Name: studying the role of alveolar macrophages in breast cancer metastasis
Uploaded: 2016-06-26T15:00:00
Description: Watch this Scientific Journal Video about Studying the Role of Alveolar Macrophages in Breast Cancer Metastasis at JoVE.com

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>
	<li>Sceneay, J., Smyth, M. J., Moller, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+pre-metastatic+niche:+finding+common+ground.">The pre-metastatic niche: finding common ground.</a> Cancer Metastasis Rev. , (2013).</li><li>Fidler, I. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+pathogenesis+of+cancer+metastasis:+the+'seed+and+soil'+hypothesis+revisited.">The pathogenesis of cancer metastasis: the 'seed and soil' hypothesis revisited.</a> Nat Rev Cancer. 3, 453-458 (2003).</li><li>Kaplan, R. N., 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=VEGFR1-positive+haematopoietic+bone+marrow+progenitors+initiate+the+pre-metastatic+niche.">VEGFR1-positive haematopoietic bone marrow progenitors initiate the pre-metastatic niche.</a> Nature. 438, 820-827 (2005).</li><li>Hiratsuka, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MMP9+induction+by+vascular+endothelial+growth+factor+receptor-1+is+involved+in+lung-specific+metastasis.">MMP9 induction by vascular endothelial growth factor receptor-1 is involved in lung-specific metastasis.</a> Cancer Cell. 2, 289-300 (2002).</li><li>Hiratsuka, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+S100A8-serum+amyloid+A3-TLR4+paracrine+cascade+establishes+a+pre-metastatic+phase.">The S100A8-serum amyloid A3-TLR4 paracrine cascade establishes a pre-metastatic phase.</a> Nat Cell Biol. 10, 1349-1355 (2008).</li><li>Yan, H. 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. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue-resident+macrophages.">Tissue-resident macrophages.</a> Nat Immunol. 14, 986-995 (2013).</li><li>Holt, P. G., Strickland, D. H., Wikstrom, M. E., Jahnsen, F. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+immunological+homeostasis+in+the+respiratory+tract.">Regulation of immunological homeostasis in the respiratory tract.</a> Nat Rev Immunol. 8, 142-152 (2008).</li><li>Sharma, S. 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. P., Massague, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+metastasis:+building+a+framework.">Cancer metastasis: building a framework.</a> Cell. 127, 679-695 (2006).</li><li>Buiting, A. M., Van Rooijen, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liposome+mediated+depletion+of+macrophages:+an+approach+for+fundamental+studies.">Liposome mediated depletion of macrophages: an approach for fundamental studies.</a> J. Drug Target. 2, 357-362 (1994).</li><li>Vadrevu, S. 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=Complement+c5a+receptor+facilitates+cancer+metastasis+by+altering+T-cell+responses+in+the+metastatic+niche.">Complement c5a receptor facilitates cancer metastasis by altering T-cell responses in the metastatic niche.</a> Cancer Res. 74, 3454-3465 (2014).</li><li>Walrath, J. C., Hawes, J. J., Van Dyke, T., Reilly, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetically+engineered+mouse+models+in+cancer+research.">Genetically engineered mouse models in cancer research.</a> Adv. Cancer Res. 106, 113-164 (2010).</li><li>Bosiljcic, M., 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+regulate+the+lung+environment--letter.">Myeloid suppressor cells regulate the lung environment--letter.</a> Cancer Res. 71, 5050-5051 (2011).</li><li>Yan, H. 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=Myeloid+Suppressor+Cells+Regulate+the+Lung+Environment-Response.">Myeloid Suppressor Cells Regulate the Lung Environment-Response.</a> Cancer Res. 71, 5052-5053 (2011).</li><li>Van Rooijen, N., Sanders, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liposome+mediated+depletion+of+macrophages:+mechanism+of+action,+preparation+of+liposomes+and+applications.">Liposome mediated depletion of macrophages: mechanism of action, preparation of liposomes and applications.</a> J. Immunol. Methods. 174, 83-93 (1994).</li></ol>

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.

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

Research

JoVE Journal

Medicine

In vivo Bioluminescence Imaging of Tumor Hypoxia Dynamics of Breast Cancer Brain Metastasis in a Mouse Model

It is well recognized that tumor hypoxia plays an important role in promoting malignant progression and affecting therapeutic response negatively. There is little knowledge about in situ, in vivo, tumor hypoxia during intracranial development of malignant brain tumors because of lack of efficient means to monitor it in these deep-seated orthotopic tumors. Bioluminescence imaging (BLI), based on the detection of light emitted by living cells expressing a luciferase gene, has been rapidly adopted for cancer research, in particular, to evaluate tumor growth or tumor size changes in response to treatment in preclinical animal studies. Moreover, by expressing a reporter gene under the control of a promoter sequence, the specific gene expression can be monitored non-invasively by BLI. Under hypoxic stress, signaling responses are mediated mainly via the hypoxia inducible factor-1&alpha; (HIF-1&alpha;) to drive transcription of various genes. Therefore, we have used a HIF-1&alpha; reporter construct, 5HRE-ODD-luc, stably transfected into human breast cancer MDA-MB231 cells (MDA-MB231/5HRE-ODD-luc). In vitro HIF-1&alpha; bioluminescence assay is performed by incubating the transfected cells in a hypoxic chamber (0.1% O2) for 24 hr before BLI, while the cells in normoxia (21% O2) serve as a control. Significantly higher photon flux observed for the cells under hypoxia suggests an increased HIF-1&alpha; binding to its promoter (HRE elements), as compared to those in normoxia. Cells are injected directly into the mouse brain to establish a breast cancer brain metastasis model. In vivo bioluminescence imaging of tumor hypoxia dynamics is initiated 2 wks after implantation and repeated once a week. BLI reveals increasing light signals from the brain as the tumor progresses, indicating increased intracranial tumor hypoxia. Histological and immunohistochemical studies are used to confirm the in vivo imaging results. Here, we will introduce approaches of in vitro HIF-1&alpha; bioluminescence assay, surgical establishment of a breast cancer brain metastasis in a nude mouse and application of in vivo bioluminescence imaging to monitor intracranial tumor hypoxia.

In vivo Bioluminescence Imaging of Tumor Hypoxia Dynamics of Breast Cancer Brain Metastasis in a Mouse Model

Bioluminescence imaging of hypoxia inducible factor-1&alpha; activity is applied to monitor intracranial tumor hypoxia development in a breast cancer brain metastasis mouse model.

It is well recognized that tumor hypoxia plays an important role in promoting malignant progression and affecting therapeutic response negatively. There ...

In vitro Mesothelial Clearance Assay that Models the Early Steps of Ovarian Cancer Metastasis

Ovarian cancer is the fifth leading cause of cancer related deaths in the United States1. Despite a positive initial response to therapies, 70 to 90 percent of women with ovarian cancer develop new metastases, and the recurrence is often fatal2. It is, therefore, necessary to understand how secondary metastases arise in order to develop better treatments for intermediate and late stage ovarian cancer. Ovarian cancer metastasis occurs when malignant cells detach from the primary tumor site and disseminate throughout the peritoneal cavity. The disseminated cells can form multicellular clusters, or spheroids, that will either remain unattached, or implant onto organs within the peritoneal cavity3 (Figure 1, Movie 1). 
All of the organs within the peritoneal cavity are lined with a single, continuous, layer of mesothelial cells4-6 (Figure 2). However, mesothelial cells are absent from underneath peritoneal tumor masses, as revealed by electron micrograph studies of excised human tumor tissue sections3,5-7 (Figure 2). This suggests that mesothelial cells are excluded from underneath the tumor mass by an unknown process. 
Previous in vitro experiments demonstrated that primary ovarian cancer cells attach more efficiently to extracellular matrix than to mesothelial cells8, and more recent studies showed that primary peritoneal mesothelial cells actually provide a barrier to ovarian cancer cell adhesion and invasion (as compared to adhesion and invasion on substrates that were not covered with mesothelial cells)9,10. This would suggest that mesothelial cells act as a barrier against ovarian cancer metastasis. The cellular and molecular mechanisms by which ovarian cancer cells breach this barrier, and exclude the mesothelium have, until recently, remained unknown. 
Here we describe the methodology for an in vitro assay that models the interaction between ovarian cancer cell spheroids and mesothelial cells in vivo (Figure 3, Movie 2). Our protocol was adapted from previously described methods for analyzing ovarian tumor cell interactions with mesothelial monolayers8-16, and was first described in a report showing that ovarian tumor cells utilize an integrin &#x2013;dependent activation of myosin and traction force to promote the exclusion of the mesothelial cells from under a tumor spheroid17. This model takes advantage of time-lapse fluorescence microscopy to monitor the two cell populations in real time, providing spatial and temporal information on the interaction. The ovarian cancer cells express red fluorescent protein (RFP) while the mesothelial cells express green fluorescent protein (GFP). RFP-expressing ovarian cancer cell spheroids attach to the GFP-expressing mesothelial monolayer. The spheroids spread, invade, and force the mesothelial cells aside creating a hole in the monolayer. This hole is visualized as the negative space (black) in the GFP image. The area of the hole can then be measured to quantitatively analyze differences in clearance activity between control and experimental populations of ovarian cancer and/ or mesothelial cells. This assay requires only a small number of ovarian cancer cells (100 cells per spheroid X 20-30 spheroids per condition), so it is feasible to perform this assay using precious primary tumor cell samples. Furthermore, this assay can be easily adapted for high throughput screening.

In vitro Mesothelial Clearance Assay that Models the Early Steps of Ovarian Cancer Metastasis

The mesothelial clearance assay described here takes advantage of fluorescently labeled cells and time-lapse video microscopy to visualize and quantitatively measure the interactions of ovarian cancer multicellular spheroids and mesothelial cell monolayers. This assay models the early steps of ovarian cancer metastasis.

Ovarian cancer is the fifth leading cause of cancer related deaths in the United States1. Despite a positive initial response to therapies, 70 to 90 ...

Multi-modal Imaging of Angiogenesis in a Nude Rat Model of Breast Cancer Bone Metastasis Using Magnetic Resonance Imaging, Volumetric Computed Tomography and Ultrasound

Angiogenesis is an essential feature of cancer growth and metastasis formation. In bone metastasis, angiogenic factors are pivotal for tumor cell proliferation in the bone marrow cavity as well as for interaction of tumor and bone cells resulting in local bone destruction. Our aim was to develop a model of experimental bone metastasis that allows in vivo assessment of angiogenesis in skeletal lesions using non-invasive imaging techniques.
For this purpose, we injected 105 MDA-MB-231 human breast cancer cells into the superficial epigastric artery, which precludes the growth of metastases in body areas other than the respective hind leg1. Following 25-30 days after tumor cell inoculation, site-specific bone metastases develop, restricted to the distal femur, proximal tibia and proximal fibula1. Morphological and functional aspects of angiogenesis can be investigated longitudinally in bone metastases using magnetic resonance imaging (MRI), volumetric computed tomography (VCT) and ultrasound (US).
MRI displays morphologic information on the soft tissue part of bone metastases that is initially confined to the bone marrow cavity and subsequently exceeds cortical bone while progressing. Using dynamic contrast-enhanced MRI (DCE-MRI) functional data including regional blood volume, perfusion and vessel permeability can be obtained and quantified2-4. Bone destruction is captured in high resolution using morphological VCT imaging. Complementary to MRI findings, osteolytic lesions can be located adjacent to sites of intramedullary tumor growth. After contrast agent application, VCT angiography reveals the macrovessel architecture in bone metastases in high resolution, and DCE-VCT enables insight in the microcirculation of these lesions5,6. US is applicable to assess morphological and functional features from skeletal lesions due to local osteolysis of cortical bone. Using B-mode and Doppler techniques, structure and perfusion of the soft tissue metastases can be evaluated, respectively. DCE-US allows for real-time imaging of vascularization in bone metastases after injection of microbubbles7.
In conclusion, in a model of site-specific breast cancer bone metastases multi-modal imaging techniques including MRI, VCT and US offer complementary information on morphology and functional parameters of angiogenesis in these skeletal lesions.

In the pathogenesis of bone metastasis, angiogenesis is a crucial process and therefore represents a target for imaging and therapy. Here, we present a rat model of site-specific breast cancer bone metastasis and describe strategies to non-invasively image angiogenesis in vivo using magnetic resonance imaging, volumetric computed tomography and ultrasound.

Angiogenesis is an essential feature of cancer growth and metastasis formation. In bone metastasis, angiogenic factors are pivotal for tumor cell ...

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 ...

An Orthotopic Murine Model of Human Prostate Cancer Metastasis

Our laboratory has developed a novel orthotopic implantation model of human prostate cancer (PCa). As PCa death is not due to the primary tumor, but rather the formation of distinct metastasis, the ability to effectively model this progression pre-clinically is of high value. In this model, cells are directly implanted into the ventral lobe of the prostate in Balb/c athymic mice, and allowed to progress for 4-6 weeks. At experiment termination, several distinct endpoints can be measured, such as size and molecular characterization of the primary tumor, the presence and quantification of circulating tumor cells in the blood and bone marrow, and formation of metastasis to the lung. In addition to a variety of endpoints, this model provides a picture of a cells ability to invade and escape the primary organ, enter and survive in the circulatory system, and implant and grow in a secondary site. This model has been used effectively to measure metastatic response to both changes in protein expression as well as to response to small molecule therapeutics, in a short turnaround time.

This orthotopic model of human prostate cancer allows for quantification of tumor size, circulating tumor cells, and formation of distinct metastasis to the lung. As cells must escape the primary organ, enter the blood stream, and implant into a secondary site, this model effectively recapitulates the scenario in humans.

Our laboratory has developed a novel orthotopic implantation model of  human prostate cancer (PCa). As PCa death is not due to the primary tumor, but  ...

Initiation of Metastatic Breast Carcinoma by Targeting of the Ductal Epithelium with Adenovirus-Cre: A Novel Transgenic Mouse Model of Breast Cancer

Breast cancer is a heterogeneous disease involving complex cellular interactions between the developing tumor and immune system, eventually resulting in exponential tumor growth and metastasis to distal tissues and the collapse of anti-tumor immunity. Many useful animal models exist to study breast cancer, but none completely recapitulate the disease progression that occurs in humans. In order to gain a better understanding of the cellular interactions that result in the formation of latent metastasis and decreased survival, we have generated an inducible transgenic mouse model of YFP-expressing ductal carcinoma that develops after sexual maturity in immune-competent mice and is driven by consistent, endocrine-independent oncogene expression. Activation of YFP, ablation of p53, and expression of an oncogenic form of K-ras was achieved by the delivery of an adenovirus expressing Cre-recombinase into the mammary duct of sexually mature, virgin female mice. Tumors begin to appear 6 weeks after the initiation of oncogenic events. After tumors become apparent, they progress slowly for approximately two weeks before they begin to grow exponentially. After 7-8 weeks post-adenovirus injection, vasculature is observed connecting the tumor mass to distal lymph nodes, with eventual lymphovascular invasion of YFP+ tumor cells to the distal axillary lymph nodes. Infiltrating leukocyte populations are similar to those found in human breast carcinomas, including the presence of &#945;&#946; and &#947;&#948; T cells, macrophages and MDSCs. This unique model will facilitate the study of cellular and immunological mechanisms involved in latent metastasis and dormancy in addition to being useful for designing novel immunotherapeutic interventions to treat invasive breast cancer.

Activation of latent mutations with adenovirus-Cre into the mammary ductal system results in a clinically relevant metastatic breast cancer. Incorporation of a YFP promoter allows tracking of distal metastatic tumor cells. This model is useful to study latent metastasis, anti-tumor immunity, and for designing novel immunotherapies to treat breast cancer.

Breast cancer is a heterogeneous disease involving complex cellular interactions between the developing tumor and immune system, eventually resulting in ...

Profiling of Estrogen-regulated MicroRNAs in Breast Cancer Cells

Estrogen plays vital roles in mammary gland development and breast cancer progression. It mediates its function by binding to and activating the estrogen receptors (ERs), ER&#945;, and ER&#946;. ER&#945; is frequently upregulated in breast cancer and drives the proliferation of breast cancer cells. The ERs function as transcription factors and regulate gene expression. Whereas ER&#945;&#39;s regulation of protein-coding genes is well established, its regulation of noncoding microRNA (miRNA) is less explored. miRNAs play a major role in the post-transcriptional regulation of genes, inhibiting their translation or degrading their mRNA. miRNAs can function as oncogenes or tumor suppressors&#160;and are also promising biomarkers. Among the miRNA assays available, microarray and quantitative real-time polymerase chain reaction (qPCR) have been extensively used to detect and quantify miRNA levels. To identify miRNAs regulated by estrogen signaling in breast cancer, their expression in ER&#945;-positive breast cancer cell lines were compared before and after estrogen-activation using both the &#181;Paraflo-microfluidic microarrays and Dual Labeled Probes-low density arrays. Results were validated using specific qPCR assays, applying both Cyanine dye-based and Dual Labeled Probes-based chemistry. Furthermore, a time-point assay was used to identify regulations over time. Advantages of the miRNA assay approach used in this study is that it enables a fast screening of mature miRNA regulations in numerous samples, even with limited sample amounts. The layout, including the specific conditions for cell culture and estrogen treatment, biological and technical replicates, and large-scale screening followed by in-depth confirmations using separate techniques, ensures a robust detection of miRNA regulations,&#160;and eliminates false positives and other artifacts. However, mutated or unknown miRNAs, or regulations at the primary and precursor transcript level, will not be detected. The method presented here represents a thorough investigation of estrogen-mediated miRNA regulation.

Molecular signaling through both estrogen and microRNAs are critical in breast cancer development and growth. Estrogen activates the estrogen receptors, which are transcription factors. Many transcription factors can regulate the expression of microRNAs, and estrogen-regulated microRNAs can be profiled using different large-scale techniques.

Estrogen plays vital roles in mammary gland development and breast cancer progression. It mediates its function by binding to and activating the estrogen ...

An Ex vivo Model to Study Hormone Action in the Human Breast

The study of hormone action in the human breast has been hampered by lack of adequate model systems. Upon in vitro culture, primary mammary epithelial cells tend to lose hormone receptor expression. Widely used hormone receptor positive breast cancer cell lines are of limited relevance to the in vivo situation. Here, we describe an ex vivo model to study hormone action in the human breast. Fresh human breast tissue specimens from surgical discard material such as reduction mammoplasties or mammectomies are mechanically and enzymatically digested to obtain tissue fragments containing ducts and lobules and multiple stromal cell types. These tissue microstructures kept in basal medium without growth factors preserve their intercellular contacts, the tissue architecture, and remain hormone responsive for several days. They are readily processed for RNA and protein extraction, histological analysis or stored in freezing medium. Fluorescence activated cell sorting (FACS) can be used to enrich for specific cell populations. This protocol provides a straightforward, standard approach for translational studies with highly complex, varied human specimens.

An Ex vivo Model to Study Hormone Action in the Human Breast

We have developed a novel ex vivo model to study hormone action in the human breast. It is based on tissue microstructures isolated from surgical breast tissue specimens which preserve tissue architecture, intercellular interactions, and paracrine signaling.

The study of hormone action in the human breast has been hampered by lack of adequate model systems. Upon in vitro culture, primary mammary epithelial ...

In Vivo and Ex Vivo Approaches to Study Ovarian Cancer Metastatic Colonization of Milky Spot Structures in Peritoneal Adipose

High-grade serous ovarian cancer (HGSC), the cause of widespread peritoneal metastases, continues to have an extremely poor prognosis; fewer than 30% of women are alive 5 years after diagnosis. The omentum is a preferred site of HGSC metastasis formation. Despite the clinical importance of this microenvironment, the contribution of omental adipose tissue to ovarian cancer progression remains understudied. Omental adipose is unusual in that it contains structures known as milky spots, which are comprised of B, T, and NK cells, macrophages, and progenitor cells surrounding dense nests of vasculature. Milky spots play a key role in the physiologic functions of the omentum, which are required for peritoneal homeostasis. We have shown that milky spots also promote ovarian cancer metastatic colonization of peritoneal adipose, a key step in the development of peritoneal metastases. Here we describe the approaches we developed to evaluate and quantify milky spots in peritoneal adipose and study their functional contribution to ovarian cancer cell metastatic colonization of omental tissues both in vivo and ex vivo. These approaches are generalizable to additional mouse models and cell lines, thus enabling the study of ovarian cancer metastasis formation from initial localization of cells to milky spot structures to the development of widespread peritoneal metastases.

In Vivo and Ex Vivo Approaches to Study Ovarian Cancer Metastatic Colonization of Milky Spot Structures in Peritoneal Adipose

We outline a protocol that implements both in vivo and ex vivo approaches to study ovarian cancer colonization of peritoneal adipose tissues, particularly the omentum. Furthermore, we present a protocol to quantitate and analyze immune cell-structures in the omentum known as milky spots, which promote metastases of peritoneal adipose.

High-grade serous ovarian cancer (HGSC), the cause of widespread peritoneal metastases, continues to have an extremely poor prognosis; fewer than 30% of ...

Utilization of the Soft Agar Colony Formation Assay to Identify Inhibitors of Tumorigenicity in Breast Cancer Cells

Given the inherent difficulties in investigating the mechanisms of tumor progression in vivo, cell-based assays such as the soft agar colony formation assay (hereafter called soft agar assay), which measures the ability of cells to proliferate in semi-solid matrices, remain a hallmark of cancer research. A key advantage of this technique over conventional 2D monolayer or 3D spheroid cell culture assays is the close mimicry of the 3D cellular environment to that seen in vivo. Importantly, the soft agar assay also provides an ideal tool to rigorously test the effects of novel compounds or treatment conditions on cell proliferation and migration. Additionally, this assay enables the quantitative assessment of cell transformation potential within the context of genetic perturbations. We recently identified peptidylarginine deiminase 2 (PADI2) as a potential breast cancer biomarker and therapeutic target. Here we highlight the utility of the soft agar assay for preclinical anti-cancer studies by testing the effects of the PADI inhibitor, BB-Cl-amidine (BB-CLA), on the tumorigenicity of human ductal carcinoma in situ (MCF10DCIS) cells.

Here, we document the use of the soft agar colony formation assay to test the effects of a peptidylarginine deiminase (PADI) enzyme inhibitor, BB-Cl-amidine, on breast cancer tumorigenicity in vitro.

Given the inherent difficulties in investigating the mechanisms of tumor progression in vivo, cell-based assays such as the soft agar colony formation ...