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

Denne forskningen har blitt støttet av Department of Defense gi TSA 140 010 til MK og BC 111038 til MMM synspunkter og meninger, og påtegninger av forfatteren (e) ikke gjenspeiler de av den amerikanske hæren eller det amerikanske forsvarsdepartementet.

Alle dyrestudier har blitt godkjent av Institutional Animal Care og bruk Committee of Texas Tech University Health Sciences Center og fulgt retningslinjene som er skissert i &quot;Guide for omsorg og bruk av forsøksdyr&quot; utgitt av National Institutes of Health. Bruke åtte til tolv uker gamle hunn BALB / c-mus som er kommersielt tilgjengelige. Injisere 1 x 10 5 4T1 eller 1 x 10 5 4T1 celler som uttrykker GFP, som kan kjøpes fra ulike leverandører, inn i melkefettpute. <p class="jove_titl...

<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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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>
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		<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>
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			<td height="21" style="height:21px;">Heat inactivated FBS</td>
			<td>Gibco (Thermo Scientific), USA</td>
			<td>10082147</td>
			<td>Media</td>
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			<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>
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			<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>
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			<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>
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			<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>
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			<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>
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		<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>
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		<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>
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			<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>
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			<td height="21" style="height:21px;">Hair removal cream (Nair)</td>
			<td>Walmart, Bentonville, AR, USA</td>
			<td></td>
			<td></td>
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			<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>
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		<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>
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		<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>
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		<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>
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		<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>
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		<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>
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		<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>
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		<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>
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		<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>
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			<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>
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		<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>
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		<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>
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		<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>
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		<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>
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		<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>
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		<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.

Injeksjon av 4T1-GFP tumorceller inn i melkefettpute fører til dannelse av musetumorer (figur 1A) som rekapitulere den metastatisk spredning av human brystcancer, som metastaser blir raskt dannet i lungene (figur 2), lever, ben og hjernen til mus 11. Den stabile transfeksjon av 4T1 celler med GFP forenkler overvåking av tumorvekst (figur 1B), sporing metastasizing kreftceller og kvantifisere metastatisk belastning (f...

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.

Studerer rolle alveolære makrofager i Breast Cancer Metastase

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 ...

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Medicine

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

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

Multi-modal Imaging av angiogenese i en Nude Rat Model of Breast Cancer Bone Metastase Bruke Magnetic Resonance Imaging, Volumetrisk computertomografi og ultralyd

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

Models of Bone Metastasis

Modeller for Bone Metastase

Bone metastases are a common occurrence in several malignancies, including breast, prostate, and lung. Once established in bone, tumors are responsible ...

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

Lever Imaging of Drug Responses i svulsten mikromiljøet i musemodeller av Breast Cancer

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

En orthotopic Murine Model of Human Prostate Cancer Metastase

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

Innvielse av metastatisk brystkreft ved målretting av Ductal Epitel med Adenovirus-Cre: A Novel transgene mus modell av brystkreft

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

Profilering av Østrogen-regulerte MicroRNAs i brystkreftceller

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

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

En<em&gt; Ex vivo</em&gt; Modell for å studere Hormone Handling i 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 ...

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

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

I Vivo og Ex Vivo tilnærminger til Study Ovarian Cancer Metastatisk Kolonisering av Milky Spot Structures i 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 ...