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
Summary
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。
Abstract
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。
Introduction
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。
Protocol
Representative Results
Discussion
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…
Disclosures
The authors have nothing to disclose.
Acknowledgements
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。
Materials
| 0.25% trypsin-EDTA | Gibco | ILT25200056 | |
| 10 mL serological pipet | Fisher Scientific | 13-678-11E | |
| 10,000 cSt Si oil | Millipore Sigma | 63148-62-9 | Used to coat spheroid array mold to facilitate removal of tissue processing gels, like Histogel, from the mold. |
| 100 mm tissue culture dish | Thermo Scientific | 130182 | |
| 15 ml conical tube | Celltreat | FL4021 | |
| 1X DMEM for Serum Free Medium | Gibco | 11965-092 | |
| 1X F12 for Serum Free Medium | Gibco | 11765-054 | |
| 1X phosphate buffered saline (PBS) | Gibco | ILT10010023 | |
| 4’,6-diamidino-2-phenylindole (DAPI) | Thermo Fisher | D1306 | |
| 40 µm filter | Fisher Scientific | 22363547 | |
| 6-well plate | Fisher Scientific | 353046 | |
| Accutase | Innovative Cell Technologies Inc. | 1449 | A gentle cell detachment enzyme composed of proteolytic and collagenolytic enzymes. |
| ACK Lysing Buffer | Thermo Scientific | A1049201 | |
| alamarBlue | Invitrogen | DAL1025 | Resazurin dye used to measure viability and proliferation of cells based on their ability to reduce resazurin to resorufin, which is highly fluorescent. |
| ALDEFLUOR assay kit | Stem Cell Tech | 1700 | 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. |
| ALDEFLUOR Diethylaminobenzaldehyde (DEAB) | Stem Cell Tech | 1705 | Diethylaminobenzaldehyde (DEAB) is an inhibitor of ALDH isozymes, used to determine non-specific ALDH staining. |
| Andor iXon x3 CCD Camera | Oxford Instruments | – | |
| Antibiotics and Antimycotics | Gibco | 15240-062 | |
| APC-isotype IgG2b | Miltenyi biotec | 130-092-217 | Isotype control to quantify non-specific staining of IgG2b antibodies. |
| B27 Supplement | Gibco | 17504044 | |
| basic Fibroblast Growth Factor | Stem Cell Technologies | 78003.1 | |
| BD Lo-Dose U-100 Insulin Syringes | Fisher Scientific | 14-826-79 | |
| BioTek Synergy HT Microplate Reader | BioTek | 7091000 | |
| CD133-APC | Miltenyi biotec | 130-113-184 | Fluorescent antibody targeting CD133, a cancer stem cell marker. |
| cellSens Dimension Software | Olympus | ||
| Cisplatin | Sigma-Aldrich | P4394 | Platinum based chemotherapy agent that functions as an alkylating agent that disrupts DNA. |
| DAPI (4',6-Diamidino-2-Phenylindole, Dihydrochloride) | Invitrogen | D1306 | |
| Epidermal Growth Factor | Gibco | PHG0311 | |
| EVOS XL Core Cell Imaging System | Life Technologies | AME3300 | |
| Fetal Bovine Serum – premium (FBS) | Atlanta Biologicals | S11150 | |
| Ficoll 400 | Sigma-Aldrich | F4375 | |
| Hemacytometer | Hausser Scientific | 1490 | |
| Histogel | Thermo Scientific | HG-4000-012 | Tissue processing gel that can penetrate and hold the specimen within the gel while preventing discoloration around the specimen upon staining. |
| Human Adipose-Derived Mesenchymal Stem Cells | Lonza | PT-5006 | |
| Human Microvascular Endothelial Cells | Lonza | CC2543 | |
| Insulin-Transferrin-Selenium Supplement | Gibco | 51500-056 | |
| Live/Dead viability kit | Invitrogen | L3224 | Kit for the fluorescence based detection of live (calcein-AM) and dead cells (Ethidium Homodimer-1). |
| MEM Non-essential Amino Acids | Gibco | 11140-050 | |
| MetaMorph 7.8 Software | Molecular Devices | – | |
| Olympus IX81 Inverted Confocal Microscope | Olympus | – | |
| Olympus IX83 Research Inverted Microscope | Olympus | ||
| Parafilm M | Thomas Scientific | 7315D35 | Thermoplastic polymer strips that serve to limit droplet evaporation in hanging drop plates while still allowing for gas exchange. |
| Perfecta 3D 384 Well Hanging Drop Plates | 3D Biomatrix | HDP1384-8 | Available through Sigma-Aldrich |
| phalloidin AlexaFluor488 | Invitrogen | A12379 | Phalloidin is a peptide to fluorescently label F-actin in fixed cells. |
| ProJet 3500 HD Max | 3D Systems | – | 3D printer |
| Sterile DI water | Fisher Scientific | 353046 | |
| Trypan Blue | Gibco | 15250061 | 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. |
| VisiJet M3 Crystal | 3D Systems | – | A biocompatible polymer material for 3D printing. |
| Yokogawa CSU-X1 Confocal Scanner Unit | Yokogawa | – |
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