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.).

Alla förfaranden som beskrivs måste utföras i enlighet med de riktlinjer och regler för användning av ryggradsdjur, inklusive förhandsgodkännande från den lokala Institutional Animal Care och användning kommittén (IACUC). 1. Framställning av lungmetastaser för Ex vivo Live Imaging (Transgena eller injektion i svansvenen) OBS: Lungmetastaser kan genereras genom användning av genetiskt manipulerade musmodeller eller genom intravenös (iv) in...

<ol>
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This manuscript describes a detailed method for ex vivo live imaging of lung metastasis in mouse models of metastasis. This imaging protocol provides a direct visualization of the dynamic and spatial tumor cell-stroma interactions within the lung microenvironment. It is a relatively easy and fast method that allows reliable imaging of lung metastasis for at least 4 hr. Movies acquired from these experiments can be used to track dynamic processes as cell motility and cellular interactions.
<p class="jove_cont...

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

Metastas är en viktig orsak för cancerrelaterad sjuklighet och dödlighet. Metastas är en flerstegsprocess och på grund av dess komplexitet, de exakta cellulära och molekylära processer som styr metastatisk spridning och tillväxt är fortfarande svårfångade. Live avbildning möjliggör visualisering av dynamiska och rumsliga interaktioner mellan celler och deras mikromiljö. Fasta tumörer metastaserar vanligen till lungorna. Emellertid den anatomiska lokaliseringen av lungorna utgör en utmaning för intravital avbildning. Detta protokoll ger en relativt enkel och snabb metod för ex vivo levande avbildning av det dynamiska samspelet mellan tumörceller och deras omgivande stroma inom lungmetastaser. Med användning av denna metod kan motiliteten av cancerceller såväl som interaktioner mellan cancerceller och stromaceller i deras mikromiljö visualiseras i realtid under flera timmar. Genom att använda transgena fluorescerande reporter möss, en fluorescerande cellinje, injicerbara fluorescerandemolekyler och / eller antikroppar, multipla komponenter i lungan mikromiljön kan visualiseras, såsom blodkärl och immunceller. Att avbilda olika celltyper, har en roterande skiva konfokalmikroskop som tillåter långsiktig kontinuerlig avbildning med snabb, fyra färgbild förvärv använts. Time-lapse filmer som sammanställts från bilder som samlats in under flera positioner och fokalplan visar interaktioner mellan levande metastaserande och immunceller under minst fyra timmar. Denna teknik kan användas för att ytterligare testa kemoterapi eller målsökande behandling. Dessutom kan detta förfarande anpassas för studiet av andra lungrelaterade patologier som kan påverka lungmikromiljö.

Using spinning-disk confocal microscopy, various mouse model systems and injectables, the metastatic microenvironment can be visualized and tracked over time. Using an MMTV-PyMT; ACTB-ECFP; c-fms-EGFP triple transgenic mouse model, different cellular components are fluorescently labeled (Figure 2A, Movie 1). The typical structure of the lung parenchyma can be visualized in the CFP channel since all cells express ECFP under the &#946;-actin promoter. Larger/multicellular lung met...

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Ex vivo Live Imaging of Lung Metastasis and Their Microenvironment

Ex vivo Live avbildning av lungmetastas och deras mikromiljö

We describe a relatively simple method for ex vivo live imaging of the tumor cell-stroma interactions within lung metastasis, utilizing fluorescent reporters in mice. Using spinning-disk confocal microscopy, this technique enables visualization of live cells for at least 4 hr and could be adapted to study other inflammatory lung conditions.

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

Ex vivo Infektion av levande vävnad med oncolytic Virus

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

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

In vivo bioluminescence avbildning av tumör Hypoxi Dynamics i bröstcancer metastaser i hjärnan i en musmodell

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 Modeller för 4D Live-cell imaging av tumör: mikromiljö Interaktioner som påverkar malign progression

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

Realtidsundersökningen av Drug svar i tumören mikromiljön i musmodeller av bröstcancer

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

Molekylär profilering av Invasive tumormicroenvironmenten i en 3-dimensionell modell av kolorektal cancer celler och<em&gt; Ex vivo</em&gt; Fibroblaster

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

Metod för Isolerad<em&gt; Ex vivo</em&gt; Lung Perfusion i en råttmodell: Lärdomar från Utveckla en Rat EVLP Program

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

Validering av Nanobody och antikroppsbaserade<em&gt; In Vivo</em&gt; Tumör Xenograft NIRF-imaging Experiment i möss med användning<em&gt; Ex vivo</em&gt; Flödescytometri och mikroskopi

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

Primärtumör och MEF Cell Isolation att studera lungmetastas

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

Utvärdering av lungmetastas i musbrösttumör Modeller av kvantitativ realtids-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

Precisionsskurna lungskivor som ett effektivt verktyg för ex vivo pulmonellkärlsstruktur och kontraktilitetsstudier

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