单元位置的Imaging-和流式细胞仪为基础的分析和3D球体黑色素瘤细胞周期
Summary
We describe two complementary methods using the fluorescence ubiquitination cell cycle indicator (FUCCI) and image analysis or flow cytometry to identify and isolate cells in the inner G1 arrested and outer proliferating regions of 3D spheroids.
Abstract
Three-dimensional (3D) tumor spheroids are utilized in cancer research as a more accurate model of the in vivo tumor microenvironment, compared to traditional two-dimensional (2D) cell culture. The spheroid model is able to mimic the effects of cell-cell interaction, hypoxia and nutrient deprivation, and drug penetration. One characteristic of this model is the development of a necrotic core, surrounded by a ring of G1 arrested cells, with proliferating cells on the outer layers of the spheroid. Of interest in the cancer field is how different regions of the spheroid respond to drug therapies as well as genetic or environmental manipulation. We describe here the use of the fluorescence ubiquitination cell cycle indicator (FUCCI) system along with cytometry and image analysis using commercial software to characterize the cell cycle status of cells with respect to their position inside melanoma spheroids. These methods may be used to track changes in cell cycle status, gene/protein expression or cell viability in different sub-regions of tumor spheroids over time and under different conditions.
Introduction
多细胞三维球状体已被被称为肿瘤模型数十年，但它只是在最近，他们已进入更常见的用法作为体外模型对许多实体癌症。它们越来越多地被用于高通量药物发现屏幕作为中间之间的复杂，昂贵和费时的体内模型和简单的，低成本的2D单层模型1-6。研究在2D培养往往无法在体内被复制。许多类型的癌症的球体模型是能够模仿的生长特性，药物敏感性，药物渗透，细胞-细胞相互作用，氧和营养物和坏死的发展是见于体内实体肿瘤6-11的有限的可及。球状体开发出坏死核心，包围芯静止或G 1被捕区域，并在球体7的周边增殖细胞。这些区域的发展可根据细胞密度，增殖率和球体12的尺寸而变化。据推测，见于这些不同子区的细胞异质性可能会导致癌症治疗电阻13,14。因此，分析细胞在这些区域中的能力分别是至关重要的理解肿瘤药物反应。
荧光泛素细胞周期指示符（FUCCI）系统是基于红色（Kusabira橙色-正）和绿色（Azami格林- AG）CDT1和geminin的荧光标记，这是降解的细胞周期15的不同阶段。因此细胞核出现红色于G1，黄S中的早期和绿色中的S / G2 / M期。我们在这里描述两个互补方法均使用FUCCI来识别细胞周期，随着使用成像软件或染料扩散流式细胞仪测定的，以确定细胞是否驻留在G1被捕中心或外细胞增生克环，和个体细胞从旋转椭圆体的边缘的距离。这些方法被开发在我们以前的出版物，在这里我们表明，黑色素瘤细胞在低氧区域中的旋转椭圆体的中心和/或在靶向治疗的存在下能够保持在G1期阻滞延长的时间段，并且可以重新在更有利的条件出现7进入细胞周期。
Protocol
Representative Results
Discussion
半自动化图像分析，鉴定球体内G1被捕区域，和增殖外层。这种方法可以在活球状体用光学部分被使用，或在固定的球体部分，以识别变化不仅在细胞周期但标记表达（通过免疫荧光），细胞死亡，或细胞形态在这些不同的区域。不同球状体区域内的细胞运动性，也可以定量 – 如果连同一个细胞跟踪图像分析步骤的现场共焦时间推移成像溶液。关键的图像分析是，高品质的Z-切片的共聚焦图像…
Divulgations
The authors have nothing to disclose.
Acknowledgements
We thank Ms. Danae Sharp and Ms. Sheena Daignault for technical assistance. We thank Dr. Atsushi Miyawaki, RIKEN, Wako-city, Japan, for providing the FUCCI constructs, Dr. Meenhard Herlyn and Ms. Patricia Brafford, The Wistar Institute, Philadelphia, for providing cell lines, the Imaging and Flow Cytometry Facility at the Centenary Institute for outstanding technical support. We thank Mr. Chris Johnson and Dr. Andrew Barlow for Volocity software technical support. N.K.H. is a Cameron fellow of the Melanoma and Skin Cancer Research Institute, Australia. K.A.B. is a fellow of the Cancer Institute New South Wales (13/ECF/1-39). W.W. is a fellow of the Cancer Institute New South Wales (11/CDF/3-39). This work was supported by project grants RG 09-08 and RG 13-06 (Cancer Council New South Wales), 570778 and 1051996 (Priority-driven collaborative cancer research scheme/Cancer Australia/Cure Cancer Australia Foundation), 08/RFG/1-27 (Cancer Institute New South Wales), and APP1003637 and APP1084893 (National Health and Medical Research Council).
Materials
| Hoechst 33342 | Life Technologies | H3570 | |
| agarose low melting point | Life Technologies | 16520-050 | For sectioning |
| noble agar | Sigma | A5431 | For making spheroids |
| agarose for spheroids | Fisher Scientific | BP1356-100 | For making spheroids |
| 0.05% trypsin/EDTA | Life Technologies | 25300-054 | |
| HBSS | Life Technologies | 14175-103 | |
| 10% formalin | Sigma | HT5014-1CS | CAUTION: Harmful, corrosive. Use Personal Protective Equipment, do not breath fumes (open in a fume cupboard). |
| live/dead near IR | Life Technologies | L10119 | |
| vibratome | Technical Products International, Inc | ||
| coulture cup | Thermo-Fisher Scientific | SIE936 | Mold for sectioning spheroids |
| hemocytometer | Sigma | Z359629 | |
| 96-well tissue culture plate | Invitro | FAL353072 | |
| collagenase | Sigma | C5138 | |
| confocal microscope | Leica | TCS SP5 | |
| Flow cytometer analyser | Becton Dickinson | LSRFortessa | |
| volocity | PerkinElmer | Imaging software | |
| flowjo | Tree Star | Flow cytometry software | |
| Vaccuum grease | Sigma | Z273554 | |
| Mounting media | Vector Laboratories | H1000 | |
| FUCCI (commercial constructs) | Life Technologies | P36238 | Transient transfection only |
| Cell strainer 70 um | In Vitro | FAL352350 | |
| Round bottom 5 mL tubes (sterile) | In Vitro | FAL352003 | |
| Round bottom 5 mL tubes (non-sterile) | In Vitro | FAL352008 |
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