这项研究是由CCSRI格兰特＃700537和＃84535 JDL和NIH / NCI授予＃CA120711 - 01A1和CA120711 - 01A1对AZ CIHR格兰特支持。所有实验均在依照法规和准则的体制动物保健和使用在加利福尼亚大学圣迭戈分校，动物保健大学的委员会，并使用在加拿大西安大略大学。

1。肿瘤接种到禽流胚胎的CAM <ol><li>准备受精的鸡胚胎壳的说明 8 。 </li><li>胚胎发育的第9天，组装微量进样器使用的是18号到1 mL注射器针头连接。剪下一块聚乙烯管（1 / 32"内径3 / 32"外径，1 / 32"壁厚）5-6英寸和仔细的油管插入针头斜面。大约4-5英寸的油管延长从针尖（图1a）。 </li><li>细胞悬液填充注射器和油管。然后，一个显微注射玻璃针管端插入，并仔细清除任何气泡。 </li><li>清扫范围与照明10,000-100,000癌细胞作为CAM（图1c）内的丸注入日，根据9个胚胎（图1b）。慢慢注入细胞，并仔细确保针在正确的地方为细胞内形成一个可见的CAM丸。凸轮表面上的滴水使用Kimwipe或其他撒施，可清洗的细胞。 </li><li>返回胚胎培养箱在38℃，湿度在60％，并允许肿瘤生长和血管（7天）。 </li></ol> 2。纳米粒子的制备<ol><li>为了准备注射到鸡胚胎CPMV纳米粒子的荧光标记;淡化（合成为8 PBS，pH值7.4，前一分钟的使用和离心机，以消除任何浓度100微克/毫升的旋涡混合的病毒粒子。根据共轭和聚集程度，也可能是有用的集合体。超声荧光标记的CPMVs存货至少1年的稳定存储在4 ° C黑暗环境中检查周期性地把一对夫妇的股票跌落到载玻片荧光检查。透射电子显微镜（TEM）也可以被用来确定颗粒保持完好后，共轭。 </li><li>简言之，一微克的病毒粒子（在最后的2μL卷），可以添加到镀铜电网。接下来，添加等体积1分钟在电子显微镜（飞利浦EM 410）2％醋酸铀。 </li></ol> 3。静脉注射病毒荧光标记的纳米粒子<ol><li> 16日，组装微量进样器，如上面所述。通过到注射器管（&gt; 200μL）绘制所需的病毒纳米粒子的体积。小心地取出任何气泡。显微注射玻璃针管端插入。确保针只要尽可能的锥形（图2a）。如果针头太钝，它不会被皮尔斯能够通过外胚层，将无法穿透血管。如果针头太锋利，针尖穿透组织时就会崩溃。 </li><li>静脉注射注入荷瘤胚胎从远端所需的站点100μL1毫克/毫升荧光右旋糖酐（MW 70000大，Invitrogen公司），可视化（图3d）。成功的CAM静脉插管是显而易见的，清除血液中注射流的路径（图2b）。评估后注射血管渗漏1小时。 </li><li>静脉注射注入胚胎含有肿瘤血管渗漏所需的站点从远端CPMV的Alexa Fluor 647（647 CPMV - AF）或聚乙二醇CPMV的Alexa Fluor 647（CPMV - PEG - 647自动对焦）为1 mg / ml的溶液50μL可视化。 </li><li>影像肿瘤立即和每一个小时后注射使用蔡司主任Z1与横河电机的旋转盘，滨松IMAGEM 9100-12 EM - CCD摄像头直立显微镜安装。 </li></ol> 4。实时活体成像<ol><li>要组装的胚胎成像单元，应用胚胎成像单元的盖子底面周长成像端口周围薄薄的一层真空润滑脂，适合到端口18毫米的玻璃盖玻片。 </li><li>盖玻片盖成像所需的区域位置的胚胎。慢慢放下盖子，直到盖玻片与胚胎接触，然后再拧上盖子的单位举行的地方（图2c）。 </li><li>将水加热至37 ° C到外面的菜含有胚胎的胚胎成像单元，然后放到一个堂堂正正的共聚焦显微镜的阶段与整个单位的内部环境室平衡至37 ° C。 </li><li>位置成像装置，旋转盘共聚焦荧光显微镜（图2E）含有胚胎。成像装置将举行到位的胚胎，并保持固定拍摄图像时的视野，从而为立体ž堆栈和时间推移图像被捕获。我们采集和分析时间推移三维图像，使用Perkin Elmer公司（前身为即兴）Volocity软件包（图3a）。采集的肿瘤高分辨率三维堆叠和周围血管可视化detaile在特定时间点的三维结构分析。收购定期的时间点的三维堆叠地图详细的结构变化，在肿瘤血管。每隔一小时注射后的图像肿瘤。 </li><li>病毒粒子的摄取定量计算平均值的Alexa Fluor（AF）的647在肿瘤或基质（非肿瘤区）的形象，如Volocity定量分析软件（Perkin Elmer公司）在选定区域内的信号。肿瘤基质的比例计算，除以平均AF 647基质的信号平均AF 647在肿瘤的信号。肿瘤/间质比高于1，表示正在采取由肿瘤的纳米颗粒。 </li></ol> 5。代表性的成果: 在这里所描述的例子，我们注入了HT - 29结肠癌细胞内形成一个约1mm大小的丸9天鸡胚胎（图1b）的凸轮。接种后，胚胎培养在培养箱7天，以允许足够的肿瘤生长和血管生成（图1c）。胚胎与低分子右旋糖酐静脉注射，以确认肿瘤血管生成，肿瘤蔡司AxioExaminer Z1直立显微镜（图2d）下的可视化。 （图3a和b）经静脉给药CPMV - 647自动对焦或CPMV PEG -自动对焦647纳米粒子，高清晰度的实时共焦成像（图2E）透露，CPMV和CPMV - PEG纳米颗粒迅速标记整个血管，但CPMV - PEG肿瘤摄取比CPMV高出约3倍，在12小时后（图3a）。纳米粒子的相对肿瘤摄取确定使用图像分析软件（Perkin Elmer公司Volocity）。选择感兴趣区域内部和外部的肿瘤（基质车厢），平均每个荧光强度测定。数据表示为肿瘤/间质比。 <img src="/files/ftp_upload/2808/2808fig1.jpg" alt="图1"/> 图1。肿瘤微量注射禽流胚胎的凸轮。 （一）显微注射器具是从组件组装表示。 （二）在9天的禽流感胚胎准备与肿瘤接种当凸轮已经蔓延到覆盖整个表面。 （c）在第16天，将有成长为肿瘤直径在1厘米（虚线），并准备注入纳米粒子。 <img src="/files/ftp_upload/2808/2808fig2.jpg" alt="图2"/> 图2纳米粒子注射液和活体成像。注射显示显微镜下解剖（一）微量注射器针头尖端的CAM静脉注入（二）微量注射器针头插入静脉（箭头表示）和纳米粒子注射到血流（通过清除看到血）。 （三）成像单元含盖玻片直接与CAM接口禽流胚胎。 （四）纳米粒子注射之前，评估肿瘤血管生成是使用荧光右旋糖酐注射后的活体成像。 （E）成像单元含有胚胎定位，内温度调节的外壳设置一个堂堂正正的共聚焦显微镜阶段至37 ° C <img src="/files/ftp_upload/2808/2808fig3.jpg" alt="图3"/> 图3纳米粒子吸收在人类肿瘤的活体可视化。 7小时后注射（一）CPMV - AF647及（b）CPMV PEG - AF647肿瘤可视化。 D）切除肿瘤禽流胚胎以供后续分析。

<ol>
	<li>Halin, C., Mora, J. R., Sumen, C., von Andrian, U. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+imaging+of+lymphocyte+trafficking.">In vivo imaging of lymphocyte trafficking.</a> Annu Rev Cell Dev Biol. 21, 581-603 (2005).</li><li>Judenhofer, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+PET-MRI:+a+new+approach+for+functional+and+morphological+imaging.">Simultaneous PET-MRI: a new approach for functional and morphological imaging.</a> Nat Med. 14, 459-465 (2008).</li><li>Weissleder, R., Pittet, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+in+the+era+of+molecular+oncology.">Imaging in the era of molecular oncology.</a> Nature. 452, 580-589 (2008).</li><li>Chambers, A. F., Ling, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selection+for+experimental+metastatic+ability+of+heterologous+tumor+cells+in+the+chick+embryo+after+DNA-mediated+transfer.">Selection for experimental metastatic ability of heterologous tumor cells in the chick embryo after DNA-mediated transfer.</a> Cancer Res. 44, 3970-3975 (1984).</li><li>Cretu, A., Fotos, J. S., Little, B. W., Galileo, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+and+rat+glioma+growth,+invasion,+and+vascularization+in+a+novel+chick+embryo+brain+tumor+model.">Human and rat glioma growth, invasion, and vascularization in a novel chick embryo brain tumor model.</a> Clin Exp Metastasis. 22, 225-236 (2005).</li><li>Zijlstra, A., Lewis, J., Degryse, B., Stuhlmann, H., Quigley, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+inhibition+of+tumor+cell+intravasation+and+subsequent+metastasis+via+regulation+of+in+vivo+tumor+cell+motility+by+the+tetraspanin+CD151.">The inhibition of tumor cell intravasation and subsequent metastasis via regulation of in vivo tumor cell motility by the tetraspanin CD151.</a> Cancer cell. 13, 221-234 (2008).</li><li>Lewis, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Viral+nanoparticles+as+tools+for+intravital+vascular+imaging.">Viral nanoparticles as tools for intravital vascular imaging.</a> Nature medicine. 12, 354-360 (2006).</li><li>Leong, H. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravital+imaging+of+embryonic+and+tumor+neovasculature+using+viral+nanoparticles.">Intravital imaging of embryonic and tumor neovasculature using viral nanoparticles.</a> Nature protocols. 5, 1406-1417 (2010).</li><li>Chatterji, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+addresses+on+an+addressable+virus+nanoblock;+uniquely+reactive+Lys+residues+on+cowpea+mosaic+virus.">New addresses on an addressable virus nanoblock; uniquely reactive Lys residues on cowpea mosaic virus.</a> Chemistry &#38; biology. 11, 855-863 (2004).</li><li>Brunel, F. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrazone+ligation+strategy+to+assemble+multifunctional+viral+nanoparticles+for+cell+imaging+and+tumor+targeting.">Hydrazone ligation strategy to assemble multifunctional viral nanoparticles for cell imaging and tumor targeting.</a> Nano letters. 10, 1093-1097 (2010).</li><li>Steinmetz, N. F., Manchester, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PEGylated+viral+nanoparticles+for+biomedicine:+the+impact+of+PEG+chain+length+on+VNP+cell+interactions+in+vitro+and+ex+vivo.">PEGylated viral nanoparticles for biomedicine: the impact of PEG chain length on VNP cell interactions in vitro and ex vivo.</a> Biomacromolecules. 10, 784-792 (2009).</li><li>Steinmetz, N. F., Cho, C. F., Ablack, A., Lewis, J. D., Manchester, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CPMV+nanoparticles+target+surface+vimentin+on+cancer+cells.">CPMV nanoparticles target surface vimentin on cancer cells.</a> Nanomedicine. , (2010).</li></ol>

禽流胚胎绒毛尿囊膜（CAM）是一个有用的模型，以评估血管动力学和人类肿瘤的药代动力学。 CAM的结构和位置，让高品质的图像采集和容纳多种癌裸鼠移植瘤无侵入性外科手术。此外，癌症肿瘤异种移植植入成为7天之内，带血管的绒毛尿囊膜，提供了一种快速，廉价和半高通量的手段来评估纳米粒子在肿瘤组织中的积累。由于植入癌裸鼠移植瘤在无壳鸡胚CAM是一个堂堂正正的epifluorescence或共聚焦显微镜，纳米粒子在肿瘤血管吸收的背景和时间信息的高清晰度光学的，可以很容易地获得。在此模型中的肿瘤异种移植物往往生长横向跨越CAM，造成大的肿瘤，而其余的在深度小于200米。这使得它们特别适合活体成像，因为标准epifluorescence的显微镜可以有效地渗透到整个肿块。相比之下，要么肤浅或原位网站​​中植入肿瘤内鼠标在三维空间中扩散，使得很难准确的本地化深纳米粒子在这些肿瘤的非侵入性技术。我们利用这个模型来评估吸收的量子点，脂质体和铁的氧化物纳米粒子在人类肿瘤异种移植的数量，突出了这种模式的潜力，适合在体内广泛的纳米粒子配方分析。

<table border="1"><tbody><tr><th>试剂名称/设备</th><th>公司</th><th>目录编号</th><th>评论</th></tr><tr><td>受精卵里窝那鸡蛋</td><td>弗雷`S孵化场，圣雅各布</td><td> N / A </td><td></td></tr><tr><td> DREMEL旋转工具</td><td> DREMEL </td><td></td><td>可用于任何模型</td></tr><tr><td> DREMEL截止车轮没有。 36 </td><td> DREMEL </td><td> 409 </td><td></td></tr><tr><td>运动员出雏</td><td>贝里山</td><td> 1550HA </td><td></td></tr><tr><td>运动员孵化器</td><td>贝里山</td><td> 1502EA </td><td></td></tr><tr><td>乙醇</td><td></td><td></td><td> 70％（体积/体积） </td></tr><tr><td>聚苯乙烯权衡船</td><td> VWR </td><td> 12577-01 </td><td></td></tr><tr><td>广场的Petri菜</td><td> Simport，VWR </td><td> 25378-115 </td><td></td></tr><tr><td> Rubbermaid的橡胶有盖的容器</td><td> Guillevin </td><td> RH3 - 228 - 00 - BLU </td><td>双方钻成孔</td></tr><tr><td>立式吸管拉马</td><td>大卫Kopf仪器</td><td>模型720 </td><td>设置:16.3（加热器）和2.3（电磁） </td></tr><tr><td>钠硼硅玻璃毛细管</td><td>萨特仪器</td><td> BF100 - 58 - 10 </td><td> （OD，1.0毫米; ID 0.58毫米; 10​​厘米长） </td></tr><tr><td> 1X贝科改良Eagle培养基（DMEM） </td><td> Invitrogen公司</td><td> 11995073 </td><td></td></tr><tr><td>贝科的</td><td> Invitrogen公司</td><td> 14190250 </td><td> pH值7.4 </td></tr><tr><td>磷酸盐缓冲液（D - PBS）（1X），液体</td><td></td><td></td><td></td></tr><tr><td>胰蛋白酶，0.05％与EDTA 4Na（1X），液体</td><td> Invitrogen公司</td><td> 25300054 </td><td></td></tr><tr><td>胎牛血清</td><td> Invitrogen公司</td><td> 12483-020 </td><td>热灭活</td></tr><tr><td>血球</td><td> Hausser科学，VWR </td><td> 15170-090 </td><td></td></tr><tr><td>离心分离</td><td> Eppendorf公司模式5810R </td><td> 5811 000.010 </td><td></td></tr><tr><td>精细点钳</td><td> VWR </td><td> 25607-856 </td><td></td></tr><tr><td>聚乙烯的R - 3603管</td><td> VWR </td><td> 63009-983 </td><td> 50英尺（1/32-inch内径，3/32-inch外径，壁厚1/32-inch </td></tr><tr><td>注射针头注射18表针</td><td>屋宇署</td><td> 305195 </td><td>盒:100个</td></tr><tr><td>注射1毫升注射器</td><td>屋宇署</td><td> 309602 </td><td>盒:100个</td></tr><tr><td>光纤显微镜照明灯</td><td> Amscope </td><td> HL250 - AY </td><td> 150W </td></tr><tr><td> kimwipes </td><td> VWR </td><td> 10805-905 </td><td></td></tr><tr><td> 五unguiculata种子（加州黑眼5） </td><td> Burpee </td><td> 51771A </td><td></td></tr><tr><td>室内生长灯</td><td>晖，供应园丁</td><td></td><td></td></tr><tr><td>甲基PEO4 - NHS酯</td><td>刺穿</td><td> PI22341 </td><td></td></tr><tr><td> MPEG - NHS 2000兆瓦，聚乙二醇琥珀酰亚胺酯</td><td> NANOCS </td><td> PEG1 - 0002 </td><td></td></tr><tr><td>的Alexa Fluor 647羧酸（琥珀酰亚胺酯） </td><td> Invitrogen公司</td><td> A20006 </td><td></td></tr><tr><td>俄勒冈州绿琥珀酰亚胺酯488 * 6 -异构体* </td><td> Invitrogen公司</td><td> O - 6149 </td><td></td></tr><tr><td>二甲基亚砜（DMSO） </td><td>西格玛</td><td> D8418 </td><td></td></tr><tr><td>磷酸磷酸氢二铵monohydrogen </td><td>西格玛</td><td> 379980 </td><td>磷酸氢二钾（phopshate缓冲区） </td></tr><tr><td>磷酸二氢二氢</td><td></td><td> P5655 </td><td>磷酸二氢钾（phopshate缓冲区） </td></tr><tr><td> Superose 6尺寸排除列</td><td> GE医疗生命科学</td><td> 17-0673-01 </td><td></td></tr><tr><td> ÄKTA资源管理器100色谱仪</td><td> GE Amersham公司法玛西亚</td><td> WS - AKTA100 </td><td></td></tr><tr><td> ÄKTA高流试剂盒</td><td> GE医疗生命科学</td><td> 18-1154-85 </td><td></td></tr><tr><td>蔗糖</td><td>西格玛</td><td> S0389 </td><td></td></tr><tr><td>超速离心机</tD&gt; <td>贝克曼</td><td></td><td></td></tr><tr><td>申银万国28钛转子</td><td>贝克曼</td><td> 342204 </td><td>摆动水桶</td></tr><tr><td> 50.2钛转子</td><td>贝克曼</td><td> 337901 </td><td>固定角度</td></tr><tr><td> Amicon超- 15离心式过滤器单元</td><td> Millipore公司</td><td> UFC910008 </td><td> 100 kDa的切断</td></tr><tr><td>渐变前</td><td> BIORAD </td><td></td><td></td></tr><tr><td>右旋糖酐，荧光，70000兆瓦，阴离子</td><td> Invitrogen公司</td><td> D1823 </td><td></td></tr><tr><td>旋转盘共聚焦荧光显微镜</td><td>法定人数;横河电机 中南大学10，横河电机</td><td> N / A </td><td></td></tr><tr><td> Epifluorescence宽场显微镜</td><td>法定人数;蔡司Axio上考官，蔡司</td><td> N / A </td><td></td></tr><tr><td>滨松IMAGEM 9100-12 EM - CCD摄像头</td><td>法定人数;滨松</td><td> N / A </td><td></td></tr><tr><td>显微镜外壳温度单位</td><td>精密塑料</td><td> N / A </td><td></td></tr><tr><td>真空润滑脂</td><td> VWR </td><td> 59344-055 </td><td></td></tr><tr><td>没有循环盖玻片。 1（18毫米） </td><td> VWR </td><td> 16004-300 </td><td></td></tr><tr><td> Volocity软件</td><td> Perkin Elmer公司</td><td></td><td></td></tr><tr><td>鸡胚外壳</td><td></td><td>自定义捏造</td><td></td></tr><tr><td>精致的剪刀</td><td> VWR </td><td> 25608-203 </td><td></td></tr><tr><td>福尔马林</td><td> Bioshop </td><td> FOR201.500 </td><td>在通风柜的使用</td></tr><tr><td>最佳的切割</td><td>费舍尔;组织TEK </td><td> 1437365 </td><td></td></tr><tr><td>温度（OCT） </td><td></td><td></td><td></td></tr><tr><td>塑胶模具</td><td>费舍尔</td><td> 22-038217 </td><td></td></tr><tr><td> VWR VistaVision HistoBond胶粘剂幻灯片</td><td> VWR </td><td> 16004-406 </td><td></td></tr><tr><td>延长黄金用DAPI </td><td> Invitrogen公司</td><td> P36931 </td><td></td></tr><tr><td>低温恒温器的一次性刀片</td><td>费舍尔</td><td> 12-634-2 </td><td></td></tr><tr><td>低温恒温器</td><td> LEICA CM 3050小号</td><td> 14047033518 </td><td></td></tr></table>

evaluation of nanoparticle uptake in tumors in real time using intravital imaging

如超声，MRI，PET和CT肿瘤显像，目前的技 ​​术无法产生高清晰度的图像，为在微观层面 1,2,3的评估在肿瘤纳米粒子吸收，突出一个合适的移植瘤模型的效用其中，进行详细的摄取分析。在这里，我们使用了高分辨率的活体成像技术来评估纳米粒子吸收在修改过的，无壳鸡胚模型人类肿瘤异种移植。鸡胚模型是特别好，适合在体内的分析，因为它支持的人类肿瘤的生长，是价格相对低廉的，不需要麻醉或手术 4,5这些。肿瘤细胞内形成7天完全吻合血管的移植瘤的植入绒毛尿囊膜（CAM）6 。由此产生的肿瘤，可以保持长达72小时与主机或肿瘤系统的影响不大的非侵入性的实时，高清晰度成像的可视化。与远端肿瘤的大小和管理配方广泛的纳米粒子可以被可视化和量化，因为他们通过血液流动，从漏肿瘤血管中渗出，并会积聚在肿瘤部位。我们在这里描述的豇豆花叶病毒（CPMV）与近红外荧光染料和/或聚合物聚乙二醇（PEG）的7，8，9,10,11装饰所得的纳米粒子的分析。静脉给药后，这些病毒的纳米粒子迅速内由内皮细胞，导致全球标签和两个外血管内的肿瘤7,12。病毒的纳米粒子的聚乙二醇化增加了他们的血浆半衰期，延长其在流通中的时间，并最终增强了通过增强通透性和保留 （EPR）的影响，10,11肿瘤的积累。纳米粒子在肿瘤积累的速度和程度是衡量随着时间的推移，使用图像分析软件。这种技术提供了一种方法来可视化和量化在人类肿瘤的纳米粒子动力学。

Watch this Scientific Journal Video about Evaluation of Nanoparticle Uptake in Tumors in Real Time Using Intravital Imaging at JoVE.com

Evaluation of Nanoparticle Uptake in Tumors in Real Time Using Intravital Imaging

我们提出了一个新颖的方法，量化动态，实时活体成像人类禽流感的胚胎模型裸鼠肿瘤血管中的纳米粒子本地化。

使用实时活体成像在肿瘤纳米粒子吸收的评价

Current technologies for tumor imaging, such as ultrasound, MRI, PET and CT, are unable to yield high-resolution images for the assessment of nanoparticle ...

evaluation-nanoparticle-uptake-tumors-real-time-using-intravital

Nanyang Technological University

Research

JoVE Journal

Medicine

18.2K 次观看.  University of Western Ontario. 我们提出了一个新颖的方法，量化动态，实时活体成像人类禽流感的胚胎模型裸鼠肿瘤血管中的纳米粒子本地化。

视频: Evaluation of Nanoparticle Uptake in Tumors in Real Time Using Intravital Imaging

Multispectral Real-time Fluorescence Imaging for Intraoperative Detection of the Sentinel Lymph Node in Gynecologic Oncology

The prognosis in virtually all solid tumors depends on the presence or absence of lymph node metastases.1-3 Surgical treatment most often combines radical excision of the tumor with a full lymphadenectomy in the drainage area of the tumor. However, removal of lymph nodes is associated with increased morbidity due to infection, wound breakdown and lymphedema.4,5 As an alternative, the sentinel lymph node procedure (SLN) was developed several decades ago to detect the first draining lymph node from the tumor.6 In case of lymphogenic dissemination, the SLN is the first lymph node that is affected (Figure 1). Hence, if the SLN does not contain metastases, downstream lymph nodes will also be free from tumor metastases and need not to be removed. The SLN procedure is part of the treatment for many tumor types, like breast cancer and melanoma, but also for cancer of the vulva and cervix.7 The current standard methodology for SLN-detection is by peritumoral injection of radiocolloid one day prior to surgery, and a colored dye intraoperatively. Disadvantages of the procedure in cervical and vulvar cancer are multiple injections in the genital area, leading to increased psychological distress for the patient, and the use of radioactive colloid.
Multispectral fluorescence imaging is an emerging imaging modality that can be applied intraoperatively without the need for injection of radiocolloid. For intraoperative fluorescence imaging, two components are needed: a fluorescent agent and a quantitative optical system for intraoperative imaging. As a fluorophore we have used indocyanine green (ICG). ICG has been used for many decades to assess cardiac function, cerebral perfusion and liver perfusion.8 It is an inert drug with a safe pharmaco-biological profile. When excited at around 750 nm, it emits light in the near-infrared spectrum around 800 nm. A custom-made multispectral fluorescence imaging camera system was used.9.
The aim of this video article is to demonstrate the detection of the SLN using intraoperative fluorescence imaging in patients with cervical and vulvar cancer. Fluorescence imaging is used in conjunction with the standard procedure, consisting of radiocolloid and a blue dye. In the future, intraoperative fluorescence imaging might replace the current method and is also easily transferable to other indications like breast cancer and melanoma.

Fluorescence imaging is a promising innovative modality for image-guided surgery in surgical oncology. In this video we describe the technical procedure for detection of the sentinel lymph node using fluorescence imaging as showcased in gynecologic oncologicy. A multispectral fluorescence camera system, together with the fluorescent agent indocyanine green, is applied.

多光谱实时荧光成像术在妇科肿瘤前哨淋巴结的检测

The prognosis in virtually all solid tumors depends on the presence or absence of lymph node metastases.1-3 Surgical treatment most often combines radical ...

科研

医学

MicroRNA Detection in Prostate Tumors by Quantitative Real-time PCR (qPCR)

MicroRNAs (miRNAs) are single-stranded, 18&#x2013;24 nucleotide long, non-coding RNA molecules. They are involved in virtually every cellular process including development1, apoptosis2, and cell cycle regulation3. MiRNAs are estimated to regulate the expression of 30% to 90% of human genes4 by binding to their target messenger RNAs (mRNAs)5. Widespread dysregulation of miRNAs has been reported in various diseases and cancer subtypes6. Due to their prevalence and unique structure, these small molecules are likely to be the next generation of biomarkers, therapeutic agents and/or targets.
Methods used to investigate miRNA expression include SYBR green I dye- based as well as Taqman-probe based qPCR. If miRNAs are to be effectively used in the clinical setting, it is imperative that their detection in fresh and/or archived clinical samples be accurate, reproducible, and specific. qPCR has been widely used for validating expression of miRNAs in whole genome analyses such as microarray studies7. The samples used in this protocol were from patients who underwent radical prostatectomy for clinically localized prostate cancer; however other tissues and cell lines can be substituted in. Prostate specimens were snap-frozen in liquid nitrogen after resection. Clinical variables and follow-up information for each patient were collected for subsequent analysis8.
Quantification of miRNA levels in prostate tumor samples. The main steps in qPCR analysis of tumors are: Total RNA extraction, cDNA synthesis, and detection of qPCR products using miRNA-specific primers. Total RNA, which includes mRNA, miRNA, and other small RNAs were extracted from specimens using TRIzol reagent. Qiagen's miScript System was used to synthesize cDNA and perform qPCR (Figure 1). Endogenous miRNAs are not polyadenylated, therefore during the reverse transcription process, a poly(A) polymerase polyadenylates the miRNA. The miRNA is used as a template to synthesize cDNA using oligo-dT and Reverse Transcriptase. A universal tag sequence on the 5' end of oligo-dT primers facilitates the amplification of cDNA in the PCR step. PCR product amplification is detected by the level of fluorescence emitted by SYBR Green, a dye which intercalates into double stranded DNA. Specific miRNA primers, along with a Universal Primer that binds to the universal tag sequence will amplify specific miRNA sequences. 
The miScript Primer Assays are available for over a thousand human-specific miRNAs, and hundreds of murine-specific miRNAs. Relative quantification method was used here to quantify the expression of miRNAs. To correct for variability amongst different samples, expression levels of a target miRNA is normalized to the expression levels of a reference gene. The choice of a gene on which to normalize the expression of targets is critical in relative quantification method of analysis. Examples of reference genes typically used in this capacity are the small RNAs RNU6B, RNU44, and RNU48 as they are considered to be stably expressed across most samples. In this protocol, RNU6B is used as the reference gene.

MicroRNA Detection in Prostate Tumors by Quantitative Real-time PCR qPCR

Quantitative Real Time polymerase chain reaction (qPCR) is a rapid and sensitive method to investigate the expression levels of various microRNA (miRNA) molecules in tumor samples. Using this method expression of hundreds of different miRNA molecules can be amplified, quantified, and analyzed from the same cDNA template.

实时定量PCR（qPCR的microRNA检测在前列腺肿瘤）

MicroRNAs (miRNAs) are single-stranded, 18&#x2013;24 nucleotide long, non-coding RNA molecules. They are involved in virtually every cellular process ...

Real-time Digital Imaging of Leukocyte-endothelial Interaction in Ischemia-reperfusion Injury (IRI) of the Rat Cremaster Muscle

Ischemia-reperfusion injury (IRI) has been implicated in a large array of pathological conditions such as cerebral stroke, myocardial infarction, intestinal ischemia as well as following transplant and cardiovascular surgery.1 Reperfusion of previously ischemic tissue, while essential for the prevention of irreversible tissue injury, elicits excessive inflammation of the affected tissue. Adjacent to the production of reactive oxygen species, activation of the complement system and increased microvascular permeability, the activation of leukocytes is one of the principle actors in the pathological cascade of inflammatory tissue damage during reperfusion.2, 3 Leukocyte activation is a multistep process consisting of rolling, firm adhesion and transmigration and is mediated by a complex interaction between adhesion molecules in response to chemoattractants such as complement factors, chemokines, or platelet-activating factor.4
While leukocyte rolling in postcapillary venules is predominantly mediated by the interaction of selectins5 with their counter ligands, firm adhesion of leukocytes to the endothelium is selectin-controlled via binding to intercellular adhesion molecules (ICAM) and vascular cellular adhesion molecules (VCAM).6, 7 
Gold standard for the in vivo observation of leukocyte-endothelial interaction is the technique of intravital microscopy, first described in 1968.8
Though various models of IRI (ischemia-reperfusion injury) have been described for various organs, 9-12 only few are suitable for direct visualization of leukocyte recruitment in the microvascular bed on a high level of image quality.8 
We here promote the digital intravital epifluorescence microscopy of the postcapillary venule in the cremasteric microcirculation of the rat 13 as a convenient method to qualitatively and quantitatively analyze leukocyte recruitment for IRI-research in striated muscle tissue and provide a detailed manual for accomplishing the technique. We further illustrate common pitfalls and provide useful tips which should enable the reader to truly appreciate, and safely perform the method.
In a step by step protocol we depict how to get started with respiration controlled anesthesia under sufficient monitoring to keep the animal firmly anesthetized for longer periods of time. We then describe the cremasteric preparation as a thin flat sheet for outstanding optical resolution and provide a protocol for leukocyte imaging in IRI that has been well established in our laboratories.

Real-time Digital Imaging of Leukocyte-endothelial Interaction in Ischemia-reperfusion Injury IRI of the Rat Cremaster Muscle

Digital intravital epifluorescence microscopy of postcapillary venules in the cremasteric microcirculation is a convenient method to gain insights into leukocyte-endothelial interaction in vivo in ischemia-reperfusion injury (IRI) of striated muscle tissue. We here provide a detailed protocol to safely perform the technique and discuss its applications and limitations.

实时数字成像大鼠提睾肌缺血再灌注损伤（IRI）的白细胞 - 内皮细胞相互作用

Ischemia-reperfusion injury (IRI) has been implicated in a large array of pathological conditions such as cerebral stroke, myocardial infarction, ...

Movement Retraining using Real-time Feedback of Performance

Any modification of movement - especially movement patterns that have been honed over a number of years - requires re-organization of the neuromuscular patterns responsible for governing the movement performance. This motor learning can be enhanced through a number of methods that are utilized in research and clinical settings alike. In general, verbal feedback of performance in real-time or knowledge of results following movement is commonly used clinically as a preliminary means of instilling motor learning. Depending on patient preference and learning style, visual feedback (e.g. through use of a mirror or different types of video) or proprioceptive guidance utilizing therapist touch, are used to supplement verbal instructions from the therapist. Indeed, a combination of these forms of feedback is commonplace in the clinical setting to facilitate motor learning and optimize outcomes.
Laboratory-based, quantitative motion analysis has been a mainstay in research settings to provide accurate and objective analysis of a variety of movements in healthy and injured populations. While the actual mechanisms of capturing the movements may differ, all current motion analysis systems rely on the ability to track the movement of body segments and joints and to use established equations of motion to quantify key movement patterns. Due to limitations in acquisition and processing speed, analysis and description of the movements has traditionally occurred offline after completion of a given testing session.
This paper will highlight a new supplement to standard motion analysis techniques that relies on the near instantaneous assessment and quantification of movement patterns and the display of specific movement characteristics to the patient during a movement analysis session. As a result, this novel technique can provide a new method of feedback delivery that has advantages over currently used feedback methods.

Retraining abnormal movement patterns following injury or disease is a key component of physical rehabilitation. Recent advances in technology have permitted accurate assessment of movement during a variety of tasks, with near instantaneous quantification of results. This provides new opportunities for modification of faulty movement patterns in real time.

的运动再培训采用实时反馈的性能

Any modification of movement - especially movement patterns that have been honed over a number of years - requires re-organization of the neuromuscular ...

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 ...

Chemotherapy-induced Vascular Toxicity - Real-time In vivo Imaging of Vessel Impairment

Certain classes of chemotherapies may exert acute vascular changes that may progress into long-term conditions that may predispose the patient to an increased risk of vascular morbidity.&#160;Yet, albeit&#160;the mounting clinical evidence, there is a paucity of clear studies of vascular toxicity and therefore the etiology of a heterogeneous group of vascular/cardiovascular disorders remains to be elucidated. Moreover, the mechanism that may underlie vascular toxicity can completely differ from the principles of chemotherapy-induced cardiotoxicity, which is related to direct myocyte injury. We have established a real-time, in vivo molecular imaging platform&#160;to evaluate&#160;the potential&#160;acute&#160;vascular toxicity of anti-cancer therapies.
We have set up a platform of in vivo, high-resolution molecular imaging&#160;in mice,&#160;suitable for visualizing vasculature within confined organs and reference blood vessels within the same individuals whereas each individual serve as its own control. Blood vessel walls were impaired after doxorubicin administration, representing a unique mechanism of vascular toxicity that may be the early event in end-organ injury. Herein, the method of fibered confocal fluorescent microscopy (FCFM) based imaging is described, which provides an innovative mode to understand physiological phenomena at the cellular and sub-cellular levels in animal subjects.

Chemotherapy-induced Vascular Toxicity - Real-time In vivo Imaging of Vessel Impairment

We herein describe the method of fibered confocal fluorescent microscopy (FCFM) based imaging, which provides an innovative mode to understand physiological phenomena at the cellular and sub-cellular levels in animal subjects.

化疗引起的血管毒性 - 实时<em&gt;在体内</em&gt;船舶减值成像

Certain classes of chemotherapies may exert acute vascular changes that may progress into long-term conditions that may predispose the patient to an ...

Real-time Cytotoxicity Assays in Human Whole Blood

A live cell-based whole blood cytotoxicity assay (WCA) that allows access to temporal information of the overall cell cytotoxicity is developed with high-throughput cell positioning technology. The targeted tumor cell populations are first preprogrammed to immobilization into an array format, and labeled with green fluorescent cytosolic dyes. Following the cell array formation, antibody drugs are added in combination with human whole blood. Propidium iodide (PI) is then added to assess cell death. The cell array is analyzed with an automatic imaging system. While cytosolic dye labels the targeted tumor cell populations, PI labels the dead tumor cell populations. Thus, the percentage of target cancer cell killing can be quantified by calculating the number of surviving targeted cells to the number of dead targeted cells. With this method, researchers are able to access time-dependent and dose-dependent cell cytotoxicity information. Remarkably, no hazardous radiochemicals are used. The WCA presented here has been tested with lymphoma, leukemia, and solid tumor cell lines. Therefore, WCA allows researchers to assess drug efficacy in a highly relevant ex&#160;vivo condition.

The whole blood cytotoxicity assay (WCA) is a cytotoxicity assay developed by incorporating high-throughput cell positioning technology with fluorescence microscopy and automated image processing. Here, we describe how lymphoma cells treated with an anti-CD20 antibody can be analyzed real-time in human whole blood to provide quantitative cellular cytotoxicity analysis.

实时的细胞毒性分析在人全血

A live cell-based whole blood cytotoxicity assay (WCA) that allows access to temporal information of the overall cell cytotoxicity is developed with ...

Real-time X-ray Imaging of Lung Fluid Volumes in Neonatal Mouse Lung

At birth, the lung undergoes a profound phenotypic switch from secretion to absorption, which allows for adaptation to breathing independently. Promoting and sustaining this phenotype is critically important in normal alveolar growth and gas exchange throughout life. Several in vitro studies have characterized the role of key regulatory proteins, signaling molecules, and steroid hormones that can influence the rate of lung fluid clearance. However, in vivo examinations must be performed to evaluate whether these regulatory factors play important physiological roles in regulating perinatal lung liquid absorption. As such, the utilization of real time X-ray imaging to determine perinatal lung fluid clearance, or pulmonary edema, represents a technological advancement in the field. Herein, we explain and illustrate an approach to assess the rate of alveolar lung fluid clearance and alveolar flooding in C57BL/6 mice at post natal day 10 using X-ray imaging and analysis. Successful implementation of this protocol requires prior approval from institutional animal care and use committees (IACUC), an in vivo small animal X-ray imaging system, and compatible molecular imaging software.

We present a protocol to assess the rate of alveolar fluid clearance or pulmonary edema in neonatal mouse lung using X-ray imaging technology.

在新生小鼠肺肺流体体积的实时X射线成像

At birth, the lung undergoes a profound phenotypic switch from secretion to absorption, which allows for adaptation to breathing independently. Promoting ...

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 ...

Extended Time-lapse Intravital Imaging of Real-time Multicellular Dynamics in the Tumor Microenvironment

In the tumor microenvironment, host stromal cells interact with tumor cells to promote tumor progression, angiogenesis, tumor cell dissemination and metastasis. Multicellular interactions in the tumor microenvironment can lead to transient events including directional tumor cell motility and vascular permeability. Quantification of tumor vascular permeability has frequently used end-point experiments to measure extravasation of vascular dyes. However, due to the transient nature of multicellular interactions and vascular permeability, the kinetics of these dynamic events cannot be discerned. By labeling cells and vasculature with injectable dyes or fluorescent proteins, high-resolution time-lapse intravital microscopy has allowed the direct, real-time visualization of transient events in the tumor microenvironment. Here we describe a method for using multiphoton microscopy to perform extended intravital imaging in live mice to directly visualize multicellular dynamics in the tumor microenvironment. This method details cellular labeling strategies, the surgical preparation of a mammary skin flap, the administration of injectable dyes or proteins by tail vein catheter and the acquisition of time-lapse images. The time-lapse sequences obtained from this method facilitate the visualization and quantitation of the kinetics of cellular events of motility and vascular permeability in the tumor microenvironment.

This protocol describes the use of multiphoton microscopy to perform extended time-lapse imaging of multicellular interactions in real time, in vivo at single cell resolution.

实时多细胞动力学延长时间推移活体成像在肿瘤微环境

In the tumor microenvironment, host stromal cells interact with tumor cells to promote tumor progression, angiogenesis, tumor cell dissemination and ...