This research has been supported by Department of Defense grant TSA 140010 to M.K. and BC 111038 to M.M.M. Views and opinions of, and endorsements by the author(s) do not reflect those of the US Army or the Department of Defense.

All animal studies have been approved by Institutional Animal Care and Use Committee of Texas Tech University Health Sciences Center and followed the guidelines outlined in the &#34;Guide for the Care and Use of Laboratory Animals&#34; published by the National Institutes of Health. Use eight to twelve week old female BALB/c mice that are commercially available. Inject 1 x 105 4T1 or 1 x 105 4T1 cells expressing GFP, which can be purchased from various vendors, into the mammary fat pad.
1. Culture of 4T1 and 4T1-GFP Cells &#38; Preparation of Tumor Cell Suspension for Injections14
<ol>
	<li>4T1 and 4T1-GFP Cell Culture 
	NOTE: Perform all steps using sterile solutions in a laminar airflow (LAF) bio-safety cabinet unless specified otherwise.
	<ol>
		<li>Maintain 4T1 and 4T1-GFP cells in RPMI medium supplemented with 10% heat inactivated fetal bovine serum and 100 U/ml penicillin G and 100 &#956;g/ml streptomycin sulfate. 
		NOTE: Determine a passage number by counting every trypsinization and plating of cells. It is important to use cells with the same passage number to all mouse experiments, as the number of passages affects cell tumorigenicity and metastatic potential.</li>
		<li>Prior to cell injections remove the T75 cm2 flask, containing tumor cells at 80 % confluency, from an incubator and aspirate medium. Add 10 ml of PBS and rotate the flask gently to wash out remaining medium, and then aspirate PBS.</li>
		<li>Add 2 ml of 0.25 % trypsin with EDTA to the flask and tilt it to distribute the solution uniformly on the surface and incubate in a humidified incubator for 3 min at 37 &#730;C with 5 % CO2.</li>
		<li>Tap the flask to facilitate detachment of cells and add 8 ml of fresh medium. Pipette up and down several times to disrupt clumps and get a single cell suspension.</li>
		<li>Centrifuge cells for 5 min. at 500 x g at RT. Aspirate the supernatant and wash cells in 10 ml of PBS.</li>
		<li>Pipette up and down several times to disrupt clumps and get a single cell suspension. Take 100 &#181;l of a cell suspension and count the cells using the cell viability analyzer as per manufacturer&#39;s instructions.</li>
		<li>Calculate number of cells required for all injections using formula: 105 cells per mouse x # of mice used in an experiment (always prepare extra cells to be on a safe side).</li>
		<li>Centrifuge cells as in 1.1.5. Remove the supernatant through aspiration and resuspend cells in the amount of fresh PBS required to obtain a final cell concentration of 1 x 106 cells/ml. Keep the cell suspension on ice until ready for injection.</li>
	</ol>
	</li>
	<li>4T1 or 4T1-GFP Cell Injection-mouse Procedures
	<ol>
		<li>The day before tumor cell injection, anesthetize mice in an induction chamber with 3 % isoflurane. Once mice are asleep and breathe regularly, place the mouse on a surgical pad with nose inside the nose cone, and connect it to the isoflurane vaporizer. Maintain anesthesia with 2.5 % isoflurane. Pinch the mouse toe to ensure that the mouse is anesthetized.</li>
		<li>Apply hair removal cream using a cotton swab to the injection site (right pectoral mammary fat pad) and wait for 2 min. Clean the site using a wet paper towel, place the mouse back into the cage and monitor an animal until it regains sufficient consciousness to maintain the sternal recumbency.</li>
		<li>On the day of injection, anesthetize the mice as in 1.2.2 and place on the surgical pad.</li>
		<li>Vortex the tube containing tumor cells to get uniform cell suspension. Aspirate 100 &#956;l of a cell suspension, containing 1 x 105 tumor cells, into a 0.5 ml insulin syringe (29 G, 12.7 mm-needle length).</li>
		<li>Lift the skin using the thumb and finger index near the 2nd and 3rd nipple, insert the needle of the syringe into the mammary fat pad just below the third nipple under the skin between the fingers and inject cells slowly to form a bubble (subcutaneous injection).</li>
		<li>Place the mouse back into the cage after injection and monitor as in 1.2.2.</li>
	</ol>
	</li>
	<li>Monitoring Tumor Growth
	<ol>
		<li>Caliper Measurements14 
		NOTE: Injected 4T1 or 4T1-GFP cells form aggressive breast tumor. Usually the palpable tumors appear ~ at day 4 - 5 after cell inoculation. 4T1-GFP cells form tumors that grow slower and give metastases later. Measure the tumor two to three times a week. If clinical status of animals deteriorates, animals lose more than 10 % body weight, tumors exceed 10 % of body weight or become ulcerated, euthanize mice immediately.
		<ol>
			<li>Anesthetize a mouse as in 1.2.1., weigh, place on the surgical pad and wet the area with 70 % ethanol.</li>
			<li>Palpate tumor in the site of injection (4 - 5 days after cell inoculation).</li>
			<li>Measure the largest diameter (D) and smaller diameter (d1) with a caliper.</li>
			<li>Calculate tumor volume using formula Volume= (D X d1 X d1)/2 mm3. Monitor mouse recovery from anesthesia as in 1.2.2.</li>
		</ol>
		</li>
		<li>Imaging (Only for 4T1 GFP Cells)
		<ol>
			<li>Anesthetize the mouse as in 1.2.1. Place the mouse on a movable stage inside the imaging instrument. Maintain anesthesia through the nose cone.</li>
			<li>Turn on the imaging instrument and the light source as per manufacturer&#39;s instructions.</li>
			<li>Under the tab &#34;Acquisition&#34; go to &#34;Lighting&#34; and switch to the empty position (no filter) for white light. Ensure that the light engine is ON and select &#39;Trans&#39; in the light table.</li>
			<li>Under the tab &#34;Microscope&#34;, select &#34;Clear&#34; emission filter and 0.5X magnification. In the &#34;Camera&#34; tab, make sure that binning for capture is &#39;1 x 1&#39; and preview &#39;4 x 4&#39;.</li>
			<li>Click on preview to locate the tumor in white light, focus to get clear image, and click capture.</li>
			<li>Go to &#34;Acquisition&#34; tab and change lighting to filter 1 (filter for GFP as per manufacturer&#39;s instruction). In the &#34;Microscope&#34; tab, change emission filter to &#34;530/20 GF&#34;. Preview and capture image in a green channel. Adjust exposure time to get the appropriate brightness.</li>
			<li>Apply pseudocolor to the image and save in appropriate folder (Figure 1).</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
2. Intranasal Administration of Liposomes10
NOTE: Perform all steps using sterile solutions in a laminar airflow (LAF) Bio-safety cabinet unless specified otherwise.
<ol>
	<li>On day 6 after tumor cell injection anesthetize mice as in 1.2.1.</li>
	<li>Vortex the clodronate or control (PBS) liposome suspension obtained from the manufacturer. Take 60 &#181;l liposome suspension using sterile pipette tip.</li>
	<li>Slowly release liposome solution (5 &#181;l each time) near the nostrils allowing the mouse to breathe in the solution, repeat until the entire dose of 60 &#181;l is administered.</li>
	<li>Let the mouse regain consciousness before placing it back in the cage.</li>
	<li>Repeat liposome administration every 3 days until the mice are sacrificed. Do not administer liposomes on a day of sacrifice.</li>
</ol>
3. Mouse Sacrifice and Tissue Collection
NOTE: Use autoclaved and sterile instruments for mouse dissection. Euthanize mice while under anesthesia through the exsanguination and removal of the vital organs.
<ol>
	<li>On day 22 (for 4T1 cells) or day 26 (for 4T1-GFP cells) after tumor cell injection anesthetize the mouse as in 1.2.1. and place on the surgical pad with a nose cone connected to vaporizer, maintain anesthesia as in 1.2.1. Pinch one of the toes to ensure that the mouse is unconscious.</li>
	<li>Pin the toes using needles to the dissection board. Spray ethanol on the mouse skin.</li>
	<li>Lift the skin using forceps and make an incision with the surgical scissors. Slowly expose the peritoneum and continue cutting skin through the thorax until the neck.</li>
	<li>Using the surgical scissors make an incision in the peritoneum and expose carefully the organs with cotton tips avoiding damage to the blood vessels.</li>
	<li>Move the organs to one side and expose the inferior vena cava. Puncture the vein and collect blood with the 29 G insulin syringe. Place collected blood into the tube and leave on ice for further processing.</li>
	<li>Cut the diaphragm to expose the rib cage, lungs and heart. Slowly cut the rib cage on both sides using the bone cutter and lift the top of ribcage. Grab the heart with forceps and cut the connective tissue connecting the heart and lungs to the chest cavity.</li>
	<li>Place the lungs in small amount of PBS in the 60 mm Petri dish on ice until ready for imaging and further processing to frozen sections for immunofluorescent microscopy or routine histology [including hematoxylin and eosin (H &#38; E) staining].</li>
</ol>
4. Lung Metastases Evaluation
<ol>
	<li>Counting and Scoring Surface Metastases
	<ol>
		<li>Place the Petri dish with the lungs under the dissection microscope. Focus to get a clear view of the lung surface.</li>
		<li>Using a dissection microscope observe the lung surface. Count metastases on the anterior and posterior sides of both lungs. 
		NOTE: The normal lung is whitish or pinkish and porous and has a sponge like structure. 4T1 metastases are solid and nonporous, sometimes appear to be similar to a drop of liquid (Figure 2A).</li>
	</ol>
	</li>
	<li>Lung imaging
	<ol>
		<li>Place the Petri dish with lungs on the movable stage inside the imaging instrument.</li>
		<li>Turn on the instrument and the biolite multispectral source. Open the software. Under the tab, &#34;Acquisition&#34;, go to &#34;Lighting&#34;, and switch to empty position (no filter) for white light. Light Engine--&#62; ON, Light table--&#62; Epi. Under the tab &#34;Microscope&#34; Select &#39;Clear&#39; emission filter and 1.66X magnification. In the &#34;Camera&#34; tab, make sure that binning for capture is &#39;1 x 1&#39; and preview &#39;4 x 4&#39;.</li>
		<li>Click on preview and focus to get a clear picture of lungs in white light and click capture.</li>
		<li>Go to &#34;Acquisition&#34; tab and change to the filter 1 (filter for GFP as per manufacturer&#39;s instruction). In the &#34;Microscope&#34; tab, change emission filter to &#34;530/20 GF&#34;. Preview and capture image in a green channel. Adjust exposure time to get the appropriate brightness.</li>
		<li>Apply pseudocolor to the image and save in appropriate folder.</li>
		<li>Flip the lungs to get image of the other side in a similar way. This procedure generates images of GFP-positive metastases that can be further quantified using appropriate software (Figure 2B)14.</li>
	</ol>
	</li>
	<li>Immunofluorescent Microscopy
	<ol>
		<li>Fix the lungs in 4% paraformaldehyde, at 4 &#176;C for 4 hr in dark.</li>
		<li>Wash the tissue twice with 10 ml PBS for 5 min to completely remove the fixative. Transfer to 18 % sucrose in PBS and incubate O/N at 4 &#176;C.</li>
		<li>Remove the tissue from the sucrose and dab off any excess.</li>
		<li>Take a cryomold, cover the bottom layer with OCT medium. Place the tissue on OCT medium. Fill the mold with OCT medium to completely cover the tissue and place on dry ice.</li>
		<li>Store block at -80 &#176;C until sectioning.</li>
		<li>Prepare 5 &#181;m thick sections with a cryostat and place on charged slides.</li>
		<li>Store unused slides in -80 &#176;C until further usage.</li>
		<li>Air dry the slides for 7 min. and wash with PBS to remove OCT. Wipe the slide with tissue or paper towel to remove excess of water, mount cover slips with mounting medium containing DAPI and visualize with GFP filter under microscope.</li>
		<li>Quantify GFP metastases (Figure 3A) with the use of appropriate software14.</li>
	</ol>
	</li>
	<li>Histology and Digital Pathology
	<ol>
		<li>Click the manual load button under the start tab and wait for the slide holding rack to eject from the scanner.</li>
		<li>Take the H&#38;E stained tissue section, clean it with tissue to remove any dirt, and place it securely in the slide holding rack.</li>
		<li>Enter the slide description in the popped-up window.</li>
		<li>Select the Scan Area tab on the SS imaging console window and adjust the green square to define the region of interest (ROI) to be scanned.</li>
		<li>Drag the blue calibration diamond to a clear portion of the slide, where tissue is absent, however, within the ROI.</li>
		<li>Next, select the Focus Points tab and click the auto select button to obtain several focus points (yellow plus signs) on the tissue in the ROI.</li>
		<li>If necessary, place additional focus points manually by double-clicking within the ROI.</li>
		<li>Proceed to the calibrate tab and select the calibrate button to begin slide calibration at the blue calibration diamond point. After the calibration is completed the image from the calibration point will be displayed.</li>
		<li>Inspect this image to ensure that it is clear and free of dirt or artifacts.</li>
		<li>Proceed to the Scan tab and select Start Scan to begin scanning of the ROI. Scanned histological section is converted to a digital slide (Figure 3B and 4A)</li>
		<li>Quantify metastatic burden as previously described (Figure 4B)14.</li>
	</ol>
	</li>
</ol>
5. Flow Cytometry (FACS) Analysis of Alveolar Macrophages in the Lungs
<ol>
	<li>Preparation of Single Cell Suspension
	<ol>
		<li>After imaging and counting of metastases, wash the lungs with sterile PBS and place to the Petri dish. Make 1 - 2 mm pieces of the lungs using surgical scissors and forceps.</li>
		<li>Transfer the lung pieces to the 15 ml conical tube with 3 ml of the digestion buffer containing 1 mg/ml collagenase P, 0.04mg/ml DNase I, and 10 &#181;g/ml trypsin inhibitor dissolved RPMI medium (sterile).</li>
		<li>Incubate the conical tube on a rotating shaker in the incubator at 37 &#176;C for 40 min.</li>
		<li>Remove the tube conical from the incubator and pipette the solution up and down to dissociate the tissue.</li>
		<li>Pass the solution through the 40 &#181;m strainer to avoid clumps and get a single cell suspension. If needed, to disintegrate better the digested tissue press tissue against the cell strainer with the help of a syringe plunger.</li>
		<li>When a single cell suspension has been achieved, collect the cells by centrifugation and lyse red blood cells with 2 ml of ACK buffer for 10 min at RT. Then, wash a pellet two times in 8 ml of RPMI.</li>
		<li>Resuspend cells in 3 ml of the complete RPMI medium and proceed to cell counting using the cell viability analyzer as per manufacturer&#39;s instructions.</li>
	</ol>
	</li>
	<li>Staining of Lung Cells for Surface Markers 
	NOTE: Perform all incubations at 4 &#176;C. Centrifuge plate at 500 x g and 4 &#176;C.
	<ol>
		<li>Pipette 1 x 106 cells in 100 &#181;l of FACS buffer (1% FBS in PBS) to individual wells of a V-bottom 96-well plate. Using a V-bottom 96 well plate facilitates handing of multiple samples.</li>
		<li>Pre-incubate the cell suspension with 1 &#181;g Mouse Fc Block purified anti-mouse CD16/CD32 in 100 &#181;l of FACS buffer for 15 min. Centrifuge the V-bottom 96 well plate to pellet the cells and remove the supernatant by flipping the plate and letting the solution drain.</li>
		<li>For surface staining, in advance, dilute the antibodies to murine antigens in one tube (master mix): BV605 CD45 (30-F11), PE CD11b (M1/70), PE/Cy7 F4/80 (BM8), APC/Cy7 CD11c (N418), PerCPCy5.5 IA/IE (MHCII) (M5/114.152), PE CD80 (16-10A1), AF 647 CD86 (GL-1) to a final concentration of 2.5 &#181;g/ml in FACS buffer containing 10 &#181;g/ml of Fc block CD16/CD32 antibody. 
		NOTE: This set of antibodies is useful for the identification and the evaluation of a functional status of alveolar macrophages and dendritic cells.</li>
		<li>Add 100 &#181;l of diluted antibodies (master mix) to the wells of the V-bottom 96 well plate and incubate at 4 &#730;C for 30 min.</li>
		<li>Centrifuge the plate to pellet cells and remove supernatant as in 5.2.2. Wash cells with 200 &#181;l of FACS buffer, centrifuge at 500 g for 5 min. Remove the supernatant as in 5.2.2. and resuspend cells in 200 &#181;l of PBS.</li>
		<li>Centrifuge the plate to pellet cells and add viability dye diluted in PBS (1:1,000). Incubate for 20 min at 4 &#730;C.</li>
		<li>Centrifuge the plate to pellet cells and remove the supernatant as in 5.2.2. Wash with 200 &#181;l of PBS and centrifuge the plate again.</li>
		<li>Resuspend cells in 100 &#181;l of 1% paraformaldehyde and store at 4 &#176;C until the acquisition by FACS instrument. Prior to acquisition transfer cell suspensions to the polypropylene FACS tubes.</li>
	</ol>
	</li>
</ol>
6. Analysis of FACS Data: Gating Strategies and the Identification of Cell Subpopulations
<ol>
	<li>Perform FACS analysis as previously reported10. Gate acquired cells/events based on forward-(FSC) and side-scatter (SSC); and then gate live and CD45+ cells. For alveolar macrophage identification, gate CD11b-negative and then CD11c+F4/80+ cells. (Figure 5A).</li>
</ol>

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The authors declare that they have no competing financial interests.

The recent insights into cancer biology and causative factors involved in carcinogenesis and tumor progression lead to development of genetically engineered mouse (GEM) models of cancer, in which tumors grow spontaneously, usually over a period of several months15. Although these tumor models appear to reflect better the natural history of human malignancies than xenografts or syngeneic models, much time required for tumor development and various degrees of malignant phenotype penetrance limit the use of these...

The premetastatic niche is an important process in cancer metastasis defined as a set of alterations that occur in the organs that are targets for metastases prior to arrival of tumor cells1,2. Therefore, therapeutic targeting of this step of cancer progression may prevent metastases to the vital organs that cause approximately 90% of cancer-associated deaths. Although the concept of the premetastatic niche, also known as the "seed and soil" theory, was introduced more than a century ago, no experimental proof has been provided until recently, when the bone marrow-derived cells were demonstrated to contribute to the premetastatic soil1,3-7. Despite these developments, the premetastatic niche remains a largely understudied aspect of cancer pathophysiology and further research to identify cellular players and mechanisms involved is needed. 
Here we report the in vivo approaches to study the role of alveolar macrophages in breast cancer metastases and the lung premetastatic niche. The alveolar macrophages arrive to the lungs early during the embryonic development and self-renew there during adulthood8. They also have important immunomodulatory and homeostatic functions including the protection of this organ from undesired inflammatory responses to the environmental innocuous antigens9. Therefore, we hypothesize that tumors exploit this physiological immunosuppression, imposed by alveolar macrophages, and, consequently, alveolar macrophages contribute to the lung premetastatic niche by suppressing antitumor immunity. This hypothesis is supported by our recent report demonstrating that the specific depletion of these cells reduces lung metastases and enhances antitumor T cell responses10. 
For these studies we apply a well-established syngeneic model of breast cancer (4T1), which mimics stage IV metastatic breast cancer11; and has been previously reported in studies of the premetastatic niche6. To track metastasizing tumor cells in vivo we use 4T1 cells expressing GFP (4T1-GFP) in conjunction with animal imaging and confocal microscopy. We focus on the lung premetastatic niche, since this organ is one of the most common targets of hematogenous metastases of human malignancies12. To investigate functions of alveolar macrophages in the premetastatic niche, we use clodronate liposomes to deplete these cells13; and evaluate impact of this depletion on lung metastases. Of note, this method specifically depletes alveolar macrophages but no other phagocytic cells in the lungs or in circulation10.

<table><tbody><tr><td>4T1 cell line&amp;nbsp;</td><td>American Type Culture Collection, Manassas, VA, USA</td><td>CRL 2539</td><td>Tumor cells</td></tr><tr><td>4T1-GFP cell line</td><td>Caliper life sciences/ Perkin Elmer, Waltham, MA, USA</td><td>BW128090</td><td>Tumor cells</td></tr><tr><td>RPMI</td><td>Corning, Corning, NY, USA</td><td>10-040-CM</td><td>Media</td></tr><tr><td>Heat inactivated FBS</td><td>Gibco (Thermo Scientific), USA</td><td>10082147</td><td>Media</td></tr><tr><td>Penicillin Streptomycin</td><td>Fisher Scientific, Waltham, MA, USA</td><td>MT-300-02-CI</td><td>Media</td></tr><tr><td>PBS</td><td>Fisher Scientific, Waltham, MA, USA</td><td>BP399-20</td><td>Dilute with distilled water</td></tr><tr><td>Trypsin 0.25% with EDTA</td><td>Hyclone, Logan, Utah, USA</td><td>SH30042.02</td><td>Tissue culture supplies</td></tr><tr><td>T75 cm&lt;sup&gt;2&lt;/sup&gt; flask</td><td>Fisher Scientific, Waltham, MA, USA</td><td>12-565-32</td><td>Tissue culture supplies</td></tr><tr><td>15 ml conical tube</td><td>BD falcon, Franklin Lakes, NJ, USA</td><td>352096</td><td>Tissue culture supplies</td></tr><tr><td>50 ml conical tube</td><td>BD falcon, Franklin Lakes, NJ, USA</td><td>352098</td><td>Tissue culture supplies</td></tr><tr><td>60 mm2 Petri dish</td><td>Fisher Scientific, Waltham, MA, USA</td><td>AS4052</td><td>For lung imaging&amp;nbsp;</td></tr><tr><td>Isoflurane (Isothesia)</td><td>Butler Schein Animal health, Dublin, OH, USA</td><td>NDC 11695-6776-2</td><td>Mouse anesthesia</td></tr><tr><td>Clodronate liposomes</td><td>Formumax Scientific Inc, Palo Alto, CA, USA</td><td>F70101C-N</td><td>Macrophages depletion</td></tr><tr><td>Control liposomes</td><td>Formumax Scientific Inc, Palo Alto, CA, USA</td><td>F70101-N</td><td>Control PBS-liposomes</td></tr><tr><td>29 gauge insulin syringes (12.7 mm and 0.5 ml capacity)- Reli-On</td><td>Walmart, Bentonville, AR, USA</td><td></td><td>For tumor cell injection</td></tr><tr><td>Hair removal cream (Nair)</td><td>Walmart, Bentonville, AR, USA</td><td></td><td></td></tr><tr><td>Paraformaldehyde solution (4%)</td><td>Affymetrix, Santa Clara, CA, USA</td><td>19943-I Lt</td><td>Dilute to 4% or 1% using 1x PBS</td></tr><tr><td>OCT compound</td><td>Fisher Scientific, Waltham, MA, USA</td><td>230-730-571</td><td>For freezing tissue in cryomolds</td></tr><tr><td>Fluoro-Gel-II with DAPI</td><td>Electron Microscopy Sciences, Hatfield, PA, USA</td><td>17985-51</td><td>Mounting medium</td></tr><tr><td>Sucrose</td><td>Sigma, St. Louis, MO, USA</td><td>S-9378</td><td>Cryopreservation</td></tr><tr><td>Collagenase P</td><td>Roche, Basel, Switzerland</td><td>11249002001</td><td>Components of tissue digestion buffer</td></tr><tr><td>Dnase I</td><td>Roche, Basel, Switzerland</td><td>10104159001</td><td>Components of tissue digestion buffer</td></tr><tr><td>Trypsin inhibitor</td><td>Sigma, St. Louis, MO, USA</td><td>T9253</td><td>Components of tissue digestion buffer</td></tr><tr><td>40 micron cell strainers</td><td>Fisher Scientific, Waltham, MA, USA</td><td>22-363-547</td><td>Used in tissue digestion to remove clumps</td></tr><tr><td>Trustain FcX-Fc Block (CD16/CD32)</td><td>Biolegend, San Diego, CA, USA</td><td>101320</td><td>Antibodies for flow cytometry</td></tr><tr><td>BV605 CD45</td><td>Biolegend, San Diego, CA, USA</td><td>103139</td><td>Antibodies for flow cytometry</td></tr><tr><td>PE CD11b</td><td>Biolegend, San Diego, CA, USA</td><td>101207</td><td>Antibodies for flow cytometry</td></tr><tr><td>PE Cy7 F4/80&amp;nbsp;</td><td>Biolegend, San Diego, CA, USA</td><td>123113</td><td>Antibodies for flow cytometry</td></tr><tr><td>APC/Cy7 CD11c</td><td>Biolegend, San Diego, CA, USA</td><td>117323</td><td>Antibodies for flow cytometry</td></tr><tr><td>PerCPcy5.5 IA/IE (MHCII)&amp;nbsp;</td><td>Biolegend, San Diego, CA, USA</td><td>107625</td><td>Antibodies for flow cytometry</td></tr><tr><td>PE CD80</td><td>Biolegend, San Diego, CA, USA</td><td>104707</td><td>Antibodies for flow cytometry</td></tr><tr><td>AF647 CD86</td><td>Biolegend, San Diego, CA, USA</td><td>105019</td><td>Antibodies for flow cytometry</td></tr><tr><td>Fixable viability Dye eflour 506</td><td>eBioscience, San Diego, CA,USA</td><td>65-0866</td><td>Antibodies for flow cytometry</td></tr><tr><td>Cryostat</td><td>Leica Biosystems, Buffalo Grove, IL, USA</td><td>CM1850</td><td>Cryosectioning</td></tr><tr><td>UVP iBox Explorer</td><td>UVP Inc, Upland, CA, USA</td><td></td><td>Mouse and lung fluorescent imaging</td></tr><tr><td>Aperio Scanscope CS</td><td>Leica Biosystems, Buffalo Grove, IL, USA</td><td></td><td>Digital pathology</td></tr><tr><td>BD LSRFortessa&amp;nbsp;</td><td>BD Biosciences, Franklin Lakes, NJ, USA</td><td></td><td>Flow cytometry/data acquisition</td></tr><tr><td>Nikon A1 confocal TE2000 microscope</td><td>Nikon Instruments Inc., Melville, NY 11747-3064, U.S.A.</td><td></td><td>Imaging and quantifying GFP fluorescence in lung cryosections</td></tr><tr><td>UVP visionworks software (Version 7.1RC3.38)</td><td>UVP Inc, Upland, CA, USA</td><td></td><td>Imaging software for iBOX</td></tr><tr><td>Aperio Imagescope software (v12.1.0.5029)&amp;nbsp;</td><td>Leica Biosystems, Buffalo Grove, IL, USA</td><td></td><td>Imaging software for analysis of digital slides</td></tr><tr><td>Flow JO software (version 9.8.1)</td><td>Flow JO LLC, Ashland, OR, USA</td><td></td><td>Analysis of flow cytometric data</td></tr><tr><td>NIS Elements AR (version 4.20.01) 64 Bit</td><td>Nikon Instruments Inc., Melville, NY 11747-3064, U.S.A.</td><td></td><td>Acquisition and analysis of lung cryosections for GFP&amp;nbsp;</td></tr></tbody></table>

studying the role of alveolar macrophages in breast cancer metastasis

This paper describes the application of the syngeneic model of breast cancer (4T1) to the studies on a role of pulmonary alveolar macrophages in cancer metastasis. The 4T1 cells expressing GFP in combination with imaging and confocal microscopy are used to monitor tumor growth, track metastasizing tumor cells, and quantify the metastatic burden. These approaches are supplemented by digital histopathology that allows the automated and unbiased quantification of metastases. In this method the routinely prepared histological lung sections, which are stained with hematoxylin and eosin, are scanned and converted to the digital slides that are then analyzed by the self-trained pattern recognition software. In addition, we describe the flow cytometry approaches with the use of multiple cell surface markers to identify alveolar macrophages in the lungs. To determine impact of alveolar macrophages on metastases and antitumor immunity these cells are depleted with the clodronate-containing liposomes administrated intranasally to tumor-bearing mice. This approach leads to the specific and efficient depletion of this cell population as confirmed by flow cytometry. Tumor volumes and lung metastases are evaluated in mice depleted of alveolar macrophages, to determine the role of these cells in the metastatic progression of breast cancer.

The injection of 4T1-GFP tumor cells into the mammary fat pad leads to the formation of mouse tumors (Figure 1A) that recapitulate the metastatic spread of human breast cancer, as metastases are rapidly formed in the lungs (Figure 2), liver, bones and brain of mice11. The stable transfection of 4T1 cells with GFP facilitates monitoring of tumor growth (Figure 1B), tracking metastasizing tumor cells and quantifying the metastati...

Watch this Scientific Journal Video about Studying the Role of Alveolar Macrophages in Breast Cancer Metastasis at JoVE.com

Studying the Role of Alveolar Macrophages in Breast Cancer Metastasis

Here we describe the model and approach to study functions of pulmonary alveolar macrophages in cancer metastasis. To demonstrate the role of these cells in metastasis, the syngeneic (4T1) model of breast cancer in conjunction with the depletion of alveolar macrophage with clodronate liposomes was used.

This paper describes the application of the syngeneic model of breast cancer (4T1) to the studies on a role of pulmonary alveolar macrophages in cancer ...

studying-the-role-of-alveolar-macrophages-in-breast-cancer-metastasis

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Medicine

9.7K Views.  Texas Tech University Health Science Center. Here we describe the model and approach to study functions of pulmonary alveolar macrophages in cancer metastasis. To demonstrate the role of these cells in metastasis, the syngeneic (4T1) model of breast cancer in conjunction with the depletion of alveolar macrophage with clodronate liposomes was used. 

Video: Studying the Role of Alveolar Macrophages in Breast Cancer Metastasis

Intra-iliac Artery Injection for Efficient and Selective Modeling of Microscopic Bone Metastasis

Intra-iliac artery (IIA) injection is an efficient approach to introduce metastatic lesions of various cancer cells in animals. Compared to the widely used intra-cardiac and intra-tibial injections, IIA injection brings several advantages. First, it can deliver a large quantity of cancer cells specifically to hind limb bones, thereby providing spatiotemporally synchronized early-stage colonization events and allowing robust quantification and swift detection of disseminated tumor cells. Second, it injects cancer cells into the circulation without damaging the local tissues, thereby avoiding inflammatory and wound-healing processes that confound the bone colonization process. Third, IIA injection causes very little metastatic growth in non-bone organs, thereby preventing animals from succumbing to other vital metastases, and allowing continuous monitoring of indolent bone lesions. These advantages are especially useful for the inspection of progression from single cancer cells to multi-cell micrometastases, which has largely been elusive in the past. When combined with cutting-edge approaches of biological imaging and bone histology, IIA injection can be applied to various research purposes related to bone metastases.

This manuscript provides the detailed procedure of intra-iliac artery (IIA) injection, a technique to deliver cancer cells specifically to hind limb tissues including bones to establish experimental bone metastases. Although initially established with breast tumor models, this protocol can be easily extended to other cancer types.

Intra-iliac artery (IIA) injection is an efficient approach to introduce metastatic lesions of various cancer cells in animals. Compared to the widely ...

Cancer Research

A Portal Vein Injection Model to Study Liver Metastasis of Breast Cancer

Breast cancer is the leading cause of cancer-related mortality in women worldwide. Liver metastasis is involved in upwards of 30% of cases with breast cancer metastasis, and results in poor outcomes with median survival rates of only 4.8 - 15 months. Current rodent models of breast cancer metastasis, including primary tumor cell xenograft and spontaneous tumor models, rarely metastasize to the liver. Intracardiac and intrasplenic injection models do result in liver metastases, however these models can be confounded by concomitant secondary-site metastasis, or by compromised immunity due to removal of the spleen to avoid tumor growth at the injection site. To address the need for improved liver metastasis models, a murine portal vein injection method that delivers tumor cells firstly and directly to the liver was developed. This model delivers tumor cells to the liver without complications of concurrent metastases in other organs or removal of the spleen. The optimized portal vein protocol employs small injection volumes of 5 - 10 &#956;l, &#8805; 32 gauge needles, and hemostatic gauze at the injection site to control for blood loss. The portal vein injection approach in Balb/c female mice using three syngeneic mammary tumor lines of varying metastatic potential was tested; high-metastatic 4T1 cells, moderate-metastatic D2A1 cells, and low-metastatic D2.OR cells. Concentrations of &#8804; 10,000 cells/injection results in a latency of ~ 20 - 40 days for development of liver metastases with the higher metastatic 4T1 and D2A1 lines, and &#62; 55 days for the less aggressive D2.OR line. This model represents an important tool to study breast cancer metastasis to the liver, and may be applicable to other cancers that frequently metastasize to the liver including colorectal and pancreatic adenocarcinomas.

A surgical procedure was developed to deliver mammary tumor cells to the murine liver via portal vein injection. This model permits investigation of late stages of liver metastasis in a fully immune competent host, including tumor cell extravasation, seeding, survival, and metastatic outgrowth in the liver.

Breast cancer is the leading cause of cancer-related mortality in women worldwide. Liver metastasis is involved in upwards of 30% of cases with breast ...

Analyzing the Communication Between Monocytes and Primary Breast Cancer Cells in an Extracellular Matrix Extract (ECME)-based Three-dimensional System

Embedded in the extracellular matrix (ECM), normal and neoplastic epithelial cells intimately communicate with hematopoietic and non-hematopoietic cells, thus greatly influencing normal tissue homeostasis and disease outcome. In breast cancer, tumor-associated macrophages (TAMs) play a critical role in disease progression, metastasis, and recurrence; therefore, understanding the mechanisms of monocyte chemoattraction to the tumor microenvironment and their interactions with tumor cells is important to control the disease. Here, we provide a detailed description of a three-dimensional (3D) co-culture system of human breast cancer (BrC) cells and human monocytes. BrC cells produced high basal levels of regulated on-activation, normal T-cell expressed and secreted (RANTES), monocyte chemoattractant protein-1 (MCP-1), and granulocyte-macrophage colony-stimulating factor (G-CSF), while in co-culture with monocytes, pro-inflammatory cytokines Interleukin (IL)-1 beta (IL-1&#946;) and IL-8 were enriched together with matrix metalloproteinases (MMP)-1, MMP-2, and MMP-10. This tumor stroma microenvironment promoted resistance to anoikis in MCF-10A 3D acini-like structures, chemoattraction of monocytes, and invasion of aggressive BrC cells. The protocols presented here provide an affordable alternative to study intra-tumor communication and are an example of the great potential that in vitro 3D cell systems provide to interrogate specific features of tumor biology related to tumor aggression.

Analyzing the Communication Between Monocytes and Primary Breast Cancer Cells in an Extracellular Matrix Extract ECME-based Three-dimensional System

Here, we describe a three-dimensional culture method to analyze the morphology of primary breast cancer cells, as well as to study their direct/indirect interactions with monocytes and the outcomes such as collagen degradation, immune cell recruitment, cell invasion, and promotion of cancer-related inflammation.

Embedded in the extracellular matrix (ECM), normal and neoplastic epithelial cells intimately communicate with hematopoietic and non-hematopoietic cells, ...

Real Time Detection of In Vitro Tumor Cell Apoptosis Induced by CD8+ T Cells to Study Immune Suppressive Functions of Tumor-infiltrating Myeloid Cells

Potentiation of the tumor-killing ability of CD8+ T cells in tumors, along with their efficient tumor infiltration, is a key element of successful immunotherapies. Several studies have indicated that tumor infiltrating myeloid cells (e.g., myeloid-derived suppressor cells (MDSCs) and tumor-associated macrophages (TAMs)) suppress cytotoxicity of CD8+ T cells in the tumor microenvironment, and that targeting these regulatory myeloid cells can improve immunotherapies. Here, we present an in vitro assay system to evaluate immune suppressive effects of monocytic-MDSCs and TAMs on the tumor-killing ability of CD8+ T cells. To this end, we first cultured na&#239;ve splenic CD8+ T cells with anti-CD3/CD28 activating antibodies in the presence or absence of suppressor cells, and then co-cultured the pre-activated T cells with target cancer cells in the presence of a fluorogenic caspase-3 substrate. Fluorescence from the substrate in cancer cells was detected by real-time fluorescence microscopy as an indicator of T-cell induced tumor cell apoptosis. In this assay, we can successfully detect the increase of tumor cell apoptosis by CD8+ T cells and its suppression by pre-culture with TAMs or MDSCs. This functional assay is useful for investigating CD8+ T cell suppression mechanisms by regulatory myeloid cells and identifying druggable targets to overcome it via high throughput screening.

Real Time Detection of In Vitro Tumor Cell Apoptosis Induced by CD8+ T Cells to Study Immune Suppressive Functions of Tumor-infiltrating Myeloid Cells

We describe here a protocol to investigate cytotoxicity of pre-activated CD8+ T cells against cancer cells by detecting apoptotic cancer cells via real-time microscopy. This protocol can investigate mechanisms behind myeloid cell-induced T cell suppression and evaluate compounds aimed at replenishing T cells via blockade of immune suppressive myeloid cells.

Potentiation of the tumor-killing ability of CD8+ T cells in tumors, along with their efficient tumor infiltration, is a key element of successful ...

Assessing Tumor Microenvironment of Metastasis Doorway-Mediated Vascular Permeability Associated with Cancer Cell Dissemination using Intravital Imaging and Fixed Tissue Analysis

The most common cause of cancer related mortality is metastasis, a process that requires dissemination of cancer cells from the primary tumor to secondary sites. Recently, we established that cancer cell dissemination in primary breast cancer and at metastatic sites in the lung occurs only at doorways called Tumor MicroEnvironment of Metastasis (TMEM). TMEM doorway number is prognostic for distant recurrence of metastatic disease in breast cancer patients. TMEM doorways are composed of a cancer cell which over-expresses the actin regulatory protein Mena in direct contact with a perivascular, proangiogenic macrophage which expresses high levels of TIE2 and VEGF, where both of these cells are tightly bound to a blood vessel endothelial cell. Cancer cells can intravasate through TMEM doorways due to transient vascular permeability orchestrated by the joint activity of the TMEM-associated macrophage and the TMEM-associated Mena-expressing cancer cell. In this manuscript, we describe two methods for assessment of TMEM-mediated transient vascular permeability: intravital imaging and fixed tissue immunofluorescence. Although both methods have their advantages and disadvantages, combining the two may provide the most complete analyses of TMEM-mediated vascular permeability as well as microenvironmental prerequisites for TMEM function. Since the metastatic process in breast cancer, and possibly other types of cancer, involves cancer cell dissemination via TMEM doorways, it is essential to employ well established methods for the analysis of the TMEM doorway activity. The two methods described here provide a comprehensive approach to the analysis of TMEM doorway activity, either in na&#239;ve or pharmacologically treated animals, which is of paramount importance for pre-clinical trials of agents that prevent cancer cell dissemination via TMEM.

We describe two methods for assessing transient vascular permeability associated with tumor microenvironment of metastasis (TMEM) doorway function and cancer cell intravasation using intravenous injection of high-molecular weight (155 kDa) dextran in mice. The methods include intravital imaging in live animals and fixed tissue analysis using immunofluorescence.

The most common cause of cancer related mortality is metastasis, a process that requires dissemination of cancer cells from the primary tumor to secondary ...

Assessing Anti-fungal Activity of Isolated Alveolar Macrophages by Confocal Microscopy

The lung is an interface where host cells are routinely exposed to microbes and microbial products. Alveolar macrophages are the first-line phagocytic cells that encounter inhaled fungi and other microbes. Macrophages and other immune cells recognize Aspergillus motifs by pathogen recognition receptors and initiate downstream inflammatory responses. The phagocyte NADPH oxidase generates reactive oxygen intermediates (ROIs) and is critical for host defense. Although NADPH oxidase is critical for neutrophil-mediated host defense1-3, the importance of NADPH oxidase in macrophages is not well defined. The goal of this study was to delineate the specific role of NADPH oxidase in macrophages in mediating host defense against A. fumigatus. We found that NADPH oxidase in alveolar macrophages controls the growth of phagocytosed A. fumigatus spores4. Here, we describe a method for assessing the ability of mouse alveolar macrophages (AMs) to control the growth of phagocytosed Aspergillus spores (conidia). Alveolar macrophages are stained in vivo and ten days later isolated from mice by bronchoalveolar lavage (BAL). Macrophages are plated onto glass coverslips, then seeded with green fluorescent protein (GFP)-expressing A. fumigatus spores. At specified times, cells are fixed and the number of intact macrophages with phagocytosed spores is assessed by confocal microscopy.

A method to evaluate the ability of isolated mouse alveolar macrophages to control the growth of phagocytosed Aspergillus spores by confocal microscopy.

The lung is an interface where host cells are routinely exposed to microbes and microbial products. Alveolar macrophages are the first-line phagocytic ...

Immunology and Infection

Methods for Culturing Human Femur Tissue Explants to Study Breast Cancer Cell Colonization of the Metastatic Niche

Bone is the most common site of breast cancer metastasis. Although it is widely accepted that the microenvironment influences cancer cell behavior, little is known about breast cancer cell properties and behaviors within the native microenvironment of human bone tissue.We have developed approaches to track, quantify and modulate human breast cancer cells within the microenvironment of cultured human bone tissue fragments isolated from discarded femoral heads following total hip replacement surgeries. Using breast cancer cells engineered for luciferase and enhanced green fluorescent protein (EGFP) expression, we are able to reproducibly quantitate migration and proliferation patterns using bioluminescence imaging (BLI), track cell interactions within the bone fragments using fluorescence microscopy, and evaluate breast cells after colonization with flow cytometry. The key advantages of this model include: 1) a native, architecturally intact tissue microenvironment that includes relevant human cell types, and 2) direct access to the microenvironment, which facilitates rapid quantitative and qualitative monitoring and perturbation of breast and bone cell properties, behaviors and interactions. A primary limitation, at present, is the finite viability of the tissue fragments, which confines the window of study to short-term culture. Applications of the model system include studying the basic biology of&#160;breast cancer and other bone-seeking malignancies within the metastatic niche, and developing therapeutic strategies to effectively target breast cancer cells in bone tissues.

Protocols are described for studying breast cancer cell migration, proliferation and colonization in a human bone tissue explant model system.

Bone is the most common site of breast cancer metastasis. Although it is widely accepted that the microenvironment influences cancer cell behavior, little ...

Examining Macrophage-Tumor Spheroid Interactions in a 3D Collagen Matrix Setup

A Macrophage-Tumor Spheroid Co-Invasion Assay

The interaction of immune and cancer cells and their respective impact on metastasis represents an important aspect of cancer research. So far, only a few protocols are available that allow an in vitro approximation of the in vivo situation. Here, we present a novel approach to observing the impact of human macrophages on the invasiveness of cancer cells, using tumor spheroids of H1299 non-small cell lung carcinoma cells embedded in a three-dimensional (3D) collagen I matrix. With this co-cultivation setup, we tested the impact of small interfering RNA (siRNA)-based depletion of regulatory factors in macrophages on the 3D invasion of cancer cells from the tumor spheroid compared to controls. This method allows the determination of different parameters, such as spheroid area or the number of invading cancer cells, and thus, to detect differences in cancer cell invasion. In this article, we present the respective setup, discuss the subsequent analysis, as well as the advantages and potential pitfalls of this method.

Here, we present a protocol to investigate the interaction of primary human monocyte-derived macrophages with a tumor spheroid in a three-dimensional (3D) collagen I matrix, with the possibility to compare the impact of soluble and physical properties of the microenvironment on cell invasion.

The interaction of immune and cancer cells and their respective impact on metastasis represents an important aspect of cancer research. So far, only a few ...

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and their important role in lung development and function, as well as their lung-localized responses to infection and inflammation. To date, no unified method for identification, isolation, and handling of alveolar macrophages from humans and mice exists. Such a method is needed for studies on these important innate immune cells in various experimental settings. The method described here, which can be easily adopted by any laboratory, is a simplified approach to harvesting alveolar macrophages from bronchoalveolar lavage fluid or from lung tissue and maintaining them in vitro. Because alveolar macrophages primarily occur as adherent cells in the alveoli, the focus of this method is on dislodging them prior to harvest and identification. The lung is a highly vascularized organ, and various cell types of myeloid and lymphoid origin inhabit, interact, and are influenced by the lung microenvironment. By using the set of surface markers described here, researchers can easily and unambiguously distinguish alveolar macrophages from other leukocytes, and purify them for downstream applications. The culture method developed herein supports both human and mouse alveolar macrophages for in vitro growth, and is compatible with cellular and molecular studies.

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

This communication describes methodologies for isolation and culture of alveolar macrophages from humans and murine models for experimental purposes.

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and ...

Alveolar Macrophage Phagocytosis and Bacteria Clearance in Mice

Alveolar macrophages (AMs) guard the alveolar space of the lung. Phagocytosis by AMs plays a critical role in the defense against invading pathogens, the removal of dead cells or foreign particles, and in the resolution of inflammatory responses and tissue remodeling, processes that are mediated by various surface receptors of the AMs. Here, we report methods for the analysis of the phagocytic function of AMs using in vitro and in vivo assays and experimental strategies to differentiate between the pattern recognition receptor-, complement receptor-, and Fc gamma receptor-mediated phagocytosis. Finally, we discuss a method to establish and characterize a P. aeruginosa pneumonia model in mice to assess bacterial clearance in vivo. These assays represent the most common methods to evaluate AM functions and can also be used to study macrophage function and bacterial clearance in other organs.

Here we report common methods to analyze the phagocytic function of murine alveolar macrophages and bacterial clearance from the lung. These methods study in vitro phagocytosis of fluorescein isothiocyanate beads and in vivo phagocytosis of Pseudomonas aeruginosa Green Fluorescent Protein. We also describe a method for clearing P. aeruginosa in mice.

Alveolar macrophages (AMs) guard the alveolar space of the lung. Phagocytosis by AMs plays a critical role in the defense against invading pathogens, the ...

Studying the Role of Alveolar Macrophages in Breast Cancer Metastasis

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