这项工作是由美国心脏协会GRA 09春季博士后奖学金（09POST2250268）和NIH R01 CA097465支持的一部分。我们高度赞赏文力博士和维卡斯Gulani博士MRI方案的测试和设置，和Yvonne帕克女士为她的肿瘤植入援助。

改编自前研究11协议。 1。的Gd-DOTA的共轭CLT1肽<ol><li>使用标准的固相肽合成法，从2 - 氯三苯甲基氯树脂（1.0毫摩尔）的Fmoc-保护的氨基酸合成肽CLT1（CGLIIQKNEC）。 </li><li>加入最后的氨基酸后，环化树脂上的线性肽与铊（III）的三氟乙酸盐（1.09克，2.0毫摩尔，2当量）的DMF（20ml）中，在0℃搅拌2小时。 </li><li>接着，共轭的PEG与赖氨酸的顺序来CLT1肽（1.0毫摩尔）在树脂中的N-末端依次用Fmoc-NH-PEG-COOH（3.0毫摩尔），FMOC-赖氨酸基（Fmoc）-OH（3.0毫摩尔）反应，以及第二批的Fmoc-赖氨酸基（Fmoc）-OH（6毫摩尔）。 </li><li>用哌啶除去Fmoc。添加DOTA-三（叔丁基）（4.58克，8.0毫摩尔，2当量至每氨基基团）与在树脂2小时的赖氨酸树枝状聚合物的四个游离胺进行反应。 </li><li>最后，切割和脱保护产品从树脂用三氟乙酸，水和triisobutylsilane（TIS）（20毫升，95/2.5/2.5）为8小时，在室温的鸡尾酒治疗。通过过滤，然后用三氟乙酸（TFA）洗涤除去树脂。加合并的滤液滴加至冷乙醚（200ml）中，离心，洗涤并用乙醚4倍。在真空下干燥，得到无色固体（1.99克，产率61％）。 </li><li>用0-40％溶剂B（0.1％TFA的乙腈溶液）在溶剂A（0.1％TFA水溶液）梯度洗脱20分钟和40-90％溶剂B在溶剂A为10分钟，使用制备型HPLC纯化粗产物。 </li><li>溶解CLT1-DL-（DOTA）4（250毫克，0.077毫摩尔）在DI水（15毫升），用1M的氢氧化钠将pH调节至6。添加的Gd（醋酸）3·4H 2 O（472毫克，0.462毫摩尔，1.5当量至DOTA单酰胺）中的部分溶液中，同时保持pH在6使用1M的NaOH。在RT搅拌反应溶液48小时。 </li><li>复杂的残余钆（III）的随ethylenediaminetetraacetic乙酸（EDTA）（90毫克，0.31毫摩尔）和纯化粗产物用尺寸排阻柱，得到最终产物CLT1-DL-（钆-DOTA）4（169毫克，57％）。 </li></ol> 2。造影剂的表征<ol><li>测量钆（III）的含量与电感耦合等离子体发射光谱法。 </li><li>获得（MALDI-TOF）的飞行时间的基质辅助激光解吸/电离质谱线性模式下与2,5 - 二羟基苯甲酸（2,5-DHB）作为基质。 </li><li>测量CLT1-DL-（钆-DOTA）4的造影剂和非定标控制剂sCLT1-DL-（的Gd-DOTA）4用不同浓度的在60兆赫（1.5 T）的水溶液中的弛豫时间与relaxometer在37°C。使用反转恢复脉冲序列测量T1 和一个卡尔-赛尔Meiboom -吉尔序列500回声测Ŧ2。计算T 1和T 2弛豫次方从1 / T 1与1 / T 2相对于钆浓度曲线的斜率Ë代理商。 </li></ol> 3。细胞培养动物模型的原位PC3前列腺肿瘤的形成与发展<ol><li>培养PC3前列腺癌细胞与绿色荧光蛋白（GFP）中以2.5×10 4个细胞/微升PBS密度补充有5％胎牛血清和青霉素/链霉素/两性霉素B，收获通过胰蛋白酶消化并重新悬浮RPMI培养基中组成型表达。 </li><li>根据批准的凯斯西储大学实验动物管理和使用委员会的动物协议的无胸腺动物核心设施，凯斯西储大学（CWRU）维持雄性NIH无胸腺裸鼠（4-5周龄）。 </li><li>手术表面喷以2％洗必泰溶液，然后铺上吸水毛巾。在手术过程中无菌手术手套戴。动物手术前，注射的ip圣阿韦坦（250毫克/千克），以anesthetize为小鼠。或者，氯胺酮/赛拉嗪/乙酰丙嗪可用于在50/5/1毫克/千克的浓度。眼用软膏施加到小鼠的麻醉过程中保持足够的水分。 </li><li>通过切割一个小切口，通过皮肤和腹膜沿下部中线以约1厘米的长度用无菌手术刀开始手术。在生存手术无菌手术单上覆盖切口周围部位。 </li><li>轻轻exteriorize和稳定的前列腺背叶，露出前列腺。注入的PC3-GFP细胞的悬浮液在PBS（20微升）与30号针的前列腺。最后伤口关闭切口autoclip 12。 </li><li>对于后处理过程的监测，监测动物每天两次持续1周。疾病的症状包括缺乏饮食，尿道口周围深色尿收集和步态不稳这可能表明感染膀胱或闭塞的主食。 </li><li>经过10天，除去叔他用主食主食卸妆。监测动物每日以评估肿瘤的大小和疼痛如体重减轻，行为的偏差，并且马达的缺陷的任何证据。安乐死这表明肿瘤大小超过肿瘤生长过程中的道德允许的极限疼痛或证据的动物。 </li></ol> 4。荧光成像和组织学的肽的肿瘤结合特异性的确认<ol><li>后肿瘤细胞接种，以便与成像开始之前大概4星期肿瘤生长。在注射前肽探针，获得的GFP荧光图像与活老鼠的荧光成像，以验证肿瘤的存在。 </li><li>四注入德克萨斯红标记的肽对荷瘤小鼠为10 nmol /小鼠的剂量。 </li><li>后来颈椎脱位牺牲小鼠2小时，收集肿瘤和主要器官，并在荧光成像仪马上图像。利用绿色滤光器的GFP（激发：444-490纳米，发射515纳米长通滤波器;采集设置：500-720在10纳米级）和红光滤光片为得克萨斯红（激发：576-621纳米;发射：635纳米长通滤波器;收购设置：630-800在10纳米级）。 10毫秒的曝光时间，GFP和150毫秒德克萨斯红。 </li><li>随即整个组织的荧光测量后，收集肿瘤组织，用福尔马林固定，冰冻切片和成5微米切片。 </li><li>用PBS清洗后，装入与1滴封固剂含有4&#39;，6 - 二脒基-2 - 苯基吲哚（DAPI），图像立即在共聚焦激光扫描显微镜。 </li></ol> 5。磁共振成像（MRI） <ol><li>之后大概4星期接种肿瘤细胞，使肿瘤长到0.3-0.6厘米，直径。使用7 T MRI扫描仪具有体积射频（RF）线圈MRI研究。 </li><li>麻醉小鼠在异氟烷诱导室中的2％异氟醚 - 氧气混合物。 OPHthalmic软膏施加到小鼠的麻醉过程中保持足够的水分。 </li><li>插入30号针入1.0米长的管道充满肝素生理盐水。将自制导管在小鼠尾静脉。 </li><li>将鼠标放到磁铁，并通过鼻锥不断吸入麻醉用1.5％异氟醚氧。 </li><li>放置连接到腹部以监测速率和呼吸的深度监测系统呼吸传感器。通过一个反馈控制系统，吹热风到磁体保持体温在37℃。 </li><li>使用本地化序列，以确定肿瘤的位置（TR / TE = 200/3.7毫秒，FOV =与矢状切面图像开始3.0厘米，切片厚度为2.2毫米，片数= 16，平均= 2，翻转角= 45°，矩阵= 128×128）。 </li><li>使用2D T1加权梯度脂肪抑制序列获得2D轴位图像获得CE-MRI（TR / TE = 151.2/1.9毫秒，FOV = 3.0厘米×3.0厘米，S虱厚度为1.2毫米，片数= 12，平均= 1，翻转角= 80°，矩阵= 128×128）。 </li><li>注射前基线MR图像采集后，开始在0.03毫摩尔GD /千克的剂量用200微升生理盐水冲洗靶向药物或控制剂注入。 </li><li>继续获取CE-MRI图像在不同时间点多达30分钟。 </li></ol> 6。图像处理与分析<ol><li>使用图像处理软件进行图像分析。 </li><li>绘制的利益（投资回报）​​，在整个肿瘤区和肾脏中的二维成像平面和测量平均信号强度。 </li><li>量化CE-MRI数据，测量肿瘤或肾对比度增强（ΔSNR）通过使用下列方程的增加后的对比度信噪比超过预对比度信噪比的计算：ΔSNR=（S 吨/σT） - （S 0 /σ0），其中，S 0和S 吨表示的信号中的量子或或肾前和对比度，σ0，σ吨后是噪声之前和之后的对比从背景气测量的标准偏差。 </li><li>通过计算学生的双尾t检验的P值，假设统计学意义为p &lt;0.05。 </li></ol>

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没有利益冲突的声明。

关键步骤 选择适当的生物标志物和靶向小肽 成功开发小尺寸的靶向造影剂，两个关键点需要考虑。首先，要选择合适的分子生物标志物是大量存在于同一点存在于正常组织中病变组织是重要的。我们选择了与癌症相关的生物标志物，凝结血浆蛋白，符合这一要求。其次，所选择的目标明确的小分子药物应具有的生物标志物高结合亲和力，使足?...

癌症相关的分子靶点有效的成像具有重要的意义，以提高癌症的早期检测和诊断的准确性。磁共振成像（MRI）是一种强有力的临床成像模态的高空间分辨率和无电离辐射1。然而，没有针对性的造影剂可用于临床肿瘤MR分子成像。创新设计有针对性的MRI造影剂的发展将大大推进磁共振肿瘤分子成像的应用。已取得显著致力为表达癌细胞表面上的生物标志物的MR成像靶向造影剂。由于MRI和这些生物标志物的低浓度的敏感性相对较低，这是一个挑战，以产生足够的对比度增强，使用小分子靶向造影剂2,3有效MR分子成像。为了得到足够的提高，各种递送系统SUCH为脂质体，纳米粒，并与顺磁性钆（Ⅲ）螯合物高负载聚合物复合物已准备加大在靶位点4,5造影剂的局部浓度。虽然这些递送系统，也能产生在动物模型显著肿瘤的增强，它们的大尺寸导致缓慢和不完全从体内消除，产生有毒的钆（III）离子，这可能会导致严重的安全问题6的长期积累。最近，一些研究表明，MRI的分子成像的限制可以通过选择合适的分子生物标记物具有高的局部表达的病变，并使用小分子试剂，可以容易地排出体外7,8来克服。这些药物的主要特点是，它们针对大​​量存在于同一点存在于正常组织中的患病组织的分子标记。高浓度造影剂可以绑定到这些目标，造成suffi为有效MR分子成像古对比度增强。因为它们的尺寸比肾小球滤过阈值时，未结合的造影剂可以很容易地从主体具有减少背景噪声排出体外。我们已经选择了一个普遍的癌症相关的生物标志物，凝结的血浆蛋白，其中大量存在于肿瘤间质，并且很少出现在正常组织9。我们合成了含小靶向肽CGLIIQKNEC（CLT1），这表现出较强的特异性结合到PC3前列腺肿瘤模型10，和四的Gd-DOTA单酰胺螯合物有针对性的造影剂。在这里，我们为MR肿瘤分子成像技术来检测肿瘤的小鼠的方法。

<table border="1">
		<tbody>
		 <tr>
			<td></td>
			<td></td>
			<td></td>
			<td>REAGENTS</td>
		 </tr>
		 <tr>
			<td>Fmoc protected amino acids</td>
			<td>EMD Chemicals Inc</td>
			<td></td>
			<td></td>
		 </tr>
		 <tr>
			<td>DOTA-tris(t-Bu)</td>
			<td>TCI America</td>
			<td></td>
			<td></td>
		 </tr>
		 <tr>
			<td>PyBOP, HOBt, HBTU</td>
			<td>Nova Biochem</td>
			<td></td>
			<td></td>
		 </tr>
		 <tr>
			<td>DIPEA, Thallium(III) trifluoroacetate, TIS</td>
			<td>Sigma-Aldrich Corp.</td>
			<td></td>
			<td></td>
		 </tr>
		 <tr>
			<td>Texas Red, succinimidyl ester, single isomer</td>
			<td>Invitrogen</td>
			<td>T20175</td>
			<td></td>
		 </tr>
		 <tr>
			<td></td>
			<td></td>
			<td></td>
			<td>EQUIPMENTS</td>
		 </tr>
		 <tr>
			<td>Agilent 1100 HPLC system</td>
			<td>Agilent</td>
			<td></td>
			<td></td>
		 </tr>
		 <tr>
			<td>ZORBAX 300SB-C18 PrepHT column</td>
			<td>Agilent</td>
			<td></td>
			<td></td>
		 </tr>
		 <tr>
			<td>ICP-OES Optima 3100XL</td>
			<td>Perkin-Elmer</td>
			<td></td>
			<td></td>
		 </tr>
		 <tr>
			<td>MALDI-TOF mass spectrometer</td>
			<td>Bruker</td>
			<td>AutoflexTM Speed</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Maestro FLEX In Vivo Imaging System</td>
			<td>Cambridge Research &amp; Instrumentation, Inc.</td>
			<td></td>
			<td></td>
		 </tr>
		 <tr>
			<td>Biospec 7T MRI scanner</td>
			<td>Bruker</td>
			<td></td>
			<td></td>
		 </tr>
		</tbody>
 </table>

mr molecular imaging of prostate cancer with a small molecular clt1 peptide targeted contrast agent

肿瘤的细胞外基质具有丰盈癌症相关的蛋白质，可以作为生物标记用于癌症的分子成像的。在这项工作中，我们证明有效MR肿瘤分子影像与靶向的Gd-DOTA单酰胺复合物作为特定于肿瘤间质凝结血浆蛋白有针对性的MRI造影剂的小分子肽。我们进行了评价非侵入性的检测前列腺肿瘤与MRI在小鼠原位PC-3前列腺癌模型中的代理的有效性的实验。靶向造影剂是有效的在0.03毫摩尔GD /公斤的低剂量产生显著肿瘤对比度增强。靶向MRI造影剂的肽前途的前列腺肿瘤MR分子成像。

图1示出合成的靶向造影剂CLT1-DL-（钆-DOTA）4和实验的整体方案的。 CLT1-DL-（的Gd-DOTA） 图4示出高得多的弛豫比临床的Gd-DOTA（表1）。在1.5 T，每钆对T 1弛豫CLT1-DL-PBS中（钆-DOTA）4（pH 7.4）中的比的Gd-DOTA 10的高约3倍。大师成像证实了强特异性德克萨斯结合红标记CLT1（CLT1-TR）对肿瘤有小的结合到正常器官和组织，而对非特定加扰的肽（sCLT1-TR）示...

Watch this Scientific Journal Video about MR Molecular Imaging of Prostate Cancer with a Small Molecular CLT1 Peptide Targeted Contrast Agent at JoVE.com

MR Molecular Imaging of Prostate Cancer with a Small Molecular CLT1 Peptide Targeted Contrast Agent

为了证明MR癌分子成像与小肽靶向特定于在小鼠前列腺癌模型的肿瘤间质凝结血浆蛋白的MRI造影剂。

前列腺癌与小分子肽CLT1靶向造影剂的磁共振分子成像

Tumor extracellular  matrix has abundance of cancer related proteins that can be used as biomarkers  for cancer molecular imaging. In this work,  we ...

mr-molecular-imaging-prostate-cancer-with-small-molecular-clt1

Research

JoVE Journal

Medicine

11.1K 次观看.  Case Western Reserve University. 为了证明MR癌分子成像与小肽靶向特定于在小鼠前列腺癌模型的肿瘤间质凝结血浆蛋白的MRI造影剂。 

视频: MR Molecular Imaging of Prostate Cancer with a Small Molecular CLT1 Peptide Targeted Contrast Agent

In vivo Near Infrared Fluorescence (NIRF) Intravascular Molecular Imaging of Inflammatory Plaque, a Multimodal Approach to Imaging of Atherosclerosis

The vascular response to injury is a well-orchestrated inflammatory response triggered by the accumulation of macrophages within the vessel wall leading to an accumulation of lipid-laden intra-luminal plaque, smooth muscle cell proliferation and progressive narrowing of the vessel lumen. The formation of such vulnerable plaques prone to rupture underlies the majority of cases of acute myocardial infarction. The complex molecular and cellular inflammatory cascade is orchestrated by the recruitment of T lymphocytes and macrophages and their paracrine effects on endothelial and smooth muscle cells.1
Molecular imaging in atherosclerosis has evolved into an important clinical and research tool that allows in vivo visualization of inflammation and other biological processes. Several recent examples demonstrate the ability to detect high-risk plaques in patients, and assess the effects of pharmacotherapeutics in atherosclerosis.4 While a number of molecular imaging approaches (in particular MRI and PET) can image biological aspects of large vessels such as the carotid arteries, scant options exist for imaging of coronary arteries.2 The advent of high-resolution optical imaging strategies, in particular near-infrared fluorescence (NIRF), coupled with activatable fluorescent probes, have enhanced sensitivity and led to the development of new intravascular strategies to improve biological imaging of human coronary atherosclerosis.
Near infrared fluorescence (NIRF) molecular imaging utilizes excitation light with a defined band width (650-900 nm) as a source of photons that, when delivered to an optical contrast agent or fluorescent probe, emits fluorescence in the NIR window that can be detected using an appropriate emission filter and a high sensitivity charge-coupled camera. As opposed to visible light, NIR light penetrates deeply into tissue, is markedly less attenuated by endogenous photon absorbers such as hemoglobin, lipid and water, and enables high target-to-background ratios due to reduced autofluorescence in the NIR window. Imaging within the NIR 'window' can substantially improve the potential for in vivo imaging.2,5
Inflammatory cysteine proteases have been well studied using activatable NIRF probes10, and play important roles in atherogenesis. Via degradation of the extracellular matrix, cysteine proteases contribute importantly to the progression and complications of atherosclerosis8. In particular, the cysteine protease, cathepsin B, is highly expressed and colocalizes with macrophages in experimental murine, rabbit, and human atheromata.3,6,7 In addition, cathepsin B activity in plaques can be sensed in vivo utilizing a previously described 1-D intravascular near-infrared fluorescence technology6, in conjunction with an injectable nanosensor agent that consists of a poly-lysine polymer backbone derivatized with multiple NIR fluorochromes (VM110/Prosense750, ex/em 750/780nm, VisEn Medical, Woburn, MA) that results in strong intramolecular quenching at baseline.10 Following targeted enzymatic cleavage by cysteine proteases such as cathepsin B (known to colocalize with plaque macrophages), the fluorochromes separate, resulting in substantial amplification of the NIRF signal. Intravascular detection of NIR fluorescence signal by the utilized novel 2D intravascular NIRF catheter now enables high-resolution, geometrically accurate in vivo detection of cathepsin B activity in inflamed plaque.
In vivo molecular imaging of atherosclerosis using catheter-based 2D NIRF imaging, as opposed to a prior 1-D spectroscopic approach,6 is a novel and promising tool that utilizes augmented protease activity in macrophage-rich plaque to detect vascular inflammation.11,12 The following research protocol describes the use of an intravascular 2-dimensional NIRF catheter to image and characterize plaque structure utilizing key aspects of plaque biology. It is a translatable platform that when integrated with existing clinical imaging technologies including angiography and intravascular ultrasound (IVUS), offers a unique and novel integrated multimodal molecular imaging technique that distinguishes inflammatory atheromata, and allows detection of intravascular NIRF signals in human-sized coronary arteries.

In vivo Near Infrared Fluorescence NIRF Intravascular Molecular Imaging of Inflammatory Plaque, a Multimodal Approach to Imaging of Atherosclerosis

We detail a new near-infrared fluorescence (NIRF) catheter for 2-dimensional intravascular molecular imaging of plaque biology in vivo. The NIRF catheter can visualize key biological processes such as inflammation by reporting on the presence of plaque-avid activatable and targeted NIR fluorochromes. The catheter utilizes clinical engineering and power requirements and is targeted for application in human coronary arteries. The following research study describes a multimodal imaging strategy that utilizes a novel in vivo intravascular NIRF catheter to image and quantify inflammatory plaque in proteolytically active inflamed rabbit atheromata.

体内近红外荧光（NIRF）血管内分子成像炎性斑块，多式联运方针成像动脉粥样硬化

The vascular response to injury is a well-orchestrated inflammatory response triggered by the accumulation of macrophages within the vessel wall leading ...

科研

医学

Contrast Enhanced Vessel Imaging using MicroCT

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

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

增强容器成像MicroCT

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

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

Current technologies for tumor imaging, such as ultrasound, MRI, PET and CT, are unable to yield high-resolution images for the assessment of nanoparticle uptake in tumors at the microscopic level1,2,3, highlighting the utility of a suitable xenograft model in which to perform detailed uptake analyses. Here, we use high-resolution intravital imaging to evaluate nanoparticle uptake in human tumor xenografts in a modified, shell-less chicken embryo model. The chicken embryo model is particularly well-suited for these in vivo analyses because it supports the growth of human tumors, is relatively inexpensive and does not require anesthetization or surgery 4,5. Tumor cells form fully vascularized xenografts within 7 days when implanted into the chorioallantoic membrane (CAM) 6. The resulting tumors are visualized by non-invasive real-time, high-resolution imaging that can be maintained for up to 72 hours with little impact on either the host or tumor systems. Nanoparticles with a wide range of sizes and formulations administered distal to the tumor can be visualized and quantified as they flow through the bloodstream, extravasate from leaky tumor vasculature, and accumulate at the tumor site. We describe here the analysis of nanoparticles derived from Cowpea mosaic virus (CPMV) decorated with near-infrared fluorescent dyes and/or polyethylene glycol polymers (PEG) 7, 8, 9,10,11. Upon intravenous administration, these viral nanoparticles are rapidly internalized by endothelial cells, resulting in global labeling of the vasculature both outside and within the tumor7,12. PEGylation of the viral nanoparticles increases their plasma half-life, extends their time in the circulation, and ultimately enhances their accumulation in tumors via the enhanced permeability and retention (EPR) effect 7, 10,11. The rate and extent of accumulation of nanoparticles in a tumor is measured over time using image analysis software. This technique provides a method to both visualize and quantify nanoparticle dynamics in human tumors.

We present a novel approach to quantify nanoparticle localization in the vasculature of human xenografted tumors using dynamic, real-time intravital imaging in an avian embryo model.

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 ...

Molecular Imaging to Target Transplanted Muscle Progenitor Cells

Duchenne muscular dystrophy (DMD) is a severe genetic neuromuscular disorder that affects 1 in 3,500 boys, and is characterized by progressive muscle degeneration1, 2. In patients, the ability of resident muscle satellite cells (SCs) to regenerate damaged myofibers becomes increasingly inefficient4. Therefore, transplantation of muscle progenitor cells (MPCs)/myoblasts from healthy subjects is a promising therapeutic approach to DMD. A major limitation to the use of stem cell therapy, however, is a lack of reliable imaging technologies for long-term monitoring of implanted cells, and for evaluating its effectiveness. Here, we describe a non-invasive, real-time approach to evaluate the success of myoblast transplantation. This method takes advantage of a unified fusion reporter gene composed of genes (firefly luciferase [fluc], monomeric red fluorescent protein [mrfp] and sr39 thymidine kinase [sr39tk]) whose expression can be imaged with different imaging modalities9, 10. A variety of imaging modalities, including positron emission tomography (PET), single-photon emission computed tomography (SPECT), magnetic resonance imaging (MRI), optical imaging, and high frequency 3D-ultrasound are now available, each with unique advantages and limitations11. Bioluminescence imaging (BLI) studies, for example, have the advantage of being relatively low cost and high-throughput. It is for this reason that, in this study, we make use of the firefly luciferase (fluc) reporter gene sequence contained within the fusion gene and bioluminescence imaging (BLI) for the short-term localization of viable C2C12 myoblasts following implantation into a mouse model of DMD (muscular dystrophy on the X chromosome [mdx] mouse)12-14. Importantly, BLI provides us with a means to examine the kinetics of labeled MPCs post-implantation, and will be useful to track cells repeatedly over time and following migration. Our reporter gene approach further allows us to merge multiple imaging modalities in a single living subject; given the tomographic nature, fine spatial resolution and ability to scale up to larger animals and humans10,11, PET will form the basis of future work that we suggest may facilitate rapid translation of methods developed in cells to preclinical models and to clinical applications.

A non-invasive means to evaluate the success of myoblast transplantation is described. The method takes advantage of a unified fusion reporter gene composed of genes whose expression can be imaged with different imaging modalities. Here, we make use of a fluc reporter gene sequence to target cells via bioluminescence imaging.

目标移植的肌肉祖细胞的分子成像

Duchenne muscular dystrophy (DMD) is a severe genetic neuromuscular disorder that affects 1 in 3,500 boys, and is characterized by progressive muscle ...

In vivo Macrophage Imaging Using MR Targeted Contrast Agent for Longitudinal Evaluation of Septic Arthritis

Macrophages are key-cells in the initiation, the development and the regulation of the inflammatory response to bacterial infection. Macrophages are intensively and increasingly recruited in septic joints from the early phases of infection and the infiltration is supposed to regress once efficient removal of the pathogens is obtained. The ability to identify in vivo macrophage activity in an infected joint can therefore provide two main applications: early detection of acute synovitis and monitoring of therapy.
In vivo noninvasive detection of macrophages can be performed with magnetic resonance imaging using iron nanoparticles such as ultrasmall superparamagnetic iron oxide (USPIO). After intravascular or intraarticular administration, USPIO are specifically phagocytized by activated macrophages, and, due to their magnetic properties, induce signal changes in tissues presenting macrophage infiltration. A quantitative evaluation of the infiltrate is feasible, as the area with signal loss (number of dark pixels) observed on gradient echo MR images after particles injection is correlated with the amount of iron within the tissue and therefore reflects the number of USPIO-loaded cells.
We present here a protocol to perform macrophage imaging using USPIO-enhanced MR imaging in an animal model of septic arthritis, allowing an initial and longitudinal in vivo noninvasive evaluation of macrophages infiltration and an assessment of therapy action.

In vivo Macrophage Imaging Using MR Targeted Contrast Agent for Longitudinal Evaluation of Septic Arthritis

We demonstrate how to perform macrophage MR imaging using ultrasmall superparamagnetic contrast agent (USPIO) in septic arthritis, allowing an initial and longitudinal in vivo non-invasive evaluation of macrophages infiltration and an assessment of therapy efficacy.

在体内的巨噬细胞成像采用MR靶向造影剂化脓性关节炎的纵向评价

Macrophages are key-cells in the initiation, the development and the regulation of the inflammatory response to bacterial infection. Macrophages 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  ...

Isolation of Cancer Stem Cells From Human Prostate Cancer Samples

The cancer stem cell (CSC) model has been considerably revisited over the last two decades. During this time CSCs have been identified and directly isolated from human tissues and serially propagated in immunodeficient mice, typically through antibody labeling of subpopulations of cells and fractionation by flow cytometry. However, the unique clinical features of prostate cancer have considerably limited the study of prostate CSCs from fresh human tumor samples. We recently reported the isolation of prostate CSCs directly from human tissues by virtue of their HLA class I (HLAI)-negative phenotype. Prostate cancer cells are harvested from surgical specimens and mechanically dissociated. A cell suspension is generated and labeled with fluorescently conjugated HLAI and stromal antibodies. Subpopulations of HLAI-negative cells are finally isolated using a flow cytometer. The principal limitation of this protocol is the frequently microscopic and multifocal nature of primary cancer in prostatectomy specimens. Nonetheless, isolated live prostate CSCs are suitable for molecular characterization and functional validation by transplantation in immunodeficient mice.

The isolation of cancer stem cells (CSCs) directly from human tissues is requisite for their biological characterization. This manuscript describes a methodology for the isolation of prostate CSCs from human tissues, while also providing tips on troubleshooting difficult steps.

癌症干细胞从人类前列腺癌样本隔离

The cancer stem cell (CSC) model has been considerably revisited over the last two decades. During this time CSCs have been identified and directly ...

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

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

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

原位胰腺癌小鼠模型的动态对比增强磁共振成像

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

Generation of Prostate Cancer Patient Derived Xenograft Models from Circulating Tumor Cells

Patient derived xenograft (PDX) models are gaining popularity in cancer research and are used for preclinical drug evaluation, biomarker identification, biologic studies, and personalized medicine strategies. Circulating tumor cells (CTC) play a critical role in tumor metastasis and have been isolated from patients with several tumor types. Recently, CTCs have been used to generate PDX experimental models of breast and prostate cancer. This manuscript details the method for the generation of prostate cancer PDX models from CTCs developed by our group. Advantages of this method over conventional PDX models include independence from surgical sample collection and generating experimental models at various disease stages. Density gradient centrifugation followed by red blood cell lysis and flow cytometry depletion of CD45 positive mononuclear cells is used to enrich CTCs from peripheral blood samples collected from patients with metastatic disease. The CTCs are then injected into immunocompromised mice; subsequently generated xenografts can be used for functional studies or harvested for molecular characterization. The primary limitation of this method is the negative selection method used for CTC enrichment. Despite this limitation, the generation of PDX models from CTCs provides a novel experimental model to be applied to prostate cancer research.

This manuscript details a method used to generate prostate cancer patient derived xenografts (PDXs) from circulating tumor cells (CTCs). The generation of PDX models from CTCs provides an alternative experimental model to study prostate cancer; the most commonly diagnosed tumor and a frequent cause of death from cancer in men.

前列腺癌患者产生派生异种移植模型从循环肿瘤细胞

Patient derived xenograft (PDX) models are gaining popularity in cancer research and are used for preclinical drug evaluation, biomarker identification, ...

A Brain Tumor/Organotypic Slice Co-culture System for Studying Tumor Microenvironment and Targeted Drug Therapies

Brain tumors are a major cause of cancer-related morbidity and mortality. Developing new therapeutics for these cancers is difficult, as many of these tumors are not easily grown in standard culture conditions. Neurosphere cultures under serum-free conditions and orthotopic xenografts have expanded the range of tumors that can be maintained. However, many types of brain tumors remain difficult to propagate or study. This is particularly true for pediatric brain tumors such as pilocytic astrocytomas and medulloblastomas. This protocol describes a system that allows primary human brain tumors to be grown in culture. This quantitative assay can be used to investigate the effect of microenvironment on tumor growth, and to test new drug therapies. This protocol describes a system where fluorescently labeled brain tumor cells are grown on an organotypic brain slice from a juvenile mouse. The response of tumor cells to drug treatments can be studied in this assay, by analyzing changes in the number of cells on the slice over time. In addition, this system can address the nature of the microenvironment that normally fosters growth of brain tumors. This brain tumor organotypic slice co-culture assay provides a propitious system for testing new drugs on human tumor cells within a brain microenvironment.

Many types of human brain tumors are localized to specific regions within the brain and are difficult to grow in culture. This protocol addresses the role of tumor microenvironment and investigates new drug treatments by analyzing fluorescent primary brain tumor cells growing in an organotypic mouse brain slice. &#160;

脑肿瘤/器官切片共培养系统研究肿瘤微环境和靶向药物治疗

Brain tumors are a major cause of cancer-related morbidity and mortality. Developing new therapeutics for these cancers is difficult, as many of these ...