这个协议描述了使用多光子显微术的实时在单细胞分辨率来执行多细胞相互作用的延长时间推移成像,体内 。
In the tumor microenvironment, host stromal cells interact with tumor cells to promote tumor progression, angiogenesis, tumor cell dissemination and metastasis. Multicellular interactions in the tumor microenvironment can lead to transient events including directional tumor cell motility and vascular permeability. Quantification of tumor vascular permeability has frequently used end-point experiments to measure extravasation of vascular dyes. However, due to the transient nature of multicellular interactions and vascular permeability, the kinetics of these dynamic events cannot be discerned. By labeling cells and vasculature with injectable dyes or fluorescent proteins, high-resolution time-lapse intravital microscopy has allowed the direct, real-time visualization of transient events in the tumor microenvironment. Here we describe a method for using multiphoton microscopy to perform extended intravital imaging in live mice to directly visualize multicellular dynamics in the tumor microenvironment. This method details cellular labeling strategies, the surgical preparation of a mammary skin flap, the administration of injectable dyes or proteins by tail vein catheter and the acquisition of time-lapse images. The time-lapse sequences obtained from this method facilitate the visualization and quantitation of the kinetics of cellular events of motility and vascular permeability in the tumor microenvironment.
从初级乳腺肿瘤的肿瘤细胞的传播已经显示出不仅涉及肿瘤细胞,但承载间质细胞包括巨噬细胞和内皮细胞。此外,肿瘤血管异常与通透性增加1。因此,确定如何肿瘤细胞,巨噬细胞和内皮细胞相互作用介导的原发肿瘤微环境的血管通透性和肿瘤细胞血管内为理解转移重要。理解血管通透性,肿瘤细胞血管内和在肿瘤微环境的多细胞相互作用的基本信令机制的动力学可提供在抗癌疗法的开发和管理的关键信息。
研究体内肿瘤血管通透性的主要手段一直是血管外染料的测量,如伊文思蓝2,高分子量的葡聚糖(155 kDa)的染料注射后3和荧光团或放射性缀合的蛋白质(包括白蛋白)4在固定的时间点。在显微镜的进步,动物模型和荧光染料已通过活体显微镜5启用细胞过程和在活体动物血管通透性的可视化。
活的动物成像采集静态图像,或短的时间推移的序列在数个时间点不允许在肿瘤微环境6,7-动态事件的完整的理解。实际上,静态图像采集在24小时的过程中显示,肿瘤血管是漏水,但血管通透性的动力学没有观察到6。因此,延长的持续时间推移成像长达12小时捕获在肿瘤微环境动态事件的动力学。
本协议描述使用较长时间推移multiphot的关于活体显微镜来研究在肿瘤微环境动态多细胞事件。在肿瘤微环境多种细胞类型标记有可注射的染料或通过使用表达荧光蛋白的转基因动物。使用尾静脉导管的血管的染料或蛋白质可成像开始之后被注入以获得在肿瘤微环境的多细胞事件的动力学数据。活细胞成像的乳腺肿瘤是通过手术准备一个皮瓣露出。图像被获取用于使用配备有多个光电倍增管(PMT)探测器8多光子显微镜长达12小时。通过使用适当的过滤器,减法算法使4 PMT检测器获取在肿瘤微环境同时9 5的荧光信号。高分辨率多光子活体显微镜捕获在肿瘤微环境肿瘤 – 基质相互作用的单细胞分辨率成像,从而导致更好ü血管通透性和肿瘤细胞血管内10-13的nderstanding。具体地,扩展的活体成像显露高度局部,瞬态血管通透性,在肿瘤细胞,巨噬细胞和内皮细胞之间的相互作用的位点选择性地发生的事件(定义为转移,TMEM 14的肿瘤微环境)13。此外,肿瘤细胞血管内仅在TMEM发生和与血管通透性13被空间和时间上相关。这些事件的动态单细胞分辨率是通过在肿瘤微环境中使用荧光标记的细胞的延长的时间推移多光子显微镜的成为可能。
在肿瘤微环境自发发生的细胞相互作用可以导致肿瘤细胞运动和血管内的变化。活肿瘤组织的高分辨率活体成像允许多细胞动力学,可以是高瞬态10,13,24的可视化。终点与分立时间点获取的可提供的在肿瘤微环境的过程的分子机制的基本信息体内测定或时间推移的图像。活体成像的研究已经被用于研究在肿瘤微环境中发生的数天的过程,生成对血管生成,肿瘤细胞迁移和响应…
The authors have nothing to disclose.
这项研究是由美国国防部的乳腺癌研究计划下授予号(ASH,W81XWH-13-1-0010),NIH CA100324,PPG CA100324,和综合成像计划的支持。
155 kDa dextran-tetramethylrhodamine isothiocyanate | Sigma Aldrich | T1287 | reconstitute at 20 mg/mL in 1 X PBS |
70 kDa dextran-Texas Red | Life Technologies | D-1830 | reconstitute at 10 mg/mL in 1 X PBS |
10 kDa dextran-fluorescein isothyocyanate | Sigma Aldrich | FD10S | reconstitute at 20 mg/mL in 1 X PBS |
Qdot 705 ITK Amino (PEG) Quantum Dots | Life Technologies | Q21561MP | Dilute 25 uL in 175 uL of 1 X PBS for injection |
MMTV-PyMT mice | Jackson Laboratory | 2374 | |
Csf1r-ECFP mice (Csf1r-Gal4/VP16,UAS-ECFP) | Jackson Laboratory | 26051 | |
Csf1r-EGFP mice | Jackson Laboratory | 18549 | |
1 x PBS | Life Technologies | ||
Isoethesia (isoflurane) | Henry Schein Animal Health | 50033 | 250 mL |
Oxygen | AirTech | ||
1 mL syringe, tuberculin slip tip | BD | 309659 | |
30G x 1 (0.3 mm X 25 mm) needle | BD | 305128 | |
Polyethylene micro medical tubing | Scientific Commodities Inc | BB31695-PE/1 | 0.28 mm I.D. X 0.64 mm O.D. |
Microscope coverglass | Corning | 2980-225 | thickness 1.5, 22 X 50 mm |
MouseOx oximeter, software and sensors | STARR Life Sciences | ||
Laboratory tape | Fisher Scientific | 159015R | |
soft rubber pad | McMaster-Carr | 8514K62 | Ultra-Soft Polyurethane Film, 3/16” Thick, 12" x 12", 40 Oo Durometer, Plain Back |
hard rubber pad | McMaster-Carr | 8568K615 | High-Strength Neoprene Rubber Sheet 1/4" Thick, 12" X 12", 50A Durometer, |
Microscope | Olympus | The microscope is a custom built two laser multiphoton microscope based on an Olympus IX-71 stand utilizing a 20x 1.05NA objective lens. | |
7-Punch set | McMaster-Carr | 3429A12 | , 1/4" to 1" Hole Diameter, for Hammer-Driven Hole Punch, |