This research is supported by funding from CBCF (to ZW).

1.准备实时成像单元格<ol><li> 细胞培养和播种 <ol><li>使用选择用于抗多西他赛（MCF-7 的TXT）和它们的非抗性亲代细胞系（MCF-7，CC）MCF-7乳腺癌细胞。详细的选择过程并且这些选择的细胞系的表征先前24描述。 </li><li>生长于10cm培养皿中的所有细胞在37℃下在由90％的Dulbecco改进的Eagle培养基（DMEM）和胎牛血清（FBS）的10％，并补充有非必需氨基酸介质。保持在5％ 的 CO 2气氛中的介质。保持在5nM的多西他赛的MCF-7 的TXT细胞。 </li><li>种子细胞盖玻片上的实时成像。通过用70％乙醇漂洗它们，然后将它们暴露于紫外线过夜消毒24毫米涂覆聚-L-赖氨酸盖玻片。放置盖玻片成35mm的6孔培养板上。 PL在每口井癿盖玻片。 </li><li>除去从10厘米的培养皿的培养基，并用PBS洗涤细胞。添加0.5毫升胰蛋白酶-EDTA的的菜分离从盘的细胞。添加5毫升培养基的培养皿中以中和胰蛋白酶的细胞的分离如下。 </li><li>计数细胞用血细胞计数器确定每单位体积电池的数目。 </li><li>加约10 5个细胞每孔的基础上每单位体积的细胞数量。轻轻摇动后，将板放回孵化器，以使细胞附着在盖玻片。 </li></ol></li><li> GFP标记α微管蛋白的表达 <ol><li>为了测定微管动力学，转导与GFP标记α微管蛋白的细胞根据制造商的协议商业GFP转导控制（ 例如，BacMam）。 </li><li>培养在井中的细胞36小时在37℃的细胞培养孵化器，以允许玉米完整粘附和60％汇合。 </li><li>计算GFP标记α微管蛋白的适当体积添加到各孔中，根据以下公式: <img alt="方程" src="/files/ftp_upload/55027/55027eq1.jpg" /> 细胞的数量是细胞在井在标签的时间的估计的总数目和所需的PPC是每个细胞的粒子数。 注:播种在MCF-7细胞应该一倍，从10 5至2×10 5细胞数为20的期望的PPC，需要为每个盖玻片的体积因此是40微升。对于不同的小区，体积算出基于上述公式可能不是最好的。确切的量应根据GFP微管蛋白的实际表达水平进行调整。 </li><li>通过倒置混合GFP标记α微管蛋白几次，以确保均匀的溶液。不要旋涡。 注:对于控制中，空（控制）试剂没有任何哺乳动物的基因元件和可用于帮助确定潜在杆状病毒介导的作用和背景荧光。非针对性的GFP转导的控制也可使用，这将照亮整个细胞。 </li><li>替换用新鲜培养基的培养基中。添加2mL的新培养基中，40微升GFP标记α微管蛋白的每个孔中。 </li><li>轻轻摇动板以允许与所述培养基GFP标记α微管蛋白的完全混合。返回板到培养培养箱孵育24小时，以允许GFP微管蛋白的表达。 </li></ol></li><li> 处理细胞多西他赛 注:此实验是测试多西他赛的微管动态不稳定性的影响。因此，该细胞与多西紫杉醇的所需浓度进行处理。 <ol><li>制备的培养基由90％的DMEM和10％FBS的和补充有非必需氨基酸。使用DMEM培养基无酚红，因为酚红可以与fluorescen干扰GFP微管蛋白的CE。添加多西他赛（范围从10nM至10μM）的所需的浓度。多西紫杉醇的储备溶液是在二甲亚砜（DMSO）的10mM。 </li><li>预暖在组织培养培养箱含多西紫杉醇的培养基至37℃。 </li><li>替换与含多西紫杉醇的培养基正常培养基并孵育在组织培养箱30分钟。应该有每孔至少为3的盖玻片为每个多西紫杉醇的浓度。 </li></ol></li></ol> 2.实时成像，以检查微管动态失稳<ol><li> 设置系统 <ol><li>在保持在37℃和5％的CO 2的腔室进行实验。由时间流逝用装有60X，在一个倒置荧光显微镜和CCD照相机1.42 NA油物镜的显微镜系统获取荧光图像。 </li><li>设置了实时成像的细胞。预暖两个样本保持器和在中至37℃。选择一个盖玻片检查。安装在试样保持器的兴趣盖玻片并用1mL在步骤1.3.1制备的含多西紫杉醇的培养基孵育它。 </li></ol></li><li> 微管动态不稳定的实时成像 <ol><li>确定将被用于成像的细胞。他们应该是平的，有表达GFP微管蛋白的荧光强度高，并有明确的微管结构。 </li><li>选择组从步骤2.2.1确定细胞的一个单元首次检验。集中在识别的小区的周边区域。设置了曝光时间的程序和图像采集的频率。由于曝光时间一般是0.5秒，图像将每两个s收购。 </li><li>允许步骤，一个额外的20分钟从1.33至2.2.3。因此，恰好在加入多西他赛之后50分钟，如在步骤1.3.3所述，开始成像（这是标准化）。 </li><li>记录微管ðynamics 2分钟，拍摄照片，每2秒。总共获得用于识别的小区60的图像。 </li><li>移动到下一个识别的小区，并重复步骤2.2.2至2.2.4。重复上述步骤，直到5细胞已被成像。当务之急是做到这一点的时间达到加成多西他赛之后70分钟，然后，如在步骤1.3.3说明。 </li><li>对于每个处理条件，重复步骤2.2.1至2.2.5 3种不同的盖玻片。总体而言，图像15细胞，这是足够的数据来衡量的微管动态不稳定。 </li></ol></li></ol> 3.图像处理和数据分析<ol><li> 图像反卷积 <ol><li>去卷积所获取的图像，以实现高品质。打开装有显微镜系统去卷积程序。 </li><li>单击"进程"，然后选择"反卷积"。图像文件拖动到"输入"窗口，点击"做。"该图像文件将被去卷积，并且反褶积文件将被自动保存为一个新文件。 </li></ol></li><li> 视频生成 <ol><li>点击反褶积图像文件与配套软件打开。 </li><li>点击"文件"，选择"另存为电影"。选择"电影格式"为"AV"和"动画风格"为"前进"。将"压缩质量"到"100％"和"帧速率"到"5张/秒。" </li><li>检查"通过时间动画"，并单击框"做吧。"视频将产生。 </li></ol></li><li> 微管缩短和增长速度的测量 <ol><li>确定将被用于测量的微管。对于每个小区，只有少数微管就能有其缩短或增长遵循清晰，完整。 </li><li>检查视频的每个帧，以确定显示微管一个的起始长度的帧第二最延长或缩短微管的帧。测量两个微管和两帧之间所经过的时间之间的长度变化。 </li><li>通过时间除以长度变化计算增长或缩短的速度（单位是微米/秒）。对于每个处理条件，遵循至少10个细胞，并测量至少20微管。 </li><li>计算每个处理条件的平均值和标准误差。分析使用Student测试条件之间的统计差异。报告在表和/或图表中的数据。 </li></ol></li></ol>

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作者宣称，他们没有竞争的经济利益。

有两种主要的方法来测量微管动态不稳定性: 在体外和体内。在体外方法中，纯化的微管蛋白是用来测量与计算机增强的时间推移微分干涉反差显微镜微管动态不稳定性。在体内方法中，显微注射荧光微管蛋白，或表达GFP的微管蛋白，掺入微管。微管的动力学（生长和缩短）然后通过时间推移在间期细胞10，20，25</su...

<html>乳腺癌死亡率的主要原因就是通过转移1，2。紫杉烷类，如多西他赛和紫杉醇，目前用作一线转移性乳腺癌2，3，4，5，6的治疗方案。他们是一群扰乱微管动力学微管靶向代理（MTA）的一部分。然而，在治疗性治疗使用紫杉烷类的最大挑战之一是在癌细胞中紫杉烷抗性，从而导致疾病复发7的发展。耐药占转移性乳腺癌7之间所有死亡的90％以上。 微管是由α-和β微管蛋白异源二聚体的聚合形成类="外部参照"&gt; 8，9。微管动力学的精确调节是许多细胞功能，包括细胞极化，细胞周期进程，细胞内运输和细胞信号传导的重要。微管和它们的动态的失调会扰乱细胞的功能，导致细胞死亡10,11。取决于它们怎样导致此失调，MTA药物可归类为微管稳定剂（即紫杉烷）或微管destabalizing剂（ 即，长春花生物碱或秋水仙碱点结合剂）20。尽管微管的质量的相反的效果，以足够的剂量，这两个类可以通过其对微管动力学21作用杀死癌细胞。 紫杉烷类通过稳定微管主轴12主要作用，导致染色体错位。在纺锤体装配检验点（SAC）的后续永久激活逮捕的有丝分裂细胞。延长的有丝分裂停滞然后导致凋亡13,14。紫杉烷与通过对β微管蛋白8，15，这是只有在组装的微管蛋白16存在的紫杉烷的结合位点的微管相互作用。 对紫杉烷抗性多种机制已经提出9,17。这些机制包括由于药物外排蛋白与紫杉类药物特异性抵抗力5，9，18，19的表达了一般多药耐药性。例如，耐紫杉类癌细胞可能已经改变了某些β桶的表达和功能同型球蛋白5，9，19，20，21，22，23。通过使用体内方法测量微管动态不稳定，我们表明，相对于非抗性，父母的MCF-7 的CC单元17时，耐多西紫杉醇的MCF-7 的TXT细胞的微管动力学是不敏感的多西他赛治疗。 为了更好地了解的MTA的功能，并在癌细胞紫杉烷抗性的确切机制，重要的是测量微管动力学。这里，我们报告这样的体内方法。通过使用实时成像结合GFP标记的微管蛋白在细胞中的表达，我们可以测量的MCF-7 的TXT和MCF-7 的CC细胞和Wi的微管动力学thout多西紫杉醇治疗。研究结果可以帮助我们设计出能够克服阻力紫杉更加有效的药物。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle&#39;s Medium (DMEM)</td>
			<td>Sigma-Aldrich</td>
			<td>D5796</td>
			<td></td>
		</tr>
		<tr>
			<td>Non-essential amino acids</td>
			<td>Life Technologies, Invitrogen</td>
			<td>11140-050</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Gibco, Invitrogen</td>
			<td>12483</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Anti (100x)</td>
			<td>Life Technologies, Invitrogen</td>
			<td>15240-062</td>
			<td></td>
		</tr>
		<tr>
			<td>docetaxel</td>
			<td>Sigma-Aldrich</td>
			<td>01885-5mg-F</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM phenol red-free</td>
			<td>Gibco, Invitrogen</td>
			<td>21063</td>
			<td></td>
		</tr>
		<tr>
			<td>CellLight Reagent *BacMam 2.0* GFP-tubulin</td>
			<td>ThermoFisher Scientific</td>
			<td>C10613</td>
			<td>Key reagent for expressing GFP tubulin in cells</td>
		</tr>
		<tr>
			<td>CellLight Reagent *BacMam 2.0* GFP</td>
			<td>ThermoFisher Scientific</td>
			<td>B10383</td>
			<td>Control</td>
		</tr>
		<tr>
			<td>Dimethyl Sulfoxide (DMSO)</td>
			<td>Sigma-Aldrich+B9:AA9</td>
			<td>472301</td>
			<td>for dissoving decetaxel</td>
		</tr>
		<tr>
			<td>22-mm glass coveslip</td>
			<td>Fisher Scientifics</td>
			<td>12-545-101</td>
			<td></td>
		</tr>
		<tr>
			<td>6-well culture plate</td>
			<td>Greiner Bio-One International</td>
			<td>6 Well Celi Culture Plate</td>
			<td></td>
		</tr>
		<tr>
			<td>DeltaVision Microscopy Imaging Systems</td>
			<td>GE Health</td>
			<td></td>
			<td>This system is equipped with weather station for controlling temperature and CO2. It also equipped with Worx Software for deconvolution and time lapse control.</td>
		</tr>
		<tr>
			<td>Trypsin-EDTA (0.25%), phenol red</td>
			<td>ThermoFisher Scientific</td>
			<td>25200056</td>
			<td></td>
		</tr>
		<tr>
			<td>Bright-Line Hemacytometer Set, Hausser Scientific</td>
			<td>Hausser Scientific, Distributed by VWR</td>
			<td>Supplier No.: 1492 VWR No.:15170-172</td>
			<td></td>
		</tr>
	</tbody>
</table>

live imaging to study microtubule dynamic instability in taxane-resistant breast cancers

Taxanes such as docetaxel belong to a group of microtubule-targeting agents (MTAs) that are commonly relied upon to treat cancer. However, taxane resistance in cancerous cells drastically reduces the effectiveness of the drugs' long-term usage. Accumulated evidence suggests that the mechanisms underlying taxane resistance include both general mechanisms, such as the development of multidrug resistance due to the overexpression of drug-efflux proteins, and taxane-specific mechanisms, such as those that involve microtubule dynamics. 
Because taxanes target cell microtubules, measuring microtubule dynamic instability is an important step in determining the mechanisms of taxane resistance and provides insight into how to overcome this resistance. In the experiment, an in vivo method was used to measure microtubule dynamic instability. GFP-tagged &#945;-tubulin was expressed and incorporated into microtubules in MCF-7 cells, allowing for the recording of the microtubule dynamics by time lapse using a sensitive camera. The results showed that, as opposed to the non-resistant parental MCF-7CC cells, the microtubule dynamics of docetaxel-resistant MCF-7TXT cells are insensitive to docetaxel treatment, which causes the resistance to docetaxel-induced mitotic arrest and apoptosis. This paper will outline this in vivo method of measuring microtubule dynamic instability.

用这里介绍的协议中，我们研究了在正常（MCF-7，CC）和耐多西紫杉醇（MCF-7 TXT）乳腺癌细胞的微管动力学的多西紫杉醇的效果。两组图像显示在微管的生长和缩短多西他赛（0.5μM）的在MCF-7 的CC和MCF-7 的TXT细胞（ 图1A）的影响。 我们还计算微管生长或缩短的速率在这些条件对两种?...

Watch this Scientific Journal Video about Live Imaging to Study Microtubule Dynamic Instability in Taxane-resistant Breast Cancers at JoVE.com

Live Imaging to Study Microtubule Dynamic Instability in Taxane-resistant Breast Cancers

In this paper, we report a protocol describing an in vivo method to measure microtubule dynamic instability in docetaxel-resistant breast cancer cells (MCF-7TXT). In this method, a deconvolution microscopy imaging system is used to detect the expression of GFP-tubulin in target cells.

实时成像来研究抗紫杉烷乳腺癌微管动态失稳

Taxanes such as docetaxel belong to a group of microtubule-targeting agents (MTAs) that are commonly relied upon to treat cancer. However, taxane ...

live-imaging-to-study-microtubule-dynamic-instability-taxane

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7.5K 次观看.  University of Alberta. In this paper, we report a protocol describing an in vivo method to measure microtubule dynamic instability in docetaxel-resistant breast cancer cells (MCF-7TXT). In this method, a deconvolution microscopy imaging system is used to detect the expression of GFP-tubulin in target cells. 

视频: Live Imaging to Study Microtubule Dynamic Instability in Taxane-resistant Breast Cancers

Utilizing Functional Genomics Screening to Identify Potentially Novel Drug Targets in Cancer Cell Spheroid Cultures

The identification of functional driver events in cancer is central to furthering our understanding of cancer biology and indispensable for the discovery of the next generation of novel drug targets. It is becoming apparent that more complex models of cancer are required to fully appreciate the contributing factors that drive tumorigenesis in vivo and increase the efficacy of novel therapies that make the transition from pre-clinical models to clinical trials.
Here we present a methodology for generating uniform and reproducible tumor spheroids that can be subjected to siRNA functional screening. These spheroids display many characteristics that are found in solid tumors that are not present in traditional two-dimension culture. We show that several commonly used breast cancer cell lines are amenable to this protocol. Furthermore, we provide proof-of-principle data utilizing the breast cancer cell line BT474, confirming their dependency on amplification of the epidermal growth factor receptor HER2 and mutation of phosphatidylinositol-4,5-biphosphate 3-kinase (PIK3CA) when grown as tumor spheroids. Finally, we are able to further investigate and confirm the spatial impact of these dependencies using immunohistochemistry.

Identifying novel drug targets that transition from pre-clinical testing to human trials is a scientific priority. To that end, here we describe a functional genomics approach for examining the impact of gene depletion on cancer cell line spheroids, which more appropriately model human cancers in vivo.

利用功能基因组筛选识别在肿瘤细胞球体培养潜在的新药物靶标

The identification of functional driver events in cancer is central to furthering our understanding of cancer biology and indispensable for the discovery ...

科研

癌症研究

A Portal Vein Injection Model to Study Liver Metastasis of Breast Cancer

Breast cancer is the leading cause of cancer-related mortality in women worldwide. Liver metastasis is involved in upwards of 30% of cases with breast cancer metastasis, and results in poor outcomes with median survival rates of only 4.8 - 15 months. Current rodent models of breast cancer metastasis, including primary tumor cell xenograft and spontaneous tumor models, rarely metastasize to the liver. Intracardiac and intrasplenic injection models do result in liver metastases, however these models can be confounded by concomitant secondary-site metastasis, or by compromised immunity due to removal of the spleen to avoid tumor growth at the injection site. To address the need for improved liver metastasis models, a murine portal vein injection method that delivers tumor cells firstly and directly to the liver was developed. This model delivers tumor cells to the liver without complications of concurrent metastases in other organs or removal of the spleen. The optimized portal vein protocol employs small injection volumes of 5 - 10 &#956;l, &#8805; 32 gauge needles, and hemostatic gauze at the injection site to control for blood loss. The portal vein injection approach in Balb/c female mice using three syngeneic mammary tumor lines of varying metastatic potential was tested; high-metastatic 4T1 cells, moderate-metastatic D2A1 cells, and low-metastatic D2.OR cells. Concentrations of &#8804; 10,000 cells/injection results in a latency of ~ 20 - 40 days for development of liver metastases with the higher metastatic 4T1 and D2A1 lines, and &#62; 55 days for the less aggressive D2.OR line. This model represents an important tool to study breast cancer metastasis to the liver, and may be applicable to other cancers that frequently metastasize to the liver including colorectal and pancreatic adenocarcinomas.

A surgical procedure was developed to deliver mammary tumor cells to the murine liver via portal vein injection. This model permits investigation of late stages of liver metastasis in a fully immune competent host, including tumor cell extravasation, seeding, survival, and metastatic outgrowth in the liver.

门静脉注射模型来研究肝转移乳腺癌

Breast cancer is the leading cause of cancer-related mortality in women worldwide. Liver metastasis is involved in upwards of 30% of cases with breast ...

Live-imaging of Breast Epithelial Cell Migration After the Transient Depletion of TIP60

The wound-healing assay is efficient and one of the most economical ways to study cell migration in vitro. Conventionally, images are taken at the beginning and end of an experiment using a phase-contrast microscope, and the migration abilities of cells are evaluated by the closure of wounds. However, cell movement is a dynamic phenomenon, and a conventional method does not allow for tracking single-cell movement. To improve current wound-healing assays, we use live-cell imaging techniques to monitor cell migration in real time. This method allows us to determine the cell migration rate based on a cell tracking system and provides a clearer distinction between cell migration and cell proliferation. Here, we demonstrate the use of live-cell imaging in wound-healing assays to study the different migration abilities of breast epithelial cells influenced by the presence of TIP60. As cell motility is highly dynamic, our method provides more insights into the processes of wound healing than a snapshot of wound closure taken with the traditional imaging techniques used for wound-healing assays.

Here, we present the real-time monitoring of cell migration in a wound-healing assay using TIP60-depleted MCF10A breast epithelial cells. The implementation of live-cell imaging techniques in our protocol allows us to analyze and visualize single-cell movement in real time and across time.

TIP60 短暂耗竭后乳腺上皮细胞迁移的活体影像学研究

The wound-healing assay is efficient and one of the most economical ways to study cell migration in vitro. Conventionally, images are taken at the ...

Long-term Live-cell Imaging to Assess Cell Fate in Response to Paclitaxel

Live-cell imaging is a powerful technique that can be used to directly visualize biological phenomena in single cells over extended periods of time. Over the past decade, new and innovative technologies have greatly enhanced the practicality of live-cell imaging. Cells can now be kept in focus and continuously imaged over several days while maintained under 37 &#176;C and 5% CO2 cell culture conditions. Moreover, multiple fields of view representing different experimental conditions can be acquired simultaneously, thus providing high-throughput experimental data. Live-cell imaging provides a significant advantage over fixed-cell imaging by allowing for the direct visualization and temporal quantitation of dynamic cellular events. Live-cell imaging can also identify variation in the behavior of single cells that would otherwise have been missed using population-based assays. Here, we describe live-cell imaging protocols to assess cell fate decisions following treatment with the anti-mitotic drug paclitaxel. We demonstrate methods to visualize whether mitotically arrested cells die directly from mitosis or slip back into interphase. We also describe how the fluorescent ubiquitination-based cell cycle indicator (FUCCI) system can be used to assess the fraction of interphase cells born from mitotic slippage that are capable of re-entering the cell cycle. Finally, we describe a live-cell imaging method to identify nuclear envelope rupture events.

Live-cell imaging provides a wealth of information on single cells or whole populations that is unattainable by fixed cell imaging alone. Here, live-cell imaging protocols to assess cell fate decisions following treatment with the anti-mitotic drug paclitaxel are described.

长期活细胞成像对紫杉醇反应细胞命运的评估

Live-cell imaging is a powerful technique that can be used to directly visualize biological phenomena in single cells over extended periods of time. Over ...

Live-Cell Imaging Assays to Study Glioblastoma Brain Tumor Stem Cell Migration and Invasion

Glioblastoma (GBM) is an aggressive brain tumor that is poorly controlled with the currently available treatment options. Key features of GBMs include rapid proliferation and pervasive invasion into the normal brain. Recurrence is thought to result from the presence of radio- and chemo-resistant brain tumor stem cells (BTSCs) that invade away from the initial cancerous mass and, thus, evade surgical resection. Hence, therapies that target BTSCs and their invasive abilities may improve the otherwise poor prognosis of this disease. Our group and others have successfully established and characterized BTSC cultures from GBM patient samples. These BTSC cultures demonstrate fundamental cancer stem cell properties such as clonogenic self-renewal, multi-lineage differentiation, and tumor initiation in immune-deficient mice. In order to improve on the current therapeutic approaches for GBM, a better understanding of the mechanisms of BTSC migration and invasion is necessary. In GBM, the study of migration and invasion is restricted, in part, due to the limitations of existing techniques which do not fully account for the in vitro growth characteristics of BTSCs grown as neurospheres. Here, we describe rapid and quantitative live-cell imaging assays to study both the migration and invasion properties of BTSCs. The first method described is the BTSC migration assay which measures the migration toward a chemoattractant gradient. The second method described is the BTSC invasion assay which images and quantifies a cellular invasion from neurospheres into a matrix. The assays described here are used for the quantification of BTSC migration and invasion over time and under different treatment conditions.

Here, we describe live-cell imaging techniques to quantitatively measure the migration and invasion of glioblastoma brain tumor stem cells over time and under multiple treatment conditions.

活体细胞成像技术研究胶质瘤脑干细胞的迁移和侵袭

Glioblastoma (GBM) is an aggressive brain tumor that is poorly controlled with the currently available treatment options. Key features of GBMs include ...

Studying Triple Negative Breast Cancer Using Orthotopic Breast Cancer Model

Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype with limited therapeutic options. When compared to patients with less aggressive breast tumors, the 5-year survival rate of TNBC patients is 77% due to their characteristic drug-resistant phenotype and metastatic burden. Toward this end, murine models have been established aimed at identifying novel therapeutic strategies limiting TNBC tumor growth and metastatic spread. This work describes a practical guide for the TNBC orthotopic model where MDA-MB-231 breast cancer cells suspended in a basement membrane matrix are implanted in the fourth mammary fat pad, which closely mimics the cancer cell behavior in humans. Measurement of tumors by caliper, lung metastasis assessment via in vivo and ex vivo imaging, and molecular detection are discussed. This model provides an excellent platform to study therapeutic efficacy and is especially suitable for the study of the interaction between the primary tumor and distal metastatic sites.

This work presets an advanced protocol to accurately assess tumor loading by detection of green fluorescent protein and bioluminescence signals as well as the integration of quantitative molecular detection technique.

研究使用正交乳腺癌模型的三阴性乳腺癌

Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype with limited therapeutic options. When compared to patients with less ...

Using the Chicken Chorioallantoic Membrane In Vivo Model to Study Gynecological and Urological Cancers

Mouse models are the benchmark tests for in vivo cancer studies. However, cost, time, and ethical considerations have led to calls for alternative in vivo cancer models. The chicken chorioallantoic membrane (CAM) model provides an inexpensive, rapid alternative that permits direct visualization of tumor development and is suitable for in vivo imaging. As such, we sought to develop an optimized protocol for engrafting gynecological and urological tumors into this model, which we present here. Approximately 7 days postfertilization, the air cell is moved to the vascularized side of the egg, where an opening is created in the shell. Tumors from murine and human cell lines and primary tissues can then be engrafted. These are typically seeded in a mixture of extracellular matrix and medium to avoid cellular dispersal and provide nutrient support until the cells recruit a vascular supply. Tumors may then grow for up to an additional 14 days prior to the eggs hatching. By implanting cells stably transduced with firefly luciferase, bioluminescence imaging can be used for the sensitive detection of tumor growth on the membrane and cancer cell spread throughout the embryo. This model can potentially be used to study tumorigenicity, invasion, metastasis, and therapeutic effectiveness. The chicken CAM model requires significantly less time and financial resources compared to traditional murine models. Because the eggs are immunocompromised and immune tolerant, tissues from any organism can potentially be implanted without costly transgenic animals (e.g., mice) required for implantation of human tissues. However, many of the advantages of this model could potentially also be limitations, including the short tumor generation time and immunocompromised/immune tolerant status. Additionally, although all tumor types presented here engraft in the chicken chorioallantoic membrane model, they do so with varying degrees of tumor growth.

We present the chicken chorioallantoic membrane model as an alternative, transplantable, in vivo model for the engraftment of gynecological and urological cancer cell lines and patient-derived tumors.

利用活体中鸡胆膜研究妇科和泌尿癌

Mouse models are the benchmark tests for in vivo cancer studies. However, cost, time, and ethical considerations have led to calls for alternative in vivo ...

Using Computer-based Image Analysis to Improve Quantification of Lung Metastasis in the 4T1 Breast Cancer Model

Breast cancer is a devastating malignancy, accounting for 40,000 female deaths and 30% of new female cancer diagnoses in the United States in 2019 alone. The leading cause of breast cancer related deaths is the metastatic burden. Therefore, preclinical models for breast cancer need to analyze metastatic burden to be clinically relevant. The 4T1 breast cancer model provides a spontaneously-metastasizing, quantifiable mouse model for stage IV human breast cancer. However, most 4T1 protocols quantify the metastatic burden by manually counting stained colonies on tissue culture plates. While this is sufficient for tissues with lower metastatic burden, human error in manual counting causes inconsistent and variable results when plates are confluent and difficult to count. This method offers a computer-based solution to human counting error. Here, we evaluate the protocol using the lung, a highly metastatic tissue in the 4T1 model. Images of methylene blue-stained plates are acquired and uploaded for analysis in Fiji-ImageJ. Fiji-ImageJ then determines the percentage of the selected area of the image that is blue, representing the percentage of the plate with metastatic burden. This computer-based approach offers more consistent and expeditious results than manual counting or histopathological evaluation for highly metastatic tissues. The consistency of Fiji-ImageJ results depends on the quality of the image. Slight variations in results between images can occur, thus it is recommended that multiple images are taken and results averaged. Despite its minimal limitations, this method is an improvement to quantifying metastatic burden in the lung by offering consistent and rapid results.

We describe a more consistent and expeditious method to quantify lung metastasis in the 4T1 breast cancer model by using Fiji-ImageJ.

利用计算机图像分析提高4T1乳腺癌模型中肺转移的定量

Breast cancer is a devastating malignancy, accounting for 40,000 female deaths and 30% of new female cancer diagnoses in the United States in 2019 alone. ...

Modeling Breast Cancer in Human Breast Tissue using a Microphysiological System

Breast cancer (BC) remains a leading cause of death for women. Despite more than $700 million invested in BC research annually, 97% of candidate BC drugs fail clinical trials. Therefore, new models are needed to improve our understanding of the disease. The NIH Microphysiological Systems (MPS) program was developed to improve the clinical translation of basic science discoveries and promising new therapeutic strategies. Here we present a method for generating MPS for breast cancers (BC-MPS). This model adapts a previously described approach of culturing primary human white adipose tissue (WAT) by sandwiching WAT between adipose-derived stem cell sheets (ASC)s. Novel aspects of our BC-MPS include seeding BC cells into non-diseased human breast tissue (HBT) containing native extracellular matrix, mature adipocytes, resident fibroblasts, and immune cells; and sandwiching the BC-HBT admixture between HBT-derived ASC sheets. The resulting BC-MPS is stable in culture ex vivo for at least 14 days. This model system contains multiple elements of the microenvironment that influence BC including adipocytes, stromal cells, immune cells, and the extracellular matrix. Thus BC-MPS can be used to study the interactions between BC and its microenvironment. 
We demonstrate the advantages of our BC-MPS by studying two BC behaviors known to influence cancer progression and metastasis: 1) BC motility and 2) BC-HBT metabolic crosstalk. While BC motility has previously been demonstrated using intravital imaging, BC-MPS allows for high-resolution time-lapse imaging using fluorescence microscopy over several days. Furthermore, while metabolic crosstalk was previously demonstrated using BC cells and murine pre-adipocytes differentiated into immature adipocytes, our BC-MPS model is the first system to demonstrate this crosstalk between primary human mammary adipocytes and BC cells in vitro.

This protocol describes the construction of an in vitro microphysiological system for studying breast cancer using primary human breast tissue with off the shelf materials.

使用微生理系统模拟人体乳房组织中的乳腺癌

Breast cancer (BC) remains a leading cause of death for women. Despite more than $700 million invested in BC research annually, 97% of candidate BC drugs ...

A Murine Ommaya Xenograft Model to Study Direct-Targeted Therapy of Leptomeningeal Disease

Leptomeningeal disease (LMD) is an uncommon type of central nervous system (CNS) metastasis to the cerebral spinal fluid (CSF). The most common cancers that cause LMD are breast and lung cancers and melanoma. Patients diagnosed with LMD have a very poor prognosis and generally survive for only a few weeks or months. One possible reason for the lack of efficacy of systemic therapy against LMD is the failure to achieve therapeutically effective concentrations of drug in the CSF because of an intact and relatively impermeable blood-brain barrier (BBB) or blood-CSF barrier across the choroid plexus. Therefore, directly administering drugs intrathecally or intraventricularly may overcome these barriers. This group has developed a model that allows for the effective delivery of therapeutics (i.e., drugs, antibodies, and cellular therapies) chronically and the repeated sampling of CSF to determine drug concentrations and target modulation in the CSF (when the tumor microenvironment is targeted in mice). The model is the murine equivalent of a magnetic resonance imaging-compatible Ommaya reservoir, which is used clinically. This model, which is affixed to the skull, has been designated as the &#34;Murine Ommaya.&#34; As a therapeutic proof of concept, human epidermal growth factor receptor 2 antibodies (clone 7.16.4) were delivered into the CSF via the Murine Ommaya to treat mice with LMD from human epidermal growth factor receptor 2-positive breast cancer. The Murine Ommaya increases the efficiency of drug delivery using a miniature access port and prevents the wastage of excess drug; it does not interfere with CSF sampling for molecular and immunological studies. The Murine Ommaya is useful for testing novel therapeutics in experimental models of LMD.

Here, we describe a murine xenograft model that functionally resembles an Ommaya reservoir in patients. We developed the Murine Ommaya to study novel therapeutics for the universally fatal leptomeningeal disease.

研究利普托梅宁格疾病直接靶向治疗的穆林·奥马亚异种格拉夫特模型

Leptomeningeal disease (LMD) is an uncommon type of central nervous system (CNS) metastasis to the cerebral spinal fluid (CSF). The most common cancers ...