或类似的经批准的镇痛药之前立即程序。 </li><li>使用异氟醚麻醉鼠标，然后移动到鼻锥和维持麻醉。 </li><li>用10％聚乙烯吡咯烷酮/碘清洁双腿拭​​子/溶液中，然后用乙醇，重复2次。脱毛腿（奈尔或类似产品）在清洁之前，如果皮毛存在。 </li><li>轻轻抓住外踝，内踝，胫骨和下半部用食指和日UMB，然后弯曲腿（屈曲和横向转动的组合，使得膝盖是可视且可访问的。 </li><li>用70％的乙醇湿润皮肤，提高知名度的基础髌韧带，这应该是作为一个独特的，厚厚的，白线可见。在牢牢把握，并进入前髁间面积在胫骨上方的腿的踝/鼠标插入28克½针髌骨下，通过中间的髌韧带。 </li><li>当插入到胫骨针，引导生长板，通过仔细稳定的，坚定的压力，轻微的钻探行动。 </li><li>穿透胫骨生长板后，中针会遇到明显阻力较小。 </li><li>使用温和的，横向移动的针，以确保针在胫骨生长板通过。运动将是有限的，如果针在胫骨内适当的位置。 </li><li>慢慢地压低柱塞注射10微升的细胞液。应该觉得在这一点上几乎没有阻力。 </li><li>斯洛伐克WLY提取针。 </li><li>按照同样的步骤，进行到下一个腿注入10μl的PBS。 </li><li>从麻醉中拔下鼠标，并继续加热垫，直到恢复。 </li><li>在接下来的24小时监控的老鼠注入额外Buprenex每12小时，如果的动物继续呈现紧张迹象的。 </li></ol> 5。成像时间课程<ol><li>萤光素酶的成像:在适用的情况下，荧光素酶的成像后的肿瘤细胞接种（虽然大多数骨转移模型中，检测到接近14天）在第7天开始，应利用。的小鼠的腹腔内注射150 mg / kg的荧光素，麻醉，和成像8分钟后，注射使用IVIS设备8。 </li><li> GFP成像:通常情况下表达GFP的肿瘤开始被探测到10-14天左右intratibial注射和16-21腔内实验使用Maestro成像设备（CRI）9。简单地说，老鼠是用异氟烷麻醉（3％），并保持在麻醉下在Maestro卡成像室通过鼻锥体。对于我们的绿色荧光蛋白表达细胞，我们用蓝色的过滤器集（498 excitation/515发射绿色荧光蛋白），在500毫秒暴露在10纳米。 </li><li> faxitron（透视）:X-射线被开始每周7至14天之间。病变在21天的腔内模型将显示在一个星期前在的intratibial注射3。我们的体外和体内的放射线照相数据获取使用Faxitron LX-60在35千伏为8秒的曝光。 </li><li>其他影像学检查:影像学技术将完全根据具体的研究。除了 ​​上述方法外，3D微型计算机断层扫描（μCT），微正电子发射断层扫描（μPET），和其他方式10,11活的动物成像的基础上提供有价值的信息。 </li></ol> 6。鼠标牺牲<ol><li>每个模型都有一个稍微不同的时间过程。 MDA-MB-231个细胞的小鼠的恶病质，或截瘫之间21和35天，一般28天左右（IT小鼠注射21〜28天之间）。 </li><li>一旦几个小鼠的病变，开始突破骨皮质，成为截瘫患者，或失去的体重在10-20％之间，牺牲所有的小鼠在研究中使用氯胺酮/甲苯​​噻嗪过量颈椎脱位（或其他IACUC批准的方法）。 </li><li>取出骨头需要进一步研究，并存储在缓存4％福尔马林。 </li><li>胸部肿瘤的小鼠是错过了注射的标志，并应被排除在研究之外。 </li></ol> 7。 体外分析<ol><li> μCT:在70％乙醇中的扫描允许的结构和组织学的参数相同的骨试样上测量。 </li><li>轮廓和分析根据个别实验设计和可用的设备10。我们的数据获得，与一个SCANCO的μCT40使用12μM分辨率的。 </li><li> Histomorphometry:使用首选的组织方法和工艺标本染色，苏木素/伊红7 </li></ol> 8。代表性的成果心腔内或intratibial接种的肿瘤细胞（ 图1）之后的小鼠每周监测由放射摄影术和荧光（或发光）。病变通常变得显而易见周2和3之间通过X-射线，而它们可以是可见的荧光和发光早在第2周，这取决于模型（ 图2）上。病变是可见的作为暗在骨孔，并随时间（ 图3）中变得更加明显。病变进行定量的MetaMorph分析X光片和荧光定量使用Maestro（CRI）的图像采集和分析软件。病变开始生长，小鼠经常成为恶病质的。牺牲时，老鼠已经失去了其初始体重在10-20％之间。的条件的<html>小鼠可以迅速改变，并且必须在白天多次监视。 3-4周的小鼠开始拖动一个或两个后肢和牺牲整个研究。图片之前采取牺牲。过量麻醉后，取血，骨代谢标志物或其他因素来衡量。鼠标和骨头切除后颈椎脱位，皮肤被剥夺体外成像（ 图3），这将使肿瘤灶的可视化至少7天前体内成像技术和清洁。一次成像完成的骨头被放置在有标签的磁带和存储在福尔马林固定和进一步的组织处理或μCT分析。组织形态计量学的样品上进行，以指示后的肿瘤负担和BV / TVμCT进行量化骨破坏的量（ 图4）上的胫骨。 机构/ 4260/4260fig1.jpg"/&gt; 图1。注射妥善安置。腹侧X光片的图（A）中示出在第 4肋间进入左心室心内接种针妥善安置的成年小鼠胸廓。解剖标志的胸骨上（见白色）与水平虚线所示。在老鼠的皮肤胸骨切迹和剑突过程作为地标，并且针插入稍左胸骨。腹侧radiographical图（B）的成年小鼠示出针的正确放置的腿之间的髁在胫骨近端，对齐。从横向的角度来看，胫骨粗隆和深骺板针的尖端放在背后。理想的位置注射的白色圆圈所示。 <img alt="图2" src="/files/ftp_upload/4260/4260fig2.jpg" /> 图2。约的时间过程中，骨转移模型。进展NCER引起的骨质疾病与X射线和GFP +的肿瘤荧光图像体内注射骨肿瘤引起的（A）和心内注射（B）。 <img alt="图3" src="/files/ftp_upload/4260/4260fig3.jpg" /> 图3。心内MDA-MB-231型顶行: 体外 GFP荧光图像显示在0天，14日，21日和28日腔内注射后，MDA-MB-231的时间过程 。需要注意的是在第14天的肿瘤病灶是唯一可见的软组织切除后， 在体外 。下排: 体外放射图像相同的股骨，说明溶骨性病变。请注意，骨质破坏通常是不可见的，直到3周后，心脏注射。 <img alt="图4" src="/files/ftp_upload/4260/4260fig4.jpg" /> 图4。量化骨转移的进展。GFP +肿瘤可视化大师成像体外蒂维AE的非荷瘤小鼠（A）和MDA-MB-231的亚克隆（B）的GFP表达的心腔内注射4周后的小鼠。肿瘤病灶与者Faxitron（D）所示的骨破坏的地区比较（C）无肿瘤的骨。微型电脑测量骨结构和矿物数据呈现为一个健康的骨骼（E）（F）骨质破坏的区域显示为空洞面积和荷瘤骨的三维图像。进一步的骨和肿瘤的细节被认为是在相应的H＆E染色的组织切片（G及H）。

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没有利益冲突的声明。

骨肿瘤引起的疾病的研究依赖于几种动物模型，其中最常用的两种心内和intratibial的接种。在许多情况下，研究转移到骨，心内是最好的选择，但是，它并不能代表整个转移过程，为了实现这一目标，因此，乳腺脂肪垫或其他原位注射，最好应使用。肿瘤细胞不容易在大多数研究中使用人类细胞从原发部位转移，如果是的话，他们主要转移到肺部。一些菌株的4T1细胞转移到骨5,6，但病变体...

<table border="1"><tbody><tr><td> 的试剂的名称 </td><td> 公司 </td><td> 目录编号 </td></tr><tr><td> MDA-MB-231人乳腺癌细胞</td><td>本来ATCC内部的克隆和选择</td><td></td></tr><tr><td> DMEM </td><td> GIBCO </td><td> 11885 </td></tr><tr><td> PBS </td><td>敏达公司</td><td> 21-040-CV </td></tr><tr><td> FBS </td><td>阿特拉斯生物</td><td> F-0050a </td></tr><tr><td>胰蛋白酶</td><td> GIBCO </td><td> 15400 </td></tr><tr><td> Faxitron </td><td> Faxitron </td><td></td></tr><tr><td>大师</td><td>国际广播电台</td><td></td></tr><tr><td> μCT扫描仪</td><td> SCANCO </td><td></td></tr><tr><td>裸鼠</td><td>哈伦</td><td>狐女 - / - </td></tr><tr><td>胰岛素注射器（300微升，28.5克） </td><td> Becton Dickinson公司</td><td> 309300 </td></tr><tr><td> IVIS </td><td>卡尺</td><td></td></tr><tr><td>辐照啮齿动物的饮食</td><td> Teklad </td><td> 2920x </td></tr></tbody></table>

models of bone metastasis

骨转移是一种常见的恶性肿瘤，包括乳腺癌，前列腺癌和肺癌的发生。一旦成立于骨，肿瘤有显着的发病率和死亡率1。因此，是一种显着的需要理解在骨的肿瘤的建立，生长和活性的分子机制控制。 体内模型中，已经建立了几个研究这些事件，每个人都有特定的优点和局限性。最常用的模型利用心内接种肿瘤细胞直接进入动脉血液供应的无胸腺（裸）BalbC小鼠。此过程可以应用到许多不同类型的肿瘤（包括PC-3前列腺癌，肺癌，和小鼠乳腺脂肪垫肿瘤），然而，在这最后，我们将重点对乳腺癌模型，MDA-MB-231。在这个模型中，我们利用骨高度选择性的克隆，最初来自曼迪博士的研究小组在圣安东尼奥2，即H转GFP的表达和克隆我们的第3组。克隆是骨转移的变体，具有高的osteotropism率和很少转移到肺，肝，肾上腺。虽然心内注射是最常见的用于研究的骨转移，在某些情况下intratibial 4或乳腺脂肪垫注射是比较合适的。腔内注射时使用的人类肿瘤细胞的目标监测的后期阶段的转移，特别是逮捕骨，生存，增殖，并建立发展成癌性骨骼疾病的肿瘤，癌细胞的能力。 intratibial注射如果侧重于肿瘤后，已转移到骨，这大致既定转移性骨病相关的癌症细胞和骨的关系进行。这些模型概括都不在转移过程中的早期步骤之前栓塞和入境进入血液循环的肿瘤细胞。如果要监控从原发部位的骨原发肿瘤的生长或转移，乳腺脂肪垫接种通常是首选;然而，极少数肿瘤细胞株将一如既往地转移到骨骼，从主站点，与4T1的骨优惠克隆的，一个小鼠乳腺癌，除5,6。 这手稿细节接种程序，并强调在接种后分析的关键步骤。具体来说，它包括细胞培养，心内和intratibial接种的肿瘤细胞接种程序，以及通过X-射线，荧光和组织形态计量学分析的简要信息，关于每周监测。

Watch this Scientific Journal Video about Models of Bone Metastasis at JoVE.com

Models of Bone Metastasis

经常用来研究癌症骨转移动物模型。在这个协议中，我们将介绍两种常见的方法接种肿瘤骨转移研究，并简要介绍了一些用来监测和量化这些模型的分析。

骨转移模型

Bone metastases are a common occurrence in several malignancies, including breast, prostate, and lung. Once established in bone, tumors are responsible ...

Universidad de Cartagena UDEC

Research

JoVE Journal

Medicine

41.9K 次观看.  Vanderbilt University. 经常用来研究癌症骨转移动物模型。在这个协议中，我们将介绍两种常见的方法接种肿瘤骨转移研究，并简要介绍了一些用来监测和量化这些模型的分析。

视频: Models of Bone Metastasis

Experimental Metastasis Assay

Metastasis is the leading cause of death in cancer patients. To understand the mechanism of metastasis, an experimental metastasis assay was established using immunodeficient mice. This article delineates the procedures involved in this assay, including sample preparation, intravenous injection, and culturing cells from lung metastases. Briefly, a pre-determined number of human cancer cells were prepared in vitro and directly injected into the circulation of immunodeficient mice through their tail veins. A small number of cells survive the turbulence in the circulation and grow as metastases in internal organs, such as lung. The injected mice are dissected after a certain period. The tissue distribution of metastases is determined under a dissecting microscope. The number of metastases in a specific tissue is counted and it directly correlates with the metastatic ability of the injected cancer cells. The arisen metastases are isolated and cultured in vitro as cell lines, which often show enhanced metastatic abilities than the parental line when injected again into immunodeficient mice. These highly metastatic derivatives become useful tools for identifying genes or molecular pathways that regulate metastatic progression.

This article describes the procedures of an experimental metastasis assay that is used to determine the metastatic potential of human cancer cell lines.

实验转移的含量

Metastasis is the leading cause of death in cancer patients.  To understand the mechanism of metastasis, an experimental metastasis assay was established ...

科研

医学

In vitro Mesothelial Clearance Assay that Models the Early Steps of Ovarian Cancer Metastasis

Ovarian cancer is the fifth leading cause of cancer related deaths in the United States1. Despite a positive initial response to therapies, 70 to 90 percent of women with ovarian cancer develop new metastases, and the recurrence is often fatal2. It is, therefore, necessary to understand how secondary metastases arise in order to develop better treatments for intermediate and late stage ovarian cancer. Ovarian cancer metastasis occurs when malignant cells detach from the primary tumor site and disseminate throughout the peritoneal cavity. The disseminated cells can form multicellular clusters, or spheroids, that will either remain unattached, or implant onto organs within the peritoneal cavity3 (Figure 1, Movie 1). 
All of the organs within the peritoneal cavity are lined with a single, continuous, layer of mesothelial cells4-6 (Figure 2). However, mesothelial cells are absent from underneath peritoneal tumor masses, as revealed by electron micrograph studies of excised human tumor tissue sections3,5-7 (Figure 2). This suggests that mesothelial cells are excluded from underneath the tumor mass by an unknown process. 
Previous in vitro experiments demonstrated that primary ovarian cancer cells attach more efficiently to extracellular matrix than to mesothelial cells8, and more recent studies showed that primary peritoneal mesothelial cells actually provide a barrier to ovarian cancer cell adhesion and invasion (as compared to adhesion and invasion on substrates that were not covered with mesothelial cells)9,10. This would suggest that mesothelial cells act as a barrier against ovarian cancer metastasis. The cellular and molecular mechanisms by which ovarian cancer cells breach this barrier, and exclude the mesothelium have, until recently, remained unknown. 
Here we describe the methodology for an in vitro assay that models the interaction between ovarian cancer cell spheroids and mesothelial cells in vivo (Figure 3, Movie 2). Our protocol was adapted from previously described methods for analyzing ovarian tumor cell interactions with mesothelial monolayers8-16, and was first described in a report showing that ovarian tumor cells utilize an integrin &#x2013;dependent activation of myosin and traction force to promote the exclusion of the mesothelial cells from under a tumor spheroid17. This model takes advantage of time-lapse fluorescence microscopy to monitor the two cell populations in real time, providing spatial and temporal information on the interaction. The ovarian cancer cells express red fluorescent protein (RFP) while the mesothelial cells express green fluorescent protein (GFP). RFP-expressing ovarian cancer cell spheroids attach to the GFP-expressing mesothelial monolayer. The spheroids spread, invade, and force the mesothelial cells aside creating a hole in the monolayer. This hole is visualized as the negative space (black) in the GFP image. The area of the hole can then be measured to quantitatively analyze differences in clearance activity between control and experimental populations of ovarian cancer and/ or mesothelial cells. This assay requires only a small number of ovarian cancer cells (100 cells per spheroid X 20-30 spheroids per condition), so it is feasible to perform this assay using precious primary tumor cell samples. Furthermore, this assay can be easily adapted for high throughput screening.

In vitro Mesothelial Clearance Assay that Models the Early Steps of Ovarian Cancer Metastasis

The mesothelial clearance assay described here takes advantage of fluorescently labeled cells and time-lapse video microscopy to visualize and quantitatively measure the interactions of ovarian cancer multicellular spheroids and mesothelial cell monolayers. This assay models the early steps of ovarian cancer metastasis.

这些模型在体外间皮间隙检测卵巢癌转移的早期步骤

Ovarian cancer is the fifth leading cause of cancer related deaths in the United States1. Despite a positive initial response to therapies, 70 to 90 ...

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 ...

Pre-clinical Evaluation of Tyrosine Kinase Inhibitors for Treatment of Acute Leukemia

Receptor tyrosine kinases have been implicated in the development and progression of many cancers, including both leukemia and solid tumors, and are attractive druggable therapeutic targets. Here we describe an efficient four-step strategy for pre-clinical evaluation of tyrosine kinase inhibitors (TKIs) in the treatment of acute leukemia. Initially, western blot analysis is used to confirm target inhibition in cultured leukemia cells. Functional activity is then evaluated using clonogenic assays in methylcellulose or soft agar cultures. Experimental compounds that demonstrate activity in cell culture assays are evaluated in vivo using NOD-SCID-gamma (NSG) mice transplanted orthotopically with human leukemia cell lines. Initial in vivo pharmacodynamic studies evaluate target inhibition in leukemic blasts isolated from the bone marrow. This approach is used to determine the dose and schedule of administration required for effective target inhibition. Subsequent studies evaluate the efficacy of the TKIs in vivo using luciferase expressing leukemia cells, thereby allowing for non-invasive bioluminescent monitoring of leukemia burden and assessment of therapeutic response using an in vivo bioluminescence imaging system. This strategy has been effective for evaluation of TKIs in vitro and in vivo and can be applied for identification of molecularly-targeted agents with therapeutic potential or for direct comparison and prioritization of multiple compounds.

Receptor tyrosine kinases are ectopically expressed in many cancers and have been identified as therapeutic targets in acute leukemia. This manuscript describes an efficient strategy for pre-clinical evaluation of tyrosine kinase inhibitors for the treatment of acute leukemia.

酪氨酸激酶抑制剂的临床前评价急性白血病的治疗

Receptor tyrosine kinases have been implicated in the development and progression of many cancers, including both leukemia and solid tumors, and 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  ...

Adjustable Stiffness, External Fixator for the Rat Femur Osteotomy and Segmental Bone Defect Models

The mechanical environment around the healing of broken bone is very important as it determines the way the fracture will heal. Over the past decade there has been great clinical interest in improving bone healing by altering the mechanical environment through the fixation stability around the lesion. One constraint of preclinical animal research in this area is the lack of experimental control over the local mechanical environment within a large segmental defect as well as osteotomies as they heal. In this paper we report on the design and use of an external fixator to study the healing of large segmental bone defects or osteotomies. This device not only allows for controlled axial stiffness on the bone lesion as it heals, but it also enables the change of stiffness during the healing process in vivo. The conducted experiments have shown that the fixators were able to maintain a 5 mm femoral defect gap in rats in vivo during unrestricted cage activity for at least 8 weeks. Likewise, we observed no distortion or infections, including pin infections during the entire healing period. These results demonstrate that our newly developed external fixator was able to achieve reproducible and standardized stabilization, and the alteration of the mechanical environment of in vivo rat large bone defects and various size osteotomies. This confirms that the external fixation device is well suited for preclinical research investigations using a rat model in the field of bone regeneration and repair.

One constraint of preclinical research in the field of bone repair is the lack of experimental control over the local mechanical environment within a healing bone lesion. We report the design and use of an external fixator for bone repair with the ability to change fixator stiffness in vivo.

可调节刚度，外固定支架的大鼠股骨截骨和节段性骨缺损模型

The mechanical environment around the healing of broken bone is very important as it determines the way the fracture will heal. Over the past decade there ...

High-frequency Ultrasound Imaging of Mouse Cervical Lymph Nodes

High-frequency ultrasound (HFUS) is widely employed as a non-invasive method for imaging internal anatomic structures in experimental small animal systems. HFUS has the ability to detect structures as small as 30 &#181;m, a property that has been utilized for visualizing superficial lymph nodes in rodents in brightness (B)-mode. Combining power Doppler with B-mode imaging allows for measuring circulatory blood flow within lymph nodes and other organs. While HFUS has been utilized for lymph node imaging in a number of mouse&#160; model systems, a detailed protocol describing HFUS imaging and characterization of the cervical lymph nodes in mice has not been reported. Here, we show that HFUS can be adapted to detect and characterize cervical lymph nodes in mice. Combined B-mode and power Doppler imaging can be used to detect increases in blood flow in immunologically-enlarged cervical nodes. We also describe the use of B-mode imaging to conduct fine needle biopsies of cervical lymph nodes to retrieve lymph tissue for histological&#160; analysis. Finally, software-aided steps are described to calculate changes in lymph node volume and to visualize changes in lymph node morphology following image reconstruction. The ability to visually monitor changes in cervical lymph node biology over time provides a simple and powerful technique for the non-invasive monitoring of cervical lymph node alterations in preclinical mouse models of oral cavity disease.

This protocol describes the application of high-frequency ultrasound (HFUS) for imaging mouse cervical lymph nodes. This technique optimizes visualization and quantification of cervical lymph node morphology, volume and blood flow. Image-guided biopsy of cervical lymph nodes and processing of lymph tissue for histological evaluation is also demonstrated.

鼠标颈淋巴结高频超声成像

High-frequency ultrasound (HFUS) is widely employed as a non-invasive method for imaging internal anatomic structures in experimental small animal ...

Quantitation of Intra-peritoneal Ovarian Cancer Metastasis

Epithelial ovarian cancer (EOC) is the leading cause of death from gynecologic malignancy in the United States. Mortality is due to diagnosis of 75% of women with late stage disease, when metastasis is already present. EOC is characterized by diffuse and widely disseminated intra-peritoneal metastasis. Cells shed from the primary tumor anchor in the mesothelium that lines the peritoneal cavity as well as in the omentum, resulting in multi-focal metastasis, often in the presence of peritoneal ascites. Efforts in our laboratory are directed at a more detailed understanding of factors that regulate EOC metastatic success. However, quantifying metastatic tumor burden represents a significant technical challenge due to the large number, small size and broad distribution of lesions throughout the peritoneum. Herein we describe a method for analysis of EOC metastasis using cells labeled with red fluorescent protein (RFP) coupled with in vivo multispectral imaging. Following intra-peritoneal injection of RFP-labelled tumor cells, mice are imaged weekly until time of sacrifice. At this time, the peritoneal cavity is surgically exposed and organs are imaged in situ. Dissected organs are then placed on a labeled transparent template and imaged ex vivo. Removal of tissue auto-fluorescence during image processing using multispectral unmixing enables accurate quantitation of relative tumor burden. This method has utility in a variety of applications including therapeutic studies to evaluate compounds that may inhibit metastasis and thereby improve overall survival.

Ovarian cancer metastasis is characterized by numerous diffuse intra-peritoneal lesions, such that accurate visual quantitation of tumor burden is challenging. Herein we describe a method for in situ and ex vivo quantitation of metastatic tumor burden using red fluorescent protein (RFP)-labeled tumor cells and optical imaging.

腹膜内卵巢癌转移的定量

Epithelial ovarian cancer (EOC) is the leading cause of death from gynecologic malignancy in the United States.  Mortality is due to diagnosis of 75% of ...

Evaluation of Lung Metastasis in Mouse Mammary Tumor Models by Quantitative Real-time PCR

Metastatic disease is the spread of malignant tumor cells from the primary cancer site to a distant organ and is the primary cause of cancer associated death 1. Common sites of metastatic spread include lung, lymph node, brain, and bone 2. Mechanisms that drive metastasis are intense areas of cancer research. Consequently, effective assays to measure metastatic burden in distant sites of metastasis are instrumental for cancer research. Evaluation of lung metastases in mammary tumor models is generally performed by gross qualitative observation of lung tissue following dissection. Quantitative methods of evaluating metastasis are currently limited to ex vivo and in vivo imaging based techniques that require user defined parameters. Many of these techniques are at the whole organism level rather than the cellular level 3-6. Although newer imaging methods utilizing multi-photon microscopy are able to evaluate metastasis at the cellular level 7, these highly elegant procedures are more suited to evaluating mechanisms of dissemination rather than quantitative assessment of metastatic burden. Here, a simple in vitro method to quantitatively assess metastasis is presented. Using quantitative Real-time PCR (QRT-PCR), tumor cell specific mRNA can be detected within the mouse lung tissue.

This protocol describes how to use quantitative Real-time PCR (QRT-PCR) to detect tumor cell specific mRNA representing metastasis within the mouse lung tissue.

评价肺转移的小鼠乳腺肿瘤模型定量实时荧光定量PCR

Metastatic disease is the spread of malignant tumor cells from the primary cancer site to a distant organ and is the primary cause of cancer associated ...

Ex vivo Live Imaging of Lung Metastasis and Their Microenvironment

Metastasis is a major cause for cancer-related morbidity and mortality. Metastasis is a multistep process and due to its complexity, the exact cellular and molecular processes that govern metastatic dissemination and growth are still elusive. Live imaging allows visualization of the dynamic and spatial interactions of cells and their microenvironment. Solid tumors commonly metastasize to the lungs. However, the anatomical location of the lungs poses a challenge to intravital imaging. This protocol provides a relatively simple and quick method for ex vivo live imaging of the dynamic interactions between tumor cells and their surrounding stroma within lung metastasis. Using this method, the motility of cancer cells as well as interactions between cancer cells and stromal cells in their microenvironment can be visualized in real time for several hours. By using transgenic fluorescent reporter mice, a fluorescent cell line, injectable fluorescently labeled molecules and/or antibodies, multiple components of the lung microenvironment can be visualized, such as blood vessels and immune cells. To image the different cell types, a spinning disk confocal microscope that allows long-term continuous imaging with rapid, four-color image acquisition has been used. Time-lapse movies compiled from images collected over multiple positions and focal planes show interactions between live metastatic and immune cells for at least 4 hr. This technique can be further used to test chemotherapy or targeted therapy. Moreover, this method could be adapted for the study of other lung-related pathologies that may affect the lung microenvironment.