Name: generation of prostate cancer patient derived xenograft models from circulating tumor cells
Uploaded: 2015-10-20T15:00:00.000+00:00
Duration: 8 min 3 s
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><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 &amp;micro;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>2 x 2 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 G 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, ...

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14.1K 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

Development and Maintenance of a Preclinical Patient Derived Tumor Xenograft Model for the Investigation of Novel Anti-Cancer Therapies

Patient derived tumor xenograft (PDTX) models provide a necessary platform in facilitating anti-cancer drug development prior to human trials. Human tumor pieces are injected subcutaneously into athymic nude mice (immunocompromised, T cell deficient) to create a bank of tumors and subsequently are passaged into different generations of mice in order to maintain these tumors from patients. Importantly, cellular heterogeneity of the original tumor is closely emulated in this model, which provides a more clinically relevant model for evaluation of drug efficacy studies (single agent and combination), biomarker analysis, resistant pathways and cancer stem cell biology. Some limitations of the PDTX model include the replacement of the human stroma with mouse stroma after the first generation in mice, inability to investigate treatment effects on metastasis due to the subcutaneous injections of the tumors, and the lack of evaluation of immunotherapies due to the use of immunocompromised mice. However, even with these limitations, the PDTX model provides a powerful preclinical platform in the drug discovery process.

Utilizing patient-derived tumors in a subcutaneous preclinical model is an excellent way to study the efficacy of novel therapies, predictive biomarker discovery, and drug resistant pathways. This model, in the drug development process, is essential in determining the fate of many novel anti-cancer therapies prior to clinical investigation.

开发和维护的临床前患者来源的肿瘤异种移植模型为新的抗癌疗法的调查

Patient derived tumor xenograft (PDTX) models provide a necessary platform in facilitating anti-cancer drug development prior to human trials. Human tumor ...

科研

癌症研究

Labeling of Breast Cancer Patient-derived Xenografts with Traceable Reporters for Tumor Growth and Metastasis Studies

The use of preclinical models to study tumor biology and response to treatment is central to cancer research. Long-established human cell lines, and many transgenic mouse models, often fail to recapitulate the key aspects of human malignancies. Thus, alternative models that better represent the heterogeneity of patients' tumors and their metastases are being developed. Patient-derived xenograft (PDX) models in which surgically resected tumor samples are engrafted into immunocompromised mice have become an attractive alternative as they can be transplanted through multiple generations,and more efficiently reflect tumor heterogeneity than xenografts derived from human cancer cell lines. A limitation to the use of PDXs is that they are difficult to transfect or transduce to introduce traceable reporters or to manipulate gene expression. The current protocol describes methods to transduce dissociated tumor cells from PDXs with high transduction efficiency, and the use of labeled PDXs for experimental models of breast cancer metastases. The protocol also demonstrates the use of labeled PDXs in experimental metastasis models to study the organ-colonization process of the metastatic cascade. Metastases to different organs can be easily visualized and quantified using bioluminescent imaging in live animals, or GFP expression during dissection and in excised organs. These methods provide a powerful tool to extend the use of multiple types of PDXs to metastasis research.

We describe a method for stable labeling of patient-derived xenografts (PDXs) with lentiviral particles expressing green-fluorescent protein and luciferase reporters. This method allows for tracking the growth of PDXs at the primary site, as well as detecting spontaneous and experimental metastases using in vivo imaging systems.

可追踪记者乳腺癌患者来源的移植瘤贴标肿瘤生长和转移的研究

The use of preclinical models to study tumor biology and response to treatment is central to cancer research. Long-established human cell lines, and many ...

Generation of Prostate Cancer Cell Models of Resistance to the Anti-mitotic Agent Docetaxel

Microtubule targeting agents (MTAs) are a mainstay in the treatment of a wide range of tumors. However, acquired resistance to chemotherapeutic drugs is a common mechanism of disease progression and a prognostic-determinant feature of malignant tumors. In prostate cancer (PC), resistance to MTAs such as the taxane Docetaxel dictates treatment failure as well as progression towards lethal stages of disease that are defined by a poor prognosis and high mortality rates. Though studied for decades, the array of mechanisms contributing to acquired resistance are not completely understood, and thus pose a significant limitation to the development of new therapeutic strategies that could benefit patients in these advanced stages of disease. In this protocol, we describe the generation of Docetaxel-resistant prostate cancer cell lines that mimic lethal features of late-stage prostate cancer, and therefore can be used to study the mechanisms by which acquired chemoresistance arises. Despite potential limitations intrinsic to a cell based model, such as the loss of resistance properties over time, the Docetaxel-resistant cell lines produced by this method have been successfully used in recent studies and offer&#160;the opportunity to advance our molecular understanding of acquired chemoresistance in lethal prostate cancer.

Resistance to cancer therapies contributes to disease progression and death. Determining the mechanistic underpinnings of resistance is crucial for improving therapeutic response. This manuscript details the protocol to generate taxane-resistant cell models of prostate cancer (PC) to help dissecting the pathways involved in progression to Docetaxel resistance in PC patients.

抗有丝分裂药物多西紫杉醇的前列腺癌细胞模型的建立

Microtubule targeting agents (MTAs) are a mainstay in the treatment of a wide range of tumors. However, acquired resistance to chemotherapeutic drugs is a ...

A Bioluminescent and Fluorescent Orthotopic Syngeneic Murine Model of Androgen-dependent and Castration-resistant Prostate Cancer

Orthotopic tumor modeling is a valuable tool for pre-clinical prostate cancer research, as it has multiple advantages over both subcutaneous and transgenic genetically engineered mouse models. Unlike subcutaneous tumors, orthotopic tumors contain more clinically accurate vasculature, tumor microenvironment, and responses to multiple therapies. In contrast to genetically engineered mouse models, orthotopic models can be performed with lower cost and in less time, involve the use of highly complex and heterogeneous mouse or human cancer cell lines, rather that single genetic alterations, and these cell lines can be genetically modified, such as to express imaging agents. Here, we present a protocol to surgically injecting a luciferase- and mCherry-expressing murine prostate cancer cell line into the anterior prostate lobe of mice. These mice developed orthotopic tumors that were non-invasively monitored in vivo and further analyzed for tumor volume, weight, mouse survival, and immune infiltration. Further, orthotopic tumor-bearing mice were surgically castrated, leading to immediate tumor regression and subsequent recurrence, representing castration-resistant prostate cancer. Although technical skill is required to carry out this procedure, this syngeneic orthotopic model of both androgen-dependent and castration-resistant prostate cancer is of great use to all investigators in the field.

The goal of this protocol is to demonstrate the intra-prostatic injection of prostate cancer cells, with subsequent castration. Orthotopic pre-clinical models of androgen-dependent and castration-resistant prostate cancer are critical to study the disease in the context of a clinically relevant tumor microenvironment and an immunocompetent host.

雄激素依赖性和去势抗性前列腺癌的荧光自体小鼠模型

Orthotopic tumor modeling is a valuable tool for pre-clinical prostate cancer research, as it has multiple advantages over both subcutaneous and ...

Patient-derived Orthotopic Xenograft Models for Human Urothelial Cell Carcinoma and Colorectal Cancer Tumor Growth and Spontaneous Metastasis

Cancer patients have poor prognoses when lymph node (LN) involvement is present in both high-grade urothelial cell carcinoma (HG-UCC) of the bladder and colorectal cancer (CRC). More than 50% of patients with muscle-invasive UCC, despite curative therapy for clinically-localized disease, will develop metastases and die within 5 years, and metastatic CRC is a leading cause of cancer-related deaths in the US. Xenograft models that consistently mimic UCC and CRC metastasis seen in patients are needed. This study aims to generate patient-derived orthotopic xenograft (PDOX) models of UCC and CRC for primary tumor growth and spontaneous metastases under the influence of LN stromal cells mimicking the progression of human metastatic diseases for drug screening. Fresh UCC and CRC tumors were obtained from consented patients undergoing resection for HG-UCC and colorectal adenocarcinoma, respectively. Co-inoculated with LN stromal cell (LNSC) analog HK cells, luciferase-tagged UCC cells were intra-vesically (IB) instilled into female non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice, and CRC cells were intra-rectally (IR) injected into male NOD/SCID mice. Tumor growth and metastasis were monitored weekly using bioluminescence imaging (BLI). Upon sacrifice, primary tumors and mouse organs were harvested, weighed, and formalin-fixed for Hematoxylin and Eosin and immunohistochemistry staining. In our unique PDOX models, xenograft tumors resemble patient pre-implantation tumors. In the presence of HK cells, both models have high tumor implantation rates measured by BLI and tumor weights, 83.3% for UCC and 96.9% for CRC, and high distant organ metastasis rates (33.3% detected liver or lung metastasis for UCC and 53.1% for CRC). In addition, both models have zero mortality from the procedure. We have established unique, reproducible PDOX models for human HG-UCC and CRC, which allow for tumor formation, growth, and metastasis studies. With these models, testing of novel therapeutic drugs can be performed efficiently and in a clinically-mimetic manner.

This protocol describes the generation of patient-derived orthotopic xenograft models by intra-vesically instilling high-grade urothelial cell carcinoma cells or intra-rectally injecting colorectal cancer cells into non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice for primary tumor growth and spontaneous metastases under the influence of lymph node stromal cells, which mimics the progression of human metastatic diseases.

患者衍生的人类泌尿细胞癌和结肠直肠癌肿瘤生长和自发转移的正交异种移植模型

Cancer patients have poor prognoses when lymph node (LN) involvement is present in both high-grade urothelial cell carcinoma (HG-UCC) of the bladder and ...

Establishment of Gastric Cancer Patient-derived Xenograft Models and Primary Cell Lines

The use of preclinical models to advance our understanding of tumor biology and investigate the efficacy of therapeutic agents is key to cancer research. Although there are many established gastric cancer cell lines and many conventional transgenic mouse models for preclinical research, the disadvantages of these in vitro and in vivo models limit their applications. Because the characteristics of these models have changed in culture, they no longer model tumor heterogeneity, and their responses have not been able to predict responses in humans. Thus, alternative models that better represent tumor heterogeneity are being developed. Patient-derived xenograft (PDX) models preserve the histologic appearance of cancer cells, retain intratumoral heterogeneity, and better reflect the relevant human components of the tumor microenvironment. However, it usually takes 4-8 months to develop a PDX model, which is longer than the expected survival of many gastric patients. For this reason, establishing primary cancer cell lines may be an effective complementary method for drug response studies. The current protocol describes methods to establish PDX models and primary cancer cell lines from surgical gastric cancer samples. These methods provide a useful tool for drug development and cancer biology research.

The current protocol describes methods to establish patient-derived xenograft (PDX) models and primary cancer cell lines from surgical gastric cancer samples. The methods provide a useful tool for drug development and cancer biology research.

建立胃癌患者衍生异种移植模型和原细胞系

The use of preclinical models to advance our understanding of tumor biology and investigate the efficacy of therapeutic agents is key to cancer research. ...

Generation of Tumor Organoids from Genetically Engineered Mouse Models of Prostate Cancer

Methods based on homologous recombination to modify genes have significantly furthered biological research. Genetically engineered mouse models (GEMMs) are a rigorous method for studying mammalian development and disease. Our laboratory has developed several GEMMs of prostate cancer (PCa) that lack expression of one or multiple tumor suppressor genes using the site-specific Cre-loxP recombinase system and a prostate-specific promoter. In this article, we describe our method for necropsy of these PCa GEMMs, primarily focusing on dissection of mouse prostate tumors. New methods developed over the last decade have facilitated the culture of epithelial-derived cells to model organ systems in vitro in three dimensions. We also detail a 3D cell culture method to generate tumor organoids from mouse PCa GEMMs. Pre-clinical cancer research has been dominated by 2D cell culture and cell line-derived or patient-derived xenograft models. These methods lack tumor microenvironment, a limitation of using these techniques in pre-clinical studies. GEMMs are more physiologically-relevant for understanding tumorigenesis and cancer progression. Tumor organoid culture is an in vitro model system that recapitulates tumor architecture and cell lineage characteristics. In addition, 3D cell culture methods allow for growth of normal cells for comparison to tumor cell cultures, rarely possible using 2D cell culture techniques. In combination, use of GEMMs and 3D cell culture in pre-clinical studies has the potential to improve our understanding of cancer biology.&#160;

We show a method for necropsy and dissection of mouse prostate cancer models, focusing on prostate tumor dissection. A step-by-step protocol for generation of mouse prostate tumor organoids is also presented.

从前列腺癌的遗传工程小鼠模型生成肿瘤有机体

Methods based on homologous recombination to modify genes have significantly furthered biological research. Genetically engineered mouse models (GEMMs) ...

遗传学

Establishment and Analysis of Three-Dimensional (3D) Organoids Derived from Patient Prostate Cancer Bone Metastasis Specimens and their Xenografts

Three-dimensional (3D) culture of organoids from tumor specimens of human patients and patient-derived xenograft (PDX) models of prostate cancer, referred to as patient-derived organoids (PDO), are an invaluable resource for studying the mechanism of tumorigenesis and metastasis of prostate cancer. Their main advantage is that they maintain the distinctive genomic and functional heterogeneity of the original tissue compared to conventional cell lines that do not. Furthermore, 3D cultures of PDO can be used to predict the effects of drug treatment on individual patients and are a step towards personalized medicine. Despite these advantages, few groups routinely use this method in part because of the extensive optimization of PDO culture conditions that may be required for different patient samples. We previously demonstrated that our prostate cancer bone metastasis PDX model, PCSD1, recapitulated the resistance of the donor patient&rsquo;s bone metastasis to anti-androgen therapy. We used PCSD1 3D organoids to characterize further the mechanisms of anti-androgen resistance. Following an overview of currently published studies of PDX and PDO models, we describe a step-by-step protocol for 3D culture of PDO using domed or floating basement membrane (e.g., Matrigel) spheres in optimized culture conditions. In vivo stitch imaging and cell processing for histology are also described. This protocol can be further optimized for other applications including western blot, co-culture, etc. and can be used to explore characteristics of 3D cultured PDO pertaining to drug resistance, tumorigenesis, metastasis and therapeutics.

Establishment and Analysis of Three-Dimensional 3D Organoids Derived from Patient Prostate Cancer Bone Metastasis Specimens and their Xenografts

Three-dimensional cultures of patient BMPC specimens and xenografts of bone metastatic prostate cancer maintain the functional heterogeneity of their original tumors resulting in cysts, spheroids and complex, tumor-like organoids. This manuscript provides an optimization strategy and protocol for 3D culture of heterogeneous patient derived samples and their analysis using IFC.

从患者前列腺癌骨转移标本及其异种移植物中提取的三维（3D）有机物的建立与分析

Three-dimensional (3D) culture of organoids from tumor specimens of human patients and patient-derived xenograft (PDX) models of prostate cancer, referred ...

Generation of an Orthotopic Xenograft of Pancreatic Cancer Cells by Ultrasound-Guided Injection

Pancreatic cancer (PCa) represents one of the deadliest cancer types worldwide. The reasons for PCa malignancy mainly rely on its intrinsic malignant behavior and high resistance to therapeutic treatments. Indeed, despite many efforts, both standard chemotherapy and innovative target therapies have substantially failed when moved from preclinical evaluation to the clinical setting. In this scenario, the development of preclinical mouse models better mimicking in vivo characteristics of PCa is urgently needed to test newly developed drugs. The present protocol describes a method to generate a mouse model of PCa, represented by an orthotopic xenograft obtained by ultrasound-guided injection of human pancreatic tumor cells. Using such a reliable and minimally invasive protocol, we also provide evidence of in vivo engraftment and development of tumor masses, which can be monitored by ultrasound (US) imaging. A noteworthy aspect of the PCa model described here is the slow development of the tumor masses over time, which allows precise identification of the starting point for pharmacological treatments and better monitoring of the effects of therapeutic interventions. Moreover, the technique described here is an example of implementation of the 3Rs principles since it minimizes pain and suffering and directly improves the welfare of animals in research.

We present a protocol to generate a minimally invasive orthotopic pancreatic cancer model by ultrasound-guided injection of human pancreatic cancer cells and the subsequent monitoring of tumor growth in vivo by ultrasound imaging.

通过超声引导注射产生胰腺癌细胞的原位异种移植物

Pancreatic cancer (PCa) represents one of the deadliest cancer types worldwide. The reasons for PCa malignancy mainly rely on its intrinsic malignant ...

Depletion of Mouse Cells from Human Tumor Xenografts Significantly Improves Downstream Analysis of Target Cells

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic patient tumors in different assays1. Human tumor xenografts represent the gold standard with respect to tumor biology, drug discovery, and metastasis research 2-4. Tumor xenografts can be derived from different types of material like tumor cell lines, tumor tissue from primary patient tumors4 or serially transplanted tumors. When propagated in vivo, xenografted tissue is infiltrated and vascularized by cells of mouse origin. Multiple factors such as the tumor entity, the origin of xenografted material, growth rate and region of transplantation influence the composition and the amount of mouse cells present in tumor xenografts. However, even when these factors are kept constant, the degree of mouse cell contamination is highly variable.
Contaminating mouse cells significantly impair downstream analyses of human tumor xenografts. As mouse fibroblasts show high plating efficacies and proliferation rates, they tend to overgrow cultures of human tumor cells, especially slowly proliferating subpopulations. Mouse cell derived DNA, mRNA, and protein components can bias downstream gene expression analysis, next-generation sequencing, as well as proteome analysis 5. To overcome these limitations, we have developed a fast and easy method to isolate untouched human tumor cells from xenografted tumor tissue. This procedure is based on the comprehensive depletion of cells of mouse origin by combining automated tissue dissociation with the benchtop tissue dissociator and magnetic cell sorting. Here, we demonstrate that human target cells can be can be obtained with purities higher than 96% within less than 20 min independent of the tumor type.

Human tumor xenografts are vascularized and infiltrated by cells of mouse origin during the growth phase in vivo. To circumvent the bias caused by these contaminating cells during downstream analysis, we developed a method allowing for comprehensive depletion of all mouse cells by magnetic cell sorting.

从人类肿瘤异种移植的小鼠细胞的消耗显着改善靶细胞下游分析

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic ...