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.
<p class...

<ol>
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H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gr-1+CD11b++myeloid+cells+tip+the+balance+of+immune+protection+to+tumor+promotion+in+the+premetastatic+lung.">Gr-1+CD11b+ myeloid cells tip the balance of immune protection to tumor promotion in the premetastatic lung.</a> Cancer Res. 70, 6139-6149 (2010).</li><li>Gao, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myeloid+progenitor+cells+in+the+premetastatic+lung+promote+metastases+by+inducing+mesenchymal+to+epithelial+transition.">Myeloid progenitor cells in the premetastatic lung promote metastases by inducing mesenchymal to epithelial transition.</a> Cancer Res. 72, 1384-1394 (2012).</li><li>Davies, L. C., Jenkins, S. J., Allen, J. E., Taylor, P. 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K., 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=Pulmonary+alveolar+macrophages+contribute+to+the+premetastatic+niche+by+suppressing+antitumor+T+cell+responses+in+the+lungs.">Pulmonary alveolar macrophages contribute to the premetastatic niche by suppressing antitumor T cell responses in the lungs.</a> J. Immunol. 194, 5529-5538 (2015).</li><li>Pulaski, B. A., Ostrand-Rosenberg, S., Coligan, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+4T1+breast+tumor+model.">Mouse 4T1 breast tumor model.</a> Current protocols in immunology. , (2001).</li><li>Gupta, G. 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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 border="0" cellpadding="0" cellspacing="0" width="1155">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:313px;">4T1 cell line&#160;</td>
			<td style="width:417px;">American Type Culture Collection, Manassas, VA, USA</td>
			<td style="width:139px;">CRL 2539</td>
			<td style="width:287px;">Tumor cells</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">4T1-GFP cell line</td>
			<td>Caliper life sciences/ Perkin Elmer, Waltham, MA, USA</td>
			<td>BW128090</td>
			<td>Tumor cells</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RPMI</td>
			<td>Corning, Corning, NY, USA</td>
			<td>10-040-CM</td>
			<td>Media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Heat inactivated FBS</td>
			<td>Gibco (Thermo Scientific), USA</td>
			<td>10082147</td>
			<td>Media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Penicillin Streptomycin</td>
			<td>Fisher Scientific, Waltham, MA, USA</td>
			<td>MT-300-02-CI</td>
			<td>Media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PBS</td>
			<td>Fisher Scientific, Waltham, MA, USA</td>
			<td>BP399-20</td>
			<td>Dilute with distilled water</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trypsin 0.25% with EDTA</td>
			<td>Hyclone, Logan, Utah, USA</td>
			<td>SH30042.02</td>
			<td>Tissue culture supplies</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">T75 cm2 flask</td>
			<td>Fisher Scientific, Waltham, MA, USA</td>
			<td>12-565-32</td>
			<td>Tissue culture supplies</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">15ml conical tube</td>
			<td>BD falcon, Franklin Lakes, NJ, USA</td>
			<td>352096</td>
			<td>Tissue culture supplies</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">50ml conical tube</td>
			<td>BD falcon, Franklin Lakes, NJ, USA</td>
			<td>352098</td>
			<td>Tissue culture supplies</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">60mm2 Petri dish</td>
			<td>Fisher Scientific, Waltham, MA, USA</td>
			<td>AS4052</td>
			<td>For lung imaging&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Isoflurane (Isothesia)</td>
			<td>Butler Schein Animal health, Dublin, OH, USA</td>
			<td>NDC 11695-6776-2</td>
			<td>Mouse anesthesia</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Clodronate liposomes</td>
			<td>Formumax Scientific Inc, Palo Alto, CA, USA</td>
			<td>F70101C-N</td>
			<td>Macrophages depletion</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Control liposomes</td>
			<td>Formumax Scientific Inc, Palo Alto, CA, USA</td>
			<td>F70101-N</td>
			<td>Control PBS-liposomes</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:313px;">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 height="21">
			<td height="21" style="height:21px;">Hair removal cream (Nair)</td>
			<td>Walmart, Bentonville, AR, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">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 height="21">
			<td height="21" style="height:21px;">OCT compound</td>
			<td>Fisher Scientific, Waltham, MA, USA</td>
			<td>230-730-571</td>
			<td>For freezing tissue in cryomolds</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fluoro-Gel-II with DAPI</td>
			<td>Electron Microscopy Sciences, Hatfield, PA, USA</td>
			<td>17985-51</td>
			<td>Mounting medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sucrose</td>
			<td>Sigma, St. Louis, MO, USA</td>
			<td>S-9378</td>
			<td style="width:287px;">Cryopreservation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Collagenase P</td>
			<td>Roche, Basel, Switzerland</td>
			<td>11249002001</td>
			<td>Components of tissue digestion buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dnase I</td>
			<td>Roche, Basel, Switzerland</td>
			<td>10104159001</td>
			<td>Components of tissue digestion buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trypsin inhibitor</td>
			<td>Sigma, St. Louis, MO, USA</td>
			<td>T9253</td>
			<td>Components of tissue digestion buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">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 height="21">
			<td height="21" style="height:21px;">Trustain FcX-Fc Block (CD16/CD32)</td>
			<td>Biolegend, San Diego, CA, USA</td>
			<td>101320</td>
			<td style="width:287px;">Antibodies for flow cytometry</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BV605 CD45</td>
			<td>Biolegend, San Diego, CA, USA</td>
			<td>103139</td>
			<td style="width:287px;">Antibodies for flow cytometry</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PE CD11b</td>
			<td>Biolegend, San Diego, CA, USA</td>
			<td>101207</td>
			<td style="width:287px;">Antibodies for flow cytometry</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PE Cy7 F4/80&#160;</td>
			<td>Biolegend, San Diego, CA, USA</td>
			<td>123113</td>
			<td style="width:287px;">Antibodies for flow cytometry</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">APC/Cy7 CD11c</td>
			<td>Biolegend, San Diego, CA, USA</td>
			<td>117323</td>
			<td style="width:287px;">Antibodies for flow cytometry</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PerCPcy5.5 IA/IE (MHCII)&#160;</td>
			<td>Biolegend, San Diego, CA, USA</td>
			<td>107625</td>
			<td style="width:287px;">Antibodies for flow cytometry</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PE CD80</td>
			<td>Biolegend, San Diego, CA, USA</td>
			<td>104707</td>
			<td style="width:287px;">Antibodies for flow cytometry</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">AF647 CD86</td>
			<td>Biolegend, San Diego, CA, USA</td>
			<td>105019</td>
			<td style="width:287px;">Antibodies for flow cytometry</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fixable viability Dye eflour 506</td>
			<td>eBioscience, San Diego, CA,USA</td>
			<td>65-0866</td>
			<td style="width:287px;">Antibodies for flow cytometry</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cryostat</td>
			<td>Leica Biosystems, Buffalo Grove, IL, USA</td>
			<td>CM1850</td>
			<td>Cryosectioning</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">UVP iBox Explorer</td>
			<td>UVP Inc, Upland, CA, USA</td>
			<td></td>
			<td>Mouse and lung fluorescent imaging</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Aperio Scanscope CS</td>
			<td>Leica Biosystems, Buffalo Grove, IL, USA</td>
			<td></td>
			<td>Digital pathology</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BD LSRFortessa&#160;</td>
			<td>BD Biosciences, Franklin Lakes, NJ, USA</td>
			<td></td>
			<td>Flow cytometry/data acquisition</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Nikon A1 confocal TE2000 microscope</td>
			<td>Nikon Instruments Inc., Melville, NY 11747-3064, U.S.A.</td>
			<td></td>
			<td style="width:287px;">Imaging and quantifying GFP fluorescence in lung cryosections</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:313px;">UVP visionworks software (Version 7.1RC3.38)</td>
			<td style="width:417px;">UVP Inc, Upland, CA, USA</td>
			<td style="width:139px;"></td>
			<td>Imaging software for iBOX</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Aperio Imagescope software (v12.1.0.5029)&#160;</td>
			<td>Leica Biosystems, Buffalo Grove, IL, USA</td>
			<td></td>
			<td style="width:287px;">Imaging software for analysis of digital slides</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">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 height="42">
			<td height="42" style="height:42px;width:313px;">NIS Elements AR (version 4.20.01) 64 Bit</td>
			<td>Nikon Instruments Inc., Melville, NY 11747-3064, U.S.A.</td>
			<td style="width:139px;"></td>
			<td style="width:287px;">Acquisition and analysis of lung cryosections for GFP&#160;</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 ...

The overall goal of these experiments is to determine to role of pulmonary alveolar macrophages in cancer metastasis using a syngenaic model of breast cancer. This method can answer some of the key questions in tumor immunology and cancer metastasis fields, such as why lungs are one of the most common target are hepathogenic cancer metastasis. The main advantage of this method that it uses very well-established breast cancer model with very specific method of the pulmioary alveolar microphages.Demonstrating this procedure will be Surya Kumari Vadrevu, a research associate in my laboratory. On the day of tumor cell injection, first place a shaved, anesthetized mouse on a surgical pad. Next, aspirate one time 10 to the fifth tumor cells in 100 microliters of PBS into a zero point five milliliter insulin syringe equipped with a 29-gauge needle.Then use the thumb and index finger to lift the skin near the second and third nipple, and insert the needle under the skin into the mammary fat pad just below the third nipple. Slowly subcutaneously inject the cells to form a bubble under the skin, and return the animal to its cage with monitoring until it is fully recovered. Four to five days post inoculation, to measure the tumors, palpagt the tumor site and use a caliper to use both the largest and smallest diameters of the tumors to determine the tumor volume.To image the injected 41GFP cells, place the anesthetized mouse on a movable stage inside the imager, and image the tumor site by fluorescence microscopy. In a laminer airflow biosafety cabinet, six days after tumor cell injection, vortex the liposomes to evenly distribute the particles, and aspirate 60 microliters of the suspension into a sterile pipette tip. Place the tip near the nostril of the anesthetized tumor-inoculated animal, and slowly release five microliters of the liposome solution, allowing the mouse to inhale the drop.It is critical to administer the liposome solution slowly while maintaining the appropriate level of anesthesia to allow the animal to breath regularly during the delivery. When the entire 60 microliters has been administered, allow the mouse to fully recover, and return the animal to its cage. To count and score the lung surface metastases, place the lungs under the dissection microscope and focus the objective until a clear view of the lung surface has been obtained.Then manually count the metasteses on the anterior and posterior sides of both lungs, and image the tumors by fluorescence microscopy. After counting and imaging the metastases, use surgical scissors and forceps to mince the lung and transfer the tissue pieces into a 15-milliliter conical tube containing three milliliters of digestion buffer. Dye test the fragments on a rotating shaker in a 37 degrees Celsius incubator for 40 minutes, and then triterate the slurry to dissociate the tissue.Next, filter the tissue suspension through a 40-micron strainer to remove any clumps, pressing the larger pieces through the strainer with a syringe plunger as necessary. When a single-cell suspension has been achieved, collect the cells by centrifugation and lyse the red blood cells with ACK buffer for 10 minutes at room temperature. Then, wash the pellet two times in eight milliliters of RPMI.After the second centrifugation, we suspend the cells in three milliliters of complete RPMI medium for counting, and spin down the cells again. Now dilute the cells to a one times 10 to the sixth cells per 100 microliters of FAX buffer concentration, and transfer 100 microliter eloquotes of cells into individual wells of a V-bottom, 96-well plate. Collect the cells in the bottom of the wells by centrifugation, then flip over the plate to let the buffer drain out.Block the cells with 100 microliters of CD16 CD32 FC block for 15 minutes at four degrees Celsius, and centrifuse the plate again. Then flip the plate to remove the supernatant, as just demonstrated, and add the antibody cocktail of interest to the appropriate wells. After 30 minutes at four degrees Celsius, pellet the cells by centrifugation, followed by a wash with 200 microliters of FAX buffer.Then re-suspend the pellets in 200 microliters of fresh PBS and stain the samples with viability dye for 20 minutes at four degrees Celsius. At the end of the incubation, wash the cells in another 200 microliters of PBS, and fix the samples in 100 microliters of one percent paraformaldehyde for storage at four degrees Celsius. Immediately prior to their analysis, transfer the cell suspensions to polypropelene flow cytometry tubes.The injection of 4T1GFP tumor cells into the mammary fat pad leads to the formation of mouse tumors that recapitulate the metastatic spread of human breast cancer, as evidenced by the rapidly forming metastases observed within the lungs. The stable transfection of 4T1 cells with GFP facilitates the tracking of metastasizing tumor cells and the quantification of the metastatic burden. Routine hastology, in conjunction with digital pathology algorithms also provide a good tool for quantifying and verifying metastases.Further, multi-color flow cycometric analysis allows the characterization of even rare cell populations, such as CD11b negative, CD11c F80 positive alveolar macrophages. The depletion of which, biclotrenate can be confirmed by the absence of these CD11c F480 positive cells in the dissociated lungs of lyposome-treated animals. After watching this video, you should have a good understanding of how to inject tumor cells into the mammary fat pad, monitor tumor growth and metastasis, and deplete alveolar macrophages, and prepare lung cells for flow cycometric analysis.

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9.5K 조회수.  Texas Tech University Health Science Center. The overall goal of these experiments is to determine to role of pulmonary alveolar macrophages in cancer metastasis using a syngenaic model of breast cancer. This method can answer some of the key questions in tumor immunology and cancer metastasis fields, such as why lungs are one of the most common target are hepathogenic cancer metastasis. The main advantage of this method that it uses very well-established breast cancer model with very specific method of the pulmioary alveolar microphage...