我们感谢伦纳德一博士在他的实验室，这些技术最初开发区域生态克里斯汀关和已故的简智彬在这项工作中所用的质粒的礼物，詹姆斯·李斯特和大卫Raible抗体染色的援助;和詹姆斯Neiswender显微注射的视频。这项工作是由美国国立卫生研究院授予R00AR056899-03 CJC

1。筛选黑色素瘤的发病修饰符<ol><li>创建网关中间条目的克隆，通过PCR扩增全长的感兴趣的基因（GOI）的开放阅读框和重组到pDONR 221中使用BP克隆酶II（Invitrogen公司）。使用多地点Gateway技术（Invitrogen）中，以重组p5E_mitfa，pME_GOI，Tol2kit＃302 p3E_SV40polyA 6和miniCoopR 5放置在miniCoopR载体（ 图1A）根据mitfa启动子基因的兴趣。 </li><li>随着25的皮克TOL2转座表达注入25皮克的每个克隆到一个细胞TG（mitfa：BRAF V600E）; p53蛋白（LF）; mitfa（LF）三重纯合子斑马鱼胚胎如前所述7。孵育注射的胚胎在28.5°C。删除任何死胚在24 HPF。 </li><li>选择转基因动物获救的黑素细胞在72 HPF放​​置在培养皿注入胚胎解剖microscopE根据入射光在白色背景（ 图1A）。 </li><li> 3 L罐中转移动物的斑马鱼设施，幼儿园的4 DPF如前所述8。 </li><li>在2个月，与黑素细胞的救援大于4毫米2（ 图1A）的至少一个区域中选择的动物。黑素细胞拯救的程度在72 hpf时，在2个月的黑素细胞的救援程度之间有很强的相关性。当胚胎与黑素细胞拯救拾取72 hpf时，它们的大部分（约80％），将至少有一个面积大于4毫米2在2个月的黑素细胞的救援。 </li><li>屏幕上所选择的动物，每周肿瘤的存在下（ 图1B）。病理组织学和形态学变化之间有很强的相关性。从良性到恶性的过渡是公认的形态学变化时，病变成了提高表面的动物。隔离荷瘤灵魂LS的研究。 </li><li>绘制黑色素瘤生存曲线的横坐标和纵坐标％的黑色素瘤生存率随着年龄的增长在几周内。动物黑素细胞表达基因的利益相比，控制动物的黑素细胞表达EGFP（增强型绿色荧光蛋白）（ 图1C）。动物注射与miniCoopR-EGFP的肿瘤发病中位数大约是18周。使用数秩检验，确定两条曲线显着差异。 </li></ol> 2。肿瘤侵袭实验<ol><li>选择孤立的斑马鱼，背面，肿瘤的发展中的背鳍（ 图2A），后脑和前缘后边界之间的区域。 </li><li>两个星期后，黑色素瘤发病安乐死的鱼的指引由美​​国兽医医学协会面板上的“安乐死”，并经大学，马萨诸塞州医学院宏titutional动物管理和使用委员会（IACUC）。具体而言，鱼被放置成菜与0.6毫克/毫升三卡因直至鳃运动停止。 </li><li>做一个切口，用解剖刀鱼的腹膜腔中，然后在4％多聚甲醛（PFA）的鱼放置在4℃下过夜进行固定。 </li><li>治疗用0.5M EDTA过夜，在4℃下脱钙。 </li><li>在录像带福尔马林固定过夜，脱水1小时，二甲苯3次，每次1小时，然后用石蜡治疗2次，每次2小时，在60°C。 </li><li>嵌入在石蜡中的组织在模具中，并使其凝固为30分钟至1小时。 </li><li>进行5μm切片，每50微米。的部分应该是通过横向和入侵最深的点可以被识别，使整个病变。按照苏木精和曙红染色。 </li><li>评估肿瘤的侵袭，通过测量肿瘤细胞是否保持​​胶原致密层以外，侵入背肌肉组织或侵入到脊柱（ 图2B，C）。确定两个样本集之间的统计学差异。 </li></ol> 3。规模的黑素细胞抗体染色<ol><li>化妆PO480毫升的0.1M的Na 2 HPO 4和20毫升0.1M的NaH 2 PO 4缓冲液。确保PH值为7.3。化妆2倍修复缓冲液含有8.0克蔗糖，0.15毫升0.2M的CaCl 2和90毫升0.2M PO 4缓冲液，pH值7.3。如果需要，调整pH值至7.3，用NaOH或HCl，然后弥补与PO 4缓冲液至100毫升。 </li><li>称取300毫克的固体PFA，并添加微量离心管中。添加5毫米的NaOH的体积等于4.5×质量，单位为毫克，PFA（ 例如 450 UL 5毫米的NaOH至100毫克PFA）。热火在60-70°C，偶尔晃动，直到PFA溶解。自旋向下剩下的颗粒物和恢复20％PFA上清液。新鲜的每一次。 </li><li>准备固定的解决方案，其中包含1个网络连接×缓冲液和4％PFA。 </li><li>在0.17毫克/毫升三卡因麻醉的鱼。 </li><li>入射光在解剖显微镜下摘下背鳞使用的是锋利的细尖镊子，小心不要损坏含黑色素细胞一半的规模（ 图3）。将尺度，直接固定到1ml的溶液，在室温下孵育，从钳子同时转动，为≥2小时。转移的鱼成鱼水的鱼和监控，以确认成功恢复。 </li><li>漂白着色的黑素细胞洗固定尺在PBST（1×PBS pH7.4的，0.1％的Triton X-100）的2倍，在室温下5分钟。加入1毫升新鲜配制的漂白剂溶液含有0.4毫升10％的KOH和0.15毫升30％H 2 O 2在5毫升用dH 2 O。 ，放在封口膜或盖锁，以防止气体从破裂的管道开放。继续漂走了，直到颜料（10分钟，是典型的）（ 图3B，C）。在PBST四次清洗，在室温下5分钟erature。 </li><li>块，用于至少30分钟，在方框溶液由1倍的PBS pH值7.4，0.2％的Triton X-100，2毫克/毫升BSA，1％DMSO，0.02％NaN 3的 2％，羔羊血清。发表NaN的3，如果发展中国家与HRP反应。 </li><li>与初级抗体孵育在方框溶液在室温下过夜。您需要有至少400微升的溶液样品，以使他们淹没。 </li><li>鳞洗净3次，在室温下30分钟，在PBST。 </li><li>从2小时至过夜，溶液在室温下在方框的二次抗体孵育。 </li><li>用PBST + 0.1μM的DAPI染色10分钟。 </li><li> 5分钟，用PBST洗涤3次。 </li><li>鳞安装在载玻片上在vectashield一滴使得秤的凹面侧朝下向滑动，并放置在他们的玻璃盖玻片。在荧光显微镜下（ 图3D，E，F，G的边缘密封的透明指甲油的幻灯片，并观察幻灯片</stronG&gt;）。 </li></ol> 4。移植含量<ol><li>准备收件人2-3个月大的casper 9鱼照射25 Gy的γ射线照射前一天，移植10。让鱼在鱼水之恢复。 </li><li>安乐死肿瘤的轴承鱼如前所述（2.2节）。 </li><li>切断肿瘤，并把它放在培养皿中，约5毫升5％FBS过滤消毒的0.9倍PBS。骰子的肿瘤，而在溶液中，用剃刀。 </li><li>在室温下工作，磨碎与P1000的移液管，以得到单一的细胞。化妆用过滤灭菌的0.9倍的PBS的体积至25ml含有5％FBS。 </li><li>通过40μM网过滤器过​​滤。 </li><li>附带在Eppendorf 5810R台式离心机在1,500 rpm（453相对离心力）10分钟。 </li><li>除去上清液，使细胞悬浮液以大约所需的浓度（假定为100毫米3肿瘤的10 7个细胞）。 </li><li>计算T他确切的细胞数用血细胞计数器，稀释细胞悬浮液，使最终的注射量是约5微升。例如，如果以被注入50,000个细胞，将细胞悬浮液应稀释至最终浓度为10000个细胞/μL。滑动管中，将细胞悬浮液以防止结块的细胞每隔几分钟。 </li><li>用100％乙醇和0.9倍的PBS洗一个26秒计（锥尖）701 N 10微升Hamilton注射器2-3倍。装入注射器5微升细胞悬浮液。 </li><li>在0.17毫克/毫升三卡因麻醉照射收件人鱼。鱼侧放在潮湿的Kimwipe地点（ 图4A）。 </li><li>稳定的鱼，另一只手将针头的斜面朝上，以45°角以上的腹腔后边界之间大约一半的后脑和前缘的背鳍的鱼到侧翼。轻轻压下柱塞（ 图4B）。/ P&gt; <li>让鱼恢复新鲜的鱼水，每天观察鱼的肿瘤细胞植入（ 图4C）。如果已发生的植入，持续增长和疾病的发展也可以观察到。 </li></ol>

<ol>
	<li>Beroukhim, R., 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=The+landscape+of+somatic+copy-number+alteration+across+human+cancers.">The landscape of somatic copy-number alteration across human cancers.</a> Nature. 463, 899-905 (2010).</li><li>Curtin, J. 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=Distinct+sets+of+genetic+alterations+in+melanoma.">Distinct sets of genetic alterations in melanoma.</a> N. Engl. J. Med. 353, 2135-2147 (2005).</li><li>Lin, W. 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=Modeling+genomic+diversity+and+tumor+dependency+in+malignant+melanoma.">Modeling genomic diversity and tumor dependency in malignant melanoma.</a> Cancer Res. 68, 664-673 (2008).</li><li>Patton, E. 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=BRAF+mutations+are+sufficient+to+promote+nevi+formation+and+cooperate+with+p53+in+the+genesis+of+melanoma.">BRAF mutations are sufficient to promote nevi formation and cooperate with p53 in the genesis of melanoma.</a> Curr. Biol. 15, 249-254 (2005).</li><li>Ceol, C. 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=The+histone+methyltransferase+SETDB1+is+recurrently+amplified+in+melanoma+and+accelerates+its+onset.">The histone methyltransferase SETDB1 is recurrently amplified in melanoma and accelerates its onset.</a> Nature. 471, 513-517 (2011).</li><li>Kwan, K. 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=The+Tol2kit:+a+multisite+gateway-based+construction+kit+for+Tol2+transposon+transgenesis+constructs.">The Tol2kit: a multisite gateway-based construction kit for Tol2 transposon transgenesis constructs.</a> Dev. Dyn. 236, 3088-3099 (2007).</li><li>Rosen, J. N., Sweeney, M. F., Mably, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microinjection+of+Zebrafish+Embryos+to+Analyze+Gene+Function.">Microinjection of Zebrafish Embryos to Analyze Gene Function.</a> J. Vis. Exp. (25), e1115 (2009).</li><li>Westerfield, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The zebrafish book. A guide of the laboratory use of zebrafish (Danio rerio). , (2000).</li><li>White, R. 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=Transparent+adult+zebrafish+as+a+tool+for+in+vivo+transplantation+analysis.">Transparent adult zebrafish as a tool for in vivo transplantation analysis.</a> Cell Stem Cell. 2, 183-189 (2008).</li><li>Traver, D., 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=Transplantation+and+in+vivo+imaging+of+multilineage+engraftment+in+zebrafish+bloodless+mutants.">Transplantation and in vivo imaging of multilineage engraftment in zebrafish bloodless mutants.</a> Nat. Immunol. 4, 1238-1246 (2003).</li></ol>

没有利益冲突的声明。

miniCoopR方法使表达的基因在斑马鱼的黑素细胞的兴趣。这种方法利用的事实，的斑马鱼mitfa基因的作用细胞自主性。出于这个原因，黑素细胞获救由miniCoopR载体是一定要包含小基因和任何感兴趣的基因，它在物理上耦合。营救黑素细胞是清晰可见，能够得到在被注射的动物，作为单细胞胚胎。特定程度的嵌合体被选中，并可以拿下超过此截止动物肿瘤发病或其他特性。的一大好处是足够的?...

<table border="1"><tbody><tr><td> 的试剂的名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td>网关重组试剂</td><td> Invitrogen公司</td><td></td><td></td></tr><tr><td> miniCoopR </td><td></td><td></td><td>参考文献5 </td></tr><tr><td> Mitfa抗体</td><td></td><td></td><td>参考文献5 </td></tr><tr><td> FITC标记的山羊抗兔IgG抗体</td><td> Invitrogen公司</td><td></td><td></td></tr><tr><td> Vectashield </td><td>矢量实验室</td><td> H-1000 </td><td></td></tr><tr><td> 卡斯帕斑马鱼</td><td></td><td></td><td>参考9 </td></tr><tr><td> 701N 10μl注射器</td><td>汉密尔顿/费舍尔</td><td> 14-824 </td><td> ＆提供; </td></tr><tr><td> 40微米的过滤器</td><td> BD猎鹰/费舍尔</td><td> 352340 </td><td></td></tr><tr><td> FBS </td><td> Invitrogen公司</td><td> 26140079 </td><td></td></tr></tbody></table>

screening for melanoma modifiers using a zebrafish autochthonous tumor model

的人类癌症基因组学研究已经取得了丰富的信息改变的基因，在肿瘤1,2,3。从这些研究中所产生的一个挑战是，许多基因被改变，它可以开车从那些在转换过程中偶然产生的肿瘤的遗传改变，很难区分。作出这样的区分是有好处的分析，可以定量衡量一个改变基因在肿瘤的发生和流程，使肿瘤的坚持和传播的效果。在这里，我们提出了一个快速的方法来筛选大量的候选黑色素瘤修饰符在斑马鱼黑色素瘤的启动和维护所需的步骤，包括一个土生土长的肿瘤模型4。在此分析是一个关键试剂的miniCoopR载体，这对新人的mitfa黑素细胞的规范因素的野生型拷贝到网关重组盒候选人梅尔阿诺马基因可重组5。 miniCoopR矢量具有mitfa的的抢救小基因的其中包含的的野生型mitfa的基因的启动子，开放阅读框和3&#39;-非翻译区的。它使我们能够使用全长度的开放阅读框的候选人黑色素瘤修饰符的结构。个人可以被注入单细胞TG（mitfa：BRAF V600E）p53蛋白（LF）; mitfa（LF）斑马鱼胚胎克隆。矢量被集成miniCoopR TOL2介导的转基因6和救援黑色素细胞。因为它们被物理地连接至的mitfa挽救小基因，候选基因的表达在获救的黑素细胞，其中的一些将改造，并发展成肿瘤。对黑色素瘤的启动和黑色素瘤细胞属性的候选基因的效果，可以使用无黑色素瘤生存曲线，入侵检测，抗体染色和移植试验测定。

一单元格的Tg（mitfa：BRAF V600E）; p53基因（如果）; mitfa（如果）斑马鱼胚胎与miniCoopR矢量含有黑色素瘤癌基因SETDB1 5或EGFP的mitfa启动子的控制下，每个注入。胚胎选择，并允许成熟的黑素细胞的救援。在2个月内黑素细胞救援与动物的年龄大于4平方毫米的选择。在动物中筛选每周黑色素瘤。为成人肿瘤发病率曲线表明，SETDB1癌基因显着加速黑...

Watch this Scientific Journal Video about Screening for Melanoma Modifiers using a Zebrafish Autochthonous Tumor Model at JoVE.com

Screening for Melanoma Modifiers using a Zebrafish Autochthonous Tumor Model

屏幕使用一个斑马鱼原地肿瘤模型的黑色素瘤的改性剂是一种快速的方式。它利用的miniCoopR允许候选黑色素的黑素细胞中的基因表达的载体。黑色素瘤生存率曲线，入侵检测，协议规模的黑素细胞抗体染色和黑色素瘤移植试验的方法获得。

对黑色素瘤修饰符一个斑马鱼原地肿瘤模型的筛选

Genomic studies of  human cancers have yielded a wealth of information about genes that are altered  in tumors1,2,3. A challenge arising from these ...

screening-for-melanoma-modifiers-using-zebrafish-autochthonous-tumor

Research

JoVE Journal

Medicine

13.6K 次观看.  University of Massachusetts Medical School. 屏幕使用一个斑马鱼原地肿瘤模型的黑色素瘤的改性剂是一种快速的方式。它利用的miniCoopR允许候选黑色素的黑素细胞中的基因表达的载体。黑色素瘤生存率曲线，入侵检测，协议规模的黑素细胞抗体染色和黑色素瘤移植试验的方法获得。

视频: Screening for Melanoma Modifiers using a Zebrafish Autochthonous Tumor Model

Multi-photon Imaging of Tumor Cell Invasion in an Orthotopic Mouse Model of Oral Squamous Cell Carcinoma

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient management strategies. Orthotopic mouse models of oral cancer have been developed to facilitate the study of factors that impact invasion and serve as model system for evaluating anti-tumor therapeutics. In these systems, visualization of disseminated tumor cells within oral cavity tissues has typically been conducted by either conventional histology or with in vivo bioluminescent methods. A primary drawback of these techniques is the inherent inability to accurately visualize and quantify early tumor cell invasion arising from the primary site in three dimensions. Here we describe a protocol that combines an established model for squamous cell carcinoma of the tongue (SCOT) with two-photon imaging to allow multi-vectorial visualization of lingual tumor spread. The OSC-19 head and neck tumor cell line was stably engineered to express the F-actin binding peptide LifeAct fused to the mCherry fluorescent protein (LifeAct-mCherry). Fox1nu/nu mice injected with these cells reliably form tumors that allow the tongue to be visualized by ex-vivo application of two-photon microscopy. This technique allows for the orthotopic visualization of the tumor mass and locally invading cells in excised tongues without disruption of the regional tumor microenvironment. In addition, this system allows for the quantification of tumor cell invasion by calculating distances that invaded cells move from the primary tumor site. Overall this procedure provides an enhanced model system for analyzing factors that contribute to SCOT invasion and therapeutic treatments tailored to prevent local invasion and distant metastatic spread. This method also has the potential to be ultimately combined with other imaging modalities in an in vivo setting.

A comprehensive overview of the techniques involved in generating a mouse model of oral cancer and quantitative monitoring of tumor invasion within the tongue through multi-photon microscopy of labeled cells is presented. This system can serve as a useful platform for the molecular assessment and drug efficacy of anti-invasive compounds.

多光子成像在口腔鳞状细胞癌原位小鼠模型的肿瘤细胞的侵袭

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient ...

科研

医学

Assessing Teratogenic Changes in a Zebrafish Model of Fetal Alcohol Exposure

Fetal alcohol syndrome (FAS) is a severe manifestation of embryonic exposure to ethanol. It presents with characteristic defects to the face and organs, including mental retardation due to disordered and damaged brain development. Fetal alcohol spectrum disorder (FASD) is a term used to cover a continuum of birth defects that occur due to maternal alcohol consumption, and occurs in approximately 4% of children born in the United States. With 50% of child-bearing age women reporting consumption of alcohol, and half of all pregnancies being unplanned, unintentional exposure is a continuing issue2. In order to best understand the damage produced by ethanol, plus produce a model with which to test potential interventions, we developed a model of developmental ethanol exposure using the zebrafish embryo. Zebrafish are ideal for this kind of teratogen study3-8. Each pair lays hundreds of eggs, which can then be collected without harming the adult fish. The zebrafish embryo is transparent and can be readily imaged with any number of stains. Analysis of these embryos after exposure to ethanol at different doses and times of duration and application shows that the gross developmental defects produced by ethanol are consistent with the human birth defect. Described here are the basic techniques used to study and manipulate the zebrafish FAS model.

In order to understand the molecular mechanisms of the ethanol-induced developmental damage, we have developed a zebrafish model of ethanol exposure and are exploring the physical, cellular, and genetic alterations that occur after ethanol exposure1. We then seek to find potential interventions and rapidly test them in this animal model.

在斑马鱼模型的胎儿酒精暴露评估致畸的变化

Fetal alcohol syndrome (FAS) is a severe manifestation of embryonic exposure to ethanol. It presents with characteristic defects to the face and organs, ...

Human Neuroendocrine Tumor Cell Lines as a Three-Dimensional Model for the Study of Human Neuroendocrine Tumor Therapy

Neuroendocrine tumors (NETs) are rare tumors, with an incidence of two per 100,&#x200A;000 individuals per year, and they account for 0.5% of all human malignancies.1 Other than surgery for the minority of patients who present with localized disease, there is little or no survival benefit of systemic therapy. Therefore, there is a great need to better understand the biology of NETs, and in particular define new therapeutic targets for patients with nonresectable or metastatic neuroendocrine tumors. 3D cell culture is becoming a popular method for drug screening due to its relevance in modeling the in vivo tumor tissue organization and microenvironment.2,3 The 3D multicellular spheroids could provide valuable information in a more timely and less expensive manner than directly proceeding from 2D cell culture experiments to animal (murine) models.
To facilitate the discovery of new therapeutics for NET patients, we have developed an in vitro 3D multicellular spheroids model using the human NET cell lines. The NET cells are plated in a non-adhesive agarose-coated 24-well plate and incubated under physiological conditions (5% CO2, 37 &deg;C) with a very slow agitation for 16-24 hr after plating. The cells form multicellular spheroids starting on the 3rd or 4th day. The spheroids become more spherical by the 6th day, at which point the drug treatments are initiated. The efficacy of the drug treatments on the NET spheroids is monitored based on the morphology, shape and size of the spheroids with a phase-contrast light microscope. The size of the spheroids is estimated automatically using a custom-developed MATLAB program based on an active contour algorithm. Further, we demonstrate a simple method to process the HistoGel embedding on these 3D spheroids, allowing the use of standard histological and immunohistochemical techniques. 
This is the first report on generating 3D spheroids using NET cell lines to examine the effect of therapeutic drugs. We have also performed histology on these 3D spheroids, and displayed an example of a single drug's effect on growth and proliferation of the NET spheroids. Our results support that the NET spheroids are valuable for further studies of NET biology and drug development.

We present a simple agarose overlay platform to grow 3D multicellular spheroids using neuroendocrine cancer cell lines. This method provides a very convenient way to examine the effect of therapeutic drugs on the neuroendocrine tumor cells. It could also help us establish human neuroendocrine tumor spheroids for cancer therapy.

人类神经内分泌肿瘤细胞株为研究人类神经内分泌肿瘤治疗的三维模型

Neuroendocrine tumors (NETs) are rare tumors, with an incidence of two per 100,&#x200A;000 individuals per year, and they account for 0.5% of all human ...

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 ...

A Zebrafish Model of Diabetes Mellitus and Metabolic Memory

Diabetes mellitus currently affects 346 million individuals and this is projected to increase to 400 million by 2030. Evidence from both the laboratory and large scale clinical trials has revealed that diabetic complications progress unimpeded via the phenomenon of metabolic memory even when glycemic control is pharmaceutically achieved. Gene expression can be stably altered through epigenetic changes which not only allow cells and organisms to quickly respond to changing environmental stimuli but also confer the ability of the cell to "memorize" these encounters once the stimulus is removed. As such, the roles that these mechanisms play in the metabolic memory phenomenon are currently being examined.
We have recently reported the development of a zebrafish model of type I diabetes mellitus and characterized this model to show that diabetic zebrafish not only display the known secondary complications including the changes associated with diabetic retinopathy, diabetic nephropathy and impaired wound healing but also exhibit impaired caudal fin regeneration. This model is unique in that the zebrafish is capable to regenerate its damaged pancreas and restore a euglycemic state similar to what would be expected in post-transplant human patients. Moreover, multiple rounds of caudal fin amputation allow for the separation and study of pure epigenetic effects in an in vivo system without potential complicating factors from the previous diabetic state. Although euglycemia is achieved following pancreatic regeneration, the diabetic secondary complication of fin regeneration and skin wound healing persists indefinitely. In the case of impaired fin regeneration, this pathology is retained even after multiple rounds of fin regeneration in the daughter fin tissues. These observations point to an underlying epigenetic process existing in the metabolic memory state. Here we present the methods needed to successfully generate the diabetic and metabolic memory groups of fish and discuss the advantages of this model.

Metabolic memory is the phenomenon by which diabetic complications persist and progress unimpeded even after euglycemia is achieved pharmaceutically. Here we describe a diabetes mellitus zebrafish model which is unique in that it allows for the examination of the mitotically transmissible epigenetic components of metabolic memory in vivo.

斑马鱼模型的糖尿病和代谢记忆

Diabetes mellitus currently affects 346 million individuals and this is projected to increase to 400 million by 2030. Evidence from both the laboratory ...

A Novel High-resolution In vivo Imaging Technique to Study the Dynamic Response of Intracranial Structures to Tumor Growth and Therapeutics

We have successfully integrated previously established Intracranial window (ICW) technology 1-4 with intravital 2-photon confocal microscopy to develop a novel platform that allows for direct long-term visualization of tissue structure changes intracranially. Imaging at a single cell resolution in a real-time fashion provides supplementary dynamic information beyond that provided by standard end-point histological analysis, which looks solely at 'snap-shot' cross sections of tissue.
Establishing this intravital imaging technique in fluorescent chimeric mice, we are able to image four fluorescent channels simultaneously. By incorporating fluorescently labeled cells, such as GFP+ bone marrow, it is possible to track the fate of these cells studying their long-term migration, integration and differentiation within tissue. Further integration of a secondary reporter cell, such as an mCherry glioma tumor line, allows for characterization of cell:cell interactions. Structural changes in the tissue microenvironment can be highlighted through the addition of intra-vital dyes and antibodies, for example CD31 tagged antibodies and Dextran molecules.
Moreover, we describe the combination of our ICW imaging model with a small animal micro-irradiator that provides stereotactic irradiation, creating a platform through which the dynamic tissue changes that occur following the administration of ionizing irradiation can be assessed.
Current limitations of our model include penetrance of the microscope, which is limited to a depth of up to 900 &mu;m from the sub cortical surface, limiting imaging to the dorsal axis of the brain. The presence of the skull bone makes the ICW a more challenging technical procedure, compared to the more established and utilized chamber models currently used to study mammary tissue and fat pads 5-7. In addition, the ICW provides many challenges when optimizing the imaging.

A Novel High-resolution In vivo Imaging Technique to Study the Dynamic Response of Intracranial Structures to Tumor Growth and Therapeutics

We describe a novel in vivo imaging technique that couples fluorescent chimeric mice with intracranial windows and high-resolution 2-photon microscopy. This imaging platform aids studies of dynamic changes in brain tissue and microvasculature, at a single-cell level, following pathological insults and is adaptable to assess intracranial drug delivery and distribution.

一种新型的高分辨率<em&gt;在体内</em&gt;成像技术研究肿瘤生长及治疗颅内结构的动态响应

We have successfully integrated previously established Intracranial window (ICW) technology 1-4 with intravital 2-photon confocal microscopy to develop a ...

A Possible Zebrafish Model of Polycystic Kidney Disease: Knockdown of wnt5a Causes Cysts in Zebrafish Kidneys

Polycystic kidney disease (PKD) is one of the most common causes of end-stage kidney disease, a devastating disease for which there is no cure. The molecular mechanisms leading to cyst formation in PKD remain somewhat unclear, but many genes are thought to be involved. Wnt5a is a non-canonical glycoprotein that regulates a wide range of developmental processes. Wnt5a works through the planar cell polarity (PCP) pathway that regulates oriented cell division during renal tubular cell elongation. Defects of the PCP pathway have been found to cause kidney cyst formation. Our paper describes a method for developing a zebrafish cystic kidney disease model by knockdown of the wnt5a gene with wnt5a antisense morpholino (MO) oligonucleotides. Tg(wt1b:GFP) transgenic zebrafish were used to visualize kidney structure and kidney cysts following wnt5a knockdown. Two distinct antisense MOs (AUG - and splice-site) were used and both resulted in curly tail down phenotype and cyst formation after wnt5a knockdown. Injection of mouse Wnt5a mRNA, resistant to the MOs due to a difference in primary base pair structure, rescued the abnormal phenotype, demonstrating that the phenotype was not due to &#8220;off-target&#8221; effects of the morpholino. This work supports the validity of using a zebrafish model to study wnt5a function in the kidney.

A Possible Zebrafish Model of Polycystic Kidney Disease: Knockdown of wnt5a Causes Cysts in Zebrafish Kidneys

We describe a method of generating a possible zebrafish model of polycystic kidney disease. We used Tg(wt1b:GFP) fish to visualize kidney structure. Knockdown of wnt5a was by morpholino injection. Pronephric cyst formation after wnt5a knockdown was observed in this GFP transgenic zebrafish.

多囊肾一种可能的斑马鱼型号：击倒<em&gt; Wnt5a的</em&gt;原因在斑马鱼肾囊肿

Polycystic kidney disease (PKD) is one of the most common causes of end-stage kidney disease, a devastating disease for which there is no cure. The ...

Three-dimensional Co-culture Model for Tumor-stromal Interaction

Cancer progression (initiation, growth, invasion and metastasis) occurs through interactions between malignant cells and the surrounding tumor stromal cells. The tumor microenvironment is comprised of a variety of cell types, such as fibroblasts, immune cells, vascular endothelial cells, pericytes and bone-marrow-derived cells, embedded in the extracellular matrix (ECM). Cancer-associated fibroblasts (CAFs) have a pro-tumorigenic role through the secretion of soluble factors, angiogenesis and ECM remodeling. The experimental models for cancer cell survival, proliferation, migration, and invasion have mostly relied on two-dimensional monocellular and monolayer tissue cultures or Boyden chamber assays. However, these experiments do not precisely reflect the physiological or pathological conditions in a diseased organ. To gain a better understanding of tumor stromal or tumor matrix interactions, multicellular and three-dimensional cultures provide more powerful tools for investigating intercellular communication and ECM-dependent modulation of cancer cell behavior. As a platform for this type of study, we present an experimental model in which cancer cells are cultured on collagen gels embedded with primary cultures of CAFs.

Here we present a protocol to co-culture in three-dimensions, which is useful for investigating multicellular interactions and extracellular matrix-dependent modulation of cancer cell behavior. In this experimental model, cancer cells are cultured on collagen gels embedded with human cancer-associated fibroblasts.

对于肿瘤基质相互作用的三维共培养模式

Cancer progression (initiation, growth, invasion and metastasis) occurs through interactions between malignant cells and the surrounding tumor stromal ...

Isolated Hepatic Perfusion as a Treatment for Liver Metastases of Uveal Melanoma

Isolated hepatic perfusion (IHP) is a procedure where the liver is surgically isolated and perfused with a high concentration of the chemotherapeutic agent melphalan. Briefly, the procedure starts with the setup of a percutaneous veno-venous bypass from the femoral vein to the external jugular vein. Via a laparotomy, catheters are then inserted into the proper hepatic artery and the caval vein. The portal vein and the caval vein, both supra- and infrahepatically, are then clamped. The arterial and venous catheters are connected to a heart lung machine and the liver is perfused with melphalan (1 mg/kg body weight) for 60 min. This way it is possible to locally perfuse the liver with a high dose of a chemotherapeutic agent, without leakage to the systemic circulation.
In previous studies including patients with isolated liver metastases of uveal melanoma, an overall response rate of 33-100% and a median survival between 9 and 13 months, have been reported. The aim of this protocol is to give a clear description of how to perform the procedure and to discuss IHP as a treatment option for liver metastases of uveal melanoma.

Here, we present a protocol how to perform an isolated liver perfusion (IHP) with melphalan and we also discuss IHP as a treatment option for liver metastases of uveal melanoma.

隔离肝脏灌注作为治疗葡萄膜黑色素瘤肝转移

Isolated hepatic perfusion (IHP) is a procedure where the liver is surgically isolated and perfused with a high concentration of the chemotherapeutic ...

Evaluation of Tumor-infiltrating Leukocyte Subsets in a Subcutaneous Tumor Model

Specialized immune cells that infiltrate the tumor microenvironment regulate the growth and survival of neoplasia.&#160; Malignant cells must elude or subvert anti-tumor immune responses in order to survive and flourish. Tumors take advantage of a number of different mechanisms of immune &#8220;escape,&#8221; including the recruitment of tolerogenic DC, immunosuppressive regulatory T cells (Tregs), and myeloid-derived suppressor cells (MDSC) that inhibit cytotoxic anti-tumor responses. Conversely, anti-tumor effector immune cells can slow the growth and expansion of malignancies: immunostimulatory dendritic cells, natural killer cells which harbor innate anti-tumor immunity, and cytotoxic T cells all can participate in tumor suppression. The balance between pro- and anti-tumor leukocytes ultimately determines the behavior and fate of transformed cells; a multitude of human clinical studies have borne this out. Thus, detailed analysis of leukocyte subsets within the tumor microenvironment has become increasingly important. Here, we describe a method for analyzing infiltrating leukocyte subsets present in the tumor microenvironment in a mouse tumor model. Mouse B16 melanoma tumor cells were inoculated subcutaneously in C57BL/6 mice. At a specified time, tumors and surrounding skin were resected en bloc and processed into single cell suspensions, which were then stained for multi-color flow cytometry. Using a variety of leukocyte subset markers, we were able to compare the relative percentages of infiltrating leukocyte subsets between control and chemerin-expressing tumors. Investigators may use such a tool to study the immune presence in the tumor microenvironment and when combined with traditional caliper size measurements of tumor growth, will potentially allow them to elucidate the impact of changes in immune composition on tumor growth. Such a technique can be applied to any tumor model in which the tumor and its microenvironment can be resected and processed.

This protocol describes a method for the detailed evaluation of leukocyte subsets within the tumor microenvironment in a mouse tumor model. Chemerin-expressing B16 melanoma cells were implanted subcutaneously into syngeneic mice. Cells from the tumor microenvironment were then stained and analyzed by flow cytometry, allowing for detailed leukocyte subset analyses.

在皮下肿瘤模型的评估肿瘤浸润白细胞亚群

Specialized immune cells that infiltrate the tumor microenvironment regulate the growth and survival of neoplasia.&#160; Malignant cells must elude or ...