This work was supported by the NIH grant (5R01 CA178386-03) and the USC ASPIRE-1 grant to HC. We would like to thank Vrushab Gowda for his assistance with manuscript.

这项研究是经全南大学和顺医院的机构审查委员会和南卡罗来纳大学。乳腺癌旁正常乳腺组织组成的TMA样品进行了全南国立大学和顺医院，韩国生物库网络的成员的生物银行提供的。 1.溶液的制备<ol><li>准备1升DNA酶和RNA酶自由水。请使用酸二乙酯（DEPC）处理过的水的所有化学解决方案。治疗1升水中，用500μL的DEPC孵育在室温下3小时。高压灭菌水以灭活DEPC。 注意:不要吸入DEPC因为它是已知的致癌物质。 </li><li>用DEPC处理过的水从10倍PBS准备1X PBS。 </li><li>制备20×SSC（溶解175.3克NaCl和在800毫升水88.2克柠檬酸钠，用盐酸的14 N溶液几滴调节pH至7.0并定容至1L用笏尔），4％多聚甲醛，95％乙醇，80％的乙醇用DEPC处理过的水。 </li><li>加入8毫升三乙醇胺和1.05 590毫升DEPC处理过的水的毫升盐酸，并添加1.5毫升乙酸酐乙酰化。 </li><li>在缓冲液制备50微克/微升蛋白酶K（50毫摩尔Tris盐酸，于DEPC pH 8.0的处理过的水）。 </li><li>制备的500微升甲酰胺，250微升20×SSC，100微升的50×Denhardt氏溶液，12.5微升叔RNA的，2.5微升鲱鱼精DNA，RVC 30微升和0.02克封闭试剂和50微升的组成的杂交缓冲DEPC的水。准备好新鲜。 </li></ol> 2.乳腺组织切片<ol><li>切5μm的福尔马林固定，石蜡包埋切片用切片机和适用于充电幻灯片14上的部分。 注意:这里使用的乳腺组织是从韩国生物库网络事先准备好的。 </li></ol> 3.组织准备<ol><li>除去来回石蜡浸渍滑入科普林缸2倍在新鲜的二甲苯10分钟每m为组织和再水合5分钟孵育的组织切片中在DDH 2 O递减的乙醇（100％，95％和80％）浓度执行在通风橱中此步骤。 </li><li>沉浸于DEPC所述组织处理水5分钟。在这一点上使周围疏水屏障笔组织切片边界。 </li><li>固定用4％PFA的组织15分钟，随后用PBS洗涤。 </li><li>浸入所述组织中的0.3％的Triton X-100 / PBS中进行10分钟，随后用PBS洗涤。 </li><li>孵育样品与在37℃下50微克/微升蛋白酶K溶液15分钟。然后孵育用三乙醇胺和乙酸酐的组织切片，在室温下5分钟，随后用PBS洗涤。没有的PBS洗涤是必需的蛋白酶K处理和三乙醇胺/乙酸酐处理之间。 </li></ol> 4.杂交<ol><li>彻底洗净INCU组织软化的组织切片在室温下用杂交缓冲液进行2至3小时。通常为120微升杂交缓冲液是足以覆盖组织切片。 </li><li>稀释5&#39;-DIG标记LNA探针在120微升杂交液15:00的终浓度。作为原料浓度通常为40纳克/微升，加入3微升的库存120微升杂交缓冲液，并在95℃下孵育混合物5分钟。 </li><li>在潮湿室孵育在54°C（Tm）的带探头一夜之间组织切片。通常情况下120微升足以覆盖一个部分。通过使用两个湿纸巾创建湿度。室的理想的尺寸是8英寸10英寸。 </li><li>注意:Tm为可能是不同的miRNA不同。 54℃是用于提的miR-489。 </li></ol> 5.严格清洗<ol><li>洗5倍SSC组织7分钟（杂交步骤后，RNA酶条件是不再需要）。 </li><li>洗净组织切片机智^ h 1X SSC在57℃7分钟两次。 </li><li>在57℃下接触，用0.2X SSC组织切片7分钟。 </li><li>洗用0.2×SSC的组织切片，在室温下7分钟。 </li><li>孵育在PBS组织切片10分钟。 </li></ol> 6.阻塞<ol><li>孵育所述组织部分与在潮湿室（为了使​​封闭缓冲液中加2ml胎牛血清，10微升吐温20和3微升的BSA在18米PBS中。保存在4封闭缓冲液阻断在室温下缓冲液1小时 C）。 </li></ol> 7.一抗孵化和发展<ol><li>稀释1:300的抗DIG-AP抗体在封闭缓冲液并在4℃孵育用抗体的组织切片过夜。 </li><li>用洗涤缓冲液洗切片3次，每次5分钟（通过显影试剂盒中提供缓冲液）。 </li><li>开发与碱性磷酸酶商业试剂盒产生CY-3荧光灯的组织切片。 </li> &lt;利&gt;清洗用洗涤缓冲液的部分5分钟。染色部用100μl2.5毫微克/微升的Hoechst染料的5分钟，然后用5分钟洗涤用洗涤缓冲液。 </li><li>安装具有根据制造商的协议安装缓冲器的部分。使用每个组织切片15微升，并擦拭组织的过度安装溶液擦拭。密封采用透明指甲油，以防止泄露的幻灯片。 </li><li>观察与10X镜头所以总放大倍率使用部分荧光显微镜100X。 </li></ol>

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There is nothing to disclose.

本研究的目的是确定在原位杂交使用的miRNA乳腺癌组织的miRNA的表达水平。必须指出，在该实验中，所有条件为乳腺癌组织优化。进一步的优化可能需要的其它类型的组织。所有解决方案必须用DEPC水进行，也可以使用所有的容器应用DEPC处理过的水冲洗，随后高压灭菌。即使是轻微的RNase污染可以用实验的最终结果产生干扰。作为miRNA的是一个非常短的序列，其有必要使用的LNA探针，以便于与...

乳腺癌是影响世界各地的女性人口中最常见的恶性肿瘤之一。超过130万例乳腺癌，每年全世界1,2报告。虽然肿瘤细胞已被传统上被视为生物学均匀和高度增殖，它已成为明显的是，乳腺癌是遗传和临床异质性。耐流行抗癌药物是先进乳腺癌的标志，导致通过它的癌症进展和远处转移3的便利在大多数患者的死亡率。 微RNA（miRNAs）是一类通过阻断mRNA翻译或介导的mRNA降解4调节基因表达的小RNA分子约22个核苷酸长的。超过2000种不同的miRNA迄今已在人类中，它们与人类疾病5识别。越来越的研究阵列甲肝Ë证实的miRNA可以作为癌基因和肿瘤抑制基因的功能，并且经常在肿瘤中6失调。通过控制细胞周期进程，衰老，凋亡和自噬的主要调节的miRNA强烈影响所有初级肿瘤发生的，与这些肿瘤细胞运动性，侵入和转移7的沿。 miRNA表达异常，已与临床意义，如分期，分化程度，预后和应对辅助治疗8也被相关。特别是，增加的miR-146表达与肺癌患者9预后差相关。另一项研究已经证明了miRNA-let7b失去表达是与恶性表型，因此，生存差6。理解表达的miRNA在细胞类型和结构是理解机制的重要组成部分，通过它miRNA表达影响细胞改变和组织表型10。发现在基质细胞被分泌某些miRNA的取在由上皮细胞并在它们的靶标特异性作用。本着同样的精神中，miR-212和miR-132是由间质分泌和调节乳腺发育11期间导管生长所需的上皮-间质相互作用。本文的总体目标是解释一个详细的协议，并通过miRNA的原位杂交技术检测乳腺癌组织中的miRNA表达。我们优化所有条件以测定乳腺癌患者样品中的miRNA-489的表达水平。为此，我们使用标有DIG在我们的实验锁核酸（LNA）探针。它的一些核苷酸的有一个LNA修饰，即，亚甲基桥连接的2&#39;氧原子和4&#39;核糖主链固定，使得它保持"锁定就位"杂交后的碱基的碳原子上。其结果，这是更为困难用于杂交复合变性12,13。

<table border="0" cellpadding="0" cellspacing="0" width="661">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">ELF-97</td>
			<td style="width:159px;">Life technology&#160;</td>
			<td style="width:119px;">E6604</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">50X Denhard&#39;t&#160;</td>
			<td style="width:159px;">Life technology&#160;</td>
			<td align="right" style="width:119px;">750018</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">t-RNA</td>
			<td style="width:159px;">Roche</td>
			<td align="right" style="width:119px;">109541</td>
			<td>Reconstitute in DEPC water</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Herring Sperm DNA</td>
			<td style="width:159px;">Promega&#160;</td>
			<td style="width:119px;">D1815</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Roche Blocking&#160;</td>
			<td style="width:159px;">Roche</td>
			<td align="right" style="width:119px;">11096176001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">RVC</td>
			<td style="width:159px;">Fisher</td>
			<td style="width:119px;">50-812-650</td>
			<td>Before use spin down it at 16.1 RCF and take supernatant&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">miR-489 probe&#160;</td>
			<td style="width:159px;">Exiqon</td>
			<td style="width:119px;">38599-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Nuclease free BSA</td>
			<td style="width:159px;">Roche&#160;</td>
			<td align="right" style="width:119px;">711454</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Primary antibody-anti DIG</td>
			<td style="width:159px;">Roche&#160;</td>
			<td align="right" style="width:119px;">11093274910</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Diethyl Pyrocarbonate&#160;</td>
			<td style="width:159px;">Sigma</td>
			<td align="right" style="width:119px;">1609478</td>
			<td></td>
		</tr>
	</tbody>
</table>

clinicopathological analysis of mirna expression in breast cancer tissues by using mirna in situ hybridization

In this article, we describe a detailed protocol for miRNA detection in breast cancer tissue using in situ hybridization with a digoxigenin-labelled LNA (Locked Nucleic Acid) probe. The probe was recognized by anti-DIG alkaline phosphatase antibodies and later developed using alkaline phosphatase substrate producing fluorescence signals. Here we utilized miRNA in situ hybridization (MISH) technique to analyze expression of miR-489 in tissues from breast cancer patients. This technique can detect the localization of miRNA of interest in individual tissue samples. This technique can be used to compare the expression of desired miRNA in tumor tissue with that in adjacent normal tissue and to identify the specific structures responsible for expressing this miRNA. This technique can be very useful in answering certain clinical questions, such as role of specific miRNA in the development of cancer. Our results indicate that mammary epithelial cells express significantly higher levels of miR-489 than adjacent tumor cells.

人乳腺癌组织用于测定的miRNA-489的表达。乳腺腺管和上皮细胞被发现表达显著较高的miRNA-489比两个相邻的患者肿瘤组织（1B 图1A和）的水平。这清楚地表明了miRNA-489的表达已在肿瘤组织中丢失了，表明miRNA的-489的肿瘤抑制活性。为了进一步证实来自同一患者这些结果，肿瘤组织和邻近的正常组织进行比较。如在图2A和2B观?...

Watch this Scientific Journal Video about Clinicopathological Analysis of miRNA Expression in Breast Cancer Tissues by Using miRNA In Situ Hybridization at JoVE.com

Clinicopathological Analysis of miRNA Expression in Breast Cancer Tissues by Using miRNA In Situ Hybridization

Here we present a protocol to detect miRNA expression in breast cancer patient samples using miRNA in situ hybridization.

利用miRNA的miRNA表达的临床病理分析乳腺癌组织<em&gt;原位</em&gt;杂交

In this article, we describe a detailed protocol for miRNA detection in breast cancer tissue using in situ hybridization with a digoxigenin-labelled LNA ...

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Clinicopathological Analysis of miRNA Expression in Breast Cancer Tissues by Using miRNA In Situ Hybridization

6.7K 次观看.  University of South Carolina. Here we present a protocol to detect miRNA expression in breast cancer patient samples using miRNA in situ hybridization.

视频: Clinicopathological Analysis of miRNA Expression in Breast Cancer Tissues by Using miRNA In Situ Hybridization

Oral Biofilm Analysis of Palatal Expanders by Fluorescence In-Situ Hybridization and Confocal Laser Scanning Microscopy

Confocal laser scanning microscopy (CLSM) of natural heterogeneous biofilm is today facilitated by a comprehensive range of staining techniques, one of them being fluorescence in situ hybridization (FISH).1,2 We performed a pilot study in which oral biofilm samples collected from fixed orthodontic appliances (palatal expanders) were stained by FISH, the objective being to assess the three-dimensional organization of natural biofilm and plaque accumulation.3,4 FISH creates an opportunity to stain cells in their native biofilm environment by the use of fluorescently labeled 16S rRNA-targeting probes.4-7,19 Compared to alternative techniques like immunofluorescent labeling, this is an inexpensive, precise and straightforward labeling technique to investigate different bacterial groups in mixed biofilm consortia.18,20 General probes were used that bind to Eubacteria (EUB338 + EUB338II + EUB338III; hereafter EUBmix),8-10 Firmicutes (LGC354 A-C; hereafter LGCmix),9,10 and Bacteroidetes (Bac303).11 In addition, specific probes binding to Streptococcus mutans (MUT590)12,13 and Porphyromonas gingivalis (POGI)13,14 were used. The extreme hardness of the surface materials involved (stainless steel and acrylic resin) compelled us to find new ways of preparing the biofilm. As these surface materials could not be readily cut with a cryotome, various sampling methods were explored to obtain intact oral biofilm. The most workable of these approaches is presented in this communication. Small flakes of the biofilm-carrying acrylic resin were scraped off with a sterile scalpel, taking care not to damage the biofilm structure. Forceps were used to collect biofilm from the steel surfaces. Once collected, the samples were fixed and placed directly on polysine coated glass slides. FISH was performed directly on these slides with the probes mentioned above. Various FISH protocols were combined and modified to create a new protocol that was easy to handle.5,10,14,15 Subsequently the samples were analyzed by confocal laser scanning microscopy. Well-known configurations3,4,16,17 could be visualized, including mushroom-style formations and clusters of coccoid bacteria pervaded by channels. In addition, the bacterial composition of these typical biofilm structures were analyzed and 2D and 3D images created.

We present a protocol for structural and compositional analysis of natural oral biofilm from orthodontic appliances with in situ hybridization (FISH) and confocal laser scanning microscopy (CLSM). Oral biofilm samples were collected from palatal expanders, scraping acrylic-resin flakes off their surface and referring them for molecular processing.

腭扩展口腔生物膜分析，荧光原位杂交和激光共聚焦显微镜

Confocal laser scanning microscopy (CLSM) of natural heterogeneous biofilm is today facilitated by a comprehensive range of staining techniques, one of ...

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 ...

Quantification of Breast Cancer Cell Invasiveness Using a Three-dimensional (3D) Model

It is now well known that the cellular and tissue microenvironment are critical regulators influencing tumor initiation and progression. Moreover, the extracellular matrix (ECM) has been demonstrated to be a critical regulator of cell behavior in culture and homeostasis in vivo. The current approach of culturing cells on two-dimensional (2D), plastic surfaces results in the disturbance and loss of complex interactions between cells and their microenvironment. Through the use of three-dimensional (3D) culture assays, the conditions for cell-microenvironment interaction are established resembling the in vivo microenvironment. This article provides a detailed methodology to grow breast cancer cells in a 3D basement membrane protein matrix, exemplifying the potential of 3D culture in the assessment of cell invasion into the surrounding environment. In addition, we discuss how these 3D assays have the potential to examine the loss of signaling molecules that regulate epithelial morphology by immunostaining procedures. These studies aid to identify important mechanistic details into the processes regulating invasion, required for the spread of breast cancer.

Quantification of Breast Cancer Cell Invasiveness Using a Three-dimensional 3D Model

This article provides detailed methodologies for the use of three-dimensional (3D) assays to quantify breast cancer cell invasion. Specifically, we discuss the procedures required to set up such assays, quantification, and data analysis, as well as methods to examine the loss of membrane integrity that occurs when cells invade.

乳腺癌细胞侵袭定量使用三维（3D）模型

It is now well known that the cellular and tissue microenvironment are critical regulators influencing tumor initiation and progression. Moreover, the ...

Carotid Artery Infusions for Pharmacokinetic and Pharmacodynamic Analysis of Taxanes in Mice

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study model. As the use of mouse models are often a vital step in preclinical drug discovery and drug development1-8, it is necessary to design a system to introduce drugs into mice in a uniform, reproducible manner. Ideally, the system should permit the collection of blood samples at regular intervals over a set time course. The ability to measure drug concentrations by mass-spectrometry, has allowed investigators to follow the changes in plasma drug levels over time in individual mice1, 9, 10. In this study, paclitaxel was introduced into transgenic mice as a continuous arterial infusion over three hours, while blood samples were simultaneously taken by retro-orbital bleeds at set time points. Carotid artery infusions are a potential alternative to jugular vein infusions, when factors such as mammary tumors or other obstructions make jugular infusions impractical. Using this technique, paclitaxel concentrations in plasma and tissue achieved similar levels as compared to jugular infusion. In this tutorial, we will demonstrate how to successfully catheterize the carotid artery by preparing an optimized catheter for the individual mouse model, then show how to insert and secure the catheter into the mouse carotid artery, thread the end of the catheter out through the back of the mouse&#8217;s neck, and hook the mouse to a pump to deliver a controlled rate of drug influx. Multiple low volume retro-orbital bleeds allow for analysis of plasma drug concentrations over time.

This method was developed with the goal of delivering a steady drug solution via the carotid artery, to assess the pharmacokinetics of novel drugs in mouse models.

颈动脉输液的紫杉烷类化合物在小鼠体内的药代动力学和药效学分析

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study ...

Mammosphere Formation Assay from Human Breast Cancer Tissues and Cell Lines

Similar to healthy tissues, many blood and solid malignancies are now thought to be organised hierarchically, with a subset of stem-like cancer cells that self-renew while giving rise to more differentiated progeny. Understanding and targeting these cancer stem cells in breast cancer, which may possess enhanced chemo- and radio-resistance compared to the non-stem tumor bulk, has become an important research area. Markers including CD44, CD24, and ALDH activity can be assessed using fluorescence activated cell sorting (FACS) to prospectively isolate cells that display enhanced tumorigenicity when implanted into immunocompromised mice: the mammosphere assay has also become widely used for its ability to retrospectively identify sphere-forming cells that develop from single stem cell-like clones. Here we outline approaches for the appropriate culturing of mammospheres from cell lines or primary patient samples, their passaging, and calculations to estimate sphere forming efficiency (SFE). First we discuss key considerations and pitfalls in the appropriate planning and interpretation of mammosphere experiments.

Floating mammosphere assays can investigate the subset of stem-like breast cancer cells that survive in suspension conditions and show enhanced tumorigenesis when implanted into mice. This protocol provides a convenient in vitro measure of sphere-forming ability, a proxy for in vivo tumorigenesis, while facilitating analysis of the stem-associated transcriptional landscape.

从人乳腺癌组织及细胞系微球体形成分析

Similar to healthy tissues, many blood and solid malignancies are now thought to be organised hierarchically, with a subset of stem-like cancer cells that ...

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 ...

Studying Pancreatic Cancer Stem Cell Characteristics for Developing New Treatment Strategies

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor initiation, metastasis and resistance to radio- and chemotherapy. Here we describe a specific methodology for culturing primary human pancreatic CSCs as tumor spheres in anchorage-independent conditions. Cells are grown in serum-free, non-adherent conditions in order to enrich for CSCs while their more differentiated progenies do not survive and proliferate during the initial phase following seeding of single cells. This assay can be used to estimate the percentage of CSCs present in a population of tumor cells. Both size (which can range from 35 to 250 micrometers) and number of tumor spheres formed represents CSC activity harbored in either bulk populations of cultured cancer cells or freshly harvested and digested tumors 1,2. Using this assay, we recently found that metformin selectively ablates pancreatic CSCs; a finding that was subsequently further corroborated by demonstrating diminished expression of pluripotency-associated genes/surface markers and reduced in vivo tumorigenicity of metformin-treated cells. As the final step for preclinical development we treated mice bearing established tumors with metformin and found significantly prolonged survival. Clinical studies testing the use of metformin in patients with PDAC are currently underway (e.g., NCT01210911, NCT01167738, and NCT01488552). Mechanistically, we found that metformin induces a fatal energy crisis in CSCs by enhancing reactive oxygen species (ROS) production and reducing mitochondrial transmembrane potential. In contrast, non-CSCs were not eliminated by metformin treatment, but rather underwent reversible cell cycle arrest. Therefore, our study serves as a successful example for the potential of in vitro sphere formation as a screening tool to identify compounds that potentially target CSCs, but this technique will require further in vitro and in vivo validation to eliminate false discoveries.

Pancreatic cancer stem cells (CSCs) can be expanded in vitro using the anchorage-independent sphere culture technique, which represents a powerful tool to study CSC biology and can serve as the first step to develop novel CSC-targeting therapies. Here the methodology for expanding, analyzing and targeting of pancreatic CSCs is provided.

研究胰腺癌干细胞特性开发新的治疗策略

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor ...

miRNA Expression Analyses in Prostate Cancer Clinical Tissues

A critical challenge in prostate cancer (PCa) clinical management is posed by the inadequacy of currently used biomarkers for disease screening, diagnosis, prognosis and treatment. In recent years, microRNAs (miRNAs) have emerged as promising alternate biomarkers for prostate cancer diagnosis and prognosis. However, the development of miRNAs as effective biomarkers for prostate cancer heavily relies on their accurate detection in clinical tissues. miRNA analyses in prostate cancer clinical specimens is often challenging owing to tumor heterogeneity, sampling errors, stromal contamination etc. The goal of this article is to describe a simplified workflow for miRNA analyses in archived FFPE or fresh frozen prostate cancer clinical specimens using a combination of quantitative real-time PCR (RT-PCR) and in situ hybridization (ISH). Within this workflow, we optimize the existing methodologies for miRNA extraction from FFPE and frozen prostate tissues and expression analyses by Taqman-probe based miRNA RT-PCR. In addition, we describe an optimized method for ISH analyses formiRNA detection in prostate tissues using locked nucleic acid (LNA)- based probes. Our optimized miRNA ISH protocol can be applied to prostate cancer tissue slides or prostate cancer tissue microarrays (TMA).

Here we describe a simplified protocol for microRNA (miRNA) expression analyses in archived Formalin-Fixed, Paraffin-Embedded (FFPE) or fresh frozen prostate cancer (PCa) clinical tissues employing quantitative real-time PCR (RT-PCR) and in situ hybridization (ISH).

在前列腺癌的临床组织中miRNA的表达分析

A critical challenge in prostate cancer (PCa) clinical management is posed by the inadequacy of currently used biomarkers for disease screening, ...

Systems Biology of Metabolic Regulation by Estrogen Receptor Signaling in Breast Cancer

With the advent of the -omics approaches our understanding of the chronic diseases like cancer and metabolic syndrome has improved. However, effective mining of the information in the large-scale datasets that are obtained from gene expression microarrays, deep sequencing experiments or metabolic profiling is essential to uncover and then effectively target the critical regulators of diseased cell phenotypes. Estrogen Receptor &#945; (ER&#945;) is one of the master transcription factors regulating the gene programs that are important for estrogen responsive breast cancers. In order to understand to role of ER&#945; signaling in breast cancer metabolism we utilized transcriptomic, cistromic and metabolomic data from MCF-7 cells treated with estradiol. In this report we described generation of samples for RNA-Seq, ChIP-Seq and metabolomics experiments and the integrative computational analysis of the obtained data. This approach is useful in delineating novel molecular mechanisms and gene regulatory circuits that are regulated by a particular transcription factor which impacts metabolism of normal or diseased cells.

Integration of data from genome-wide sequencing experiments and metabolomics experiments is a challenge. In this paper we report, for the first time, generation, analysis and integration of transcriptome, cistrome and metabolome data from breast cancer cells treated with estradiol.

乳腺癌代谢调控的系统生物学雌激素受体信号

With the advent of the -omics approaches our understanding of the chronic diseases like cancer and metabolic syndrome has improved. However, effective ...