这项工作是由哈里里家族基金会和TJ马爹利基金会的支持。

1。采收和从手术标本人前列腺癌组织处理<ol><li>制备含有15毫升Roswell公园纪念研究所（RPMI）含10％胎牛血清（FBS）和1％青霉素/链霉素1640培养基250毫升聚苯乙烯锥形管中。 </li><li>病理学服务人员从收获前列腺切除和姑息手术标本原发性和转移性前列腺癌样本，分别根据机构审查委员会批准的协议。转移性样本可以从淋巴结清扫术中主要手术来获得，在此期间在诊断除去单个转移灶，其中包括姑息性外科手术，例如脊髓减压，和肺中的椎骨。 <ol><li>收获过量块状组织不需要用于临床诊断为第一的50ml聚苯乙烯锥形管从步骤1.1，并立即储存于4℃。该harveste三维组织至少应为4-5厘米2的面积和1-2厘米的厚度。 </li></ol></li><li>实验室人员处理从筹备组织学检查细胞悬浮液的产生病理的服务获得大量的组织。 <ol><li>散装组织处理不应超过从手术切除24小时，以保证最大的细胞活力。 </li><li>工作在生物安全柜消毒的手术刀和镊子，采取从肉眼可见的肿瘤结节2-4毫米厚的水平组织切片。立即存储部分在第二50毫升聚苯乙烯锥形管从步骤1.1。 </li><li>当宏观结节不从前列腺可视化的样本，收集来自后叶和过渡区随机2-4毫米厚的横向组织切片。 </li></ol></li><li>用无菌手术刀和镊子分离外科组织的一部分，并且固定在10％福尔马林之前属化细胞悬液。存档和分析该材料在组织学证实的前列腺癌组织中的存在。 </li></ol> 2。从组织切片悬浮细胞的生成<ol><li>工作在一个无菌的生物安全柜，组织切片转移到含1ml无菌1X磷酸盐缓冲盐水（PBS）中的60×15毫米培养皿中。 <ol><li>用无菌手术刀和镊子机械磨碎的样品。 </li><li>没有消化酶在这个协议。 </li></ol></li><li>转移1毫升悬浮到无菌的50ml锥形管中。 <ol><li>加入1 ml至培养皿，并重复研磨步骤，直到所述组织部分被完全离解的，一般为3至4倍。 </li><li>重复此过程连续，直到所有的组织切片已经分离。 </li></ol></li><li>重悬50毫升聚苯乙烯锥形管中的内容物用5ml的血清学圆周率pette和涡以最大速度（约3,200 rpm）离心1分钟。 </li><li>通过一个35微米的细胞滤网过滤所得悬浮液，并收集到一个第二无菌50毫升聚苯乙烯锥形管。 </li><li>通过离心分离，在450×g离心10分钟，生成的颗粒。 </li><li>弃上清，重悬沉淀，用5ml的红细胞裂解缓冲液，并温育5分钟，在室温的溶液中。通过离心除去红血细胞裂解液5分钟，在450×g离心于RT。 </li><li>弃去上清液，沉淀重悬在1×PBS中补充有5％FBS，并使用血细胞计数器或自动细胞计数器量化可行的（台盼蓝阴性）细胞的数量。产生一个1×10 6细胞/ ml的悬浮液，并将其存储在冰中不超过1小时。 <ol><li>的可行性和细胞的产量可能肿瘤标本之间有很大的不同。在前列腺切除术的设定，生存能力可能之间45-70％，和fi范围已经2-4毫米从宏观的肿瘤结节厚的水平组织切片可以预计产量约3×10 6活细胞。 </li></ol></li></ol> 3。细胞的流式细胞仪抗体染色<ol><li>标签515毫升聚苯乙烯锥形管，如下图所示。使用CD45和CD31抗原的标记，以分别排除造血细胞和内皮元素。 <ol><li>未染色</li><li> 2A的IgGκ-PE + IgG的1κ-FITC </li><li> HLAI-PE +的IgG 1κ-FITC </li><li> 2A的IgGκ-PE + CD31-FITC + CD45-FITC </li><li> HLAI-PE + CD45-FITC + CD31-FITC </li></ol></li><li>从上一节（步骤2.7）分发量化细胞悬液进五管在步骤3.1。加入抗体稀释度为1:250，并在冰上孵育的细胞悬液30分钟。 </li><li>离心细胞，在450×g离心3分钟，在4℃，并丢弃上清液。 </li&gt; <li>通过再悬浮每个粒料用无菌的1×PBS洗涤细胞在补充有10％FBS，然后离心以450×g离心3分钟，在4℃下</li><li>重悬在1×PBS中的1毫升溶液含4&#39;，6 - 二脒基-2 - 苯基吲哚（DAPI）在10微克/毫升的浓度将细胞。 </li><li>通过35微米过滤器过滤帽的最终解决方案为12厘米x 75厘米聚苯乙烯管。 </li></ol> 4。前列腺肿瘤干细胞的流式细胞仪分离<ol><li>利用流式细胞仪进行细胞分选。 </li><li>设置使用细胞管＃1，＃2，＃3补偿控制，和＃4。 </li><li>创建，使用前向和侧向散射参数排除碎片和细胞群的大门。 </li><li>从管＃3和＃管4确定用悬浮液FITC和PE门，分别为。 </li><li>门可行的（DAPI阴性）使用太平洋蓝色的通道细胞。 </li><li>采用管＃5丢弃的CD31阳性且CD45阳性细胞和收集两个HLAI阴性和HLAI阳性人群成含2ml RPMI的补充有10％FBS的无菌的15毫升的聚苯乙烯锥形管中。 </li><li>离心机的排序，在450×g离心5分钟，细胞悬浮液，然后重新悬浮小球在200-500微升的RPMI补加有10％胎牛血清。 </li><li>使用血球或自动细胞计数器量化可行的（台盼蓝染色阴性）细胞。 </li></ol>

<ol>
	<li>Hanahan, D., Weinberg, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hallmarks+of+cancer:+the+next+generation.">Hallmarks of cancer: the next generation.</a> Cell. 144, 646-674 (2011).</li><li>Magee, J. A., Piskounova, E., Morrison, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+stem+cells:+impact,+heterogeneity,+and+uncertainty.">Cancer stem cells: impact, heterogeneity, and uncertainty.</a> Cancer Cell. 21, 283-296 (2012).</li><li>Nguyen, L. V., Vanner, R., Dirks, P., Eaves, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+stem+cells:+an+evolving+concept.">Cancer stem cells: an evolving concept.</a> Nat. Rev. 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没有利益冲突的声明。

本协议描述的CSCs的从新鲜人前列腺癌组织中的隔离。几个重要的考虑因素影响本协议的成功结果。 高数可行前列腺癌细胞的恢复依赖于外科手术样品小心总的评估。在我们的经验，致瘤性的前列腺细胞的成功分离，最好保证当肉眼可见的肿瘤结节观察和处理10。 然而，从前列腺切除标本派生时下原发病常常是多灶性和微观。在这些情况下，当可?...

瘤内的异质性被认为是癌症1的标志。事实上，肿瘤内的异质性的几种机制已经描述了，包括基因突变，并与微环境相互作用。此外，一些癌症可能含有与癌症干细胞（肿瘤干细胞）亚群表现出的肿瘤起始能力和自我更新的串行移植实验2-5的属性蜂窝层次。在血液学6，乳腺癌7，和脑8恶性肿瘤最初描述，肿瘤干细胞也已经研究了前列腺癌9-12以及其他类型的肿瘤2-5。 肿瘤干细胞通常被认为是一个异质群体2-5中的细胞部分。因此，肿瘤干细胞的功能和分子特征是在从大量人群的充实队伍。因此，在过去的二十年里遇到几个CSC富集hodologies已经设计出通常涉及标记人群通过流式细胞仪分离。另外，在肿瘤干细胞的研究的显著考虑的是，人体组织的层次组织可以通过实验操作，例如在培养物或免疫缺陷小鼠中连续传代被打乱。其结果是，从人体组织的CSCs的直接分离已经成为在CSC领域的一个重要方法。 前列腺癌是癌症的发病率和死亡率在美国和世界各地13的主要原因。因此，前列腺肿瘤干细胞的分离和生物学特性是显著的兴趣。前列腺肿瘤干细胞先前已经从富集的细胞系，来自患者的移植，以及低传来自患者的悬浮培养9,10,12,14。 我们最近报道的前列腺肿瘤干细胞直接从人的手术S中的隔离amples凭借自己HLAI阴性细胞表面表型10。在这里，我们详细地实施了这些细胞的分离的过程。前列腺肿瘤是从手术标本采集后制成细胞悬液。然后将细胞用抗HLAI抗体以及基质和生存能力标记染色，肿瘤干细胞是通过荧光激活细胞分选术（FACS）分离。孤立的CSCs可以被用于需要活细胞测定法。

<table border="0" cellpadding="0" cellspacing="0" width="779">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">RPMI</td>
			<td style="width:195px;">Gibco Life Technologies</td>
			<td style="width:187px;">11875-093</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">Fetal bovine serum</td>
			<td style="width:195px;">Gibco Life Technologies</td>
			<td style="width:187px;">10437-028</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">Penicillin Streptomycin</td>
			<td style="width:195px;">Gibco Life Technologies</td>
			<td style="width:187px;">15140-122</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">PBS</td>
			<td style="width:195px;">Corning cell gro</td>
			<td style="width:187px;">21-031-CM</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">60 mm-15 mm cell culture dish</td>
			<td style="width:195px;">Corning</td>
			<td style="width:187px;">3295</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">35 &#181;m Cell Strainer</td>
			<td style="width:195px;">BD Falcon</td>
			<td style="width:187px;">352340</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">50 ml conial tube</td>
			<td style="width:195px;">Crystalgen</td>
			<td style="width:187px;">23-2263</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">15 ml conical tune</td>
			<td style="width:195px;">Crystalgen</td>
			<td style="width:187px;">23-2265</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">Red blood cell lysing buffer</td>
			<td style="width:195px;">Sigma</td>
			<td style="width:187px;">R7757</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">HLA class I (W6/32) PE antibody</td>
			<td style="width:195px;">Abcam</td>
			<td style="width:187px;">ab43545</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">CD31 FITC antibody</td>
			<td style="width:195px;">ebioscience</td>
			<td style="width:187px;">11-0319-42</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">CD45 FITC antibody</td>
			<td style="width:195px;">Abcam</td>
			<td style="width:187px;">ab27287</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">IgG2a&#954; PE antibody</td>
			<td style="width:195px;">BD Pharminogen</td>
			<td style="width:187px;">555574</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">IgG1&#954; FITC antibody</td>
			<td style="width:195px;">BD Pharminogen</td>
			<td style="width:187px;">551954</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">DAPI</td>
			<td style="width:195px;">Invitrogen&#160;</td>
			<td style="width:187px;">d3571</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:232px;">12 x 75 mm polystyrene tubes with cell strainer cap</td>
			<td style="width:195px;">BD Falcon&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;</td>
			<td style="width:187px;">352235</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">Vortex Mixer</td>
			<td style="width:195px;">Crystalgen</td>
			<td style="width:187px;">CG-BV1000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">10% Neutral Buffer Formalin</td>
			<td style="width:195px;">Fisher</td>
			<td style="width:187px;">RBBP-0480</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation of cancer stem cells from human prostate cancer samples

癌症干细胞（CSC）型号已大大超过重新审视过去二十年。在这段时间的CSCs已经确定并直接分离自人组织和串联传播在免疫缺陷小鼠中，通常是通过细胞和分馏通过流式细胞术的亚群的抗体标记。然而，前列腺癌的独特临床特征有很大限制从人类的肿瘤标本前列腺肿瘤干细胞的研究。我们最近报道的前列腺肿瘤干细胞直接从人体组织凭借自己的HLA I类（HLAI）阴性表型的隔离。前列腺癌的细胞是从手术标本收集和机械解离。细胞悬液生成和标有荧光标记的HLAI和间质抗体。 HLAI阴性细胞亚群用流式细胞仪最终分离。该协议的主要限制是经常微观和多灶性性质原发癌在前列腺切除标本。尽管如此，独立的活前列腺肿瘤干细胞是适合的分子特征和功能验证通过移植免疫缺陷小鼠。

<img alt="图1" fo:content-width="5in" fo:src="/files/ftp_upload/51332/51332fig1highres.jpg" src="/files/ftp_upload/51332/51332fig1.jpg" /> 图1。人类前列腺癌肿瘤的收获和流式细胞仪曲线图，展示从人类前列腺癌的特定人群（A）肿瘤结节收获生成细胞悬液。经处理的样品的肿瘤含量用常规苏木精和曙红染色证实。 （B）细胞为DAPI染色阳性被排除隔离只存活细胞。 （ <str...

Watch this Scientific Journal Video about Isolation of Cancer Stem Cells From Human Prostate Cancer Samples at JoVE.com

Isolation of Cancer Stem Cells From Human Prostate Cancer Samples

肿瘤干细胞（肿瘤干细胞）直接从人体组织中的隔离是必要的其生物学特性。这个手稿描述了从人体组织前列腺肿瘤干细胞的分离的方法，同时还提供疑难排解困难的步骤提示。

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 ...

isolation-of-cancer-stem-cells-from-human-prostate-cancer-samples

Research

JoVE Journal

Medicine

14.0K 次观看.  Icahn School of Medicine at Mount Sinai. 肿瘤干细胞（肿瘤干细胞）直接从人体组织中的隔离是必要的其生物学特性。这个手稿描述了从人体组织前列腺肿瘤干细胞的分离的方法，同时还提供疑难排解困难的步骤提示。 

视频: Isolation of Cancer Stem Cells From Human Prostate Cancer Samples

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

Isolation, Characterization and Comparative Differentiation of Human Dental Pulp Stem Cells Derived from Permanent Teeth by Using Two Different Methods

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human pulp-derived stem cells (hDPSCs) possess high proliferation potential with multi-lineage differentiation capacity compare to the ordinary source of adult stem cells1-8; therefore, hDPSCs could be the good candidates for autologous transplantation in tissue engineering and regenerative medicine. Along with these benefits, possessing the mesenchymal stem cells (MSC) features, such as immunolodulatory effect, make hDPSCs more valuable, even in the case of allograft transplantation6,9,10. Therefore, the primary step for using this source of stem cells is to select the best protocol for isolating hDPSCs from pulp tissue. In order to achieve this goal, it is crucial to investigate the effect of various isolation conditions on different cellular behaviors, such as their common surface markers &amp; also their differentiation capacity.
Thus, here we separate human pulp tissue from impacted third molar teeth, and then used both existing protocols based on literature, for isolating hDPSCs,11-13 i.e. enzymatic dissociation of pulp tissue (DPSC-ED) or outgrowth from tissue explants (DPSC-OG). In this regards, we tried to facilitate the isolation methods by using dental diamond disk. Then, these cells characterized in terms of stromal-associated Markers (CD73, CD90, CD105 &amp; CD44), hematopoietic/endothelial Markers (CD34, CD45 &amp; CD11b), perivascular marker, like CD146 and also STRO-1. Afterwards, these two protocols were compared based on the differentiation potency into odontoblasts by both quantitative polymerase chain reaction (QPCR) &amp; Alizarin Red Staining. QPCR were used for the assessment of the expression of the mineralization-related genes (alkaline phosphatase; ALP, matrix extracellular phosphoglycoprotein; MEPE &amp; dentin sialophosphoprotein; DSPP).14

The method described isolation and characterization of human Dental Pulp Stem Cells (hDPSCs) by using either enzymatic dissociation of pulp (DPSC-ED) or direct outgrowth of stem cells from pulp tissue explants (DPSC-OG). Then followed by in vitro comparative differentiation of both types of hDPSCs into odontoblasts.

人牙髓干细胞的分离，鉴定和比较性恒牙使用两种不同的方法，得出

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human ...

Time-lapse Imaging of Primary Preneoplastic Mammary Epithelial Cells Derived from Genetically Engineered Mouse Models of Breast Cancer

Time-lapse imaging can be used to compare behavior of cultured primary preneoplastic mammary epithelial cells derived from different genetically engineered mouse models of breast cancer. For example, time between cell divisions (cell lifetimes), apoptotic cell numbers, evolution of morphological changes, and mechanism of colony formation can be quantified and compared in cells carrying specific genetic lesions. Primary mammary epithelial cell cultures are generated from mammary glands without palpable tumor. Glands are carefully resected with clear separation from adjacent muscle, lymph nodes are removed, and single-cell suspensions of enriched mammary epithelial cells are generated by mincing mammary tissue followed by enzymatic dissociation and filtration. Single-cell suspensions are plated and placed directly under a microscope within an incubator chamber for live-cell imaging. Sixteen 650 &mu;m x 700 &mu;m fields in a 4x4 configuration from each well of a 6-well plate are imaged every 15 min for 5 days. Time-lapse images are examined directly to measure cellular behaviors that can include mechanism and frequency of cell colony formation within the first 24 hr of plating the cells (aggregation versus cell proliferation), incidence of apoptosis, and phasing of morphological changes. Single-cell tracking is used to generate cell fate maps for measurement of individual cell lifetimes and investigation of cell division patterns. Quantitative data are statistically analyzed to assess for significant differences in behavior correlated with specific genetic lesions.

Time-lapse imaging is used to assess behavior of primary preneoplastic mammary epithelial cells derived from genetically engineered mouse models of breast cancer risk to determine if there are correlations between specific behavioral parameters and distinct genetic lesions.

延时成像的主要癌前乳腺上皮细胞来源于遗传工程小鼠乳腺癌模型

Time-lapse  imaging can be used to compare behavior of cultured primary preneoplastic  mammary epithelial cells derived from different genetically ...

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for progression-free and overall survival. Therefore, translational research needs to provide further molecular evidence to improve targeted therapies for malignant melanomas. In the past, oncogenic mechanisms related to melanoma were extensively studied in established cell lines. On the way to more personalized treatment regimens based on individual genetic profiles, we propose to use patient-derived cell lines instead of generic cell lines. Together with high quality clinical data, especially on patient follow-up, these cells will be instrumental to better understand the molecular mechanisms behind melanoma progression.
Here, we report the establishment of primary melanoma cultures from dissected fresh tumor tissue. This procedure includes mincing and dissociation of the tissue into single cells, removal of contaminations with erythrocytes and fibroblasts as well as primary culture and reliable verification of the cells' melanoma origin.
Recent reports revealed that melanomas, like the majority of tumors, harbor a small subpopulation of cancer stem cells (CSCs), which seem to exclusively fuel tumor initiation and progression towards the metastatic state. One of the key markers for CSC identification and isolation in melanoma is CD133. To isolate CD133+ CSCs from primary melanoma cultures, we have modified and optimized the Magnetic-Activated Cell Sorting (MACS) procedure from Miltenyi resulting in high sorting purity and viability of CD133+ CSCs and CD133- bulk, which can be cultivated and functionally analyzed thereafter.

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

This article describes the preparation of freshly obtained melanoma tissue into primary cell cultures, and how to remove contaminations of erythrocytes and fibroblasts from the tumor cells. Finally, we describe how CD133+ putative melanoma stem cells are sorted from the CD133- bulk using Magnetic Activated Cell Sorting (MACS).

来自患者的细胞培养和分离的CD133<sup&gt; +</sup&gt;假定的肿瘤干细胞的恶性黑色素瘤

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for ...

Formation of Human Prostate Epithelium Using Tissue Recombination of Rodent Urogenital Sinus Mesenchyme and Human Stem Cells

Progress in prostate cancer research is severely limited by the availability of human-derived and hormone-na&iuml;ve model systems, which limit our ability to understand genetic and molecular events underlying prostate disease initiation. Toward developing better model systems for studying human prostate carcinogenesis, we and others have taken advantage of the unique pro-prostatic inductive potential of embryonic rodent prostate stroma, termed urogenital sinus mesenchyme (UGSM). When recombined with certain pluripotent cell populations such as embryonic stem cells, UGSM induces the formation of normal human prostate epithelia in a testosterone-dependent manner. Such a human model system can be used to investigate and experimentally test the ability of candidate prostate cancer susceptibility genes at an accelerated pace compared to typical rodent transgenic studies. Since Human embryonic stem cells (hESCs) can be genetically modified in culture using inducible gene expression or siRNA knock-down vectors prior to tissue recombination, such a model facilitates testing the functional consequences of genes, or combinations of genes, which are thought to promote or prevent carcinogenesis.
The technique of isolating pure populations of UGSM cells, however, is challenging and learning often requires someone with previous expertise to personally teach. Moreover, inoculation of cell mixtures under the renal capsule of an immunocompromised host can be technically challenging. Here we outline and illustrate proper isolation of UGSM from rodent embryos and renal capsule implantation of tissue mixtures to form human prostate epithelium. Such an approach, at its current stage, requires in vivo xenografting of embryonic stem cells; future applications could potentially include in vitro gland formation or the use of induced pluripotent stem cell populations (iPSCs).

To unravel the earliest molecular mechanisms underlying prostate cancer initiation, novel and innovative human model systems and approaches are desperately needed. The potential of pre-prostatic urogenital sinus mesenchyme (UGSM) to induce pluripotent stem cell populations to form human prostate epithelium is a powerful experimental tool in prostate research.

人类前列腺上皮细胞形成的使用鼠类泌尿生殖窦间质细胞和人类干细胞的组织重组

Progress in prostate cancer research is severely  limited by the availability of human-derived and hormone-na&iuml;ve model systems,  which limit our ...

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  ...

Enrichment for Chemoresistant Ovarian Cancer Stem Cells from Human Cell Lines

Cancer stem cells (CSCs) are defined as a subset of slow cycling and undifferentiated cells that divide asymmetrically to generate highly proliferative, invasive, and chemoresistant tumor cells. Therefore, CSCs are an attractive population of cells to target therapeutically. CSCs are predicted to contribute to a number of types of malignancies including those in the blood, brain, lung, gastrointestinal tract, prostate, and ovary. Isolating and enriching a tumor cell population for CSCs will enable researchers to study the properties, genetics, and therapeutic response of CSCs. We generated a protocol that reproducibly enriches for ovarian cancer CSCs from ovarian cancer cell lines (SKOV3 and OVCA429). Cell lines are treated with 20 &#181;M cisplatin for 3 days. Surviving cells are isolated and cultured in a serum-free stem cell media containing cytokines and growth factors. We demonstrate an enrichment of these purified CSCs by analyzing the isolated cells for known stem cell markers Oct4, Nanog, and Prom1 (CD133) and cell surface expression of CD177 and CD133. The CSCs exhibit increased chemoresistance. This method for isolation of CSCs is a useful tool for studying the role of CSCs in chemoresistance and tumor relapse.

Cancer stem cells (CSCs) are&#160;implicated in tumor relapse due to chemoresistance. We have optimized a protocol for selection and expansion of CSCs from ovarian cancer cell lines. By treating cells with the chemotherapeutic cisplatin and culturing&#160;in a stem cell promoting media we enrich for non-adherent CSC cultures.

丰富的人力细胞系化疗耐药性卵巢癌干细胞

Cancer stem cells (CSCs) are defined as a subset of slow cycling and undifferentiated cells that divide asymmetrically to generate highly proliferative, ...

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 ...

Isolation and Propagation of Circulating Tumor Cells from a Mouse Cancer Model

Cancer metastasis is the foremost cause of cancer-associated deaths. Recent studies have shown that circulating tumor cells (CTCs) are important in cancer metastasis. Indeed, the number of CTCs correlates with tumor size. Here, a detailed description is provided of a methodology for isolation and propagation of CTCs from a syngeneic mouse model of hepatocellular carcinoma (HCC) which allows for downstream analysis of potentially important molecular mechanisms of solid organ tumor metastasis. This method is efficient and reproducible. It is a non-invasive technique and, therefore, has potential to replace the invasive biopsy of tissues from humans which may be associated with complications. Therefore, the method discussed here allows for the isolation and propagation of CTCs from whole blood samples such that they can be examined and characterized. This has potential for future adaptation for clinical applications such as diagnosis, and personalized targeted therapy.

Circulating tumor cells (CTCs) have been shown to play an important role in tumor metastasis. Here, a method for the isolation and propagation of CTCs from the whole blood of a syngeneic mouse tumor model of hepatocellular carcinoma (HCC) metastasis is described.

从小鼠癌症模型的循环肿瘤细胞的分离和繁殖

Cancer metastasis is the foremost cause of cancer-associated deaths. Recent studies have shown that circulating tumor cells (CTCs) are important in cancer ...

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).