Name: physiologic patient derived 3d spheroids for anti-neoplastic drug screening to target cancer stem cells
Uploaded: 2019-07-05T12:30:00
Description: Watch this Scientific Journal Video about Physiologic Patient Derived 3D Spheroids for Anti-neoplastic Drug Screening to Target Cancer Stem Cells at JoVE.com

这项工作主要支持 DOD OCRP 早期职业调查员奖 W81XWH-13-1-0134 (GM), DOD 飞行员奖 W81XWH-16-1-0426 (GM), 国防部调查员启动奖 W81XWH-17-OCRP-IIRA (GM), 里夫金卵巢癌和密歇根州卵巢癌中心联盟。本出版物中报告的研究得到了国家卫生研究院国家癌症研究所的支持,奖励编号为P30CA046592。CMN由国家科学基金会研究生研究奖学金资助,奖学金号为1256260。教育部由教育部国家需要领域研究生援助(GAANN)研究金提供支持。

所有患者样本均根据经批准的 IRB 协议从同意的患者那里收集,其样本在肿瘤去散、腹水收集后脱认。
1. 在384孔挂落板中从小细胞数生成球形
<ol><li>将悬挂的滴板放入装有无菌脱离子 (DI) 水和声波的声波器中 20 分钟。</li>
	<li>用戴手套的手,从声波器中取出盘子,用运行的DI水清洗。</li>
	<li>让盘子在0.1%的普鲁酮酸浴中24小时,以防止蛋白质吸附和球体附着在井中。</li>
	<li>用戴手...

<ol>
	<li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> All cancers Source: Globocan. , (2018).</li><li>Prasad, V., Mailankody, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Research+and+Development+Spending+to+Bring+a+Single+Cancer+Drug+to+Market+and+Revenues+After+Approval.">Research and Development Spending to Bring a Single Cancer Drug to Market and Revenues After Approval.</a> JAMA Internal Medicine. 177, 1569-1575 (2017).</li><li>Raghavan, 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=Personalized+Medicine+Based+Approach+to+Model+Patterns+of+Chemoresistance+and+Tumor+Recurrence+Using+Ovarian+Cancer+Stem+Cell+Spheroids.">Personalized Medicine Based Approach to Model Patterns of Chemoresistance and Tumor Recurrence Using Ovarian Cancer Stem Cell Spheroids.</a> Clinical Cancer Research. 23 (22), 6934-6945 (2017).</li><li>Jordan, C. T., Guzman, M. L., Noble, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+Stem+Cells.">Cancer Stem Cells.</a> New England Journal of Medicine. 355, 1253-1261 (2006).</li><li>Ishiguro, T., 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=Establishment+and+Characterization+of+an+In+Vitro+Model+of+Ovarian+Cancer+Stem-like+Cells+with+an+Enhanced+Proliferative+Capacity.">Establishment and Characterization of an In Vitro Model of Ovarian Cancer Stem-like Cells with an Enhanced Proliferative Capacity.</a> Cancer Research. 76, 150-160 (2016).</li><li>P&#225;ez, 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=Cancer+Dormancy:+A+Model+of+Early+Dissemination+and+Late+Cancer+Recurrence.">Cancer Dormancy: A Model of Early Dissemination and Late Cancer Recurrence.</a> Clinical Cancer Research. 18, 645-653 (2012).</li><li>Ahmed, N., Stenvers, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Getting+to+Know+Ovarian+Cancer+Ascites:+Opportunities+for+Targeted+Therapy-Based+Translational+Research.">Getting to Know Ovarian Cancer Ascites: Opportunities for Targeted Therapy-Based Translational Research.</a> Frontiers in Oncology. 3, (2013).</li><li>Pradhan, S., Clary, J. M., Seliktar, D., Lipke, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+three-dimensional+spheroidal+cancer+model+based+on+PEG-fibrinogen+hydrogel+microspheres.">A three-dimensional spheroidal cancer model based on PEG-fibrinogen hydrogel microspheres.</a> Biomaterials. 115, 141-154 (2017).</li><li>Varna, M., Bertheau, P., Legr&#232;s, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+Microenvironment+in+Human+Tumor+Xenografted+Mouse+Models.">Tumor Microenvironment in Human Tumor Xenografted Mouse Models.</a> Journal of Analytical Oncology. 3, 159-166 (2014).</li><li>Huang, S. B., 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=An+integrated+microfluidic+cell+culture+system+for+high-throughput+perfusion+three-dimensional+cell+culture-based+assays:+Effect+of+cell+culture+model+on+the+results+of+chemosensitivity+assays.">An integrated microfluidic cell culture system for high-throughput perfusion three-dimensional cell culture-based assays: Effect of cell culture model on the results of chemosensitivity assays.</a> Lab on a Chip. 13, 1133-1143 (2013).</li><li>Zang, R., Li, D., Tang, I. C., Wang, J., Yang, S. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-Based+Assays+in+High-Throughput+Screening+for+Drug+Discovery.">Cell-Based Assays in High-Throughput Screening for Drug Discovery.</a> International Journal of Biotechnology for Wellness Industries. , (2012).</li><li>Murphy, B., 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=Evaluation+of+alternative+in+vivo+drug+screening+methodology:+a+single+mouse+analysis.">Evaluation of alternative in vivo drug screening methodology: a single mouse analysis.</a> Cancer Research. 76, 5798-5809 (2016).</li><li>Hidalgo, 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=Patient-derived+Xenograft+models:+An+emerging+platform+for+translational+cancer+research.">Patient-derived Xenograft models: An emerging platform for translational cancer research.</a> Cancer Discovery. 4, 998-1013 (2014).</li><li>Damiati, S., Kompella, U. B., Damiati, S. A., Kodzius, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidic+devices+for+drug+delivery+systems+and+drug+screening.">Microfluidic devices for drug delivery systems and drug screening.</a> Genes. 9, (2018).</li><li>Caliari, S. R., Burdick, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Practical+Guide+to+Hydrogels+for+Cell+Culture.">A Practical Guide to Hydrogels for Cell Culture.</a> Nature Methods. 14, 69-81 (2016).</li><li>Mehta, G., Hsiao, A. Y., Ingram, M., Luker, G. D., Takayama, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Opportunities+and+challenges+for+use+of+tumor+spheroids+as+models+to+test+drug+delivery+and+efficacy.">Opportunities and challenges for use of tumor spheroids as models to test drug delivery and efficacy.</a> Journal of Control Release. , (2012).</li><li>Mehta, P., Novak, C., Raghavan, S., Ward, M., Mehta, G., Papaccio, G., Desiderio, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Self-Renewal+and+CSCs+In+Vitro+Enrichment:+Growth+as+Floating+Spheres.">Self-Renewal and CSCs In Vitro Enrichment: Growth as Floating Spheres.</a> Cancer Stem Cells: Methods and Protocols. , 61-75 (2018).</li><li>Raghavan, 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=Formation+of+stable+small+cell+number+three-dimensional+ovarian+cancer+spheroids+using+hanging+drop+arrays+for+preclinical+drug+sensitivity+assays.">Formation of stable small cell number three-dimensional ovarian cancer spheroids using hanging drop arrays for preclinical drug sensitivity assays.</a> Gynecologic Oncology. 138, 181-189 (2015).</li><li>Raghavan, 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=Comparative+analysis+of+tumor+spheroid+generation+techniques+for+differential+in+vitro+drug+toxicity.">Comparative analysis of tumor spheroid generation techniques for differential in vitro drug toxicity.</a> Oncotarget. 7, 16948-16961 (2016).</li><li>Kim, 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=Evaluating+Tumor+Evolution+via+Genomic+Profiling+of+Individual+Tumor+Spheroids+in+a+Malignant+Ascites.">Evaluating Tumor Evolution via Genomic Profiling of Individual Tumor Spheroids in a Malignant Ascites.</a> Scientific Reports. 8, 1-11 (2018).</li><li>Silva, I. 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=Aldehyde+dehydrogenase+in+combination+with+CD133+defines+angiogenic+ovarian+cancer+stem+cells+that+portend+poor+patient+survival.">Aldehyde dehydrogenase in combination with CD133 defines angiogenic ovarian cancer stem cells that portend poor patient survival.</a> Cancer Research. 71, 3991-4001 (2011).</li><li>Pulaski, H. L., 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=Identifying+alemtuzumab+as+an+anti-myeloid+cell+antiangiogenic+therapy+for+the+treatment+of+ovarian+cancer.">Identifying alemtuzumab as an anti-myeloid cell antiangiogenic therapy for the treatment of ovarian cancer.</a> Journal of Translational Medicine. 7, 49 (2009).</li><li>Jager, L. 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=Effect+of+enzymatic+and+mechanical+methods+of+dissociation+on+neural+progenitor+cells+derived+from+induced+pluripotent+stem+cells.">Effect of enzymatic and mechanical methods of dissociation on neural progenitor cells derived from induced pluripotent stem cells.</a> Advances in Medical Sciences. 61, 78-84 (2016).</li><li>Ivanov, D. P., Grabowska, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spheroid+arrays+for+high-throughput+single-cell+analysis+of+spatial+patterns+and+biomarker+expression+in+3D.">Spheroid arrays for high-throughput single-cell analysis of spatial patterns and biomarker expression in 3D.</a> Scientific Reports. 7, 41160 (2017).</li></ol>

用于 3D 球形形成的 384 孔挂落板平台是任何细胞生物学或癌症生物学实验室轻松实现的工具。该生理学平台支持研究细胞系,以及生理相关3D培养系内的初级患者样本,同时允许高通量药物筛选。该平台还确保培养条件高度可调,从而严格控制电镀密度、细胞共培养比、细胞外组件和介质组成。此外,该生理学平台使实验高度适应下游分析技术,需要大或小细胞计数,如qRT-PCR、FACS和各种成像方法。虽然易...

2018年全球癌症相关死亡人数达到980万,突显出需要开发改进的治疗方法。不幸的是,开发抗癌药物的成本正在增加,开发一种药物的成本约为6.5亿美元,这表明需要改进开发抗癌新药的战略。癌症干细胞(CSCs),其特点是增加化学抵抗力3,自我更新的能力,和播种新的肿瘤4的能力被认为是导致肿瘤复发4,转移5,和化学抗药性4,6,所有这些都有助于肿瘤的恶性能力,从而造成高死亡率。在卵巢癌中,这些细胞在腹腔的恶性腹水液中被发现富集,这种病症与不良的临床结果1相关。由于CSC的恶性能力,已经推动开发新的CSC靶向药物,用于与传统化疗。CSC靶向药物的开发面临若干挑战,包括:1) 在体外扩展和维护CSC的困难;2) 患者样本稀缺;3)文化平台的生理相关性;4)患者之间药物敏感性的异质性。该协议概述了高吞吐量 3D 文化平台的实现,该平台可以克服这些挑战。特别是,该系统允许使用少量患者衍生的卵巢CSC进行快速药物筛查,并且高度适用于下游分析技术。我们的平台是研究卵巢癌和CSC的理想选择,在复杂的3D环境中研究其他癌症和分化细胞类型也很有价值。
复杂的三维(3D)模型对于研究肿瘤微环境(TME)至关重要,肿瘤微环境是由癌细胞、非癌症支持细胞和细胞外基质(ECM)蛋白4组成的3D利基。与传统的体外2D细胞培养相比,这种3D环境具有独特的细胞形态、细胞和细胞-基质相互作用、细胞分化、细胞迁移、细胞密度和扩散梯度。所有这些因素最终导致3D培养物内药物反应的差异,表现出耐药性增加和生理相关性7,8 。由于3D TME在CSC分化和化学抗性中的作用,在生理微环境中筛选CSC靶向药物至关重要。提高CSC药物筛选平台的生理相关性,有可能改善患者特定药物筛选、药物开发、治疗策略的制定,并最终获得临床结果。同样重要的是,用于药物筛选的平台具有高通量,并与下游分析方法兼容,以尽量减少有前途的药物9的成本、时间和临床翻译时间。
目前,复杂的TME最好通过体内模型(如鼠群合并肿瘤模型、细胞系衍生异种移植和患者衍生异种移植(PDX)模型12进行药物筛选应用,因为它们提供生理条件。然而,这些模型的低通量性质,以及它们需要的成本、时间和技术技能集限制了它们在快速、高通量药物筛选应用中的效用13。作为这些体内模型的替代品,许多体外3D模型使用水凝胶8,微流体装置或"片上器官"设备10、14和非粘附培养物3,8也已经开发,因为他们在成本,时间和所需的技能集的低进入门槛。
水凝胶培养平台对基质组成、机械特性和基质结构的精细控制具有优势;然而,它们可以抑制高密度细胞培养14。此外,从水凝胶中采集细胞会使下游分析复杂化,因为收获方法15的潜在有害影响。另一方面,微流体设备是微尺度设备,可在同一设备内进行输出检测,并在生理相关尺度上进行细胞培养,同时减少试剂消耗、缩短反应时间、减少浪费,快速扩散14.这些特性使它们成为研究药物毒性、疗效和药代动力学的有希望的平台。然而,高效、可量化、可重复和用户友好的3D细胞培养以及笨重且昂贵的泵送系统的挑战限制了微流体在高通量研究中的应用。有效的检测设置和潜在的困难跨领域的实施也阻碍了微流体系统10的广泛采用。
相反,在旋转混合器(螺母)、超低附着板和悬滴的非附着条件下生成的球体不包括用户定义的矩阵组件。这些方法对于研究卵巢癌特别相关,因为非粘附条件代表球体在围肠腔5内生长的条件。在这些非粘附培养方法中,与超低附着板生成的球体相比,螺母和悬挂滴球体表现出更高的压实、重塑和化学抵抗力,这表明生理上增加相关性16,17,18,19。由于从较小的孔径和最少的所需细胞数量提高高通量筛查能力,悬挂滴板中的球形生成是药物筛选的理想平台。在这里,我们在384孔挂滴板中提供了一个可调的3D生理平台,易于实施,高度易于下游分析,使其成为卵巢癌和卵巢CSC高通量药物筛查的理想选择。
我们的 3D 生理平台提供 3D 培养的所有优势,包括生理细胞-细胞接触、扩散梯度、细胞密度和自然产生的 ECM 蛋白,这些蛋白质可能有助于实现现实的药物反应16 17,18,19.此外,通过使用患者衍生的CSC生成这些球体,我们能够同时确定患者对药物1的具体反应,同时进行许多技术复制,以克服患者肿瘤中可能存在的异质性样本20.此外,3D文化已被证明加强CSC种群的维持3,16,因此代表丰富的CSC种群在腹水7。这与简单的下游分析相结合,包括流动性分析的可行性和CSC比例允许对CSC靶向药物疗效进行最佳评估。最后,该生理平台在实验过程中与多个时间点的成像相容,细胞死亡和增殖评价,细胞组织和形态学与免疫组织化学,在条件性ELISA上的可溶性信号培养基、带流式细胞测定的细胞表型,以及PCR后的基因表达。

<table>
	<tbody>
		<tr>
			<td>0.25% trypsin-EDTA</td>
			<td>Gibco</td>
			<td>ILT25200056</td>
			<td></td>
		</tr>
		<tr>
			<td>10 mL serological pipet</td>
			<td>Fisher Scientific</td>
			<td>13-678-11E</td>
			<td></td>
		</tr>
		<tr>
			<td>10,000 cSt Si oil</td>
			<td>Millipore Sigma</td>
			<td>63148-62-9</td>
			<td>Used to coat spheroid array mold to facilitate removal of tissue processing gels, like Histogel, from the mold.</td>
		</tr>
		<tr>
			<td>100 mm tissue culture dish</td>
			<td>Thermo Scientific</td>
			<td>130182</td>
			<td></td>
		</tr>
		<tr>
			<td>15 ml conical tube</td>
			<td>Celltreat</td>
			<td>FL4021</td>
			<td></td>
		</tr>
		<tr>
			<td>1X DMEM for Serum Free Medium</td>
			<td>Gibco</td>
			<td>11965-092</td>
			<td></td>
		</tr>
		<tr>
			<td>1X F12 for Serum Free Medium</td>
			<td>Gibco</td>
			<td>11765-054</td>
			<td></td>
		</tr>
		<tr>
			<td>1X phosphate buffered saline (PBS)</td>
			<td>Gibco</td>
			<td>ILT10010023</td>
			<td></td>
		</tr>
		<tr>
			<td>4&#8217;,6-diamidino-2-phenylindole (DAPI)</td>
			<td>Thermo Fisher</td>
			<td>D1306</td>
			<td></td>
		</tr>
		<tr>
			<td>40 &#181;m filter</td>
			<td>Fisher Scientific</td>
			<td>22363547</td>
			<td></td>
		</tr>
		<tr>
			<td>6-well plate</td>
			<td>Fisher Scientific</td>
			<td>353046</td>
			<td></td>
		</tr>
		<tr>
			<td>Accutase</td>
			<td>Innovative Cell Technologies Inc.</td>
			<td>1449</td>
			<td>A gentle cell detachment enzyme composed of proteolytic and collagenolytic enzymes.</td>
		</tr>
		<tr>
			<td>ACK Lysing Buffer</td>
			<td>Thermo Scientific</td>
			<td>A1049201</td>
			<td></td>
		</tr>
		<tr>
			<td>alamarBlue</td>
			<td>Invitrogen</td>
			<td>DAL1025</td>
			<td>Resazurin dye used to measure viability and proliferation of cells based on their ability to reduce resazurin to resorufin, which is highly fluorescent.</td>
		</tr>
		<tr>
			<td>ALDEFLUOR assay kit</td>
			<td>Stem Cell Tech</td>
			<td>1700</td>
			<td>Kit to identify stem and progenitor cells that express high levels of aldehyde dehydrogenase , an indicator of cancer stem cells. The kit is composed of ALDEFLUOR Reagent, DEAB, Hydrochloric Acid, Dimethylsulphoxide, and ALDEFLUOR Assay Buffer.</td>
		</tr>
		<tr>
			<td>ALDEFLUOR Diethylaminobenzaldehyde (DEAB)</td>
			<td>Stem Cell Tech</td>
			<td>1705</td>
			<td>Diethylaminobenzaldehyde (DEAB) is an inhibitor of ALDH isozymes, used to determine non-specific ALDH staining.</td>
		</tr>
		<tr>
			<td>Andor iXon x3 CCD Camera</td>
			<td>Oxford Instruments</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibiotics and Antimycotics</td>
			<td>Gibco</td>
			<td>15240-062</td>
			<td></td>
		</tr>
		<tr>
			<td>APC-isotype IgG2b</td>
			<td>Miltenyi biotec</td>
			<td>130-092-217</td>
			<td>Isotype control to quantify non-specific staining of IgG2b antibodies.</td>
		</tr>
		<tr>
			<td>B27 Supplement</td>
			<td>Gibco</td>
			<td>17504044</td>
			<td></td>
		</tr>
		<tr>
			<td>basic Fibroblast Growth Factor</td>
			<td>Stem Cell Technologies</td>
			<td>78003.1</td>
			<td></td>
		</tr>
		<tr>
			<td>BD Lo-Dose U-100 Insulin Syringes</td>
			<td>Fisher Scientific</td>
			<td>14-826-79</td>
			<td></td>
		</tr>
		<tr>
			<td>BioTek Synergy HT Microplate Reader</td>
			<td>BioTek</td>
			<td>7091000</td>
			<td></td>
		</tr>
		<tr>
			<td>CD133-APC</td>
			<td>Miltenyi biotec</td>
			<td>130-113-184</td>
			<td>Fluorescent antibody targeting CD133, a cancer stem cell marker.</td>
		</tr>
		<tr>
			<td>cellSens Dimension Software</td>
			<td>Olympus</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cisplatin</td>
			<td>Sigma-Aldrich</td>
			<td>P4394</td>
			<td>Platinum based chemotherapy agent that functions as an alkylating agent that disrupts DNA.</td>
		</tr>
		<tr>
			<td>DAPI (4&#39;,6-Diamidino-2-Phenylindole, Dihydrochloride)</td>
			<td>Invitrogen</td>
			<td>D1306</td>
			<td></td>
		</tr>
		<tr>
			<td>Epidermal Growth Factor</td>
			<td>Gibco</td>
			<td>PHG0311</td>
			<td></td>
		</tr>
		<tr>
			<td>EVOS XL Core Cell Imaging System</td>
			<td>Life Technologies</td>
			<td>AME3300</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum - premium (FBS)</td>
			<td>Atlanta Biologicals</td>
			<td>S11150</td>
			<td></td>
		</tr>
		<tr>
			<td>Ficoll 400</td>
			<td>Sigma-Aldrich</td>
			<td>F4375</td>
			<td></td>
		</tr>
		<tr>
			<td>Hemacytometer</td>
			<td>Hausser Scientific</td>
			<td>1490</td>
			<td></td>
		</tr>
		<tr>
			<td>Histogel</td>
			<td>Thermo Scientific</td>
			<td>HG-4000-012</td>
			<td>Tissue processing gel that can penetrate and hold the specimen within the gel while preventing discoloration around the specimen upon staining.</td>
		</tr>
		<tr>
			<td>Human Adipose-Derived Mesenchymal Stem Cells</td>
			<td>Lonza</td>
			<td>PT-5006</td>
			<td></td>
		</tr>
		<tr>
			<td>Human Microvascular Endothelial Cells</td>
			<td>Lonza</td>
			<td>CC2543</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin-Transferrin-Selenium Supplement</td>
			<td>Gibco</td>
			<td>51500-056</td>
			<td></td>
		</tr>
		<tr>
			<td>Live/Dead viability kit</td>
			<td>Invitrogen</td>
			<td>L3224</td>
			<td>Kit for the fluorescence based detection of live (calcein-AM) and dead cells (Ethidium Homodimer-1).</td>
		</tr>
		<tr>
			<td>MEM Non-essential Amino Acids</td>
			<td>Gibco</td>
			<td>11140-050</td>
			<td></td>
		</tr>
		<tr>
			<td>MetaMorph 7.8 Software</td>
			<td>Molecular Devices</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Olympus IX81 Inverted Confocal Microscope</td>
			<td>Olympus</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Olympus IX83 Research Inverted Microscope</td>
			<td>Olympus</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Parafilm M</td>
			<td>Thomas Scientific</td>
			<td>7315D35</td>
			<td>Thermoplastic polymer strips that serve to limit droplet evaporation in hanging drop plates while still allowing for gas exchange.</td>
		</tr>
		<tr>
			<td>Perfecta 3D 384 Well Hanging Drop Plates</td>
			<td>3D Biomatrix</td>
			<td>HDP1384-8</td>
			<td>Available through Sigma-Aldrich</td>
		</tr>
		<tr>
			<td>phalloidin AlexaFluor488</td>
			<td>Invitrogen</td>
			<td>A12379</td>
			<td>Phalloidin is a peptide to fluorescently label F-actin in fixed cells.</td>
		</tr>
		<tr>
			<td>ProJet 3500 HD Max</td>
			<td>3D Systems</td>
			<td>-</td>
			<td>3D printer</td>
		</tr>
		<tr>
			<td>Sterile DI water</td>
			<td>Fisher Scientific</td>
			<td>353046</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan Blue</td>
			<td>Gibco</td>
			<td>15250061</td>
			<td>Azo dye used to differentiate between live and dead cells based on its ability to pass through the damaged membrane of dead cells, but not the intact membrane of live cells.</td>
		</tr>
		<tr>
			<td>VisiJet M3 Crystal</td>
			<td>3D Systems</td>
			<td>-</td>
			<td>A biocompatible polymer material for 3D printing.</td>
		</tr>
		<tr>
			<td>Yokogawa CSU-X1 Confocal Scanner Unit</td>
			<td>Yokogawa</td>
			<td>-</td>
			<td></td>
		</tr>
	</tbody>
</table>

physiologic patient derived 3d spheroids for anti-neoplastic drug screening to target cancer stem cells

在本协议中,我们概述了在384孔挂液滴内生成肿瘤球体的程序,以便在具有生理代表性的微环境中对抗癌疗法进行高通量筛查。我们概述了患者衍生的癌症干细胞球体的形成,以及这些球体的操作,以便在药物治疗后进行彻底分析。具体来说,我们描述了球体形态、增殖、活力、药物毒性、细胞表型和细胞定位数据的收集。该协议主要侧重于使用 384 well 悬垂平台轻松实现的分析技术,使其成为高通量药物筛选的理想选择。虽然我们强调该模型在卵巢癌研究和癌症干细胞研究中的重要性,但 384 well 平台适用于其他癌症类型和疾病模型的研究,将该平台的实用性扩展到许多领域。通过通过易于实施的具有生理代表性的3D培养技术,提高个性化药物筛选速度和筛查结果的质量,预计该平台将有助于开发新的治疗药物和患者特定疗法治疗策略,因此具有广泛的临床影响。

用细胞系或患者衍生的CSC形成的类球体可以在悬挂液滴内用一系列小细胞数形成(图2A)。球形能可靠地形成,每口井只有10个细胞,这样可以保存稀有的患者样本。这些球体内的细胞被其他细胞以3维包围,就像它们在体内一样,允许生理细胞-细胞接触和扩散率。球体内的肿瘤细胞增殖,导致球体随时间而扩大大小(图2B)。由于更具攻击性的患者细胞或细胞系比?...

Watch this Scientific Journal Video about Physiologic Patient Derived 3D Spheroids for Anti-neoplastic Drug Screening to Target Cancer Stem Cells at JoVE.com

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

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

该协议描述了患者衍生球体的生成,以及下游分析,包括增殖定量、细胞毒性测试、流式细胞测定、免疫荧光染色和共聚焦成像,以便评估药物候选人作为抗肿瘤治疗的潜力。该协议支持精密医学,用于识别每个患者和疾病阶段的特定药物。

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

我们的协议为3D细胞培养提供了一种简单的方法，并且非常适合下游分析。这有助于评估异构患者样本及其特定的药物反应。该技术的优点是，球体可以从小细胞数中形成，既可保存稀缺的原样品，又可对患者特异性药物反应进行高通量筛选。悬滴模型创造了一个三D药物扩散的生理环境，并做与肿瘤阶段相对应的反应。这允许开发有针对性的疗法和患者特定的治疗。该方法是研究卵巢癌、异质性和化疗抗性发展的理想之选。然而，它也可以用来研究其他癌症和抗药细胞种群。电镀球体时，移液器精确体积非常重要。这确保了球体之间的一致性。球体及其各自的液滴本质上是脆弱的，在没有正确处理的情况下很容易丢失，因此我们将展示如何适当地对悬挂落板进行板式、维护和分析。演示这个程序将是迈克尔·布雷根泽，一个研究生从我们的实验室。首先，用四到五毫升的解洗脱压水填充六井板的每个井，将悬挂的落盘夹在盖子和板底之间。在悬挂落盘边缘周围加入800至1000微升水，以提供潮湿的环境并最大限度地减少蒸发。接下来，收集2D生长细胞，用三足动物分离它们，然后加入六或八毫升含有FBS的介质。用10毫升血清移液器吸气细胞，并将它们沉积在15毫升锥形管中。使用血细胞计计算细胞数。根据手稿指示准备细胞悬浮液，并轻轻地与移液器混合，以确保均匀分布。将移液器的尖端以 45 度的角度放入井中，将移液器 20 微升悬浮液放入每个悬挂落井中。将六井板的盖子放回去，并使用弹性热塑性带密封边缘。在标准的二氧化碳加湿培养箱中孵育，每两到三天给挂滴喂食一次，在每个含球类的井中加入两到三个微升的细胞培养基。为了量化细胞的增殖和生存能力，在指定用于增殖分析和根据手稿方向孵育的井中加入两个微升的过滤雷萨祖林溶液。孵育期后，打开生物安全柜中的挂滴三明治，将盖有盖的384井板固定到板读卡器上。将它放在板读卡器中并读取板。在弹出窗口中保存实验，将数据导出到电子表格中。将板放回六井底座，并放在培养箱中。一旦读取了当天的所有时间点，在孵化前重新密封板。要准备用于流式细胞分析的球体，请使用 1000 微升移液器从每孔收集它们，并将它们存入 15 毫升锥形管中进行分解。等离子细胞悬浮成5个微离心管，使每个管至少含有5万个细胞。在微离心中以 400 次 G 离心管，5 分钟，然后在 100 微升的 Aldefluor 缓冲液中吸升液和重新悬浮颗粒。根据手稿指示对管子进行标记。在 APC ISO 管中加入 0.5 微升 APC 等型抗体，在 ALDH CD133 管中加入 1 微升 CD133 抗体。然后将五微升的DEAB试剂和0.5微升的ALDH添加到DEAB管中，在ALDH CD133管中加入1微升ALDH。漩涡所有管几秒钟，并在37摄氏度孵育45分钟。孵育后，再次涡流所有管，并在400次G下离心5分钟。根据手稿方向标记 FACS 管，并用冰填充绝缘泡沫容器。从微离心管中吸出上流液。然后重新挂起 400 微升 FACS 缓冲液中的未污染控制，并在 400 微升 FAPI 缓冲液中重新挂起其余单元。将管子放在冰上，直到它们可以在流动的圆度仪上进行分析。使用 FlowJo 分析流式细胞仪中的数据。双击未污染的文件，将 Y 轴设置为侧散点高度或 SSCH，将 X 轴设置为向前散射高度或 FSCH。单击每个轴旁边的 T 按钮以调整比例并最大化不同细胞群之间的分离。接下来，单击多边形浇注按钮，在单元格总体周围绘制多边形门并标记总体单元格。双击工作区中的单元格总体，然后将 FSC 轴更改为 FSC 宽度，将 SSC 轴更改为 FSC 高度。选择矩形门，仅围绕最左侧密集的单元格群体周围绘制一个矩形，跨越整个 Y 轴。标记此门单单元。然后右键单击并复制嵌套的单单元格门中的此单元格，并将其粘贴到工作区的每个样本下。双击嵌套在 DAPI 样本下的单细胞门，以查看该样本管中的单个细胞总体。将 FSC 轴通道更改为 DAPI 区域通道，然后将 SSC 轴通道更改为直方图。单击 DAPI 轴旁边的 T 按钮，然后单击自定义轴以调整比例并最大化 DAPI 正峰和 DAPI 负峰之间的分离。单击弹出窗口中的"应用"并选择"范围门"按钮。将它铺在 DAPI 负峰上，并为此门实时单元格添加标签。复制活细胞门并将其粘贴在单细胞门下，在 APC ISO DEAB 和 ALDH CD133 管下，选择每个管中相同部分的活细胞。然后双击嵌套在 APC ISO 样本文件下的实时单元格总体，然后将 X 轴切换到 ALDH 区域，将 Y 轴切换到 APC 区域。应用象限门并调整栅极的交点，例如大约 0.5% 的人口位于绘图窗口的左上角。然后根据需要命名象限，然后复制并粘贴象限门到嵌套在 DEAB 文件下的活细胞群，并调整垂直线，使大约 0.15% 的单元格群位于 ALDH 正象限内。最后，复制并粘贴象限门到ALDH CD133文件活细胞种群和评估癌症干细胞比例的基础上ALDH和CD133比例。该协议可用于患者衍生的癌症干细胞的高通量分析。每井只有10个细胞可以形成可靠的球体，随着细胞的增殖，球体的大小会扩大。增殖能力可以很容易地量化与雷萨祖林为基础的荧光测定。药物对球状形态的影响可以通过相位对比成像来可视化，并通过比较未经治疗和治疗的细胞中的雷萨祖林荧光来量化。细胞死亡可以通过添加钙素-AM和同性I来验证球体，随后在共声显微镜上成像。最后，球体可以收获并分散到单个细胞悬浮液中，用于流式细胞学分析，从而能够辨别不同药物治疗对特定细胞群的有效性。为了避免液滴丢失、中等蒸发和球状尺寸的变化，重要的是小心处理您的板，精确移液器，并完全密封悬挂滴板。为了评估药物治疗导致的基因表达和可溶性信号的变化，单细胞RNA测序和酶相关免疫吸附测定，可用于促进治疗的发展。该技术允许肿瘤学、精密医学和药物开发领域的研究，通过小活检的高通量药物筛选，探索患者内部和患者之间的异质性以及化疗耐药性。执行此协议时，请务必佩戴所有标准个人防护设备。包括手套、安全眼镜、实验室外套、裤子和近趾鞋。

Research

JoVE Journal

Cancer Research

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

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

Modeling Osteosarcoma Using Li-Fraumeni Syndrome Patient-derived Induced Pluripotent Stem Cells

应用 Fraumeni 综合征诱导多潜能干细胞建模骨肉瘤

Li-Fraumeni syndrome (LFS) is an autosomal dominant hereditary cancer disorder. Patients with LFS are predisposed to a various type of tumors, including ...

Drug Screening of Primary Patient Derived Tumor Xenografts in Zebrafish

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 ...

Patient-Derived Tumor Explants As a "Live" Preclinical Platform for Predicting Drug Resistance in Patients

患者源性肿瘤外植体作为预测患者耐药性的"实时"临床前平台

An understanding of drug resistance and the development of novel strategies to sensitize highly resistant cancers rely on the availability of suitable ...

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

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 ...

Exploring Mitochondrial Energy Metabolism of Single 3D Microtissue Spheroids Using Extracellular Flux Analysis

Three-dimensional (3D) cellular aggregates, termed spheroids, have become the forefront of in vitro cell culture in recent years. In contrast to culturing ...

Pooled shRNA Library Screening to Identify Factors that Modulate a Drug Resistance Phenotype

合并的shRNA文库筛选，以确定调节耐药性表型的因素

Understanding clinically relevant driver mechanisms of acquired chemo-resistance is crucial for elucidating ways to circumvent resistance and improve ...

Establishing 3-Dimensional Spheroids from Patient-Derived Tumor Samples and Evaluating their Sensitivity to Drugs

从患者来源的肿瘤样本中建立三维微球并评估其对药物的敏感性

Despite remarkable advances in understanding tumor biology, the vast majority of oncology drug candidates entering clinical trials fail, often due to a ...

Orthotopic Implantation of Patient-Derived Cancer Cells in Mice Recapitulates Advanced Colorectal Cancer

小鼠患者来源的癌细胞原位植入概括了晚期结直肠癌

Over the last decade, more sophisticated preclinical colorectal cancer (CRC) models have been established using patient-derived cancer cells and 3D ...

Generation of 3D Tumor Spheroids for Drug Evaluation Studies

生成用于药物评估研究的 3D 肿瘤微球

In recent decades, in addition to monolayer-cultured cells, three-dimensional tumor spheroids have been developed as a potentially powerful tool for the ...