Name: extended time-lapse intravital imaging of real-time multicellular dynamics in the tumor microenvironment
Uploaded: 2016-06-12T14:00:00
Description: Watch this Scientific Journal Video about Extended Time-lapse Intravital Imaging of Real-time Multicellular Dynamics in the Tumor Microenvironment at JoVE.com

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。

中描述的所有程序必须按照与使用脊椎动物，包括医药机构动物护理和使用委员会的爱因斯坦医学院的事先批准的准则和规定进行。 1.生成荧光标记的肿瘤及肿瘤相关巨噬细胞<ol><li>通过杂交自发，原地，遗传工程小鼠乳腺癌模型中，小鼠乳腺肿瘤病毒长末端重复与荧光报告[ 即转基因小鼠驱动多瘤中间T抗原（MMTV-PyMT），增强型绿色荧光产生的荧光标记的肿瘤细胞蛋白（EGFP）...

<ol>
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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...

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。

<table border="0" cellpadding="0" cellspacing="0" width="637">
	<tbody>
		<tr height="63">
			<td height="63" style="height:63px;width:248px;">155 kDa dextran-tetramethylrhodamine isothiocyanate</td>
			<td style="width:95px;">Sigma Aldrich</td>
			<td style="width:128px;">T1287</td>
			<td style="width:167px;">reconstitute at 20 mg/mL in 1 X PBS</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:248px;">70 kDa dextran-Texas Red</td>
			<td style="width:95px;">Life Technologies</td>
			<td style="width:128px;">D-1830</td>
			<td>reconstitute at 10 mg/mL in 1 X PBS</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:248px;">10 kDa dextran-fluorescein isothyocyanate</td>
			<td style="width:95px;">Sigma Aldrich</td>
			<td style="width:128px;">FD10S</td>
			<td>reconstitute at 20 mg/mL in 1 X PBS</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:248px;">Qdot 705 ITK Amino (PEG) Quantum Dots</td>
			<td style="width:95px;">Life Technologies</td>
			<td style="width:128px;">Q21561MP</td>
			<td>Dilute 25 uL in 175 uL of 1 X PBS for injection</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:248px;">MMTV-PyMT mice</td>
			<td style="width:95px;">Jackson Laboratory</td>
			<td align="right" style="width:128px;">2374</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:248px;">Csf1r-ECFP mice (Csf1r-Gal4/VP16,UAS-ECFP)</td>
			<td style="width:95px;">Jackson Laboratory</td>
			<td align="right" style="width:128px;">26051</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:248px;">Csf1r-EGFP mice</td>
			<td style="width:95px;">Jackson Laboratory</td>
			<td align="right" style="width:128px;">18549</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:248px;">1 x PBS</td>
			<td style="width:95px;">Life Technologies</td>
			<td style="width:128px;"></td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:248px;">Isoethesia (isoflurane)</td>
			<td style="width:95px;">Henry Schein Animal Health</td>
			<td align="right" style="width:128px;">50033</td>
			<td>250 mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:248px;">Oxygen</td>
			<td style="width:95px;">AirTech</td>
			<td style="width:128px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:248px;">1 mL syringe, tuberculin slip tip</td>
			<td style="width:95px;">BD</td>
			<td align="right" style="width:128px;">309659</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:248px;">30G x 1 (0.3 mm X 25 mm) needle</td>
			<td style="width:95px;">BD</td>
			<td align="right" style="width:128px;">305128</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:248px;">Polyethylene micro medical tubing&#160;</td>
			<td style="width:95px;">Scientific Commodities Inc</td>
			<td style="width:128px;">BB31695-PE/1</td>
			<td>0.28 mm I.D. X 0.64 mm O.D.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:248px;">Microscope coverglass</td>
			<td style="width:95px;">Corning</td>
			<td style="width:128px;">2980-225</td>
			<td>thickness 1.5, 22 X 50 mm</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:248px;">MouseOx oximeter, software and sensors</td>
			<td style="width:95px;">STARR Life Sciences</td>
			<td style="width:128px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:248px;">Laboratory tape</td>
			<td style="width:95px;">Fisher Scientific</td>
			<td style="width:128px;">159015R</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:248px;">soft rubber pad</td>
			<td style="width:95px;">McMaster-Carr</td>
			<td>8514K62</td>
			<td>Ultra-Soft Polyurethane Film, 3/16&#8221; Thick, 12&#34; x 12&#34;, 40 Oo Durometer, Plain Back</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:248px;">hard rubber pad</td>
			<td style="width:95px;">McMaster-Carr</td>
			<td>8568K615</td>
			<td>High-Strength Neoprene Rubber Sheet 1/4&#34; Thick, 12&#34; X 12&#34;, 50A Durometer,</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:248px;">Microscope</td>
			<td style="width:95px;">Olympus</td>
			<td style="width:128px;"></td>
			<td>The microscope is a custom built two laser multiphoton microscope based on an Olympus IX-71 stand utilizing a 20x 1.05NA objective lens.&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:248px;">7-Punch set</td>
			<td style="width:95px;">McMaster-Carr</td>
			<td style="width:128px;">3429A12</td>
			<td>, 1/4&#34; to 1&#34; Hole Diameter, for Hammer-Driven Hole Punch,&#160;</td>
		</tr>
	</tbody>
</table>

extended time-lapse intravital imaging of real-time multicellular dynamics in the tumor microenvironment

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.

延长的时间推移活体显微镜使在肿瘤微环境的多细胞过程的单细胞分辨率的成像。通过荧光标记的肿瘤细胞，巨噬细胞，血管空间和可视化使用二次谐波生成信号的胶原纤维网络，在肿瘤微环境多个隔间成像期间被同时跟踪。标记的荧光蛋白的肿瘤细胞可以在转基因小鼠如已在MMTV-PyMT-Dendra2小鼠已经完成，用标记与Dendra2（ 图1A）的乳腺肿瘤细胞中产生。通过?...

Watch this Scientific Journal Video about Extended Time-lapse Intravital Imaging of Real-time Multicellular Dynamics in the Tumor Microenvironment at JoVE.com

Extended Time-lapse Intravital Imaging of Real-time Multicellular Dynamics in the Tumor Microenvironment

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 。

实时多细胞动力学延长时间推移活体成像在肿瘤微环境

In the tumor microenvironment, host stromal cells interact with tumor cells to promote tumor progression, angiogenesis, tumor cell dissemination and ...

Research

JoVE Journal

Medicine

Multispectral Real-time Fluorescence Imaging for Intraoperative Detection of the Sentinel Lymph Node in Gynecologic Oncology

多光谱实时荧光成像术在妇科肿瘤前哨淋巴结的检测

The prognosis in virtually all solid tumors depends on the presence or absence of lymph node metastases.1-3 Surgical treatment most often combines radical ...

科研

医学

Evaluation of Nanoparticle Uptake in Tumors in Real Time Using Intravital Imaging

使用实时活体成像在肿瘤纳米粒子吸收的评价

Current technologies for tumor imaging, such as ultrasound, MRI, PET and CT, are unable to yield high-resolution images for the assessment of nanoparticle ...

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 ...

Real-time Digital Imaging of Leukocyte-endothelial Interaction in Ischemia-reperfusion Injury (IRI) of the Rat Cremaster Muscle

Real-time Digital Imaging of Leukocyte-endothelial Interaction in Ischemia-reperfusion Injury IRI of the Rat Cremaster Muscle

实时数字成像大鼠提睾肌缺血再灌注损伤（IRI）的白细胞 - 内皮细胞相互作用

Ischemia-reperfusion injury (IRI) has been implicated in a large array of pathological conditions such as cerebral stroke, myocardial infarction, ...

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 ...

Time-lapse Imaging of Primary Preneoplastic Mammary Epithelial Cells Derived from Genetically Engineered Mouse Models of Breast Cancer

延时成像的主要癌前乳腺上皮细胞来源于遗传工程小鼠乳腺癌模型

Time-lapse  imaging can be used to compare behavior of cultured primary preneoplastic  mammary epithelial cells derived from different genetically ...

Transplantation into the Anterior Chamber of the Eye for Longitudinal, Non-invasive In vivo Imaging with Single-cell Resolution in Real-time

Transplantation into the Anterior Chamber of the Eye for Longitudinal, Non-invasive In vivo Imaging with Single-cell Resolution in Real-time

纵向，非侵入性的移植到眼睛的前房<em在体内</em&gt;单细胞分辨率的实时成像

Intravital imaging has emerged as an indispensable tool in biological research.  In the process, many imaging techniques have been developed to study ...

Chemotherapy-induced Vascular Toxicity - Real-time In vivo Imaging of Vessel Impairment

Chemotherapy-induced Vascular Toxicity - Real-time In vivo Imaging of Vessel Impairment

化疗引起的血管毒性 - 实时<em&gt;在体内</em&gt;船舶减值成像

Certain classes of chemotherapies may exert acute vascular changes that may progress into long-term conditions that may predispose the patient to an ...

Confocal Time Lapse Imaging as an Efficient Method for the Cytocompatibility Evaluation of Dental Composites

焦时间推移成像为牙科复合材料的细胞相容性评价的有效方法

It is generally accepted that in vitro cell material interaction is a useful criterion in the evaluation of dental material biocompatibility. The ...

Real-time X-ray Imaging of Lung Fluid Volumes in Neonatal Mouse Lung

在新生小鼠肺肺流体体积的实时X射线成像

At birth, the lung undergoes a profound phenotypic switch from secretion to absorption, which allows for adaptation to breathing independently. Promoting ...