This work was supported by a grant from the Jan Kornelis de Cock foundation.

1，创建模具硅胶用于肿瘤模拟夹杂物<ol><li>收集所希望的形状和尺寸可以作为肿瘤模拟夹杂物， 如珠子或弹子模型的固体物品。 </li><li>彻底清洁肿瘤模型。以确保容易地移除从硅酮模具中，肿瘤模型可喷施抗粘附喷涂或覆盖有一层薄薄的凡士林或蜂蜡的。 </li><li>放置在一个单独的薄壁方（塑料）中的每个模型表面光滑。如果需要的话，注视模型到盒子的底部以保持其位置。用一个框，比肿瘤模型本身，以避免浪费硅过量稍大。 </li><li>倒入所需的有机硅组分A的量在搅拌碗，并在10加有机硅组分B：1的比例（重量）。彻底混合这两种成分。任选地，真空泵可用于从硅氧烷混合物中除去气泡。 </li><li>轻轻婆乌尔在塑料盒中硅的混合物，防止截留气泡。聚硅氧烷混合物应在45分钟进行处理，以获得最佳结果。 </li><li>让切割模具，除去肿瘤模型之前硅氧烷混合物固化为至少6小时。任选地，所述有机硅模具可切割以Z字形图案，以允许它适合背部一起干净。 3天之后，得到的聚硅氧烷的最大强度。 </li></ol> 2创建的Tris-缓冲盐溶液<ol><li>创建Tris缓冲盐水（TBS）加入6.1克（50毫摩尔）的Tris和8.8克（150毫摩尔）氯化钠800毫升去离子水的溶液。 </li><li>加入将1.0g（15毫摩尔）的NaN 3的方框血红蛋白（步骤3.3和4.4）的氧化和抑制细菌生长。注意：NaN 3的是一种严重的毒。这可能是致命的皮肤或吞食接触。该化合物的毒性与可溶性碱金属氰化物和致死剂量的对于成人是约0.7g。始终遵循安全指示由制造商提供。 </li><li>将pH调节到7.4，并把该卷到1000毫升去离子水。 </li></ol> 3，建立荧光包裹<ol><li>添加2克琼脂糖至50ml TBS从步骤2琼脂糖的较高熔点相比明胶（步骤4.2）将防止夹杂物的溶解和当放置在熔化明胶漏出荧光染料。任选地，加入的琼脂糖的量可以被改变为1或3克，得到较软的或可触知的肿瘤的夹杂物，分别。 </li><li>加热使用微波，直到沸点达到琼脂糖​​浆。彻底搅拌直到琼脂糖完全溶解。 </li><li>添加1.1克（17微摩尔）的血红蛋白和5ml脂肪乳20％溶解在50毫升的TBS的琼脂糖混合物在不断搅拌下，以类似的周围乳房假体组织（步骤4）的光学特性。 </li><li>加入20.0毫克（250.8微摩尔的荧光色素吲哚花青绿83.8毫升去离子水）。确保染料完全溶解。 </li><li>吸管5.0毫升从该溶液中，并把它添加到琼脂糖混合物以获得14微米的最终浓度。任选地，可以使用其他荧光染料相比ICG如果需要的话用自己的最佳浓度。 </li><li>轻轻地填充在步骤1与使用注射器（ 图1A）的热琼脂糖混合物中创建的硅氧烷模具。重复这个过程，直到所有的模具都充满。 </li><li>让荧光夹杂物固化在室温约1小时。由覆盖有铝箔的整个模具保护从光的夹杂物。 </li><li>凝固后，轻轻地打开模具和压出的夹杂物（ 图1B）。或者，使用注射器的针尖对夹杂物的表面上应用小水珠融化的琼脂糖混合物。通过重复此过程数次的同一位置上，小督铁道部马刺可以创建模拟浸润性肿瘤。 </li><li>避光和脱水琼脂糖夹杂物通过它们包装在铝箔和它们在潮湿的储存容器储存于4℃。 注：较低或较高的荧光染料浓度比使用已知浓度的优化都将会导致减少的荧光信号强度。看似有悖常理降低信号强度随上述最佳荧光染料浓度的染料浓度是由于这种现象称为淬火。当评估在幻影的荧光染料的最大深度渗透，用最适浓度是必要的。 </li></ol> 4，创建乳房幻影<ol><li>获取一个杯形的模具制造所需的尺寸和体积， 如玻璃或塑料碗的乳房假体。模具应具有平滑的表面，以防止凝胶的形式附着于模具。的模具VOLUM电子500毫升将创建一个具有相当规模的乳房假体。 </li><li>要创建一个乳房假体与500毫升的体积，加入50克明胶250开花至500ml的TBS（步骤2）。加热在不断搅拌下将明胶浆液至50℃。 </li><li>一旦明胶完全溶解后，让明胶混合物逐渐冷却并在35℃的恒定温度下用热水浴维持它。 </li><li>在恒定搅拌下，加入5.5克（85毫摩尔），牛血红蛋白和25ml脂肪乳20％，分别模拟吸收的光子和散射在组织。 </li><li>预冷的杯形模具中在4℃下至少1小时。接着，倒入模具中的凝胶混合物以对应于琼脂糖肿瘤模拟夹杂物（ 图1C）的预定深度的水平。让明胶混合物固化在4℃下进行30分钟至1小时。 </li><li>凝固后，放置一个肿瘤模拟荧光琼脂糖夹杂物的表面上<html>幻影暂时注视列入用小针。最多3肿瘤模拟荧光灯的夹杂物可以在一个单一的乳房假体被引入。足够的空间（至少5厘米）应保持个人肿瘤模拟夹杂物（ 图1D）之间。 </li><li>在剩下的模具量倒入温热的明胶混合的其余部分，允许两层的坚持而不产生折射文物。标记在模具上的荧光肿瘤模拟夹杂物的位置。让幻影凝固在o / n 4℃。 </li><li>一旦固化，取出用于夹杂物临时固定的针头，轻轻将其从模具中（ 图1E）的乳房假体。通过将其包装在铝箔避光和脱水的乳房假体，它在湿润的储存容器储存于4℃。 </li></ol><html>茜1“FO：内容宽度=”5英寸“SRC =”/文件/ ftp_upload / 51776 / 51776fig1highres.jpg“WIDTH =”500“/&gt; 图1的序列的步骤创建一个包含荧光肿瘤模拟夹杂乳房假体的。创建所需的形状和尺寸的模具硅胶后，将模具填充有使用注射器（A）的熔化琼脂糖混合物。不同大小和形状的肿瘤模拟夹杂物产生的在目前的研究（B）。接着，薄薄的一层熔胶的混合物倒入一个定制的涂木乳腺模具（C）。凝固后，将肿瘤模拟夹杂物被定位时，暂时固着，并覆盖着熔化明胶混合物（D）的另一个层。凝固后，乳房假体是轻轻地从模具中（E）中删除。幻像然后可以应用用于模拟各种NIRF成像应用（F）。REF =“/文件/ ftp_upload / 51776 / 51776fig1highres.jpg”目标=“_ blank将”&gt;请点击这里查看该图的放大版本。 5，设置NIRF摄像系统<ol><li>需要术中应用程序中的NIRF摄像系统，模拟针对性NIRF成像在乳腺癌手术。目前可用于研究几个NIRF成像系统进行实时术NIRF成像。虽然这些设备之间的一些差异存在，它们都包含一个激励光源（用于荧光肿瘤夹杂物的激发）和用于检测发射的光子的高度敏感的成像装置。 </li><li>请务必使用足够的波长的激发光源。对于含有ICG肿瘤模拟夹杂物，使用的激励光源（ 如激光），其发射750和800纳米之间的光子。如果一个备选的荧光染料的情况下，激发波长应调整符合的毫安<html>生产商产生的说明。 </li><li>若NIRF相机系统包含一个发射滤波器滤除不需要的背景信号，确保正确的过滤器使用。对于含有ICG肿瘤模拟夹杂物，用800和850纳米之间的发射滤波器。替代性的荧光染料，可能需要不同的发射器，根据不同的制造商的说明进行操作。 注意：请确保有激发和发射波长，以防止过饱和的图像之间的零重叠。此外，该图像捕获时间可能要进行调整，以获得最佳的荧光图像。在深层次的荧光夹杂物或弱荧光信号的情况下，图像采集时间可以增加多达几秒钟到最小。在浅的夹杂物或强的荧光信号的情况下，采集时间可以降低到几毫秒，以允许在实时视频速率荧光成像。 </li></ol>E“&gt; 6。仿真技术在乳腺癌手术NIRF成像中的应用<ol><li>以从容器中组织模拟乳房假体，并将其放置在平坦的非荧光面。接着，放置上述的乳房假体的NIRF成像装置中，留下了足够的工作距离为肿瘤模拟夹杂物的切除。 </li><li>利用本地化NIRF成像和/或幻象乳房触诊肿瘤模拟荧光包容。万一没有荧光信号可被检测，则夹杂物或者定位在假想的检测或图像采集时间应增加太深。 </li><li>一旦列入本地化​​，切开假体丰胸和消除肿瘤，包括模拟实时NIRF指导下，采用传统的手术器械。可选地，包含可切下单独引导通过目视检查和乳房假体的触诊来模拟的护理标准。 </li><li>拆除后直接的肿瘤模拟夹杂物，像术腔的任何剩余的荧光活性指示切除不充分的。 </li><li>在任何情况下，剩余的荧光活动，消费NIRF直接指导下，纳入剩余直到没有荧光信号离开了。 </li><li>形象切除假体片段，模拟NIRF引导宏观边缘状态的评估。至此，切片3幽灵组织 - 5毫米斑和图像的斑块相应。荧光信号到达入手术切缘表示手术切缘阳性的存在。 </li></ol>

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作者什么都没有透露。

我们通过使用乳房形幻影集成肿瘤模拟夹杂模拟NIRF制导BCS的潜在的临床应用。术中肿瘤的定位，NIRF引导下肿瘤切除术，手术的程度的评估，以及手术切缘术后评估中使用自定义构建NIRF摄像系统中的所有可行的。非侵入性的检测荧光肿瘤模拟夹杂物仅是为夹杂物定位在虚线组织在2厘米或更小的深度是可行的。术，但是，在有限的信号的穿透深度在很大程度上解决了由BCS手术的性质，其中，所述外...

保乳手术（BCS），其次是放射治疗是乳腺癌患者的标准治疗为T 1-T 2乳腺癌1,2。不准确的手术结果的阳性手术切缘在20至40％的谁接受BCS，因此需要附加的外科手术或放射治疗3,4,5病人的程度术评估。虽然广泛切除邻近健康乳房组织可能会降低手术切缘阳性的频率，这也将阻碍美容效果，并增加合并症6,7。因此，需要新的技术，提供了对原发肿瘤的位置和手术的程度术的反馈。光学成像，特别是近红外荧光（NIRF）成像，可以通过提供的外科医生用的r中前和术中肿瘤的定位工具减少以下BCS阳性手术切缘的频率EAL时间。最近，我们小组汇报了卵巢癌患者先在人类试验的肿瘤靶向荧光成像，显示出这项技术来检测原发肿瘤及腹腔转移癌具有较高的敏感性8的可行性。在继续到临床研究中的乳腺癌患者，但是，各种肿瘤靶向NIRF成像应用中的BCS的可行性已经可以使用临床前体模进行评价。 下面的研究方案介绍含有荧光肿瘤模拟夹杂9组织模拟乳房假体的使用NIRF成像。幽灵提供了一个廉价和灵活的工具来模拟前和术中肿瘤定位，实时NIRF引导肿瘤切除术，对手术切缘状况进行评估，并检测残留病灶。凝胶状幻影具有类似于人体组织的弹性属性，并且可以使用常规s中被切urgical仪器。在模拟的手术过程中，外科医生通过触觉信息（在可触知的夹杂物的情况下）和手术区域的视觉检查指导。此外，NIRF成像应用，以提供对手术的程度实时术中反馈的外科医生。 但是应当强调的是，NIRF成像需要使用的荧光染料。理想地，荧光染料，应使用能发出在近红外光谱范围内的光子（650 - 900纳米），以尽量减少由分子在组织生理学上丰富的吸收的光子和散射（ 例如 ，血红蛋白，脂质，弹性蛋白，胶原，和水） 10,11。而且，自体荧光（ 即，在由于在活细胞中的生化反应组织中的固有荧光活性）被最小化，在近红外光谱范围内，产生最佳的肿瘤-背景比11。由共轭NIRF染料肿瘤精华素泰德部分（ 例如，单克隆抗体），可用于术中成像应用而获得的靶向递送的荧光染料。 由于人的眼睛是不敏感的，在近红外光谱范围内点亮，高灵敏度的摄像机装置所需的NIRF成像。已经开发了几个NIRF成像系统，术中使用迄今12。在当前的研究中，我们使用一个自定义的构建是在术中的合作与应用慕尼黑工业大学开发NIRF成像系统。该系统允许同时获取彩色图像和荧光图像。以提高荧光图像的精度，校正方案为光强变化的组织实施。详细描述通过Themelis 等人提供的。13

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Bovine hemoglobin</td>
			<td>Sigma-Aldrich, Zwijndrecht, The Netherlands</td>
			<td>H2500</td>
			<td>Simulates absorption of photons in tissue&#160;</td>
		</tr>
		<tr>
			<td>Intralipid 20%</td>
			<td>Sigma-Aldrich, Zwijndrecht, The Netherlands</td>
			<td>I141</td>
			<td>Simulates scattering of photons in tissue</td>
		</tr>
		<tr>
			<td>Silicone A translucent 40 (2-components poly-addition silicone)</td>
			<td>NedForm, Geleen, The Netherlands</td>
			<td>N/A</td>
			<td>Package consists of components A and B, that should be mixed one on one (A:B=10:1).&#160; Link to manufacturers page: http://tinyurl.com/ncjq7jx</td>
		</tr>
		<tr>
			<td>Gelatine 250 Bloom</td>
			<td>Sigma-Aldrich, Zwijndrecht, The Netherlands</td>
			<td>48724</td>
			<td>Construction of breast-shaped phantoms</td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>Hispanagar, Burgos, Spain</td>
			<td>N/A</td>
			<td>Construction of tumor-simulating inclusions</td>
		</tr>
		<tr>
			<td>Tris</td>
			<td>Sigma-Aldrich, Zwijndrecht, The Netherlands</td>
			<td>T1503&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Hcl</td>
			<td>Sigma-Aldrich, Zwijndrecht, The Netherlands</td>
			<td>258148</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma-Aldrich, Zwijndrecht, The Netherlands</td>
			<td>S9888</td>
			<td></td>
		</tr>
		<tr>
			<td>NaH3</td>
			<td>Merck, Darmstadt, Germany</td>
			<td>822335</td>
			<td>CAUTION: severe poison. The toxicity of this compound is comparable to that of soluble alkali cyanides and the lethal dose for an adult human is about 0.7 grams.</td>
		</tr>
		<tr>
			<td colspan="2">Examples of NIRF imaging devices for intraoperative application:</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>T2 NIRF imaging platform&#160;</td>
			<td>SurgVision BV, Heerenveen, The Netherlands</td>
			<td>N/A</td>
			<td>Customized NIRF imaging system used in the current study. More details available at www.surgvision.com</td>
		</tr>
		<tr>
			<td>Photodynamic Eye</td>
			<td>Hamamatsu Photonics Deutschland GmbH, Herrsching am Ammersee, Germany</td>
			<td>PC6100</td>
			<td>www.iht-ltd.com</td>
		</tr>
		<tr>
			<td>FLARE imaging system kit</td>
			<td>The FLARE Foundation Inc, Wayland, MA, USA</td>
			<td>N/A</td>
			<td>www.theflarefoundation.org</td>
		</tr>
		<tr>
			<td>Fluobeam</td>
			<td>Fluoptics, Grenoble, France</td>
			<td>N/A</td>
			<td>www.fluoptics.com</td>
		</tr>
		<tr>
			<td>Artemis handheld camera</td>
			<td>Quest Medical Imaging BV, Middenmeer, the Netherlands</td>
			<td>N/A</td>
			<td>www.quest-mi.com</td>
		</tr>
		<tr>
			<td colspan="2">Examples of NIRF fluorescent dyes for intraoperative application:</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Indocyanine green</td>
			<td>ICG-PULSION,&#160; Feldkirchen, Germany</td>
			<td>PICG0025DE&#160;&#160;</td>
			<td>Clinical grade fluorescent dye for NIRF imaging used in the current study. More details available at www.pulsion.com</td>
		</tr>
		<tr>
			<td>IRDye 800CW NHS Ester</td>
			<td>LI-COR Biosciences, Lincoln, NE, USA</td>
			<td>929-70021</td>
			<td>www.licor.com</td>
		</tr>
	</tbody>
</table>

tissue-simulating phantoms for assessing potential near-infrared fluorescence imaging applications in breast cancer surgery

不准确的术中肿瘤的定位及手术切缘状态结果在保乳手术（BCS）的次优结果的评价。光学成像，特别是近红外荧光（NIRF）成像，可以通过提供的外科医生用在实时前和术中肿瘤的定位工具降低阳性手术切缘的下面BCS的频率。在目前的研究中，NIRF引导BCS的潜在利用组织模拟乳房假体的标准化和培训的原因进行了评价。 乳房假体与媲美正常乳腺组织的光学特性来模拟乳腺癌保乳手术。肿瘤模拟含有荧光染料吲哚菁绿（ICG）的夹杂物被结合在体模在预定的位置和成像前和术中肿瘤的定位，实时NIRF引导肿瘤切除术，NIRF制导评估手术的程度，以及手术切缘术后评估。定制的NIRF相机作为一种临床原型成像的目的。 含肿瘤模拟夹杂物的乳房假体提供了一个简单，价格低廉，用途广泛的工具来模拟并评估术中肿瘤成像。凝胶状幻影具有类似于人体组织的弹性属性，并且可以使用常规的外科手术器械进行切割。此外，该模体含有血红蛋白和脂肪乳用于模仿吸收的光子和散射，分别建立类似于人类乳腺组织均匀的光学特性。 NIRF成像的主要缺点是有限的穿透深度的光子穿过组织，从而阻碍深层肿瘤（非侵袭性）成像反射照明策略传播时。

从本研究结果已经预先别处9报告。 我们的数据表明，NIRF成像可应用于检测荧光肿瘤模拟夹杂物在组织模拟乳房假体，模拟NIRF制导保乳手术的乳腺癌患者。使用我们的幻象模型，我们发现术中肿瘤的定位，NIRF引导肿瘤切除术，手术腔边缘的术评估，并检测残留病是可行的（图2）。简单地说，一共有4乳房假体的制作，都包含两个荧光夹杂有明显的尺寸和/...

Watch this Scientific Journal Video about Tissue-simulating Phantoms for Assessing Potential Near-infrared Fluorescence Imaging Applications in Breast Cancer Surgery at JoVE.com

Tissue-simulating Phantoms for Assessing Potential Near-infrared Fluorescence Imaging Applications in Breast Cancer Surgery

Near-infrared fluorescence (NIRF) imaging may improve therapeutic outcome of breast cancer surgery by enabling intraoperative tumor localization and evaluation of surgical margin status. Using tissue-simulating breast phantoms containing fluorescent tumor-simulating inclusions, potential clinical applications of NIRF imaging in breast cancer patients can be assessed for standardization and training purposes.

组织鬼怪模拟，以评估乳癌手术潜在的近红外荧光成像中的应用

Inaccuracies in intraoperative tumor localization and evaluation of surgical margin status result in suboptimal outcome of breast-conserving surgery ...

tissue-simulating-phantoms-for-assessing-potential-near-infrared

Research

JoVE Journal

Medicine

12.0K 次观看.  University Medical Center Groningen. Near-infrared fluorescence (NIRF) imaging may improve therapeutic outcome of breast cancer surgery by enabling intraoperative tumor localization and evaluation of surgical margin status. Using tissue-simulating breast phantoms containing fluorescent tumor-simulating inclusions, potential clinical applications of NIRF imaging in breast cancer patients can be assessed for standardization and training purposes. 

视频: Tissue-simulating Phantoms for Assessing Potential Near-infrared Fluorescence Imaging Applications in Breast Cancer Surgery

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

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

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

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

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

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

Profiling of Estrogen-regulated MicroRNAs in Breast Cancer Cells

Estrogen plays vital roles in mammary gland development and breast cancer progression. It mediates its function by binding to and activating the estrogen receptors (ERs), ER&#945;, and ER&#946;. ER&#945; is frequently upregulated in breast cancer and drives the proliferation of breast cancer cells. The ERs function as transcription factors and regulate gene expression. Whereas ER&#945;&#39;s regulation of protein-coding genes is well established, its regulation of noncoding microRNA (miRNA) is less explored. miRNAs play a major role in the post-transcriptional regulation of genes, inhibiting their translation or degrading their mRNA. miRNAs can function as oncogenes or tumor suppressors&#160;and are also promising biomarkers. Among the miRNA assays available, microarray and quantitative real-time polymerase chain reaction (qPCR) have been extensively used to detect and quantify miRNA levels. To identify miRNAs regulated by estrogen signaling in breast cancer, their expression in ER&#945;-positive breast cancer cell lines were compared before and after estrogen-activation using both the &#181;Paraflo-microfluidic microarrays and Dual Labeled Probes-low density arrays. Results were validated using specific qPCR assays, applying both Cyanine dye-based and Dual Labeled Probes-based chemistry. Furthermore, a time-point assay was used to identify regulations over time. Advantages of the miRNA assay approach used in this study is that it enables a fast screening of mature miRNA regulations in numerous samples, even with limited sample amounts. The layout, including the specific conditions for cell culture and estrogen treatment, biological and technical replicates, and large-scale screening followed by in-depth confirmations using separate techniques, ensures a robust detection of miRNA regulations,&#160;and eliminates false positives and other artifacts. However, mutated or unknown miRNAs, or regulations at the primary and precursor transcript level, will not be detected. The method presented here represents a thorough investigation of estrogen-mediated miRNA regulation.

Molecular signaling through both estrogen and microRNAs are critical in breast cancer development and growth. Estrogen activates the estrogen receptors, which are transcription factors. Many transcription factors can regulate the expression of microRNAs, and estrogen-regulated microRNAs can be profiled using different large-scale techniques.

在乳腺癌细胞雌激素调节小分子RNA的剖析

Estrogen plays vital roles in mammary gland development and breast cancer progression. It mediates its function by binding to and activating the estrogen ...

Voluntary Breath-hold Technique for Reducing Heart Dose in Left Breast Radiotherapy

Breath-holding techniques reduce the amount of radiation received by cardiac structures during tangential-field left breast radiotherapy. With these techniques, patients hold their breath while radiotherapy is delivered, pushing the heart down and away from the radiotherapy field. Despite clear dosimetric benefits, these techniques are not yet in widespread use. One reason for this is that commercially available solutions require specialist equipment, necessitating not only significant capital investment, but often also incurring ongoing costs such as a need for daily disposable mouthpieces. The voluntary breath-hold technique described here does not require any additional specialist equipment. All breath-holding techniques require a surrogate to monitor breath-hold consistency and whether breath-hold is maintained. Voluntary breath-hold uses the distance moved by the anterior and lateral reference marks (tattoos) away from the treatment room lasers in breath-hold to monitor consistency at CT-planning and treatment setup. Light fields are then used to monitor breath-hold consistency prior to and during radiotherapy delivery.

The current priority in breast cancer radiotherapy is to reduce cardiac doses without compromising target tissue coverage. The voluntary breath-hold technique described here is a simple, inexpensive solution to this problem and capable of being instituted widely without the need for specialized equipment.

自愿屏气技术减少心脏剂量的左乳房放射治疗

Breath-holding techniques reduce the amount of radiation received by cardiac structures during tangential-field left breast radiotherapy. With these ...

In Vivo and Ex Vivo Approaches to Study Ovarian Cancer Metastatic Colonization of Milky Spot Structures in Peritoneal Adipose

High-grade serous ovarian cancer (HGSC), the cause of widespread peritoneal metastases, continues to have an extremely poor prognosis; fewer than 30% of women are alive 5 years after diagnosis. The omentum is a preferred site of HGSC metastasis formation. Despite the clinical importance of this microenvironment, the contribution of omental adipose tissue to ovarian cancer progression remains understudied. Omental adipose is unusual in that it contains structures known as milky spots, which are comprised of B, T, and NK cells, macrophages, and progenitor cells surrounding dense nests of vasculature. Milky spots play a key role in the physiologic functions of the omentum, which are required for peritoneal homeostasis. We have shown that milky spots also promote ovarian cancer metastatic colonization of peritoneal adipose, a key step in the development of peritoneal metastases. Here we describe the approaches we developed to evaluate and quantify milky spots in peritoneal adipose and study their functional contribution to ovarian cancer cell metastatic colonization of omental tissues both in vivo and ex vivo. These approaches are generalizable to additional mouse models and cell lines, thus enabling the study of ovarian cancer metastasis formation from initial localization of cells to milky spot structures to the development of widespread peritoneal metastases.

In Vivo and Ex Vivo Approaches to Study Ovarian Cancer Metastatic Colonization of Milky Spot Structures in Peritoneal Adipose

We outline a protocol that implements both in vivo and ex vivo approaches to study ovarian cancer colonization of peritoneal adipose tissues, particularly the omentum. Furthermore, we present a protocol to quantitate and analyze immune cell-structures in the omentum known as milky spots, which promote metastases of peritoneal adipose.

在体内和体外途径研究银河系结构现货卵巢癌转移定植腹膜脂肪

High-grade serous ovarian cancer (HGSC), the cause of widespread peritoneal metastases, continues to have an extremely poor prognosis; fewer than 30% of ...

Generation of Organ-conditioned Media and Applications for Studying Organ-specific Influences on Breast Cancer Metastatic Behavior

Breast cancer preferentially metastasizes to the lymph node, bone, lung, brain and liver in breast cancer patients. Previous research efforts have focused on identifying factors inherent to breast cancer cells that are responsible for this observed metastatic pattern (termed organ tropism), however much less is known about factors present within specific organs that contribute to this process. This is in part because of a lack of in vitro model systems that accurately recapitulate the organ microenvironment. To address this, an ex vivo model system has been established that allows for the study of soluble factors present within different organ microenvironments. This model consists of generating conditioned media from organs (lymph node, bone, lung, and brain) isolated from normal athymic nude mice. The model system has been validated by demonstrating that different breast cancer cell lines display cell-line specific and organ-specific malignant behavior in response to organ-conditioned media that corresponds to their in vivo metastatic potential. This model system can be used to identify and evaluate specific organ-derived soluble factors that may play a role in the metastatic behavior of breast and other types of cancer cells, including influences on growth, migration, stem-like behavior, and gene expression, as well as the identification of potential new therapeutic targets for cancer. This is the first ex vivo model system that can be used to study organ-specific metastatic behavior in detail and evaluate the role of specific organ-derived soluble factors in driving the process of cancer metastasis.

This manuscript describes an ex vivo model system comprised of organ-conditioned media derived from the lymph node, bone, lung, and brain of mice. This model system can be used to identify and study organ-derived soluble factors and their effects on the organ tropism and metastatic behavior of cancer cells.

器官空调，媒体和应用的产生对乳腺癌转移行为研究器官特异性的影响

Breast cancer preferentially metastasizes to the lymph node, bone, lung, brain and liver in breast cancer patients. Previous research efforts have focused ...

Combined Near-infrared Fluorescent Imaging and Micro-computed Tomography for Directly Visualizing Cerebral Thromboemboli

Direct thrombus imaging visualizes the root cause of thromboembolic infarction. Being able to image thrombus directly allows far better investigation of stroke than relying on indirect measurements, and will be a potent and robust vascular research tool. We use an optical imaging approach that labels thrombi with a molecular imaging thrombus marker&#160;&#8212; a Cy5.5 near-infrared fluorescent (NIRF) probe that is covalently linked to the fibrin strands of the thrombus by the fibrin-crosslinking enzymatic action of activated coagulation factor XIIIa during the process of clot maturation. A micro-computed tomography (microCT)-based approach uses thrombus-seeking gold nanoparticles (AuNPs) functionalized to target the major component of the clot: fibrin. This paper describes a detailed protocol for the combined in vivo microCT and ex vivo NIRF imaging of thromboemboli in a mouse model of embolic stroke. We show that in vivo microCT and fibrin-targeted glycol-chitosan AuNPs (fib-GC-AuNPs) can be used for visualizing both in situ thrombi and cerebral embolic thrombi. We also describe the use of in vivo microCT-based direct thrombus imaging to serially monitor the therapeutic effects of tissue plasminogen activator-mediated thrombolysis. After the last imaging session, we demonstrate by ex vivo NIRF imaging the extent and the distribution of residual thromboemboli in the brain. Finally, we describe quantitative image analyses of microCT and NIRF imaging data. The combined technique of direct thrombus imaging allows two independent methods of thrombus visualization to be compared: the area of thrombus-related fluorescent signal on ex vivo NIRF imaging vs. the volume of hyperdense microCT thrombi in vivo.

This protocol describes the application of combined near-infrared fluorescent (NIRF) imaging and micro-computed tomography (microCT) for visualizing cerebral thromboemboli. This technique allows the quantification of thrombus burden and evolution. The NIRF imaging technique visualizes fluorescently labeled thrombus in excised brain, while the microCT technique visualizes thrombus inside living animals using gold-nanoparticles.

结合近红外荧光成像和微计算机断层扫描直接可视化脑血栓

Direct thrombus imaging visualizes the root cause of thromboembolic infarction. Being able to image thrombus directly allows far better investigation of ...

Studying the Role of Alveolar Macrophages in Breast Cancer Metastasis

This paper describes the application of the syngeneic model of breast cancer (4T1) to the studies on a role of pulmonary alveolar macrophages in cancer metastasis. The 4T1 cells expressing GFP in combination with imaging and confocal microscopy are used to monitor tumor growth, track metastasizing tumor cells, and quantify the metastatic burden. These approaches are supplemented by digital histopathology that allows the automated and unbiased quantification of metastases. In this method the routinely prepared histological lung sections, which are stained with hematoxylin and eosin, are scanned and converted to the digital slides that are then analyzed by the self-trained pattern recognition software. In addition, we describe the flow cytometry approaches with the use of multiple cell surface markers to identify alveolar macrophages in the lungs. To determine impact of alveolar macrophages on metastases and antitumor immunity these cells are depleted with the clodronate-containing liposomes administrated intranasally to tumor-bearing mice. This approach leads to the specific and efficient depletion of this cell population as confirmed by flow cytometry. Tumor volumes and lung metastases are evaluated in mice depleted of alveolar macrophages, to determine the role of these cells in the metastatic progression of breast cancer.

Here we describe the model and approach to study functions of pulmonary alveolar macrophages in cancer metastasis. To demonstrate the role of these cells in metastasis, the syngeneic (4T1) model of breast cancer in conjunction with the depletion of alveolar macrophage with clodronate liposomes was used.

研究肺泡巨噬细胞的作用乳腺癌转移

This paper describes the application of the syngeneic model of breast cancer (4T1) to the studies on a role of pulmonary alveolar macrophages in cancer ...

A Quantitative Sensory Testing Paradigm to Obtain Measures of Pain Processing in Patients Undergoing Breast Cancer Surgery

Chronic pain following surgery, persistent postsurgical pain, is an important highly prevalent condition contributing to significant symptom burden and lower quality of life. Persistent postsurgical pain is relatively refractory to treatment hence generating a high need for preventive strategies and treatments. Therefore, the identification of patients at risk of developing persistent pain is an area of active ongoing research. Recently it was demonstrated that peri-operative disruptions in central pain processing may be able to predict persistent postsurgical pain at long term follow-up in breast cancer patients. The aim of the current report is to present a short protocol to obtain pain thresholds to different stimuli at multiple sites and a measure of endogenous analgesia in breast cancer patients. We have used this method successfully in a clinical context and detail some representative results from a clinical study.

Persistent postsurgical pain may be related to changes in pain processing. By assessing pain in response to standardized stimuli, changes in pain processing can be elucidated. We present methods to obtain pain thresholds to different stimuli and a measure of endogenous analgesia in patients undergoing breast cancer surgery.

一种定量感官检测方法在乳腺癌手术患者疼痛处理中的研究

Chronic pain following surgery, persistent postsurgical pain, is an important highly prevalent condition contributing to significant symptom burden and ...

Skeletal Muscle Neurovascular Coupling, Oxidative Capacity, and Microvascular Function with 'One Stop Shop' Near-infrared Spectroscopy

Exercise represents a major hemodynamic stress that demands a highly coordinated neurovascular response in order to match oxygen delivery to metabolic demand. Reactive hyperemia (in response to a brief period of tissue ischemia) is an independent predictor of cardiovascular events and provides important insight into vascular health and vasodilatory capacity. Skeletal muscle oxidative capacity is equally important in health and disease, as it determines the energy supply for myocellular processes. Here, we describe a simple, non-invasive approach using near-infrared spectroscopy to assess each of these major clinical endpoints (reactive hyperemia, neurovascular coupling, and muscle oxidative capacity) during a single clinic or laboratory visit. Unlike Doppler ultrasound, magnetic resonance images/spectroscopy, or invasive catheter-based flow measurements or muscle biopsies, our approach is less operator-dependent, low-cost, and completely non-invasive. Representative data from our lab taken together with summary data from previously published literature illustrate the utility of each of these end-points. Once this technique is mastered, application to clinical populations will provide important mechanistic insight into exercise intolerance and cardiovascular dysfunction.

Skeletal Muscle Neurovascular Coupling, Oxidative Capacity, and Microvascular Function with &#039;One Stop Shop&#039; Near-infrared Spectroscopy

Here, we describe a simple, non-invasive approach using near-infrared spectroscopy to assess reactive hyperemia, neurovascular coupling and skeletal muscle oxidative capacity in a single clinic or laboratory visit.

"一站式" 近红外光谱对骨骼肌神经血管的耦合、氧化能力及微血管功能的作用

Exercise represents a major hemodynamic stress that demands a highly coordinated neurovascular response in order to match oxygen delivery to metabolic ...