Name: ex vivo live imaging of lung metastasis and their microenvironment
Uploaded: 2016-02-03T14:00:00
Description: Watch this Scientific Journal Video about Ex vivo Live Imaging of Lung Metastasis and Their Microenvironment at JoVE.com

We thank Nguyen H. Nguyen for her technical help and Audrey O&#8217;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.).

中描述的所有程序必须按照与使用脊椎动物，包括由当地机构动物护理和使用委员会（IACUC）批准的准则和规定进行。 1.产生肺转移瘤防爆体内实时成像（转基因或尾静脉注射） 注：肺转移可通过利用遗传工程小鼠模型或通过静脉内（IV）注射癌细胞产生。 <ol><li>通过杂交的基因工程肿瘤小鼠模型成转基因记者鼠标，例如，生成用?...

<ol>
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J., van Rheenen, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+hallmarks+of+cancer+in+living+mice.">Imaging hallmarks of cancer in living mice.</a> Nat Rev Cancer. 14 (6), 406-418 (2014).</li><li>Looney, M. R., Thornton, E. E., Sen, D., Lamm, W. J., Glenny, R. W., Krummel, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stabilized+imaging+of+immune+surveillance+in+the+mouse+lung.">Stabilized imaging of immune surveillance in the mouse lung.</a> Nat Methods. 8 (1), 91-96 (2011).</li><li>Thornton, E. E., Krummel, M. F., Looney, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live+imaging+of+the+lung.">Live imaging of the lung.</a> Cur Protoc Cytom. , (2012).</li><li>Thornton, E. E., Looney, M. 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A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+tumor-stroma+interactions+during+chemotherapy+reveals+contributions+of+the+microenvironment+to+resistance.">Imaging tumor-stroma interactions during chemotherapy reveals contributions of the microenvironment to resistance.</a> Cancer cell. 21 (4), (2012).</li><li>Cheng, N., Lambert, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mammary+transplantation+of+stromal+cells+and+carcinoma+cells+in+C57BL/6J+mice.">Mammary transplantation of stromal cells and carcinoma cells in C57BL/6J mice.</a> J Vis Exp. (54), (2011).</li><li>Al-Mehdi, A. B., Tozawa, K., Fisher, A. B., Shientag, L., Lee, A., Muschel, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravascular+origin+of+metastasis+from+the+proliferation+of+endothelium-attached+tumor+cells:+a+new+model+for+metastasis.">Intravascular origin of metastasis from the proliferation of endothelium-attached tumor cells: a new model for metastasis.</a> Nat Med. 6 (1), 100-102 (2000).</li><li>Wong, C. W., Song, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravascular+location+of+breast+cancer+cells+after+spontaneous+metastasis+to+the+lung.">Intravascular location of breast cancer cells after spontaneous metastasis to the lung.</a> Am J Pathol. 161 (3), 749-753 (2002).</li><li>Liang, C. -. C., Park, A. Y., Guan, J. -. 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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 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&#8211;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.

<table>
	<tbody>
		<tr>
			<td>MMTV-PyMT/FVB mice</td>
			<td>Jackson Laboratory</td>
			<td>2374</td>
			<td>Female mice</td>
		</tr>
		<tr>
			<td>ACTB-ECFP/FVB mice</td>
			<td>UCSF Werb lab</td>
			<td></td>
			<td>Female mice</td>
		</tr>
		<tr>
			<td>c-fms-EGFP/FVB mice</td>
			<td>UCSF Werb lab</td>
			<td></td>
			<td>Female mice</td>
		</tr>
		<tr>
			<td>FVB mice</td>
			<td>Jackson Laboratory</td>
			<td>1800</td>
			<td>Female mice</td>
		</tr>
		<tr>
			<td>GFP+ VO-PyMT cells</td>
			<td>UCSF Werb lab</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>70,000 kDa Dextran, rhodamine-conjugated</td>
			<td>Invitrogen</td>
			<td>D1818</td>
			<td>Dilute to&#160; 4mg/ml in 1 x PBS and store at -20&#160; &#176;C. Use 0.4 mg per animal.&#160;</td>
		</tr>
		<tr>
			<td>10,000 kDa Dextran, Alexa Fluor 647 conjugated</td>
			<td>Invitrogen</td>
			<td>D22914</td>
			<td>Dilute to&#160; 4mg/ml in 1 x PBS and store at -20&#160; &#176;C. Use 0.4 mg per animal.&#160;</td>
		</tr>
		<tr>
			<td>Anti-mouse Gr-1 antibody Alexa Fluor 647</td>
			<td>UCSF Monoclonal antibody core</td>
			<td></td>
			<td>Stock 1mg/ml. Use 7 ug per animal.</td>
		</tr>
		<tr>
			<td>Anesthetic</td>
			<td></td>
			<td></td>
			<td>Anesthesia approved by IACUC, used for anesthesia and/or euthanesia</td>
		</tr>
		<tr>
			<td>1X PBS</td>
			<td>UCSF cell culture facility</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PBS, USP sterile&#160;</td>
			<td>Amresco INC</td>
			<td>K813-500ML</td>
			<td>Ultra pure grade for i.v. injection</td>
		</tr>
		<tr>
			<td>Styrofoam platform</td>
			<td></td>
			<td></td>
			<td>Will be used as dissection board</td>
		</tr>
		<tr>
			<td>Fine scissors sharp&#160;</td>
			<td>Fine Science Tools</td>
			<td>14060-11</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>Roboz Surgical Store</td>
			<td>RS-5135</td>
			<td></td>
		</tr>
		<tr>
			<td>Hot bead sterilizer</td>
			<td>Fine Science Tools</td>
			<td>18000-45</td>
			<td>Turn ON 30min before use</td>
		</tr>
		<tr>
			<td>Air</td>
			<td>UCSF</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Oxygen</td>
			<td>UCSF</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Carbon dioxide</td>
			<td>UCSF</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1 mL syringe without needle&#160;</td>
			<td>BD</td>
			<td>309659</td>
			<td></td>
		</tr>
		<tr>
			<td>27 G x 1/2 needle&#160;&#160;</td>
			<td>BD</td>
			<td>305109</td>
			<td>for i.v. injection</td>
		</tr>
		<tr>
			<td>20 G x 1 needle, short bevel&#160;&#160;</td>
			<td>BD</td>
			<td>305178</td>
			<td></td>
		</tr>
		<tr>
			<td>Low-melting-temperature agarose&#160;</td>
			<td>Lonza</td>
			<td>50111</td>
			<td>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 &#176;C water bath until used for inflation.</td>
		</tr>
		<tr>
			<td>RPMI-1640 medium without phenol red</td>
			<td>Life Technologies</td>
			<td>11835-030</td>
			<td></td>
		</tr>
		<tr>
			<td>24 well Imaging plate&#160;</td>
			<td>E&#38;K scientific</td>
			<td>EK-42892</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass cover slides, 15 mm&#160;</td>
			<td>Fisher Scientific</td>
			<td>22-031-144</td>
			<td></td>
		</tr>
		<tr>
			<td>Digital CO2 and temperature controller</td>
			<td>Okolab</td>
			<td>DGTCO2BX</td>
			<td>http://www.oko-lab.com</td>
		</tr>
		<tr>
			<td>Climate chamber</td>
			<td>Okolab</td>
			<td></td>
			<td>http://www.oko-lab.com</td>
		</tr>
		<tr>
			<td>Cell Observer spinning disk confocal microscope</td>
			<td>Zeiss</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Zen software</td>
			<td>Zeiss</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Inverted microscope</td>
			<td>Carl Zeiss Inc</td>
			<td>Zeiss Axiovert 200M</td>
			<td></td>
		</tr>
		<tr>
			<td>ICCD camera</td>
			<td>Stanford Photonics</td>
			<td>XR-Mega-10EX S-30</td>
			<td></td>
		</tr>
		<tr>
			<td>Spinning disk confocal scan-head</td>
			<td>Yokogawa Corporation</td>
			<td>CSU-10b</td>
			<td></td>
		</tr>
		<tr>
			<td>Imaris</td>
			<td>Bitplane</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>mManager</td>
			<td>Vale lab, UCSF</td>
			<td></td>
			<td>Open-source software</td>
		</tr>
	</tbody>
</table>

ex vivo live imaging of lung metastasis and their microenvironment

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。

使用纺丝盘共焦显微镜，各种小鼠模型系统和注射剂，转移微环境可以可视化并随时间跟踪。使用MMTV-PyMT; ACTB-ECFP;的c-FMS-EGFP三重转基因小鼠模型，不同的细胞组分的荧光标记（图2A，电影1）。肺实质的典型结构可以在CFP通道由于所有细胞中的β肌动蛋白启动子下表达的ECFP可视化。这些表现为大部分肺组织结构中细胞的大/多肺转移是很容易解决。髓细胞可视化在...

Watch this Scientific Journal Video about Ex vivo Live Imaging of Lung Metastasis and Their Microenvironment at JoVE.com

Ex vivo Live Imaging of Lung Metastasis and Their Microenvironment

体外肺转移及微环境的实时成像

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。

Metastasis is a major cause for cancer-related morbidity and mortality. Metastasis is a multistep process and due to its complexity, the exact cellular ...

Research

JoVE Journal

Medicine

Ex vivo Live Imaging of Lung Metastasis and Their Microenvironment

Ex Vivo Infection of Live Tissue with Oncolytic Viruses

Ex Vivo Infection of Live Tissue with Oncolytic Viruses

前溶瘤病毒的活组织的体内感染

Oncolytic Viruses (OVs) are novel therapeutics that selectively replicate in and kill tumor cells1. Several clinical trials evaluating the effectiveness ...

科研

医学

In vivo Bioluminescence Imaging of Tumor Hypoxia Dynamics of Breast Cancer Brain Metastasis in a Mouse Model

In vivo Bioluminescence Imaging of Tumor Hypoxia Dynamics of Breast Cancer Brain Metastasis in a Mouse Model

在小鼠模型体内生物发光成像乳腺癌脑转移的肿瘤乏氧动力学

It is well recognized that tumor hypoxia plays an important role in promoting malignant progression and affecting therapeutic response negatively. There ...

MAME Models for 4D Live-cell Imaging of Tumor: Microenvironment Interactions that Impact Malignant Progression

MAME的模型四维活细胞成像肿瘤微环境的相互作用，影响的恶性进展

We have developed 3D coculture models, which we term MAME (mammary architecture and microenvironment engineering), and used them for live-cell imaging in ...

Live Imaging of Drug Responses in the Tumor Microenvironment in Mouse Models of Breast Cancer

实时成像在乳腺癌小鼠模型中的肿瘤微环境对药物的反应

The tumor microenvironment plays a pivotal role in tumor initiation, progression, metastasis, and the response to anti-cancer therapies. Three-dimensional ...

Molecular Profiling of the Invasive Tumor Microenvironment in a 3-Dimensional Model of Colorectal Cancer Cells and Ex vivo Fibroblasts

Molecular Profiling of the Invasive Tumor Microenvironment in a 3-Dimensional Model of Colorectal Cancer Cells and Ex vivo Fibroblasts

在结直肠癌细胞的三维模型的入侵肿瘤微环境的分子剖析和<em&gt;体外</em&gt;成纤维细胞

Invading colorectal cancer (CRC) cells have acquired the capacity to break free from their sister cells, infiltrate the stroma, and remodel the ...

Method of Isolated Ex Vivo Lung Perfusion in a Rat Model: Lessons Learned from Developing a Rat EVLP Program

Method of Isolated Ex Vivo Lung Perfusion in a Rat Model: Lessons Learned from Developing a Rat EVLP Program

的隔离方法<em&gt;离体</em&gt;肺灌注的大鼠模型：教训制定鼠EVLP课程中学

The number of acceptable donor lungs available for lung transplantation is severely limited due to poor quality. Ex-Vivo Lung Perfusion (EVLP) has allowed ...

Validation of Nanobody and Antibody Based In Vivo Tumor Xenograft NIRF-imaging Experiments in Mice Using Ex Vivo Flow Cytometry and Microscopy

Validation of Nanobody and Antibody Based In Vivo Tumor Xenograft NIRF-imaging Experiments in Mice Using Ex Vivo Flow Cytometry and Microscopy

纳米抗体和基于抗体的验证<em&gt;在体内</em&gt;肿瘤异种移植NIRF成像的小鼠实验使用<em&gt;离体</em&gt;流式细胞仪和显微镜

This protocol outlines the steps required to perform ex vivo validation of in vivo near-infrared fluorescence (NIRF) xenograft imaging experiments in mice ...

Primary Tumor and MEF Cell Isolation to Study Lung Metastasis

原发灶和MEF细胞分离，研究肺转移

In breast tumorigenesis, the metastatic stage of the disease poses the greatest threat to the affected individual. Normal breast cells with altered ...

Evaluation of Lung Metastasis in Mouse Mammary Tumor Models by Quantitative Real-time PCR

评价肺转移的小鼠乳腺肿瘤模型定量实时荧光定量PCR

Metastatic disease is the spread of malignant tumor cells from the primary cancer site to a distant organ and is the primary cause of cancer associated ...

Precision Cut Lung Slices as an Efficient Tool for Ex vivo Pulmonary Vessel Structure and Contractility Studies

Precision Cut Lung Slices as an Efficient Tool for Ex vivo Pulmonary Vessel Structure and Contractility Studies

精密切肺切片作为 离体 肺血管结构和收缩力研究的有效工具

The visualization of murine lung tissue provides valuable structural and cellular information regarding the underlying airway and vasculature. However, ...