这项工作成为可能，补助JK从卓越中心安大略省和安大略研究基金。 我们想拍摄期间感谢Siddika Pardhan和彼得·弗格森博士的技术援助。

注意：下面的协议被设计为与贴壁细胞使用。修改都需要该方法适用于在悬浮液中生长的非贴壁细胞。在协议中所描述的CAM实验被设计用于与细胞表达的荧光标记物（ 例如 ，GFP，RFP， 等等 ）的使用。九日龄鸡胚上需要的实验方案为所述CAM实验的第2天。 1.准备和肿瘤细胞与反义寡核苷酸转染（艾滋病服务） <ol><li>从包含感兴趣的细胞T75烧瓶（S）吸中，洗1XPBS，并加2ml 0.25％胰蛋白酶/ EDTA的。加入10 ml完全生长培养基到每个烧瓶中，并在溶液中转移细胞到50ml管中。计数细胞并调节音量，以使最终浓度为1.0×10每毫升5个细胞。 </li><li>标签两组T25烧瓶：一组将包含8瓶，标记1-8，并为USED为实时代谢测定实验 ;和第二组也将包含8瓶和将被用于鸡胚胎的CAM实验 。在溶液中加2ml细胞到每个烧瓶中，并发生在培养箱中（37℃，5％CO 2的O / N）。 </li><li>第二天早上（第0天，时间0），标号35毫升管如下：对照（非靶向ASO或siRNA），&#39;靶的兴趣“（即 ，靶向ASO或siRNA），和脂质体2000（或类似的转染试剂）。稀释股票ASO解到适当的浓度与在相应的管无血清培养基中。 <ol><li>添加所需的转染试剂的量，以适当的管中并孵育在无血清培养基中5分钟。转染试剂溶液添加到ASO管并孵育20分钟。 </li></ol></li><li>加入250微升的控制的ASO转染试剂溶液向每个烧瓶1-4中各组中，与250微升“的目标利率的”ASO转染试剂溶液每每组5-8瓶的。孵育4小时（37ᵒC，5％的CO 2）。 <ol><li>在培养过程中，开展2.1节所述的步骤。培养后，加入3 ml完全培养基的每个烧瓶中所述CAM实验组中，并返回到培养箱O / N（37ᵒC，5％的CO 2）。根据第2.2节的描述过程中的代谢实验组。处理CAM实验根据第4节的说明进行设置。 </li></ol></li></ol> 2.生物传感器芯片制备及细胞种植<ol><li>在无菌环境中，仔细清洗6的生物传感器芯片用400μlPBS中。消毒芯片用400μl的70％乙醇20分钟。用400微升PBS洗三次。 <ol><li>将里面的无菌组织文化性的芯片Ë菜肴以防止污染。将三个芯片在标有“控制”一盘菜，三个在标有“目标的景点一盘。 </li></ol></li><li>标签2 50ml试管为“控制”和“目标的景点。用PBS洗涤细胞，并添加0.25％胰蛋白酶/ EDTA的适当体积。等到单层分离并从每个烧瓶中收集细胞。存款解决方案到相应的管。 <ol><li>计数细胞并调节音量，以使最终浓度为6.0×10 5细胞/ ml。离心机和重悬细胞，如果初始浓度太低。 </li></ol></li><li>加入250微升的细胞溶液以每生物传感器芯片中的相应组（总共每片1.25×10 5个细胞）的。在一个时间执行此任务一组，以减少混淆的芯片的危险。放置装有芯片湿度腔室内部以防止蒸发无菌菜肴，并放置湿度茶MBER在孵化器O / N（37ᵒC，5％的CO 2）。 </li><li>第二天早上（第1天），小心地从每个芯片吸出培养基，并用250微升生长培养基补充有0.2％胎牛血清（FBS）和1×青霉素和链霉素（P / S）取代。孵育4小时（37ᵒC，5％的CO 2）。 </li></ol> 3.代谢测量系统准备和方案准备<ol><li>在孵化，准备必要的药品解决方案，并为即将到来的检测代谢测量系统。 注：新陈代谢测量系统共包含六个模块。每个模块可容纳每个实验一筹。 <ol><li>从各“控制”和“目标的兴趣&#39;基团以接收车辆的测定法的持续时间中的一个芯片;：细分测定成以下的实验组从每组两个芯片来接收药物治疗所界定的时间周期的一个中SSAY，随后车辆在清除期。 </li><li>用于药物致敏研究，修改长度，定时和浓度的药物治疗，以适合所考虑的特定的药物。 注：对于顺铂，被凭经验用A549细胞中确定的下列步骤，并且可以根据细胞系而改变。完成所有阶段以4微升/分钟默认流量。 6小时生长培养基+ 0.2％FBS和1×P / S的 6μM顺铂媒体24小时+ 0.2％FBS 1倍P / S 生长培养基+ 0.2％FBS和1×P / S中的24小时 18小时生长培养基+ 0.2％FBS和1×P / S的 4小时0.1％的Triton-X的 注：从这些步骤的偏差，需要的计算来确定的基础上，期望的流速和试验持续时间中，药物溶液的所需体积。下面的章节是基于以上所述的步骤。 </li></ol></li><li>标签和填补14 50ml试管用50毫升生长培养基+ 0.2％FBS的ð1倍P / S。放置6在一个代谢测量系统管架，在另一个6，并在第三个试管架还包含管与顺铂的其余两个。密封第一两个机架具有空气可渗透膜以防止蒸发。 </li><li>准备6μM顺铂培养基+ 0.2％FBS与1倍P / S的解决方案（适当的量）。免除顺铂方案所需的量分成四个标有50ml试管。 <ol><li>将管在第三代谢测定管机架的其余四个槽。确保将管放置在对应于所需biomodules适当的槽。封用的空气可渗透膜的管中。 </li></ol></li><li>准备和标记6 50ml试管并用各20ml的培养基中0.1％的Triton-X溶液填充它们。所述的Triton-X溶液将溶解任何残留的细胞，并提供一个“零”值的每个芯片。将管在第四代谢测量单元管架和密封机智h的空气permable膜，以防止过度蒸发。 </li><li>加载芯片到biomodules并开始实验。 </li></ol> 4. CAM实验药物治疗和注射注：该步骤和程序，准备鸡胚的CAM实验在别处描述19 <ol><li>开始处理的CAM实验 48小时转染后（第2天）。子分割“控制”和“目标利益团体分成两个瓶中的车辆，以及两个顺铂。吸从烧瓶的培养基，并补充有是3毫升的新鲜培养基，或6μM顺铂。孵育6小时（37ᵒC，5％的CO 2）。 </li><li>吸出培养基，然后洗净，trypsinize细胞;收集的胰蛋白酶的细胞中溶液分为四个适当标记的15ml试管。 <ol><li>离心机和洗涤细胞三次，用PBS。重新悬浮在5ml PBS中的的细胞沉淀ð计数细胞。 </li><li>调节音量，以使最终的细胞浓度为1.0×10 6细胞/ ml。转移的细胞溶液倒入标5毫升管注射，并置于冰上。倒置试管之前轻轻注射，以确保细胞是充分混合。 </li></ol></li><li>设置共24鸡胚（9日龄）注射（每组6只）一边。执行使用连接到一个柔性管连接到1ml注射器的玻璃针注射。执行注射者必须失明的治疗组。 <ol><li>使用立体显微镜，注入1.0×10 5个细胞中的100微升的体积为所述CAM的静脉循环。使用缓慢，脉动技术，细胞的最大音量注入。轻轻地轻拍注射部位用软擦拭，以阻止血液的流动和吸收任何泄漏。标签每个胚胎适当的容器在每次注射。 </li></ol></li><li>确认存在和DISTRI通过使用荧光显微镜可视化它们下列荧光细胞在CAM的脉管bution注射。请注意其中的肿瘤细胞数量出现低或分布不佳任何胚胎。 </li><li>将鸡胚胎在湿度室中，并将它们存储在培养箱（37℃，60％湿度）。等待7-9天历数转移灶之前。 </li></ol> 5，转移灶枚举<ol><li>在7-9天的潜伏期，监控胚胎并丢弃任何死的。保证室内湿度在孵化期间保持湿润，在任何时候。 </li><li>用荧光立体显微镜在20X放大倍率，扫描胚胎的CAM的整个上表面以规则的，格子状图案。 </li><li>算转移灶的数目在每个视场，则相符的数量为每胚胎的最终总。这样做对各组中的6胚胎将提供一个平均数ö每处理F转移灶。 </li></ol>

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作者什么都没有透露。

由于与临床试验相关的固有的成本和风险，有必要开发更好的和更严格的临床前测试方法，以充分评估新的抗癌治疗方案。当前常用的技术都表现出弱点，这可能限制它们的潜在有希望的治疗靶标/剂之间进行区分的能力，以及那些可能不太有效。我们设计了一个协议，以评估新的抗癌方法，解决了许多传统方法的不足之处。 所描述的协议的一个特别重要的特征是，以实时测?...

获得抗性靶向和/或细胞毒性癌症治疗的一个重要的临床问题，可导致治疗失败，复发，并增加患者的死亡率1。给定异质性的高水平，在大多数肿瘤中，它是一个具有足够高的细胞数的肿瘤将包含细胞抗单一或组合疗法靶向在其上的那些细胞赖以生存2,3-分子途径的子集的数学确定性。这样的肿瘤细胞可以在治疗过程中可靠地选择，从而导致疾病复发。新疗法，能同时针对不同的癌细胞存活机制之前或治疗介导的选择之后发展，因此是临床上重要的。 肿瘤基因组高水平的基因组不稳定和突变是区分癌细胞非肿瘤宿主细胞4根本特征。因此，一个usefu升的策略来增加DNA损伤化疗的效果，并防止耐药性的发展是积极抑制DNA修复中的肿瘤细胞5。这是调查和各种新的DNA修复的目标的一个活跃的领域正在探索在临床前设置。一些小分子或它们的靶的反义基抑制剂已经被开发并正在接受测试6-8。该目标是确定最有希望的临床前候选并评估其安全性和有效性的临床试验。 临床试验的成本高，失败的风险（为各种原因，包括亚最佳的临床前评估）是难以克服的障碍中的新疗法9发展进步。使用适当的和严格的临床前模型，以充分评估新的治疗靶点和候选药物可能会降低的临床试验10高故障率</suP&gt;。 一些常用的临床前的方法来评估的新的抗癌疗法的功效是：a）测量能力，减少肿瘤细胞增殖在体外 （细胞增殖测定）中的肿瘤细胞的能力，二）治疗诱导减少的形式的组织培养的菌落（菌落形成测定法）中的肿瘤细胞的代谢活性，三）治疗诱导的减少在体外 （氧化还原染料的转换）中，d）治疗诱导的诱导体外肿瘤细胞死亡（凋亡，坏死，自噬相关联在体内有丝分裂及其他）11，和e）治疗诱导的减少生长人和小鼠异种移植物的12-14或消融。 列出的体外方法的一个主要弱点是，他们没有提供候选疗法的效果持续的实时评估。相反，他们只在选定的，广泛分隔时间点提供信息在治疗的过程中。这样的测量已经减少容量以准确地反映了肿瘤细胞应答的大小和定时。 体内小鼠异种移植模型也受限于成本高，时间来完成的长度，和亚最佳剂量和治疗时间（调度）的风险。此外，有证据表明，啮齿动物异种移植物模型是临床疗效在人类有限的预测相比， 在体外评估的原代人肿瘤细胞的应答，并建立了人肿瘤细胞系对候选治疗性干预15,16。 我们设计了一种新的组合协议来评估新的药物组合临床前，在解决了上面列出的更多的一般的步骤的弱点的方式。代替扩散，集落形成，或氧化还原染料转化测定中，我们利用了代谢测定单元来分析细胞粘附，呼吸，并实时酸化在整个治疗期间17。同时，我们通过使用鸡胚绒毛尿囊膜（CAM）模型的侵袭和转移18,19的研究体内治疗相结合的效果。我们使用这些方法来评价反义寡核苷酸（ASO）靶向BRCA2能增强常用化疗药物顺铂的有效性的能力。

<table border="0" cellpadding="0" cellspacing="0" >
	<tbody>
		<tr >
			<td >Name of Material/ Equipment</td>
			<td >Company</td>
			<td >Catalog Number</td>
			<td >Comments/Description</td>
		</tr>
		<tr >
			<td >A549 cells</td>
			<td >ATCC</td>
			<td >CCL-185</td>
			<td>http://www.atcc.org/products/all/CCL-185.aspx</td>
		</tr>
		<tr >
			<td >AeraSeal film&#160;</td>
			<td >Carl Roth GmbH</td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >AMEM</td>
			<td >Wisent Bioproducts</td>
			<td >210-011-QK</td>
			<td></td>
		</tr>
		<tr >
			<td >Antisense oligodeoxynucleotides</td>
			<td >Avecia</td>
			<td ></td>
			<td>BRCA2 target sequence: 5&#39; - UAAGGAACGUCAAGAGAUAC - 3&#39; (bases 7241-7259 )</td>
		</tr>
		<tr >
			<td >Axio Zoom V16 Microscope</td>
			<td >Carl Zeiss</td>
			<td ></td>
			<td>http://www.zeiss.ca/microscopy/en_ca/products/stereo-zoom-microscopes/axio-zoom-v16.html</td>
		</tr>
		<tr >
			<td >Bionas Biosensor Chips</td>
			<td >Bionas GmbH and Micronas GmbH</td>
			<td >BIS8001D</td>
			<td></td>
		</tr>
		<tr >
			<td >Bionas Discovery 2500 System&#160;</td>
			<td >Bionas GmbH</td>
			<td ></td>
			<td>http://www.bionas-discovery.com/prodservices/instruments/system2500/</td>
		</tr>
		<tr >
			<td >Cisplatin</td>
			<td >Sigma Aldrich</td>
			<td >479306</td>
			<td></td>
		</tr>
		<tr >
			<td >Fertilized chicken eggs</td>
			<td >Sourced locally</td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >Fetal bovine serum&#160;</td>
			<td >Gibco - Life Technologies</td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >Lipofectamine 2000&#160;</td>
			<td >Invitrogen - Life Technologies&#160;</td>
			<td >12566014</td>
			<td>http://www.lifetechnologies.com/ca/en/home/life-science/protein-expression-and-analysis/transfection-selection/lipofectamine-2000.html</td>
		</tr>
		<tr >
			<td >PBS</td>
			<td >Wisent Bioproducts</td>
			<td >311-010-CL</td>
			<td></td>
		</tr>
		<tr >
			<td >Trypsin (0.25%)/EDTA</td>
			<td >Wisent Bioproducts</td>
			<td >325-043-CL</td>
			<td></td>
		</tr>
	</tbody>
</table>

evaluating the effectiveness of cancer drug sensitization in vitro and in vivo

由于异质性和固有的人类癌症，单一药剂的治疗或组合治疗方案靶向相同途径突变的水平高，很可能会失败。必须强调在通路是负责对疗法的固有和/或适应性阻力的抑制。调查的一个活跃的领域是开发和DNA修复抑制剂促进的作用，和防止抗，常用的化疗和放疗的测试。我们使用了一种新的协议，以评估BRCA2抑制的有效性，以敏感的肿瘤细胞对DNA损伤药物顺铂的装置。肿瘤细胞代谢（酸化和呼吸）实时一段72小时的划定上的分一秒基础治疗效果进行监测。在组合中，我们使用的是鸡胚胎绒毛尿囊膜（CAM）外渗及浸润模型进行转移频率的评估。此协议解决了一些常用的体外和体内方法来评价新的癌症治疗方案的弱点。它可以在除了常见的方法，例如细胞增殖试验，细胞死亡测定法中使用，并且在体内小鼠异种移植物的研究，以更紧密地辨别之间候选目标和剂，并仅选择最有前途的候选者的进一步发展。

结果与数字改编许可，从发表的作品22。 BRCA2诱导抑制呼吸的下降顺铂治疗的肿瘤细胞 与BRCA2靶向ASO和顺铂表现在呼吸早期的和不可逆转的下降，相比于它接收到的控制ASO与顺铂单独处理细胞A549人类肺癌细胞; 24小时顺铂治疗的BRCA2 ASO和控制ASO呼吸作用的差值处理的细胞后为39％（图1A）。重要的是，这减少呼吸开始在细胞粘附?...

Watch this Scientific Journal Video about Evaluating the Effectiveness of Cancer Drug Sensitization In Vitro and In Vivo at JoVE.com

Evaluating the Effectiveness of Cancer Drug Sensitization In Vitro and In Vivo

Here, real-time monitoring of tumor cell metabolism, combined with an in vivo chicken embryo chorio-allantoic membrane (CAM) model of metastasis, are used to evaluate novel anti-cancer targets/agents for their ability to sensitize tumor cells to DNA damaging chemotherapeutics.

评估癌症药物过敏的有效性<em&gt;在体外</em&gt;和<em&gt;在体内</em

Due to the high level of heterogeneity and mutations inherent in human cancers, single agent therapies, or combination regimens which target the same ...

evaluating-effectiveness-cancer-drug-sensitization-vitro

Research

JoVE Journal

Medicine

Evaluating the Effectiveness of Cancer Drug Sensitization In Vitro and In Vivo

8.2K 次观看.  Western University. Here, real-time monitoring of tumor cell metabolism, combined with an in vivo chicken embryo chorio-allantoic membrane (CAM) model of metastasis, are used to evaluate novel anti-cancer targets/agents for their ability to sensitize tumor cells to DNA damaging chemotherapeutics. 

视频: Evaluating the Effectiveness of Cancer Drug Sensitization In Vitro and In Vivo

An In Vitro System to Study Tumor Dormancy and the Switch to Metastatic Growth

Recurrence of breast cancer often follows a long latent period in which there are no signs of cancer, and metastases may not become clinically apparent until many years after removal of the primary tumor and adjuvant therapy. A likely explanation of this phenomenon is that tumor cells have seeded metastatic sites, are resistant to conventional therapies, and remain dormant for long periods of time 1-4.
The existence of dormant cancer cells at secondary sites has been described previously as quiescent solitary cells that neither proliferate nor undergo apoptosis 5-7. Moreover, these solitary cells has been shown to disseminate from the primary tumor at an early stage of disease progression 8-10 and reside growth-arrested in the patients' bone marrow, blood and lymph nodes 1,4,11. Therefore, understanding mechanisms that regulate dormancy or the switch to a proliferative state is critical for discovering novel targets and interventions to prevent disease recurrence. However, unraveling the mechanisms regulating the switch from tumor dormancy to metastatic growth has been hampered by the lack of available model systems.
in vivo and ex vivo model systems to study metastatic progression of tumor cells have been described previously 1,12-14. However these model systems have not provided in real time and in a high throughput manner mechanistic insights into what triggers the emergence of solitary dormant tumor cells to proliferate as metastatic disease. We have recently developed a 3D in vitro system to model the in vivo growth characteristics of cells that exhibit either dormant (D2.OR, MCF7, K7M2-AS.46) or proliferative (D2A1, MDA-MB-231, K7M2) metastatic behavior in vivo . We demonstrated that tumor cells that exhibit dormancy in vivo at a metastatic site remain quiescent when cultured in a 3-dimension (3D) basement membrane extract (BME), whereas cells highly metastatic in vivo readily proliferate in 3D culture after variable, but relatively short periods of quiescence. Importantly by utilizing the 3D in vitro model system we demonstrated for the first time that the ECM composition plays an important role in regulating whether dormant tumor cells will switch to a proliferative state and have confirmed this in in vivo studies15-17. Hence, the model system described in this report provides an in vitro method to model tumor dormancy and study the transition to proliferative growth induced by the microenvironment.

An In Vitro System to Study Tumor Dormancy and the Switch to Metastatic Growth

A modified 3-D in vitro system is presented in which growth characteristics of several tumor cell lines in reconstituted basement membrane correlate with the dormant or proliferative behavior of the tumor cells at a metastatic secondary site in vivo.

一个<em在体外系统来研究肿瘤休眠和转移性增长

Recurrence of breast cancer often follows a long latent period in which there are no signs of cancer, and metastases may not become clinically apparent ...

科研

医学

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

The tumor microenvironment plays a pivotal role in tumor initiation, progression, metastasis, and the response to anti-cancer therapies. Three-dimensional co-culture systems are frequently used to explicate tumor-stroma interactions, including their role in drug responses. However, many of the interactions that occur in vivo in the intact microenvironment cannot be completely replicated in these in vitro settings. Thus, direct visualization of these processes in real-time has become an important tool in understanding tumor responses to therapies and identifying the interactions between cancer cells and the stroma that can influence these responses. Here we provide a method for using spinning disk confocal microscopy of live, anesthetized mice to directly observe drug distribution, cancer cell responses and changes in tumor-stroma interactions following administration of systemic therapy in breast cancer models. We describe procedures for labeling different tumor components, treatment of animals for observing therapeutic responses, and the surgical procedure for exposing tumor tissues for imaging up to 40 hours. The results obtained from this protocol are time-lapse movies, in which such processes as drug infiltration, cancer cell death and stromal cell migration can be evaluated using image analysis software.

We describe a method for imaging response to anti-cancer treatment in vivo and at single cell resolution.

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 ...

Slow-release Drug Delivery through Elvax 40W to the Rat Retina: Implications for the Treatment of Chronic Conditions

Diseases of the retina are difficult to treat as the retina lies deep within the eye. Invasive methods of drug delivery are often needed to treat these diseases. Chronic retinal diseases such as retinal oedema or neovascularization usually require multiple intraocular injections to effectively treat the condition. However, the risks associated with these injections increase with repeated delivery of the drug. Therefore, alternative delivery methods need to be established in order to minimize the risks of reinjection. Several other investigations have developed methods to deliver drugs over extended time, through materials capable of releasing chemicals slowly into the eye. In this investigation, we outline the use of Elvax 40W, a copolymer resin, to act as a vehicle for drug delivery to the adult rat retina. The resin is made and loaded with the drug. The drug-resin complex is then implanted into the vitreous cavity, where it will slowly release the drug over time. This method was tested using 2-amino-4-phosphonobutyrate (APB), a glutamate analogue that blocks the light response of the retina. It was demonstrated that the APB was slowly released from the resin, and was able to block the retinal response by 7 days after implantation. This indicates that slow-release drug delivery using this copolymer resin is effective for treating the retina, and could be used therapeutically with further testing.

This paper details how Elvax 40W can be used as a slow-release method for drug delivery to the adult rat retina. The protocol for preparing, loading, and delivering the drug-resin complex to the eye is described.

通过40W的Elvax缓释给药的大鼠视网膜：对慢性疾病的治疗

Diseases of the retina are difficult to treat as the retina lies deep within the eye. Invasive methods of drug delivery are often needed to treat these ...

Carotid Artery Infusions for Pharmacokinetic and Pharmacodynamic Analysis of Taxanes in Mice

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study model. As the use of mouse models are often a vital step in preclinical drug discovery and drug development1-8, it is necessary to design a system to introduce drugs into mice in a uniform, reproducible manner. Ideally, the system should permit the collection of blood samples at regular intervals over a set time course. The ability to measure drug concentrations by mass-spectrometry, has allowed investigators to follow the changes in plasma drug levels over time in individual mice1, 9, 10. In this study, paclitaxel was introduced into transgenic mice as a continuous arterial infusion over three hours, while blood samples were simultaneously taken by retro-orbital bleeds at set time points. Carotid artery infusions are a potential alternative to jugular vein infusions, when factors such as mammary tumors or other obstructions make jugular infusions impractical. Using this technique, paclitaxel concentrations in plasma and tissue achieved similar levels as compared to jugular infusion. In this tutorial, we will demonstrate how to successfully catheterize the carotid artery by preparing an optimized catheter for the individual mouse model, then show how to insert and secure the catheter into the mouse carotid artery, thread the end of the catheter out through the back of the mouse&#8217;s neck, and hook the mouse to a pump to deliver a controlled rate of drug influx. Multiple low volume retro-orbital bleeds allow for analysis of plasma drug concentrations over time.

This method was developed with the goal of delivering a steady drug solution via the carotid artery, to assess the pharmacokinetics of novel drugs in mouse models.

颈动脉输液的紫杉烷类化合物在小鼠体内的药代动力学和药效学分析

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study ...

In Vivo and Ex Vivo Approaches to Study Ovarian Cancer Metastatic Colonization of Milky Spot Structures in Peritoneal Adipose

High-grade serous ovarian cancer (HGSC), the cause of widespread peritoneal metastases, continues to have an extremely poor prognosis; fewer than 30% of women are alive 5 years after diagnosis. The omentum is a preferred site of HGSC metastasis formation. Despite the clinical importance of this microenvironment, the contribution of omental adipose tissue to ovarian cancer progression remains understudied. Omental adipose is unusual in that it contains structures known as milky spots, which are comprised of B, T, and NK cells, macrophages, and progenitor cells surrounding dense nests of vasculature. Milky spots play a key role in the physiologic functions of the omentum, which are required for peritoneal homeostasis. We have shown that milky spots also promote ovarian cancer metastatic colonization of peritoneal adipose, a key step in the development of peritoneal metastases. Here we describe the approaches we developed to evaluate and quantify milky spots in peritoneal adipose and study their functional contribution to ovarian cancer cell metastatic colonization of omental tissues both in vivo and ex vivo. These approaches are generalizable to additional mouse models and cell lines, thus enabling the study of ovarian cancer metastasis formation from initial localization of cells to milky spot structures to the development of widespread peritoneal metastases.

In Vivo and Ex Vivo Approaches to Study Ovarian Cancer Metastatic Colonization of Milky Spot Structures in Peritoneal Adipose

We outline a protocol that implements both in vivo and ex vivo approaches to study ovarian cancer colonization of peritoneal adipose tissues, particularly the omentum. Furthermore, we present a protocol to quantitate and analyze immune cell-structures in the omentum known as milky spots, which promote metastases of peritoneal adipose.

在体内和体外途径研究银河系结构现货卵巢癌转移定植腹膜脂肪

High-grade serous ovarian cancer (HGSC), the cause of widespread peritoneal metastases, continues to have an extremely poor prognosis; fewer than 30% of ...

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.

研究胰腺癌干细胞特性开发新的治疗策略

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor ...

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 ...

Forskolin-induced Swelling in Intestinal Organoids: An In Vitro Assay for Assessing Drug Response in Cystic Fibrosis Patients

Recently-developed cystic fibrosis transmembrane conductance regulator (CFTR)-modulating drugs correct surface expression and/or function of the mutant CFTR channel in subjects with cystic fibrosis (CF). Identification of subjects that may benefit from these drugs is challenging because of the extensive heterogeneity of CFTR mutations, as well as other unknown factors that contribute to individual drug efficacy. Here, we describe a simple and relatively rapid assay for measuring individual CFTR function and response to CFTR modulators in vitro. Three dimensional (3D) epithelial organoids are grown from rectal biopsies in standard organoid medium. Once established, the organoids can be bio-banked for future analysis. For the assay, 30-80 organoids are seeded in 96-well plates in basement membrane matrix and are then exposed to drugs. One day later, the organoids are stained with calcein green, and forskolin-induced swelling is monitored by confocal live cell microscopy at 37 &#176;C. Forskolin-induced swelling is fully CFTR-dependent and is sufficiently sensitive and precise to allow for discrimination between the drug responses of individuals with different and even identical CFTR mutations. In vitro swell responses correlate with the clinical response to therapy. This assay provides a cost-effective approach for the identification of drug-responsive individuals, independent of their CFTR mutations. It may also be instrumental in the development of future CFTR modulators.

Forskolin-induced Swelling in Intestinal Organoids: An In Vitro Assay for Assessing Drug Response in Cystic Fibrosis Patients

This protocol describes an assay for measuring CFTR function and CFTR modulator responses in cultured tissue from subjects with cystic fibrosis (CF). Biopsy-derived intestinal organoids swell in a cAMP-driven fashion, a response that is defective (or strongly reduced) in CF organoids and can be restored by exposure to CFTR modulators.

毛喉素诱导在肠组织体溶胀：一个<em&gt;体外</em&gt;检测方法的评估囊性纤维化患者对药物的反应

Recently-developed cystic fibrosis transmembrane conductance regulator (CFTR)-modulating drugs correct surface expression and/or function of the mutant ...

Optimized Analysis of In Vivo and In Vitro Hepatic Steatosis

Establishing a system of procedures to qualitatively and quantitatively characterize in vivo and in vitro hepatic steatosis is important for metabolic study in the liver. Here, numerous assays are described to comprehensively measure the phenotype and parameters of hepatic steatosis in mouse and hepatocyte models. 
Combining the physiological, histological, and biochemical methods, this system can be used to assess the progress of hepatic steatosis. In vivo, the measurements of body weight and nuclear magnetic resonance (NMR) provide a general understanding of mice in a non-invasive manner. Hematoxylin and Eosin (H&amp;E) and Oil Red O staining determine the histological morphology and lipid deposition of liver tissue under nutrient overload conditions, such as high-fat diet feeding. Next, the total lipid contents are isolated by chloroform/methanol extraction, which are followed by a biochemical analysis for triglyceride and cholesterol. Moreover, mouse primary hepatocytes are treated with high glucose plus insulin to stimulate lipid accumulation, an efficient in vitro model to mimic diet-induced hyperglycemia and hyperinsulinemia in vivo. Then, the lipid deposition is measured by Oil Red O staining and chloroform/methanol extraction. Oil Red O staining determines intuitive hepatic steatotic phenotypes, while the lipid extraction analysis determines the parameters that can be analyzed statistically. The present protocols are of interest to scientists in the fields of fatty liver diseases, insulin resistance, and type 2 diabetes.

Optimized Analysis of In Vivo and In Vitro Hepatic Steatosis

Here, optimized methods to generate in vivo and in vitro models of hepatic steatosis and to analyze the steatotic phenotypes and related physiological parameters are described.

优化分析<em&gt;在体内</em&gt;和<em&gt;体外</em&gt;脂肪肝

Establishing a system of procedures to qualitatively and quantitatively characterize in vivo and in vitro hepatic steatosis is important for metabolic ...

Preparation of Herbal Medicine: Er-Xian Decoction and Er-Xian-containing Serum for In Vivo and In Vitro Experiments

Traditional herbal medicine, an alternative medicine in the clinical setting, has received increased attention in recent years. Before delivery to the body, an additional extraction procedure is commonly required to release the active constituents from raw herbs. Water decoction is a classical extraction procedure that is still broadly used in the clinical settings. Here, we propose a detailed protocol for er-xian decoction (EXD) in order to apply herbal decoctions to experimental studies. The calculation of an animal-appropriate dose is described, as well as the four main steps of EXD: soaking, water decoction, filtration, and concentration. In addition, serum-containing EXD is introduced to rats as a means of in vitro validation. Here, rats were orally administered EXD for three days. Blood samples were then collected, inactivated, centrifuged, and filtered. The serum, diluted with the culture medium, can be utilized to treat cells or tissues in vitro. For example, EXD was applied to both in vivo and in vitro studies and demonstrated that EXD enhances osteogenesis. This protocol can be used as a reference for the preparation and application of herbal medicines.

Preparation of Herbal Medicine: Er-Xian Decoction and Er-Xian-containing Serum for In Vivo and In Vitro Experiments

Here, we present the preparation of an er-xian decoction (EXD) in four steps&#8212;soaking, decoction, filtration, and concentration&#8212;and demonstrate the administration of a prepared EXD-containing serum to rats. These methods are applicable to the in vivo and in vitro study of herbal decoctions such as traditional Chinese medicines.

草药的制备：洱西汤和含茜素的血清<em&gt;体内</em&gt;和<em&gt;体外</em&gt;实验

Traditional herbal medicine, an alternative medicine in the clinical setting, has received increased attention in recent years. Before delivery to the ...