通过高分辨率共聚焦显微镜对3D肿瘤球体入侵的影响表征

Summary

使用组蛋白脱乙酰雅酶（HDAC）抑制剂的三维（3D）入侵测定中，压伤抑制剂对胶质瘤癌细胞迁移的影响以高分辨率共聚焦显微镜为特征。

Abstract

癌症研究中的药物发现和开发越来越多地基于3D格式的药物屏幕。针对癌细胞的迁移和侵入性潜力，以及由此而转移的疾病传播的新型抑制剂正在被发现，并被视为高侵入性癌症（如胶质瘤）的补充疗法。因此，在添加药物后生成数据，以便对3D环境中的细胞进行详细分析。此处描述的方法，将球体入侵检测与高分辨率图像捕获和由共聚焦激光扫描显微镜 （CLSM） 进行数据分析相结合，可详细描述潜在抗迁移抑制剂的影响MI-192对胶质瘤细胞。球状物从细胞系生成，用于低附着96孔板中的入侵测定，然后准备进行CLSM分析。由于易用性和可重复性，描述的工作流程比其他常用的球形生成技术更可取。与传统广域方法相比，共聚焦显微镜获得的图像分辨率增强，可以识别和分析以下三维环境中迁移细胞的不同形态变化。用止血药物MI-192治疗。

Introduction

用于临床前药物发现和潜在抗癌药物的开发的三维球形技术正日益受到传统药物筛选的青睐;因此，有较多的米格雷静态 – 迁移和入侵预防 – 药物的发展。癌症治疗这些发展背后的理由很明确：3D球形测定是筛选潜在抗癌药物的更现实的方法，因为它们比细胞单层培养更忠实地模仿3D肿瘤结构，更准确地概括药物-肿瘤相互作用（动力学），并允许在肿瘤相关环境中描述药物活性。此外，许多癌症类型对化学毒性药物的抗药性上升，以及癌症患者因癌细胞迁移到遥远肿瘤部位的能力而发生转移而死亡率高，支持了化疗药物的加入在未来的临床癌症^试验1中，将癌细胞的迁移潜力作为辅助治疗。高侵入性癌症尤其如此，如高等级胶质细胞瘤（GBM）。GBM 管理包括手术、放疗和化疗。然而，即使结合治疗，大多数患者在初步诊断后1年内复发，中位存活期为11-15^{个月2，3。}在过去几年中，3D 技术领域取得了巨大进步：旋转式系统、微结构结构和 3D 支架以及其他单个检测正在不断改进，以便进行大规模的常规测试^{4 5，6，7}.但是，必须以有意义的方式分析从这些检测中获得的结果，因为使用 2D 图像分析系统分析 3D 生成数据的尝试通常会妨碍数据解释。

尽管在图像采集速度和光毒性降低方面更可取，但大多数广域系统仍然受到^分辨率8的限制。因此，除了与药物疗效相关的数据读出外，如果使用宽场系统成像，药物作用对迁移细胞的3D细胞结构的详细影响将不可避免地丧失。相反，共聚焦激光扫描显微镜 （CLSM） 可捕获高质量光学切片图像，这些图像可在使用计算机软件在 3D 采集后重建和渲染。因此，CLSM 易于适用于成像复杂的 3D 细胞结构，从而能够询问抗压格雷抑制剂对 3D 结构的影响，并深入分析细胞迁移机制。这无疑将指导未来的止血药物开发。这里描述了球体生成、药物治疗、染色方案以及高分辨率共聚焦显微镜表征的组合工作流程。

Protocol

1. 生成细胞类球体 第1天 根据被调查的细胞系的要求准备标准培养基。 使用无菌处理技术在组织培养罩中执行所有组织培养相关的步骤。 胰腺化并计数癌细胞。每盘使用20 mL的细胞悬浮液。将细胞悬浮液保存在明确标记的无菌通用管中。 向每个井添加预定数量的单元。所需的细胞初始数量和最终球体大小取决于被调查的细胞系的增?…

Representative Results

三维球形技术正在促进对药物-肿瘤相互作用的理解，因为它更具有癌症特定环境的代表性。球体的生成可以通过多种方式实现;该协议使用了低粘附性 96 孔板。在测试了来自不同制造商的几种产品后，选择此处使用的板材，因为它们在成功的球形生产和均匀性方面始终表现最佳。替换步骤，其中生长培养物被胶原蛋白基质替换，是协议的关键点;必须非常小心，以消除大部…

Discussion

介绍了一种利用高分辨率共聚焦显微镜鉴定压凝药物活性的癌细胞球体的新方法。使用低粘附板比其他技术，如挂^滴15，促进了产生可重复和均匀的球体用于胶原蛋白迁移和入侵测定的手段。该协议中的关键点是，在细胞球体嵌入胶原体基质之前，从96孔板中去除生长介质，并仔细处理此后含有球体的胶原蛋白塞。数字微^流体16等新技术现已推出，虽然价格较高?…

Materials

Collagen I, rat tail, 100 mg	Corning	354236	for glioma invasion assay; this is offered by many distributors/manufacturers and will need to be determined for both the type of assay intended and cell lines used. For glioma cancer cell lines Collagen rat tail type 1 ( e.g. Corning) is the preferred choice. Collagen should be stored at 4oC, in the dark, until required. It is not advisable to mix collagen from different batches as this may affect the consistency of the polymerized collagen.
Coverslips	various	various	for microscopy imaging
DMEM powder	Sigma	D5648	needed at 5X concentration for collagen solution for glioma invasion assay;  this may be purchased in powdered form, made up in double distilled water and, depending upon final composition of the growth medium, completed with any additives required. The complete 5X solution should be filtered through a syringe filter system (0.22 microns) before use.
Foetal calf serum	Sigma	F7524-500ML	needed for cell culture of glioma cell lines
Glass slides	various	various	for microscopy imaging
High glucose DMEM	Gibco	41965062	needed for cell culture of glioma cell lines
Inhibitor	Tocris	various	various – according to experimental design; inhibitors can be purchased from manufacturers such as Selleckchem and Tocris. These manufacturers offer detailed description of inhibitor characteristics, links to associated references and suggestion of working concentrations. As with all inhibitors, they may be potentially toxic and should be handled according to health and safety guidelines. Inhibitors are prepared as stock solutions as recommended by the manufacturer. As an example we used the migrastatic inhibitor MI-192 to demonstrate the use of such inhibitors. We have tested a range of migrastatic inhibitors in this way with comparable results.
Mountant	various	various	for microscopic imaging
NaOH (1 M)	various	various	NaOH can be either purchased at the required molarity or prepared from solid form. The prepared solution should be filter sterilized using a syringe filter system. One M NaOH is corrosive and care should be taken during solution preparation.
Paraformaldehyde	various	various	for fixing spheroids and cells; make up at 4%, caution health hazard, ensure that health and safety regulations are adhered to for collagen solution for invasion assay
Pastettes (graduated pipette, 3ml)	various	various	for invasion assay, solution removal
PBS, sterile for tissue culture	Sigma	D1408-500ML	needed for cell culture of glioma cell lines and washes for staining
Pen/strep (antibiotics)	Sigma	P4333	needed for cell culture of glioma cell lines
Primary antibody, secondary antibody, DAPI, Phalloidin	various	various	there are many manufacturers for these reagents, for secondary labelled antibodies we recommend Alexa Fluor (Molecular Probes). Here we used for primary antibodies mouse anti-acetylated tubulin antibody (1/100, Abcam). For secondary antibodies we used 1/500 anti-mouse Alexa Fluor 488 conjugated antibody, Molecular Probes. For nuclear stain we used DAPI (many manufacturers) and the actin stain Phalloidin (many manufacturers) both used at recommended dilution of 1/500.
Sodium bicarbonate	Sigma	S5761	needed for collagen solution for glioma invasion assay at 5X concentration
Sodium pyruvate	Sigma	P5280	needed for collagen solution for glioma invasion assay at 5X concentration
Trypsin	Sigma	T4049	for trypsinisation
Ultra low attachment plates	Sigma/Nunc	CLS7007-24EA	for glioma invasion assay; a low adherent plate is required, with 96-well plates preferred to allow for large-scale screening of compounds under investigation. There are several low adherence plates commercially available; it is advisable to test a variety of plates for optimum spheroid generation. In our experience Costar Ultra Low Cluster with lid, round bottom, works best for the generation of spheroids from glioma cancer cells in terms of 100% spheroid formation and reproducibility. These plates were also successfully used for the generation of glioma spheroids from patient-derived material, bladder and ovarian cancer cells in our laboratory. In addition, stem or progenitor neurospheres can be used in these plates to facilitate the generation of standardized neurosphere-spheroids
Stripettes (serological pipettes, sterile, 5ml and 10 ml)	various e.g. Costar	CLS4488-50; CLS4487-50	for tissue culture and collagen preparation
various multichannel (50 – 250 uL) and single channel pipettes (10 uL, 50 uL, 200 uL 1 mL)	various	various	for cell and spheroid handling
Widefield microscopy	various	various	for observation of spheroid generation and spheroid imaging; here wide-field fluorescence images were captured using an EVOS FL cell imaging system (Thermo Fisher Scientific)
Zeiss LSM 880 CLSM equipped with a Plan Apochromat 63x 1.4 NA oil objective	Zeiss	quote from manufacturer	Confocal images were captured using a Zeiss LSM 880 CLSM equipped with a Plan Apochromat 63x 1.4 NA oil objective. Diode 405nm, 458/488/514 nm argon multiline and HeNe 594nm lasers were used to excite Phalloidin 594, Alexa Fluor 488, and DAPI respectively. For each image a single representative optical section were captured, with all settings, both pre- and post-image capture, maintained for comparative purposes. All images were subsequently processed using the associated Zen imaging software and Adobe Photoshop.