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.).

Alle beschriebenen Verfahren muss mit Richtlinien und Vorschriften für den Einsatz von Wirbeltieren, einschließlich der vorherigen Zustimmung durch die lokalen Institutional Animal Care und Use Committee (IACUC) entsprechend durchgeführt werden. 1. Erzeugung von Lungenmetastasen für Ex-Live-Imaging (Transgene oder Schwanzveneninjektion) vivo HINWEIS: Lungenmetastasen kann durch Verwendung gentechnisch veränderter Mausmodelle oder durch in...

<ol>
	<li>Chaffer, C. L., Weinberg, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+perspective+on+cancer+cell+metastasis.">A perspective on cancer cell metastasis.</a> Science. 331 (6024), 1559-1564 (2011).</li><li>Plaks, V., Kong, N., Werb, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+cancer+stem+cell+niche:+how+essential+is+the+niche+in+regulating+stemness+of+tumor+cells.">The cancer stem cell niche: how essential is the niche in regulating stemness of tumor cells.</a> Cell stem cell. 16 (3), 225-238 (2015).</li><li>Nguyen, D. X., Bos, P. D., Massague, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metastasis:+from+dissemination+to+organ-specific+colonization.">Metastasis: from dissemination to organ-specific colonization.</a> Nat Rev Cancer. 9 (4), 274-284 (2009).</li><li>Egeblad, M., Ewald, A. J., 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=Visualizing+stromal+cell+dynamics+in+different+tumor+microenvironments+by+spinning+disk+confocal+microscopy.">Visualizing stromal cell dynamics in different tumor microenvironments by spinning disk confocal microscopy.</a> Dis Model Mech. 1 (2-3), 155-167 (2008).</li><li>Ellenbroek, S. I. 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. R., 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=Spatiotemporally+separated+antigen+uptake+by+alveolar+dendritic+cells+and+airway+presentation+to+T+cells+in+the+lung.">Spatiotemporally separated antigen uptake by alveolar dendritic cells and airway presentation to T cells in the lung.</a> J Exp Med. 209 (6), 1183-1199 (2012).</li><li>Mendoza, A., Hong, S. -. H., 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=Modeling+metastasis+biology+and+therapy+in+real+time+in+the+mouse+lung.">Modeling metastasis biology and therapy in real time in the mouse lung.</a> J Clin Invest. 120 (8), 2979-2988 (2010).</li><li>Lelkes, E., Headley, M. B., Thornton, E. E., Looney, M. R., 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=The+spatiotemporal+cellular+dynamics+of+lung+immunity.">The spatiotemporal cellular dynamics of lung immunity.</a> Trends Immunol. 35 (8), 379-386 (2014).</li><li>Guy, C. T., Cardiff, R. D., Muller, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+mammary+tumors+by+expression+of+polyomavirus+middle+T+oncogene:+a+transgenic+mouse+model+for+metastatic+disease.">Induction of mammary tumors by expression of polyomavirus middle T oncogene: a transgenic mouse model for metastatic disease.</a> Mol Cell Biol. 12 (3), 954-961 (1992).</li><li>Hadjantonakis, A. -. K., Macmaster, S., Nagy, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryonic+stem+cells+and+mice+expressing+different+GFP+variants+for+multiple+non-invasive+reporter+usage+within+a+single+animal.">Embryonic stem cells and mice expressing different GFP variants for multiple non-invasive reporter usage within a single animal.</a> BMC Biotechnol. 2, (2002).</li><li>Casbon, A. -. J., Reynaud, D., 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=Invasive+breast+cancer+reprograms+early+myeloid+differentiation+in+the+bone+marrow+to+generate+immunosuppressive+neutrophils.">Invasive breast cancer reprograms early myeloid differentiation in the bone marrow to generate immunosuppressive neutrophils.</a> Proc Natl Acad Sci USA. 112 (6), 566-575 (2015).</li><li>Donovan, J., Brown, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Parenteral+injections.">Parenteral injections.</a> Curr Protoc Immunol. , (2006).</li><li>Halpern, J., Lynch, C. 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=The+application+of+a+murine+bone+bioreactor+as+a+model+of+tumor:+bone+interaction.">The application of a murine bone bioreactor as a model of tumor: bone interaction.</a> Clin Exp Metastas. 23 (7-8), 345-356 (2006).</li><li>Sasmono, R. T., Oceandy, D., 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=A+macrophage+colony-stimulating+factor+receptor-green+fluorescent+protein+transgene+is+expressed+throughout+the+mononuclear+phagocyte+system+of+the+mouse.">A macrophage colony-stimulating factor receptor-green fluorescent protein transgene is expressed throughout the mononuclear phagocyte system of the mouse.</a> Blood. 101 (3), 1155-1163 (2003).</li><li>Ewald, A. J., Werb, Z., Egeblad, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic,+long-term+in+vivo+imaging+of+tumor-stroma+interactions+in+mouse+models+of+breast+cancer+using+spinning-disk+confocal+microscopy.">Dynamic, long-term in vivo imaging of tumor-stroma interactions in mouse models of breast cancer using spinning-disk confocal microscopy.</a> Cold Spring Harb Protoc. (2), (2011).</li><li>Bonnans, C., Lohela, M., Werb, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+imaging+of+myeloid+cells+dynamics+in+ApcMin/++intestinal+tumors+by+spinning+disk+confocal+microscopy.">Real-time imaging of myeloid cells dynamics in ApcMin/+ intestinal tumors by spinning disk confocal microscopy.</a> J Vis Exp. (92), (2014).</li><li>Nakasone, E. S., Askautrud, H. 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. -. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+scratch+assay:+a+convenient+and+inexpensive+method+for+analysis+of+cell+migration+in+vitro.">In vitro scratch assay: a convenient and inexpensive method for analysis of cell migration in vitro.</a> Nat protoc. 2 (2), 329-333 (2007).</li><li>Nelson, K., Bobba, C., Ghadiali, S., Hayes, D. J., Black, S. M., Whitson, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+of+ex+vivo+lung+perfusion+as+a+platform+for+transplantation+research.">Animal models of ex vivo lung perfusion as a platform for transplantation research.</a> World J Exp Med. 4 (2), (2014).</li><li>Magness, S. T., Bataller, R., Yang, L., Brenner, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+dual+reporter+gene+transgenic+mouse+demonstrates+heterogeneity+in+hepatic+fibrogenic+cell+populations.">A dual reporter gene transgenic mouse demonstrates heterogeneity in hepatic fibrogenic cell populations.</a> Hepatology. 40 (5), 1151-1159 (2004).</li><li>Motoike, T., Loughna, S., 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=Universal+GFP+reporter+for+the+study+of+vascular+development.">Universal GFP reporter for the study of vascular development.</a> Genesis. 28 (2), (2000).</li><li>Srivastava, M. K., Andersson, 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=Myeloid+suppressor+cells+and+immune+modulation+in+lung+cancer.">Myeloid suppressor cells and immune modulation in lung cancer.</a> Immunotherapy. 4 (3), (2012).</li><li>Craig, A., Mai, J., Cai, S., Jeyaseelan, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutrophil+recruitment+to+the+lungs+during+bacterial+pneumonia.">Neutrophil recruitment to the lungs during bacterial pneumonia.</a> Infect Immun. 77 (2), 568-575 (2009).</li><li>Kreisel, D., Nava, R. G., 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=In+vivo+two-photon+imaging+reveals+monocyte-dependent+neutrophil+extravasation+during+pulmonary+inflammation.">In vivo two-photon imaging reveals monocyte-dependent neutrophil extravasation during pulmonary inflammation.</a> Proc Natl Acad Sci USA. 107 (42), 18073-18078 (2010).</li></ol>

Dieses Manuskript beschreibt ein detailliertes Verfahren zur ex vivo Echtzeit-Bildgebung von Lungenmetastasen in Maus-Modellen der Metastasierung. Diese Bildgebungsprotokoll stellt eine direkte Visualisierung der Dynamik und räumliche Tumorzell-Stroma-Interaktionen innerhalb der Lunge Mikroumgebung. Es ist eine relativ einfache und schnelle Methode, die für mindestens 4 h zuverlässige Abbildung von Lungenmetastasen ermöglicht. Filme aus diesen Experimenten erworben kann verwendet werden, um dynamische Proze...

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

Metastasierung ist eine Hauptursache für krebsbedingten Morbidität und Mortalität. Metastasierung ist ein mehrstufiger Prozess und aufgrund seiner Komplexität, die genauen zellulären und molekularen Prozesse, die metastatische Verbreitung und Wachstum sind immer noch schwer zu regieren. Echtzeit-Bildgebung ermöglicht die Visualisierung der dynamischen und räumlichen Wechselwirkungen von Zellen und ihrer Mikroumgebung. Soliden Tumoren metastasieren häufig in die Lunge. Jedoch stellt die anatomische Lage der Lungen eine Herausforderung für die intravital Bildgebung. Dieses Protokoll stellt eine relativ einfache und schnelle Methode zur ex vivo Echtzeit-Bildgebung der dynamischen Wechselwirkungen zwischen Tumorzellen und ihrer Umgebung Stroma in Lungenmetastasen. Mit dieser Methode kann die Motilität von Krebszellen als auch Wechselwirkungen zwischen Krebszellen und Stromazellen in ihrer Mikroumgebung für mehrere Stunden in Echtzeit visualisiert werden. transgenen fluoreszierenden Reporter-Mäuse Durch die Verwendung eines fluoreszierenden Zelllinie, injizierbare fluoreszenzmarkiertenMoleküle und / oder Antikörper, mehrere Komponenten der Lunge Mikroumgebung visualisiert, wie Blutgefäße und Immunzellen werden. Um das Bild zu den verschiedenen Zelltypen, eine sich drehende Scheibe konfokalen Mikroskop, das langfristige kontinuierliche Bildgebung mit schnellen, vierfarbige Bildaufnahme ermöglicht verwendet wurde. Zeitraffer-Filme aus Bildern zusammengestellt mehrere Positionen und Brennebenen gesammelt über zeigen Wechselwirkungen zwischen Live-metastatischen und Immunzellen für mindestens 4 Stunden. Diese Technik kann weiter zu testen Chemotherapie oder gezielte Therapie verwendet werden. Darüber hinaus könnte dieses Verfahren für die Untersuchung von anderen Lungenbezogenen Pathologien angepasst werden, dass die Lunge Mikroumgebung beeinflussen.

Mit Spinnen-Scheibe konfokalen Mikroskopie, verschiedene Mausmodellsystemen und Injektionen kann der metastatischen Mikroumgebung sichtbar gemacht und im Laufe der Zeit verfolgt werden. Mit Hilfe eines MMTV-PyMT; ACTB-ECFP; c-fms-EGFP dreifach transgenen Mausmodell, verschiedenen zellulären Komponenten werden fluoreszenzmarkierten (2A, Film 1). Die typische Struktur des Lungenparenchym kann in der GFP-Kanal sichtbar gemacht werden, da alle Zellen unter der β-Actin-Promotors e...

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

Ex-vivo-Live-Imaging von Lungenmetastasen und deren Mikromilieu

Wir beschreiben ein relativ einfaches Verfahren zur ex vivo Echtzeit-Bildgebung der Tumorzell Stroma Wechselwirkungen innerhalb Lungenmetastasen unter Verwendung fluoreszierenden Reportern in Mäusen. Verwendung von Spinnplatten konfokale Mikroskopie, ermöglicht diese Technik Visualisierung von lebenden Zellen für mindestens 4 h und angepasst werden könnten andere entzündliche Lungenerkrankungen zu untersuchen.

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 von lebendem Gewebe mit onkolytischen Viren

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

Forschung

Medizin

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-Biolumineszenz-Bildgebung von Tumorhypoxie Dynamics of Breast Cancer Hirnmetastasen in einem Mausmodell

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 Modelle für die 4D-Bildgebung lebender Zellen von Tumor: Mikroumgebung Wechselwirkungen, die Einfluss auf maligne 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

Live-Imaging of Drug Responses in der Tumor-Mikroumgebung in Mausmodellen von Brustkrebs

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

Molecular Profiling von der invasiven Tumor-Microenvironment in einem 3-Dimensional Model of Colorectal Cancer Cells und<em&gt; Ex-vivo-</em&gt; Fibroblasten

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

Methode Isolated<em&gt; Ex vivo</em&gt; Lung Perfusion in einem Rattenmodell: Lehren aus Entwicklung einer Ratte EVLP Programm gelernt

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

Validierung der Nanobody und Antikörper-basierte<em&gt; In-vivo-</em&gt; Tumorxenotransplantat NIRF-Bildgebung Experimente bei Mäusen verwenden<em&gt; Ex vivo</em&gt; Durchflusszytometrie und Mikroskopie

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

Primärtumor und MEF Zellisolation zu Lungenmetastasen Studieren

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

Beurteilung von Lungenmetastasen in Maus-Mammatumormodelle durch quantitative Echtzeit-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

Präzisionsschnitt-Lungenschnitte als effizientes Werkzeug für Ex-vivo-Studien zur Struktur und Kontraktilität von Lungengefäßen

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