We thank Dr. Jordi Ochando from the Flow Cytometry Shared Resources at the Mount Sinai Medical Center for their assistance in flow cytometry analysis. We thank Dr. Rumana Huq from the Microscopy Shared Resource Facility at the Mount Sinai Medical Center for their imaging assistance. The authors thank the TJ Martell Foundation for its support in this project.

该协议已在我们的机构，从体制研究伦理委员会的批准，已进行并符合对人类福利的所有机构，国家和国际准则。 从晚期前列腺癌1.采集外周血注意：选择治疗转移性前列腺癌。获得书面患者同意，并记录患者的临床特征，包括年龄在隔离和以前的化疗和激素治疗。转移性肿瘤患者将有可能拥有的CTC的外周血中含量最高。 <ol><li>知情同意书后，并在适当的机构审查委员会批准的协议采集血液样本。戴乳胶手套和试验服整个过程。 </li><li>连接25摹蝴蝶针和管针持有人。用酒精棉签擦拭的面积二头肌和前臂的肘静脉，然后应用止血带患者的上臂。 </li><li>将蝴蝶针插入肘静脉推采血管为持针器开始收集。收集大约10毫升的血液中含有乙二胺四乙酸（EDTA）以防止凝固收集管。 </li><li>从患者除去蝴蝶针和用消毒纱布垫施加压力超过现场静脉穿刺的1分钟。盖部位用无菌绷带。 </li></ol> 2.分离单个核细胞注意：从第1步收集的血液将包含外周血单核细胞（PBMC）（例如，淋巴细胞和单核细胞），除了单核的CTC。 <ol><li>吸移管的全血到50ml聚苯乙烯锥形管用5 ml的血清吸液管和添加Hanks平衡盐溶液（HBSS）以1：1的比例。轻轻吸管混合均匀化。 </li><li>加入15mL可商制备的溶液（菲可 - 帕克）由低速密度梯度离心来分离血液成分为空50毫升聚苯乙烯锥形管中。 </li><li>轻轻吸取全血和HBSS混合物，从步骤2.1，到聚苯乙烯管从步骤2.2，以形成不同的顶层。设置吸管以减少溶液混合的最低设置，这将确保有效的分离。 </li><li>离心机在400×g离心30分钟，在室温（25℃）。设置减速到最低设置，以防止分离后的溶液混合。加速度可以被设置为任何速度。 </li><li>离心后，确定在顶部和分离溶液等离子体之间PBMC和的CTC夹层的薄白灰色条纹在管的底部。 </li><li>收集来自白灰色条纹在步骤2.5的细胞分化成使用塑料移液管50毫升的聚苯乙烯管中。 </li><li>加入HBSS至50ml聚苯乙烯锥形管PBMC和检验中心和c再次entrifuge在400 XG 10分钟RT洗。 </li><li>再次滗析液重悬沉淀在50ml的HBSS和离心机在400×g离心3分钟，在室温洗。 </li><li>弃去上清液，重悬其余沉淀用5ml红细胞裂解缓冲液孵育5分钟，在室温的溶液中。 </li><li>除去离心的红细胞裂解缓冲液在400×g离心3分钟，在RT。 </li><li>弃上清，重悬沉淀在1ml的PBS 1×补充有10％胎牛血清（FBS），以阻止前抗体染色。 </li></ol> 3.染色单个核细胞CD45-FITC抗体进行荧光活化细胞分选（FACS） <ol><li>量化使用血球或自动细胞计数器上可行的（台盼蓝阴性）细胞（来自步骤2.11）的数量。制备1×10 6个细胞/ ml的悬浮液（在PBS中1×有10％FBS），并放置在冰上1小时。 </li><li>分发量化来自前一步骤的两试管细胞悬浮液。标签管作为对照和CD45染色。 </li><li> 250：在控制管，在一个稀释的1加IgG1κ-FITC（2.5微克/毫升）。在CD45染色管中，加入CD45 FITC（2.5微克/毫升）的共轭初级抗体的稀释度为1：250和在冰上孵育细胞悬浮液30分钟。 CD45抗原标记用于排除造血细胞。 </li><li>离心细胞，在400×g离心3分钟，在4℃下，并弃去上清液。 </li><li>洗涤细胞两次，悬浮每个粒料用无菌1×PBS中补充了10％FBS的随后离心，在400×g离心3分钟，在4℃。 </li><li>悬浮在1毫升溶液含4&#39;，6-二脒基-2-苯基吲哚（DAPI）以10μg/ ml的浓度的PBS 1×的细胞。 </li><li>过滤器通过35微米的过滤器盖的最终解决方案，为12毫米×75毫米聚苯乙烯管。 </li></ol> 4.隔离前列腺检验中心通过FACS 注：利用流式细胞仪来收集实时CD45阴性检验中心。 <ol><li>采用细胞标记，管道组补偿控制“控制”。 </li><li>创建排除碎片和集群使用适当的正向散射和侧向散射参数细胞的大门。 </li><li>建立使用从标记的管中，细胞悬浮液的FITC门“CD45-FITC”。 </li><li>门可行的（DAPI阴性）使用太平洋蓝色的通道细胞。 </li><li>收集CD45阴性人口到无菌的15 ml的聚苯乙烯锥形管含有2毫升罗斯韦尔园区纪念研究所（RPMI）1640补充有10％FBS中。 </li><li>离心机的排序前列腺肿瘤细胞悬浮液在400×g离心3分钟，然后重悬在粒料200 - 500微升的RPMI补充有10％FBS中。 </li></ol> 5.注射的CTC入小鼠移植瘤生长和监控注意：开展所有动物的程序符合批准的机构动物保护委员会的协议。该协议已在我们的制度下，根据和遵守所有相关的监管和体制机构，法规和准则经我们的动物保护委员会的特定动物使用协议进行的。 <ol><li>混合排序前列腺肿瘤细胞悬浮液与细胞外基质以1：1的比例，并置于冰上。 </li><li>麻醉小鼠之前皮下植入的腔室在1L供给5％（体积/体积）吸入异氟烷的氧气/分钟。通过检查角膜和脚趾反射小鼠丧失确保适当的麻醉。 </li><li>使用地下25针和1ml注射器，注入250微升前列腺肿瘤细胞和细胞外基质的细胞悬浮液皮下注射到一个8-10周龄雄性免疫缺陷小鼠的两个上侧翼。注入无论四氯化碳浓度的250微升作为喷射体积是第important值。在小鼠最大SQ施用是不超过1毫升&#160;</li><li>通过执行鼠标注射部位为皮下结节密度生长每周触诊并通过每周测量小鼠体重两次监测小鼠。 </li><li>安乐死的小鼠通过二氧化碳窒息颈椎脱位和收获皮下人前列腺癌异种移植物当肿瘤大​​小超过0.5厘米和小于1厘米的最大直径，以确保有足够的肿瘤组织进行分子分析和连续传代。安乐死小鼠困扰或体重减轻15％的迹象，尽管未见肿瘤是显而易见的。 </li></ol>

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The authors declare that they have no competing financial interests.

这份手稿描述了从检验中心的新一代前列腺癌PDX模型的方法。对于PDX一代的使用检验中心的 时相比，现有方法的模型有几个潜在的重要的优点。首先，访问集合的CTC的外周血使实验模型的产生来自同一患者在不同疾病阶段。第二，血液采集代表一个更安全和更便宜的方法相比，需要外科手术收集的肿瘤细胞的现有方法以分离肿瘤细胞。 CTC富集多种方法已经被描?...

A correction was made to <a href="http://www.jove.com/video/53182">Generation of Prostate Cancer Patient Derived Xenograft Models from Circulating Tumor Cells</a>. There was a spelling error in one of the authors' surname. The author's name was corrected from:
Veronica Rodriquez-Bravo
to:
Veronica Rodriguez-Bravo

A correction was made to Generation of Prostate Cancer Patient Derived Xenograft Models from Circulating Tumor Cells.

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

来自患者的异种移植物是用于癌症研究日益流行的实验模型。它们可用于生物标志物和生物学途径，药物疗效的临床前评估，并创建化身个性化癌症疗法1,2的表征。此前，其他研究小组已经开发出PDX机型无论是通过植入或注射单个肿瘤细胞悬液或全肿瘤植到免疫小鼠1。这些PDX 模型需要外科收集新鲜实体瘤，恶性腹水或从患者经历外科手术这既昂贵又暴露病人医源性发病的危险性增加胸腔积液。 在癌症研究显著最近的发展一直循环肿瘤细胞检测，分离和鉴定。从原发肿瘤肿块这些肿瘤细胞逃逸，并进入循环在那里他们在转移和复发发挥关键作用，癌症的最常见的原因有关的死亡率3。的CTC从几个实体瘤类型的评估和表征提供了临床信息进行诊断，预后，监测和残留病3。多种目前使用的方法依赖于任一物理性质，生物标志物的表达，或的CTC的功能特性可以用来有效地分离的CTC 4。现有宏观尺度四氯化碳的分离方法包括密度梯度离心，带过滤器的孔的物理过滤和分离针对表面分子。最广​​泛使用的CTC隔离方法是基于的CTC的基于抗体的捕获。两个阳性和阴性选择的细胞表面标记物可以用来从外周血分离的CTC。阳性选择为在外围循环的CTC通常使用上皮标记（例如，EpCAM的），其一个重新表达的CTC而不是造血干细胞。这种方法的缺点是，的CTC转移性潜力经常经历上皮至间质转变（EMT），该下调上皮表面标志3。为了分离的CTC转移性潜力，负选择方法，它采用了造血表面标记，CD45，耗尽白细胞的正常细胞群可以使用5。 前列腺癌是最常见的诊断的癌症和癌症相关死亡的男性6的一个主要原因。肿瘤进展和侵略性的机制尚未完全了解，因此，产生和实验模型概括前列腺癌的分子异质性的表征是显著兴趣的。前列腺癌的PDX机型已经 由人前列腺癌细胞的植入先前产生成immunocom承诺小鼠7,8。然而，这样的模式的产生受到阻碍低植入率前列腺癌到免疫小鼠，这主要归因于疾病的性质无痛。最近，的CTC已经用于生成乳腺癌9，肺癌10和前列腺癌11 PDX模型。概念验证的这些研究引入生成的需要手术样品收集PDX模型独立的可能性。在本文中，我们详细描述了这个新的实验模型的生成方法。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Roswell Park Memorial Institute (RPMI) 1640</td>
			<td>Gibco Life Technologies</td>
			<td>11875-093</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>Gibco Life Technologies</td>
			<td>10437-028</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin Streptomycin</td>
			<td>Gibco Life Technologies</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline (PBS)</td>
			<td>Corning Cell Gro</td>
			<td>21-031-CM</td>
			<td></td>
		</tr>
		<tr>
			<td>35 &#181;m Cell Strainer</td>
			<td>BD Falcon</td>
			<td>352340</td>
			<td></td>
		</tr>
		<tr>
			<td>50 ml polystyrene conical tube</td>
			<td>Crystalgen</td>
			<td>23-2263</td>
			<td></td>
		</tr>
		<tr>
			<td>Red blood cell lysing buffer</td>
			<td>Sigma</td>
			<td>R7757</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Invitrogen</td>
			<td>d3571</td>
			<td></td>
		</tr>
		<tr>
			<td>Ficoll-Paque Plus</td>
			<td>GE Healthcare</td>
			<td>17-1440</td>
			<td></td>
		</tr>
		<tr>
			<td>12 mm x 75 mm Polystyrene tubes with cell strainer cap</td>
			<td>BD Falcon</td>
			<td>352235</td>
			<td></td>
		</tr>
		<tr>
			<td>BD Vacutainer Lavender Blood Collection Tubes with EDTA</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>BD Winged Blood Collection Set with Push Button Retract Needle 23 gauge</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>BD Vacutainer One Use Needle Holder</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable Latex Tourniquet</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Latex or non-latex gloves</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>alcohol swabs</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>2x2 cotton gauze pads</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Adhesive bandage</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>25 gauge needle</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1 ml syringe</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

该协议将导致从孤立的CD45阴性前列腺癌的CTC的产生PDX机型。根据我们的协议中使用的负选择方法，有必要用DAPI染色来排除死细胞。 CD45阴性细胞通过流式细胞术检测到的百分比是可变的并且取决于患者（图1A）的肿瘤负荷。使用CD45和DAPI未分选的细胞的免疫荧光染色（鉴别细胞核）揭示了CD45阴性细胞中，由白色箭头（图1B）所指示的。 在图1C中，皮下结节密?...

Watch this Scientific Journal Video about Generation of Prostate Cancer Patient Derived Xenograft Models from Circulating Tumor Cells at JoVE.com

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

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, ...

generation-prostate-cancer-patient-derived-xenograft-models-from

Research

JoVE Journal

Medicine

14.0K 次观看.  Icahn School of Medicine at Mount Sinai. 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.

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

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 ...

科研

医学

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 ...

Adaptation of Semiautomated Circulating Tumor Cell (CTC) Assays for Clinical and Preclinical Research Applications

The majority of cancer-related deaths occur subsequent to the development of metastatic disease. This highly lethal disease stage is associated with the presence of circulating tumor cells (CTCs). These rare cells have been demonstrated to be of clinical significance in metastatic breast, prostate, and colorectal cancers. The current gold standard in clinical CTC detection and enumeration is the FDA-cleared CellSearch system (CSS). This manuscript outlines the standard protocol utilized by this platform as well as two&#160;additional adapted protocols that describe the detailed process of user-defined marker optimization for protein characterization of patient CTCs and a comparable protocol for CTC capture in very low volumes of blood, using standard CSS reagents, for studying in vivo preclinical mouse models of metastasis. In addition, differences in CTC quality between healthy donor blood spiked with cells from tissue culture versus patient blood samples are highlighted. Finally, several commonly discrepant items that can lead to CTC misclassification errors are outlined. Taken together, these protocols will provide a useful resource for users of this platform interested in preclinical and clinical research pertaining to metastasis and CTCs.

Adaptation of Semiautomated Circulating Tumor Cell CTC Assays for Clinical and Preclinical Research Applications

Circulating tumor cells (CTCs) are prognostic in several metastatic cancers. This manuscript describes the gold standard CellSearch system (CSS) CTC enumeration platform and highlights common misclassification errors. In addition, two&#160;adapted protocols are described for user-defined marker characterization of CTCs and CTC enumeration in preclinical mouse models of metastasis using this technology.

半自动适应循环肿瘤细胞（CTC）分析临床和临床前研究中的应用

The majority of cancer-related deaths occur subsequent to the development of metastatic disease. This highly lethal disease stage is associated with the ...

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 ...

In vitro Method to Observe E-selectin-mediated Interactions Between Prostate Circulating Tumor Cells Derived From Patients and Human Endothelial Cells

Metastasis is a process in which tumor cells shed from the primary tumor intravasate blood vascular and lymphatic system, thereby, gaining access to extravasate and form a secondary niche.&#160;The extravasation of tumor cells from the blood vascular system can be studied using endothelial cells (ECs) and tumor cells obtained from different cell lines.&#160;Initial studies were conducted using static conditions but it has been well documented that ECs behave differently under physiological flow conditions.&#160;Therefore, different flow chamber assemblies are currently being used to studying cancer cell interactions with ECs.&#160;Current flow chamber assemblies offer reproducible results using either different cell lines or fluid at different shear stress conditions.&#160;However, to observe and study interactions with rare cells such as circulating tumor cells (CTCs), certain changes are required to be made to the conventional flow chamber assembly.&#160;CTCs are a rare cell population among millions of blood cells. Consequently, it is difficult to obtain a pure population of CTCs.&#160;Contamination of CTCs with different types of cells normally found in the circulation is inevitable using present enrichment or depletion techniques.&#160;In the present report, we describe a unique method to fluorescently label circulating prostate cancer cells and study their interactions with ECs in a self-assembled flow chamber system.&#160;This technique can be further applied to observe interactions between prostate CTCs and any protein of interest.

In vitro Method to Observe E-selectin-mediated Interactions Between Prostate Circulating Tumor Cells Derived From Patients and Human Endothelial Cells

Our report describes a unique method to visualize and analyze CTC/EC interactions in prostate cancer under physiological flow conditions.

在体外的方法来观察派生病人和人内皮细胞前列腺循环肿瘤细胞之间的E-选择素介导的相互作用

Metastasis is a process in which tumor cells shed from the primary tumor intravasate blood vascular and lymphatic system, thereby, gaining access to ...

Generation of Induced Pluripotent Stem Cells from Muscular Dystrophy Patients: Efficient Integration-free Reprogramming of Urine Derived Cells

Dystrophic cardiomyopathy is a poorly understood consequence of muscular dystrophy. Generating induced Pluripotent Stem Cells (iPSCs) from patients with muscular dystrophy is an invaluable cellular source for in&#160;vitro disease model systems and can be used for drug screening studies. Patient-derived urine cells have been used in successful reprogramming into induced pluripotent stem cells in order to model dystrophic cardiomyopathy1. Addressing the safety concerns of integrating vector systems, we present a protocol using a non-integrating Sendai virus vector for transduction of Yamanaka factors into urine cells collected from patients with muscular dystrophy. This protocol generates fully reprogrammed clones within 2&#8211;3 weeks. The pluripotent cells are vector-free by passage-13. These dystrophic iPSCs can be differentiated into cardiomyocytes and used either to study disease mechanisms or for drug screening.

This protocol entails detailed procedures for isolation of urine derived cells from muscular dystrophy patients; their efficient and rapid reprogramming through Sendai virus transduction.

从生成性肌营养不良患者诱导多能干细胞的：尿源性细胞的有效整合，免费重编程

Dystrophic cardiomyopathy is a poorly understood consequence of muscular dystrophy. Generating induced Pluripotent Stem Cells (iPSCs) from patients with ...

Isolation and Immortalization of Patient-derived Cell Lines from Muscle Biopsy for Disease Modeling

The generation of patient-specific cell lines represents an invaluable tool for diagnostic or translational research, and these cells can be collected from skin or muscle biopsy tissue available during the patient&#8217;s diagnostic workup. In this protocol, we describe a technique for live cell isolation from small amounts of muscle or skin tissue for primary cell culture. Additionally, we provide a technique for the immortalization of myogenic cell lines and fibroblast cell lines from primary cells. Once cell lines are immortalized, substantial expansion of patient-derived cells can be achieved. Immortalized cells are amenable to many downstream applications, including drug screening and in vitro correction of the genetic mutation. Altogether, these protocols provide a reliable tool to generate and preserve patient-derived cells for downstream applications.

This protocol describes techniques for live cell isolation and primary culture of myogenic and fibroblast cell lines from muscle or skin tissue. A technique for the immortalization of these cell lines is also described. Altogether, these protocols provide a reliable tool to generate and preserve patient-derived cells for downstream applications.

从肌肉活检患者来源的细胞系疾病建模隔离和永生化

The generation of patient-specific cell lines represents an invaluable tool for diagnostic or translational research, and these cells can be collected ...

In vitro Enrichment of Ovarian Cancer Tumor-initiating Cells

Evidence suggests that small subpopulations of tumor cells maintain a unique self-renewing and differentiation capacity and may be responsible for tumor initiation and/or relapse. Clarifying the mechanisms by which these tumor-initiating cells (TICs) support tumor formation and progression could lead to the development of clinically favorable therapies. Ovarian cancer is a heterogeneous and highly recurrent disease. Recent studies suggest TICs may play an important role in disease biology. We have identified culture conditions that enrich for TICs from ovarian cancer cell lines. Growing either adherent cells or non-adherent &#8216;floater&#8217; cells in a low attachment plate with serum free media in the presence of growth factors supports the propagation of ovarian cancer TICs with stem cell markers (CD133 and ALDH activity) and increased tumorigenicity without the need to physically separate the TICs from other cell types within the culture. Although the presence of floater cells is not common for all cell lines, this population of cells with innate low adherence may have high tumorigenic potential.Compared to adherent cells grown in the presence of serum, TICs readily form spheres, are significantly more tumorigenic in mice, and express putative stem cell markers. The conditions are easy to establish in a timely manner and can be used to study signaling pathways important for maintaining stem characteristics, and to identify drugs or combinations of drugs targeting TICs. The culture conditions described herein are applicable for a variety of ovarian cancer cells of epithelial origin and will be critical in providing new information about the role of TICs in tumor initiation, progression, and relapse.

In vitro Enrichment of Ovarian Cancer Tumor-initiating Cells

Tumor-initiating cells (TICs) may represent a viable therapeutic target for the treatment of ovarian cancer, a highly recurrent and fatal disease. We present a protocol for culture conditions that enrich for this highly tumorigenic population of cells.

在卵巢癌肿瘤起始细胞体外富集

Evidence suggests that small subpopulations of tumor cells maintain a unique self-renewing and differentiation capacity and may be responsible for tumor ...

Establishment of a Human Multiple Myeloma Xenograft Model in the Chicken to Study Tumor Growth, Invasion and Angiogenesis

Multiple myeloma (MM), a malignant plasma cell disease, remains incurable and novel drugs are required to improve the prognosis of patients. Due to the lack of the bone microenvironment and auto/paracrine growth factors human MM cells are difficult to cultivate. Therefore, there is an urgent need to establish proper in vitro and in vivo culture systems to study the action of novel therapeutics on human MM cells. Here we present a model to grow human multiple myeloma cells in a complex 3D environment in vitro and in vivo. MM cell lines OPM-2 and RPMI-8226 were transfected to express the transgene GFP and were cultivated in the presence of human mesenchymal cells and collagen type-I matrix as three-dimensional spheroids. In addition, spheroids were grafted on the chorioallantoic membrane (CAM) of chicken embryos and tumor growth was monitored by stereo fluorescence microscopy. Both models allow the study of novel therapeutic drugs in a complex 3D environment and the quantification of the tumor cell mass after homogenization of grafts in a transgene-specific GFP-ELISA. Moreover, angiogenic responses of the host and invasion of tumor cells into the subjacent host tissue can be monitored daily by a stereo microscope and analyzed by immunohistochemical staining against human tumor cells (Ki-67, CD138, Vimentin) or host mural cells covering blood vessels (desmin/ASMA).
In conclusion, the onplant system allows studying MM cell growth and angiogenesis in a complex 3D environment and enables screening for novel therapeutic compounds targeting survival and proliferation of MM cells.

Human multiple myeloma (MM) cells require the supportive microenvironment of mesenchymal cells and extracellular matrix components for survival and proliferation. We established an in vivo chicken embryo model with engrafted human myeloma and mesenchymal cells to study effects of cancer drugs on tumor growth, invasion and angiogenesis.

建立一个人类多发性骨髓瘤异种移植模型中的鸡，研究肿瘤的生长，侵袭和血管生成

Multiple myeloma (MM), a malignant plasma cell disease, remains incurable and novel drugs are required to improve the prognosis of patients. Due to the ...

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.