This study was also financed by MOCA (Multidisciplinair Oncologisch Centrum Antwerpen) grant 2014.

1.外来体隔离注意:从血浆样品外来体的分离，根据制造商的协议，并通过加入RNA酶处理与市售的试剂盒进行的，（所有这些步骤必须以个人和环境的保护设备及以下对生物样品的操纵特定的法律程序执行）。 <ol><li>取各1ml样品的血浆，保存于-80℃，并放置在冰上。 </li><li>离心样品以2,000×g下在室温下20分钟;这段话是为了去除细胞和碎片强制性的。 </li><li>传送包含清洁等离子体上清至新的1.5毫升管。 </li><li>离心新管以10,000×g下在室温下20分钟。 </li><li>传送包含清洁等离子体上清至新的1.5毫升管。 </li><li>在37℃下为100ng / ml RNA酶的添加到上清液孵育管为在10分钟内为了降低循环的RNA。 </li><li>传输所有处理的血浆，以新的2ml管中，加入0.5倍体积的1X PBS的。 </li><li>涡样品以混合溶液。 </li><li>添加0.05体积的蛋白酶K溶液（括在试剂盒中）到样品中。 </li><li>涡旋样品，然后在37℃下进行10分钟。 </li><li>添加0.2体积外来体沉淀缓冲液的样品，并通过倒置混合该溶液。 </li><li>在2℃孵育样品8℃下进行30分钟，然后在10,000×g下离心样品在RT 5分钟。 </li><li>吸弃上清。沉淀包含隔离外来体。 </li><li>选择的缓冲液添加到外来体颗粒以悬浮外来体。缓冲区的选择取决于计划的下游分析，在这种情况下: <ol><li>添加100μl的1×PBS中的为了进行TEM分析。 </li><li>加入346.5微升裂解特定的BUFFER提取RNA（从商业套件）（注意:化学危险品）。 </li><li>加入100微升用于蛋白提取的裂解缓冲液（Tris-盐酸50mM的pH值7.6 - 氯化钠300mM的 - 的Triton X-100 0.5％ - PMSF 1mM的 - 亮抑酶肽/抑肽酶10微克/毫升），以便执行Western印迹分析。 （注意:化学危险品）。 </li></ol></li><li>储存在-20℃的悬浮外来体。 </li></ol> 2.外来体表征蛋白质印迹分析注意:为了表征的分离的纳米囊泡，用抗ALIX和TSG101（公知的外来体标记）抗体western印迹建议。 <ol><li>在裂解缓冲液外来体颗粒再悬浮后，将其放置在冰上1小时，以溶解该外来体膜。 </li><li>离心溶胞产物以12,000×g进行10分钟，将上清转移至新管，而不会干扰沉淀。 </li><li>量化的总蛋白质外体conten通过Bradford法或其他方法T和负载于Tris /甘氨酸SDS每孔蛋白的50微克 - 8％聚丙烯酰胺凝胶。 （注意:化学危险品）。 </li><li>分离在8％SDS-PAGE凝胶电泳的蛋白（和适当的分子量标记）与运行缓冲液（25mM的Tris，192毫甘氨酸，0.1％SDS）中1小时，并在120伏。30分钟（注意:化学危害）。 </li><li> （:化学危险警告）通过用4℃下在恒定的50伏1小时Ç冷转移缓冲液（25mM的Tris，192毫甘氨酸，20％甲醇）电印迹转移的蛋白质到硝酸纤维素膜上。 </li><li>在缓慢搅拌以在1×TBS中-Tween 0.1％5％无脂干牛奶1小时阻断膜。 </li><li>洗膜4次，快速搅拌5分钟用1X TBS - 吐温0.1％。 </li><li>添加抗ALIX和TSG101（每膜一个）在适当的缓冲液中的一抗（相应地从制造商的抗体数据表），并孵育膜O / N;缓慢搅拌。 </li><li>洗膜在快速搅拌下用1×TBS 5分钟5次 - 吐温0.1％，以除去过量的抗体。 </li><li>适当的第二抗体加入适当的缓冲液（相应地从制造商的抗体数据表），并孵育所述膜在缓慢搅拌1小时。 </li><li>洗膜在快速搅拌下用1×TBS 5分钟5次 - 吐温0.1％，以除去过量的抗体。 </li><li>检测化学发光分析11所选择的蛋白质。 </li></ol> 3.外来体表征TEM分析注意:为了形象化所述分离的外泌体，进行TEM（透射电子显微镜）分析。 <ol><li>广场〜10微克悬浮在1X PBS上的封口膜完整的外来的样品（以前由Bradford分析量化），并把在样品上滴聚乙烯醇缩甲醛碳包镍网格1小时。 </li> &lt;LI&gt; 1倍PBS滴把它定​​位在三个40微升洗电网的3倍。 </li><li>通过定位其在五个30微升超纯水滴洗网格5倍。 </li><li> 10分钟:通过添加2.5％戊二醛（化学危险小心）的下降修复样本。 </li><li> 1倍PBS滴把它定​​位在三个40微升洗电网的3倍。 </li><li>添加2％醋酸铀下降（注意:化学危险品），并把它的顶部电网15分钟，以对比样本。 </li><li>嵌入0.13％甲基纤维素的一滴样品（注意:化学危险）和0.4％乙酸双氧铀和孵化的下拉10分钟的顶部的网格。 </li><li>轻轻取出液体过量的一吸收纸和干燥格5分钟与涂布的一面朝上。 </li><li>审查通过透射型电子显微镜12内的网格。 </li></ol> 4.外来体RNA提取和定量注意:我们决定为PErFORM从使用商业试剂盒外来体的样品的总RNA提取，根据制造商的协议:（注意:化学危险，审查该制造商的协议和准则）。 <ol><li>在346.5微升裂解液的外来体颗粒再悬浮后，加入3.5微升β巯基乙醇（注意:化学危险品）和涡大力;这段话是为了破坏脂膜强制性的。 </li><li>裂解物添加到在一个收集管和离心洗涤塔在11000×g的1分钟。离心后，弃去柱，将滤液转移到一个新的1.5毫升的无RNase的管。 </li><li>添加350微升70％乙醇（小心:化学危险），并通过涡旋5秒混合。 </li><li>加载在解决列，离心裂解液在8000×g下30秒。离心后，弃去流过液和柱转移至新的收集管。 </li><li>加载350微升脱盐buffe的r和离心机在11000×g的1分钟。离心后，弃去流穿，并返回该列到同一个收集管。 </li><li>上塔的二氧化硅膜中添加95微升DNA酶I反应混合物（10微升重组DNA酶I + 90微升DNA酶缓冲液中，对于每个样品），并在室温在室温下孵育15分钟。 （注意:化学危险品） </li><li>在11000×g下:（化学危险警告），以列和离心1分钟加入洗涤缓冲液200微升。放置柱到一个新的收集管。 </li><li>在11000×g下添加600微升洗涤缓冲液对柱子和离心1分钟。离心后，弃去流过液和列放置到相同收集管。 </li><li>在11000×g下加入250微升洗涤缓冲液对柱子和离心2分钟。将列到1.5毫升的无RNA酶管。 </li><li>洗脱在11000在40微升RNA酶的水中的RNA和离心机<html> 5;克1分钟，以便在这小体积高的RNA浓度。 </li><li>立即置于冰上洗脱的RNA，以防止在-80℃下潜在的降解或存储。 </li><li>量化RNA含量。注意:在此，将RNA量化用1微升样品的通过分光光度计进行。 </li></ol> 5. Retrotrascription外来体miRNA的注意:由于在外来体的miRNA内容所描述的有限数量，因此决定通过使用用于每个miRNA的个体测定商业试剂盒来执行选择的miRNA的反转录，根据制造商的协议。对于每个样品，8的miRNA相关的非小细胞肺癌的状态被选择（的miR-30b中;的miR-30c中;的miR-103;的miR-122;的miR-195;的miR-203;的miR-221;的miR-222;和miR-1228- 3P为内对照）。 <ol><li>准备反转录主混合物（RT混合物）。每个反应制备7微升RT混合物，正如所描述https://www.jove.com/files/ftp_upload/53900/Table1.xlsx&quot;&gt;Table 1（点击此处下载）（注意:化学危险品）。 注意:在这个实验中，9个不同的RT混合物中每个样品制备。 </li><li>轻轻混匀并储存在冰的RT混合物。新增7微升每个反应管RT混合。 </li><li>加入5微升每个反应管中的样品RNA（总RNA的10微克），轻轻混匀。 </li><li>从每个实验设定到相应的反应管:（化学危险小心）加入3微升的5倍逆转录引物。 </li><li>混合慢，关闭该管，并在冰上孵育5分钟。 </li><li>加载反应管进入热循环，并使用以下参数值（HOLD启动逆转录反应在16℃下30分钟;在42℃下保持30分钟;在85℃下保持5分钟; HOLD∞4 C）。 </li></ol> 6.外来体miRNA的实时定量PCR分析注意:此分析是使用实时定量PCR（qPCR的）市售的试剂盒，每个样品三次生物学和技术重复进行。 <ol><li>对于每一个反应，准备实时PCR混合物的17.67微升（10微升PCR预混+ 7.67μL无核酸酶水的），混合，并置于冰上。 （注意:化学危险品） </li><li>加入1μl，每个PCR管，20倍定量PCR引物（注意:化学危险品）。 </li><li>放置18.67微升的qPCR混合物的各孔板。 </li><li>添加1.33微升反转录产物进入相应孔板。 </li><li>关闭板旋转它（300 GX 5秒）。 </li><li>插入该板在一个实时定量PCR系统和使用以下PCR条件启动运行:HOLD在95℃下进行10分钟; 40个循环的95℃15秒，在60℃下60秒的。 ΔΔCT13 -根据公式2的过程和正常化的数据。 </li></ol>

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All authors declare no competing financial interests.

用该组合协议是可能的从非小细胞肺癌患者血浆执行外来体miRNA的分析。这些数据可反映疾病​​的状态，但是这需要通过进一步研究加以证实。 在这篇文章中所使用的协议是基于切体隔离商业套件。其原因是，其它已知的分离方法（ 例如 ，密度梯度超速离心）需要更多的人工程序，从而导致有限的标准化。然而，该协议的局限性之一是，与用于外来体隔离密度梯度?...

非小细胞肺癌（非小细胞肺癌）患者生存率低的主要原因是在先进的疾病和不良的早期检测1治疗的疗效有限。在EGFR基因活化突变，已在非小细胞肺癌的患者的亚群被发现是已知赋予灵敏度酪氨酸激酶抑制剂（TKI中）。不幸的是，大多数EGFR突变的非小细胞肺癌患者的发展TKI阻力位2。特别是在这种情况下，正确的诊断是必需的。在一般情况下，由于肿瘤的定位，在非小细胞肺癌获得活检组织并不总是可行的。因此，一些研究是使用非侵入性的方法来获得这些生物数据正在进行。外来体被描述为可能，以获得与非侵入性技术3的诊断和预后值进行调查液体活检组件。 外来体是纳米囊泡 - 电子商务（40 100纳米直径）ndocytic原点由既在生理和病理条件4种不同类型的细胞释放。它们可以携带蛋白质，脂质，mRNA和微小RNA。此外，一些研究表明，肿瘤衍生的外来体，其可影响肿瘤细胞，血管生成，基质细胞和胞外基质重构和耐药5的生长和存活的多效性作用。 微RNA（miRNA）是所涉及的转录后调控，结合靶mRNA的3&#39;-UTR以及导致mRNA的降解或到非翻译6短的非编码RNA。 miRNA的选择性排序成外来体已经描述。在这种情况下，最近证实， 在体外和体内中，miR-21（公知的miRNA用致癌效应）的姜黄素治疗7后由慢性髓性白血病细胞系释放外来的选择性包装。 该外来体miRNA谱是类似的原发肿瘤的miRNA谱和这个特征可在早期诊断和预后3被利用。具体地说，是表征，通过实时PCR一种可能性，与疾病的分子轮廓，相关选择的miRNA 例如 ，除其他8 EGFR突变。 这里，描述了非小细胞肺癌相关的miRNA 8-9的选定面板的分析，认为是从非小细胞肺癌患者的血液中释放的外来体分离。获得来自患者的知情同意报告后，12个NSCLC患者和6名健康对照的血浆，进行了分析。外来体分离按照生产商的方案用市售的试剂盒进行。外来体分离之前，血浆样品用RNA酶处理，以降低污染物循环的miRNA。我们决定来执行该分析用市售试剂盒，由于其他TECHNI有限标准化疑问句（ 例如 ，超离心）的临床样品时这是特别重要的。这种试剂盒包含一个蛋白酶K处理，这将降低RNA酶，并因此降低外来体裂解后降解外来体的miRNA的风险。被敏感，特异的实时PCR分析进行分析的microRNA;的mir-1228-3p用作内源的控制和数据进行标准化根据公式2 - ΔΔCT。控制值被用作基准和结果在对数标度示出。

<table border="0" cellpadding="0" cellspacing="0" width="884">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Total exosome isolation kit (from plasma)</td>
			<td style="width:159px;">&#160;Invitrogen</td>
			<td style="width:159px;">4484450</td>
			<td style="width:265px;">Use Proteinase K treatment</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ribonuclease A</td>
			<td>Sigma-Aldrich</td>
			<td>R4875-100MG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ALIX Antibody (3A9)</td>
			<td>Cell Signaling</td>
			<td>2171S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TSG-101 Antibody (C-2)</td>
			<td>SantaCruz Biotecnology</td>
			<td>SC-7964&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-Mouse HRP 1ml&#160;&#160;</td>
			<td>Cell Signaling</td>
			<td>7076P2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RNAspin Illustra mini kit 50</td>
			<td>GE HealthCare</td>
			<td>25-0500-71</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Taqman MicroRNA Reverse Transcription Kit</td>
			<td>Life Technologies</td>
			<td>4366596</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">hsa-miR-1228-3p TaqMan MicroRNA assay</td>
			<td>Life Technologies</td>
			<td>002919</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">hsa-miR-30b TaqMan MicroRNA assay</td>
			<td>Life Technologies</td>
			<td>000602</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">hsa-miR-30c TaqMan MicroRNA assay</td>
			<td>Life Technologies</td>
			<td>000419</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">hsa-miR-122 TaqMan MicroRNA assay</td>
			<td>Life Technologies</td>
			<td>002245</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">hsa-miR-195 TaqMan MicroRNA assay</td>
			<td>Life Technologies</td>
			<td>000494</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">hsa-miR-203 TaqMan MicroRNA assay</td>
			<td>Life Technologies</td>
			<td>000507</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">hsa-miR-103 TaqMan MicroRNA assay</td>
			<td>Life Technologies</td>
			<td>000439</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">hsa-miR-221 TaqMan MicroRNA assay</td>
			<td>Life Technologies</td>
			<td>000524</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">hsa-miR-222 TaqMan MicroRNA assay</td>
			<td>Life Technologies</td>
			<td>002276</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">TaqMan Universal Master Mix II, no UNG</td>
			<td>Life Technologies</td>
			<td>4440043</td>
			<td></td>
		</tr>
	</tbody>
</table>

exosomal mirna analysis in non-small cell lung cancer (nsclc) patients' plasma through qpcr: a feasible liquid biopsy tool

The discovery of alterations in the EGFR and ALK genes, amongst others, in NSCLC has driven the development of targeted-drug therapy using selective tyrosine kinase inhibitors (TKIs). To optimize the use of these TKIs, the discovery of new biomarkers for early detection and disease progression is mandatory. These plasma-isolated exosomes can be used as a non-invasive and repeatable way for the detection and follow-up of these biomarkers. One ml of plasma from 12 NSCLC patients, with different mutations and treatments (and 6 healthy donors as controls), were used as exosome sources. After RNAse treatment, in order to degrade circulating miRNAs, the exosomes were isolated with a commercial kit and resuspended in specific buffers for further analysis. The exosomes were characterized by western blotting for ALIX and TSG101 and by transmission electron microscopy (TEM) analysis, the standard techniques to obtain biochemical and dimensional data of these nanovesicles. Total RNA extraction was performed with a high yield commercial kit. Due to the limited miRNA-content in exosomes, we decided to perform retro-transcription PCR using an individual assay for each selected miRNA. A panel of miRNAs (30b, 30c, 103, 122, 195, 203, 221, 222), all correlated with NSCLC disease, were analyzed taking advantage of the remarkable sensitivity and specificity of Real-Time PCR analysis; mir-1228-3p was used as endogenous control and data were processed according to the formula 2-&#916;&#916;ct 13. Control values were used as baseline and results are shown in logarithmic scale.

共有NSCLC患者12血浆和6名健康对照的与从血浆中分离的外来体商业试剂盒进行了处理。每个样品用蛋白质印迹分析，以评估所述外来体标记ALIX（96 kDa的）和TSG101（43 kDa）的处理。这些结果的一个例子示出了用于在图1 3的样品，这表明从血浆样品外来体隔离与此协议是可行的。 为了表征生物物理的样品外体，进行​...

Watch this Scientific Journal Video about Exosomal miRNA Analysis in Non-small Cell Lung Cancer NSCLC Patients' Plasma Through qPCR: A Feasible Liquid Biopsy Tool at JoVE.com

Exosomal miRNA Analysis in Non-small Cell Lung Cancer NSCLC Patients' Plasma Through qPCR: A Feasible Liquid Biopsy Tool

This protocol describes the feasibility to perform miRNA profiling in exosomes, released in plasma of NSCLC patients, through a commercial exosome isolation kit with Proteinase K and RNAse treatments, in order to avoid circulating miRNAs contamination and evaluate their biomarker features in NSCLC.

外来体miRNA的分析非小细胞肺癌（NSCLC）患者的血浆通过定量PCR:一种可行的液体活检工具

The discovery of alterations in the EGFR and ALK genes, amongst others, in NSCLC has driven the development of targeted-drug therapy using selective ...

exosomal-mirna-analysis-non-small-cell-lung-cancer-nsclc-patients

Research

JoVE Journal

Medicine

Exosomal miRNA Analysis in Non-small Cell Lung Cancer (NSCLC) Patients' Plasma Through qPCR: A Feasible Liquid Biopsy Tool

11.0K 次观看.  University of Palermo. This protocol describes the feasibility to perform miRNA profiling in exosomes, released in plasma of NSCLC patients, through a commercial exosome isolation kit with Proteinase K and RNAse treatments, in order to avoid circulating miRNAs contamination and evaluate their biomarker features in NSCLC.

视频: Exosomal miRNA Analysis in Non-small Cell Lung Cancer NSCLC Patients' Plasma Through qPCR: A Feasible Liquid Biopsy Tool

Multi-photon Imaging of Tumor Cell Invasion in an Orthotopic Mouse Model of Oral Squamous Cell Carcinoma

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient management strategies. Orthotopic mouse models of oral cancer have been developed to facilitate the study of factors that impact invasion and serve as model system for evaluating anti-tumor therapeutics. In these systems, visualization of disseminated tumor cells within oral cavity tissues has typically been conducted by either conventional histology or with in vivo bioluminescent methods. A primary drawback of these techniques is the inherent inability to accurately visualize and quantify early tumor cell invasion arising from the primary site in three dimensions. Here we describe a protocol that combines an established model for squamous cell carcinoma of the tongue (SCOT) with two-photon imaging to allow multi-vectorial visualization of lingual tumor spread. The OSC-19 head and neck tumor cell line was stably engineered to express the F-actin binding peptide LifeAct fused to the mCherry fluorescent protein (LifeAct-mCherry). Fox1nu/nu mice injected with these cells reliably form tumors that allow the tongue to be visualized by ex-vivo application of two-photon microscopy. This technique allows for the orthotopic visualization of the tumor mass and locally invading cells in excised tongues without disruption of the regional tumor microenvironment. In addition, this system allows for the quantification of tumor cell invasion by calculating distances that invaded cells move from the primary tumor site. Overall this procedure provides an enhanced model system for analyzing factors that contribute to SCOT invasion and therapeutic treatments tailored to prevent local invasion and distant metastatic spread. This method also has the potential to be ultimately combined with other imaging modalities in an in vivo setting.

A comprehensive overview of the techniques involved in generating a mouse model of oral cancer and quantitative monitoring of tumor invasion within the tongue through multi-photon microscopy of labeled cells is presented. This system can serve as a useful platform for the molecular assessment and drug efficacy of anti-invasive compounds.

多光子成像在口腔鳞状细胞癌原位小鼠模型的肿瘤细胞的侵袭

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient ...

科研

医学

In vitro Mesothelial Clearance Assay that Models the Early Steps of Ovarian Cancer Metastasis

Ovarian cancer is the fifth leading cause of cancer related deaths in the United States1. Despite a positive initial response to therapies, 70 to 90 percent of women with ovarian cancer develop new metastases, and the recurrence is often fatal2. It is, therefore, necessary to understand how secondary metastases arise in order to develop better treatments for intermediate and late stage ovarian cancer. Ovarian cancer metastasis occurs when malignant cells detach from the primary tumor site and disseminate throughout the peritoneal cavity. The disseminated cells can form multicellular clusters, or spheroids, that will either remain unattached, or implant onto organs within the peritoneal cavity3 (Figure 1, Movie 1). 
All of the organs within the peritoneal cavity are lined with a single, continuous, layer of mesothelial cells4-6 (Figure 2). However, mesothelial cells are absent from underneath peritoneal tumor masses, as revealed by electron micrograph studies of excised human tumor tissue sections3,5-7 (Figure 2). This suggests that mesothelial cells are excluded from underneath the tumor mass by an unknown process. 
Previous in vitro experiments demonstrated that primary ovarian cancer cells attach more efficiently to extracellular matrix than to mesothelial cells8, and more recent studies showed that primary peritoneal mesothelial cells actually provide a barrier to ovarian cancer cell adhesion and invasion (as compared to adhesion and invasion on substrates that were not covered with mesothelial cells)9,10. This would suggest that mesothelial cells act as a barrier against ovarian cancer metastasis. The cellular and molecular mechanisms by which ovarian cancer cells breach this barrier, and exclude the mesothelium have, until recently, remained unknown. 
Here we describe the methodology for an in vitro assay that models the interaction between ovarian cancer cell spheroids and mesothelial cells in vivo (Figure 3, Movie 2). Our protocol was adapted from previously described methods for analyzing ovarian tumor cell interactions with mesothelial monolayers8-16, and was first described in a report showing that ovarian tumor cells utilize an integrin &#x2013;dependent activation of myosin and traction force to promote the exclusion of the mesothelial cells from under a tumor spheroid17. This model takes advantage of time-lapse fluorescence microscopy to monitor the two cell populations in real time, providing spatial and temporal information on the interaction. The ovarian cancer cells express red fluorescent protein (RFP) while the mesothelial cells express green fluorescent protein (GFP). RFP-expressing ovarian cancer cell spheroids attach to the GFP-expressing mesothelial monolayer. The spheroids spread, invade, and force the mesothelial cells aside creating a hole in the monolayer. This hole is visualized as the negative space (black) in the GFP image. The area of the hole can then be measured to quantitatively analyze differences in clearance activity between control and experimental populations of ovarian cancer and/ or mesothelial cells. This assay requires only a small number of ovarian cancer cells (100 cells per spheroid X 20-30 spheroids per condition), so it is feasible to perform this assay using precious primary tumor cell samples. Furthermore, this assay can be easily adapted for high throughput screening.

In vitro Mesothelial Clearance Assay that Models the Early Steps of Ovarian Cancer Metastasis

The mesothelial clearance assay described here takes advantage of fluorescently labeled cells and time-lapse video microscopy to visualize and quantitatively measure the interactions of ovarian cancer multicellular spheroids and mesothelial cell monolayers. This assay models the early steps of ovarian cancer metastasis.

这些模型在体外间皮间隙检测卵巢癌转移的早期步骤

Ovarian cancer is the fifth leading cause of cancer related deaths in the United States1. Despite a positive initial response to therapies, 70 to 90 ...

A Simple Method of Mouse Lung Intubation

A simple procedure to intubate mice for pulmonary function measurements would have several advantages in longitudinal studies with limited numbers or expensive animal. One of the reasons that this is not done more routinely is that it is relatively difficult, despite there being several published studies that describe ways to achieve it. In this paper we demonstrate a procedure that eliminates one of the major hurdles associated with this intubation, that of visualizing the trachea during the entire time of intubation. The approach uses a 0.5 mm fiberoptic light source that serves as an introducer to direct the intubation cannula into the mouse trachea. We show that it is possible to use this procedure to measure lung mechanics in individual mice over a time course of at least several weeks. The technique can be set up with relatively little expense and expertise, and it can be routinely accomplished with relatively little training. This should make it possible for any laboratory to routinely carry out this intubation, thereby allowing longitudinal studies in individual mice, thereby minimizing the number of mice needed and increasing the statistical power by using each mouse as its own control.

This paper describes a striaghforward and efficient method of intubating mice for pulmonary function measurements or pulmonary instillation, that allows the mice to recover and be studied at later times. The procedure involves an inexpensive fiberoptic light source that directly illuminates the trachea.

一个简单的方法小鼠肺插管

A  simple procedure to intubate mice for pulmonary function measurements would  have several advantages in longitudinal studies with limited numbers or  ...

A Murine Model of Myocardial Ischemia-reperfusion Injury through Ligation of the Left Anterior Descending Artery

Acute or chronic myocardial infarction (MI) are cardiovascular events resulting in high morbidity and mortality. Establishing the pathological mechanisms at work during MI and developing effective therapeutic approaches requires methodology to reproducibly simulate the clinical incidence and reflect the pathophysiological changes associated with MI. Here, we describe a surgical method to induce MI in mouse models that can be used for short-term ischemia-reperfusion (I/R) injury as well as permanent ligation. The major advantage of this method is to facilitate location of the left anterior descending artery (LAD) to allow for accurate ligation of this artery to induce ischemia in the left ventricle of the mouse heart. Accurate positioning of the ligature on the LAD increases reproducibility of infarct size and thus produces more reliable results. Greater precision in placement of the ligature will improve the standard surgical approaches to simulate MI in mice, thus reducing the number of experimental animals necessary for statistically relevant studies and improving our understanding of the mechanisms producing cardiac dysfunction following MI. This mouse model of MI is also useful for the preclinical testing of treatments targeting myocardial damage following MI.

We introduce a surgical method to induce experimental ischemia/reperfusion (I/R) injury to simulate myocardial infarction (MI) in mouse models that allows for more clarity in positioning of the ligation on the left anterior descending artery (LAD) to increase the reproducibility of MI experiments in mice.

心肌缺血再灌注损伤的通过左前降支结扎小鼠模型

Acute or chronic myocardial infarction (MI) are cardiovascular events resulting in high morbidity and mortality. Establishing the pathological mechanisms ...

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 ...

Quantitative Mass Spectrometric Profiling of Cancer-cell Proteomes Derived From Liquid and Solid Tumors

In-depth analyses of cancer cell proteomes are needed to elucidate oncogenic pathomechanisms, as well as to identify potential drug targets and diagnostic biomarkers. However, methods for quantitative proteomic characterization of patient-derived tumors and in particular their cellular subpopulations are largely lacking. Here we describe an experimental set-up that allows quantitative analysis of proteomes of cancer cell subpopulations derived from either liquid or solid tumors. This is achieved by combining cellular enrichment strategies with quantitative Super-SILAC-based mass spectrometry followed by bioinformatic data analysis. To enrich specific cellular subsets, liquid tumors are first immunophenotyped by flow cytometry followed by FACS-sorting; for solid tumors, laser-capture microdissection is used to purify specific cellular subpopulations. In a second step, proteins are extracted from the purified cells and subsequently combined with a tumor-specific, SILAC-labeled spike-in standard that enables protein quantification. The resulting protein mixture is subjected to either gel electrophoresis or Filter Aided Sample Preparation (FASP) followed by tryptic digestion. Finally, tryptic peptides are analyzed using a hybrid quadrupole-orbitrap mass spectrometer, and the data obtained are processed with bioinformatic software suites including MaxQuant. By means of the workflow presented here, up to 8,000 proteins can be identified and quantified in patient-derived samples, and the resulting protein expression profiles can be compared among patients to identify diagnostic proteomic signatures or potential drug targets.

In-depth analyses of cancer cell proteomes facilitate identification of novel drug targets and diagnostic biomarkers. We describe an experimental workflow for quantitative analysis of (phospho-)proteomes in cancer cell subpopulations derived from liquid and solid tumors. This is achieved by combining cellular enrichment strategies with quantitative Super-SILAC-based mass spectrometry.

对源自液体和固体肿瘤癌症细胞定量蛋白质组质谱剖析

In-depth analyses of cancer cell proteomes are needed to elucidate oncogenic pathomechanisms, as well as to identify potential drug targets and diagnostic ...

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, ...

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.

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 ...

Phosphorus-31 Magnetic Resonance Spectroscopy: A Tool for Measuring In Vivo Mitochondrial Oxidative Phosphorylation Capacity in Human Skeletal Muscle

Skeletal muscle mitochondrial oxidative phosphorylation (OXPHOS) capacity, which is critically important in health and disease, can be measured in vivo and noninvasively in humans via phosphorus-31 magnetic resonance spectroscopy (31PMRS). However, the approach has not been widely adopted in translational and clinical research, with variations in methodology and limited guidance from the literature. Increased optimization, standardization, and dissemination of methods for in vivo 31PMRS would facilitate the development of targeted therapies to improve OXPHOS capacity and could ultimately favorably impact cardiovascular health. 31PMRS produces a noninvasive, in vivo measure of OXPHOS capacity in human skeletal muscle, as opposed to alternative measures obtained from explanted and potentially altered mitochondria via muscle biopsy. It relies upon only modest additional instrumentation beyond what is already in place on magnetic resonance scanners available for clinical and translational research at most institutions. In this work, we outline a method to measure in vivo skeletal muscle OXPHOS. The technique is demonstrated using a 1.5 Tesla whole-body MR scanner equipped with the suitable hardware and software for 31PMRS, and we explain a simple and robust protocol for in-magnet resistive exercise to rapidly fatigue the quadriceps muscle. Reproducibility and feasibility are demonstrated in volunteers as well as subjects over a wide range of functional capacities.

Phosphorus-31 Magnetic Resonance Spectroscopy: A Tool for Measuring In Vivo Mitochondrial Oxidative Phosphorylation Capacity in Human Skeletal Muscle

This work demonstrates the feasibility of an in vivo phosphorus-31 magnetic resonance spectroscopy (31PMRS) technique to quantify mitochondrial oxidative phosphorylation (OXPHOS) capacity in human skeletal muscle.

磷31磁共振波谱分析:一种测量工具<em&gt;在体内</em&gt;线粒体氧化磷酸化能力人骨骼肌

Skeletal muscle mitochondrial oxidative phosphorylation (OXPHOS) capacity, which is critically important in health and disease, can be measured in vivo ...