用水凝胶微室阵列 (HMCA) 板进行肿瘤细胞侵袭和抗转移药物筛选的分析
Summary
针对入侵检测的性能, 提出了一种基于 HMCA 的成像板。该板块有助于形成三维 (3D) 肿瘤旋转椭球体和测量癌细胞侵入细胞外基质 (ECM)。通过半自动分析实现了入侵检测的定量化。
Abstract
癌症转移是已知的导致90% 的癌症致死率。转移是一个多级过程, 启动与渗透/侵入肿瘤细胞到邻近组织。因此, 入侵是转移的关键一步, 使入侵过程的研究和开发的抗转移药物, 具有非常重要的意义。为了满足这一需求, 需要开发 3D的体外模型, 以模拟实体肿瘤及其微环境的结构, 一方面最接近体内状态, 但同时具有可重现性、鲁棒性和适用于高产量和高含量测量。目前, 大多数入侵检测都依赖于成熟的微流控技术, 它们足够用于研究, 但不能用于高通量药物筛选。在每个井中使用具有独立单独旋转椭球体的板型设备的其他检测是材料消耗, 每个条件的样品尺寸都很低。目前的协议的目标是提供一个简单的和可重现的仿生3D 细胞系统, 以分析大种群肿瘤旋转椭球体的侵袭能力。我们开发了一种基于 HMCA 成像板的3D 入侵检测模型, 用于肿瘤侵袭和抗转移药物发现的研究。该器件可生产多种均匀的旋转椭球体 (每个条件下的高样本大小), 同时在单元素分辨率下连续、同时观察和测量旋转椭球体的介质抗转移药物的高通量筛选。这个平台是在这里提供的 HeLa 和 MCF7 旋转椭球体的例证单细胞和集体入侵的生产。比较了 ECM 成分透明质酸 (HA) 对旋转椭球体周围胶原的侵袭能力的影响。最后, 我们引入漆黄素 (入侵抑制剂) 对 HeLa 旋转椭球体和一氧化氮 (NO) (入侵激活剂) MCF7 旋转椭球体。通过内部软件对结果进行分析, 实现半自动、简单、快速的分析, 便于多参数检测。
Introduction
癌症死亡主要归因于转移细胞传播到遥远的地方。许多癌症治疗的努力重点是瞄准或预防转移性菌落的形成和系统性转移性疾病1的进展。癌细胞迁移是肿瘤转移过程中的重要一步, 因此, 对肿瘤侵袭性叶栅的研究是非常重要的, 也是寻找抗转移治疗的前提条件。
动物模型作为研究转移性疾病的工具被发现是非常昂贵的, 并不总是代表在人类的肿瘤。此外, 细胞外微环境的拓扑结构、力学和成分对癌细胞的行为有强烈的影响2。由于体内模型本身缺乏分离和控制导致癌症侵袭和转移的特定参数的能力, 因此需要对体外模型进行可控仿生。
为了转移到遥远的器官, 癌细胞必须表现出迁徙和侵入性表型特征, 这些性状可以被治疗。然而, 由于大多数体外肿瘤模型不模仿实体肿瘤3的实际特征, 因此检测生理相关表型非常具有挑战性。此外, 在肿瘤内存在的表型异质性, 决定了在单元素分辨率下分析肿瘤迁移的必要性, 以发现表现性定向疗法, 例如, 通过靶向转移起始细胞异质肿瘤中的人口4。
细胞运动和集体迁移的研究主要在同质平面表面上皮细胞的单层培养中进行。这些用于癌症细胞迁移的常规细胞模型是基于通过细胞膜和 ECM 组件5侵入的单个细胞的种群分析, 但在这种系统中, 单个细胞之间的内在差异不能研究。通过支架或无支架微结构生成3D 旋转椭球体被认为是研究肿瘤细胞生长和肿瘤侵袭6、7、8的一种优越手段。但是, 大多数3D 系统不适合高吞吐量格式, 并且在每个微井9中生成独立的单个旋转椭球体时, 通常无法实现椭球体之间的交互。最近涉及癌症迁移的研究基于微流控设备3,10,11,12, 然而, 这些复杂的微流控工具很难生产, 不能用于抗侵入性药物的高通量筛选 (HTS)。
两种主要表型, 集体和个体细胞迁移, 在肿瘤细胞中发挥作用, 克服 ECM 屏障和入侵邻近组织, 已证明13,14, 每个显示不同的形态学特征、生化、分子和遗传机制。此外, 在每个表型中观察到两种形式的迁移肿瘤细胞, 成纤维细胞样和变形虫。由于入侵表型和迁移模式, 主要是由形态学属性定义的, 因此需要对细胞模型进行基于成像的检测和检查所有形式的肿瘤侵袭和迁移细胞。
目前的方法的总体目标是提供一个简单和可重现的仿生 3D体外细胞系统, 以分析肿瘤旋转椭球体大种群的侵袭能力。在这里, 我们介绍了基于 HMCA 的6孔成像板用于肿瘤侵袭和抗转移治疗的研究。该技术能够在水凝胶微室 (MC) 结构中形成大量均匀的3D 肿瘤旋转椭球体 (450)。将各种 ECM 组件添加到椭球体阵列中, 以使细胞侵入周围环境。通过对同一个体旋转椭球体/入侵细胞的短期和长期观察, 对入侵过程进行持续监测, 并在任何时候促进形态学表征、荧光染色和特定旋转椭球体的检索。由于许多旋转椭球体共享空间和媒介, 通过个体旋转椭球体之间的可溶性分子相互作用及其对彼此的影响是可能的。使用内部 MATLAB 代码进行半自动图像分析, 从而能够更快、更高效地收集大量数据。该平台能够以高效的方式对众多旋转椭球体/入侵细胞进行准确、同时的测量, 从而实现对抗入侵药物的中等吞吐量筛选。
Protocol
1. HMCA 板压花 注: 本协议中使用的硅油 (PDMS) 图章和 HMCA 成像板的设计和制造的完整过程在我们前面的文章15,16中详细介绍。PDMS 图章 (负形状) 用于浮雕 HMCA (正形), 由大约450个 MCs (图 1A) 组成。如图 1B所示, 每个 MCs 都有一个被截断的倒置方形金字塔的形状 (高度: 190 µm, 小底座面积:90 µm x 90 …
Representative Results
独特的 HMCA 成像板用于3D 肿瘤旋转椭球体的侵袭性测定。整个测试, 开始与椭球体形成和结束与入侵过程和额外的操作, 在同一板块内执行。对于椭球体的形成, HeLa 细胞被加载到阵列盆地中, 并通过重力沉降到水凝胶 MCs 中。水凝胶 MCs 具有非附着性/低附着特性, 促进细胞细胞相互作用和3D 肿瘤旋转椭球体的形成。图 2A说明了在 MCs 中解决的单元格, 使?…
Discussion
据记载, 活生物体的特点是其复杂的3D 多细胞组织与常用的2D 单层培养细胞截然不同, 强调使用细胞模型更好地模仿功能的关键需要和生物药物筛选的过程。最近, 多细胞旋转椭球体、器官型文化、organoids 和片上器官已被引入8用于标准化药物发现。然而, 3D 多细胞模型的复杂性极大地危及了其鲁棒性、并行性和数据分析, 这对检测效率至关重要。
入侵能力的?…
Divulgations
The authors have nothing to disclose.
Acknowledgements
这项工作得到了佩雷斯和朱蒂丝 Weisbrodt 的遗赠的支持。
Materials
| 6 Micro-well Glass Bottom Plates with 14 mm micro-well #1.5 cover glass | Cellvis | P06-14-1.5-N | Commercial glass bottom plates which are used for HMCA embossing |
| UltraPure Low Melting Point Agarose | Invitrogen | 16520100 | A solution of 6% agarose is warmed up to 80°C before use, a solution of 1% agarose is warmed to 37°C |
| Trypsin EDTA solution B | Biological Industries | 03-052-1A | Warmed to 37°C before use |
| DMEM medium, high glucose | Biological Industries | 01-055-1A | Warmed to 37°C before use |
| Special Newborn Calf Serum (NBCS) | Biological Industries | 04-122-1A | Heat Inactivated |
| DPBS (10X), no calcium, no magnesium | Biological Industries | 02-023-5A | Kept on ice before use |
| NaOH, anhydrous | Sigma-Aldrich | S5881-500G | Used for the preparation of 1M NaOH solution |
| Cultrex Type I collagen from rat tail, 5mg/ml | Trevigen | 3440-100-01 | Kept on ice before use |
| Hyaluronic acid sodium salt | Sigma-Aldrich | H5542-10MG | Kept on ice before use |
| Fisetin | Sigma-Aldrich | F505-100MG | Added to the culture medium, invasion inhibitor |
| DETA/NO | Enzo Life Sciences | alx-430-014-m005 | Added to the culture medium, nitric oxide donor |
| PI | Sigma-Aldrich | P4170 | Used at very low concenrtation without the need for washing |
| Dymax 5000-EC UV flood lamp complete system with light shield & Dymax 400 Watt EC power supply | Dymax Corporation | PN 39823 | Used for HMCA plate sterilization by UV |
| Inverted IX81 microscope | Olympus | Used for automatic image acquisition | |
| Incubator for microscope | Life Imaging Services | Essential for time lapse experiments with image acquisition, pre adjusted to 37°C, 5% CO2 and keeping a humidified atmosphere | |
| Sub-micron motorized stage type SCAN-IM | Marzhauser Wetzlar GmbH | Used to predetermine image acquisition areas, for automatic image acquisition | |
| 14-bit, ORCA II C4742-98 cooled camera | Hamamatsu Photonics | Highly sensitive, used for imaging | |
| Fluorescent filter cube for PI detection | Chroma Technology Corporation | Filter cube specifications: excitation filter 530-550 nm, dichroic mirror 565 nm long pass and emission filter 600-660 nm | |
| The Olympus Cell^P operating software | Olympus | Software used to control microscope, motorized stage, camera and image acquisition | |
| Matlab R2014B analysis software | Mathworks | Used to develop in house graphic user interface for image analysis | |
| Excel software | Microsoft | Used for data management, calculation, plot creation and statistics |
References
- Guan, X. Cancer metastases: challenges and opportunities. Acta pharmaceutica Sinica. B. 5 (5), 402-418 (2015).
- Sapudom, J., et al. The phenotype of cancer cell invasion controlled by fibril diameter and pore size of 3D collagen networks. Biomaterials. 52, 367-375 (2015).
- Portillo-Lara, R., Annabi, N. Microengineered cancer-on-a-chip platforms to study the metastatic microenvironment. Lab on a chip. 16 (21), 4063-4081 (2016).
- Gkountela, S., Aceto, N. Stem-like features of cancer cells on their way to metastasis. Biology Direct. 11 (1), 33 (2016).
- Kramer, N., et al. In vitro cell migration and invasion assays. Mutation Research/Reviews in Mutation Research. 752 (1), 10-24 (2013).
- Guzman, A., Sánchez Alemany, V., Nguyen, Y., Zhang, C. R., Kaufman, L. J. A novel 3D in vitro metastasis model elucidates differential invasive strategies during and after breaching basement membrane. Biomaterials. 115, 19-29 (2017).
- Lee, E., Song, H. -. H. G., Chen, C. S. Biomimetic on-a-chip platforms for studying cancer metastasis. Current opinion in chemical engineering. 11, 20-27 (2016).
- Mittler, F., Obeïd, P., Rulina, A. V., Haguet, V., Gidrol, X., Balakirev, M. Y. High-Content Monitoring of Drug Effects in a 3D Spheroid Model. Frontiers in Oncology. 7, 293 (2017).
- Evensen, N. A., et al. Development of a High-Throughput Three-Dimensional Invasion Assay for Anti-Cancer Drug Discovery. PLoS ONE. 8 (12), e82811 (2013).
- Aw Yong, K. M., Li, Z., Merajver, S. D., Fu, J. Tracking the tumor invasion front using long-term fluidic tumoroid culture. Scientific Reports. 7 (1), 10784 (2017).
- Mi, S., et al. Microfluidic co-culture system for cancer migratory analysis and anti-metastatic drugs screening. Scientific Reports. 6 (1), 35544 (2016).
- Chung, S., Sudo, R., Mack, P. J., Wan, C. -. R., Vickerman, V., Kamm, R. D. Cell migration into scaffolds under co-culture conditions in a microfluidic platform. Lab on a chip. 9 (2), 269-275 (2009).
- Krakhmal, N. V., Zavyalova, M. V., Denisov, E. V., Vtorushin, S. V., Perelmuter, V. M. Cancer Invasion: Patterns and Mechanisms. Acta naturae. 7 (2), 17-28 (2015).
- Lintz, M., Muñoz, A., Reinhart-King, C. A. The Mechanics of Single Cell and Collective Migration of Tumor Cells. Journal of Biomechanical Engineering. 139 (2), 21005 (2017).
- Afrimzon, E., et al. Hydrogel microstructure live-cell array for multiplexed analyses of cancer stem cells, tumor heterogeneity and differential drug response at single-element resolution. Lab on a Chip. 16 (6), 1047-1062 (2016).
- Shafran, Y., et al. Nitric oxide is cytoprotective to breast cancer spheroids vulnerable to estrogen-induced apoptosis. Oncotarget. 8 (65), 108890-108911 (2017).
- Sato, H., Idiris, A., Miwa, T., Kumagai, H. Microfabric Vessels for Embryoid Body Formation and Rapid Differentiation of Pluripotent Stem Cells. Scientific Reports. 6 (1), 31063 (2016).
- Lee, K., et al. Gravity-oriented microfluidic device for uniform and massive cell spheroid formation. Biomicrofluidics. 6 (1), 14114 (2012).
- Zaretsky, I., et al. Monitoring the dynamics of primary T cell activation and differentiation using long term live cell imaging in microwell arrays. Lab on a Chip. 12 (23), 5007 (2012).
- Khan, N., Syed, D. N., Ahmad, N., Mukhtar, H. Fisetin: a dietary antioxidant for health promotion. Antioxidants & redox signaling. 19 (2), 151-162 (2013).
- Lee, G. H., et al. Networked concave microwell arrays for constructing 3D cell spheroids. Biofabrication. 10 (1), 15001 (2017).
- Vinci, M., Box, C., Eccles, S. A. Three-dimensional (3D) tumor spheroid invasion assay. Journal of visualized experiments: JoVE. (99), e52686 (2015).
- Toh, Y. -. C., Raja, A., Yu, H., van Noort, D. A 3D Microfluidic Model to Recapitulate Cancer Cell Migration and Invasion. Bio-ingénierie. 5 (2), 29 (2018).
- Sugimoto, M., Kitagawa, Y., Yamada, M., Yajima, Y., Utoh, R., Seki, M. Micropassage-embedding composite hydrogel fibers enable quantitative evaluation of cancer cell invasion under 3D coculture conditions. Lab on a Chip. 18 (9), 1378-1387 (2018).
- Yamamoto, S., Hotta, M. M., Okochi, M., Honda, H. Effect of Vascular Formed Endothelial Cell Network on the Invasive Capacity of Melanoma Using the In Vitro 3D Co-Culture Patterning Model. PLoS ONE. 9 (7), e103502 (2014).
- Lee, S. -. H., Moon, J. J., West, J. L. Three-dimensional micropatterning of bioactive hydrogels via two-photon laser scanning photolithography for guided 3D cell migration. Biomaterials. 29 (20), 2962-2968 (2008).
- Gschwind, A., Zwick, E., Prenzel, N., Leserer, M., Ullrich, A. Cell communication networks: epidermal growth factor receptor transactivation as the paradigm for interreceptor signal transmission. Oncogene. 20 (13), 1594-1600 (2001).
- Jiang, K., Dong, C., Xu, Y., Wang, L. Microfluidic-based biomimetic models for life science research. RSC Advances. 6 (32), 26863-26873 (2016).
- Mason, B. N., Starchenko, A., Williams, R. M., Bonassar, L. J., Reinhart-King, C. A. Tuning three-dimensional collagen matrix stiffness independently of collagen concentration modulates endothelial cell behavior. Acta biomaterialia. 9 (1), 4635-4644 (2013).
- Raub, C. B., Putnam, A. J., Tromberg, B. J., George, S. C. Predicting bulk mechanical properties of cellularized collagen gels using multiphoton microscopy. Acta Biomaterialia. 6 (12), 4657-4665 (2010).
- Paszek, M. J., et al. Tensional homeostasis and the malignant phenotype. Cancer Cell. 8 (3), 241-254 (2005).
- Rao, S. S., DeJesus, J., Short, A. R., Otero, J. J., Sarkar, A., Winter, J. O. Glioblastoma Behaviors in Three-Dimensional Collagen-Hyaluronan Composite Hydrogels. ACS Applied Materials & Interfaces. 5 (19), 9276-9284 (2013).
- Kreger, S. T., Voytik-Harbin, S. L. Hyaluronan concentration within a 3D collagen matrix modulates matrix viscoelasticity, but not fibroblast response. Matrix Biology. 28 (6), 336-346 (2009).
- Chanmee, T., Ontong, P., Itano, N. Hyaluronan: A modulator of the tumor microenvironment. Cancer Letters. 375 (1), 20-30 (2016).
- Zhao, Y., et al. Modulating Three-Dimensional Microenvironment with Hyaluronan of Different Molecular Weights Alters Breast Cancer Cell Invasion Behavior. ACS Applied Materials & Interfaces. 9 (11), 9327-9338 (2017).
- Wu, M., et al. A novel role of low molecular weight hyaluronan in breast cancer metastasis. The FASEB Journal. 29 (4), 1290-1298 (2015).
- Fisher, G. J. Cancer resistance, high molecular weight hyaluronic acid, and longevity. Journal of cell communication and signaling. 9 (1), 91-92 (2015).
Citer Cet Article
Ravid-Hermesh, O., Zurgil, N., Shafran, Y., Afrimzon, E., Sobolev, M., Hakuk, Y., Bar-On Eizig, Z., Deutsch, M. Analysis of Cancer Cell Invasion and Anti-metastatic Drug Screening Using Hydrogel Micro-chamber Array (HMCA)-based Plates. J. Vis. Exp. (140), e58359, doi:10.3791/58359 (2018).