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

이 연구는 보너스 번호 (ASH, W81XWH-13-1-0010)에서 국방부의 유방암 연구 프로그램, NIH CA100324, PPG CA100324 및 통합 이미징 프로그램에 의해 지원되었다.

설명하는 모든 절차는 의학 기관 동물 관리 및 사용위원회의 알버트 아인슈타인 대학의 사전 승인을 포함한 척추 동물의 사용에 대한 지침과 규정에 따라 수행해야합니다. 1. 생성 형광 표지 된 종양 및 종양 관련 대 식세포 <ol><li> 강화 된 녹색 형광 마우스 유방 종양 바이러스 긴 터미널 반복, 즉 형광 기자 [와 형질 전환 마우스와 폴리오 중간에 T 항원 (MMTV-PyMT)를 구동하는 자연, 토...

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종양 미세 환경에서 자연적으로 발생하는 세포 상호 작용은 종양 세포 운동성 및 intravasation 변화로 이어질 수있다. 살아있는 종양 조직의 고해상도 생체 내에 촬상 매우 과도 10,13,24 될 수 다세포 역학의 시각화를 허용한다. 종양 미세 환경에서 프로세스의 분자 메커니즘에 필수적인 정보를 제공 할 수 있습니다 이산 시점으로 얻은 생체 내 분석 또는 시간 경과 이미지의 최?...

차 유방 종양에서 종양 세포의 보급은 대 식세포 및 혈관 내피 세포를 포함하는 기질 세포뿐만 아니라 종양 세포를 포함하지만, 호스팅하는 것으로 나타났다. 또한, 종양 혈관 투과성 증대 1 이상이다. 따라서, 종양 세포, 대 식세포 및 내피 세포가 일차 종양 미세 혈관 투과성 및 종양 세포 intravasation을 중재 상호 작용하는 방법을 결정하는 단계 이해 전이에 중요하다. 혈관 투과성, 종양 세포 intravasation 종양 미세 환경에 다세포 상호 작용의 기본 신호 메커니즘의 동역학을 이해하는 항암 치료제의 개발 및 관리에 중요한 정보를 제공 할 수있다. 생체 내에서 종양 혈관 투과성 연구의 주요 수단은 에반스 블루 2, 고 분자량의 덱스 트란 (155 kDa의) 혈관 외 염료로 측정했다염료의 주입 후 일정한 시간 점 (3)과 형광 또는 (알부민을 포함) 추적자 공역 단백질 4. 현미경의 발전은, 동물 모델 및 형광 염료는 생체 내에 현미경 (5)를 통해 세포 과정과 살아있는 동물의 혈관 투과성의 시각화를 사용할 수있다. 여러 시점에 걸쳐 정적 인 이미지, 또는 짧은 시간 경과 시퀀스의 인수와 라이브 동물 이미징은 종양 미세 환경 6,7 동적 이벤트의 완전한 이해를 허용하지 않습니다. 실제로, 24 시간의 과정을 통해 정적 이미지 획득은 종양 혈관 그러나 혈관 투과성의 역학은 6 관찰되지 않았다 새는 것을 보여 주었다. 따라서 12 시간까지 연장 된 연속 시간 경과 영상은 종양 미세 환경에서 동적 이벤트의 반응 속도를 캡처합니다. 이 프로토콜은 확장 된 시간 경과 multiphot의 사용을 설명합니다생체 내에 현미경에 종양 미세 환경에서 동적 다세포 이벤트를 공부합니다. 종양 미세 환경의 여러 세포 유형은 주사 염료 또는 형광 단백질을 발현하는 형질 전환 동물을 사용하여 표시되어 있습니다. 혈관 염료 또는 단백질 촬상 개시 후 주입 될 수있는 꼬리 정맥 카테터를 사용하여 종양 미세 환경에 다세포 이벤트 운동 데이터를 획득한다. 생균 이미징 유방 종양 피부 플랩 수술 준비를 통해 노출된다. 이미지는 다수의 광전자 증 배관 튜브 (PMT) 검출기 (8)를 구비 한 다 광자 현미경을 이용하여 12 시간 동안 획득된다. 적절한 필터를 사용하여 감산 알고리즘 종양 미세 구 동시에 5 형광 신호를 획득하도록 4 PMT 검출기를 가능하게한다. 고해상도의 광자 생체 내에 현미경 더 이어지는 종양 미세 환경에서 종양 - 기질 상호 작용의 단일 셀의 고해상도 영상을 캡처 U혈관 투과성 및 종양 세포 intravasation 10-13 nderstanding. 특히 종양 세포, 대 식세포 및 내피 세포 간의 상호 작용 부위에서 선택적으로 발생하는 확장 된 생체 내에 촬상 밝혀 고도로 국부 일시적인 혈관 투과성 이벤트가도 13의 (전이 TMEM 14 종양 미세 환경으로 정의). 또한, 종양 세포는 intravasation TMEM 발생 공간적 및 시간적 혈관 투과성 (13)와 상관된다. 이러한 이벤트의 역학의 단일 셀 해상도는 종양 미세 환경에 형광 표지 된 세포의 확장 시간 경과 광자 현미경의 사용을 통해 가능하게되었다.

<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.

연장 시간 경과 생체 내에 현미경 종양 미세 환경에서 다세포 프로세스의 단일 셀 해상도 이미징 할 수 있습니다. 형광 표지 된 종양 세포, 대 식세포, 혈관 공간, 제 2 고조파 발생 신호를 이용하여 콜라겐 섬유 네트워크를 시각화함으로써 종양 미세 환경에서 다수의 구획을 동시에 촬상 동안 추적. Dendra2 (도 1a)로 표지 유방 종양 세포, MMTV-PyMT-Dendra2 쥐에서 수?...

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

이 프로토콜은 단일 셀 해상도 생체 내에서 실시간으로 상호 작용 다세포 장시간 경과 이미징을 수행하는 다 광자 현미경의 사용을 설명한다.

종양 미세 환경에서 실시간 다세포 역학의 확장 시간 경과 생체 내에 영상

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

Gynecologic 종양학의 센티널 림프절의 Intraoperative 감지 Multispectral 실시간 형광 이미징

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 ...

연구

의학

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

Intravital 이미징을 사용하여 실시간으로 종양의 Nanoparticle의 이해 평가

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

종양의 4D 라이브 세포 이미징을위한 MAME 모델 : Microenvironment 상호 작용 영향을 미치는 악성 진행

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

쥐 Cremaster 근육의 국소 빈혈 reperfusion의 - 상해에서 백혈구 - 내피 상호 작용의 실시간 디지털 이미징 (IRI)

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

유방암의 마우스 모델에서 종양 Microenvironment에 약물 응답의 영상을 라​​이브

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

기본 Preneoplastic 유방 상피 세포의 시간 경과 영상은 유방암의 유전자 변형 마우스 모델에서 파생

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

길이, 비 침습적을위한 안구의 앞쪽 상공 회의소에 이식<em&gt; 생체에</em실시간으로 단일 셀 해상도&gt; 영상

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

화학 요법에 의한 혈관 독성 - 실시간<em&gt; 생체 내</em&gt; 선박 손상의 영상

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

치과 복합 재료의 Cytocompatibility 평가하기위한 효율적인 방법으로 공 촛점 시간 경과 영상

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

신생아 마우스 폐에서 폐 유체 볼륨의 실시간 X 선 영상

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