The authors would like to thank Dr. Javed Mahmood for assistance with culturing MDA-MB-231 cells and implanting the MDA-MB-231 xenografts, Linyu Fan for preparing the CT-liposomes. Shawn Stapleton is grateful for funding from the Natural Sciences and Engineering Research Postgraduate Scholarships Program and the Terry Fox Foundation Strategic Initiative for Excellence in Radiation Research for the 21st Century (EIRR21) at CIHR. This study was supported by grants from the Terry Fox New Frontiers Program (020005) and the Canadian Institutes of Health Research (102569).

所有的体内实验是由大学健康网络机构动物护理和使用委员会批准的方案下进行的。 1.动物模型<ol><li> 5至7×10 6个 MDA-MB-231乳腺癌肿瘤细胞在含10％胎牛血清（FBS）和100倍稀释青霉素-链霉素的一起之间培养。 </li><li>收获细胞时，他们使用的是0.05％胰蛋白酶EDTA溶液80％汇合。 3-5分钟后中和胰蛋白酶EDTA的DMEM的3倍量。取细胞的15微升等分，并使用血球计数。离心细胞向在200×g离心5分钟沉淀，并在HBS以每毫升10×10 6个细胞的浓度重悬。 </li><li>植入皮下（SC）由每个8至12周大的雌性SCID小鼠的后肢注射1到2×10 6个细胞瘤（N = 5）。使用标准的25针摹注射。 </li><li>监视器ŧumor用卡尺增长（体积= 0.5×长×宽2），并开始进行测量，一旦SC肿瘤已经达到了量&gt; 200 立方毫米（约7-9天）。 </li></ol> 2. CT-脂质体的制备与表征<ol><li>脂质体制剂<ol><li>溶解脂质组分（200毫摩尔/ L），用于对CT-脂质体，其中包括1,2-二棕榈酰-sn-甘油基-3-磷酸胆碱（DPPC），胆固醇（CH），和1,2-二硬脂酰-sn-甘油-3- -phosphoethanolamine-N - 聚（乙二醇）2000（DSPE-PEG2000）的无水乙醇在70℃下以55的摩尔比:40:5的DPPC CH:DSPE-PEG2000。 </li><li>通过在70℃下保持热蒸发乙醇，然后加入CT造影剂碘海醇（300毫克/毫升碘）到该溶液中，使得最终的脂质浓度为100mM。 </li><li>保持在70℃4小时频繁涡旋该溶液中。 </li><li>为了获得单层囊，挤出样品5恬通过两个ES，在250 psi的压力层叠200nm的孔尺寸的膜，并再次通过5个循环通过两个层叠80nm的孔径的膜，在400磅使用10ml的脂质挤出机挤出。吸取10毫升脂质体的体积到挤压机在每个挤压周期的开始，并收集到每个挤压周期后的无菌锥形管或玻璃管形瓶中。 </li><li>使用100 kDa的分子量切断对250倍体积过量的0.02毫米的HEPES缓冲的盐水溶液（HBS，pH值7.4）（MWC）透析袋透析16小时除去未包封碘海醇。例如，地点1毫升的透析袋用250毫升HBS的外袋在烧杯内的脂质体溶液。 </li><li>集中根据制造商的说明使用75万nomical MWC商业切向流系统对CT-脂质体。集中到约55毫克ml的最终碘浓度-1。 </li></ol></li><li>脂质体表征<ol><li>通过使用10倍体积的过量的乙醇以释放ioxehol然后稀释用100倍体积的过量的去离子水破裂对CT-脂质体测量包封率（ 即 10微升脂质体的使用的乙醇100微升，然后稀释破裂至10毫升的最终体积）。 </li><li>使用带有检测的UV光谱仪上在245纳米的波长确定碘海醇的浓度。通过采取释放碘海醇剂的比例量剂的量计算出的封装效率制备过程中加入。 </li><li>使用根据制造商的说明，动态光散射粒度分析仪系统测量的流体动力学直径和zeta电位。稀释由200倍（ 即 5微升脂质体在1ml最终体积的）对CT-脂质体溶液在去离子水中，以促进测量。 </li></ol></li></ol> 3.肿瘤微循环和CT-脂质体的CT成像分配注:按照制造商的说明，如果使用不同的软件版本或设备执行体积扫描。 <ol><li>麻醉，用2％异氟烷以医用空气或氧气混合各小鼠和按捏脚趾和观察没有反应确认。适用软膏的眼睛，防止干燥时的麻醉下。用胶带爪子薄塑料板固定在俯卧的动物。 </li><li>将一个自定义的27克导管，连接到PE10管20厘米，到侧尾静脉并在插好几块胶带固定。 </li><li>制备1ml注射器包含对CT-脂质体中的至少200微升。制备1ml注射器用生理盐水使用冲洗导管。最后，制备1ml注射器具有至少150微升游离碘海醇与盐水混合（9:1体积比）。 </li><li>把鼠标放在微CT扫描仪床上俯卧。用激光定位系统以放置致瘤R IN大约每个扫描相同的方向。 </li><li>放置在CT脂质体注射器在注射器泵和导管连接到注射器。设置的每秒10微升泵速率。 </li><li>通过使用CT扫描仪控制台软件进行明暗校准扫描初始化系统。选择每个感兴趣的成像协议的亮暗扫描选项，从下拉菜单中亮暗，然后按下扫描按钮开始校准。 </li><li>执行前的任何造影剂注射的肿瘤的体积解剖显微CT。再看CT扫描仪控制台软件指标，以确保CT扫描仪的安全联锁装置已经被清除。在CT扫描器控制台选择扫描选择80千伏70毫安的管电流的X射线能量，并且随着时间的推移16秒捕获1000图像的预测。按下扫描按钮开始扫描。 </li><li>使用注射泵在400毫克碘kg的浓度注入CT-脂质体的大丸剂-1。设置泵注入大约150微升（假设将25g小鼠）的体积。按下泵注入"开始"按钮。手动冲洗导管与盐水的50微升（两次导管的体积），以确保在注射整个剂量和导管是明确的。 </li><li>等待10分钟的CT-脂质体的注射后，然后执行使用在3.5中描述的相同的方法和设置的第二解剖扫描。 </li><li>使用在3.3中描述的相同的喷射率设定:（1体积比9）通过设置在注射器泵注入100μl的游离碘海醇与盐水混合的体积执行一个DCE-CT扫描。 <ol><li>于CT扫描器控制台选择使用80千伏，90毫安的管能量的x射线能量设定，并捕获416图像投影每一秒用于第一30秒和随后的获取每10秒5分钟动态扫描。捕获5秒DCE-CT数据，然后按下该注射液的启动按钮ñ泵。 </li><li>在DCE-CT扫描后进行解剖体积微型CT扫描。 </li></ol></li><li>捕获解剖CT图像的CT-脂质体的48和72小时后喷射之间，使用相同的立体CT设置在3.5步骤所述。 </li><li>重建使用GPU-重建软件解剖CT和DCE-CT数据。 <ol><li>加载图像进重建软件。选择感兴趣的区域，以通过使用鼠标绘制的ROI在图像重建。设置重建成像的保存位置和文件名，选择输出文件类型为".MAT"。 注:该软件会自动设置重建像素大小为0.153 0.153点¯x0.153点¯xmm 3的对解剖扫描和 0.153 0.153点¯x点¯x0.462毫米3 DCE-CT扫描。点击"开始重建"按钮。 </li></ol></li><li>使用CT-脂质体的预喷射和10分钟后注射扫描来计算血浆体积分数如前所述3。此外，使用预喷射和碘海醇的5分钟注射后扫描如前所述7来计算间隙体积分数。 </li><li>通过导入DCE-CT数据转换为提供给肿瘤体积内找出感兴趣区域（ROI）的区域能力的软件获得时间强度曲线（TICS资本流动）。然后计算在ROI的平均CT增强作为时间的函数。在这个实验中的定制软件的开发是为了确定一个投资回报率和计算TIC。 </li><li>通过使用二室示踪剂动力学模型拟合测定国际信托获得灌注和血管通透性的定量估计。配件可使用的DCE-CT分析软件来执行，并使用等离子体体积分数的先验估计和插页体积分数为在两隔室示踪剂动力学模型的固定参数。使用先前报道的方法来获取血浆的先验估计一个和间质体积分数14。 </li></ol> 4.肿瘤组织间液压力的空间测量<ol><li>为了测量IFP通过50厘米PE20聚乙烯管的地下25脊椎针头连接到压力换能器和对IFP采集系统。冲洗整个系统用硫酸肝素/盐水溶液（1:10）。用消毒后再使用70％的异丙醇针。 </li><li>打开采集系统，并启动IFP采集软件和加载设置文件到系统收​​购毫米汞柱IFP测量校准。点击获取按钮连续收集数据IFP。 </li><li>执行的CT-脂质体的48和72小时后喷射之间IFP测量（这对应于肿瘤的CT-脂质体的峰积累的大致时间），使用在4.8中描述的方法。附加IFP针对CT-IFP机器人。 </li><li>执行校准扫描对准的坐标系对CT-IFP机器人和CT扫描仪。添加基准标记附着到CT-IFP机器人和在四个不同位置的基准标记进行的四立体CT扫描。 <ol><li>启动CT-IFP机器人控制器软件，初始化机器人，并通过输入X，Y，Z目标位置，然后点击"进入"按钮，机器人移动到三个位置。 </li><li>以CT扫描在下面的X，Y，Z坐标:（1）0,0,0; （2）-10,0,0; （3）0,7,0;和（4）0,0,10。选择一个90千伏为10 mA，16使用CT扫描仪软件，然后按"开始"开始扫描秒扫描。在3.10中描述重建扫描。 </li></ol></li><li>启动CT-IFP机器人比对软件。点击"注册数据"区域中加载的"添加"按钮，然后在4.3得到的4个重建登记扫描，然后点击"打开"。 注:基准标记的像素位置将被自动输入到softwa回覆。 <ol><li>点击"计算转换"按钮，然后点击"应用转换"按钮。这产生将被用于转换对CT-IFP机器人坐标系到CT扫描器的坐标系对准数据。校准完成后，贴上动物平台，以CT-IFP机器人。 </li></ol></li><li>麻醉，用2％异氟烷以医用空气或氧气混合各小鼠和按捏脚趾和观察没有反应确认。固定对CT-IFP机器人平台对动物和定位鼠标，使得肿瘤的CT-IFP机器人系统进​​行访问。固定用胶带，使得它不IFP针插入期间移动至肿瘤。 </li><li>执行前，插入针IFP解剖显微CT扫描。重建用3.10描述的步骤的CT数据。 </li><li>加载针预先插入CT数据到对CT-IFP机器人对准软件。调整窗口和水平可视化的肿瘤。点击次在任何图像肿瘤电子边缘，然后在第二个边缘的位置。 注:该软件将计算出的一系列位置的沿在两个位置之间的直线。注意x，y和一系列从列表5-8均匀间隔的位置中的Z坐标。 </li><li>冲洗以插入之前肝素盐溶液针准备IFP系统。 </li><li>进入第一预确定针的位置在X，Y，Z，进入对CT-IFP机器人控制软件和压移动到"走"按钮，将机器人移动到所需的位置。点击"插入针"按钮，插入针头进入组织。 <ol><li>插入针确保IFP针和捏和释放PE20管组织之间良好的流体连通，并指出，IFP测量的增加和返回的IFP采集软件到预先捏值之后。拒绝不回到基线测量。 </li></ol></li><li>获得一个插入针解剖CT扫描，然后点击CT-IFP机器人控制软件的"回退针"按钮，退离组织的针。拒绝任何IFP测量其中的IFP值不针撤出后返回到针预先插入值。这意味着针可能在测量过程中已被堵塞。重复步骤对每个针位4.8〜4.10。 </li><li>通过计算相对于在针的插入后的容积CT扫描确定的肿瘤体积的质量中心针端口的x，y和z位置确定肿瘤体积内的针的位置。 </li><li>返回动物的笼子里所有的测量完成后。不要让动物无人值守，并注意观察他们，直到意识已恢复，他们能够保持胸骨斜卧。 </li></ol>

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The authors have nothing to disclose

本文中所呈现用于基于图像的测量方法能够肿瘤微循环特性，IFP，和CT-脂质积累的空间分布的确定。以前的尝试涉及这些属性都依赖于执行在多个肿瘤携带动物散装测量，因此缺乏灵敏度阐明负责该已普遍被观察到纳米尺寸的药物输送系统15在肿瘤内积聚的异质性的机制。 DCE-CT提供了测量在肿瘤微循环特性的肿瘤内变化的工具，立体CT提供的CT-脂质沉积动力学的准确描绘，与CT-IFP机器人系...

测量纳米颗粒的药物递送系统的肿瘤内积累可以提供，以确定是否细胞毒性药物的适当浓度已在肿瘤内实现的​​一个重要工具。 "图象能够"脂质体系统的发展允许非侵入性和定量的体内检测用成像方式如正电子发射断层扫描（PET）1，光学荧光2，和计算机断层扫描（CT）3的药物递送载体的， 4和磁共振成像（MRI）5。成像已被用于确定药代动力学和脂质体递送系统的生物分布和以显示跨学科和肿瘤内的异质性在纳米颗粒累积6,7的程度。然而，纳米粒子成像本身并不能识别已经给他们可怜的积累和分配贡献了生物屏障。这方面的知识是最重要的，将r更有效的配方憩的发展和战略，以提高肿瘤内积聚8。它已被证实的治疗策略可以应用于调节从而提高纳米颗粒运输9特定生物屏障。此外，纳米颗粒制剂已经开发了专门克服特定生物传输阻挡10。在这两种情况下，生物屏障的测量可以被用来指导使用合适的纳米颗粒的药物递送策略的。 肿瘤微循环和升高IFP被认为是纳米颗粒的肿瘤内蓄积的两个关键因素，如脂质体，在实体瘤9,11。然而，有助于脂质体积累穷人服务的其他障碍包括致密的细胞外基质，防渗血管和实体组织的压力12。这些障碍在一个时空相关方式，与异常血流和升高的组织间隙液压作为驱动纳米颗粒的初始传送和外渗的两个重要因素。如先前所讨论的，建立肿瘤微循环，升高的IFP和脂质体的肿瘤内蓄积之间的关系是当务之急脂质体的成像数据的适当解释。在此定量的方法来衡量实体瘤，肿瘤微循环，IFP升高和积累纳米之间的关系呈现。这是通过使用容积CT成像，肿瘤微循环使用动态对比增强计算机断层成像执行的CT脂质体的造影剂的肿瘤内分布的共定位测量完成，使用图像引导的机器人针定位系统，肿瘤IFP称为对CT-IFP机器人13。

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			<td height="21" style="height:21px;width:632px;">MDA-MB-231 metastatic breast adenocarcinoma tumor cells&#160;</td>
			<td style="width:471px;">ATCC</td>
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			<td height="21" style="height:21px;">Dulbecco&#39;s Modified Eagle Medium (DMEM)&#160;</td>
			<td style="width:471px;">Life Technologies</td>
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			<td height="21" style="height:21px;">Fetal Bovine Serum (FBS)</td>
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			<td height="21" style="height:21px;">HyClone Penicillin-Streptomycin 100x Solution</td>
			<td>GE Healthcare Life Sciences</td>
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			<td height="21" style="height:21px;">Trypsin-EDTA (0.05%), phenol red</td>
			<td>ThermoFisher Scientific</td>
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			<td height="21" style="height:21px;">1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC)</td>
			<td>Avanti Lipids Inc., USA</td>
			<td>850355P</td>
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			<td height="21" style="height:21px;">1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-poly(ethylene glycol) 2000 (DSPE-PEG2000)</td>
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			<td height="21" style="height:21px;">Omnipaque (Iohexol) 300 mg of iodine/mL&#160;</td>
			<td>GE Healthcare, CA</td>
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			<td height="21" style="height:21px;">80 nm pore size Track-Etch polycarbonate membranes</td>
			<td style="width:471px;">Whatman Inc., USA</td>
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			<td height="21" style="height:21px;">200 nm pore size Track-Etch polycarbonate membranes</td>
			<td style="width:471px;">Whatman Inc., USA</td>
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			<td height="21" style="height:21px;">10 mL Lipex Extruder&#160;</td>
			<td>Nothern Lipids Inc, CA</td>
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			<td height="21" style="height:21px;width:632px;">Dialysis Bag Molecular Weight Cut Off (MWCO) of 8 kDa</td>
			<td>Spectrum Labs, USA&#160;</td>
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			<td height="21" style="height:21px;">750,000 Nomical Molecular Weight Cut Off (NMWC) Tangential flow column&#160;</td>
			<td>MidGee ultrafiltration cartridge, GE Healthcare, CA</td>
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			<td height="21" style="height:21px;">Peristaltic pump&#160;</td>
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			<td height="21" style="height:21px;">eXplore Locus Ultra micro-CT system&#160;</td>
			<td>GE Healthcare, CA</td>
			<td style="width:119px;"></td>
			<td style="width:299px;">Manipulated using CT-Console Software</td>
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			<td height="21" style="height:21px;width:632px;">AxRecon GPU-based Reconstruction</td>
			<td>&#160;Acceleware Corp. CA</td>
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			<td height="21" style="height:21px;width:632px;">27G Catheter SURFLO Winged Infusion Set</td>
			<td>Terumo Medical Products, USA</td>
			<td style="width:119px;">SV*27EL</td>
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			<td height="21" style="height:21px;width:632px;">PE20 polyethylyne tubing</td>
			<td>Becton Dickinson, USA</td>
			<td style="width:119px;">427406</td>
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			<td height="21" style="height:21px;">Pen tip 25G &#215; 3.5&#8242;&#8242; Whitacre spinal needle&#160;</td>
			<td>Becton Dickinson, USA</td>
			<td style="width:119px;">405140</td>
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			<td height="21" style="height:21px;">P23XL&#160; pressure transducer&#160;</td>
			<td>Harvard Apparatus, CA</td>
			<td style="width:119px;">P23XL</td>
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			<td height="21" style="height:21px;width:632px;">PowerLab 4/35, Bridge Amp, with LabChart Pro 7.0</td>
			<td>ADInstruments Pty Ltd., USA</td>
			<td style="width:119px;">PL3504, FE221</td>
			<td>IFP acquisition system and acquisition software</td>
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			<td height="42" style="height:42px;width:632px;">CT-Sabre Small Animall Intervention system (CT-IFP Robot)</td>
			<td style="width:471px;">Parallax Innovations, CA</td>
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			<td style="width:299px;">Manipulated using CT-IFP robot Control Software</td>
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			<td style="width:299px;">Custom Matlab software</td>
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			<td height="21" style="height:21px;width:632px;">Matlab 2013b</td>
			<td style="width:471px;">Mathworks, USA</td>
			<td style="width:119px;"></td>
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spatial measurements of perfusion, interstitial fluid pressure and liposomes accumulation in solid tumors

脂质体的异质肿瘤内积累是其功效的关键决定因素。既混沌肿瘤微循环和高架IFP被链接到基于纳米技术的药物递送系统的异质肿瘤内分布，如脂质体。在本研究中，肿瘤微循环，升高的IFP，和纳米颗粒的积累之间的关系，通过体内实验进行了研究。这是通过使用动态对比增强计算机断层扫描（DCE-CT）和使用连接到微CT扫描仪的新型图像引导的机器人针放置系统肿瘤IFP的测量肿瘤微循环的评估来实现的。脂质体的肿瘤内积聚是由纳米颗粒脂质体制剂稳定地包封造影剂碘海醇（CT-脂质体）的基于图像的CT评价来确定。 CT成像允许的空间分布的共定位肿瘤血流动力学，IFP和CT-脂质体蓄积在乳腺癌的个体皮下异种移植物小鼠模型。测量导致发现该灌注和血​​浆体积分数是脂质体的肿瘤内分布的强介体。此外，结果表明，IFP起着通过调节血流介导的脂质体分布的间接作用。

上述协议应产生的CT-脂质体与碘海醇的包封浓度，平均脂质体直径，和55毫克毫升-1ζ电位，分别91.8±0.3 nm和-45.5±2.5毫伏， 图1a包括代表DCE-CT成像结果，产生一个时间序列，显示在碘海醇的肿瘤内蓄积的经时变化的体积数据。选择肿瘤内的投资回报率产生可使用示踪动力学建模方法以获得灌注，血管通透性，血浆体积分数，和间质体积分数（ 图1b...

Watch this Scientific Journal Video about Spatial Measurements of Perfusion, Interstitial Fluid Pressure and Liposomes Accumulation in Solid Tumors at JoVE.com

Spatial Measurements of Perfusion, Interstitial Fluid Pressure and Liposomes Accumulation in Solid Tumors

The heterogeneous intra-tumoral accumulation of liposomes has been linked to an abnormal tumor microenvironment. Herein methods are presented to measure tumor microcirculation by perfusion imaging and elevated interstitial fluid pressure (IFP) using an image-guided robotic system. Measurements are compared to the intra-tumoral accumulation of liposomes, determined using volumetric micro-CT imaging.

灌注的空间测量，实体肿瘤间质流体压力和脂质体积累

The heterogeneous intra-tumoral accumulation of liposomes is a critical determinant of their efficacy. Both the chaotic tumor microcirculation and ...

spatial-measurements-perfusion-interstitial-fluid-pressure-liposomes

Research

JoVE Journal

Medicine

7.6K 次观看.  University of Toronto. The heterogeneous intra-tumoral accumulation of liposomes has been linked to an abnormal tumor microenvironment. Herein methods are presented to measure tumor microcirculation by perfusion imaging and elevated interstitial fluid pressure (IFP) using an image-guided robotic system. Measurements are compared to the intra-tumoral accumulation of liposomes, determined using volumetric micro-CT imaging.

视频: Spatial Measurements of Perfusion, Interstitial Fluid Pressure and Liposomes Accumulation in Solid Tumors

Establishment and Propagation of Human Retinoblastoma Tumors in Immune Deficient Mice

Culturing retinoblastoma tumor cells in defined stem cell media gives rise to primary tumorspheres that can be grown and maintained for only a limited time. These cultured tumorspheres may exhibit markedly different cellular phenotypes when compared to the original tumors. Demonstration that cultured cells have the capability of forming new tumors is important to ensure that cultured cells model the biology of the original tumor. 
Here we present a protocol for propagating human retinoblastoma tumors in vivo using Rag2-/- immune deficient mice. Cultured human retinoblastoma tumorspheres of low passage or cells obtained from freshly harvested human retinoblastoma tumors injected directly into the vitreous cavity of murine eyes form tumors within 2-4 weeks. These tumors can be harvested and either further passaged into murine eyes in vivo or grown as tumorspheres in vitro. Propagation has been successfully carried out for at least three passages thus establishing a continuing source of human retinoblastoma tissue for further experimentation. 
Wesley S. Bond and Lalita Wadhwa are co-first authors.

A method is described to propagate human retinoblastoma tumors in mice. Tumor cells are directly injected into the eyes of immune deficient mice. Secondary tumors have been successfully established using both cells directly harvested from human tumors and cultured tumorspheres.

在免疫缺陷小鼠的建立和传播人类视网膜母细胞瘤肿瘤

Culturing retinoblastoma tumor cells in defined stem cell media gives rise to primary tumorspheres that can be grown and maintained for only a limited ...

科研

医学

Femoral Arterial and Venous Catheterization for Blood Sampling, Drug Administration and Conscious Blood Pressure and Heart Rate Measurements

In multiple fields of study, access to the circulatory system in laboratory studies is necessary. Pharmacological studies in rats using chronically implanted catheters permit a researcher to effectively and humanely administer substances, perform repeated blood sampling and assists in conscious direct measurements of blood pressure and heart rate. Once the catheter is implanted long-term sampling is possible. Patency and catheter life depends on multiple factors including the lock solution used, flushing regimen and catheter material. This video will demonstrate the methodology of femoral artery and venous catheterization of the rat. In addition the video will demonstrate the use of the femoral venous and arterial catheters for blood sampling, drug administration and use of the arterial catheter in taking measurements of blood pressure and heart rate in a conscious freely-moving rat. A tether and harness attached to a swivel system will allow the animal to be housed and have samples taken by the researcher with minimal disruption to the animal. To maintain patency of the catheter, careful daily maintenance of the catheter is required using lock solution (100 U/ml heparinized saline), machine-ground blunt tip syringe needles and the use of syringe filters to minimize potential contamination. With careful aseptic surgical techniques, proper catheter materials and careful catheter maintenance techniques, it is possible to sustain patent catheters and healthy animals for long periods of time (several weeks).

Chronic catheterization of blood vessels in the rat is often required for administration of substances, obtain blood sample over a period of time or for direct conscious blood pressure measurements. Femoral arterial catheterization of the rat and corresponding measurements of blood pressure in the conscious animal will be demonstrated.

股动脉和静脉导管采血，药品监督管理局和意识的血压和心率测量

In multiple fields of study, access to the circulatory system in laboratory studies is necessary. Pharmacological studies in rats using chronically ...

Identification of Sleeping Beauty Transposon Insertions in Solid Tumors using Linker-mediated PCR

Genomic, proteomic, transcriptomic, and epigenomic analyses of human tumors indicate that there are thousands of anomalies within each cancer genome compared to matched normal tissue. Based on these analyses it is evident that there are many undiscovered genetic drivers of cancer1. Unfortunately these drivers are hidden within a much larger number of passenger anomalies in the genome that do not directly contribute to tumor formation. Another aspect of the cancer genome is that there is considerable genetic heterogeneity within similar tumor types. Each tumor can harbor different mutations that provide a selective advantage for tumor formation2. Performing an unbiased forward genetic screen in mice provides the tools to generate tumors and analyze their genetic composition, while reducing the background of passenger mutations. The Sleeping Beauty (SB) transposon system is one such method3. The SB system utilizes mobile vectors (transposons) that can be inserted throughout the genome by the transposase enzyme. Mutations are limited to a specific cell type through the use of a conditional transposase allele that is activated by Cre Recombinase. Many mouse lines exist that express Cre Recombinase in specific tissues. By crossing one of these lines to the conditional transposase allele (e.g. Lox-stop-Lox-SB11), the SB system is activated only in cells that express Cre Recombinase. The Cre Recombinase will excise a stop cassette that blocks expression of the transposase allele, thereby activating transposon mutagenesis within the designated cell type. An SB screen is initiated by breeding three strains of transgenic mice so that the experimental mice carry a conditional transposase allele, a concatamer of transposons, and a tissue-specific Cre Recombinase allele. These mice are allowed to age until tumors form and they become moribund. The mice are then necropsied and genomic DNA is isolated from the tumors. Next, the genomic DNA is subjected to linker-mediated-PCR (LM-PCR) that results in amplification of genomic loci containing an SB transposon. LM-PCR performed on a single tumor will result in hundreds of distinct amplicons representing the hundreds of genomic loci containing transposon insertions in a single tumor4. The transposon insertions in all tumors are analyzed and common insertion sites (CISs) are identified using an appropriate statistical method5. Genes within the CIS are highly likely to be oncogenes or tumor suppressor genes, and are considered candidate cancer genes. The advantages of using the SB system to identify candidate cancer genes are: 1) the transposon can easily be located in the genome because its sequence is known, 2) transposition can be directed to almost any cell type and 3) the transposon is capable of introducing both gain- and loss-of-function mutations6. The following protocol describes how to devise and execute a forward genetic screen using the SB transposon system to identify candidate cancer genes (Figure 1).

Identification of Sleeping Beauty Transposon Insertions in Solid Tumors using Linker-mediated PCR

A method of identifying unknown drivers of carcinogenesis using an unbiased approach is described. The method uses the Sleeping Beauty transposon as a random mutagen directed to specific tissues. Genomic mapping of transposon insertions that drive tumor formation identifies novel oncogenes and tumor suppressor genes

鉴定<em&gt;睡美人</em&gt;转座子插入连接器介导的PCR实体瘤

Genomic, proteomic, transcriptomic, and epigenomic analyses of human tumors indicate that there are thousands of anomalies within each cancer genome ...

In Vivo Optical Imaging of Brain Tumors and Arthritis Using Fluorescent SapC-DOPS Nanovesicles

We describe a multi-angle rotational optical imaging (MAROI) system for in vivo monitoring of physiopathological processes labeled with a fluorescent marker. Mouse models (brain tumor and arthritis) were used to evaluate the usefulness of this method. Saposin C (SapC)-dioleoylphosphatidylserine (DOPS) nanovesicles tagged with CellVue Maroon (CVM) fluorophore were administered intravenously. Animals were then placed in the rotational holder (MARS) of the in vivo imaging system. Images were acquired in 10&#176; steps over 380&#176;. A rectangular&#160;region of interest (ROI) was placed across the full image width at the model disease site. Within the ROI, and for every image, mean fluorescence intensity was computed after background subtraction. In the mouse models studied, the labeled nanovesicles were taken up in both the orthotopic and transgenic brain tumors, and in the arthritic sites (toes and ankles). Curve analysis of the multi angle image ROIs determined the angle with the highest signal. Thus, the optimal angle for imaging each disease site was characterized. The MAROI method applied to imaging of fluorescent compounds is a noninvasive, economical, and precise tool for in vivo quantitative analysis of the disease states in the described mouse models.

In Vivo Optical Imaging of Brain Tumors and Arthritis Using Fluorescent SapC-DOPS Nanovesicles

We describe a multi-angle rotational optical imaging (MAROI) system for in vivo quantitation of a fluorescent marker delivered by saposin C (SapC)-dioleoylphosphatidylserine (DOPS) nanovesicles. Employing mouse models of cancer and arthritis, we demonstrate how the MAROI signal curve analysis can be used for the precise mapping and biological characterization of disease processes.

在脑肿瘤和关节炎的用荧光SAPC-DOPS纳米囊泡体光学成像

We describe a multi-angle rotational optical imaging (MAROI) system for in vivo monitoring of physiopathological processes labeled with a fluorescent ...

Quantitative Mass Spectrometric Profiling of Cancer-cell Proteomes Derived From Liquid and Solid Tumors

In-depth analyses of cancer cell proteomes are needed to elucidate oncogenic pathomechanisms, as well as to identify potential drug targets and diagnostic biomarkers. However, methods for quantitative proteomic characterization of patient-derived tumors and in particular their cellular subpopulations are largely lacking. Here we describe an experimental set-up that allows quantitative analysis of proteomes of cancer cell subpopulations derived from either liquid or solid tumors. This is achieved by combining cellular enrichment strategies with quantitative Super-SILAC-based mass spectrometry followed by bioinformatic data analysis. To enrich specific cellular subsets, liquid tumors are first immunophenotyped by flow cytometry followed by FACS-sorting; for solid tumors, laser-capture microdissection is used to purify specific cellular subpopulations. In a second step, proteins are extracted from the purified cells and subsequently combined with a tumor-specific, SILAC-labeled spike-in standard that enables protein quantification. The resulting protein mixture is subjected to either gel electrophoresis or Filter Aided Sample Preparation (FASP) followed by tryptic digestion. Finally, tryptic peptides are analyzed using a hybrid quadrupole-orbitrap mass spectrometer, and the data obtained are processed with bioinformatic software suites including MaxQuant. By means of the workflow presented here, up to 8,000 proteins can be identified and quantified in patient-derived samples, and the resulting protein expression profiles can be compared among patients to identify diagnostic proteomic signatures or potential drug targets.

In-depth analyses of cancer cell proteomes facilitate identification of novel drug targets and diagnostic biomarkers. We describe an experimental workflow for quantitative analysis of (phospho-)proteomes in cancer cell subpopulations derived from liquid and solid tumors. This is achieved by combining cellular enrichment strategies with quantitative Super-SILAC-based mass spectrometry.

对源自液体和固体肿瘤癌症细胞定量蛋白质组质谱剖析

In-depth analyses of cancer cell proteomes are needed to elucidate oncogenic pathomechanisms, as well as to identify potential drug targets and diagnostic ...

Intrauterine Telemetry to Measure Mouse Contractile Pressure In Vivo

A complex integration of molecular and electrical signals is needed to transform a quiescent uterus into a contractile organ at the end of pregnancy. Despite the discovery of key regulators of uterine contractility, this process is still not fully understood. Transgenic mice provide an ideal model in which to study parturition. Previously, the only method to study uterine contractility in the mouse was ex vivo isometric tension recordings, which are suboptimal for several reasons. The uterus must be removed from its physiological environment, a limited time course of investigation is possible, and the mice must be sacrificed. The recent development of radiometric telemetry has allowed for longitudinal, real-time measurements of in vivo intrauterine pressure in mice. Here, the implantation of an intrauterine telemeter to measure pressure changes in the mouse uterus from mid-pregnancy until delivery is described. By comparing differences in pressures between wild type and transgenic mice, the physiological impact of a gene of interest can be elucidated. This technique should expedite the development of therapeutics used to treat myometrial disorders during pregnancy, including preterm labor.

Intrauterine Telemetry to Measure Mouse Contractile Pressure In Vivo

This manuscript provides a protocol for implanting telemeters in the mouse for the purpose of measuring intrauterine pressures during pregnancy.

宫内遥测来衡量鼠标收缩压力<em&gt;在体内</em

A complex integration of molecular and electrical signals is needed to transform a quiescent uterus into a contractile organ at the end of pregnancy. ...

Ex Situ Normothermic Machine Perfusion of Donor Livers

In contrast to conventional static cold preservation (0-4 &#176;C), ex situ machine perfusion may provide better preservation of donor livers. Continuous perfusion of organs provides the opportunity to improve organ quality and allows ex situ viability assessment of donor livers prior to transplantation. This video article provides a step by step protocol for ex situ normothermic machine perfusion (37 &#176;C) of human donor livers using a device that provides a pressure and temperature controlled pulsatile perfusion of the hepatic artery and continuous perfusion of the portal vein. The perfusion fluid is oxygenated by two hollow fiber membrane oxygenators and the temperature can be regulated between 10 &#176;C and 37 &#176;C. During perfusion, the metabolic activity of the liver as well as the degree of injury can be assessed by biochemical analysis of samples taken from the perfusion fluid. Machine perfusion is a very promising tool to increase the number of livers that are suitable for transplantation.

Ex Situ Normothermic Machine Perfusion of Donor Livers

Here we present a protocol describing oxygenated ex situ machine perfusion of donor liver grafts. This article contains a step by step protocol to procure and prepare the liver graft for machine perfusion, prepare the perfusion fluid, prime the perfusion machine and perform oxygenated normothermic machine perfusion of the liver graft.

供肝易地常温灌注机

In contrast to conventional static cold preservation (0-4 &#176;C), ex situ machine perfusion may provide better preservation of donor livers. Continuous ...

Functional Human Liver Preservation and Recovery by Means of Subnormothermic Machine Perfusion

There is currently a severe shortage of liver grafts available for transplantation. Novel organ preservation techniques are needed to expand the pool of donor livers. Machine perfusion of donor liver grafts is an alternative to traditional cold storage of livers and holds much promise as a modality to expand the donor organ pool. We have recently described the potential benefit of subnormothermic machine perfusion of human livers. Machine perfused livers showed improving function and restoration of tissue ATP levels. Additionally, machine perfusion of liver grafts at subnormothermic temperatures allows for objective assessment of the functionality and suitability of a liver for transplantation. In these ways a great many livers that were previously discarded due to their suboptimal quality can be rescued via the restorative effects of machine perfusion and utilized for transplantation. Here we describe this technique of subnormothermic machine perfusion in detail. Human liver grafts allocated for research are perfused via the hepatic artery and portal vein with an acellular oxygenated perfusate at 21 &#176;C.

We describe a method of ex vivo machine perfusion of human liver grafts at subnormothermic temperature (21 &#176;C).

功能人类肝脏保存和恢复由Subnormothermic机灌注手段

There is currently a severe shortage of liver grafts available for transplantation.  Novel organ preservation techniques are needed to expand the pool of ...

Imaging Features of Systemic Sclerosis-Associated Interstitial Lung Disease

Early diagnosis of systemic sclerosis-related interstitial lung disease (SSc-ILD) is important to enable treatment to be administered with minimal delay. However, diagnosing SSc-ILD is challenging because key symptoms are non-specific. High-resolution computed tomography (HRCT) of the chest is recognized as a sensitive imaging method for diagnosing and assessing SSc-ILD. Exposure of patients to ionizing radiation may be considered as a limitation, although methodological steps may be taken to moderate this. We present practical recommendations for performing HRCT scans and interpreting the results. Key features of SSc-ILD on HRCT include a non-specific interstitial pneumonia (NSIP) pattern with peripheral ground-glass opacities and extensive traction bronchiectasis. Despite similarities between SSc-ILD and idiopathic pulmonary fibrosis (IPF), HRCT can be used to differentiate between these conditions: in SSc-ILD compared with IPF, there is a greater proportion of ground-glass opacity and fibrosis is less coarse. A dilated, air-filled esophagus with diameter &#62;10 mm, suggestive of esophageal dysmotility is commonly seen in SSc-ILD. Pulmonary artery size greater than the adjacent ascending aorta suggests coexistent pulmonary hypertension. Nodules must be monitored due to the increased risk of lung cancer. A large extent of disease on HRCT (&#8805;20%) or a high fibrosis score suggests an increased risk of mortality. HRCT is central to diagnosing SSc-ILD, and serial assessments can be helpful in monitoring disease progression or treatment response.

Here, we present practical recommendations for performing thoracic high-resolution computed tomography for diagnosing and assessing systemic sclerosis-related interstitial lung disease.

全身硬化-相关间交性肺病的成像特征

Early diagnosis of systemic sclerosis-related interstitial lung disease (SSc-ILD) is important to enable treatment to be administered with minimal delay. ...

On-Site Sampling and Extraction of Brain Tumors for Metabolomics and Lipidomics Analysis

Despite the variety of tools available for cancer diagnosis and classification, methods that enable fast and simple characterization of tumors are still in need. In recent years, mass spectrometry has become a method of choice for untargeted profiling of discriminatory compound as potential biomarkers of a disease. Biofluids are generally considered as preferable matrices given their accessibility and easier sample processing while direct tissue profiling provides more selective information about a given cancer. Preparation of tissues for the analysis via traditional methods is much more complex and time-consuming, and, therefore, not suitable for fast on-site analysis. The current work presents a protocol combining sample preparation and extraction of small molecules on-site, immediately after tumor resection. The sampling device, which is of the size of an acupuncture needle, can be inserted directly into the tissue and then transported to the nearby laboratory for instrumental analysis. The results of metabolomics and lipidomics analyses demonstrate the capability of the approach for the establishment of phenotypes of tumors related to the histological origin of the tumor, malignancy, and genetic mutations, as well as for the selection of discriminating compounds or potential biomarkers. The non-destructive nature of the technique permits subsequent performance of routinely used tests e.g., histological tests, on the same samples used for SPME analysis, thus enabling attainment of more comprehensive information to support personalized diagnostics.

The manuscript presents on-site sampling of human brain tumors with solid phase microextraction followed by their biochemical profiling towards the discovery of biomarkers.

代谢组学和利皮多米分析脑肿瘤的现场采样与提取

Despite the variety of tools available for cancer diagnosis and classification, methods that enable fast and simple characterization of tumors are still ...