我们描述用于离体肺转移中的肿瘤细胞-基质相互作用的实时成像的相对简单的方法,利用荧光报告小鼠。使用纺丝盘共焦显微镜,该技术使活细胞的可视化的至少4小时,并可以适用于研究其它炎性肺部疾病。
转移是癌症相关的发病率和死亡率的一个主要原因。转移是一个多步骤的过程,并且由于其复杂性,支配转移性传播和增长的确切细胞和分子过程仍然难以捉摸。实时成像使细胞微环境的动态性和空间相互作用的可视化。实体瘤通常转移至肺部。然而,肺的解剖位置姿势到活体成像的一个挑战。这个协议提供了用于离体肿瘤细胞和肺转移中其周围基质之间的动态相互作用的实时成像的相对简单和快速的方法。使用这种方法,在其微环境的癌细胞的癌症细胞和基质细胞之间的运动,以及相互作用可以实时可视化数小时。通过使用转基因荧光报道小鼠,荧光细胞系,可注射荧光标记的分子和/或抗体,肺微环境的多个组件可以被可视,如血管和免疫细胞。到图象的不同类型的细胞,一旋转盘共聚焦显微镜,它允许快速,四色图像采集长期连续成像已被使用。从收集到的多个位置和焦平面图像编译时间推移电影显示活转移性和免疫细胞之间的相互作用至少4小时。这种技术可以进一步用于测试化疗或靶向治疗。此外,这种方法可以适用于其它肺相关疾病可能影响肺微环境的研究。
The deadliest aspect of cancer is metastasis, which accounts for more than 90% of cancer-related morbidity and mortality1. Metastasis is a multistep process and due to its complexity, the exact cellular and molecular mechanisms that govern metastatic dissemination and growth are still elusive. To metastasize, tumor cells in the primary tumor must detach from their neighboring cells and basement membrane, cross through the extracellular matrix, intravasate, travel via blood or lymphatic vessels, extravasate at the secondary site, and finally, survive and establish secondary tumors. In addition to the properties of the tumor cells, the contribution from the microenvironment, which includes the adjacent stroma along with the normal counterparts of the cancer cells, is crucial for the seeding and establishment of metastatic lesions2.
Traditional methods to study metastatic seeding and growth examine static states, as tissues are excised and sectioned for histology. These data only generate a snapshot of this highly dynamic process. Although some useful information can be gained from these studies, the complicated process by which tumor and stromal cells interact during metastatic formation cannot be adequately assessed by these methods. Furthermore, it is not possible to gain insights into tumor or stromal cell migration patterns, which are important in establishing a colony at the distant site. In order to effectively study the metastatic process, it is essential to visualize various interactions between cancer cells and their microenvironment in a continuous manner and at real time.
The lung is a common site for metastases from solid tumors as breast, colorectal, pancreatic cancer, melanoma and sarcoma3. Intravital imaging was previously used to study cell-cell interaction in various primary tumor and metastatic models4,5. Methods of lung imaging in mice, including intravital imaging, lung section imaging, and an ex vivo pulmonary metastasis assay have been published6–9. Intravital imaging of mouse lungs utilizes a thoracic suction window to stabilize the lungs6. This method is used for time-lapse imaging of the lung microcirculation and alveolar spaces. The anatomical location of the lungs poses a challenge to intravital imaging. In order to access the lungs, the chest cavity must be opened which leads to loss of negative pressure and collapsed lungs. This method only allows the visualization of a small part of the lungs and is technically demanding; an unnecessary complication in studies that examine processes that are independent of blood flow. Moreover, this method also requires gating out movement caused by breathing. This is done either by collecting images between breaths or during post image acquisition analyses10. The alternative ex vivo lung section imaging provides stability and depth, and also prepares lung parenchyma for immunostaining7. However, the lengthy sectioning process leads to an extensive delay between the time of animal sacrifice and the start of the imaging session. Moreover, the process of sectioning a mouse lung causes considerable amount of cell death8, thus interfering with the quality and quantity of imaging samples and perhaps needlessly altering tumor-stroma interactions. In order to technically bridge between the methods of intravital imaging and lung section imaging, while exploiting the advantages of the two techniques, a relatively fast and easy method for ex vivo lung imaging was developed. This method was achieved by imaging of non-sectioned whole lung lobes. Using this method, the motility of cancer cells as well as interactions between cancer cells and stromal cells in their microenvironment can be visualized in real time for several hours.
这份手稿描述了转移的小鼠模型体外肺转移的实时成像的详细方法。该成像协议提供了肺的微环境中的动态和空间肿瘤细胞 – 基质相互作用的直接可视化。它是一个相对容易的和快速的方法,它允许为至少4小时的肺转移的可靠的成像。从这些实验中获得的影可以用来跟踪动态过程如细胞运动和细胞相互作用。
被描述为肺转移的产生方法有两种:一种基因工程小鼠模型?…
The authors have nothing to disclose.
We thank Nguyen H. Nguyen for her technical help and Audrey O’Neill for support with the Zeiss Cell Observer spinning-disk confocal microscope. This work was supported by a Department of Defense postdoctoral fellowship (W81XWH-11-01-0139) and the Weizmann Institute of Science-National Postdoctoral Award Program for Advancing Women in Science (to V.P.).
MMTV-PyMT/FVB mice | Jackson Laboratory | 2374 | Female mice |
ACTB-ECFP/FVB mice | UCSF Werb lab | Female mice | |
c-fms-EGFP/FVB mice | UCSF Werb lab | Female mice | |
FVB mice | Jackson Laboratory | 1800 | Female mice |
GFP+ VO-PyMT cells | UCSF Werb lab | ||
70,000 kDa Dextran, rhodamine-conjugated | Invitrogen | D1818 | Dilute to 4mg/ml in 1 x PBS and store at -20 °C. Use 0.4 mg per animal. |
10,000 kDa Dextran, Alexa Fluor 647 conjugated | Invitrogen | D22914 | Dilute to 4mg/ml in 1 x PBS and store at -20 °C. Use 0.4 mg per animal. |
Anti-mouse Gr-1 antibody Alexa Fluor 647 | UCSF Monoclonal antibody core | Stock 1mg/ml. Use 7 ug per animal. | |
Anesthetic | Anesthesia approved by IACUC, used for anesthesia and/or euthanesia | ||
1X PBS | UCSF cell culture facility | ||
PBS, USP sterile | Amresco INC | K813-500ML | Ultra pure grade for i.v. injection |
Styrofoam platform | Will be used as dissection board | ||
Fine scissors sharp | Fine Science Tools | 14060-11 | |
Forceps | Roboz Surgical Store | RS-5135 | |
Hot bead sterilizer | Fine Science Tools | 18000-45 | Turn ON 30min before use |
Air | UCSF | ||
Oxygen | UCSF | ||
Carbon dioxide | UCSF | ||
1 mL syringe without needle | BD | 309659 | |
27 G x 1/2 needle | BD | 305109 | for i.v. injection |
20 G x 1 needle, short bevel | BD | 305178 | |
Low-melting-temperature agarose | Lonza | 50111 | To make 10 ml of solution, weigh 0.2 g of agarose, add to 10 ml 1 x PBS, and heat to dissolve. Agarose will solidify at room temperature, so maintain in a 37 °C water bath until used for inflation. |
RPMI-1640 medium without phenol red | Life Technologies | 11835-030 | |
24 well Imaging plate | E&K scientific | EK-42892 | |
Glass cover slides, 15 mm | Fisher Scientific | 22-031-144 | |
Digital CO2 and temperature controller | Okolab | DGTCO2BX | http://www.oko-lab.com |
Climate chamber | Okolab | http://www.oko-lab.com | |
Cell Observer spinning disk confocal microscope | Zeiss | ||
Zen software | Zeiss | ||
Inverted microscope | Carl Zeiss Inc | Zeiss Axiovert 200M | |
ICCD camera | Stanford Photonics | XR-Mega-10EX S-30 | |
Spinning disk confocal scan-head | Yokogawa Corporation | CSU-10b | |
Imaris | Bitplane | ||
mManager | Vale lab, UCSF | Open-source software |