前列腺癌患者产生派生异种移植模型从循环肿瘤细胞
Summary
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.
Abstract
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.
Introduction
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。
Protocol
Representative Results
Discussion
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?…
Divulgations
The authors have nothing to disclose.
Acknowledgements
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.
Materials
| Roswell Park Memorial Institute (RPMI) 1640 | Gibco Life Technologies | 11875-093 | |
| Fetal Bovine Serum (FBS) | Gibco Life Technologies | 10437-028 | |
| Penicillin Streptomycin | Gibco Life Technologies | 15140-122 | |
| Phosphate Buffered Saline (PBS) | Corning Cell Gro | 21-031-CM | |
| 35 µm Cell Strainer | BD Falcon | 352340 | |
| 50 ml polystyrene conical tube | Crystalgen | 23-2263 | |
| Red blood cell lysing buffer | Sigma | R7757 | |
| DAPI | Invitrogen | d3571 | |
| Ficoll-Paque Plus | GE Healthcare | 17-1440 | |
| 12 mm x 75 mm Polystyrene tubes with cell strainer cap | BD Falcon | 352235 | |
| BD Vacutainer Lavender Blood Collection Tubes with EDTA | |||
| BD Winged Blood Collection Set with Push Button Retract Needle 23 gauge | |||
| BD Vacutainer One Use Needle Holder | |||
| Disposable Latex Tourniquet | |||
| Latex or non-latex gloves | |||
| alcohol swabs | |||
| 2×2 cotton gauze pads | |||
| Adhesive bandage | |||
| 25 gauge needle | |||
| 1 ml syringe |
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