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.).

All procedures described must be performed in accordance with guidelines and regulations for the use of vertebrate animals, including prior approval by the local Institutional Animal Care and Use Committee (IACUC).
1. Generation of Lung Metastases for Ex vivo Live Imaging (Transgenic or Tail Vein Injection)
NOTE: Lung metastases can be generated by utilizing genetically engineered mouse models or by intravenous (i.v) injection of cancer cells.
<ol>
	<li>Generate lung metastases for imaging by crossing a genetically engineered tumor mouse model into a transgenic reporter mouse, e.g., cross the breast cancer mouse model, mouse mammary tumor virus long terminal repeat-polyoma middle T antigen (MMTV-PyMT)11 into ACTB-ECFP mouse model12. 
	NOTE: The ACTB-ECFP model expresses enhanced cyan fluorescent protein (ECFP) under the &#946;-actin promoter such that all cells fluoresce in the blue, CFP channel. However, cancer cells are by far the most prominent and appear as a bulk of ECFP-positive cells under the microscope. The MMTV-PyMT mouse model develops a progressive disease, in which mammary tumor growth is associated with the dissemination of cancer cells to the periphery, especially to the lungs. In MMTV-PyMT mice on the FVB/n background, micrometastases can be observed around 10-11 weeks of age. Generally, these progress to macrometastases at around 14 weeks of age13. 
	OR</li>
	<li>Generate experimental metastases using primary cells or syngeneic cell lines. Use in vitro manipulated primary tumor cells or cell lines (e.g., transduction) followed by i.v. injection14.
	<ol>
		<li>Briefly, in this protocol, inject a green fluorescent protein (GFP)-expressing (+) MMTV-PyMT cell line into fluorescent reporter mice (ACTB-ECFP) or wildtype mice. Then, visualize these cells referred to as VO-PyMT cells15 using the green, GFP channel. 
		NOTE: The original VO-PyMT cell line was derived at the Vanderbilt Orthopaedics in Nashville, TN. VO stands for Vanderbilt Orthopaedics.</li>
		<li>Following the injection of 106 cells (in 200 &#181;l), observe cancer cell extravasation immediately and up to a few hours after injection; observe micrometastases between 1-3 weeks after injection and detect macrometastases 3 weeks after injection15. 
		NOTE: Fewer cells can be injected to prolong the time from injection to metastatic growth.</li>
	</ol>
	</li>
</ol>
2. Labeling of Components of Interest in the Metastatic Microenvironment (Transgenic and/or Injectables)
NOTE: Labeling can be achieved by transgenic mice and/or by various injectables. Make sure to use different fluorescent colors for the labeling of various cell types.
<ol>
	<li>Label components of the metastatic microenvironment using transgenic mice. Cross the previously mentioned mouse tumor model (e.g.,&#160;MMTV-PyMT x ACTB-ECFP) into a transgenic mouse model in which the stromal cells of interest are labeled by a fluorescent protein which is not ECFP, e.g.,&#160;c-fms-EGFP4,16. 
	NOTE: In addition to visualization of cancer cells in the CFP channel, this enables visualization of myeloid cells in the GFP channel4. 
	AND/OR</li>
	<li>Label various components of the metastatic microenvironment using injectables into transgenic fluorescent reporter mice or (non-fluorescent) wildtype mice. 
	NOTE: Several compounds can be injected to label various components of the metastatic microenvironment, e.g., an AF647-conjugated Gr-1 antibody is used here to label neutrophils and some monocytes13 and different molecular weight dextrans are used to label lung capillaries. For preparation of these injectables see step 4. </li>
</ol>
3. Preparation of Materials Before Dissection
<ol>
	<li>2% Agarose
	<ol>
		<li>Weigh 0.2 g of agarose and add to 10 ml 1 x PBS. Heat the solution to dissolve the agarose. Agarose will solidify at RT, so maintain it in a 37 &#176;C water bath until used for inflation.</li>
	</ol>
	</li>
	<li>CO2 and temperature controller
	<ol>
		<li>Check ddH2O in the humidification chamber. Refill when needed. Insert configuration plate into temperature stage plate holder (climate chamber). Turn on the CO2 controller and set CO2 at 5%. Make sure the airflow rate is set at 0.4 Nl/min.</li>
		<li>Open the air and CO2 valves. Turn on the temperature controller. Make sure the temperature of the climate chamber and the lid are set at 37 &#176;C.</li>
		<li>Release air pressure on CO2 meter. Check CO2 increasing, equilibration can take up to 30 min.</li>
	</ol>
	</li>
	<li>Spinning disk confocal microscope 
	NOTE: Details of the microscope set-up have been previously described4,17. 
	<ol>
		<li>Turn on the lasers (the argon laser for 488 nm excitation and the solid-state 405 nm, 561 nm and 640 nm lasers). Turn on the microscope, the camera, the spinning disk control unit, the AOTF, the laser control unit and the camera controller.</li>
		<li>Open the microscope shutter, turn on the computer running the microscope and open the software.</li>
	</ol>
	</li>
	<li>Preparation of the tools and dissection platform.
	<ol>
		<li>Turn on the hot bead sterilizer and let it reach 250 &#176;C. Clean 2 pairs of surgical scissors and forceps with water and soap. Sterilize the tools for at least 30 sec. Let the tools cool off. Use a polystyrene lid as dissection platform. Cover it with a piece of lab soaker.</li>
	</ol>
	</li>
</ol>
4. Preparation of Injections
NOTE: Depending on the half-life and the preferred response, inject fluorescently labeled antibodies and/or fluorescent molecules either immediately prior to animal sacrifice or a couple of hours to days before.
<ol>
	<li>To image Gr1-positive neutrophils and monocytes, prepare a syringe with 7 &#181;l of stock AF647-conjugated Gr-1 antibody (1 mg/ml) into 100 &#181;l of sterile PBS under the hood. Place a 27 G &#189; needle on the syringe.</li>
	<li>To image lung capillaries, prepare a second and third syringe with 100 &#181;l of either 70 kD rhodamine-conjugated dextran (4 mg/ml) or 10 kD AF647-conjugated dextran (4 mg/ml). Place a 27 G &#189; needle on the syringes.</li>
	<li>Inject the AF647-conjugated antibody solution i.v. 5 hr prior to lungs&#8217; excision.</li>
	<li>Inject one or both dextran solutions i.v. immediately prior to lungs&#8217; excision.</li>
</ol>
5. Preparation of Lungs for Ex vivo Live Imaging
NOTE: Try to work as sterile and careful as possible to avoid unnecessary challenges of the immune cells within the lungs.
<ol>
	<li>Inject the mouse intraperitoneal (i.p.) with a lethal overdose of an anesthetic permitted by the animal protocol approved by IACUC, e.g., 1 ml of 2.5% Avertin. Wait for the mouse to stop breathing and be completely non-responsive to noxious stimuli (hind paw pinch). 
	NOTE: Cervical dislocation and carbon dioxide euthanasia should be avoided as it can detrimentally affect lung cell viability.</li>
	<li>Immobilize the mouse on a dissection board and sterilize the mouse with 70% ethanol.</li>
	<li>Use surgical scissors to first make a transverse epigastric incision through the skin, followed by a similar incision through the peritoneum. Hold the dissection board in a vertical position and cut the descending aorta, so that blood pools down in the abdomen and not in the chest cavity.</li>
	<li>Snip a small opening in the diaphragm to release vacuum. Cut along the 10th and 12th rib to excise the diaphragm and get visual access to the lungs.</li>
	<li>Use surgical scissors to cut the skin up to the trachea over the ribcage but leave the ribcage intact. Separate the skin from the ribcage. Expose the trachea by removing the surrounding connective tissue, being careful not to damage the trachea itself (Figure 1A).</li>
	<li>Snip a small opening approximately 1 mm in diameter in the exposed trachea parallel to the cartilaginous rings, as close to the larynx as possible (Figure 1B). Be careful not to cut completely through the trachea.</li>
	<li>Take a 20 G needle and gently insert the needle 4-5 mm into the trachea without any counter force (Figure 1D). The end of the needle should be visible through the trachea (Figure 1C). Use forceps to stabilize the needle in the trachea. Alternatively, a suture may be tied around the trachea to hold the needle in place. 
	NOTE: By inserting too deep, the carina may be traumatized or only one side of the lungs might be inflated.</li>
	<li>Fill a syringe with 400 &#181;l of 37 &#176;C 2% low-melting-temperature agarose (taken directly from a constant temperature bath). Make sure the dissection board is standing up and slowly instill the warm agarose through the needle into the lungs, use ~400 &#181;l to inflate the lungs. 
	NOTE: Watch the lungs inflating inside the ribcage. Do not over-inflate the lung as it will rupture.</li>
	<li>Once the lungs are inflated, filling ~&#8532; of the rib cage, detach the syringe and keep the needle inside the trachea to prevent any agarose from leaking.</li>
	<li>Pour approximately 50 ml of 20 &#176;C PBS over the inflated lungs to allow the agarose inside the lungs to set and solidify. Slowly remove the needle and close the trachea with forceps to prevent any non-solidified agarose from leaking.</li>
	<li>Expose the lungs by performing a sternotomy and subsequently excise the lungs. For excision of the lungs, hold on to the trachea while cutting through the trachea completely. Gently pull the trachea up, cut away the connective tissue and esophagus while pulling the lungs out of the chest cavity until the lungs is separated from the mouse (Figure 1E).</li>
	<li>Immerse the lungs in warm RPMI-1640 to wash off excessive blood and gently separate the lobes by using scissors and forceps to cut the lobes&#8217; main stem bronchus at hilum (Figure 1F).</li>
	<li>Place the lobes, with the flat surface down to maximize imaging surface, in a well of a 24-well imaging plate (Figure 1G). Add 100 &#181;l of 37 &#176;C RPMI-1640 on top of the lobes. Place several 15 mm circular microscope cover slides on top of the lobes to prevent it from floating.</li>
	<li>Pour warm PBS into the surrounding wells to prevent the RPMI-1640 media from evaporating. Insert the 24-well plate into the equilibrated climate chamber and maintain the lung lobes at 37 &#176;C with air and 5% CO2. Insert the climate chamber on the stage of the confocal microscope. 
	NOTE: Other gas mixtures (e.g., 5% O2, 5% CO2 in N2 to examine cell behavior under conditions of hypoxia/lower oxygen) could also be considered. </li>
</ol>
<img alt="Figure 1" src="/files/ftp_upload/53741/53741fig1.jpg" /> 
Figure 1. Protocol for preparation of lungs for live imaging. (A) Exposure of the trachea after preparation of the mouse. (B) Small snip made in the exposed trachea parallel to the cartilaginous rings. (C) 20 G needle inserted 4-5 mm into the trachea. (D) Instillation of 400 &#181;l 2% low-melting-temperature agarose into the lungs. (E) Inflated lungs separated from the mouse. (F) Lobes separated after inflation. (G) Lobes placed in a well of a 24-well imaging plate. <a href="https://www.jove.com/files/ftp_upload/53741/53741fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
6. Acquisition and Analysis of Images
NOTE: Images can be acquired with a variety of spinning disk confocal microscopes supported by various software programs. In this protocol, either &#181;Manager with a custom-made spinning disk confocal microscope or Zen with a commercially-available spinning disk confocal microscope is used for image acquisition, while Imaris is used for movie editing and analysis.
<ol>
	<li>Acquire images using &#181;Manager. A detailed step by step protocol for the acquisition of images using &#181;Manager software is previously described18. 
	OR</li>
	<li>Acquire images using image analysis software such as Zen (see Figure S1).
	<ol>
		<li>Click on the &#8216;Locate&#8217; tab, and choose objective (10x or 20x) in the &#8216;Light Path&#8217; tool (Figure S1A, red box). Subsequently, click on &#8216;Eyes &#8211; DAPI&#8217; to look at the CFP channel through the eyepiece (Figure S1A, blue box). Localize the sample manually using the microscope. Click &#8216;All Off&#8217;after the tissue is center of the field of view.</li>
		<li>Click on the &#8216;Acquisition&#8217; tab to set all the parameters for image acquisition.</li>
		<li>In the &#8216;Channels&#8217; tool, click the &#8216;+&#8217; button (Figure S1B, red box). A pop-up menu appears and search for the dye(s) present in the sample in the &#8216;Dye Database&#8217; (Figure S1B). Select the dye and click &#8216;Add&#8217;. 
		NOTE: The program will set all filters to be optimized. A dye can be deleted by selecting it followed by clicking the trash can button (Figure S1B, yellow box).</li>
		<li>In the &#8216;Acquisition Mode&#8217; menu, set &#8216;Binning&#8217; to 5x5. Double click on ECFP in the channels menu to select it. Lower the laser power to 20% so the sample will not be bleached while setting up the parameters for image acquisition.</li>
		<li>Check the &#8216;Tiles&#8217; box in the &#8216;Experiment Manager&#8217; section and the tiles tool appears in the &#8216;Multidimensional Acquisition&#8217; tool group (Figure S1C). Click on &#8216;Advanced Setup&#8217; button to view the live image from the camera. Click on the &#8216;Add&#8217; button in the &#8216;Positions&#8217; section to add 4 to 6 positions to the experiment. To delete a position, select that position and click on the trash can button.</li>
		<li>In the &#8216;Acquisition Parameter&#8217; tool group, open the &#8216;Focus Strategy&#8217; tool, and select &#8216;Absolute Fixed Z-position&#8217; from the dropdown list.</li>
		<li>Check the Z-Stack box in the &#8216;Experiment Manager&#8217; section and the Z-Stack tool appears in the &#8216;Multidimensional Acquisition&#8217; tool group (Figure S1D). Double click on one of the positions in the &#8216;Positions&#8217; section and press &#8216;Live&#8217;. Manually set first and set last position of the imaging field. Set the interval at 4 &#181;m. 
		NOTE: The program will determine the number of slices for the chosen range and interval. Ideally, 5-7 slices are convenient to allow sufficient visualization and rapid image acquisition.</li>
		<li>Check the &#8216;Time Series&#8217; box in the &#8216;Experiment Manager&#8217; section. Set desired &#8216;Duration&#8217; and &#8216;Interval&#8217; times in the &#8216;Time Series&#8217; tool that appeared in the &#8216;Multidimensional Acquisition&#8217; tool group (Figure S1E).</li>
		<li>In the &#8216;Acquisition Mode&#8217; menu, set &#8216;Binning&#8217; to 2x2. Double click on a fluorophore in the channels menu to select it and increase the laser power to 100%. Press &#8216;Live&#8217; and adjust the &#8216;Exposure Time&#8217;. Repeat this for every fluorophore.</li>
		<li>Check the &#8216;Enable Auto Save&#8217; box. Select a folder and type in the name of the file. All the acquired images will be automatically saved in this folder.</li>
		<li>Click on &#8216;Start Experiment&#8217; in the &#8216;Experiment Manager&#8217; section to start image acquisition.</li>
	</ol>
	</li>
	<li>After image acquisition, compile the raw data in Imaris software. Convert images to .ims files and adjustments can be made. A detailed step by step protocol for conversion of files, making adjustments and saving movies using Imaris is previously described18.</li>
	<li>When saving the movie, set the &#8216;Frame Rate&#8217; to 5 frames per second (fps).</li>
</ol>

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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>

The authors have no conflicts of interest to disclose. All animal experiments were conducted in accordance with IACUC approved protocols, UCSF.

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.
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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&amp;nbsp; 4mg/ml in 1 x PBS and store at -20&amp;nbsp; &amp;deg;C. Use 0.4 mg per animal.&amp;nbsp;</td></tr><tr><td>10,000 kDa Dextran, Alexa Fluor 647 conjugated</td><td>Invitrogen</td><td>D22914</td><td>Dilute to&amp;nbsp; 4mg/ml in 1 x PBS and store at -20&amp;nbsp; &amp;deg;C. Use 0.4 mg per animal.&amp;nbsp;</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&amp;nbsp;</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&amp;nbsp;</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&amp;nbsp;</td><td>BD</td><td>309659</td><td></td></tr><tr><td>27 G x 1/2 needle&amp;nbsp;&amp;nbsp;</td><td>BD</td><td>305109</td><td>for i.v. injection</td></tr><tr><td>20 G x 1 needle, short bevel&amp;nbsp;&amp;nbsp;</td><td>BD</td><td>305178</td><td></td></tr><tr><td>Low-melting-temperature agarose&amp;nbsp;</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 &amp;deg;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&amp;nbsp;</td><td>E&amp;amp;K scientific</td><td>EK-42892</td><td></td></tr><tr><td>Glass cover slides, 15 mm&amp;nbsp;</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

Metastasis is a major cause for cancer-related morbidity and mortality. Metastasis is a multistep process and due to its complexity, the exact cellular and molecular processes that govern metastatic dissemination and growth are still elusive. Live imaging allows visualization of the dynamic and spatial interactions of cells and their microenvironment. Solid tumors commonly metastasize to the lungs. However, the anatomical location of the lungs poses a challenge to intravital imaging. This protocol provides a relatively simple and quick method for ex vivo live imaging of the dynamic interactions between tumor cells and their surrounding stroma within lung metastasis. 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. By using transgenic fluorescent reporter mice, a fluorescent cell line, injectable fluorescently labeled molecules and/or antibodies, multiple components of the lung microenvironment can be visualized, such as blood vessels and immune cells. To image the different cell types, a spinning disk confocal microscope that allows long-term continuous imaging with rapid, four-color image acquisition has been used. Time-lapse movies compiled from images collected over multiple positions and focal planes show interactions between live metastatic and immune cells for at least 4 hr. This technique can be further used to test chemotherapy or targeted therapy. Moreover, this method could be adapted for the study of other lung-related pathologies that may affect the lung microenvironment.

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

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.

Ex vivo Live Imaging of Lung Metastasis and Their Microenvironment

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

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12.3K Views.  University of California. 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.

Video: Ex vivo Live Imaging of Lung Metastasis and Their Microenvironment

Long-term High-Resolution Intravital Microscopy in the Lung with a Vacuum Stabilized Imaging Window

Metastasis to secondary sites such as the lung, liver and bone is a traumatic event with a mortality rate of approximately 90% 1. Of these sites, the lung is the most difficult to assess using intravital optical imaging due to its enclosed position within the body, delicate nature and vital role in sustaining proper physiology. While clinical modalities (positron emission tomography (PET), magnetic resonance imaging (MRI) and computed tomography (CT)) are capable of providing noninvasive images of this tissue, they lack the resolution necessary to visualize the earliest seeding events, with a single pixel consisting of nearly a thousand cells. Current models of metastatic lung seeding postulate that events just after a tumor cell&#39;s arrival are deterministic for survival and subsequent growth. This means that real-time intravital imaging tools with single cell resolution 2 are required in order to define the phenotypes of the seeding cells and test these models. While high resolution optical imaging of the lung has been performed using various ex vivo preparations, these experiments are typically single time-point assays and are susceptible to artifacts and possible erroneous conclusions due to the dramatically altered environment (temperature, profusion, cytokines, etc.) resulting from removal from the chest cavity and circulatory system 3. Recent work has shown that time-lapse intravital optical imaging of the intact lung is possible using a vacuum stabilized imaging window 2,4,5 however, typical imaging times have been limited to approximately 6 hr. Here we describe a protocol for performing long-term intravital time-lapse imaging of the lung utilizing such a window over a period of 12 hr. The time-lapse image sequences obtained using this method enable visualization and quantitation of cell-cell interactions, membrane dynamics and vascular perfusion in the lung. We further describe an image processing technique that gives an unprecedentedly clear view of the lung microvasculature.

This protocol describes the use of multiphoton microscopy to perform long-term high-resolution, single cell imaging of the intact lung in real time using a vacuum stabilized imaging window.

Metastasis to secondary sites such as the lung, liver and bone is a traumatic event with a mortality rate of approximately 90% 1. Of these sites, the lung ...

Cancer Research

Practical Considerations in Studying Metastatic Lung Colonization in Osteosarcoma Using the Pulmonary Metastasis Assay

The pulmonary metastasis assay (PuMA) is an ex vivo lung explant and closed cell culture system that permits researchers to study the biology of lung colonization in osteosarcoma (OS) by fluorescence microscopy. This article provides a detailed description of the protocol, and discusses examples of obtaining image data on metastatic growth using widefield or confocal fluorescence microscopy platforms. The flexibility of the PuMA model permits researchers to study not only the growth of OS cells in the lung microenvironment, but also to assess the effects of anti-metastatic therapeutics over time. Confocal microscopy allows for unprecedented, high-resolution imaging of OS cell interactions with the lung parenchyma. Moreover, when the PuMA model is combined with fluorescent dyes or fluorescent protein genetic reporters, researchers can study the lung microenvironment, cellular and subcellular structures, gene function, and promoter activity in metastatic OS cells. The PuMA model provides a new tool for osteosarcoma researchers to discover new metastasis biology and assess the activity of novel anti-metastatic, targeted therapies.

The goal of this article is to provide a detailed description of the protocol for the pulmonary metastasis assay (PuMA). This model permits researchers to study metastatic osteosarcoma (OS) cell growth in lung tissue using a widefield fluorescence or confocal laser-scanning microscope.

The pulmonary metastasis assay (PuMA) is an ex vivo lung explant and closed cell culture system that permits researchers to study the biology of lung ...

The Establishment of a Lung Colonization Assay for Circulating Tumor Cell Visualization in Lung Tissues

Metastasis is the major cause of cancer death. The role of circulating tumor cells (CTCs) in promoting cancer metastasis, in which lung colonization by CTCs critically contributes to early lung metastatic processes, has been vigorously investigated. As such, animal models are the only approach that captures the full systemic process of metastasis. Given that problems occur in previous experimental designs for examining the contributions of CTCs to blood vessel extravasation, we established an in vivo lung colonization assay in which a long-term-fluorescence cell-tracer, carboxyfluorescein succinimidyl ester (CFSE), was used to label suspended tumor cells and lung perfusion was performed to clear non-specifically trapped CTCs prior to lung removal, confocal imaging, and quantification. Polymeric fibronectin (polyFN) assembled on CTC surfaces has been found to mediate lung colonization in the final establishment of metastatic tumor tissues. Here, to specifically test the requirement of polyFN assembly on CTCs for lung colonization and extravasation, we performed short term lung colonization assays in which suspended Lewis lung carcinoma cells (LLCs) stably expressing FN-shRNA (shFN) or scramble-shRNA (shScr) and pre-labeled with 20 &#956;M of CFSE were intravenously inoculated into C57BL/6 mice. We successfully demonstrated that the abilities of shFN LLC cells to colonize the mouse lungs were significantly diminished in comparison to shScr LLC cells. Therefore, this short-term methodology may be widely applied to specifically demonstrate the ability of CTCs within the circulation to colonize the lungs.

An animal model is needed to decipher the role of circulating tumor cells (CTCs) in promoting lung colonization during cancer metastasis. Here, we established and successfully performed an in vivo assay to specifically test the requirement of polymeric fibronectin (polyFN) assembly on CTCs for lung colonization.

Metastasis is the major cause of cancer death. The role of circulating tumor cells (CTCs) in promoting cancer metastasis, in which lung colonization by ...

Improved Visualization of Lung Metastases at Single Cell Resolution in Mice by Combined In-situ Perfusion of Lung Tissue and X-Gal Staining of lacZ-Tagged Tumor Cells

Metastasis is the main cause of death in the majority of cancer types and consequently a main focus in cancer research. However, the detection of micrometastases by radiologic imaging and the success in their therapeutic eradication remain limited.
While animal models have proven to be invaluable tools for cancer research1, the monitoring/visualization of micrometastases remains a challenge and inaccurate evaluation of metastatic spread in preclinical studies potentially leads to disappointing results in clinical trials2. Consequently, there is great interest in refining the methods to finally allow reproducible and reliable detection of metastases down to the single cell level in normal tissue. The main focus therefore is on techniques, which allow the detection of tumor cells in vivo, like micro-computer tomography (micro-CT), positron emission tomography (PET), bioluminescence or fluorescence imaging3,4. We are currently optimizing these techniques for in vivo monitoring of primary tumor growth and metastasis in different osteosarcoma models. Some of these techniques can also be used for ex vivo analysis of metastasis beside classical methods like qPCR5, FACS6 or different types of histological staining. As a benchmark, we have established in the present study the stable transfection or transduction of tumor cells with the lacZ gene encoding the bacterial enzyme &beta;-galactosidase that metabolizes the chromogenic substrate 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-Gal) to an insoluble indigo blue dye7 and allows highly sensitive and selective histochemical blue staining of tumor cells in mouse tissue ex vivo down to the single cell level as shown here. This is a low-cost and not equipment-intensive tool, which allows precise validation of metastasis8 in studies assessing new anticancer therapies9-11. A limiting factor of X-gal staining is the low contrast to e.g. blood-related red staining of well vascularized tissues. In lung tissue this problem can be solved by in-situ lung perfusion, a technique that was recently established by Borsig et al.12 who perfused the lungs of mice under anesthesia to clear them from blood and to fix and embed them in-situ under inflation through the trachea. This method prevents also the collapse of the lung and thereby maintains the morphology of functional lung alveoli, which improves the quality of the tissue for histological analysis. In the present study, we describe a new protocol, which takes advantage of a combination of X-gal staining of lacZ-expressing tumor cells and in-situ perfusion and fixation of lung tissue. This refined protocol allows high-sensitivity detection of single metastatic cells in the lung and enabled us in a recent study to detect "dormant" lung micrometastases in a mouse model13, which was originally described to be non-metastatic14.

Improved Visualization of Lung Metastases at Single Cell Resolution in Mice by Combined In-situ Perfusion of Lung Tissue and X-Gal Staining of lacZ-Tagged Tumor Cells

The novel protocol reported in the present study allows selective detection of lung metastases at single cell resolution in mice by combined in-situ lung perfusion and fixation and X-Gal staining of lacZ-tagged tumor cells.

Metastasis is the main cause of death in the majority of cancer types and consequently a main focus in cancer research. However, the detection of ...

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

The tumor microenvironment plays a pivotal role in tumor initiation, progression, metastasis, and the response to anti-cancer therapies. Three-dimensional co-culture systems are frequently used to explicate tumor-stroma interactions, including their role in drug responses. However, many of the interactions that occur in vivo in the intact microenvironment cannot be completely replicated in these in vitro settings. Thus, direct visualization of these processes in real-time has become an important tool in understanding tumor responses to therapies and identifying the interactions between cancer cells and the stroma that can influence these responses. Here we provide a method for using spinning disk confocal microscopy of live, anesthetized mice to directly observe drug distribution, cancer cell responses and changes in tumor-stroma interactions following administration of systemic therapy in breast cancer models. We describe procedures for labeling different tumor components, treatment of animals for observing therapeutic responses, and the surgical procedure for exposing tumor tissues for imaging up to 40 hours. The results obtained from this protocol are time-lapse movies, in which such processes as drug infiltration, cancer cell death and stromal cell migration can be evaluated using image analysis software.

We describe a method for imaging response to anti-cancer treatment in vivo and at single cell resolution.

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

Combined Use of Tail Vein Metastasis Assays and Real-Time In Vivo Imaging to Quantify Breast Cancer Metastatic Colonization and Burden in the Lungs

Metastasis is the main cause of cancer-related deaths and there are limited therapeutic options for patients with metastatic disease. The identification and testing of novel therapeutic targets that will facilitate the development of better treatments for metastatic disease requires preclinical in vivo models. Demonstrated here is a syngeneic mouse model for assaying breast cancer metastatic colonization and subsequent growth. Metastatic cancer cells are stably transduced with viral vectors encoding firefly luciferase and ZsGreen proteins. Candidate genes are then stably manipulated in luciferase/ZsGreen-expressing cancer cells and then the cells are injected into mice via the lateral tail vein to assay metastatic colonization and growth. An in vivo imaging device is then used to measure the bioluminescence or fluorescence of the tumor cells in the living animals to quantify changes in metastatic burden over time. The expression of the fluorescent protein allows the number and size of metastases in the lungs to be quantified at the end of the experiment without the need for sectioning or histological staining. This approach offers a relatively quick and easy way to test the role of candidate genes in metastatic colonization and growth, and provides a great deal more information than traditional tail vein metastasis assays. Using this approach, we show that simultaneous knockdown of Yes associated protein (YAP) and transcriptional co-activator with a PDZ-binding motif (TAZ) in breast cancer cells leads to reduced metastatic burden in the lungs and that this reduced burden is the result of significantly impaired metastatic colonization and reduced growth of metastases.

The described approach combines experimental tail vein metastasis assays with in vivo live animal imaging to allow real-time monitoring of breast cancer metastasis formation and growth in addition to the quantification of metastasis number and size in the lungs.

Metastasis is the main cause of cancer-related deaths and there are limited therapeutic options for patients with metastatic disease. The identification ...

A Permanent Window for Investigating Cancer Metastasis to the Lung

Metastasis, accounting for ~90% of cancer-related mortality, involves the systemic spread of cancer cells from primary tumors to secondary sites such as the bone, brain, and lung. Although extensively studied, the mechanistic details of this process remain poorly understood. While common imaging modalities, including computed tomography (CT), positron emission tomography (PET), and magnetic resonance imaging (MRI), offer varying degrees of gross visualization, each lacks the temporal and spatial resolution necessary to detect the dynamics of individual tumor cells. To address this, numerous techniques have been described for intravital imaging of common metastatic sites. Of these sites, the lung has proven especially challenging to access for intravital imaging owing to its delicacy and critical role in sustaining life. Although several approaches have previously been described for single-cell intravital imaging of the intact lung, all involve highly invasive and terminal procedures, limiting the maximum possible imaging duration to 6-12 h. Described here is an improved technique for the permanent implantation of a minimally invasive thoracic optical Window for High-Resolution Imaging of the Lung (WHRIL). Combined with an adapted approach to microcartography, the innovative optical window facilitates serial intravital imaging of the intact lung at single-cell resolution across multiple imaging sessions and spanning multiple weeks. Given the unprecedented duration of time over which imaging data can be gathered, the WHRIL can facilitate the accelerated discovery of the dynamic mechanisms underlying metastatic progression and numerous additional biologic processes within the lung.

Here, we present a protocol for the surgical implantation of a permanently indwelling optical window for the murine thorax, which enables high-resolution, intravital imaging of the lung. The permanence of the window makes it well-suited to the study of dynamic cellular processes in the lung, especially those that are slowly evolving, such as metastatic progression of disseminated tumor cells.

Metastasis, accounting for ~90% of cancer-related mortality, involves the systemic spread of cancer cells from primary tumors to secondary sites such as ...

A Label-Free Segmentation Approach for Intravital Imaging of Mammary Tumor Microenvironment

The ability to visualize complex and dynamic physiological interactions between numerous cell types and the extracellular matrix (ECM) within a live tumor microenvironment is an important step toward understanding mechanisms that regulate tumor progression. While this can be accomplished through current intravital imaging techniques, it remains challenging due to the heterogeneous nature of tissues and the need for spatial context within the experimental observation. To this end, we have developed an intravital imaging workflow that pairs collagen second harmonic generation imaging, endogenous fluorescence from the metabolic co-factor NAD(P)H, and fluorescence lifetime imaging microscopy (FLIM) as a means to non-invasively compartmentalize the tumor microenvironment into basic domains of the tumor nest, the surrounding stroma or ECM, and the vasculature. This non-invasive protocol details the step-by-step process ranging from the acquisition of time-lapse images of mammary tumor models to post-processing analysis and image segmentation. The primary advantage of this workflow is that it exploits metabolic signatures to contextualize the dynamically changing live tumor microenvironment without the use of exogenous fluorescent labels, making it advantageous for human patient-derived xenograft (PDX) models and future clinical use where extrinsic fluorophores are not readily applicable.

The intravital imaging method described here utilizes collagen second harmonic generation and endogenous fluorescence from the metabolic co-factor NAD(P)H to non-invasively segment an unlabeled tumor microenvironment into tumor, stromal, and vascular compartments for in-depth analysis of 4D intravital images.

The ability to visualize complex and dynamic physiological interactions between numerous cell types and the extracellular matrix (ECM) within a live tumor ...

In Vivo Imaging to Measure Spontaneous Lung Metastasis of Orthotopically-injected Breast Tumor Cells

Metastasis remains the primary cause of cancer-related death. The succession of events that characterize the metastatic cascade presents multiple opportunities for therapeutic intervention, and the ability to accurately model them in mice is critical to evaluate their effects. Here, a step-by-step protocol is presented for the establishment of orthotopic primary breast tumors and the subsequent monitoring of the establishment and growth of metastatic lesions in the lung using in vivo bioluminescence imaging. This methodology allows for the evaluation of treatment or its biological effects along the entire range of metastatic development, from primary tumor escape to outgrowth in the lungs. Breast orthotopic tumors are generated in mice via injection of a luciferase-labeled cell suspension in the 4th mammary gland. Tumors are allowed to grow and disseminate for a specific amount of time and are then surgically resected. Upon resection, spontaneous lung metastasis is detected, and the growth over time is monitored using in vivo bioluminescence imaging. At the desired experimental endpoint, lung tissue can be collected for downstream analysis. The treatment of established, clinically evident metastasis is critical to improve outcomes for stage IV cancer patients, and it can be evaluated through tail vein models of experimental lung metastasis. However, metastatic dissemination occurs early in breast cancer, and many patients have latent, subclinical disseminated disease after surgery. Utilization of spontaneous models such as this one provides the opportunity to study the whole spectrum of the disease, especially the systemic effects driven by treatment of the primary tumor such as pre-metastatic niche priming, and evaluate treatments on dormant and subclinical disease after surgery.

In Vivo Imaging to Measure Spontaneous Lung Metastasis of Orthotopically-injected Breast Tumor Cells

The manuscript describes a methodology for the establishment as well as longitudinal growth monitoring of spontaneous lung metastasis from orthotopically-injected breast tumors, amenable to intervention at all stages of the metastatic cascade.

Metastasis remains the primary cause of cancer-related death. The succession of events that characterize the metastatic cascade presents multiple ...

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.

This protocol describes the use of multiphoton microscopy to perform extended time-lapse imaging of multicellular interactions in real time, in vivo at single cell resolution.

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

Ex vivo Live Imaging of Lung Metastasis and Their Microenvironment

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Chapters in this video

Long-term High-Resolution Intravital Microscopy in the Lung with a Vacuum Stabilized Imaging Window

Practical Considerations in Studying Metastatic Lung Colonization in Osteosarcoma Using the Pulmonary Metastasis Assay

The Establishment of a Lung Colonization Assay for Circulating Tumor Cell Visualization in Lung Tissues

Improved Visualization of Lung Metastases at Single Cell Resolution in Mice by Combined In-situ Perfusion of Lung Tissue and X-Gal Staining of lacZ-Tagged Tumor Cells

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

Combined Use of Tail Vein Metastasis Assays and Real-Time In Vivo Imaging to Quantify Breast Cancer Metastatic Colonization and Burden in the Lungs

A Permanent Window for Investigating Cancer Metastasis to the Lung

A Label-Free Segmentation Approach for Intravital Imaging of Mammary Tumor Microenvironment

In Vivo Imaging to Measure Spontaneous Lung Metastasis of Orthotopically-injected Breast Tumor Cells

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