我们承认由美国国家癌症研究所（NCI）R01 CA128908和斯坦福大学医学学术研究奖学金的支持。没有其他潜在的利益冲突与本文相关的报道。

1。肿瘤模型的建立<ol><li>文化H460细胞（美国典型培养物保藏中心），在补充有10％牛胎儿血清，1％青霉素/链霉素（Invitrogen公司Life Technologies公司）的RPMI 1640培养基中。应当指出的细胞株，培养基，接种的位置，数目每只小鼠的异种移植物，和其他方面的考虑，选择的都是进行调整以适应一个特定的研究的目标。在这里，我们将只提出一个具体的项目设计，作为一个例子。 </li><li>保持在5％CO 2的潮湿气氛中的细胞系，在37℃和改变到新鲜培养基中每隔一天。 </li><li>当75％汇合的单层细胞的形成，用胰蛋白酶和离解成单细胞悬液，进一步的细胞培养物的细胞，分离的单层。 </li><li>挂起约1×10 6个 H460细胞在磷酸盐缓冲盐水（PBS，Invitrogen公司）和植入皮下裸小鼠（雌性无胸腺裸鼠（ 女/女 ），4 - 6周龄，Charles River实验室，公司）的左侧和右侧的肩膀。 </li><li>允许肿瘤的生长为150 - 200毫米3。 H460肿瘤异种移植，大约需要2周的时间，这种规模的增长。标准卡尺测量进行跟踪肿瘤大小。 </li><li>当肿瘤达到理想的规模，现在已经准备好荷瘤小鼠的治疗和活体成像，通过PET和CLI。 </li></ol> 2。 PET <ol><li>执行的PET研究根据这个时间表或任何变化，它取决于特定的项目（ 图1），12。有许多因素可影响的时间表的设计，包括，但不限于，肿瘤异种移植的细胞系，抗癌药物，和给药方案的选择。在这里，我们将只提出一个具体的成像时间表。 CLI研究是根据进行后立即执行相应的PET的PET的研究，使用CLI的那些相同的时间表。还应该指出的是，在PET研究的目的主要是用于验证的CLI的结果。对于普通用户而言，只希望使用其他投资工具进行放射性标记的探针成像，无PET是必要的。但是，如果一个人的欲望PET验证，它应该强调的是，PET和CLI工具必须位于非常接近的验证是成功的，因为半衰期短，18 F（109.77分）。 </li><li>将小鼠随机分为治疗组和对照组（N≥3）。贝伐单抗的20毫克/公斤0和2天2次注射治疗在治疗组小鼠。第0天被定义由第一注射。请注意，在第-1天的预扫描，应执行通过PET和CLI。 </li><li>荷瘤小鼠的小动物PET是R4的啮齿动物模型扫描仪（西门子医疗解决方案A，公司）。 </li><li>麻醉所有小鼠用2％异氟烷（Aerrane巴克斯特）和3&#39;-脱氧-3&#39;-（18）F-氟（18 F-FLT; 7.3 - 8.0注射活度[198 - 215微居里]）通过尾静脉。的PET探针是在注射前，将在PBS中稀释。 </li><li> 1小时后，再次麻醉小鼠麻醉小鼠放置俯卧和领域的中心附近的小动物PET扫描仪的图。 </li><li>获得三分钟的静态扫描和重建图像的2维有序子集期望最大算法。背景校正是没有必要的。 </li><li>在肿瘤的衰减校正全身冠状面图像绘制地区的利益（投资回报率;冠状位和横断切片5个像素）。获取每分钟从感兴趣区的每个像素的最大计数和转换通过使用每毫升每分钟计数的校准常数。组织密度为1克/毫升的假设，转换投资回报率进行计数每克每分钟。确定图像ROI衍生％ID / g值除以每克注射剂量每分钟的计数。衰减校正是没有必要的。 </li></ol> 3。 CLI <ol><li> CLI是要执行的IVIS Spectrum系统（卡尺生命科学版）。利用活体成像软件3.0（卡尺生命科学版）进行图像采集与分析。使用18设定的窄带发射过滤器（490 - 850纳米）的波长分辨光谱成像是要执行。同样，为每个鼠标，执行CLI后立即PET放射性衰变的量最小化，如果包含在协议中的PET的研究。 </li><li>异氟醚麻醉下，将动物在一个不透光的室。多小鼠可以同时放置，以提高吞吐量。 </li><li>获取图像，用3分钟的曝光时间（F /停止= 1，分级= 4）。使用相同的照明设置（灯电压，过滤器的f /停止，视野，BINNING）获得的所有图像。使用的背部皮肤的面积，计算出的信号强度的背景组织。归一化荧光发射的光子每秒每平方厘米每球面度（P / S /厘米2 / SR）。 </li></ol> 4。代表性的成果 CLI和PET图像之间的视觉比较，可以很容易地进行。后统一在图像比例尺相同的方式和CLI和PET的影像侧的一面可 ​​以看到，这代表面板（ 图2A），CLI和PET透露H460裸鼠移植瘤的治疗组小鼠从治疗前显着下降的信号到第3天，提示显着的治疗效果。作为比较，在未经处理的小鼠中观察到中等程度的增加，以不变的信号的周期（数据未示出）在同一时间。仅通过视觉检查可以观察到，有一个很好的一致性与肿瘤的对比，视觉从CLI和PET化。事实上，这种视觉的相关性有足够的分辨率，显示中央坏死继发肿瘤的抗癌治疗（请比较CLI和PET图像从第3天）。为了验证影像学检查结果量化，并可以进行相关性分析。 量化的CLI和PET图像和简单的通过线性回归拟合，结果表明两种模式确实有很好的相关性（ 图2B，R 2 = 0.9309，为18 F-FLT探测治疗组）。值得一提的是，在所有我们的CLI和PET成像研究，不同的肿瘤模型和不同的抗癌药物的斜坡拟合也非常接近，因此建议甚至所有数据的线性回归非常适合集团化（数据未显示）。这两个代表性的图片是改编自我们之前发表的12。 er.within页的"永远"&gt; <img src="/files/ftp_upload/4341/4341fig1.jpg" alt="图1"/> 图1 PET和CLI研究的实验设计原理。肿瘤植入双侧肩区域和允许增长到150至200毫米3荷瘤小鼠进行体内成像通过PET和CLI在天-1，1，和3。贝伐单抗的治疗进行2次注射20毫克/公斤0和2天。 <img src="/files/ftp_upload/4341/4341fig2.jpg" alt="图2"/> 图2。 （B）（A）H460移植瘤的治疗与贝伐单抗治疗前（预扫描）和治疗后（第3天）荷瘤小鼠的体内的 CLI和PET图像。CLI和PET相应的定量分析结果（N = 3）和的相关性。 （6）改编的图片。arge.jpg"目标="_blank"&gt;点击这里查看大图。

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没有利益冲突的声明。

CLI是一种新兴的一个有希望的分子成像技术，已经发现在许多基础科学的研究应用和临床使用4,5,15,16,17潜力。 CLI的主要优点是比传统的核医学成像方式，如PET干其使用的其他投资工具，它更容易使用，采集时间短和高吞吐量，显着更便宜，更广泛地提供给研究人员。此外，CLI除了OI一般是使用β-发光标记的分子成像探针，其中许多已通过美国食品和药物管理局（FDA），不像传统的OI剂。这些独特的优良品质，CLI已迅速获得从分子成像领域的关注。然而，其在临床前和临床应用的潜力还没有得到充分的研究。 癌症治疗监测的领域之一CLI可以有一些显着的效用。这是一个非常重要的领域，关键是探针的发展，药物筛选，甚至剪裁癌症治疗的患者。目前，临床前癌症治疗监测进行核成像方式，如PET几乎完全通过。因此，CLI提供了一个非常有吸引力的替代PET，特别是考虑到CLI和PET图像之间有很好的相关性。然而，CLI用于治疗监测的另一个优点在于，CLI可以图像不仅如32 P，90 Y，和131 I的β+器，但也β -发射器，所有这些都是临床相关的事实。 然而，CLI也不是没有缺陷。该上OI仪器的依赖决定了，CLI患有如在活组织中的信号衰减和散射的光学成像固有的一些缺点，是。此外，特定的频谱CR还导致在有限的信号强度，并随后，更深的信号从主体表面，较低的灵敏度，和量化越差能力6。然而，可以被视为是显着的，而缺点，可以在很大程度上绕过这些障碍，在临床前研究中，通过采用小动物如小鼠。更重要的是，至少有一对夫妇，有可能有利于从CLI癌症治疗监测的临床领域。表面的皮肤病炎症性疾病和癌症等疾病的监测，可以作为一个很好的例子。此外，疾病实体是深尚未访问的电荷耦合器件或基于光纤的技术可以使用，以及优异的灵敏度和定量能力的CLI。然而，另一个令人兴奋的可能性是使用CLI，以帮助医生获得肿瘤的解剖和功能信息，在手术室。最近的两个概念证明的学生模具已经证明检测和肿瘤切除术，术中影像学引导CLI 18,19小鼠。

<table border="1"&gt;<tr&gt;<td<strong&gt;名称</strong</td&gt;<td<strong&gt;公司</strong</td&gt;<td<strong&gt;产品目录号</strong</td&gt;</tr&gt;<tr&gt;<td&gt; H460细胞系</td&gt;<td&gt;美国典型培养物保藏</td&gt;<tdATCC编号:HTB-177</td&gt;</tr&gt;<tr&gt;<td&gt; RPMI 1640中等</td&gt;<tdInvitrogen生命技术</td&gt;<td&gt; 12633-012</td&gt;</tr&gt;<tr&gt;<td&gt;胎牛血清</td&gt;<tdInvitrogen生命技术</td&gt;<td&gt; 10091-148</td&gt;</tr&gt;<tr&gt;<td&gt;青霉素/链霉素</td&gt;<tdInvitrogen生命技术</td&gt;<td&gt; 15640-055</td&gt;</tr&gt;<tr&gt;<td&gt;磷酸盐缓冲盐水</td&gt;<tdInvitrogen生命技术</td&gt;<td&gt; 10010-023</td&gt;</tr&gt;<tr&gt;<td&gt;女裸鼠</td&gt;<td&gt; Charles River实验室，公司</td&gt;<td&gt;应变代号:088</td&gt;</tr&gt;<tr&gt;<td&gt;贝伐单抗（商品名Avastin）</td&gt;<td&gt;基因泰克/罗氏</td&gt;<td&gt; N / A</td&gt;</tr&gt;<tr&gt;<td&gt; MicroPET的啮齿动物R4</td&gt;<td西门子医疗解决方案（美国）公司</td&gt;<td&gt; N / A</td&gt;</tr&gt;<tr&gt;<td&gt;异氟醚（Aerrane）</td&gt;<td&gt;巴克斯特</td&gt;<td&gt;巴克斯特号码:AHN3637</td&gt;</tr&gt;<tr&gt;<td&gt; IVIS频谱</td&gt;<td卡尺生命科学</td&gt;<td&gt; N / A</td&gt;</tr&gt;</table

cerenkov luminescence imaging (cli) for cancer therapy monitoring

在分子成像，正电子发射断层扫描（PET）和光学成像（OI）的两个最重要的，从而最广泛使用的方式1-3。 PET的特点是其出色的灵敏度和定量分析能力，而OI值得注意的是无辐射，成本相对较低，扫描时间短，高吞吐量，基础研究人员和广泛的可用性。然而，这两种方式也有自己的缺点。 PET的空间分辨率差，成本高，而患有成骨不全临床前应用大多局限于有明显的散射光信号通过活组织的厚度，因为其有限的组织穿透力沿。 最近发现的切伦科夫发光成像（CLI）4-6 PET和OI之间的桥梁，又出现了。 CLI是一种新的成像方式，充分利用切伦科夫辐射（CR）图像放射性核素与其他投资工具。俄罗斯诺贝尔LAUReate阿列克谢耶维奇的切伦科夫和他的同事们最初发现于1934年CR。它是在电介质7,8在一个超光速行进时的带电粒子发射的电磁辐射的一种形式。无论是正电子或电子的带电粒子，通过移位它的原子中的电子的介质，扰动电磁场。发出的在通过中断光子是流离失所的电子返回到基态。例如，一个18 F衰变估计，以产生一个平均3光子在水5。 由于它的出现，CLI研究已在各种临床前的应用，包括在体内肿瘤的成像，报告基因显像，放射性示踪剂的发展，多模态成像，等等4,5,9,10,11供其使用。为什么CLI了巨大的成功，最重要的原因是，这项新技术利用低COST和广泛的可用性的OI图像的放射性核素，只有更昂贵和缺乏提供核成像方式，如PET成像。 在这里，我们介绍的方法使用CLI监测癌症的药物治疗。我们的研究小组最近研究这个新的应用程序，并验证了其可行性的证明了概念研究12。我们表明，CLI和PET具有优异的相关性在不同的肿瘤异种移植和成像探针。这CLI基本上是可视化的放射性核素作为PET的总体原则，CR是一致的。我们选择了贝伐单抗（商品名Avastin，基因泰克/罗氏）作为我们治疗剂，因为它是一个著名的血管生成抑制剂13,14。可以预见在不久的将来，这项技术的成熟，有一​​个显著影响临床前药物开发，筛选，以及接受治疗的患者的治疗监测。

Watch this Scientific Journal Video about Cerenkov Luminescence Imaging CLI for Cancer Therapy Monitoring at JoVE.com

Cerenkov Luminescence Imaging CLI for Cancer Therapy Monitoring

切伦科夫发光成像（CLI）监测的临床前癌症治疗中的使用说明在这里。这种方法利用的切伦科夫辐射（CR）和光学成像（OI）的放射性标记的探针可视化，从而提供了一种替代PET在临床前治疗的监测和药物筛选。

癌症治疗监测的切伦科夫发光成像（CLI）

In  molecular imaging, positron emission tomography (PET) and optical imaging (OI)  are two of the most important and thus most widely used modalities1-3. ...

cerenkov-luminescence-imaging-cli-for-cancer-therapy-monitoring

Nanyang Technological University

Research

JoVE Journal

Medicine

Cerenkov Luminescence Imaging (CLI) for Cancer Therapy Monitoring

13.6K 次观看.  Stanford University. 切伦科夫发光成像（CLI）监测的临床前癌症治疗中的使用说明在这里。这种方法利用的切伦科夫辐射（CR）和光学成像（OI）的放射性标记的探针可视化，从而提供了一种替代PET在临床前治疗的监测和药物筛选。

视频: Cerenkov Luminescence Imaging CLI for Cancer Therapy Monitoring

Monitoring Tumor Metastases and Osteolytic Lesions with Bioluminescence and Micro CT Imaging

Following intracardiac delivery of MDA-MB-231-luc-D3H2LN cells to Nu/Nu mice, systemic metastases developed in the injected animals. Bioluminescence imaging using IVIS Spectrum was employed to monitor the distribution and development of the tumor cells following the delivery procedure including DLIT reconstruction to measure the tumor signal and its location.
Development of metastatic lesions to the bone tissues triggers osteolytic activity and lesions to tibia and femur were evaluated longitudinally using micro CT. Imaging was performed using a Quantum FX micro CT system with fast imaging and low X-ray dose. The low radiation dose allows multiple imaging sessions to be performed with a cumulative X-ray dosage far below LD50. A mouse imaging shuttle device was used to sequentially image the mice with both IVIS Spectrum and Quantum FX achieving accurate animal positioning in both the bioluminescence and CT images. The optical and CT data sets were co-registered in 3-dimentions using the Living Image 4.1 software. This multi-mode approach allows close monitoring of tumor growth and development simultaneously with osteolytic activity.

An experimental mouse model of bone metastasis was established following intracardiac delivery of luciferase expressing mammary tumor cells. Tumor development and resulted osteolytic lesion were monitored longitudinally with bioluminescence and micro CT imaging.

生物发光和微型CT成像监测肿瘤转移和溶骨性病变

Following intracardiac delivery of MDA-MB-231-luc-D3H2LN cells to Nu/Nu mice, systemic metastases developed in the injected animals. Bioluminescence ...

科研

医学

Modeling and Imaging 3-Dimensional Collective Cell Invasion

A defining characteristic of cancer malignancy is invasion and metastasis 1. In some cancers (e.g. glioma 2), local invasion into surrounding healthy tissue is the root cause of disease and death. For other cancers (e.g. breast, lung, etc.), it is the process of metastasis, in which tumor cells move from a primary tumor mass, colonize distal sites and ultimately contribute to organ failure, that eventually leads to morbidity and mortality 3. It has been estimated that invasion and metastasis are responsible for 90&#37; of cancer deaths 4. As a result, there has been intense interest in identifying the molecular processes and critical protein mediators of invasion and metastasis for the purposes of improving diagnosis and treatment 5.
A challenge for cancer scientists is to develop invasion assays that sufficiently resemble the in vivo situation to enable accurate disease modeling 6. Two-dimensional cell motility assays are only informative about one aspect of invasion and do not take into account extracellular matrix (ECM) protein remodeling which is also a critical element. Recently, research has refined our understanding of tumor cell invasion and revealed that individual cells may move by elongated or rounded modes 7. In addition, there has been greater appreciation of the contribution of collective invasion, in which cells invade in strands, sheets and clusters, particularly in highly differentiated tumors that maintain epithelial characteristics, to the spread of cancer 8.
We present a refined method 9 for examining the contributions of candidate proteins to collective invasion 10. In particular, by engineering separate pools of cells to express different fluorescent proteins, it is possible to molecularly dissect the activities and proteins required in leading cells versus those required in following cells. The use of RNAi provides the molecular tool to experimentally disassemble the processes involved in individual cell invasion as well as in different positions of collective invasion. In this procedure, mixtures of fluorescently-labeled cells are plated on the bottom of a Transwell insert previously filled with Matrigel ECM protein, then allowed to invade "upwards" through the filter and into the Matrigel. Reconstruction of z-series image stacks, obtained by confocal imaging, into three-dimensional representations allows for visualization of collectively invading strands and analysis of the representation of fluorescently-labeled cells in leading versus following positions.

Models of tumor cell invasion into three-dimensional extracellular matrix better reflect the in vivo situation than two-dimensional motility assays. Using matrix invasion assays combined with confocal imaging of fluorescently-labeled cells, detailed information on invasion modes and the distinct contributions of leading versus following cells can be obtained.

3维建模和成像集体细胞的侵袭

A defining characteristic of cancer malignancy is invasion and metastasis 1. In some cancers (e.g. glioma 2), local invasion into surrounding healthy ...

Protocols for Assessing Radiofrequency Interactions with Gold Nanoparticles and Biological Systems for Non-invasive Hyperthermia Cancer Therapy

Cancer therapies which are less toxic and invasive than their existing counterparts are highly desirable. The use of RF electric-fields that penetrate deep into the body, causing minimal toxicity, are currently being studied as a viable means of non-invasive cancer therapy. It is envisioned that the interactions of RF energy with internalized nanoparticles (NPs) can liberate heat which can then cause overheating (hyperthermia) of the cell, ultimately ending in cell necrosis.
In the case of non-biological systems, we present detailed protocols relating to quantifying the heat liberated by highly-concentrated NP colloids. For biological systems, in the case of in vitro experiments, we describe the techniques and conditions which must be adhered to in order to effectively expose cancer cells to RF energy without bulk media heating artifacts significantly obscuring the data. Finally, we give a detailed methodology for in vivo mouse models with ectopic hepatic cancer tumors.

We describe the protocols used to investigate the interactions of 13.56 MHz radiofrequency (RF) electric-fields with gold nanoparticle colloids in both non-biological and biological systems (in vitro/vivo). These interactions are being investigated for applications in cancer therapy.

协议的射频评估与互动金纳米粒子和生物系统的非侵入性癌症热疗治疗

Cancer therapies  which are less toxic and invasive than their existing counterparts are highly  desirable. The use of RF electric-fields that penetrate ...

Cerenkov Luminescence Imaging of Interscapular Brown Adipose Tissue

Brown adipose tissue (BAT), widely known as a &#8220;good fat&#8221; plays pivotal roles for thermogenesis in mammals. This special tissue is closely related to metabolism and energy expenditure, and its dysfunction is one important contributor for obesity and diabetes. Contrary to previous belief, recent PET/CT imaging studies indicated the BAT depots are still present in human adults. PET imaging clearly shows that BAT has considerably high uptake of 18F-FDG under certain conditions. In this video report, we demonstrate that Cerenkov luminescence imaging (CLI) with 18F-FDG can be used to optically image BAT in small animals. BAT activation is observed after intraperitoneal injection of norepinephrine (NE) and cold treatment, and depression of BAT is induced by long anesthesia. Using multiple-filter Cerenkov luminescence imaging, spectral unmixing and 3D imaging reconstruction are demonstrated. Our results suggest that CLI with 18F-FDG is a practical technique for imaging BAT in small animals, and this technique can be used as a cheap, fast, and alternative imaging tool for BAT research.

In this video report, we show the application of Cerenkov Luminescence Imaging (CLI) for interscapular brown adipose tissue in mice under activated and depressed conditions.

肩胛间褐色脂肪组织的切伦科夫发光成像

Brown adipose tissue (BAT), widely known as a &#8220;good fat&#8221; plays pivotal roles for thermogenesis in mammals. This special tissue is closely ...

Murine Model for Non-invasive Imaging to Detect and Monitor Ovarian Cancer Recurrence

Epithelial ovarian cancer is the most lethal gynecologic malignancy in the United States. Although patients initially respond to the current standard of care consisting of surgical debulking and combination chemotherapy consisting of platinum and taxane compounds, almost 90% of patients recur within a few years. In these patients the development of chemoresistant disease limits the efficacy of currently available chemotherapy agents and therefore contributes to the high mortality. To discover novel therapy options that can target recurrent disease, appropriate animal models that closely mimic the clinical profile of patients with recurrent ovarian cancer are required. The challenge in monitoring intra-peritoneal (i.p.) disease limits the use of i.p. models and thus most xenografts are established subcutaneously. We have developed a sensitive optical imaging platform that allows the detection and anatomical location of i.p. tumor mass. The platform includes the use of optical reporters that extend from the visible light range to near infrared, which in combination with 2-dimensional X-ray co-registration can provide anatomical location of molecular signals. Detection is significantly improved by the use of a rotation system that drives the animal to multiple angular positions for 360 degree imaging, allowing the identification of tumors that are not visible in single orientation. This platform provides a unique model to non-invasively monitor tumor growth and evaluate the efficacy of new therapies for the prevention or treatment of recurrent ovarian cancer.

We describe a non-invasive animal imaging platform that allows the detection, quantification, and monitoring of ovarian cancer growth and recurrence. This intra-peritoneal xenograft model mimics the clinical profile of patients with ovarian cancer.

小鼠模型的非侵入性成像检测和监测卵巢癌复发

Epithelial ovarian cancer is the most lethal gynecologic malignancy in the United States. Although patients initially respond to the current standard of ...

Dynamic Lung Tumor Tracking for Stereotactic Ablative Body Radiation Therapy

Physicians considering stereotactic ablative body radiation therapy (SBRT) for the treatment of extracranial cancer targets must be aware of the sizeable risks for normal tissue injury and the hazards of physical tumor miss. A first-of-its-kind SBRT platform achieves high-precision ablative radiation treatment through a combination of versatile real-time imaging solutions and sophisticated tumor tracking capabilities. It uses dual-diagnostic kV x-ray units for stereoscopic open-loop feedback of cancer target intrafraction movement occurring as a consequence of respiratory motions and heartbeat. Image-guided feedback drives a gimbaled radiation accelerator (maximum 15 x 15 cm field size) capable of real-time &#177;4 cm pan-and-tilt action. Robot-driven &#177;60&#176; pivots of an integrated &#177;185&#176; rotational gantry allow for coplanar and non-coplanar accelerator beam set-up angles, ultimately permitting unique treatment degrees of freedom. State-of-the-art software aids real-time six dimensional positioning, ensuring irradiation of cancer targets with sub-millimeter accuracy (0.4 mm at isocenter). Use of these features enables treating physicians to steer radiation dose to cancer tumor targets while simultaneously reducing radiation dose to normal tissues. By adding respiration correlated computed tomography (CT) and 2-[18F] fluoro-2-deoxy-&#7429;-glucose (18F-FDG) positron emission tomography (PET) images into the planning system for enhanced tumor target contouring, the likelihood of physical tumor miss becomes substantially less1. In this article, we describe new radiation plans for the treatment of moving lung tumors.

Stereotactic ablative body radiation therapy (SBRT) involves the precise delivery of high-dose radiation to cancer tumor targets. A novel SBRT platform offers a first-of-its-kind gimbaled radiation accelerator mounted within a pivoting O-ring gantry capable of dynamic image-guided tumor tracking. Here, we describe dynamic tumor tracking for lung targets.

动态肺肿瘤跟踪烧蚀立体定向放射身体治疗

Physicians considering stereotactic ablative body radiation therapy (SBRT) for the treatment of extracranial cancer targets must be aware of the sizeable ...

An Organotypic High Throughput System for Characterization of Drug Sensitivity of Primary Multiple Myeloma Cells

In this work we describe a novel approach that combines ex vivo drug sensitivity assays and digital image analysis to estimate chemosensitivity and heterogeneity of patient-derived multiple myeloma (MM) cells. This approach consists in seeding primary MM cells freshly extracted from bone marrow aspirates into microfluidic chambers implemented in multi-well plates, each consisting of a reconstruction of the bone marrow microenvironment, including extracellular matrix (collagen or basement membrane matrix) and stroma (patient-derived mesenchymal stem cells) or human-derived endothelial cells (HUVECs). The chambers are drugged with different agents and concentrations, and are imaged sequentially for 96 hr through bright field microscopy, in a motorized microscope equipped with a digital camera. Digital image analysis software detects live and dead cells from presence or absence of membrane motion, and generates curves of change in viability as a function of drug concentration and exposure time. We use a computational model to determine the parameters of chemosensitivity of the tumor population to each drug, as well as the number of sub-populations present as a measure of tumor heterogeneity. These patient-tailored models can then be used to simulate therapeutic regimens and estimate clinical response.

We describe a high-throughput drug sensitivity assay for primary multiple myeloma cells. It consists of a reconstruction of the bone marrow microenvironment (including extracellular matrix and stroma) in multi-well plates, and a non-invasive method for longitudinal quantification of cell viability.

一个器官高通量系统原发性多发性骨髓瘤细胞的药物敏感性的表征

In this work we describe a novel approach that combines ex vivo drug sensitivity assays and digital image analysis to estimate chemosensitivity and ...

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 ...

Quantitative Visualization of Leukocyte Infiltrate in a Murine Model of Fulminant Myocarditis by Light Sheet Microscopy

Light-sheet fluorescence microscopy (LSFM), in combination with chemical clearing protocols, has become the gold standard for analyzing fluorescently labelled structures in large biological specimens, and is down to cellular resolution. Meanwhile, the constant refinement of underlying protocols and the enhanced availability of specialized commercial systems enable us to investigate the microstructure of whole mouse organs and even allow for the characterization of cellular behavior in various live-cell imaging approaches. Here, we describe a protocol for the spatial whole-mount visualization and quantification of the CD45+ leukocyte population in inflamed mouse hearts. The method employs a transgenic mouse strain (CD11c.DTR)that has recently been shown to serve as a robust, inducible model for the study of the development of fulminant fatal myocarditis, characterized by lethal cardiac arrhythmias. This protocol includes myocarditis induction, intravital antibody-mediated cell staining, organ preparation, and LSFM with subsequent computer-assisted image post-processing. Although presented as a highly-adapted method for our particular scientific question, the protocol represents the blueprint of an easily adjustable system that can also target completely different fluorescent structures in other organs and even in other species.

Here, we describe a light-sheet microscopy approach to visualize the cardiac CD45+ leukocyte infiltrate in a murine model of aseptic fulminant myocarditis, which is induced by the intratracheal diphtheria toxin treatment of CD11c.DTR mice.

通过轻型显微镜对小鼠急性心肌炎模型中白细胞浸润的定量可视化

Light-sheet fluorescence microscopy (LSFM), in combination with chemical clearing protocols, has become the gold standard for analyzing fluorescently ...

Mechanical Micronization of Lipoaspirates for Regenerative Therapy

Stromal vascular fraction (SVF) has become a regenerative tool for various diseases; however, legislation strictly regulates the clinical application of cell products using collagenase. Here, we present a protocol to generate an injectable mixture of SVF cells and native extracellular matrix from adipose tissue by a purely mechanical process. Lipoaspirates are put into a centrifuge and spun at 1,200 x g for 3 min. The middle layer is collected and separated into two layers (high-density fat at the bottom and low-density fat on the top). The upper layer is directly emulsified by intersyringe shifting, at a rate of 20 mL/s for 6x to 8x. The emulsified fat is centrifuged at 2,000 x g for 3 min, and the sticky substance under the oil layer is collected and defined as the extracellular matrix (ECM)/SVF-gel. The oil on the top layer is collected. Approximately 5 mL of oil is added to 15 mL of high-density fat and emulsified by intersyringe shifting, at a rate of 20 mL/s for 6x to 8x. The emulsified fat is centrifuged at 2,000 x g for 3 min, and the sticky substance is also ECM/SVF-gel. After the transplantation of the ECM/SVF-gel into nude mice, the graft is harvested and assessed by histologic examination. The result shows that this product has the potential to regenerate into normal adipose tissue. This procedure is a simple, effective mechanical dissociation procedure to condense the SVF cells embedded in their natural supportive ECM for regenerative purposes.

Here, we present a protocol to obtain stromal vascular fraction from adipose tissue through a series of mechanical processes, which include emulsification and multiple centrifugations.

再生疗法用脂质吸入剂的机械微化

Stromal vascular fraction (SVF) has become a regenerative tool for various diseases; however, legislation strictly regulates the clinical application of ...