Name: generation of prostate cancer patient derived xenograft models from circulating tumor cells
Uploaded: 2015-10-20T15:00:00
Description: Watch this Scientific Journal Video about Generation of Prostate Cancer Patient Derived Xenograft Models from Circulating Tumor Cells at JoVE.com

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...

<ol>
	<li>Hidalgo, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patient-derived+xenograft+models:+an+emerging+platform+for+translational+cancer+research.">Patient-derived xenograft models: an emerging platform for translational cancer research.</a> Cancer discovery. 4, 998-1013 (2014).</li><li>Siolas, D., Hannon, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patient-derived+tumor+xenografts:+transforming+clinical+samples+into+mouse+models.">Patient-derived tumor xenografts: transforming clinical samples into mouse models.</a> Cancer research. 73, 5315-5319 (2013).</li><li>Joosse, S. A., Gorges, T. M., Pantel, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biology,+detection,+and+clinical+implications+of+circulating+tumor+cells.">Biology, detection, and clinical implications of circulating tumor cells.</a> EMBO molecular medicine. 1, 1-11 (2014).</li><li>Yu, M., Stott, S., Toner, M., Maheswaran, S., Haber, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circulating+tumor+cells:+approaches+to+isolation+and+characterization.">Circulating tumor cells: approaches to isolation and characterization.</a> The Journal of cell biology. 192, 373-382 (2011).</li><li>Liu, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Negative+enrichment+by+immunomagnetic+nanobeads+for+unbiased+characterization+of+circulating+tumor+cells+from+peripheral+blood+of+cancer+patients.">Negative enrichment by immunomagnetic nanobeads for unbiased characterization of circulating tumor cells from peripheral blood of cancer patients.</a> Journal of translational medicine. 9, 1-9 (2011).</li><li>Siegel, R., Naishadham, D., Jemal, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+statistics+,+2013.">Cancer statistics , 2013.</a> CA: a cancer journal for clinicians. 63, 11-30 (2013).</li><li>Domingo-Domenech, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Suppression+of+acquired+docetaxel+resistance+in+prostate+cancer+through+depletion+of+notch-+and+hedgehog-dependent+tumor-initiating+cells.">Suppression of acquired docetaxel resistance in prostate cancer through depletion of notch- and hedgehog-dependent tumor-initiating cells.</a> Cancer cell. 22, 373-388 (2012).</li><li>Klein, K. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progression+of+metastatic+human+prostate+cancer+to+androgen+independence+in+immunodeficient+SCID+mice.">Progression of metastatic human prostate cancer to androgen independence in immunodeficient SCID mice.</a> Nature Medicine. 3, 402-408 (1997).</li><li>Yu, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+therapy.+Ex+vivo+culture+of+circulating+breast+tumor+cells+for+individualized+testing+of+drug+susceptibility.">Cancer therapy. Ex vivo culture of circulating breast tumor cells for individualized testing of drug susceptibility.</a> Science. 345, 216-220 (2014).</li><li>Hodgkinson, C. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumorigenicity+and+genetic+profiling+of+circulating+tumor+cells+in+small-cell+lung+cancer.">Tumorigenicity and genetic profiling of circulating tumor cells in small-cell lung cancer.</a> Nature medicine. 20, 897-903 (2014).</li><li>Vidal, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Targetable+GATA2-IGF2+Axis+Confers+Aggressiveness+in+Lethal+Prostate+Cancer.">A Targetable GATA2-IGF2 Axis Confers Aggressiveness in Lethal Prostate Cancer.</a> Cancer cell. 27, 223-239 (2015).</li><li>Quintana, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+tumour+formation+by+single+human+melanoma+cells.">Efficient tumour formation by single human melanoma cells.</a> Nature. 456, 593-598 (2008).</li><li>DeRose, Y. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+grafts+derived+from+women+with+breast+cancer+authentically+reflect+tumor+pathology,+growth,+metastasis+and+disease+outcomes.">Tumor grafts derived from women with breast cancer authentically reflect tumor pathology, growth, metastasis and disease outcomes.</a> Nature. 17, 1514-1520 (2011).</li></ol>

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, ...

Research

JoVE Journal

Medicine

Live Imaging of Drug Responses in the Tumor Microenvironment in Mouse Models of Breast Cancer

实时成像在乳腺癌小鼠模型中的肿瘤微环境对药物的反应

The tumor microenvironment plays a pivotal role in tumor initiation, progression, metastasis, and the response to anti-cancer therapies. Three-dimensional ...

科研

医学

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

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

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

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

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

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

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

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

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

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 ...

In vitro Enrichment of Ovarian Cancer Tumor-initiating Cells

In vitro Enrichment of Ovarian Cancer Tumor-initiating 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

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 ...