这项工作得到了 NIH R01CA187780 的部分支持。

注: 形和神经元慢报告系统被购买作为预先包装, 集中慢准备。具备流式细胞术的基本知识。此外, 为充分利用该协议, 用户将需要访问的流量式细胞仪与高通量的能力 (接受96井板作为样本来源)。
1. 慢转录报告系统
注: 在所有流细胞分析中, 使用父母、non-transduced、细胞或向量转 (荧光) 细胞来建立基线荧光。另外, 请注意, 在所有需要机械研磨的步骤中, 要温柔。苛刻的研磨可以杀死大量的脆弱的 GSCs 和影响流式细胞仪的结果。
<ol><li>板 1x106细胞在2毫升的完整神经干细胞生长培养基中的 6-井板。</li>
	<li>传感器细胞通过添加慢记者在多重感染 (莫伊) 等于5。</li>
	<li>添加2µL 的凝聚, 最终浓度为8µg/毫升。</li>
	<li>孵育细胞在37° c 和 5% CO2过夜。</li>
	<li>第二天, 替换生长培养基以去除未绑定病毒。将板放回孵化器中37° c 和 5% CO2 , 并孵育24小时。</li>
	<li>收获0.5 毫升的总体积;在15毫升锥形管中, 在室温下旋转 360 x g 的5分钟的细胞, 并通过吸入去除上清液。在剩余的细胞中加入0.5 毫升的新鲜神经干细胞生长培养基, 并将板块返回到孵化器继续扩张。</li>
	<li>加入200µL 离解试剂, 孵育5分钟, 在37° c。</li>
	<li>通过轻轻移来分离细胞。 注意: 苛刻的研磨可能导致严重的 GSCs, 完全离解通常达到 20-30 倍。</li>
	<li>为了最大限度地减少细胞的附着或聚集, 增加800µL 的平衡盐溶液 (HBSS) 的最终体积为1毫升。</li>
	<li>将每个细胞悬浮液的200µL 转移到一个96井板的井中。</li>
	<li>在这个过程中, 使用台式流式细胞仪与96井板能力配备了蓝色激光励磁和能力检测绿色荧光蛋白。对每次获得的至少1万活细胞进行流细胞分析。</li>
	<li>用流式细胞仪测定 GFP 阳性细胞的百分率。</li>
</ol>2. 克隆的选择、扩展和验证
<ol><li>平板细胞在100µL 的神经干细胞培养基的密度0.7 细胞每井 96-井板。</li>
	<li>培养克隆在11天在37° c 和 5% CO2。此步骤高度依赖于单元格类型, 并且可能需要根据所使用的单元格行进行调整。作为一般规则, 球直径≥100µm 是一个很好的迹象表明存在的克隆 GSCs 在球。</li>
	<li>使用装有 FITC 过滤器的荧光显微镜, 将含有单干细胞的水井标记为有1-5% 的细胞呈 GFP 阳性。 注: 在这一点上, 确定 GFP 阳性细胞的确切百分比并不太重要。每一个 subclones 将稍后通过流式细胞仪进行评估。这一步是为了减少要分析的克隆的总数量, 重点是由未分化的, 胶质-阴性, GSC 的球。</li>
	<li>展开选定的记者克隆, 直到有足够数量的细胞进行流式细胞仪分析。一个 sub-confluent 的 6-井板含有≤ 1.5x105细胞/毫升应提供足够数量的细胞。 注意: GSCs 的隔离和扩展的详细过程可用17。</li>
</ol>3. 胶质蛋白的测定: 流式细胞仪的 GFP 表达
<ol><li>从每个记者克隆和 non-transduced 控制中收获一个分细胞 (0.5 毫升), 以确定 GFP 阳性细胞的确切百分比。考虑到含有〜 1-5% GFP 阳性细胞的记者克隆。</li>
	<li>在室温下旋转 360 x g 细胞5分钟, 用吸入去除上清。</li>
	<li>对每个颗粒, 加入200µL 细胞离解试剂和孵化管在一个水浴设置为37° c。</li>
	<li>研制细胞轻轻地达到一个单一的细胞悬浮 (通常在20到30倍)。</li>
	<li>为了最小化细胞的连接或聚集, 添加800µL HBSS 到最后的体积1毫升。</li>
	<li>每一个 96-井板从每个细胞悬浮转移200µL。</li>
	<li>对于这个过程, 使用台式流式细胞仪与 96-井板能力。对每次获得的至少1万活细胞进行流细胞分析。</li>
</ol>4. 埃尔达自我更新法评估克隆能力
注意: 对于对照, 父母、non-transduced 以及 GFP 表达的慢-转细胞, 都应用于确定 GSC 分化报告者的相对克隆潜能, 并将其与原来的 GSC 文化派生.
<ol><li>如上文所述, 游离分化--记者 subclones 成单细胞悬浮液。</li>
	<li>96-井板细胞在100µL 完全神经干细胞生长培养基的细胞密度范围内, 每井5至500细胞。</li>
	<li>孵育细胞9至11天在37° c 和 5% CO2。</li>
	<li>在光显微镜下直接可视化球, 对正井进行评分。一个井应该被认为是 "积极的", 如果它包含至少一个单一的大干细胞。</li>
	<li>插入数据: 总井分析和正面井的数量使用埃尔达在线接口可利用在 http://bioinf.wehi.edu.au/software/elda/index.html。</li>
</ol>5. 药物库稀释制剂
<ol><li>从-80 ° c 的存储器中取出库板, 用铝箔盖 (保护光敏化合物)。室温解冻约30分钟至一小时。</li>
	<li>使用12声道多通道 pipettor 将库化合物稀释到完全神经干细胞介质中的0.2 毫米。在生长培养基中稀释亚甲基亚砜至10% 注: 当添加到细胞时, 亚砜的最终浓度应为0.1%。在培养中较高浓度的亚甲基亚砜可能会产生毒性。因此, 在治疗前, 建议在使用的细胞系中测试对亚砜的敏感性。</li>
	<li>使用亚甲基亚砜作为车辆控制处理在每个板块的左、右栏中发现的细胞 (1 和12栏;井 A 到 H)。</li>
	<li>用铝箔将稀释后的库板盖住, 并将原有的库板归还给-80 ° c 的冰箱进行长期贮存。</li>
</ol>6. 药物筛
注意: 应使用亚砜处理的细胞来设置基线荧光和调整浇口。
<ol><li>板 5x103细胞在一个 96-井板99µL 完全神经干细胞生长培养基。</li>
	<li>使用12声道多通道 pipettor, 在最终浓度为2µM (1 µL 0.2 毫米药物到99µL 的细胞悬浮) 或与亚砜 (控制) 的稀释库化合物处理细胞。在37° c 和 5% CO2中孵育72小时的板。</li>
	<li>使用12声道多通道 pipettor 增加150µL 细胞离解试剂到每个井和孵化20分钟, 在37° c。</li>
	<li>通过轻轻研制与12声道多通道 pipettor 分离细胞, 直到单细胞悬浮达到 (通常在20到30倍)。完全取消球所需的时间量将因 GSC 线而异。虽然推荐的细胞离解试剂 (见材料) 是安全和孵化45分钟没有影响的可行性在多个 GSC 线测试, 验证每 GSC 线将用于筛选。 注: 每井的最终体积应约250µL (100 µL 细胞悬浮液 + 150 µL 细胞离解试剂)。</li>
	<li>用流式细胞仪测定表达胶质蛋白的细胞百分率: GFP 报告者。使用标准的浇口策略。首先, 剧情向前和侧面驱散得到细胞大小和生存能力的一般感觉。然后在可行的单细胞种群上放置一扇门。这是绿色荧光 (eGFP) 将被确定的人口。任何化合物, 导致在超过三标准偏差超过控制 (亚砜处理) GFP 阳性细胞的百分比增加被认为是一个积极的 "命中"。这个门槛应根据具体的应用和预期的严格度来调整。 注: 对于高吞吐量屏幕建议使用单个 GSC 记者克隆。在命中识别之后, 每个命中应该被验证反对另外的报告人 subclones 从同一 GSC 线。为了增加对真实命中的信心也建议测试化合物对记者 subclones 隔离从不同的 GSC 球线。</li>
</ol>

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作者没有什么可透露的。

虽然以前的大多数 GSCs 研究主要集中在定义它们的标记上, 但在这项研究中, 我们决定采取逆向方法。我们主要关注 GSCs 产生的分化后代 (例如, 表达形和神经元标记的细胞)。在这里, 我们展示了一个基于细胞的高通量药物筛选系统的利用, 这是建立在人胶质蛋白的启动依赖的 GFP 表达。所有的实验都是利用患者衍生的干细胞胶质瘤细胞系进行的。加利et al17中描述了描述这些...

最近的研究表明, 肿瘤中含有少量的细胞, 称为癌症干细胞 (协会) 或肿瘤启动细胞, 它们负责肿瘤的进展、转移以及对化疗和无线电治疗的抵抗力1,2. 肿瘤干细胞及其分化后代的存在被认为是促进瘤内异质性的重要因素, 因此是治疗癌症的主要障碍3。肿瘤细胞的层次, 由癌症干细胞理论提供, 启发了新的治疗癌症的策略的发展4。一种靶向肿瘤干细胞的方法是识别和抑制在胚胎发育过程中已知需要的信号通路。事实上, 我们和其他人以前发表过多篇论文, 描述了对神经干细胞相关信号通路的持续需求, 在胶质母细胞瘤中的声波刺猬和凹槽5,6,7。这项工作有助于巩固的理由, 几个肾小球临床试验。第二种靶向肿瘤干细胞的方法是促进其分化。这种方法得到了很多的支持, 由于良好的结果, 从前临床研究和治疗急性早幼粒细胞白血病的维甲酸 (ATRA, 维生素 a 模拟)。这里 ATRA 被发现去除成熟块和促进肿瘤细胞分化8。最近, Piccirillo 和同事已经优雅地表明, BMP-4 促进 GSC 分化为星形胶质细胞具有显著的抗大膜效应体外和体内9。
目前研究的基本原理是基于针对 GSCs 的 "逆向工程" 方法。鉴于大紫荆存在巨大的异质性, 且分化程度差是癌症的标志之一, 我们问是否可以促进一种更有利的表型分化成星形胶质细胞样的状态。在这里, 我们没有先验知识的信号通路, 维持 GSCs 在一个给定的肿瘤标本, 而是目标, 以达到预期的表型 (如胶质细胞阳性)。
本报告描述的程序, 建立 GSC 分化记者线从 GSC 丰富的文化转介到 GSC 克隆选择。使用的胶质母细胞瘤干细胞线是建立在实验室的安吉洛教授 Vescovi 从诊断原发性胶质瘤在医院圣拉斐尔-米兰, 意大利。这些线路已在多个出版物中广泛研究6、10、11、12、13、14。强烈建议那些有兴趣在实验室中实施这些技术的个人, 确定记者在他们计划研究的细胞中对癌症干细胞自我更新能力的相关性 (这对任何记者都是如此)。为完成此15、16提供了一个在该字段中接受的体外克隆分析的详细协议。最后, 给出了一个详细的协议, 描述了在流式细胞仪的药物屏幕上区分记者线的使用。值得注意的是, 类似于这里描述的形微分系统, 我们已经成功地建立并验证了 GSC 记者的 MAP2:GFP (神经元分化) 报告者的线。因此, 本文所描述的方法可以用于研究细胞分化为各种细胞谱系。
本报告中的一些数字可以在最近的出版物中找到: "阿曲库磺和其他神经肌肉阻滞剂促进形分化和消耗胶质母细胞瘤干细胞18。

<table>
	<tbody>
		<tr>
			<td>ESGRO Complete Accutase</td>
			<td>EMD Millipore</td>
			<td>SF006</td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethyl Sulfoxide (DMSO)</td>
			<td>Sigma Aldrich</td>
			<td>D2650</td>
			<td></td>
		</tr>
		<tr>
			<td>HBSS (Hank&#39;s Balanced Salt Solution)</td>
			<td>Sigma Aldrich</td>
			<td>H6648</td>
			<td></td>
		</tr>
		<tr>
			<td>Human GFAP Differentiation Reporter (pGreenZeo, Virus)</td>
			<td>SBI (System Biosciences)</td>
			<td>SR10015VA-1</td>
			<td></td>
		</tr>
		<tr>
			<td>50 ml sterile disposable reagent reservoirs</td>
			<td>Corning</td>
			<td>4870</td>
			<td></td>
		</tr>
		<tr>
			<td>6 well plate</td>
			<td>Thermo Fisher Scientific</td>
			<td>130184</td>
			<td></td>
		</tr>
		<tr>
			<td>96 well plate</td>
			<td>Falcon</td>
			<td>353072</td>
			<td></td>
		</tr>
		<tr>
			<td>Biolite T25 cm&#178; Flask Vented</td>
			<td>Thermo Fisher Scientific</td>
			<td>130189</td>
			<td></td>
		</tr>
		<tr>
			<td>Biolite T75 cm&#178; Flask Vented</td>
			<td>Thermo Fisher Scientific</td>
			<td>130190</td>
			<td></td>
		</tr>
		<tr>
			<td>15 ml Centrifuge tubes</td>
			<td>Celltreat</td>
			<td>229411</td>
			<td></td>
		</tr>
		<tr>
			<td>1.5ml Microcentrifuge tubes</td>
			<td>Fisher Scientific</td>
			<td>05-408-129</td>
			<td></td>
		</tr>
		<tr>
			<td>Ovation Multi Channel Pipette, 12 Channel, 0.5 - 20uL</td>
			<td>VistaLab Technologies</td>
			<td>1060-0020</td>
			<td></td>
		</tr>
		<tr>
			<td>Ovation Multi Channel Pipette, 12 Channel, 5-250uL</td>
			<td>VistaLab Technologies</td>
			<td>1060-0250</td>
			<td></td>
		</tr>
		<tr>
			<td>Multi 12-channel pipette tips 25 &#956;l</td>
			<td>VistaLab Technologies</td>
			<td>4060-1002</td>
			<td></td>
		</tr>
		<tr>
			<td>Multi 12-channel pipette tips 250 &#956;l</td>
			<td>VistaLab Technologies</td>
			<td>4060-9025</td>
			<td></td>
		</tr>
		<tr>
			<td>Guava easyCyte 5HT Benchtop Flow Cytometer</td>
			<td>EMD Millipore</td>
			<td>0500-4005</td>
			<td></td>
		</tr>
		<tr>
			<td>NIH Clinical Collections 1 and 2 small molecule libraries</td>
			<td>Evotec</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>For the preparation of neural stem cell media (500 mL)</td>
			<td></td>
			<td></td>
			<td>Final concentration</td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>GoldBio.com</td>
			<td>A-421-250</td>
			<td>0.20%</td>
		</tr>
		<tr>
			<td>DMEM/F12 10X</td>
			<td>Corning</td>
			<td>90-091-PB</td>
			<td>1X</td>
		</tr>
		<tr>
			<td>Heparin sodium salt</td>
			<td>Sigma Aldrich</td>
			<td>H3149</td>
			<td>0.0002%</td>
		</tr>
		<tr>
			<td>HEPES 1M</td>
			<td>Sigma Aldrich</td>
			<td>H4036</td>
			<td>5.4 mM</td>
		</tr>
		<tr>
			<td>Insulin-Transferrin- Selenium (ITS -G) (100X)</td>
			<td>Life Technologies</td>
			<td>41400-045</td>
			<td>1X</td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>Sigma Aldrich</td>
			<td>S-5761</td>
			<td>14.5 mM</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin (10,000 U/mL) 100X</td>
			<td>Gibco</td>
			<td>15140-122</td>
			<td>1X</td>
		</tr>
		<tr>
			<td>Progesterone</td>
			<td>Sigma Aldrich</td>
			<td>P8783</td>
			<td>16 nM</td>
		</tr>
		<tr>
			<td>Putrescine</td>
			<td>Sigma Aldrich</td>
			<td>P5780</td>
			<td>4.8 &#181;M</td>
		</tr>
		<tr>
			<td>Basic FGF (FGF2), Human</td>
			<td>GoldBio</td>
			<td>1140-02-50</td>
			<td>10 ng/ml</td>
		</tr>
		<tr>
			<td>EGF, Human</td>
			<td>GoldBio</td>
			<td>1150-04-100</td>
			<td>20 ng/ml</td>
		</tr>
		<tr>
			<td>Bottle-Top Filter, 150ml, 33mm, 0.22um, Pes, S, Ind</td>
			<td>Corning</td>
			<td>431160</td>
			<td>Use to filter sterlize media</td>
		</tr>
	</tbody>
</table>

flow cytometry-based drug screening system for the identification of small molecules that promote cellular differentiation of glioblastoma stem cells

胶质母细胞瘤是成人最常见和最致命的原发性脑瘤, 仅在美国每年造成大约1.4万人死亡。术后存活中位数少于15月, 最大手术切除、放疗、莫化疗。发展更有效的大紫荆治疗所固有的挑战已变得越来越明显, 包括其不屈的侵袭性, 它对标准治疗的抵抗, 其遗传复杂性和分子适应性, 和亚大紫荆与正常干细胞有表型相似的细胞, 这里称为胶质母细胞瘤干细胞 (GSCs)。由于 GSCs 是肿瘤生长和进展所必需的, differentiation-based 治疗是这些不治之症的一种可行的治疗方式。
下面的协议描述了建立一个高通量筛选平台的程序集合, 旨在识别促进 GSC 形分化的小分子。在该系统的核心是一个胶质纤维酸性蛋白 (胶质细胞) 分化的记者-构造。本议定书载有下列一般程序: (1) 建立 GSC 分化的记者行;(2) 测试/验证记者对 GSC 自我更新/克隆能力的相关性;和 (3) 高容量流式细胞仪基础药物筛选。
筛选平台提供了一种简单而廉价的方法来识别促进 GSCs 分化的小分子。此外, 利用 FDA 批准的药物库, 有可能识别能更快地进行用途的制剂。此外, 促进癌症干细胞分化的疗法有望与目前的 "护理标准" 治疗方法协同工作, 这些疗法已被证明是针对和消除主要分化的癌细胞。

三独立的患者衍生球线转与慢形记者编码的绿色荧光蛋白 (GFP) 融合在帧与 Zeocin 电阻卡带和驱动的人胶质细胞启动器元素 (图 1)。接下来的单个克隆是通过在96井板 (图 2) 中的每井电镀0.7 细胞而分离的, 随后是流细胞测定表达 GFP 的细胞百分比 (图 3)。干细胞克隆, 从单个细胞衍生, 含有 5% gfp 阳性细胞被称?...

Watch this Scientific Journal Video about Flow Cytometry-based Drug Screening System for the Identification of Small Molecules That Promote Cellular Differentiation of Glioblastoma Stem Cells at JoVE.

Flow Cytometry-based Drug Screening System for the Identification of Small Molecules That Promote Cellular Differentiation of Glioblastoma Stem Cells

流动 Cytometry-based 药物筛选系统对促进细胞分化胶质母细胞瘤小分子的鉴定

提出了一种有效的筛选方案, 以识别小分子, 促进形分化的胶质母细胞瘤干细胞 (GSCs)。该检测是基于一个干细胞分化的记者, 即增强 GFP (eGFP) 的表达是由人胶质细胞促进剂驱动。

Glioblastoma (GBM) is the most common and most lethal primary brain tumor in adults, causing roughly 14,000 deaths each year in the U.S. alone. Median ...

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Research

JoVE Journal

Cancer Research

8.2K 次观看.  Case Western Reserve University School of Medicine. 提出了一种有效的筛选方案, 以识别小分子, 促进形分化的胶质母细胞瘤干细胞 (GSCs)。该检测是基于一个干细胞分化的记者, 即增强 GFP (eGFP) 的表达是由人胶质细胞促进剂驱动。

视频: Flow Cytometry-based Drug Screening System for the Identification of Small Molecules That Promote Cellular Differentiation of Glioblastoma Stem Cells

Generation of High-Throughput Three-Dimensional Tumor Spheroids for Drug Screening

Cancer cells have routinely been cultured in two dimensions (2D) on a plastic surface. This technique, however, lacks the true environment a tumor mass is exposed to in vivo. Solid tumors grow not as a sheet attached to plastic, but instead as a collection of clonal cells in a three-dimensional (3D) space interacting with their neighbors, and with distinct spatial properties such as the disruption of normal cellular polarity. These interactions cause 3D-cultured cells to acquire morphological and cellular characteristics which are more relevant to in vivo tumors. Additionally, a tumor mass is in direct contact with other cell types such as stromal and immune cells, as well as the extracellular matrix from all other cell types. The matrix deposited is comprised of macromolecules such as collagen and fibronectin.
In an attempt to increase the translation of research findings in oncology from bench to bedside, many groups have started to investigate the use of 3D model systems in their drug development strategies. These systems are thought to be more physiologically relevant because they attempt to recapitulate the complex and heterogeneous environment of a tumor. These systems, however, can be quite complex, and, although amenable to growth in 96-well formats, and some now even in 384, they offer few choices for large-scale growth and screening. This observed gap has led to the development of the methods described here in detail to culture tumor spheroids in a high-throughput capacity in 1536-well plates. These methods represent a compromise to the highly complex matrix-based systems, which are difficult to screen, and conventional 2D assays. A variety of cancer cell lines harboring different genetic mutations are successfully screened, examining compound efficacy by using a curated library of compounds targeting the Mitogen-Activated Protein Kinase or MAPK pathway. The spheroid culture responses are then compared to the response of cells grown in 2D, and differential activities are reported. These methods provide a unique protocol for testing compound activity in a high-throughput 3D setting.

Across a wide variety of disease indications, more physiologically relevant models are being developed and implemented into drug discovery programs. The new model system described here demonstrates how three-dimensional tumor spheroids can be cultured and screened in a high-throughput 1536-well plate-based system to search for new oncology drugs.

高通量三维肿瘤球体药物筛选的生成

Cancer cells have routinely been cultured in two dimensions (2D) on a plastic surface. This technique, however, lacks the true environment a tumor mass is ...

科研

癌症研究

Live-Cell Imaging Assays to Study Glioblastoma Brain Tumor Stem Cell Migration and Invasion

Glioblastoma (GBM) is an aggressive brain tumor that is poorly controlled with the currently available treatment options. Key features of GBMs include rapid proliferation and pervasive invasion into the normal brain. Recurrence is thought to result from the presence of radio- and chemo-resistant brain tumor stem cells (BTSCs) that invade away from the initial cancerous mass and, thus, evade surgical resection. Hence, therapies that target BTSCs and their invasive abilities may improve the otherwise poor prognosis of this disease. Our group and others have successfully established and characterized BTSC cultures from GBM patient samples. These BTSC cultures demonstrate fundamental cancer stem cell properties such as clonogenic self-renewal, multi-lineage differentiation, and tumor initiation in immune-deficient mice. In order to improve on the current therapeutic approaches for GBM, a better understanding of the mechanisms of BTSC migration and invasion is necessary. In GBM, the study of migration and invasion is restricted, in part, due to the limitations of existing techniques which do not fully account for the in vitro growth characteristics of BTSCs grown as neurospheres. Here, we describe rapid and quantitative live-cell imaging assays to study both the migration and invasion properties of BTSCs. The first method described is the BTSC migration assay which measures the migration toward a chemoattractant gradient. The second method described is the BTSC invasion assay which images and quantifies a cellular invasion from neurospheres into a matrix. The assays described here are used for the quantification of BTSC migration and invasion over time and under different treatment conditions.

Here, we describe live-cell imaging techniques to quantitatively measure the migration and invasion of glioblastoma brain tumor stem cells over time and under multiple treatment conditions.

活体细胞成像技术研究胶质瘤脑干细胞的迁移和侵袭

Glioblastoma (GBM) is an aggressive brain tumor that is poorly controlled with the currently available treatment options. Key features of GBMs include ...

Flow Cytometric Detection of Newly-formed Breast Cancer Stem Cell-like Cells After Apoptosis Reversal

Cancer recurrence has long been studied by oncologists while the underlying mechanisms remain unclear. Recently, we and others found that a phenomenon named apoptosis reversal leads to increased tumorigenicity in various cell models under different stimuli. Previous studies have been focused on tracking this process in vitro and in vivo; however, the isolation of real reversed cells has yet to be achieved, which limits our understanding on the consequences of apoptosis reversal. Here, we take advantage of a Caspase-3/7 Green Detection dye to label cells with activated caspases after apoptotic induction. Cells with positive signals are further sorted out by fluorescence-activated cell sorting (FACS) for recovery. Morphological examination under confocal microscopy helps confirm the apoptotic status before FACS. An increase in tumorigenicity can often be attributed to the elevation in the percentage of cancer stem cell (CSC)-like cells. Also, given the heterogeneity of breast cancer, identifying the origin of these CSC-like cells would be critical to cancer treatment. Thus, we prepare breast non-stem cancer cells before triggering apoptosis, isolating caspase-activated cells and performing the apoptosis reversal procedure. Flow cytometry analysis reveals that breast CSC-like cells re-appear in the reversed group, indicating breast CSC-like cells are transited from breast non-stem cancer cells during apoptosis reversal. In summary, this protocol includes the isolation of apoptotic breast cancer cells and detection of changes in CSC percentage in reversed cells by flow cytometry.

Here, we present a protocol to isolate apoptotic breast cancer cells by fluorescence-activated cell sorting and further detect the transition of breast non-stem cancer cells to breast cancer stem cell-like cells after apoptosis reversal by flow cytometry.

细胞凋亡逆转后新形成乳腺癌干细胞样细胞的流式细胞仪检测

Cancer recurrence has long been studied by oncologists while the underlying mechanisms remain unclear. Recently, we and others found that a phenomenon ...

Physiologic Patient Derived 3D Spheroids for Anti-neoplastic Drug Screening to Target Cancer Stem Cells

In this protocol, we outline the procedure for generation of tumor spheroids within 384-well hanging droplets to allow for high-throughput screening of anti-cancer therapeutics in a physiologically representative microenvironment. We outline the formation of patient derived cancer stem cell spheroids, as well as, the manipulation of these spheroids for thorough analysis following drug treatment. Specifically, we describe collection of spheroid morphology, proliferation, viability, drug toxicity, cell phenotype and cell localization data. This protocol focuses heavily on analysis techniques that are easily implemented using the 384-well hanging drop platform, making it ideal for high throughput drug screening. While we emphasize the importance of this model in ovarian cancer studies and cancer stem cell research, the 384-well platform is amenable to research of other cancer types and disease models, extending the utility of the platform to many fields. By improving the speed of personalized drug screening and the quality of screening results through easily implemented physiologically representative 3D cultures, this platform is predicted to aid in the development of new therapeutics and patient-specific treatment strategies, and thus have wide-reaching clinical impact.

This protocol describes generation of patient-derived spheroids, and downstream analysis including quantification of proliferation, cytotoxicity testing, flow cytometry, immunofluorescence staining and confocal imaging, in order to assess drug candidates&#8217; potential as anti-neoplastic therapeutics. This protocol supports precision medicine with identification of specific drugs for each patient and stage of disease.

生理患者衍生的3D类血球体用于抗肿瘤药物筛选,以靶向癌症干细胞

In this protocol, we outline the procedure for generation of tumor spheroids within 384-well hanging droplets to allow for high-throughput screening of ...

Differentiation of Mouse Breast Epithelial HC11 and EpH4 Cells

Cadherins play an important role in the regulation of cell differentiation as well as neoplasia. Here we describe the origins and methods of the induction of differentiation of two mouse breast epithelial cell lines, HC11 and EpH4, and their use to study complementary stages of mammary gland development and neoplastic transformation.
The HC11 mouse breast epithelial cell line originated from the mammary gland of a pregnant Balb/c mouse. It differentiates when grown to confluence attached to a plastic Petri dish surface in medium containing fetal calf serum and Hydrocortisone, Insulin and Prolactin (HIP medium). Under these conditions, HC11 cells produce the milk proteins &#946;-casein and whey acidic protein (WAP), similar to lactating mammary epithelial cells, and form rudimentary mammary gland-like structures termed &#34;domes&#34;.
The EpH4 cell line was derived from spontaneously immortalized mouse mammary gland epithelial cells isolated from a pregnant Balb/c mouse. Unlike HC11, EpH4 cells can fully differentiate into spheroids (also called mammospheres) when cultured under three-dimensional (3D) growth conditions in HIP medium. Cells are trypsinized, suspended in a 20% matrix consisting of a mixture of extracellular matrix proteins produced by Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells, plated on top of a layer of concentrated matrix coating a plastic Petri dish or multiwell plate, and covered with a layer of 10% matrix-containing HIP medium. Under these conditions, EpH4 cells form hollow spheroids that exhibit apical-basal polarity, a hollow lumen, and produce &#946;-casein and WAP.
Using these techniques, our results demonstrated that the intensity of the cadherin/Rac signal is critical for the differentiation of HC11 cells. While Rac1 is necessary for differentiation and low levels of activated RacV12 increase differentiation, high RacV12 levels block differentiation while inducing neoplasia. In contrast, EpH4 cells represent an earlier stage in mammary epithelial differentiation, which is inhibited by even low levels of RacV12.

We describe techniques for differentiation induction of two breast epithelial lines, HC11 and EpH4. While both require fetal calf serum, insulin, and prolactin to produce milk proteins, EpH4 cells can fully differentiate into mammospheres in three-dimensional culture. These complementary models are useful for signal transduction studies of differentiation and neoplasia.

小鼠乳腺皮下HC11和EpH4细胞的分化

Cadherins play an important role in the regulation of cell differentiation as well as neoplasia. Here we describe the origins and methods of the induction ...

Establishment and Characterization of Small Bowel Neuroendocrine Tumor Spheroids

Small bowel neuroendocrine tumors (SBNETs) are rare cancers originating from enterochromaffin cells of the gut. Research in this field has been limited because very few patient derived SBNET cell lines have been generated. Well-differentiated SBNET cells are slow growing and are hard to propagate. The few cell lines that have been established are not readily available, and after time in culture may not continue to express characteristics of NET cells. Generating new cell lines could take many years since SBNET cells have a long doubling time and many enrichment steps are needed in order to eliminate the rapidly dividing cancer-associated fibroblasts. To overcome these limitations, we have developed a protocol to culture SBNET cells from surgically removed tumors as spheroids in extracellular matrix (ECM). The ECM forms a 3-dimensional matrix that encapsulates SBNET cells and mimics the tumor micro-environment for allowing SBNET cells to grow. Here, we characterized the growth rate of SBNET spheroids and described methods to identify SBNET markers using immunofluorescence microscopy and immunohistochemistry to confirm that the spheroids are neuroendocrine tumor cells. In addition, we used SBNET spheroids for testing the cytotoxicity of rapamycin.

Neuroendocrine tumors (NETs) originate from neuroendocrine cells of the neural crest. They are slow growing and challenging to culture. We present an alternative strategy to grow NETs from the small bowel by culturing them as spheroids. These spheroids have small bowel NET markers and can be used for drug testing.

小肠神经内分泌肿瘤类球体的建立与特征

Small bowel neuroendocrine tumors (SBNETs) are rare cancers originating from enterochromaffin cells of the gut. Research in this field has been limited ...

Identification of Transcription Factor Regulators using Medium-Throughput Screening of Arrayed Libraries and a Dual-Luciferase-Based Reporter

Transcription factors can alter the expression of numerous target genes that influence a variety of downstream processes making them good targets for anti-cancer therapies. However, directly targeting transcription factors is often difficult and can cause adverse side effects if the transcription factor is necessary in one or more adult tissues. Identifying upstream regulators that aberrantly activate transcription factors in cancer cells offers a more feasible alternative, particularly if these proteins are easy to drug. Here, we describe a protocol that can be used to combine arrayed medium-scale lentiviral libraries and a dual-luciferase-based transcriptional reporter assay to identify novel regulators of transcription factors in cancer cells. Our approach offers a quick, easy, and inexpensive way to test hundreds of genes in a single experiment. To demonstrate the use of this approach, we performed a screen of an arrayed lentiviral RNAi library containing several regulators of Yes-associated protein (YAP) and transcriptional co-activator with PDZ-binding motif (TAZ), two transcriptional co-activators that are the downstream effectors of the Hippo pathway. However, this approach could be modified to screen for regulators of virtually any transcription factor or co-factor and could also be used to screen CRISPR/CAS9, cDNA, or ORF libraries.

To identify novel regulators of transcription factors, we developed an approach to screen arrayed lentiviral or retroviral RNAi libraries using a dual-luciferase-based transcriptional reporter assay. This approach offers a quick and relatively inexpensive way to screen hundreds of candidates in a single experiment.

使用阵列库的中等通量筛选和基于双 Luciferase 的分录报告器识别转录因子稳压器

Transcription factors can alter the expression of numerous target genes that influence a variety of downstream processes making them good targets for ...

Drug Screening of Primary Patient Derived Tumor Xenografts in Zebrafish

Patient derived xenograft models are critical in defining how different cancers respond to drug treatment in an in vivo system. Mouse models are the standard in the field, but zebrafish have emerged as an alternative model with several advantages, including the ability for high-throughput and low-cost drug screening. Zebrafish also allow for in vivo drug screening with large replicate numbers that were previously only obtainable with in vitro systems. The ability to rapidly perform large scale drug screens may open up the possibility for personalized medicine with rapid translation of results back to clinic. Zebrafish xenograft models could also be used to rapidly screen for actionable mutations based on tumor response to targeted therapies or to identify new anti-cancer compounds from large libraries. The current major limitation in the field has been quantifying and automating the process so that drug screens can be done on a larger scale and be less labor-intensive. We have developed a workflow for xenografting primary patient samples into zebrafish larvae and performing large scale drug screens using a fluorescence microscope equipped imaging unit and automated sampler unit. This method allows for standardization and quantification of engrafted tumor area and response to drug treatment across large numbers of zebrafish larvae. Overall, this method is advantageous over traditional cell culture drug screening as it allows for growth of tumor cells in an in vivo environment throughout drug treatment, and is more practical and cost-effective than mice for large scale in vivo drug screens.

Zebrafish xenograft models allow for high-throughput drug screening and fluorescent imaging of human cancer cells in an in vivo microenvironment. We developed a workflow for large scale, automated drug screening on patient-derived leukemia samples in zebrafish using an automated fluorescence microscope equipped imaging unit.

斑马鱼原发性患者衍生肿瘤异种移植物的药物筛选

Patient derived xenograft models are critical in defining how different cancers respond to drug treatment in an in vivo system. Mouse models are the ...

Co-culture of Glioblastoma Stem-like Cells on Patterned Neurons to Study Migration and Cellular Interactions

Glioblastomas (GBMs), grade IV malignant gliomas, are one of the deadliest types of human cancer because of their aggressive characteristics. Despite significant advances in the genetics of these tumors, how GBM cells invade the healthy brain parenchyma is not well understood. Notably, it has been shown that GBM cells invade the peritumoral space via different routes; the main interest of this paper is the route along white matter tracts (WMTs). The interactions of tumor cells with the peritumoral nervous cell components are not well characterized. Herein, a method has been described that evaluates the impact of neurons on GBM cell invasion. This paper presents an advanced co-culture in vitro assay that mimics WMT invasion by analyzing the migration of GBM stem-like cells on neurons. The behavior of GBM cells in the presence of neurons is monitored by using an automated tracking procedure with open-source and free-access software. This method is useful for many applications, in particular, for functional and mechanistic studies as well as for analyzing the effects of pharmacological agents that can block GBM cell migration on neurons.

Here, we present an easy-to-use co-culture assay to analyze glioblastoma (GBM) migration on patterned neurons. We developed a macro in FiJi software for easy quantification of GBM cell migration on neurons, and observed that neurons modify GBM cell invasive capacity.

胶质母细胞瘤干细胞在模式神经元上共同培养，以研究迁移和细胞相互作用

Glioblastomas (GBMs), grade IV malignant gliomas, are one of the deadliest types of human cancer because of their aggressive characteristics. Despite ...

In Vivo Immunogenicity Screening of Tumor-Derived Extracellular Vesicles by Flow Cytometry of Splenic T Cells

Immunogenic cell death of tumors, caused by chemotherapy or irradiation, can trigger tumor-specific T cell responses by releasing danger-associated molecular patterns and inducing the production of type I interferon. Immunotherapies, including checkpoint inhibition, primarily rely on preexisting tumor-specific T cells to unfold a therapeutic effect. Thus, synergistic therapeutic approaches that exploit immunogenic cell death as an intrinsic anti-cancer vaccine may improve their responsiveness. However, the spectrum of immunogenic factors released by cells under therapy-induced stress remains incompletely characterized, especially regarding extracellular vesicles (EVs). EVs, nano-scale membranous particles emitted from virtually all cells, are considered to facilitate intercellular communication and, in cancer, have been shown to mediate cross-priming against tumor antigens. To assess the immunogenic effect of EVs derived from tumors under various conditions, adaptable, scalable, and valid methods are sought-for. Therefore, herein a relatively easy and robust approach is presented to assess EVs' in vivo immunogenicity. The protocol is based on flow cytometry analysis of splenic T cells after in vivo immunization of mice with EVs, isolated by precipitation-based assays from tumor cell cultures under therapy or steady-state conditions. For example, this work shows that oxaliplatin exposure of B16-OVA murine melanoma cells resulted in the release of immunogenic EVs that can mediate the activation of tumor-reactive cytotoxic T cells. Hence, screening of EVs via in vivo immunization and flow cytometry identifies conditions under which immunogenic EVs can emerge. Identifying conditions of immunogenic EV release provides an essential prerequisite to testing EVs' therapeutic efficacy against cancer and exploring the underlying molecular mechanisms to ultimately unveil new insights into EVs' role in cancer immunology.

In Vivo Immunogenicity Screening of Tumor-Derived Extracellular Vesicles by Flow Cytometry of Splenic T Cells

This manuscript describes how to assess in vivo immunogenicity of tumor cell-derived extracellular vesicles (EVs) using flow cytometry. EVs derived from tumors undergoing treatment-induced immunogenic cell death seem particularly relevant in tumor immunosurveillance. This protocol exemplifies the assessment of oxaliplatin-induced immunostimulatory tumor EVs but can be adapted to various settings.

体内 通过脾T细胞流式细胞术筛选肿瘤来源的细胞外囊泡的免疫原性

Immunogenic cell death of tumors, caused by chemotherapy or irradiation, can trigger tumor-specific T cell responses by releasing danger-associated ...

流动 Cytometry-based 药物筛选系统对促进细胞分化胶质母细胞瘤小分子的鉴定

摘要

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此视频中的章节

高通量三维肿瘤球体药物筛选的生成

活体细胞成像技术研究胶质瘤脑干细胞的迁移和侵袭

细胞凋亡逆转后新形成乳腺癌干细胞样细胞的流式细胞仪检测

生理患者衍生的3D类血球体用于抗肿瘤药物筛选,以靶向癌症干细胞

小鼠乳腺皮下HC11和EpH4细胞的分化

小肠神经内分泌肿瘤类球体的建立与特征

使用阵列库的中等通量筛选和基于双 Luciferase 的分录报告器识别转录因子稳压器

斑马鱼原发性患者衍生肿瘤异种移植物的药物筛选

胶质母细胞瘤干细胞在模式神经元上共同培养，以研究迁移和细胞相互作用

体内通过脾T细胞流式细胞术筛选肿瘤来源的细胞外囊泡的免疫原性

流动 Cytometry-based 药物筛选系统对促进细胞分化胶质母细胞瘤小分子的鉴定

摘要

探索更多视频

此视频中的章节

高通量三维肿瘤球体药物筛选的生成

活体细胞成像技术研究胶质瘤脑干细胞的迁移和侵袭

细胞凋亡逆转后新形成乳腺癌干细胞样细胞的流式细胞仪检测

生理患者衍生的3D类血球体用于抗肿瘤药物筛选,以靶向癌症干细胞

小鼠乳腺皮下HC11和EpH4细胞的分化

小肠神经内分泌肿瘤类球体的建立与特征

使用阵列库的中等通量筛选和基于双 Luciferase 的分录报告器识别转录因子稳压器

斑马鱼原发性患者衍生肿瘤异种移植物的药物筛选

胶质母细胞瘤干细胞在模式神经元上共同培养，以研究迁移和细胞相互作用

体内 通过脾T细胞流式细胞术筛选肿瘤来源的细胞外囊泡的免疫原性

体内通过脾T细胞流式细胞术筛选肿瘤来源的细胞外囊泡的免疫原性