Name: flow cytometry-based drug screening system for the identification of small molecules that promote cellular differentiation of glioblastoma stem cells
Uploaded: 2018-01-10T14:00:00.000+00:00
Duration: 10 min 28 s
Description: 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.

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>

<ol>
	<li>Maher, E. 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=Malignant+glioma:+genetics+and+biology+of+a+grave+matter.">Malignant glioma: genetics and biology of a grave matter.</a> Genes Dev. 15 (11), 1311-1333 (2001).</li><li>Bonavia, R., Inda, M. M., Cavenee, W. K., Furnari, F. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterogeneity+maintenance+in+glioblastoma:+a+social+network.">Heterogeneity maintenance in glioblastoma: a social network.</a> Cancer Res. 71 (12), 4055-4060 (2011).</li><li>Bao, S., 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=Glioma+stem+cells+promote+radioresistance+by+preferential+activation+of+the+DNA+damage+response.">Glioma stem cells promote radioresistance by preferential activation of the DNA damage response.</a> Nature. 444 (7120), 756-760 (2006).</li><li>Sul, J., Fine, H. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Malignant+gliomas:+new+translational+therapies.">Malignant gliomas: new translational therapies.</a> Mt Sinai J Med. 77 (6), 655-666 (2010).</li><li>Bar, E. E., Chaudhry, A., Farah, M. H., Eberhart, C. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hedgehog+signaling+promotes+medulloblastoma+survival+via+Bc/II.">Hedgehog signaling promotes medulloblastoma survival via Bc/II.</a> Am J Pathol. 170 (1), 347-355 (2007).</li><li>Chu, Q., Orr, B. A., Semenkow, S., Bar, E. E., Eberhart, C. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prolonged+inhibition+of+glioblastoma+xenograft+initiation+and+clonogenic+growth+following+in+vivo+Notch+blockade.">Prolonged inhibition of glioblastoma xenograft initiation and clonogenic growth following in vivo Notch blockade.</a> Clin Cancer Res. 19 (12), 3224-3233 (2013).</li><li>Schreck, K. C., 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+Notch+target+Hes1+directly+modulates+Gli1+expression+and+Hedgehog+signaling:+a+potential+mechanism+of+therapeutic+resistance.">The Notch target Hes1 directly modulates Gli1 expression and Hedgehog signaling: a potential mechanism of therapeutic resistance.</a> Clin Cancer Res. 16 (24), 6060-6070 (2010).</li><li>Warrell, R. P., 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=Differentiation+therapy+of+acute+promyelocytic+leukemia+with+tretinoin+(all-trans-retinoic+acid).">Differentiation therapy of acute promyelocytic leukemia with tretinoin (all-trans-retinoic acid).</a> N Engl J Med. 324 (20), 1385-1393 (1991).</li><li>Piccirillo, S. G., 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=Bone+morphogenetic+proteins+inhibit+the+tumorigenic+potential+of+human+brain+tumour-initiating+cells.">Bone morphogenetic proteins inhibit the tumorigenic potential of human brain tumour-initiating cells.</a> Nature. 444 (7120), 761-765 (2006).</li><li>Bar, 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=Cyclopamine-mediated+hedgehog+pathway+inhibition+depletes+stem-like+cancer+cells+in+glioblastoma.">Cyclopamine-mediated hedgehog pathway inhibition depletes stem-like cancer cells in glioblastoma.</a> Stem Cells. 25 (10), 2524-2533 (2007).</li><li>Bar, E. E., Lin, A., Mahairaki, V., Matsui, W., Eberhart, C. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypoxia+increases+the+expression+of+stem-cell+markers+and+promotes+clonogenicity+in+glioblastoma+neurospheres.">Hypoxia increases the expression of stem-cell markers and promotes clonogenicity in glioblastoma neurospheres.</a> Am J Pathol. 177 (3), 1491-1502 (2010).</li><li>Kahlert, U. 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=CD133/CD15+defines+distinct+cell+subpopulations+with+differential+in+vitro+clonogenic+activity+and+stem+cell-related+gene+expression+profile+in+in+vitro+propagated+glioblastoma+multiforme-derived+cell+line+with+a+PNET-like+component.">CD133/CD15 defines distinct cell subpopulations with differential in vitro clonogenic activity and stem cell-related gene expression profile in in vitro propagated glioblastoma multiforme-derived cell line with a PNET-like component.</a> Folia Neuropathol. 50 (4), 357-368 (2012).</li><li>Lim, K. S., 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=Inhibition+of+monocarboxylate+transporter-4+depletes+stem-like+glioblastoma+cells+and+inhibits+HIF+transcriptional+response+in+a+lactate-independent+manner.">Inhibition of monocarboxylate transporter-4 depletes stem-like glioblastoma cells and inhibits HIF transcriptional response in a lactate-independent manner.</a> Oncogene. 33 (35), 4433-4441 (2014).</li><li>Kahlert, U. 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=ZEB1+Promotes+Invasion+in+Human+Fetal+Neural+Stem+Cells+and+Hypoxic+Glioma+Neurospheres.">ZEB1 Promotes Invasion in Human Fetal Neural Stem Cells and Hypoxic Glioma Neurospheres.</a> Brain Pathol. 25 (6), 724-732 (2014).</li><li>Hu, Y., Smyth, G. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ELDA:+extreme+limiting+dilution+analysis+for+comparing+depleted+and+enriched+populations+in+stem+cell+and+other+assays.">ELDA: extreme limiting dilution analysis for comparing depleted and enriched populations in stem cell and other assays.</a> J Immunol Methods. 347 (1-2), 70-78 (2009).</li><li>Meyer, 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=Single+cell-derived+clonal+analysis+of+human+glioblastoma+links+functional+and+genomic+heterogeneity.">Single cell-derived clonal analysis of human glioblastoma links functional and genomic heterogeneity.</a> Proc Natl Acad Sci U S A. 112 (3), 851-856 (2015).</li><li>Galli, 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=Isolation+and+characterization+of+tumorigenic,+stem-like+neural+precursors+from+human+glioblastoma.">Isolation and characterization of tumorigenic, stem-like neural precursors from human glioblastoma.</a> Cancer Res. 64 (19), 7011-7021 (2004).</li><li>Spina, R., Voss, D. M., Asnaghi, L., Sloan, A., Bar, E. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Atracurium+Besylate+and+other+neuromuscular+blocking+agents+promote+astroglial+differentiation+and+deplete+glioblastoma+stem+cells.">Atracurium Besylate and other neuromuscular blocking agents promote astroglial differentiation and deplete glioblastoma stem cells.</a> Oncotarget. 7 (1), 459-472 (2016).</li></ol>

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&#039;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&amp;sup2; Flask Vented</td><td>Thermo Fisher Scientific</td><td>130189</td><td></td></tr><tr><td>Biolite T75 cm&amp;sup2; 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 &amp;mu;l</td><td>VistaLab Technologies</td><td>4060-1002</td><td></td></tr><tr><td>Multi 12-channel pipette tips 250 &amp;mu;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>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;For the preparation of neural stem cell media (500 mL)&lt;/strong&gt;</td><td></td><td></td><td>&lt;strong&gt;Final concentration&lt;/strong&gt;</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 &amp;micro;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 ...

flow-cytometry-based-drug-screening-system-for-identification-small

Universidad San Sebastian

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

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 ...

科研

癌症研究

Identification and Isolation of Slow-Dividing Cells in Human Glioblastoma Using Carboxy Fluorescein Succinimidyl Ester (CFSE)

Tumor heterogeneity represents a fundamental feature supporting tumor robustness and presents a central obstacle to the development of therapeutic strategies1. To overcome the issue of tumor heterogeneity, it is essential to develop assays and tools enabling phenotypic, (epi)genetic and functional identification and characterization of tumor subpopulations that drive specific disease pathologies and represent clinically relevant targets. It is now well established that tumors exhibit distinct sub-fractions of cells with different frequencies of cell division, and that the functional criteria of being slow cycling is positively associated with tumor formation ability in several cancers including those of the brain, breast, skin and pancreas as well as leukemia2-8. The fluorescent dye carboxyfluorescein succinimidyl ester (CFSE) has been used for tracking the division frequency of cells in vitro and in vivo in blood-borne tumors and solid tumors such as glioblastoma2,7,8. The cell-permeant non-fluorescent pro-drug of CFSE is converted by intracellular esterases into a fluorescent compound, which is retained within cells by covalently binding to proteins through reaction of its succinimidyl moiety with intracellular amine groups to form stable amide bonds9. The fluorescent dye is equally distributed between daughter cells upon divisions, leading to the halving of the fluorescence intensity with every cell division. This enables tracking of cell cycle frequency up to eight to ten rounds of division10. CFSE retention capacity was used with brain tumor cells to identify and isolate a slow cycling subpopulation (top 5% dye-retaining cells) demonstrated to be enriched in cancer stem cell activity2. 
This protocol describes the technique of staining cells with CFSE and the isolation of individual populations within a culture of human glioblastoma (GBM)-derived cells possessing differing division rates using flow cytometry2. The technique has served to identify and isolate a brain tumor slow-cycling population of cells by virtue of their ability to retain the CFSE labeling.

Identification and Isolation of Slow-Dividing Cells in Human Glioblastoma Using Carboxy Fluorescein Succinimidyl Ester CFSE

This video protocol demonstrates the application of the fluorescent dye carboxyfluorescein succinimidyl ester (CFSE) for the identification and separation of different sub-populations of cells in human glioblastoma based on frequency of cell division.

慢分裂的细胞在人脑胶质瘤羧基荧光素琥珀酰亚胺酯（CFSE）的分离与鉴定

Tumor heterogeneity represents a fundamental feature supporting tumor robustness and presents a central obstacle to the development of therapeutic ...

医学

Processing of Primary Brain Tumor Tissue for Stem Cell Assays and Flow Sorting

Brain tumors are typically comprised of morphologically diverse cells that express a variety of neural lineage markers. Only a relatively small fraction of cells in the tumor with stem cell properties, termed brain tumor initiating cells (BTICs), possess an ability to differentiate along multiple lineages, self-renew, and initiate tumors in vivo. We applied culture conditions originally used for normal neural stem cells (NSCs) to a variety of human brain tumors and found that this culture method specifically selects for stem-like populations. Serum-free medium (NSC) allows for the maintenance of an undifferentiated stem cell state, and the addition of bFGF and EGF allows for the proliferation of multi-potent, self-renewing, and expandable tumorspheres.
To further characterize each tumor's BTIC population, we evaluate cell surface markers by flow cytometry. We may also sort populations of interest for more specific characterization. Self-renewal assays are performed on single BTICs sorted into 96 well plates; the formation of tumorspheres following incubation at 37 &deg;C indicates the presence of a stem or progenitor cell. Multiple cell numbers of a particular population can also be sorted in different wells for limiting dilution analysis, to analyze self-renewal capacity. We can also study differential gene expression within a particular cell population by using single cell RT-PCR.
The following protocols describe our procedures for the dissociation and culturing of primary human samples to enrich for BTIC populations, as well as the dissociation of tumorspheres. Also included are protocols for staining for flow cytometry analysis or sorting, self-renewal assays, and single cell RT-PCR.

The identification of brain tumor initiating cells (BTICs), the rare cells within a heterogeneous tumor possessing stem cell properties, provides new insights into human brain tumor pathogenesis. We have refined specific culture conditions to enrich for BTICs, and we routinely use flow cytometry to further enrich these populations. Self-renewal assays and transcript analysis by single cell RT-PCR can subsequently be performed on these isolated cells.

加工的主要脑肿瘤组织的干细胞检测和流量排序

Brain tumors are typically comprised of morphologically diverse cells that express a variety of neural lineage markers. Only a relatively small fraction ...

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 ...

A Rapid Screening Workflow to Identify Potential Combination Therapy for GBM using Patient-Derived Glioma Stem Cells

The glioma stem cells (GSCs) are a small fraction of cancer cells which play essential roles in tumor initiation, angiogenesis, and drug resistance in glioblastoma (GBM), the most prevalent and devastating primary brain tumor. The presence of GSCs makes the GBM very refractory to most of individual targeted agents, so high-throughput screening methods are required to identify potential effective combination therapeutics. The protocol describes a simple workflow to enable rapid screening for potential combination therapy with synergistic interaction. The general steps of this workflow consist of establishing luciferase-tagged GSCs, preparing matrigel coated plates, combination drug screening, analyzing, and validating the results.

快速筛选工作流程，利用患者衍生胶质瘤干细胞确定GBM的潜在组合疗法

The glioma stem cells (GSCs) are a small fraction of cancer cells which play essential roles in tumor initiation, angiogenesis, and drug resistance in ...

Optimization of High Grade Glioma Cell Culture from Surgical Specimens for Use in  Clinically Relevant Animal Models and 3D Immunochemistry

Glioblastomas, the most common and aggressive form of astrocytoma, are refractory to therapy, and molecularly heterogeneous. The ability to establish cell cultures that preserve the genomic profile of the parental tumors, for use in patient specific in vitro and in vivo models, has the potential to revolutionize the preclinical development of new treatments for glioblastoma tailored to the molecular characteristics of each tumor.
Starting with fresh high grade astrocytoma tumors dissociated into single cells, we use the neurosphere assay as an enrichment method for cells presenting cancer stem cell phenotype, including expression of neural stem cell markers, long term self-renewal in vitro, and the ability to form orthotopic xenograft tumors. This method has been previously proposed, and is now in use by several investigators. Based on our experience of dissociating and culturing 125 glioblastoma specimens, we arrived at the detailed protocol we present here, suitable for routine neurosphere culturing of high grade astrocytomas and large scale expansion of tumorigenic cells for preclinical studies. We report on the efficiency of successful long term cultures using this protocol&#160;and suggest affordable alternatives for culturing dissociated glioblastoma cells that fail to grow as neurospheres. We also describe in detail a protocol for preserving the neurospheres 3D architecture for immunohistochemistry. Cell cultures enriched in CSCs, capable of generating orthotopic xenograft models that preserve the molecular signatures and heterogeneity of GBMs, are becoming increasingly popular for the study of the biology of GBMs and for the improved design of preclinical testing of potential therapies.

Protocol for propagation of dissociated high grade glioma surgical specimens in serum-free neurosphere medium to select for cells with cancer stem cell phenotype. For specimens that fail to grow as neurospheres, an alternative protocol is suggested. A paraffin embedding technique for maintaining the 3D neurosphere architecture for immunocytochemistry is described.

优化高等级胶质瘤细胞培养从外科标本用于临床相关的动物模型和3D免疫化学

Glioblastomas, the most common and aggressive form of astrocytoma, are refractory to therapy, and molecularly heterogeneous. The ability to establish cell ...

Spheroid Drug Sensitivity Screening in Glioma Stem Cell Lines

In vitro drug sensitivity screens are important tools in the discovery of anti-cancer drug combination therapies. Typically, these in vitro drug screens are performed on cells grown in a monolayer. However, these two-dimensional (2D) models are considered less accurate compared to three-dimensional (3D) spheroid cell models; this is especially true for glioma stem cell lines. Cells grown in spheres activate different signaling pathways and are considered more representative of in vivo models than monolayer cell lines. This protocol describes a method for in vitro drug screening of spheroid lines; mouse and human glioma stem cell lines are used as an example. This protocol describes a 3D spheroid drug sensitivity and synergy assay that can be used to determine if a drug or drug combination induces cell death and if two drugs synergize. Glioma stem cell lines are&#160;modified to express RFP. Cells are plated in low attachment round well bottom 96 plates, and spheres are allowed to form overnight. Drugs are added, and the growth is monitored by measuring the RFP signal over time using the Incucyte live imaging system,&#160;a fluorescence microscope embedded in the tissue culture incubator. Half maximal inhibitory concentration (IC50), median lethal dose (LD50), and synergy score are subsequently calculated to evaluate sensitivities to drugs alone or in combination. The three-dimensional nature of this assay&#160;provides a more accurate reflection of tumor growth, behavior, and drug sensitivities in vivo, thus forming the basis for further preclinical investigation.

In vitro drug sensitivity screens are important tools for discovering anti-cancer drug combinations. Cells grown in spheres activate different signaling pathways and are considered more representative of in vivo models than monolayer cell lines. This protocol describes a method for in vitro drug screening for spheroid lines.

In vitro drug sensitivity screens are important tools in the discovery of anti-cancer drug combination therapies. Typically, these in vitro drug screens ...

Isolation and Flow Cytometric Analysis of Glioma-infiltrating Peripheral Blood Mononuclear Cells

Our laboratory has recently demonstrated that natural killer (NK) cells are capable of eradicating orthotopically implanted mouse GL26 and rat CNS-1 malignant gliomas soon after intracranial engraftment if the cancer cells are rendered deficient in their expression of the &#946;-galactoside-binding lectin galectin-1 (gal-1). More recent work now shows that a population of Gr-1+/CD11b+ myeloid cells is critical to this effect. To better understand the mechanisms by which NK and myeloid cells cooperate to confer gal-1-deficient tumor rejection we have developed a comprehensive protocol for the isolation and analysis of glioma-infiltrating peripheral blood mononuclear cells (PBMC). The method is demonstrated here by comparing PBMC infiltration into the tumor microenvironment of gal-1-expressing GL26 gliomas with those rendered gal-1-deficient via shRNA knockdown. The protocol begins with a description of&#160;how to culture and prepare GL26 cells for inoculation into the syngeneic C57BL/6J mouse brain. It then explains the steps involved in the isolation and flow cytometric analysis of glioma-infiltrating PBMCs from the early brain tumor microenvironment. The method is adaptable to a number of in vivo experimental designs in which temporal data on immune infiltration into the brain is required. The method is sensitive and highly reproducible, as glioma-infiltrating PBMCs can be isolated from intracranial tumors as soon as 24 hr post-tumor engraftment with similar cell counts observed from time point matched tumors throughout independent experiments. A single experimentalist can perform the method from brain harvesting to flow cytometric analysis of glioma-infiltrating PBMCs in roughly 4-6 hr depending on the number of samples to be analyzed. Alternative glioma models and/or cell-specific detection antibodies may also be used at the experimentalists&#8217; discretion to assess the infiltration of several other immune cell types of interest without the need for alterations to the overall procedure.

Presented here is a straightforward method for the isolation and flow cytometric analysis of glioma-infiltrating peripheral blood mononuclear cells that yields time-dependent quantitative data on the number and activation status of immune cells entering the early brain tumor microenvironment.

分离和胶质瘤浸润外周血单个核细胞的流式细胞仪分析

Our laboratory has recently demonstrated that natural killer (NK) cells are capable of eradicating orthotopically implanted mouse GL26 and rat CNS-1 ...

免疫与感染

Cellular Bioluminescence Based Assay: A High Throughput Method for Combinatorial Drug Screening

Source: Hu, Z. et al. <a href="https://www-jove-com.bdigitaluss.remotexs.co/v/62312/a-rapid-screening-workflow-to-identify-potential-combination-therapy">A Rapid Screening Workflow to Identify Potential Combination Therapy for GBM using Patient-Derived Glioma Stem Cells.</a>&#160;J. Vis. Exp.&#160;(2021)
In this video, we describe a method highlighting the utility of bioluminescence assay for sensitive and rapid screening of drugs. These drugs can be tested alone or in combination to assess the synergism in their mode of action.

基于细胞生物发光的检测:一种用于组合药物筛选的高通量方法

Source: Hu, Z. et al. A Rapid Screening Workflow to Identify Potential Combination Therapy for GBM using Patient-Derived Glioma Stem Cells.&#160;J. Vis. ...

JoVE Encyclopedia of Experiments

Cancers of the Nervous System

In vitro studies

Luciferase-tagged Glioma Stem Cell Model: A Lentivirus-mediated Method to Prepare Luciferase-tagged Glioma Stem Cells for In Vitro Therapeutic Studies

Source: Hu, Z. et al. <a href="https://www-jove-com.bdigitaluss.remotexs.co/v/62312/a-rapid-screening-workflow-to-identify-potential-combination-therapy">A Rapid Screening Workflow to Identify Potential Combination Therapy for GBM using Patient-Derived Glioma Stem Cells.</a> J. Vis. Exp. (2021)
This video demonstrates the establishment of luciferase-tagged glioma stem cells via lentivirus-mediated gene delivery. Luciferase acts as a reporter gene and helps facilitate rapid drug screening experiments.

荧光素酶标记的神经胶质瘤干细胞模型:一种慢病毒介导的制备荧光素酶标记的神经胶质瘤干细胞用于体外治疗研究的方法

Source: Hu, Z. et al. A Rapid Screening Workflow to Identify Potential Combination Therapy for GBM using Patient-Derived Glioma Stem Cells. J. Vis. Exp. ...