我们感谢罗杰·埃里克森博士对他的支持和帮助准备手稿。 这项工作是由美国国家癌症研究所美国国立卫生研究院的支持 （批准号RO1CA177984; RO1CA138642），对前列腺癌（BX001604）VA计划项目。

福尔马林固定，石蜡包埋（FFPE）的或新鲜冷冻的前列腺癌样品从SFVAMC获得。样品从前列腺癌患者在谁SFVAMC接受前列腺癌根治术。书面知情同意，所有患者和研究经加州大学旧金山分校人类研究委员会。可替代地，前列腺癌组织的微阵列从商业来源采购并用于miRNA的分析由ISH。收集临床病理及随访资料分析，前列腺癌患者。 1.组织样本<ol><li>切的前列腺癌组织样品成使用切片机和染色用H＆E按照制造商的协议10微米的部分。 </li><li>检查染色切片用于前列腺癌病灶的标识以及邻近的正常腺上皮。 注:委员会认证的病理学家应检讨H＆E染色切片和马的rk为肿瘤和正常区域。使用标记幻灯片中的miRNA的后续部分导游分析肿瘤与正常区域。 </li></ol> 2. miRNA的表达分析定量实时荧光定量PCR 注意:此流程包括以下步骤:激光捕获显微切割，总RNA（包括miRNA与mRNA）的分离，使用如在以下部分中详细描述的的TaqMan微小RNA表达测定成熟miRNA的含量测定。 <ol><li> RNA提取<ol><li>前列腺癌FFPE组织提取的miRNA <ol><li>激光捕获显微切割（LCM） 注:执行步骤2.1.1.1.1- 2.1.1.1.4在通风橱。 <ol><li>对于LCM准备组织切片（10微米的部分）。 Deparrafinize组织切片在二甲苯浸泡（2次），每10分钟，然后通过将幻灯片的每个在梯度乙醇（100％，95％，90％，80％，70％）5分钟，然后用蒸馏水补湿组织水（5分钟）。 </li><li>以下补液，染色切片用苏木精30秒，然后用水。 </li><li>地方的组织在梯度乙醇（70％，95％90％，80％，70％）（每次5分钟）和二甲苯（5分钟）。 </li><li>在通风橱中干燥载玻片，然后将在LCM的仪器为显微切割。 </li><li>按照制造商的说明24执行microdissections与LCM系统。使用病理学家标记的幻灯片作为指南，前列腺癌病灶的标识以及邻近的正常组织。使用激光束在10-20微米的直径脉冲70-90毫瓦的功率。 </li><li>用红外激光脉冲的兴趣到LCM上限捕捉区域。从2-5个盖帽为每个样品的miRNA萃取结合细胞。为确保提取的RNA完整性，立即处理捕获的细胞miRNA的提取。 </li><li>备选地，裂解细胞中miRNA的提取缓冲液（根据制造商的协议）和STO再裂解液在-80ºC。 </li></ol></li><li> miRNA的提取和分析，从LCM显微组织<ol><li>从使用商业试剂盒按照制造商的说明，显微切割的FFPE组织中提取总RNA。 注:RNA的激光捕获miscrodissection的收益率通常较低。因此，RNA被洗脱到小体积（20-30微升）和用于使用以下制造商的说明的Taqman微小RNA表达分析表达谱（2.2节中也有描述）。优化的输入核糖核酸为实时PCR检测成熟miRNA的表明5微升洗脱的RNA是最佳的每个miRNA特异性逆转录反应。作为内源对照，RNU48 / RNU24被使用。对于对照反应，2微升洗脱的RNA用于逆转录反应。 </li></ol></li></ol></li><li>前列腺癌冷冻组织提取的miRNA <ol><li> Homoge通过使用研钵和研杵研磨该组织在液氮中冷冻nize切除前列腺组织。使用冷却的刮刀转移匀浆组织到一个离心管中。通过在液氮中，以便匀浆组​​织浸渍可以容易地转移保持砂浆/杵和抹刀在相同的温度。 </li><li>添加商业的异硫氰酸胍 - 苯酚试剂（1毫升/ 0.1克​​组织）的均质化的组织和在室温下孵育5分钟。 注意:在商业异硫氰酸胍 - 酚试剂均化组织可以储存在-80ºC数月。 </li><li>加三氯甲烷匀浆（0.3毫升氯仿/毫升），并剧烈摇晃30秒。孵育的样品在RT 3-5分钟，随后离心以10,000×g离心20分钟，在4ºC。 </li><li>上层水相转移到一个新的无菌无RNase 1.5毫升试管中。 </li><li>添加异丙醇等体积。混合和孵育10英里n将在室温然后离心在12,000×g离心20分钟，在4℃到沉淀的RNA。 </li><li>吸出上清液，并用1mL 70％乙醇洗涤RNA沉淀。离心机在12,000rpm离心10分钟，在4ºC。 </li><li>小心吸出上清液。干燥核糖核酸粒料在RT 5-10分钟。在50-100微升无核酸酶水溶解RNA。 </li><li>使用NanoDrop分光光度计进行的RNA定量，并确定与一个生物分析RNA的完整性。调整RNA浓度为10毫微克/微升。用10-50毫微克的RNA的miRNA-特异基因反应，接着通过实时PCR分析（2.2节）。 </li></ol></li></ol></li><li>定量实时PCR检测miRNA表达分析 注:测定成熟miRNA使用两步骤RT-PCR方案如下所述。 <ol><li>反转录（RT） <ol><li>反向使用的miRNA逆转录试剂盒按照制造商的说明由RNA转录的cDNA。使用10-50纳克的总RNA与miRNA的从微RNA测定法和RT试剂盒特异性引物。使用RNU48 / RNU24 / RNU6B作为对照。稀释的cDNA 1:5至1:10（取决于所分析的miRNA的丰度）并如以下步骤描述在实时PCR检测使用。 </li></ol></li><li>实时PCR检测成熟miRNA的<ol><li>扩增PCR产物利用微RNA检测具有快速通用预混按照制造商的说明cDNA样品。规范化样品RNU48 / RNU24 / RNU6B控制。使用比较Ct（阈值循环）方法来计算上快速实时PCR系统中的基因表达的相对变化。一式三份每个样品分析。 </li></ol></li></ol></li></ol> 3. miRNA的表达分析通过原位杂交（ISH） <ol><li>组织的前处理<ol><li>切从使用切片机FFPE前列腺癌组织块5微米的部分。 </li><lI&gt;孵育载玻片在56℃下1小时固定的组织切片。 注意:幻灯片可以存储在RT几个星期的miRNA分析。 </li><li>浸泡载玻片用二甲苯（2×），每次15分钟，Deparrafinize组织。 </li><li>通过将载玻片5分钟，在每一个分级的乙醇（100％（2次），95％，90％，80％，70％），然后加入蒸馏水（5分钟）再水合组织。 </li><li>固定用溶于PBS的4％多聚甲醛的幻灯片在室温下20分钟。 </li><li>在室温下洗涤​​用PBS（2×）的幻灯片每次5分钟。 </li><li>治疗的幻灯片用10微克/毫升蛋白酶K在37ºC为在预热的蛋白酶K缓冲液10分钟（5毫摩尔Tris-HCL pH7.4的1mM EDTA中，1毫摩尔NaCl）。 </li><li>下列蛋白酶K处理，冲洗用0.2％的甘氨酸的幻灯片在PBS中持续30秒。 </li><li>在PBS（1×），在室温下5分钟洗幻灯片。 </li><li>固定用溶于PBS的4％多聚甲醛的幻灯片15分钟。 </li><li>冲洗幻灯片PBS 5分钟。 </li></ol></li><li>杂交<ol><li>预杂交用3-4小时，在湿盒预杂交溶液在55ºC幻灯片。使用浸泡在50％甲酰胺/ 50％5×SSC组织实验室湿巾以保持腔室加湿。 </li><li>使用5&#39;洋地黄毒苷标记的探针，在20-50纳米杂交缓冲液中的浓度。稀的miRNA特异性探针（20-50纳米）和小核RNA U6对照探针（20纳米），热在90ºC4分钟，将其放置在冰上，并加入到冰冷的杂交缓冲液（2-4毫微克/微升）。 </li><li>除去预杂交液，加入杂交液（探针+杂交缓冲液，每幻灯片100微升），孵育12-16小时，在探针特异性杂交​​的温度（杂交温度= Tm为探针 - 21℃）。在湿润的腔室（50％甲酰胺/ 50％5×SSC）的执行杂交。 </li></ol></li><li>洗涤步骤<ol><li> 10分钟，45ºC洗涤用2×SSC的幻灯片。 </li><li>洗吨他幻灯片1.5倍SSC在45ºC10分钟。 </li><li>在37ºC每次20分钟，洗涤0.2×SSC（2×）的幻灯片。 </li><li>孵育载玻片用1×阻断溶液1-2小时，在室温下</li></ol></li><li>发现<ol><li>孵育载玻片以1:100 PBS稀释的AP缀合的抗洋地黄毒苷抗体为1-4小时或过夜，在4ºC。 </li><li>在室温下洗涤​​用PBS（3×）的幻灯片，每次10分钟。 </li><li>洗载玻片（2次）与碱性磷酸酶缓冲液（100mM的Tris pH为9.5，50mM的MgCl 2的，100 mM氯化钠，0.1％吐温-20）在室温下，每次5分钟。 </li><li>孵育在BM紫AP底载玻片在黑暗中在RT 1-20小时。 </li><li>冲洗载玻片用PBS含有0.1％Tween-20和洗涤（2×）水。 </li><li>安装使用含水封装介质的幻灯片，并在显微镜下检查。 </li></ol></li></ol>

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The authors have no financial disclosures.

在这篇文章中，我们描述了miRNA表达一个简化的工作流程分析在存档FFPE和新鲜冷冻前列腺癌的临床组织。在前列腺癌，一些研究表明，在前列腺癌的发生，发展和转移的microRNA中起重要作用。然而，矛盾的结果往往是获得一个特定的miRNA 22以来的miRNA的提取和分析方法有很大的出入。鉴于越来越多的证据支持的miRNA作为替代的前列腺癌生物标志物的前列腺癌的预后，诊断和治疗的潜在的应用...

前列腺癌是一种常见的诊断男性恶性肿瘤即癌症相关死亡的男子居于领先地位的原因之一。在美国，估计有220800例新病例和27540例死亡将在2015年1报告。 前列腺癌是一种异质性疾病与高度可变的疾病课程 - 肿瘤可好逸恶劳或非常积极。在前列腺癌的临床管理的一个重要挑战是目前常用的方法/生物标志物的疾病筛查，诊断，预后和治疗2的不足所造成。当前的筛选方法包括前列腺特异性抗原（PSA）测试和直肠指检（DRE），随后前列腺活检3。前列腺特异性抗原（PSA）是使用最广泛的前列腺癌生物标志物已显著革命性临床管理和改进的存活率4。然而，由于粘合剂包括紫胶固有的局限性k的特异性，PSA为基础的筛选，导致过度的诊断和治疗上的疾病。鉴于此，积极努力正在指向一个搜索备用前列腺癌生物标志物，特别是那些可以预测疾病的攻击性和实现更好的治疗决策4,5。在过去的几年里，微RNA（miRNA）已成为有前途的替代前列腺癌生物标志物。 微RNA（miRNA）构成一个进化上保守类小的非编码RNA，它通过与同源的mRNA靶的3&#39;-非翻译区（UTR）序列特异性相互作用的基因表达抑制转录后。据估计，&gt;的mRNA的60％是保守的miRNA 6的目标。 miRNA基因位于基因间隔区或内含子或蛋白/非蛋白质的外显子编码基因7内。这些基因被RNA聚合酶Ⅱ优先转录成初步RY的miRNA（PRI-miRNA的，几千个碱基长）的形式发夹状茎环二级结构。这些PRI-miRNA的加工成miRNA前体（的pre-miRNA，60-75核苷酸长），其出口到细胞质和进一步加工成成熟miRNA（18-25核苷酸长）8-10。的miRNA调节关键的细胞过程，包括增殖，发育，分化和凋亡11。研究表明miRNA的表达谱在各种人类恶性肿瘤包括前列腺癌12-15普遍失调。 miRNA的表达谱已报道被广泛失调在原发性和转移性前列腺癌。改变的miRNA表达已与前列腺癌的进展，攻击性和复发突出的miRNA 12,14,16-19的预后潜力。越来越多的证据体表明miRNA的前列腺癌症的发生，发展，进步起到重要的作用机理离子和转移。总体而言，微RNA正在成为有前途的替代生物标志物前列腺癌的诊断和预后，可在前列腺肿瘤12分层正常，癌组织和援助加以区分。此外，miRNA是发展的有效的治疗方法预防前列腺癌20的重要目标。 由于它们的小尺寸和抗内源性RNase活性，miRNA是，可以在福尔马林固定的组织21和前列腺活检22容易地检测的生物标志物的稳定。此外，miRNA的表达谱进行了比较在冷冻和福尔马林固定的组织，并已被发现是强烈相关21。然而，miRNA表达谱的前列腺癌临床组织往往是具有挑战性的，由于肿瘤的异质性，抽样误差，间质污染等miRNA的有效生物标记物的前列腺癌严重RELI的发展ES它们在临床组织中精确检测。在这里，我们描述了在我们的实验室进行miRNA表达谱的存档FFPE和新鲜冷冻前列腺癌的临床标本使用简化的工作流程。我们使用定量实时PCR的组合和在对miRNA的原位杂交分析临床标本的，前者得到更多量化信息，后者用于可视化潜在的miRNA标志物的差异表达的组织中的阵列。在这个工作流程中，我们优化的miRNA提取从FFPE和冷冻前列腺组织的现有方法，表达由基于miRNA的RT-PCR和miRNA的原位杂交技术利用锁核酸（LNA）的Taqman探针分析为基础的探针23。 LNA基于探头提供增加的灵敏度和特异性相比DNA-或RNA-基于探针和使所有miRNA序列的鲁棒检测，无论其GC含量也允许discrimina化miRNA家族的。我们已优化的miRNA ISH方法可应用于前列腺癌组织切片或前列腺癌组织微阵列（TMA），后者提供到加速miRNA的生物标志物发现的潜力。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Microtome</td>
			<td>Leica Biosystems&#160;</td>
			<td>RM2255</td>
			<td></td>
		</tr>
		<tr>
			<td>Arcturus Autopix for LCM</td>
			<td>Arcturus/ Life Technologies</td>
			<td>LCM1621/LCM1110</td>
			<td>Alternatively, Arcutus Xt system from Life Technolgies can be used.&#160;</td>
		</tr>
		<tr>
			<td>CapSure Macro LCM Caps</td>
			<td>Life Technologies</td>
			<td>LCM0211</td>
			<td></td>
		</tr>
		<tr>
			<td>miRNeasy FFPE Kit&#160;</td>
			<td>Qiagen</td>
			<td align="right">217504</td>
			<td></td>
		</tr>
		<tr>
			<td>7500 Fast Real Time PCR System&#160;</td>
			<td>Applied Biosystems/ Life Technologies</td>
			<td align="right">4351106</td>
			<td></td>
		</tr>
		<tr>
			<td>Taqman MicroRNA Reverse Transcription kit&#160;</td>
			<td>Applied Biosystems/ Life Technologies</td>
			<td align="right">4366596</td>
			<td></td>
		</tr>
		<tr>
			<td>Taqman Fast Universal PCR master mix&#160;</td>
			<td>Applied Biosystems/ Life Technologies</td>
			<td align="right">4352042</td>
			<td></td>
		</tr>
		<tr>
			<td>DIG labeled LNA probe for U6</td>
			<td>Exiqon</td>
			<td>99002-01</td>
			<td></td>
		</tr>
		<tr>
			<td>BM Purple AP substrate</td>
			<td>Roche</td>
			<td align="right">11442074001</td>
			<td></td>
		</tr>
		<tr>
			<td>Pre-hybridization solution&#160;</td>
			<td>Biochain</td>
			<td><a href="http://www.biochain.com/coa/K21910XXCA.pdf">K2191050-1</a></td>
			<td></td>
		</tr>
		<tr>
			<td>Hybridization solution&#160;</td>
			<td>Biochain</td>
			<td><a href="http://www.biochain.com/coa/K21910XXCA.pdf">K2191050-2</a></td>
			<td></td>
		</tr>
		<tr>
			<td>Blocking solution&#160;</td>
			<td>Biochain</td>
			<td><a href="http://www.biochain.com/coa/K21910XXCA.pdf">K2191050-8</a></td>
			<td></td>
		</tr>
		<tr>
			<td>AP-conjugated anti-digoxigenin antibody</td>
			<td>Biochain</td>
			<td><a href="http://www.biochain.com/coa/K21910XXCA.pdf">K2191050-7</a></td>
			<td></td>
		</tr>
		<tr>
			<td>Aqueous mounting media&#160;</td>
			<td>Vector Laboratories&#160;</td>
			<td>H-5501&#160;&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Trizol (guanidine isothiocyanate-phenol reagent)&#160;</td>
			<td>Life Technologies</td>
			<td><a href="https://www.lifetechnologies.com/order/catalog/product/15596018">15596-018</a></td>
			<td></td>
		</tr>
		<tr>
			<td>Harris hematoxylin</td>
			<td>Statlab</td>
			<td>SL200</td>
			<td></td>
		</tr>
		<tr>
			<td>Eosin&#160;</td>
			<td>Statlab</td>
			<td>SL201&#160;</td>
			<td></td>
		</tr>
	</tbody>
</table>

mirna expression analyses in prostate cancer clinical tissues

在前列腺癌（PCA）的临床管理的一个重要挑战是目前使用的生物标志物的疾病筛查，诊断，预后和治疗的不足所造成。近年来，微RNA（miRNA）已成为有前途的替代标志物前列腺癌的诊断和预后。然而，miRNA的有效生物标记前列腺癌的发展在很大程度上依赖于他们在临床组织中准确的检测。 miRNA的分析了前列腺癌的临床标本往往是具有挑战性的，由于肿瘤的异质性，抽样误差，间质污染等。这篇文章的目的是描述一个简化的工作流程在存档FFPE和新鲜冷冻前列腺癌的临床标本的miRNA分析使用的组合定量实时PCR（RT-PCR），原位杂交（ISH）。在这个流程中，我们优化了miRNA的提取从FFPE现有的方法和冷冻前列腺Tissu酒店ES和表达分析了基于miRNA的RT-PCR的Taqman探针。此外，我们描述了ISH优化方法分析了使用锁核酸（LNA），前列腺组织formiRNA探测 - 基于探针。我们已优化的miRNA ISH方法可应用于前列腺癌组织切片或前列腺癌组织微阵列（TMA）。

的miR-203的LCM原发性前列腺癌临床标本表达谱，通过RT-PCR分析（图1） RT-PCR分析相对的miR-203表达在LCM原发性前列腺癌组织和匹配的邻近的正常区域进行与在赛尼等人 15 RNU48所述的用作对照。下面的表总结了在前列腺癌肿瘤组织相对于邻近的正常组织的相对的miR-203的表达。 在显微的miR-383的表达通过RT-PCR比较分析VS激光捕获显微切...

Watch this Scientific Journal Video about miRNA Expression Analyses in Prostate Cancer Clinical Tissues at JoVE.com

miRNA Expression Analyses in Prostate Cancer Clinical Tissues

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, ...

mirna-expression-analyses-in-prostate-cancer-clinical-tissues

Research

JoVE Journal

Medicine

10.8K 次观看.  University of California San Francisco. 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).

视频: miRNA Expression Analyses in Prostate Cancer Clinical Tissues

Evaluation of Nanoparticle Uptake in Tumors in Real Time Using Intravital Imaging

Current technologies for tumor imaging, such as ultrasound, MRI, PET and CT, are unable to yield high-resolution images for the assessment of nanoparticle uptake in tumors at the microscopic level1,2,3, highlighting the utility of a suitable xenograft model in which to perform detailed uptake analyses. Here, we use high-resolution intravital imaging to evaluate nanoparticle uptake in human tumor xenografts in a modified, shell-less chicken embryo model. The chicken embryo model is particularly well-suited for these in vivo analyses because it supports the growth of human tumors, is relatively inexpensive and does not require anesthetization or surgery 4,5. Tumor cells form fully vascularized xenografts within 7 days when implanted into the chorioallantoic membrane (CAM) 6. The resulting tumors are visualized by non-invasive real-time, high-resolution imaging that can be maintained for up to 72 hours with little impact on either the host or tumor systems. Nanoparticles with a wide range of sizes and formulations administered distal to the tumor can be visualized and quantified as they flow through the bloodstream, extravasate from leaky tumor vasculature, and accumulate at the tumor site. We describe here the analysis of nanoparticles derived from Cowpea mosaic virus (CPMV) decorated with near-infrared fluorescent dyes and/or polyethylene glycol polymers (PEG) 7, 8, 9,10,11. Upon intravenous administration, these viral nanoparticles are rapidly internalized by endothelial cells, resulting in global labeling of the vasculature both outside and within the tumor7,12. PEGylation of the viral nanoparticles increases their plasma half-life, extends their time in the circulation, and ultimately enhances their accumulation in tumors via the enhanced permeability and retention (EPR) effect 7, 10,11. The rate and extent of accumulation of nanoparticles in a tumor is measured over time using image analysis software. This technique provides a method to both visualize and quantify nanoparticle dynamics in human tumors.

We present a novel approach to quantify nanoparticle localization in the vasculature of human xenografted tumors using dynamic, real-time intravital imaging in an avian embryo model.

使用实时活体成像在肿瘤纳米粒子吸收的评价

Current technologies for tumor imaging, such as ultrasound, MRI, PET and CT, are unable to yield high-resolution images for the assessment of nanoparticle ...

科研

医学

Cell Population Analyses During Skin Carcinogenesis

Cancer development is a multiple-step process involving many cell types including cancer precursor cells, immune cells, fibroblasts and endothelial cells. Each type of cells undergoes signaling and functional changes during carcinogenesis. The current challenge for many cancer researchers is to dissect these changes in each cell type during the multiple-step process in vivo. In the last few years, the authors have developed a set of procedures to isolate different cell populations during skin cancer development using K14creER/R26-SmoM2YFP mice. The procedure is divided into 6 parts: 1) generating appropriate mice for the study (K14creER+ and R26-SmoM2YFP+ mice in this protocol); 2) inducing SmoM2YFP expression in mouse skin; 3) preparing mouse skin biopsies; 4) isolating epidermis from skin; 5) preparing single cells from epidermis; 6) labeling single cell populations for flow cytometry analysis. Generation of sufficient number of mice with the right genotype is the limiting step in this protocol, which may take up to two months. The rest of steps take a few hours to a few days. Within this protocol, we also include a section for troubleshooting. Although we focus on skin cancer, this protocol may be modified to apply for other animal models of human diseases.

Carcinogenesis is a process involving interactions between cancer cells and the cancer microenvironment. To dissect the molecular events, one needs to isolate different cell populations at different stages during cancer development. Using a mouse model for basal cell carcinoma, we describe a protocol for cell analyses during carcinogenesis.

在皮肤癌变细胞群分析

Cancer development is  a multiple-step process involving many cell types including cancer precursor  cells, immune cells, fibroblasts and endothelial ...

Microarray-based Identification of Individual HERV Loci Expression: Application to Biomarker Discovery in Prostate Cancer

The prostate-specific antigen (PSA) is the main diagnostic biomarker for prostate cancer in clinical use, but it lacks specificity and sensitivity, particularly in low dosage values1&#8203;&#8203;. &#8216;How to use PSA&#39; remains a current issue, either for diagnosis as a gray zone corresponding to a concentration in serum of 2.5-10 ng/ml which does not allow a clear differentiation to be made between cancer and noncancer2 or for patient follow-up as analysis of post-operative PSA kinetic parameters can pose considerable challenges for their practical application3,4. Alternatively, noncoding RNAs (ncRNAs) are emerging as key molecules in human cancer, with the potential to serve as novel markers of disease, e.g. PCA3 in prostate cancer5,6 and to reveal uncharacterized aspects of tumor biology. Moreover, data from the ENCODE project published in 2012 showed that different RNA types cover about 62% of the genome. It also appears that the amount of transcriptional regulatory motifs is at least 4.5x higher than the one corresponding to protein-coding exons. Thus, long terminal repeats (LTRs) of human endogenous retroviruses (HERVs) constitute a wide range of putative/candidate transcriptional regulatory sequences, as it is their primary function in infectious retroviruses. HERVs, which are spread throughout the human genome, originate from ancestral and independent infections within the germ line, followed by copy-paste propagation processes and leading to multicopy families occupying 8% of the human genome (note that exons span 2% of our genome). Some HERV loci still express proteins that have been associated with several pathologies including cancer7-10. We have designed a high-density microarray, in Affymetrix format, aiming to optimally characterize individual HERV loci expression, in order to better understand whether they can be active, if they drive ncRNA transcription or modulate coding gene expression. This tool has been applied in the prostate cancer field (Figure 1).

Human endogenous retroviruses (HERV), which occupy 8% of the human genome, retain scarce coding capacities but a hundred thousand long terminal repeats (LTRs). A custom Affymetrix microarray was designed to identify individual HERV locus expression and was used on prostate cancer tissues as a proof of concept for future clinical studies.

应用生物标志物发现在前列腺癌:个别赫尔维位点表达微阵列鉴定

The prostate-specific antigen (PSA) is the main diagnostic biomarker for prostate cancer in clinical use, but it lacks specificity and sensitivity, ...

An Orthotopic Murine Model of Human Prostate Cancer Metastasis

Our laboratory has developed a novel orthotopic implantation model of human prostate cancer (PCa). As PCa death is not due to the primary tumor, but rather the formation of distinct metastasis, the ability to effectively model this progression pre-clinically is of high value. In this model, cells are directly implanted into the ventral lobe of the prostate in Balb/c athymic mice, and allowed to progress for 4-6 weeks. At experiment termination, several distinct endpoints can be measured, such as size and molecular characterization of the primary tumor, the presence and quantification of circulating tumor cells in the blood and bone marrow, and formation of metastasis to the lung. In addition to a variety of endpoints, this model provides a picture of a cells ability to invade and escape the primary organ, enter and survive in the circulatory system, and implant and grow in a secondary site. This model has been used effectively to measure metastatic response to both changes in protein expression as well as to response to small molecule therapeutics, in a short turnaround time.

This orthotopic model of human prostate cancer allows for quantification of tumor size, circulating tumor cells, and formation of distinct metastasis to the lung. As cells must escape the primary organ, enter the blood stream, and implant into a secondary site, this model effectively recapitulates the scenario in humans.

人类前列腺癌转移的原位小鼠模型

Our laboratory has developed a novel orthotopic implantation model of  human prostate cancer (PCa). As PCa death is not due to the primary tumor, but  ...

Isolation of Cancer Stem Cells From Human Prostate Cancer Samples

The cancer stem cell (CSC) model has been considerably revisited over the last two decades. During this time CSCs have been identified and directly isolated from human tissues and serially propagated in immunodeficient mice, typically through antibody labeling of subpopulations of cells and fractionation by flow cytometry. However, the unique clinical features of prostate cancer have considerably limited the study of prostate CSCs from fresh human tumor samples. We recently reported the isolation of prostate CSCs directly from human tissues by virtue of their HLA class I (HLAI)-negative phenotype. Prostate cancer cells are harvested from surgical specimens and mechanically dissociated. A cell suspension is generated and labeled with fluorescently conjugated HLAI and stromal antibodies. Subpopulations of HLAI-negative cells are finally isolated using a flow cytometer. The principal limitation of this protocol is the frequently microscopic and multifocal nature of primary cancer in prostatectomy specimens. Nonetheless, isolated live prostate CSCs are suitable for molecular characterization and functional validation by transplantation in immunodeficient mice.

The isolation of cancer stem cells (CSCs) directly from human tissues is requisite for their biological characterization. This manuscript describes a methodology for the isolation of prostate CSCs from human tissues, while also providing tips on troubleshooting difficult steps.

癌症干细胞从人类前列腺癌样本隔离

The cancer stem cell (CSC) model has been considerably revisited over the last two decades. During this time CSCs have been identified and directly ...

Ex Vivo Treatment Response of Primary Tumors and/or Associated Metastases for Preclinical and Clinical Development of Therapeutics

The molecular analysis of established cancer cell lines has been the mainstay of cancer research for the past several decades. Cell culture provides both direct and rapid analysis of therapeutic sensitivity and resistance. However, recent evidence suggests that therapeutic response is not exclusive to the inherent molecular composition of cancer cells but rather is greatly influenced by the tumor cell microenvironment, a feature that cannot be recapitulated by traditional culturing methods. Even implementation of tumor xenografts, though providing a wealth of information on drug delivery/efficacy, cannot capture the tumor cell/microenvironment crosstalk (i.e., soluble factors) that occurs within human tumors and greatly impacts tumor response. To this extent, we have developed an ex vivo (fresh tissue sectioning) technique which allows for the direct assessment of treatment response for preclinical and clinical therapeutics development. This technique maintains tissue integrity and cellular architecture within the tumor cell/microenvironment context throughout treatment response providing a more precise means to assess drug efficacy.

Ex Vivo Treatment Response of Primary Tumors and/or Associated Metastases for Preclinical and Clinical Development of Therapeutics

Established cancer cell lines and xenografts have been the mainstay of cancer research for the past several decades. However, recent evidence suggests that therapeutic response is greatly influenced by the tumor cell microenvironment. Therefore, we have developed an ex vivo analysis of primary tumor specimens for drug development purposes.

原发性肿瘤和/或转移相关的临床前和治疗的临床开发的离体处理响应

The molecular analysis of established cancer cell lines has been the mainstay of cancer research for the past several decades. Cell culture provides both ...

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 ...

Generation of Prostate Cancer Patient Derived Xenograft Models from Circulating Tumor Cells

Patient derived xenograft (PDX) models are gaining popularity in cancer research and are used for preclinical drug evaluation, biomarker identification, biologic studies, and personalized medicine strategies. Circulating tumor cells (CTC) play a critical role in tumor metastasis and have been isolated from patients with several tumor types. Recently, CTCs have been used to generate PDX experimental models of breast and prostate cancer. This manuscript details the method for the generation of prostate cancer PDX models from CTCs developed by our group. Advantages of this method over conventional PDX models include independence from surgical sample collection and generating experimental models at various disease stages. Density gradient centrifugation followed by red blood cell lysis and flow cytometry depletion of CD45 positive mononuclear cells is used to enrich CTCs from peripheral blood samples collected from patients with metastatic disease. The CTCs are then injected into immunocompromised mice; subsequently generated xenografts can be used for functional studies or harvested for molecular characterization. The primary limitation of this method is the negative selection method used for CTC enrichment. Despite this limitation, the generation of PDX models from CTCs provides a novel experimental model to be applied to prostate cancer research.

This manuscript details a method used to generate prostate cancer patient derived xenografts (PDXs) from circulating tumor cells (CTCs). The generation of PDX models from CTCs provides an alternative experimental model to study prostate cancer; the most commonly diagnosed tumor and a frequent cause of death from cancer in men.

前列腺癌患者产生派生异种移植模型从循环肿瘤细胞

Patient derived xenograft (PDX) models are gaining popularity in cancer research and are used for preclinical drug evaluation, biomarker identification, ...

Working with Human Tissues for Translational Cancer Research

Medical research for human benefit is greatly impeded by the necessity for human tissues and subjects. However, upon obtaining consent for human specimens, precious samples must be handled with the greatest care in order to ensure integrity of organs, tissues, and cells to the highest degree. Unfortunately, tissue processing by definition requires extraction of tissues from the host, a change which can cause great cellular stress and have major repercussions on subsequent analyses. These stresses could result in the specimen being no longer representative of the site from which it was retrieved. Therefore, a strict protocol must be adhered to while processing these specimens to ensure representativeness. The desired assay(s) must also be taken into consideration in order to ensure that an optimal technique is used for sample processing. Outlined here is a protocol for tissue retrieval, processing and various analyses which may be performed on processed tissue in order to maximize downstream production from limited tissue samples.

Translational cancer research is dependent on extraction of human tissues. Much work has gone into optimizing extraction methods for ex vivo analysis. Here, we describe tissue processing methods allowing for maximal data output from limited samples.

与人体组织转化为癌症研究工作

Medical research for human benefit is greatly impeded by the necessity for human tissues and subjects. However, upon obtaining consent for human ...

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.

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 ...